Jordan Journal of Biological Sciences (JJBS) (ISSN: 1995–6673 (Print); 2307-7166 (Online)): An International Peer- Reviewed Open Access Research Journal financed by the Scientific Research Support Fund, Ministry of Higher Education and Scientific Research, Jordan and published quarterly by the Deanship of Research and Graduate Studies, The Hashemite University, Jordan.

Editor-in-Chief

Assistant Chief Dr. Tahtamouni Lubna H. Developmental Biology, The Hashemite University

Editorial Board (Arranged alphabetically) Professor Abdalla, Shtaywy S Human Physiology, Tafila Technical University

Professor Lahham, Jamil N. Plant Taxonomy, Yarmouk University

Professor Al-Hadidi, Hakam F. Toxicology and Clinical Pharmacology

Professor Bashir, Nabil A. Biochemistry and Molecular Genetics

Jordan University of Science and Technology

Professor Khyami-Horani, Hala. Microbial Biotechnology, The University of Jordan

Professor Tarawneh, Khaled A. Molecular Microbiology, Mutah University

Submission Address Editorial Board Support Team Professor Abu-Elteen, Khaled H Language Editor Publishing Layout The Hashemite University Dr. Qusai Al-Debyan Eng.Mohannad Oqdeh P.O. Box 330127, Zarqa, 13115, Jordan Phone: +962-5-3903333 ext. 5157 E-Mail: jjbs@hu.edu.jo

Jordan Journal of Biological Sciences (JJBS) http://jjbs.hu.edu.jo

��المجلة األردنية للعلوم الحياتية��

Professor Abu-Elteen, Khaled H. Medical Mycology ,The Hashemite University

Jordan University of Science and Technology

Jordan University of Science and TechnologyApplied Microbiology

Professor Sallal, Abdul-Karim J.

Professor Elkarmi, Ali Z. Bioengineering, The Hashemite University

International Advisory Board

Prof. Abdel-Hafez, Sami K. Yarmouk University, Jordan

Prof. Abdul-Haque, Allah Hafiz National Institute for Biotechnology and Genetic Engineering, Pakistan

Prof. Al-Najjar, Tariq Hasan Ahmad The University of Jordan, Jordan

Prof. Bamburg, James Colorado State University, U.S.A.

Prof. Garrick, Michael D State University of New York at Buffalo, U.S.A.

Prof. Gurib-Fakim, Ameenah F 0T 0TCenter for Phytotherapy and Research,Ebene,Mauritius.

Prof. Hanawalt ,Philip C Stanford University Stanford , U.S.A

Prof. Hunaiti, Abdelrahim A. University of Jordan. Jordan

Prof. Kaviraj, Anilava India University of Kalyani,Kenya

Prof. Matar, Ghassan M American University of Beirut, Lebanon

Prof. Nasher, Abdul Karim Sanna' University, Yemen

Prof. Qoronfleh,Mohammad Walid Director of Biotechnology Biomedical Research Institute . Qatar

Prof. Schatten, Gerald University of Pittsburgh School of Medicine,U.S.A

Prof. Stanway, Glyn University of Essex, England

Prof. Waitzbauer, Wolfgang University of Vienna, Austria

Associate Editorial Board

Professor Al-Hindi, Adnan I. Dr. Fass, Uwe W. The Islamic University of Gaza, Palestine Oman Medical College,Sulatante of Oman

Professor Al-Homida, Abdullah S. Dr. Gammoh, Noor King Saud University, Saudi Arabia The University of Edinburgh, UK

Professor Kachani, Malika Western University of Health Sciences, USA

Jordan Journal of Biological Sciences (JJBS) http://jjbs.hu.edu.jo

��المجلة األردنية للعلوم الحياتية��

Prof. Abuharfeil, Nizar M Jordan University of science and Technology, Jordan

Prof. Amr,Zuhair Jordan University of science and Technology, Jordan

Prof. El Makawy, Aida, I National Research Center ,Giza, Egypt

Prof. Ghannoum, Mahmoud A. University Hospital of Cleveland and Case Western Reserve University, U.S.A.

Prof. Hamad, Mawieh, University of Sharjah, U.A,E

Prof. Hassanali, Ahmed Kenyatta University, Nairobi,Kenya

Prof. Ismail,Naim The Hashemite University, Jordan

Prof. Kilbane, John J Intertek, Houston, Texas, U.S.A.

Prof. Martens, Jochen Institute Fur Zoologie, Germany

Prof. Na'was,Tarek E Lebanese American University,Lebanon

Prof. Sadiq, May Fouad George Yarmouk University, Jordan

Prof. Shakhanbeh, Jumah Mutie Mutah University ,Jordan

Prof. Tamimi,Samih Mohammad University of Jordan, Jordan

Prof. Wan Yusoff, Wan Mohtar University Kebangsaan Malaysia, Malaysia

Instructions to Authors Scope Study areas include cell biology, genomics, microbiology, immunology, molecular biology, biochemistry, embryology, immunogenetics, cell and tissue culture, molecular ecology, genetic engineering and biological engineering, bioremediation and biodegradation, bioinformatics, biotechnology regulations, gene therapy, organismal biology, microbial and environmental biotechnology, marine sciences. The JJBS welcomes the submission of manuscript that meets the general criteria of significance and academic excellence. All articles published in JJBS are peer-reviewed. Papers will be published approximately one to two months after acceptance. Type of Papers The journal publishes high-quality original scientific papers, short communications, correspondence and case studies. Review articles are usually by invitation only. However, Review articles of current interest and high standard will be considered. Submission of Manuscript Manuscript, or the essence of their content, must be previously unpublished and should not be under simultaneous consideration by another journal. The authors should also declare if any similar work has been submitted to or published by another journal. They should also declare that it has not been submitted/ published elsewhere in the same form, in English or in any other language, without the written consent of the Publisher. The authors should also declare that the paper is the original work of the author(s) and not copied (in whole or in part) from any other work. All papers will be automatically checked for duplicate publication and plagiarism. If detected, appropriate action will be taken in accordance with International Ethical Guideline. By virtue of the submitted manuscript, the corresponding author acknowledges that all the co-authors have seen and approved the final version of the manuscript. The corresponding author should provide all co-authors with information regarding the manuscript, and obtain their approval before submitting any revisions. Electronic submission of manuscripts is strongly recommended, provided that the text, tables and figures are included in a single Microsoft Word file. Submit manuscript as e-mail attachment to the Editorial Office at: JJBS@hu.edu.jo. After submission, a manuscript number will be communicated to the corresponding author within 48 hours. Peer-review Process It is requested to submit, with the manuscript, the names, addresses and e-mail addresses of at least 4 potential reviewers. It is the sole right of the editor to decide whether or not the suggested reviewers to be used. The reviewers’ comments will be sent to authors within 6-8 weeks after submission. Manuscripts and figures for review will not be returned to authors whether the editorial decision is to accept, revise, or reject. All Case Reports and Short Communication must include at least one table and/ or one figure. Preparation of Manuscript The manuscript should be written in English with simple lay out. The text should be prepared in single column format. Bold face, italics, subscripts, superscripts etc. can be used. Pages should be numbered consecutively, beginning with the title page and continuing through the last page of typewritten material. The text can be divided into numbered sections with brief headings. Starting from introduction with section 1. Subsections should be numbered (for example 2.1 (then 2.1.1, 2.1.2, 2.2, etc.), up to three levels. Manuscripts in general should be organized in the following manner: Title Page The title page should contain a brief title, correct first name, middle initial and family name of each author and name and address of the department(s) and institution(s) from where the research was carried out for each author. The title should be without any abbreviations and it should enlighten the contents of the paper. All affiliations should be provided with a lower-case superscript number just after the author's name and in front of the appropriate address. The name of the corresponding author should be indicated along with telephone and fax numbers (with country and area code) along with full postal address and e-mail address.

Abstract The abstract should be concise and informative. It should not exceed 350 words in length for full manuscript and Review article and 150 words in case of Case Report and/ or Short Communication. It should briefly describe the purpose of the work, techniques and methods used, major findings with important data and conclusions. No references should be cited in this part. Generally non-standard abbreviations should not be used, if necessary they should be clearly defined in the abstract, at first use. Keywords Immediately after the abstract, about 4-8 keywords should be given. Use of abbreviations should be avoided, only standard abbreviations, well known in the established area may be used, if appropriate. These keywords will be used for indexing. Abbreviations Non-standard abbreviations should be listed and full form of each abbreviation should be given in parentheses at first use in the text.

Introduction Provide a factual background, clearly defined problem, proposed solution, a brief literature survey and the scope and justification of the work done. Materials and Methods Give adequate information to allow the experiment to be reproduced. Already published methods should be mentioned with references. Significant modifications of published methods and new methods should be described in detail. Capitalize trade names and include the manufacturer’s name and address. Subheading should be used. Results Results should be clearly described in a concise manner. Results for different parameters should be described under subheadings or in separate paragraph. Results should be explained, but largely without referring to the literature. Table or figure numbers should be mentioned in parentheses for better understanding. Discussion The discussion should not repeat the results, but provide detailed interpretation of data. This should interpret the significance of the findings of the work. Citations should be given in support of the findings. The results and discussion part can also be described as separate, if appropriate. The Results and Discussion sections can include subheadings, and when appropriate, both sections can be combined Conclusions This should briefly state the major findings of the study. Acknowledgment A brief acknowledgment section may be given after the conclusion section just before the references. The acknowledgment of people who provided assistance in manuscript preparation, funding for research, etc. should be listed in this section. Tables and Figures Tables and figures should be presented as per their appearance in the text. It is suggested that the discussion about the tables and figures should appear in the text before the appearance of the respective tables and figures. No tables or figures should be given without discussion or reference inside the text. Tables should be explanatory enough to be understandable without any text reference. Double spacing should be maintained throughout the table, including table headings and footnotes. Table headings should be placed above the table. Footnotes should be placed below the table with superscript lowercase letters. Each table should be on a separate page, numbered consecutively in Arabic numerals. Each figure should have a ca ption. The caption should be concise and typed separately, not on the figure area. Figures should be self-explanatory. Information presented in the figure should not be repeated in the table. All symbols and abbreviations used in the illustrations should be defined clearly. Figure legends should be given below the figures.

Instructions to Authors Scope Study areas include cell biology, genomics, microbiology, immunology, molecular biology, biochemistry, embryology, immunogenetics, cell and tissue culture, molecular ecology, genetic engineering and biological engineering, bioremediation and biodegradation, bioinformatics, biotechnology regulations, gene therapy, organismal biology, microbial and environmental biotechnology, marine sciences. The JJBS welcomes the submission of manuscript that meets the general criteria of significance and academic excellence. All articles published in JJBS are peer-reviewed. Papers will be published approximately one to two months after acceptance. Type of Papers The journal publishes high-quality original scientific papers, short communications, correspondence and case studies. Review articles are usually by invitation only. However, Review articles of current interest and high standard will be considered. Submission of Manuscript Manuscript, or the essence of their content, must be previously unpublished and should not be under simultaneous consideration by another journal. The authors should also declare if any similar work has been submitted to or published by another journal. They should also declare that it has not been submitted/ published elsewhere in the same form, in English or in any other language, without the written consent of the Publisher. The authors should also declare that the paper is the original work of the author(s) and not copied (in whole or in part) from any other work. All papers will be automatically checked for duplicate publication and plagiarism. If detected, appropriate action will be taken in accordance with International Ethical Guideline. By virtue of the submitted manuscript, the corresponding author acknowledges that all the co-authors have seen and approved the final version of the manuscript. The corresponding author should provide all co-authors with information regarding the manuscript, and obtain their approval before submitting any revisions. Electronic submission of manuscripts is strongly recommended, provided that the text, tables and figures are included in a single Microsoft Word file. Submit manuscript as e-mail attachment to the Editorial Office at: JJBS@hu.edu.jo. After submission, a manuscript number will be communicated to the corresponding author within 48 hours. Peer-review Process It is requested to submit, with the manuscript, the names, addresses and e-mail addresses of at least 4 potential reviewers. It is the sole right of the editor to decide whether or not the suggested reviewers to be used. The reviewers’ comments will be sent to authors within 6-8 weeks after submission. Manuscripts and figures for review will not be returned to authors whether the editorial decision is to accept, revise, or reject. All Case Reports and Short Communication must include at least one table and/ or one figure. Preparation of Manuscript The manuscript should be written in English with simple lay out. The text should be prepared in single column format. Bold face, italics, subscripts, superscripts etc. can be used. Pages should be numbered consecutively, beginning with the title page and continuing through the last page of typewritten material. The text can be divided into numbered sections with brief headings. Starting from introduction with section 1. Subsections should be numbered (for example 2.1 (then 2.1.1, 2.1.2, 2.2, etc.), up to three levels. Manuscripts in general should be organized in the following manner: Title Page The title page should contain a brief title, correct first name, middle initial and family name of each author and name and address of the department(s) and institution(s) from where the research was carried out for each author. The title should be without any abbreviations and it should enlighten the contents of the paper. All affiliations should be provided with a lower-case superscript number just after the author's name and in front of the appropriate address. The name of the corresponding author should be indicated along with telephone and fax numbers (with country and area code) along with full postal address and e-mail address.

References References should be listed alphabetically at the end of the manuscript. Every reference referred in the text must be also present in the reference list and vice versa. In the text, a reference identified by means of an author’s name should be followed by the year of publication in parentheses ( e.g.( Brown,2009)). For two authors, both authors’ names followed by the year of publication (e.g.( Nelson and Brown, 2007)). When there are more than two authors, only the first author's name followed by "et al." and the year of publication ( e.g. ( Abu-Elteen et al., 2010)). When two or more works of an author has been published during the same year, the reference should be identified by the letters "a", "b", "c", etc., placed after the year of publication. This should be followed both in the text and reference list. e.g., Hilly, (2002a, 2002b); Hilly, and Nelson, (2004). Articles in preparation or submitted for publication, unpublished observations, personal communications, etc. should not be included in the reference list but should only be mentioned in the article text ( e.g., Shtyawy,A., University of Jordan, personal communication). Journal titles should be abbreviated according to the system adopted in Biological Abstract and Index Medicus, if not included in Biological Abstract or Index Medicus journal title should be given in full. The author is responsible for the sccuracy and completeness of the references and for their correct textual citation. Failure to do so may result in the paper being withdraw from the evaluation process. Example of correct reference form is given as follows:- Reference to a journal publication:

Ogunseitan OA. 1998. Protein method for investigating mercuric reductase gene expression in aquatic environments. Appl Environ Microbiol., 64 (2): 695-702.

Govindaraj S and Ranjithakumari B D. 2013. Composition and larvicidal activity of Artemisia vulgaris L. stem essential oil against Aedes aegypti. Jordan J Biol Sci., 6(1): 11-16. Hilly MO, Adams MN and Nelson SC. 2009. Potential fly-ash utilization in agriculture. Progress in Natural Sci., 19: 1173-1186.

Reference to a book: Brown WY and White SR.1985. The Elements of Style, third ed. MacMillan, New York.

Reference to a chapter in an edited book: Mettam GR and Adams LB. 2010. How to prepare an electronic version of your article. In: Jones BS and Smith RZ (Eds.), Introduction to the Electronic Age. Kluwer Academic Publishers, Netherlands, pp. 281–304. Conferences and Meetings:Embabi NS. 1990. Environmental aspects of distribution of mangrove in the United Arab Emirates. Proceedings of the First ASWAS Conference. University of the United Arab Emirates. Al-Ain, United Arab Emirates. Theses and Dissertations:El-Labadi SN. 2002. Intestinal digenetic trematodes of some marine fishes from the Gulf of Aqaba. MSc dissertation, The Hashemite University, Zarqa, Jordan. Nomenclature and Units Internationally accepted rules and the international system of units (SI) should be used. If other units are mentioned, please give their equivalent in SI. For biological nomenclature, the conventions of the International Code of Botanical Nomenclature, the International Code of Nomenclature of Bacteria, and the International Code of Zoological Nomenclature should be followed. Scientific names of all biological creatures (crops, plants, insects, birds, mammals, etc.) should be mentioned in parentheses at first use of their English term. Chemical nomenclature, as laid down in the International Union of Pure and Applied Chemistry and the official recommendations of the IUPAC-IUB Combined Commission on Biochemical Nomenclature should be followed. All biocides and other organic compounds must be identified by their Geneva names when first used in the text. Active ingredients of all formulations should be likewise identified.

Math formulae All equations referred to in the text should be numbered serially at the right-hand side in parentheses. Meaning of all symbols should be given immediately after the equation at first use. Instead of root signs fractional powers should be used. Subscripts and superscripts should be presented clearly. Variables should be presented in italics. Greek letters and non-Roman symbols should be described in the margin at their first use. To avoid any misunderstanding zero (0) and the letter O, and one (1) and the letter l should be clearly differentiated. For simple fractions use of the solidus (/) instead of a horizontal line is recommended. Levels of statistical significance such as: *P <0.05, **P <0.01 and ***P <0.001 do not require any further explanation. Copyright Submission of a manuscript clearly indicates that: the study has not been published before or is not under consideration for publication elsewhere (except as an abstract or as part of a published lecture or academic thesis); its publication is permitted by all authors and after accepted for publication it will not be submitted for publication anywhere else, in English or in any other language, without the written approval of the copyright-holder. The journal may consider manuscripts that are translations of articles originally published in another language. In this case, the consent of the journal in which the article was originally published must be obtained and the fact that the article has already been published must be made clear on submission and stated in the abstract. It is compulsory for the authors to ensure that no material submitted as part of a manuscript infringes existing copyrights, or the rights of a third party. Ethical Consent All manuscripts reporting the results of experimental investigation involving human subjects should include a s tatement confirming that each subject or subject's guardian obtains an informed consent, after the approval of the experimental protocol by a l ocal human ethics committee or IRB. When reporting experiments on animals, authors should indicate whether the institutional and national guide for the care and use of laboratory animals was followed. Plagiarism The JJBS hold no responsibility for plagiarism. If a published paper is found later to be extensively plagiarized and is found to be a d uplicate or redundant publication, a note of retraction will be published, and copies of the correspondence will be sent to the authors’ head of institute. Galley Proofs The Editorial Office will send proofs of the manuscript to the corresponding author as an e-mail attachment for final proof reading and it will be the responsibility of the corresponding author to return the galley proof materials appropriately corrected within the stipulated time. Authors will be asked to check any typographical or minor clerical errors in the manuscript at this stage. No other major alteration in the manuscript is allowed. After publication authors can freely access the full text of the article as well as can download and print the PDF file. Publication Charges There are no page charges for publication in Jordan Journal of Biological Sciences, except for color illustrations. Reprints Twenty (20) reprints are provided to corresponding author free of charge within two weeks after the printed journal date. For orders of more reprints, a reprint order form and prices will be sent with article proofs, which should be returned directly to the Editor for processing. Disclaimer Articles, communication, or editorials published by JJBS represent the sole opinions of the authors. The publisher shoulders no responsibility or liability what so ever for the use or misuse of the information published by JJBS.

References References should be listed alphabetically at the end of the manuscript. Every reference referred in the text must be also present in the reference list and vice versa. In the text, a reference identified by means of an author’s name should be followed by the year of publication in parentheses ( e.g.( Brown,2009)). For two authors, both authors’ names followed by the year of publication (e.g.( Nelson and Brown, 2007)). When there are more than two authors, only the first author's name followed by "et al." and the year of publication ( e.g. ( Abu-Elteen et al., 2010)). When two or more works of an author has been published during the same year, the reference should be identified by the letters "a", "b", "c", etc., placed after the year of publication. This should be followed both in the text and reference list. e.g., Hilly, (2002a, 2002b); Hilly, and Nelson, (2004). Articles in preparation or submitted for publication, unpublished observations, personal communications, etc. should not be included in the reference list but should only be mentioned in the article text ( e.g., Shtyawy,A., University of Jordan, personal communication). Journal titles should be abbreviated according to the system adopted in Biological Abstract and Index Medicus, if not included in Biological Abstract or Index Medicus journal title should be given in full. The author is responsible for the sccuracy and completeness of the references and for their correct textual citation. Failure to do so may result in the paper being withdraw from the evaluation process. Example of correct reference form is given as follows:- Reference to a journal publication:

Ogunseitan OA. 1998. Protein method for investigating mercuric reductase gene expression in aquatic environments. Appl Environ Microbiol., 64 (2): 695-702.

Govindaraj S and Ranjithakumari B D. 2013. Composition and larvicidal activity of Artemisia vulgaris L. stem essential oil against Aedes aegypti. Jordan J Biol Sci., 6(1): 11-16. Hilly MO, Adams MN and Nelson SC. 2009. Potential fly-ash utilization in agriculture. Progress in Natural Sci., 19: 1173-1186.

Reference to a book: Brown WY and White SR.1985. The Elements of Style, third ed. MacMillan, New York.

Reference to a chapter in an edited book: Mettam GR and Adams LB. 2010. How to prepare an electronic version of your article. In: Jones BS and Smith RZ (Eds.), Introduction to the Electronic Age. Kluwer Academic Publishers, Netherlands, pp. 281–304. Conferences and Meetings:Embabi NS. 1990. Environmental aspects of distribution of mangrove in the United Arab Emirates. Proceedings of the First ASWAS Conference. University of the United Arab Emirates. Al-Ain, United Arab Emirates. Theses and Dissertations:El-Labadi SN. 2002. Intestinal digenetic trematodes of some marine fishes from the Gulf of Aqaba. MSc dissertation, The Hashemite University, Zarqa, Jordan. Nomenclature and Units Internationally accepted rules and the international system of units (SI) should be used. If other units are mentioned, please give their equivalent in SI. For biological nomenclature, the conventions of the International Code of Botanical Nomenclature, the International Code of Nomenclature of Bacteria, and the International Code of Zoological Nomenclature should be followed. Scientific names of all biological creatures (crops, plants, insects, birds, mammals, etc.) should be mentioned in parentheses at first use of their English term. Chemical nomenclature, as laid down in the International Union of Pure and Applied Chemistry and the official recommendations of the IUPAC-IUB Combined Commission on Biochemical Nomenclature should be followed. All biocides and other organic compounds must be identified by their Geneva names when first used in the text. Active ingredients of all formulations should be likewise identified.

Indexing JJBS is indexed and abstracted by:

CABI DOAJ ( Directory of Open Access Journals

EBSCO Google Scholar CAS ( Chemical Abstract Service) Journal Seek

ETH- Citations HINARI Open J-Gat Index Copernicus SCImago NDL Japanese Periodicals Index

Zoological RecordsSCIRUS Scopus OAJSE

ISC (Islamic World Science Citation Center)

0T

AGORA

0T

(United Nation's FAO database)

Directory of Research Journal Indexing

(DRJI)

0T

SHERPA/RoMEO

0T

(UK)

0T

International Institute of Organized Research (I2OR) Database

المجلة األردنية للعلوم الحياتيةJordan Journal of Biological Sciences (JJBS)

http://jjbs.hu.edu.joThe Hashemite University

Deanship of Scientific Research and Graduate StudiesTRANSFER OF COPYRIGHT AGREEMENT

Journal publishers and authors share a common interest in the protection of copyright: authors principally because they want their creative works to be protected from plagiarism and other unlawful uses, publishers because they need to protect their work and investment in the production, marketing and distribution of the published version of the article. In order to do so effectively, publishers request a formal written transfer of copyright from the author(s) for each article published. Publishers and authors are also concerned that the integrity of the official record of publication of an article (once refereed and published) be maintained, and in order to protect that reference value and validation process, we ask that authors recognize that distribution (including through the Internet/WWW or other on-line means) of the authoritative version of the article as published is best administered by the Publisher.

To avoid any delay in the publication of your article, please read the terms of this agreement, sign in the space provided and return the complete form to us at the address below as quickly as possible.

Article entitled:--------------------------------------------------------------------------------------------------------------------------------

Corresponding author: -----------------------------------------------------------------------------

To be published in the journal: Jordan Journal of Biological Sciences (JJBS)

I hereby assign to the Hashemite University the copyright in the manuscript identified above and any supplemental tables, illustrations or other information submitted therewith (the "article") in all forms and media (whether now known or hereafter developed), throughout the world, in all languages, for the full term of copyright and all extensions and renewals thereof, effective when and if the article is accepted for publication. This transfer includes the right to adapt thepresentation of the article for use in conjunction with computer systems and programs, including reproduction or publication in machine-readable form and incorporation in electronic retrieval systems.Authors retain or are hereby granted (without the need to obtain further permission) rights to use the article for traditionalscholarship communications, for teaching, and for distribution within their institution. I am the sole author of the manuscript

I am signing on behalf of all co-authors of the manuscript

The article is a ‘work made for hire’ and I am signing as an authorized representative of the employing company/institution

Please mark one or more of the above boxes (as appropriate) and then sign and date the document in black ink.

Signed:___________________________________________ Name printed: _____________________________________________________Title and Company (if employer representative) : ___________________________________________________________________________Date: _________________________________

Data Protection: By submitting this form you are consenting that the personal information provided herein may be used by the Hashemite University and its affiliated institutions worldwide to contact you concerning the publishing of your article.

Please return the completed and signed original of this form by mail or fax, or a scanned copy of the signed original by e-mail, retaining a copy for your files, to:

Hashemite University Zarqa 13115 JordanFax: +962 5 3903338Email: jjbs@hu.edu.jo

Indexing JJBS is indexed and abstracted by:

CABI DOAJ ( Directory of Open Access Journals

EBSCO Geogle Scholar CAS ( Chemical Abstract Service) Journal Seek

ETH- Citations HINARI Open J-Gat Index Copernicus SCImago NDL Japanese Periodicals Index

Zoological RecordsSCIRUS Scopus OAJSE

ISC (Islamic World Science Citation Center)

0T

AGORA

0T

(United Nation's FAO database)

Directory of Research Journal Indexing

(DRJI)

0T

SHERPA/RoMEO

0T

(UK)

0T

International Institute of Organized Research (I2OR) Database

EDITORIAL PREFACE

It is my pleasure to present the tenth volume of the Jordan Journal of Biological Sciences (JJBS) to the audience. JJBS is a refereed, peer reviewed quarterly international journal issued by the Jordanian Ministry of Higher Education and Scientific Research Support Fund in cooperation with The Hashemite University, Zarqa, Jordan. This journal publishes papers in Biological Sciences encompassing all the branches at molecular, cellular and organismal levels.

A group of distinguished scholars have agreed to serve on the Editorial Board. Without the service and dedication of these eminent scholars, JJBS would have never existed. Now, the Editorial Board is encouraged by the continuous growth of the journal and its formation into a true multidisciplinary publication. I am also honored to have the privilege of working with all members of the international advisory board served by a team of highly reputable researchers from different countries across the globe. I am also delighted with our team of national and international reviewers who are actively involved in research in different biological sciences and who provide authors with high quality reviews and helpful comments to improve their manuscripts.

JJBS has been indexed by SCOPUS, CABI’s Full-Text Repository, EBSCO, Zoological Records and National Library of Medicine’s MEDLINE\ Pub Med system and others. I would like to reaffirm that the success of the journal depends on the quality of reviewing and, equally, the quality of the research papers published.

In addition to being a hard-copy journal, JJBS is an open access journal which means that all contents are freely available the users and their institutions free of charge. Users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles in this journal without asking for prior permission from the publisher or the author. This is in accordance with the BOAI definition of open access.

At the end of this preface, I would like to thank our readers and authors for their continuing interest in JJBS, and each member of our editorial and review boards for their continued hard work, support and dedication, which made it possible to bring another new issue of JJBS to the multidisciplinary international audience. My thanks are also extended to the Hashemite University and Jordanian Scientific Research Support Fund for their continuous support to Jordan Journal of Biological Sciences. I very much appreciate your support as we strive to make JJBS one of the most leading and authoritative journals in the field of Biological Sciences.

September,2017Prof. Abu-Elteen, Khaled HEditor-in-ChiefThe Hashemite University, Zarqa, Jordan

JJBS Volume 10, Number 3, September .2017 ISSN 1995-6673

Jordan Journal of Biological Sciences

CONTENTS Original Articles

145 - 149 Role of Oral Glucose Tolerance Test in Detection of Hyperglycemia among Non-diabetic Patients with Acute Myocardial Infarction Mahir A. Jassim

151 - 158 On some Records of Dragonflies (Insecta: Odonata: Anisoptera) from the West Bank (Palestine) Shadi H. Adawi, Khalid R. Qasem, Mubarak M. Zawahra and Elias N. Handal

159 – 165 Assessment of Antigenotoxic Effect of Nanoselenium and Metformin on Diabetic Rats Abeer H. Abd El-Rahim, Omaima M Abd-Elmoneim and Naglaa A. Hafiz

167 – 176 Avifauna Diversity of Bahr Al-Najaf Wetlands and the Surrounding Areas, Iraq Mudhafar A. Salim and Salwan Ali Abed

177 - 183 Inhibitory Effect of Crude Ethanol and Water Extracts of Phytolacca dodecandra (L’ Herit) on Embryonic Development of Anopheles gambiae (Diptera: Culicidae) Jared O. Yugi and Joyce J. Kiplimo

185 - 191 Comparative Anatomy of Stem, Petiole and Flower Stalks and its significance in the Taxonomy of Some Members of Cucurbits Chimezie Ekeke, Ikechukwu O. Agbagwa and Alozie C. Ogazie

193 - 197 Estimation of Median Lethal Dose of Commercial Formulations of Some Type II Pyrethroids Prabhu N. Saxena and Brijender Bhushan

199 - 203

Morphometric Relationships of the Endangered Ticto barb Pethia ticto (Hamilton, 1822) in the Ganges River (NW Bangladesh) through Multi-Linear Dimensions Fairuz Nawer, Md. Yeamin Hossain, Md. Alomgir Hossen, Dalia Khatun, Most. Farida Parvin, Jun Ohtomi and Md. Ariful Islam

205 - 212 Antibacterial Activities (Bacitracin A and Polymyxin B) of Lyophilized Extracts from Indigenous Bacillus subtilis Against Staphylococcus aureus Marwan Y. Hussain, Adnan A. Ali-Nizam and Samir M. Abou-Isba

213 - 217 Antibacterial and Cytotoxic Activity of the Bark of Phoenix paludosa in Different Solvents Saikat Ranjan Paul, Md. Raad Sayeed1, Md. Lukman Hakim

Short Communication

219 - 220 Eragrostis nairii Kalidass C. (Poaceae): New Record for Southern Western Ghats, India Moorthy Pavithra , Manogaran Parthipan, Iwar Kanivalan and Arumugam Rajendran

JJBS Volume 10, Number 3,September 2017

ISSN 1995-6673 Pages 145 - 149

Jordan Journal of Biological Sciences

Role of Oral Glucose Tolerance Test in Detection of Hyperglycemia among Non-diabetic Patients with Acute

Myocardial Infarction Mahir A. Jassim*

Department of Physiology, College of Medicine, Anbar University, Anbar Governorate, Iraq.

Received March 7, 2017 Revised May 10, 2017 Accepted May 29, 2017

Abstract

Previously undetected glucose abnormalities are not unusual in patients with Acute Myocardial Infarction (AMI). The aim of the present study is to assess the prevalence of Diabetes Mellitus (DM) and Impaired Glucose Tolerance (IGT) in patients presented with myocardial infarction without known history of DM and to examine whether Oral Glucose Tolerance Test (OGTT) is superior to Fasting Plasma Glucose (FPG) and Glycosylated hemoglobin (HbA1c) as screening tools. We enrolled 112 patients who were admitted to the Coronary Care Unit (CCU) at Al-Ramadi Teaching Hospital with acute myocardial infarction and no previous history of diabetes mellitus. An oral glucose tolerance test (OGTT) was done at discharge (mean hospital stay was 5 days). Fasting Plasma Glucose (FPG) and Glycosylated hemoglobin (HbA1c) were estimated at admission. At discharge, 66 patients (58.9%), 31(27.7%), and 15(13.4%) were classified as having normal glucose tolerance, impaired glucose tolerance (IGT), or type 2 diabetes, respectively. If Fasting Plasma Glucose (FPG) was used alone, only 6 (5.3%) patients were considered diabetic and 8 (7.1%) patients with IFG were identified. HbA1c identified only 2 (1.7%) patients with DM and only 4 (3.5%) patients with increased risk of DM. Thus, a FPG test alone identified only 30% of the patients with abnormal OGTT. In the present study, a fasting glucose and HbA1c test failed to identify more than (70%) and (87%), respectively, of the patients with abnormal glucose tolerance. The prevalence of DM and IGT in patients discharged from the CCU after a myocardial infarction without known history DM diagnosis was high (41%). Glucose abnormalities were common in patients with AMI. In comparison to OGTT, FPG or HbA1c alone failed to identify the majority of patients with IGT or T2DM screened for undiagnosed glucose abnormalities in patients with MI. The oral glucose tolerance test showed superiority to other means for the screening of glucose intolerance and, thus, should be considered as a routine test after a myocardial infarction in subjects without known DM.

Keywords: Hyperglycemia, Oral glucose tolerance test, Acute Myocardial infarction.

* Corresponding author. e-mail: drmahiraltalah@yahoo.com.

1. Introduction

Numerous studies have demonstrated that hyper-glycemia is not unusual among patients with acute myocardial infarction (Bartnik et al., 2004; Hu et al., 2006). It is encountered in up to 50% of all ST elevation myocardial infarcted (STEMI) patients, whereas previously diagnosed DM is present in only 20% to 25% of STEMI patients (Wahab et al., 2002).

Hyperglycemia during AMI was thought to be "stress hyperglycemia," which develops due to a highly complex interplay between hormones (such as catecholamine, growth hormones, and cortisol) and cytokines, ultimately leading to excessive hepatic glucose production and insulin resistance. (Dungan et al., 2009).

Stress hyperglycemia is a medical term referring to transient elevation of blood glucose in hospitalized patients due to the stress of illness without evidence of previous diabetes(fasting glucose >6·9 mmol/L or random

glucose >11·1 mmol/L without evidence of previous diabetes) (Moghissi et al., 2009). These patients are of particular interest to clinicians because they are reported to have a worse prognosis than those with diabetes despite higher blood glucose levels on admission in the latter population (Kosiborod et al., 2005; Ishihara et al., 2005; Ga˛sior et al., 2008; Ishihara et al., 2009; Capes et al., 2000). It has also been identified as a major independent predictor of both in-hospital congestive heart failure and mortality in STEMI (Zeller et al., 2005). Thus, patients with acute MI represent an opportunity for targeted screening for diabetes and institution of effective management strategies aimed to improve cardiovascular outcome (Onyebuchi et al., 2008).

The routine performance of an oral glucose tolerance test to diagnose diabetes during the acute phase MI is still the subject of ongoing debate (Yicong et al., 2012)

European guidelines on diabetes, pre-diabetes, and cardiovascular diseases (Rydén et al., 2007) recommend the performance of an oral glucose tolerance test in

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 146

patients with established cardiovascular disease. Furthermore, the guidelines on management of acute myocardial infarction in patients presenting with persistent ST-segment elevation (Van de Werf et al., 2008) specify that an OGTT should be performed before or shortly after hospital discharge. Another recent guideline issued by the American Diabetes Association (ADA) and the World Health Organization (WHO) recommend the use of HbA1c as an alternative (WHO, 2011; American Diabetes Association, 2010).

The aim of the present study is to assess the prevalence of DM and IGT in patients after a myocardial infarction without known DM and to compare FPG and HbA1c with OGTT as screening tools for detection of glucose abnormalities in those patients.

2. Patient and Method

The present study encompassed 126 patients who were admitted to the Coronary Care Unit (CCU) at Al-Ramadi Teaching Hospital during the period from February -2013 till January 2014 for the first time with acute myocardial infarction (irrespective to lesion topography) without previous history of diabetes mellitus.

The diagnosis of AMI was based on the WHO MI diagnosis criteria (Alpert et al., 2000). The criteria, as revised in 2000, are elevated cardiac troponin accompanied by either typical symptoms, pathological Q waves, ST segment changes (elevation or depression) or coronary intervention are diagnostic of MI. Of those 126 patients, only 14 patients met the exclusion criteria that were septicemia, endocrine disorders, comorbid conditions, and on drugs affecting blood glucose levels, such as Beta blockers, thiazides and glucocorticoids.

The details of the present study patients’ age, gender, height, weight, (BMI), smoking, history of hypertension and other associated clinical features are shown in the results section along with the data related to the family history of diabetes.

Random and fasting plasma glucose were measured on admission; blood samples were sent as well for HbA1c (NycoCard™ HbA1c).

The present study was approved by the Ethics committee at Al-Anbar Medical College, all the patients were informed about the aims of the study, procedures and possible risks before their enrollment and giving their written consent.

We performed the OGTT according to the WHO instructions. Briefly, 75 gram of glucose was given to the patients dissolved in 200 ml of water after overnight fast. Blood was aspirated at the beginning of the procedure and every half an hour for two hours for measurement of plasma glucose According to the WHO recommendation undiagnosed diabetes was defined as a FPG of ≥7.0mmol/l (126 mg/dl) or a plasma glucose value of ≥11.1mmol/l 200 mg/dL 2 h after loading with 75 g glucose (post load glucose [PLG]).

Subjects with fasting plasma glucose < 7.0mmol/l (126 mg/dl) or 2-h plasma glucose ≥7.8 mmol/l (140mg/dl) and <11.1mmol/l(200mg/dl) were classified as Impaired Glucose Tolerance (IGT), while the diagnostic limits for impaired fasting glucose was 6.1 to 6.9 mmol/l (110mg/dl to 125mg/dl) (WHO, 2008).

A diagnosis of prediabetes (a risk factor for later diabetes) may be made in cases where A1C measures 5.7-6.4%, while normal blood glucose profile in someone without diagnosed diabetes is reflected by an A1C result below 5.7% (Moghissi et al., 2009; Ellahham., 2010). 2.1. Statistical Analysis

Analysis of data was carried out using Statistical Package for Social Sciences (SPSS) version 21.0. Frequencies and relative frequencies were calculated for each predictor; standard statistical tests (such as chi-square, Fisher's Exact test and t-test) were used when appropriate. All P values less than 0.05 were considered statistically significant.

3. Results

The mean age of the patients in the present study was 54 years with the age range of (58.9± 10.0). The majority of the patients were male. There was nearly equal number with history of hypertension (Hx.HT) and without it, while history of smoking was identified in exactly equal number of patients to those who do not have it. In contrast, less patients had high Body Mass Index (BMI) >30. The present study population consisted of nearly equal number of patient with and without family history of DM (FHDM). More numerical details can be seen in Table 1.

Table 1. Baseline characteristics of the study group

Clinicopathological variables No. of patients %

Age

(x ± sd)

<55

>55

58.9± 10.0

58 (52%)

54 (48%)

Sex Male

Female

66 (59%)

46 (41%)

Hx.HT +ve

-ve

54 (48%)

58 (52%)

Smoking +ve

-ve

56 (50%)

56 (50%)

FHDM +ve

-ve

55 (49%)

57 (51%)

BMI(kg/m2) <30

>30

63 (56%)

49 (44%)

The 2-hr OGTT results (Table 2) identified diabetes in 15 patients (13.4%), IGT in 31 patients (27.9%), and normal glucose tolerance in 66 patients (58.9%).

Table 2. Prevalence of glucose abnormalities among study population using OGTT

Test DM (n,%) IGT(n,%) NGT(n,%)

OGTT (2-h PG) 15(13.4%) 31(27.7%) 66 (58.9%)

When the fasting plasma glucose criteria (Table 3) were applied, however, only 6 (5.3%) patients were diagnosed as having diabetes, 8 (7.1%) with IFG and normal fasting plasma glucose in 98 (87.5%) patients.

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 147

Table 3. Prevalence of glucose abnormalities among study population using fasting plasma glucose criteria in comparison with 2-hr OGTT results

Test DM (n,%)

IGT/ IFG

NGT/ NFG, Sensitivity Specificity

OGTT (2-h PG)

15(13.4%) 31(27.7%) 66(58.9%) 100% 100%

FPS 6(5.3%) 8(7.1%) 98(87.5%) 30.4% 100%

The HbA1C results (Table 4) identified diabetes in 2

(1.7%) patients (HbA1c _>6.5%), 4 (3.5%) patients with increased risk for diabetes (HbA1c 5.7–6.4%) and 106 (94.6%) patients were normal HbA1c. Giving that oral glucose tolerance test is the gold standard, the HbA1C and FPG show sensitivity of 13.4%, and 30.4%, respectively, while they demonstrated a specificity of 100%.

Table 4 Prevalence of glucose abnormalities among study population using HbA1C criteria in comparison with 2-hr OGTT results

Test .DM (n,%)

IGT / Increased risk for DM by HbA1C (n,%)

NGT/

Normal HbA1C

(n,%)

Sensitivity Specificity

OGTT (2-h PG)

15(13.4%) 31(27.7%) 66(58.9%) 100% 100%

HbA1C 2 (1.7%) 4 (3.5%) 106(94.6%) 13.04% 100%

In the present study, among acute MI patients, more men were included than women (59%), but no significant differences were observed regarding the incidence of DM or IGT between genders.

Being old (> 55 years) was strongly associated with development of DM and IFG (p =0.008) in comparison with those younger than 55 years, in the same way, history of smoking was also correlated with glucose abnormalities (p =0.007).

Additionally, a positive family history of diabetes mellitus (FHDM) (p=0.0001) and BMI greater than 30kg/m2 (p =0.001) were found as powerful predictors that help in diagnosis of Diabetes Mellitus (DM) and Impaired Glucose Tolerance (IGT) in patients presented with myocardial infarction without known history of DM. Statistical analysis showed no significant influence of hypertension on the risk of glucose abnormalities (p =0.78)

Table 5 shows variables that were found as independent predictors of disturbances in glucose metabolism at 120 min of OGTT immediately before discharge.

Table 5. Independent predictors of disturbances in glucose metabolism at 120 min of OGTT immediately before discharge

predictors NGT

N= 66

IGT

N= 31

DM

N= 15

P value

Age

(x ±sd)

<55

>55

58.9±10.0

42(63.6)

24(36.4)

61.5±7.4

12 (38.7)

19 (61.3)

68.9±5.2

4 (26.6)

11(73.3)

0.008

Sex

Male

Female

39(59.1)

27(40.9)

19 (61.3)

12 (38.7)

8 (53.4)

7 (46.6)

0.87

Hx.HT

+ve

-ve

30(45.5)

36(54.5)

16 (51.6)

15 (48.4)

8 (53.4)

7 (46.6)

0.78

Smoking

+ve

-ve

25(37.9)

41(62.1)

22 (72.4)

9 (27.6%)

9 (60.0)

6 (40.0)

0.007

FHDM

+ve

-ve

22(33.3)

44(66.7)

23 (72.4)

8 (27.6)

10 (66.7)

5 (33.3)

0.0001

BMI <30

>30

56(84.8)

10(15.2)

3 (9.7)

28(90.3)

4 (26.7)

11(73.3)

0.001

4. Discussion

There is increasing recognition of the significant relationship between diabetes and cardiovascular disease, and the contribution that each of these conditions makes to the risk of morbidity and mortality from both diseases (Gholap, et al., 2012; Ryden, et al., 2013).

In recent years, several studies have reported an increased prevalence of prior undiagnosed abnormal glucose tolerance (i.e., Impaired Glucose Tolerance (IGT) and diabetes) in patients with AMI (Bartnik et al., 2004; Norhammar et al., 2002; Hashimoto et al., 2005; Ramachandran et al., 2005).

Our objective in the present study was to clarify the prevalence of unrecognized abnormal glucose tolerance in our own population of patients with acute MI in Ramadi teaching hospital and identify the best method for screening.

The prevalence of disturbances in glucose metabolism in acute myocardial infarction in our study was 41% which is comparable to other published data. The Euro Heart Survey (Bartnik et al., 2004) reported the combined incidence of new-onset IGT and DM to be 58%, while in The China Heart Survey (Hu et al., 2006), it was 45%.

In the present study, we found that pre-discharge OGTT was a more sensitive method for detecting undiagnosed diabetes and impaired glucose metabolism than FPG and HbA1c. These results confirm the important role of an OGTT in detecting previously undiagnosed diabetes.

Our finding is consistent with the European guidelines on diabetes, pre-diabetes, and cardiovascular diseases which recommend the performance of an OGTT (due to its high sensitivity) in patients with established cardiovascular disease (Rydén et al., 2007). Furthermore, the European guidelines on the management of acute myocardial infarction in patients presenting with persistent ST-segment elevation specify that an OGTT should be performed before or shortly after hospital discharge (Van de Werf et al., 2008).

On the other hand, a scientific statement from the American Heart Association Diabetes Committee of the

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 148

Council on Nutrition, Physical Activity, and Metabolism does not encourage routine use of the OGTT for screening during the hospital stay (Deedwania et al., 2008).

The Joint British Societies’ 2005 clinical guidelines on the prevention of cardiovascular disease recommended that a fasting glucose measurement can be done as an alternative to an OGTT in patients who have had an acute cardiovascular event (JBS 2, 2005).

Despite the conflict of results emerging from different studies, we believe that OGTT is superior to FBG test and HbA1c.

There may be several reasons for the limited sensitivity of HbA1c to detect undiagnosed diabetes. As HbA1c correlates with the mean blood glucose over the previous 8-12 weeks, it requires regularly elevated glucose levels to increase. Consequently, HbA1c levels rise above the diagnostic threshold at a later stage than direct glucose level measurement with an OGTT (de Mulder et al., 2012).

With regard to IFG, it is well known that this impairment occurs in the late stage of diabetes as the pathophysiology of type 2 DM is characterized by insulin resistance firstly then progress to impaired insulin secretion (Dan Longo et al., 2015). As insulin resistance and compensatory hyperinsulinemia progress, the pancreatic islets are unable to sustain the hyperinsulinemic state. As a result, postprandial glucose intolerance then develops. A further decline in insulin secretion and an increase in hepatic glucose production lead to impaired fasting hyperglycemia. Accordingly, relying on IFG will result in missing many cases in their early stage of diabetes development.

5. Conclusion

Our results clarify the fact that hyperglycemia occurs frequently in patients with acute MI and previously undiagnosed diabetes mellitus. FPG and HbA1c leaves a majority of patients with IGT or T2DM undetected compared with an OGTT. As the OGTT is a straightforward, non-invasive and affordable test that can be performed during clinical recovery, we would advocate that it should become a standard of care in all patients admitted with acute MI, at least in those with old age, positive family history of DM and BMI ≥30 kg/m2. This will result in early recognition of diabetes and pre-diabetes and, subsequently, earlier lifestyle and medical interventions. Study Limitations 1. The number of patients included is relatively small and

selected from patients who participated in a single-center; the true prevalence of undiagnosed diabetes should be confirmed in a larger number of patients from multiple hospitals.

2. We cannot exclude the possibility that reduction in daily calorie intake or decrease in daily carbohydrate consumption within the first days of myocardial infarction might produce false negative results in OGTT tests performed in this period.

Acknowledgment

The authors of the present study thank the members of the coronary care unit and biochemistry department of the

central laboratory in Al-Ramadi Teaching Hospital for their help.

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© 2014 Jordan Journal of Biological Sciences. All rights reserved - Volume 8, Number 4334

Number 4

247 - 256Cryopreservation and Genetic Stability Assessment of Threatened Medicinal Plant (Ziziphora tenuior L.) Grown Wild in JordanHasan Al- Baba, Rida A. Shibli , Muhanad Akash, Tamara S. Al-Qudah, Reham W. Tahtamouni and Hamdan Al- Ruwaiei

257 - 262Production and Characterization of a Recombinant Camel Full Heavy Chain Antibody against Human IgESana’ H. Yousef , Abdelrahman M. Rawashdeh, Raida W. Khalil2, Sami K. Abdel-Hafez1,3and Khaled M. Al-Qaoud

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269 - 272A New Record of Two Species of Hydra in Iraq: An Ecological and A Histological Study Luay A. Ali, Yahya A. Shekha, Sherwan T. Ahmad and Falah M. Aziz

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281 - 286Effect of Faba Bean (Vicia faba L.) Varieties on Yield Attributes at Sinana and Agarfa Districts of Bale Zone, Southeastern EthiopiaAshenafi Mitiku and Mekuria Wolde

289 - 299The Phytochemical and Antimicrobial Properties of the Extracts Obtained from Trametes elegans Collected from Osengere in Ibadan, NigeriaSamuel I. Awala and Victor O. Oyetayo

301 - 306The Potential Allelopathic Effects of Varthemia iphionoides and the Identification of Phenolic AllelochemicalsSaeid M. Abu-Romman , Moawiya A. Haddad and Khaldoun J. Al-Hadid

307 - 313Identifying Selection Signatures Related to Domestication Process in Barley (Hordeum vulgare L.) Landraces of Jordan Using Microsatellite MarkersNidal A. Odat, Maen K. Hasan, Maher S. Obeidat, Mohamad A. Shatnawi1, Saeid M. Abu-Romman, Issam M. Qrunfleh, and Muhannad I. Massadeh

315 - 318Phlomis brachydon Essential Oil Against Bacterial BiofilmSameeh Al- Sarayreh , Arwa Rawashdeh, Ibrahim Al-Tarawneh, and Mahmoud Al-Qudah

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JJBS Volume 10, Number 3,September 2017

ISSN 1995-6673 Pages 151 - 157

Jordan Journal of Biological Sciences

On some Records of Dragonflies (Insecta: Odonata: Anisoptera) from the West Bank (Palestine)

Shadi H. Adawi1,2, Khalid R. Qasem¹, Mubarak M. Zawahra³ and Elias N. Handal2,*

1Salfit Primary Health Care Center, Ministry of Health;

2Palestine Museum of Natural History, Bethlehem University, Bethlehem,

3Environmental Quality Authority, Palestine

Received January 29, 2017 Revised May 9, 2017 Accepted May 29, 2017

Abstract

Three families with thirteen species were collected/observed from 35 localities representing various habitats and water bodies in the West Bank (State of Palestine) over the past four years. These are Brachythemis impartita, Crocothemis erythraea, Orthetrum brunneum, O. chrysostigma, O. teniolatum, Sympetrum fonscolombii S. meridionale, Trithemis annulata, T. arteriosa, Paragomphus genei, Anax imperator, A. pathenope, and A. ephippiger. There may have been a decline in dragonfly diversity in the area due to human population growth accompanied by habitat destruction especially around springs.

Keywords: Dragonflies, Odonata, Anisoptera, West Bank, Palestine, Indicator species.

* Corresponding author. e-mail: eliashandal93@gmail.com.

1. Introduction

About 5700 species of Odonata were described worldwide with most of them being tropical (Ellenrieder, 2004). Odonata are classified into three suborders: Anisoptera (true dragonflies), Zygoptera (damselflies) and Anisozygoptera (a very small suborder considered intermediate between damselflies and dragonflies). Anisoptera has ten families with Libellulidae being the largest with 140 genera and over 962 described species (Tennessen, 2003). The dragonfly fauna of Palestine were studied over the past decades (Morton, 1924; Schmidt, 1939; De Marmel, 1995; Schneider 1986; Dumont 1991). Dumont (1991) gave a comprehensive account of the dragonflies of the Levant, with localities from historic Palestine. Family Libellulidae is the largest family with 35 species in historic Palestine, 10 species detected in the West Bank (Boudot et al., 2009). Dragonflies are susceptible to climate change, use of insecticides, and habitat modification, with several species at risk and are listed in the IUCN Red List as endangered or threatened (Schneider, 1982, 2004).

Qumsiyeh et al. (2014) and Salman et al. (2014) pointed out the urgent need to study the biodiversity in the

occupied Palestinian territories, especially since habitat destruction is accelerating and affecting many species. This communication documents the collected dragonflies of the families Libellulidae, Gomphidae, and Aeshnidae at the recently established Palestine Museum of Natural History (PMNH) (Qumsiyeh et al., 2017).

2. Materials and Methods

The occupied Palestinian territory of the West Bank is located in central Palestine and characterized by geological and environmental diversity and, thus, includes four biogeographic regions: Mediterranean (including coastal and semi-coastal regions), Irano-Turanian, Saharo-Arabian, and Ethiopian/Sudanese. Significant biodiversity is thus noted in a small area of a few thousand square kilometers.

Specimens were collected from 35 localities representing the diverse habitats and biogeographic regions within the Occupied Palestinian Territory by the PMNH team during field trips from April 2012 until September 2016 (Table 1). Dragonflies were collected by means of insect nets, and deposited at PMNH collection. Identification was based on previous literature (Schneider 1986; Dumont, 1991; Dijkstra, 2006).

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 152

Table 1. List of visited localities and their coordinates

Locality N E Locality N E

Ain Al Hilweh 32° 21' 38.4726" 35° 32' 8.2386" Jiftlik 32° 8' 33.2442" 35° 29' 47.3382"

Ain Al Sakout 32° 21' 55.3572" 35° 32' 50.1822" Kafer al- Deek 32° 3' 41.1552" 35° 5' 16.2342"

Ain Al Shamsat 32° 22' 47.0922" 35° 30' 40.4058" Kifl Haris 32° 7' 23.9268" 35° 8' 52.065"

Ain Al Sultan 31° 52' 45.3216" 35° 26' 50.0532" Nahaleen 31° 41' 18.693" 35° 7' 0.9834"

Ain Fasayil 32° 1' 28.0842" 35° 26' 38.2338" Naqura- Nablus 32° 15' 58.4706" 35° 11' 59.4666"

Ain Hijla 31° 49' 29.4312" 35° 30' 38.0124" Nawama 31° 53' 27.6072" 35° 25' 59.9196"

Ain Qinia 31° 55' 34.683" 35° 8' 59.0166" Qarawat Bani Hassan 32° 8' 48.4182" 35° 3' 20.0556"

Ain Samiya 31° 59' 19.6614" 35° 20' 2.8818" Qbatia 32° 24' 8.2836" 35° 18' 3.1494"

Ain Yabroud 31° 57' 5.2842" 35° 15' 6.0984" Salfit 32° 4' 57.7416" 35° 8' 49.9776"

Al Makhrour 31° 43' 8.0538" 35° 9' 32.5398" Wadi Al Badan 32° 15' 22.2006" 35° 19' 16.9212"

Al Walaja 31° 43' 49.8432" 35° 9' 14.619" Wadi Al Hakeem 32° 0' 59.6262" 35° 5' 7.5834"

Artas 31° 41' 17.7504" 35° 11' 9.873" Wadi Al Muqatta' 32° 29' 51.9462" 35° 16' 14.3832"

Bardala 32° 23' 29.9328" 35° 28' 58.5192" Wadi Al Qelt 31° 51' 3.8952" 35° 25' 53.817"

Bethlehem 31° 43' 3.6258" 35° 12' 20.4006" Wadi- Al Zarqa 32° 3' 37.4688" 35° 5' 7.2744"

Deir Istiya 32° 7' 32.5122" 35° 8' 45.7296" Wadi Fukeen 31° 42' 24.714" 35° 6' 13.3986"

Ednha 31° 33' 42.588" 34° 59' 12.6312" Wadi Qana 32° 9' 30.5382" 35° 6' 41.8242"

Husan 31° 42' 41.6232" 35° 7' 39.9144" Za'tara 31° 40' 6.9096" 35° 14' 53.5848"

Iskaka 32° 6' 16.038" 35° 13' 23.127"

3. Results

Thirteen species belonging to seven genera (Orthetrum, Trithemis, Crocothemis, Sympetrum, Brachythemis, Paragomphus, and Anax) in three families (Libellulidae, Gomphidae and Aeshnidae) were recorded during the present study. We did not study the damselflies and focused on the dragonflies.

3.1. Family Libellulidae

3.1.1. Brachythemis impartita (Karsch, 1890) Fig.1A

Material examined (2♂♂): Ain Al Sakout (PMNH-E-10255, ♂, 21.9.2016) Ain Al Sakout. (PMNH-E-10254, ♂, 3.8.2015).

Remarks Specimens were collected from a single habitat in the

Jordan Valley. In this site, it was associated with O. chrysostigma, T. arteriosa, T. annulata and S. fonscolombii. The taxonomic status of this species was revised by Dijkstra, and Matushkina (2009). Brachythemis impartita is distributed from north and south of the Sahara, extending to Eurasia eastwards, while B. leucosticta is wide spread across most of tropical Africa and Madagascar (Dijkstra and Matushkina, 2009). All previous records of this species in the Levant should be assigned to B. impartita.

3.1.2. Crocothemis erythraea (Brullé, 1832) Fig.1 B

Material examined (25♂♂, 5♀♀): Ain Al Sultan (PMNH-E-10256, ♀, 29.8.2016). Ain Al Zarqa (PMNH-E-10257, ♂, 19.9.2016; PMNH-E-10258, ♂, 19.9.2016). Ain Fasayel (PMNH-E-10081, ♀, 4.1.2015). Ain Qinia (PMNH-E-10049, ♂, 1.8.2014; PMNH-E-10063, ♂, 15.8.2014; PMNH-E-10065, ♂, 3.8.2015; PMNH-E-

10074, ♂, 3.8.2015; PMNH-E-10090, ♂, 3.8.2015). Ain Samia (PMNH-E-10075, ♀, 26.8.2015). Ain Yabroud (PMNH-E-10220, ♀, 5.10.2016; PMNH-E-10221, ♀, 5.10.2016). Artas (PMNH-E-10070, ♂, 18.7.2015; PMNH-E-10077, ♂, 13.8.2014). Deir Istia (PMNH-E-10158, ♂, 4.6.2016; PMNH-E-10159, ♂, 4.6.2016). Wadi Qana (PMNH-E-10185, ♂, 21.7.2016; PMNH-E-10188, ♂, 4.6.2016; PMNH-E-10189, ♂, 4.6.2016; PMNH-E-10202, ♂, 21.7.2016). Nawama (PMNH-E-10048, ♂, 24.4.2015). Qarawa (PMNH-E-10167, ♂, 29.6.2016; PMNH-E-10177, ♂, 14.7.2016). Salfit (PMNH-E-10168, ♂, 19.6.2016). Wadi Al Muqatta' (PMNH-E-10095, ♂, 8.4.2016). Wadi Fukeen (PMNH-E-10050, ♂, 6.6.2015; PMNH-E-10064, ♂, 29.7.2015; PMNH-E-10072, ♂, 7.3.2016; PMNH-E-10082, ♂, 27.5.2015).

Remarks We collected specimens from 14 different locations

throughout the West Bank, including springs and irrigation canals. It was associated with O. chrysostigma, T. arteriosa, T. annulata, S. meridionale and S. fonscolombii. It has a pan-African and Mediterranean distribution reaching Iraq (Schmidt, 1939; Dumont, 1991; Schneider, 2013; Kalkman, 2003; Katbeh-Bader et al., 2004; Dia et al., 2011 and Amr et al., 2013).

3.1.3. Orthetrum brunneum (Fonscolombe, 1837) Fig.1 C

Material examined (2♂♂): Ain Al Hilweh (PMNH-E-10259, ♂, 21.9.2016). Ain Yabroud (PMNH-E-10213, ♂, 5.10.2016).

Remarks Specimens were collected from two locations. It is a

widespread species in central and southern Europe, North Africa and the Levant (Dumont, 1991; Boudot et al., 2009; Schneider, 2013; Amr et al., 2013). It was found along with O. chrysostigma and C. erythraea.

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3.1.4. Orthetrum chrysostigma (Burmeister, 1839) Fig.1 D

Material examined (63♂♂,1♀): Ain Al Hilweh (PMNH-E-10262, ♂, 21.9.2016; PMNH-E-10263, ♂, 21.9.2016). Ain Al Shamsat (PMNH-E-10264, ♂, 21.9.2016; PMNH-E-10265, ♂, 21.9.2016). Ain Al Sultan (PMNH-E-10260, ♂, 29.8.2016; PMNH-E-10261, ♂, 29.8.2016). Ain Hijla (PMNH-E-10046, ♂, 18.4.2014). Ain Qinia (PMNH-E-10030, ♂, 3.8.2015; PMNH-E-10034, ♂, 15.8.2014; PMNH-E-10039, ♂, 20.5.2015; PMNH-E-10040, ♂, 1.8.2014; PMNH-E-10041, ♂, 15.8.2014; PMNH-E-10045, ♂, 15.8.2014; PMNH-E-10054, ♂, 20.5.2015; PMNH-E-10061, ♂, 3.8.2015). Ain Al Sakout (PMNH-E-10227, ♂, 10.10.2016; PMNH-E-10231, ♂, 10.10.2016; PMNH-E-10239, ♂, 10.10.2016; PMNH-E-10242, ♂, 10.10.2016; PMNH-E-10245, ♂, 10.10.2016; PMNH-E-10246, ♂, 10.10.2016). Ain Samia (PMNH-E-10084, ♂, 7.9.2015). Ain Yabroud (PMNH-E-10214, ♂, 5.10.2016; PMNH-E-10215, ♂, 5.10.2016; PMNH-E-10216, ♂, 5.10.2016; PMNH-E-10217, ♂, 5.10.2016; PMNH-E-10218, ♂, 5.10.2016; PMNH-E-10219, ♂, 5.10.2016; PMNH-E-10222, ♂, 5.10.2016; PMNH-E-10223, ♂, 5.10.2016; PMNH-E-10224, ♂, 5.10.2016). Al Walaja (PMNH-E-10056, ♂, 8.8.2014). Deiristia (PMNH-E-10144, ♂, 4.6.2016; PMNH-E-10145, ♂, 4.6.2016; PMNH-E-10146, ♂, 4.6.2016; PMNH-E-10184, ♂, 20.7.2016; PMNH-E-10186, ♂, 21.7.2016; PMNH-E-10199, ♂, 21.7.2016; PMNH-E-10200, ♂, 21.7.2016). Iskaka (PMNH-E-10164, ♂, 26.6.2-16). Nahaleen (PMNH-E-10033, ♂, 8.6.2015; PMNH-E-10035, ♂, 8.6.2015; PMNH-E-10062, ♂, 8.6.2015). Qarawa (PMNH-E-10165, ♂, 29.6.2016; PMNH-E-10166, ♂, 29.6.2016; PMNH-E-10175, ♂, 14.7.2016; PMNH-E-10196, ♂, 14.7.2016). Salfit (PMNH-E-10047, ♂, no date; PMNH-E-10161, ♂, 19.6.2016; PMNH-E-10162, ♂, 19.6.2016; PMNH-E-10169, ♂, 19.6.2016; PMNH-E-10178, ♂, 17.7.2016; PMNH-E-10204, ♂, 16.8.2016; PMNH-E-10205, ♂, 16.8.2016). Tal Al Sultan (PMNH-E-10121, ♀, 25.7.2016). Wadi Al Badan (PMNH-E-10032, ♂, 16.9.2015). Wadi Al Hakeem - Abood (PMNH-E-10038, ♂, 27.7.2015). Wadi Al Qelt (PMNH-E-10036, ♂, 4.4.2015). Wadi Fukeen (PMNH-E-10031, ♂, 6.6.2015; PMNH-E-10037, ♂, 6.6.2015; PMNH-E-10042, ♂, 6.6.2015). Wadi Qana (PMNH-E-10043, ♂, 18.5.2013; PMNH-E-10044, ♂, 18.5.2013). Remarks

This is a common and widespread species collected from most visited habitats, noted perching near water bodies especially around springs. This species has a wide distribution range extending North Africa, southern Europe to western Asia (Dumont, 1991; Boudot et al., 2009). It was the most common species in 19 sites, and was found along all other species at different habitats ranging from 1-5 species.

3.1.5. Orthetrum taeniolatum (Schneider, 1845)

Material examined (7♂♂,1♀): Ain Qinia (PMNH-E-10060, ♂, 15.8.2014; PMNH-E-10076, ♂, 15.8.2014; PMNH-E-10083, ♂, 3.8.2015). Deri Istia (PMNH-E-10147, ♂, 4.6.2016). Wadi Qana. (PMNH-E-10148, ♂, 4.6.2016; PMNH-E-10183, ♂, 20.7.2016; PMNH-E-

10195, ♂, 4.6.2016). Wadi Al Qelt (PMNH-E-10079, ♀, 4.4.2015).

Remarks This species collected from different habitats in West

Bank, including arid and humid areas. It was reported from the Greek Islands, Cyprus, Turkey, Jordan, Lebanon, Syria, Sinai, and Iraq (Dumont, 1991; Kalkman, 2003; Katbeh-Bader et al., 2004; Amr et al., 2013; Dia et al., 2011; Schneider, 2013). It was collected from four different sites, along with 1-4 other species.

3.1.6. Sympetrum fonscolombii (Selys, 1840) Fig.1 E

Material examined (1♂, 6♀♀): Ain Al Sakout (PMNH-E-10270, ♀, 21.9.2016). Wadi Al Zarqa (PMNH-E-10271, ♀, 19.9.2016; PMNH-E-10272, ♀, 19.9.2016). Wadi Fukeen (PMNH-E-10207, ♂, 7.3.2016). Za'tara (PMNH-E-10267, ♀, 5.9.2016; PMNH-E-10268, ♀, 5.9.2016; PMNH-E-10269, ♀, 5.9.2016).

Remarks Collected specimens from three different sites

including springs, open ponds and irrigation canals. It was found along with O. chrysostigma, T. arteriosa, T. annulata, C. erythraea andS. meridionale. It is distributed along Central and southern Europe, North Africa, and southwestern Asia (Dumont, 1991; Katbeh-Bader et al., 2004; Boudot et al., 2009). 3.1.7. Sympetrum meridionale (Selys, 1841) Fig.1 F

Material examined (3♂♂): Wadi Al Zarqa (PMNH-E-10275, ♂, 19.9.2016). Za'tara (PMNH-E-10273, ♂, 5.9.2016; PMNH-E-10274, ♂, 5.9.2013). Remarks

We collected specimens from two locations: one in the center of the West Bank and the other in the arid region of Bethlehem. It was found to coexist with O. chrysostigma, T. arteriosa, T. annulata, C. erythraea and S. fonscolombii. Its distribution range extends along southern Europe, North Africa, the Levant reaching Iraq (Schmidt, 1939; Dumont, 1991; Kalkman, 2003; Katbeh-Bader et al., 2004; Boudot et al., 2009; Dia et al., 2011; Amr et al., 2013; Schneider, 2013).

3.1.8. Trithemis annulata (Palisot de Beauvois, 1805) Fig.1 G

Material examined (31♂♂): Ain Al Sakout (PMNH-E-10277, ♂, 21.9.2016; PMNH-E-10278, ♂, 21.9.2016; PMNH-E-10279, ♂, 21.9.2016). Ain Al Sultan (PMNH-E-10276, ♂, 29.8.2016). Ain Al Zarqa (PMNH-E-10280, ♂, 19.9.2016). Ain Al Sakout (PMNH-E-10226, ♂, 10.10.2016; PMNH-E-10228, ♂, 10.10.2016; PMNH-E-10230, ♂, 10.10.2016; PMNH-E-10233, ♂, 10.10.2016; PMNH-E-10234, ♂, 10.10.2016; PMNH-E-10235, ♂, 10.10.2016; PMNH-E-10236, ♂, 10.10.2016; PMNH-E-10237, ♂, 10.10.2016; PMNH-E-10238, ♂, 10.10.2016; PMNH-E-10240, ♂, 10.10.2016; PMNH-E-10244, ♂, 10.10.2016; PMNH-E-10247, ♂, 10.10.2016; PMNH-E-10248, ♂, 10.10.2016; PMNH-E-10249, ♂, 10.10.2016; PMNH-E-10250, ♂, 10.10.2016; PMNH-E-10251, ♂, 10.10.2016; PMNH-E-10252, ♂, 10.10.2016; PMNH-E-10253, ♂, 10.10.2016). Deir Istia (PMNH-E-10149, ♂,

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4.6.2016; PMNH-E-10150, ♂, 4.6.2016; PMNH-E-10151, ♂, 4.6.2016; PMNH-E-10201, ♂, 21.7.2016). Husan (PMNH-E-10017, ♂, no date). Salfit (PMNH-E-10206, ♂, 16.8.2016). Wadi Fukeen (PMNH-E-10096, ♂, 29.7.2015). Wadi Qana (PMNH-E-10097, ♂, 1.6.2016). Remarks

This species was found in seven different areas in the West Bank. It was found along with O. chrysostigma, T. arteriosa C. erythraea, S. meridionale, S. fonscolombii and B. impartita. It is known from southern Europe, North Africa, the Levant, and northern Arabia to Iraq (Schmidt, 1939; Dumont, 1991; Kalkman, 2003; Katbeh-Bader et al., 2004; Boudot et al., 2009; Amr et al., 2013; Schneider, 2013; Dia et al., 2011).

3.1.9. Trithemis arteriosa (Burmeister, 1839) Fig.1 H

Material examined (71♂♂): Ain Al Sakout (PMNH-E-10281, ♂, 21.9.2016). Ain Hijla (PMNH-E-10058, ♂, 18.4.2014). Ain Najjar (PMNH-E-10142, ♂, no date). Ain Qinia (PMNH-E-10001, ♂, 15.8.2014; PMNH-E-10005, ♂, 15.8.2014; PMNH-E-10006, ♂, 15.8.2014; PMNH-E-10024, ♂, 15.8.2014; PMNH-E-10025, ♂, 15.8.2014; PMNH-E-10027, ♂, 3.8.2015). Ain Al Sakout (PMNH-E-10229, ♂, 10.10.2016; PMNH-E-10232, ♂, 10.10.2016; PMNH-E-10241, ♂, 10.10.2016; PMNH-E-10243, ♂, 10.10.2016). Al Makhrour (PMNH-E-10029, ♂, 3.5.2015). Bardala (PMNH-E-10085, ♂, 18.4.2014). Deiristia (PMNH-E-10152, ♂, 4.6.2016; PMNH-E-10153, ♂, 4.6.2016; PMNH-E-10154, ♂, 4.6.2016; PMNH-E-10155, ♂, 4.6.2016; PMNH-E-10156, ♂, 4.6.2016; PMNH-E-10163, ♂, 28.5.2016; PMNH-E-10187, ♂, 21.7.2016; PMNH-E-10190, ♂, 4.6.2016; PMNH-E-10191, ♂, 4.6.2016; PMNH-E-10192, ♂, 4.6.2016; PMNH-E-10193, ♂, 4.6.2016; PMNH-E-10194, ♂, 4.6.2016; PMNH-E-10197, ♂, 21.7.2016; PMNH-E-10198, ♂, 21.7.2016). Edna (PMNH-E-10011, ♂, 23.8.2014; PMNH-E-10015, ♂, 23.8.2014). Husan (PMNH-E-10009, ♂, no date). Kefel Haris (PMNH-E-10171, ♂, 11.7.2016; PMNH-E-10172, ♂, 11.7.2016; PMNH-E-10173, ♂, 11.7.2016). Kufer aldeek (PMNH-E-10157, ♂, 4.6.2016). Kefer aldeek (PMNH-E-10174, ♂, 11.7.2016). Nablus (PMNH-E-10007, ♂, Jul.2014; PMNH-E-10089, ♂, Apr.2011). Qarawa (PMNH-E-10176, ♂, 14.7.2016). Salfit (PMNH-E-10002, ♂, Aug.2014; PMNH-E-10010, ♂, 22.8.2014; PMNH-E-10012, ♂, 22.8.2014; PMNH-E-10019, ♂, no date; PMNH-E-10020, ♂, Nov.2014; PMNH-E-10023, ♂, 22.8.2014; PMNH-E-10028, ♂, Aug.2014; PMNH-E-10160, ♂, 19.6.2016; PMNH-E-10170, ♂, 19.6.2016; PMNH-E-10179, ♂, 17.7.2016; PMNH-E-10180, ♂, 17.7.2016; PMNH-E-10181, ♂, 17.7.2016; PMNH-E-10182, ♂, 17.7.2016). Wadi Al Muqatta' (PMNH-E-10208, ♂, 8.4.2016). Wadi Fukeen (PMNH-E-10003, ♂, 27.5.2015; PMNH-E-10004, ♂, 6.6.2015; PMNH-E-10008, ♂, 29.8.2014; PMNH-E-10013, ♂, 6.6.2015; PMNH-E-10014, ♂, 6.6.2015; PMNH-E-10016, ♂, 6.6.2015; PMNH-E-10018, ♂, 6.6.2015; PMNH-E-10021, ♂, 29.7.2015; PMNH-E-10026, ♂, 6.6.2015; PMNH-E-10059, ♂, 29.7.2015; PMNH-E-10068, ♂, 6.6.2015; PMNH-E-10069, ♂, 29.8.2014; PMNH-E-10071, ♂, 7.3.2016; PMNH-E-10073, ♂, 7.3.2016; PMNH-E-10106,

♂, 7.5.2015). Wadi Qana (PMNH-E-10057, ♂, 18.5.2013; PMNH-E-10067, ♂, 18.5.2013). Remarks

This is a common species in all Palestinian areas and was collected from 16 different sites, including swimming and irrigation pools. It was associated with five other species including (O. chrysostigma, T. annulata, C. erythraea, S. meridionale and S. fonscolombii). The species is found in Africa and the circum-Mediterranean region (Dumont, 1991; Kalkman, 2003; Katbeh-Bader et al., 2004; Boudot et al., 2009; Dia et al., 2011; Schneider, 2013).

3.2. Family Gomphidae

3.2.1. Paragomphus genei (Sélys, 1841)

Material examined (1♂): Wadi Al Qelt (PMNH-E-10115, ♂, 4.4.2015).

Remarks Collected specimen from one site in West Bank

(northern the Dead Sea). It is distributed in Western Mediterranean region and in tropical Africa, (Dumont, 1991; Katbeh-Bader et al., 2004; Boudot et al., 2009).

3.3. Family Aeshnidae

3.3.1. Anax imperator (Leach, 1815) Fig.1 I

Material examined (4♂): Naqura (PMNH-E-10143, ♂, 31.7.2012). Bethlehem (PMNH-E-1010052,♂ 20.7.2015). Wadi Al Muqatta’ (PMNH-E-10086, ♂, 8.4.2016, PMNH-E-10123, ♂, 5.9.2016). Remarks

Collected specimens from three different sites including springs, open ponds and irrigation canals. It is distributed along Europe, North Africa, and in Mediterranean region (Dumont, 1991; Katbeh-Bader et al., 2004; Boudot et al., 2009).

3.3.2. Anax pathenope (Sélys, 1839)

Fig.1 J Material examined (1♂): Qabatia (PMNH-E-10053,

7.9.2015).

Remarks Collected specimens from one site in the northern of

West Bank, around open ponds and irrigation canals. It is distributed along southern Europe, North Africa, and in Mediterranean region. (Dumont, 1991; Katbeh-Bader et al., 2004; Boudot et al., 2009).

3.3.3. Hemianax ephippiger (Burmeister, 1839) Fig.1 K

Material examined (2♂♂): Jiftlik (PMNH-E-10053, 27.3.2013). Wadi Al Qelt (PMNH-E-10087, 7.3.2016).

Remarks Collected specimens from one site in the northern of

West Bank, around open ponds and irrigation canals. It is distributed along southern Europe, North Africa, and in Mediterranean region. (Dumont, 1991; Katbeh-Bader et al., 2004; Boudot et al., 2009).

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Figure 1.A. Brachythemis impartita B. Crocothemis erythraea C. Orthetrum brunneum D. Orthetrum chrysostigma E. Sympetrum fonscolombii F. Sympetrum meridionale G. Trithemis annulata H. Trithemis arteriosa I. Anax imperator J. Anax pathenope K. Hemianax ephippiger. Scale is 10 mm.

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 156

4. Discussion

The nine species of Libellulidae family recorded from the Palestinian areas include four widespread African and circum-Mediterranean species (Dument, 1991; Schneider et al., 1986). In historic Palestine and nearby areas, family Libellulidae is represented by more than 30 species (Borton, 1924; Boudot et al., 2009; Dument, 1991; Schneider et al., 1986). In an unpublished doctoral thesis, Schneider (1986) reported other species from the area of the West Bank collected between 1979 and 1983 including Epallage fatime, Coenagrium syriacum, Ischnura evansi, I. fountainei, I. elegens ebneri. Psudagrion sublacteum, P. syriacum, Gomphus davidi, Onichogomphus lefebvrii, Orthetrum anceps, O. trinacrium, Eurothemis edwardsi, Zygonyx torridus, and Pseudagrion sublacteum. Thus, a total of 27species of dragonflies were reported in this area over this period of time (1979 until today). We were able to find only 13 species. This could have two explanations: our sampling may not have been covering all the areas visited by Schneider (1986) or that there has been a significant decline in species richness of dragonflies in this area since the work of Schneider (1986, collected 1979-1983). Schneider (1982) examined man-induced changes on the dragonfly fauna of the Jordan Valley and warned of impending dangers. On the eastern banks of the Jordan River, many species of the dragonflies of Jordan disappeared in the past fifty years (Erythromanalindenii zernyi and Agriocnemis sania) or underwent retraction in their distribution ranges (Cabpteryx syriaca) (Katbeh-Bader et al., 2004).

Thus, the weight of the evidence points to man induced changes. Many streams from which earlier records existed have been altered so that water is removed directly from the spring source (no more open stream in the area). Salman et al. (2014) commented on the potential impact of these practices on the declining amphibian populations. Dragonfly fauna in other parts of the world are affected by these factors (Watson et al., 1982; Ward et al., 1982; Wright et al., 1995; Clark et al., 1996; Stewart et al., 1998; Bell, 1971). Further, the population in the West Bank has more than doubled since Schneider’s study (1986) both by natural increase of the native Palestinian population and by settler population increase (from less than 200,000 then to over 750,000 now). This has resulted in habitat destruction and pollution that impacts water sources for both humans and animals (Hammd and Qumsiyeh, 2013; Qumsiyeh et al., 2014; Salman et al., 2014; Handal et al., 2015). More studies are needed especially in the Northern parts of the West Bank and the Jordan Valley.

At the global level, many dragonfly species have shown a dramatic decline in their distribution and abundance patterns since the second half of the 20th century (Westfall and May, 1996; Sahlen et al., 2004; Inoue, 2004). The causes of such decline are mainly due to habitat destruction, eutrophication, acidification, pollution and water mismanagement (channelization, dam's construction, and modifications of the structure of rivers). Effects of water extraction and pollution of natural water resources on the natural populations of dragonflies in the Palestinian regions should be a first priority.

The present study documents species composition and distribution in the Palestinian areas. Further assessment is

required to study the other families of both dragonflies and damselflies all over the West Bank especially with a focus to habitat changes and threats to populations.

Acknowledgements

We are grateful for Prof. Mazin B. Qumsiyeh and Prof. Zuhair S. Amr for their support, encouragement, and editing this manuscript. We thank the Environmental Quality Authority for field and other logistical assistance. We also thank two anonymous referees for their input, which improved the present study. Funding for the present work was solely internal (Palestine Museum of Natural History).

References

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JJBS Volume 10, Number 3,September 2017

ISSN 1995-6673 Pages 159 - 165

Jordan Journal of Biological Sciences

Assessment of Antigenotoxic Effect of Nanoselenium and Metformin on Diabetic Rats

Abeer H. Abd El-Rahim*, Omaima M Abd-Elmoneim and Naglaa A. Hafiz

Cell Biology Department, National Research Center, El Tahrir Street, 12622 Dokki, Giza, Egypt

Received March 5, 2017 Revised July 24, 2017 Accepted August 4, 2017

Abstract

The present study was conducted to investigate the protective role of selenium nanoparticles on type-2 diabetes mellitus (T2DM) in male rats treated with metformin drug compared to the control. Chromosomal aberrations, DNA fragmentation, micronucleus tests as well as comet assay were carried out. Rats were divided into four groups: group1, normal control in which the rats were received normal saline solution, group 2, control diabetic group in which the diabetic rats were induced using 45mg/Kg body weight Streptozotocin (STZ). Group 3, Metformin treated group in which the diabetic rats were treated with a standard oral hypoglycemic agent, metformin (100 mg/kg body weight (b.wt) while, Group 4, selenium nanoparticles (SeNPs) treated group, in which the rats were treated with nanoselenium stabilized in liposome (0.1 mg/kg b. wt) orally plus metformin. Rats were sacrificed after 21 days following the last injection. The results demonstrated that diabetes caused increase in chromosomal aberrations, micronucleus formation, DNA fragmentation as well as DNA damage using comet assay, but these parameters decrease in treated animals with metformin. In addition, metformin + nanoselenium combined treatment caused decreased in all studied parameters relative to metformin alone. In conclusion, it appeared that SeNPs combined with metformin have antidiabetic effect in experimental model of T2DM. This effect could be ascribed to their ability in decreasing frequencies of chromosomal aberrations, decreasing the number of micronucleus as well as decreasing the percentages of DNA damage. Thus, SeNPs may benefit in clinical purposes especially in enhancing the effect of T2DM.

Keywords: Selenium nanoparticles, Diabetes, Bone marrow, DNA damage, Rats.

* Corresponding author. e-mail: abeerabdel_rahim@yahoo.com.

1. Introduction

Diabetes mellitus is the most common endocrine disease in which there is an increase in the level of blood sugar over a prolonged period (hyperglycemia) (World Health Organization (WHO), 2014). There are many symptoms of this disease, such as increased ingestion of food, increased thirst, muscular proteolysis, postprandial sickness and damage of many organs, such as eyes, nerves, kidneys and heart, these symptoms affect the human life as a whole. If untreated, diabetes causes many problems, such as cardiovascular diseases, kidney failure and eye damage (WHO, 2013), diabetic ketoacidosis and nonketetotic hyperosmolar coma (Kitabchi et al., 2009).

There are three types of diabetes mellitus: type 1, which results from a failure in the pancreas (WHO, 2013). Type 2 (the most common type of diabetes) in which the cells do not respond to insulin property, and the number of diagnosed cases of this type has increased over the past few decades (Olansky et al., 2003), and it forms about 90% of the diabetes cases (Shi and Frank, 2014)

distributed equally between women and men (Vos et al., 2012) and the number is expected to reach 592 million in 2035 (IDF, 2014). Type 3 is gestational diabetes and this type occurs in pregnant women who have diabetes history in their family (WHO, 2013).

The treatment of type 2 diabetes depends on using many drugs, such as sulfonylureas, Insulin, peroxisome proliferator-activated receptor- γ agonists and metformin.

Metformin (dimethylbiguanide) is the most described oral drug used in the treatment of type 2 diabetes (The American Society of Health-System Pharmacists, 2016; Maruthur et al., 2016) to control the blood sugar level with or without Insulin in conjunction with life modification, such as weight control, special diet and physical activity (Sardas et al., 2001). Metformin decreases the resistance of insulin, hepatic glucose output and enhances the uptaking of glucose (Bailey and Turner, 1996; Nisbet et al., 2004); these to decrease fasting and blood glucose by 20 to 40 percent, decreasing of hemoglobin and body weight and increase high density lipoprotein (HDL) (Howlett and Bailey, 1999).

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 160

Selenium (Se) is an important element for health with important biological and biochemical functions due to its antioxidant properties (Chunying et al., 2006). Se is a basic component of Selenoprotein which reduces oxidative stress (Rayman, 2012, Ahmed et al., 2016).

High selenium level in blood plasma has been correlated with prevention of several diseases, such as cancers, cardiovascular diseases, muscle disorders as well as diabetes mellitus (Navarro-Alarco´n and Lo´pez-Martı´nez, 2000).

The relation between selenium and T2DM is controversial (Wang et al., 2016). So untreated diabetes causes oxidative stress which leads to complications of the disease and the antioxidant intake is considered as being favorable for the therapy of diabetes (Mueller et al., 2009), and results in decreasing insulin, homeostasis model of assessment-insulin resistance, serum high-sensitivity C-reactive protein, and increasing the quantitative insulin sensitivity check index score as well as the concentration of the total antioxidant capacity (Farrokhian et al., 2016). It is clear that Selenium plays a protective role against T2DM (Steinbrenner and Sies, 2009).

Furthermore, it has been found that the half-life of some therapeutics can be improved by conjugating them to nanocarriers (Makhluf et al., 2008; Liu et al., 2012). It is obvious that selenium nanoparticles (SeNPs) have been considered a promising tool in drug therapies of type 2 diabetes; this is due to its low toxicity and high therapeutic properties (Rao et al., 2014).

SeNPs have been widely studied because they have advanced biological activity and high photoelectric performance, antioxidant activity, anticancer, and immune-system enhancements (Gao et al., 2002; Gates et al., 2002). By comparing Se with SeNPs, it was found that SeNPs can serve as a potential chemopreventive agent with reduced risk of Se toxicity (Zhang et al., 2005; Wang et al., 2007).

The purpose of the present study is to investigate the action of nanoselenium, combined with metformin, on the induction of chromosomal aberrations, formation of micronuclei and DNA fragmentation in streptozotocin induced T2DM rats compared to those healthy controls.

2. Materials and Methods

2.1. Experimental Animals For the present study, adult female albino rats of

Westar Strain weighing 180- 200 g were obtained from the Holding Company for Biological Products and Vaccines (VACSERA, Cairo, Egypt). After an acclimatization period of one week, the animals were classified into four groups of equal average body weight and housed in wire-bottomed cages in room under standard condition of illumination with 12 hours light-dark cycle at 25+1 C̊. They were provided with water and rodent chow ad libitum. All animals received care in compliance with the Egyptian rules of animal experiments which were approved by the ethical committee of medical research of the National Research Centre, Cairo, Egypt.

2.2. Induction of Diabetes

Rats were fasted for 18 – 24 h before induction diabetes by using streptozotocin (STZ) from Sigma. St. Louis, MO, USA. (CAS no 18883-66-4). The rats received a single intra-peritoneal (i. p) injection of 45 mg/Kg/wt of STZ, which was freshly prepared and dissolved in 0.05 M citrate buffer, pH 4.5 as described previously (Wei et al., 2003). Blood glucose was contoured every 2 days using an Accu-chek blood glucose meter (Roche Diagnostics, Basel, Switzerland). Rats with blood glucose levels > 15 Mm (200mg/dl) for 7 consecutive days were considered diabetic. 2.3. Experimental Design

Rats were divided into four groups: group1, a normal control group in which the rats received normal saline solution; group 2, a diabetic control group in which the rats were diabetic using Streptozotocin (45mg/Kg b.wt) and left untreated; group 3, a group treated with Metformin in which the diabetic rats were treated with a standard oral hypoglycemic agent, metformin from Merck, CAS no 657-24-9, USA (100 mg/kg b.wt) (Kosegawa et al., 1999); and group 4, selenium nanoparticles (SeNPs)-treated group, in which the rats were treated with nanoselenium stabilized in liposome (0.1 mg/kg b. wt) orally (Loeschner et al., 2014) in addition to metformin treatment. After 14 days of daily treatment, overnight fasting animals euthanized under mild ether anesthesia. 2.4. Biosynthesis of Selenium Nanoparticles

Nanoselenium was prepared according to the method of Dwivedi et al.(2011) by a simple wet chemical method. Sodium selenosulphate (Na2SeSO3) (Sigma. St. Louis, MO, USA) precursor reacted with different organic carboxylic acids in aqueous medium by using polyvinyl alcohol to stabilize selenium nanoparticles. Then, the synthesized nanoparticles were separated from their solution by centrifugation at 15,000 rpm and redispersed in aqueous medium with a sonicator. 2.5. Characterization of Selenium Nanoparticles

Characterization of selenium nanoparticles was carried out by examination of a sample under Transmission Electron Microscope (TEM) (JEOL-100 CX). 2.6. Liposome Preparation

Elemental selenium nanopartiacles in molar ratio7:2 were used to prepare neutral mutilamellar vesicles using the method of Kim et al. (1985). Briefly, 10 mg of high purity L-alpha-diapalmitoyl phosphatidyl choline (DPPC) from (Lipoid KG-Germany) and 1 mg of selenium were transferred to 50 ml round bottom flask. Then, 15 ml of Chloroform was added, and the flask was shaken until all lipids dissolved in Chloroform. The solvent evaporated under vacuum using rotary evaporator until a thin dry film of lipids was formed. The flask left under vacuum for 12 h to ensure the evaporation of all traces of chloroform. 10 ml of buffer (10 mM Trizma, pH 7) added to the flask in mechanically shaken for 1 h at temperature of 45 ̊C. The suspension centrifuged at 8000 rpm for 20 min and the supernatant discarded. The liposome re-suspended in 10 ml buffer solution. Control liposome was prepared following the same classical method as before using only

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161

aliquots of 10 mg of DPPC. The concentration of free selenium/ml of buffer was adjusted to be 1/mg/ml. 2.7. DNA Fragmentation Assay

DNA fragmentation was measured by spectrophotometer using diphenylamine (DPA) method, according to the method of (Perandones et al., 1993) with some modifications. Samples of liver were homogenized in lyses buffer containing 5mM Tris–HCl, pH 8.0, 20mM EDTA and 0.5% Triton X-100. Then, centrifuged at 1500×g for 20 min. Pellets were resuspended in 0.5N perchloric acid and 5.5N perchloric acid was added to supernatant, centrifuged again at 1500×g for 10 min to remove proteins. Samples were heated at 90 ◦C and after cool reacted with diphenylamine (DPA) for 16–20h at room temperature. Absorbance was measured at 600 nm using a UV-double beam spectrophotometer (Shimdazu 160 A). DNA fragmentation in samples = (fragmented DNA in supernatant.)/[(fragmented DNA in supernatant intact + DNA in pellet)] were expressed as percentage of total DNA appearing in the supernatant fraction. 2.8. Detection of Oxidative DNA Damage (Comet Assay)

According to the method of Singh et al. (1988), 0.5 g of crushed samples were transferred to 1 ml ice-cold PBS. This suspension was stirred for 5 min and filtered. Cell suspension (100 μl) was mixed with 600 μl of low-melting agarose (0.8% in PBS). 100 μl of this mixture was spread on pre-coated slides. The coated slides were immersed in lyses buffer (0.045 M TBE, pH 8.4, containing 2.5% SDS) for 15 min. The slides were placed in electrophoresis chamber containing the same TBE buffer, but devoid of SDS. The electrophoresis conditions were 2 V/cm for 2 min and 100 mA. Staining was done using 20μg/ml ethidium bromide at 4°C. A total of 100 randomly captured comets from each slide was examined at 400 x magnification using a fluorescence microscope connected to CCD camera to an image analysis system [komet 5 image analysis software developed by Kinetic Imaging, Ltd. (Liverpool, UK)]. A computerized image analysis system acquires images, computes the integrated intensity profiles for each cell, estimates the comet cell components and, then, evaluates the range of derived parameters. To quantify the DNA damage, Tail Length (TL), the percentage of migrated DNA (Tail DNA %) and Tail Moment (TM) were evaluated. Tail length (length of DNA migration) is related directly to the DNA fragment size and presented in micrometers. It was calculated from the centre of the cell. Finally, the program calculates tail moment. 2.9. Bone Marrow Chromosomal Aberration Assay

At first, the mice were injected with 4 mg/kg b.wt. colchicine two hours before sacrifice. Metaphase cells were prepared according to the standard technique of Preston et al. (1987). Bone marrow cells were aspirated

from both femurs of each animal; then, the cells were centrifuged at 1000 rpm for 10 min. and resuspended in pre-warmed (37°C), hypotonic solution (0.075M potassium chloride) for 20 min at 37°C. The samples were centrifuged and fixed in cold 3:1 methanol: glacial acetic acid. Each sample was washed five times fixative. The slides were stained in 10% buffered Giemsa (pH 7.0), air-dried and mounted in DPX. Chromosome aberrations were identified according to criteria described by Savage (1975). 2.10. The Micronucleus Test

Bone marrow slides were prepared according to the method described by Hayashi et al. (1983). The bone marrow was washed with 1 ml of fetal calf serum and then smeared on clean slides. The slides were left to air-dry and then fixed in methanol for 5 minutes followed by staining in May-Grunwald- Gemisa for 5 minutes then washed in distilled water and mounted. For each animal, at least 2000 polychromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei. 2.11. Statistical Analysis

Statistical analysis was carried out with SPSS software. Data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan's post hoc test for comparison between different treatments. The values were expressed as mean ± S.E and differences were considered as significant when < P 0.05.

3. Results

3.1. Chromosomes Examination

The analysis of bone marrow chromosomes in diabetic animals depicted various types of chromosomal aberrations, which included gap, break, deletions, fragments, centromeric attenuations and endomitosis as structural aberrations as well as polyploidy and prediploidy as numerical aberrations.

The results in table 1 represent the chromosomal aberrations analysis and showed that the frequencies of structural and numerical chromosomal aberrations were significantly increased (P< 0.05) in diabetic animals (STZ-challenged group) comparing to control group. In contrast, the diabetic animals treated with Metformin had significant (P< 0.05) decreases in the frequencies of all structural and numerical chromosomal aberrations than diabetic group. In addition, metformin/nanoselenium group had the lowest frequencies of most structural aberrations (gap, fragment, endomitosis, C.A, total structural aberrations) and total numerical chromosome aberrations relative to Metformin group.

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 162

Table 1. Effect of metformin and nanoselenium treatments on the frequency of chromosome aberrations in STZ- induced diabetic rats

Total aberrations

Numerical aberrations Structural aberrations Treatment

Total Polyploidy Preidiploidy Total Endomitosis Centromeric ttenuation Fragment Deletion Break Gap

3.84±0.30P

d 1.67±0.21bP

c 0.33±0.21P

b 1.33±0.21P

b 2.17±0.40P

d 0.67±0.21P

c 0.50±0.22P

c 0.17±0.16P

c 0.33±0.21P

cP 0.33±0.21P

c 0.67±0.21P

c Control

25.50±0.56P

a 7.17±0.30P

a 2.50±0.22P

a 4.67±0.21P

a 18.33±0.42P

aP 3.67±0.21P

a 4.50±0.22P

a 2.50±0.22P

a 2.67±0.33P

a 2.67±0.21P

a 3.67±0.21P

a D

11.34±0.42P

b 2.17±0.16P

b 0.67±0.21P

b 1.50±0.22P

b 9.17±0.47P

b 2.00±0.26P

b 2.33±0.21P

b 1.50±0.22P

b 1.33±0.21P

b 1.50±0.22P

b 2.00±0.26P

b

D+M

5.83±4.77P

c 1.33±0.21P

c 0.33±0.21P

b 1.17±0.16P

b 4.50±0.34P

c 1.17±0.30P

c 0.83±0.16P

c 0.50±0.22P

c 1.17±0.23P

b 1.46±0.19P

b 1.17±0.30P

c D+M+Se

D: Diabetic rats by using streptozotocin (STZ); D+M: Diabetic group treated with metformin drug; D+M+Se: Diabetic group treated with metformin and nanoselenium particles. All data are expressed as mean ± SEM. Means bearing different letters superscripts are significantly different at (P < 0.05).

3.2. Influence of Diabetes on the DNA Damage (Comet Assay)

The DNA damage resulted from diabetes was investigated by comet assy. the lengths of the comets (DNA tails) depended on the effect of diabetic in rats, longer tails indicating more DNA damage.

Figure 1 shows representative examples of diabetic rats. Control liver rat cells showed no tails (Figure A). Tails appeared in diabetic rats (Figure B) and were substantially less in diabetic treatment one (Figures C and D). The mean value of tail length in Diabetic group was increased rapidly and significantly more than that of their controls, as shown in table 2, and more DNA damage was observed in the tail when compared with control group (Figure 1).

As shown in table 2, the extent of DNA damage, measured in TM, increased rapidly in diabetic group when compared to control. Treated groups with metformin or metformin+ nanoselenium showed a significant decrease in comet TM values when compared to diabetic group (P<0.05) but is still a significant increase compared with the control.

The maximum extent of DNA damage, increased rapidly in diabetic group when compared to control. Then, the extent of damage decreased significantly (P<0.05) in both treated groups with metformin or metformin+nanoslenium but it did not reach the control level.

Figure 1. Effect of metformin, nanoselenium treatments in STZ- induced diabetic rats on bone marrow DNA as detected by comet assay. (A) Normal untreated cell; (B) diabetic cell, (c) diabetic rat treated with metformin and (D) diabetic rat treated with nanoselenium and metformin.

Table 2. Comet parameters in liver cells of metformin, nanoselenium treatments in STZ- induced diabetic rats as measured by comet assay.

D: Diabetic rats by using streptozotocin (STZ); D+M: Diabetic group treated with metformin drug; D+M+Se: Diabetic group treated with metformin and nanoselenium particles. All data are expressed as mean ± SEM. Means bearing different letters superscripts are significantly different at (P < 0.05).

3.3. Micronucleus Test

The effect of diabetes and treatments with metformin and nanoselenium on micronucleated polychromatic erythrocytes (MnPCEs) formation in the bone marrow cells of male rats is shown in Figure 2. In this Figure, it is demonstrated that there were few formations of MN in rats belong to control group (3.33±0.21), but MN formation significantly increased (P<0.05) in diabetic animals (26.00±0.25) comparing to control group. Meanwhile, treated groups with metformin significantly reduced the MNPCE when compared with diabetic group. Additionally, MNPCE in the combined metformin and nanoselenium (14.00±0.25) significantly reduced more than the group treated with metformin alone (20.67±0.33).

Figure 2. Frequencies of micronucleated polychromatic erythrocytes (MPCEs) in rat bone marrow cells in all experimental groups.

Tail moment (unit)

DNA %

Tail length µm

Untailed %

Tailed % Treatment

2.84± 0.41P

d 1.33± 0.22P

d 2.16± 0.09P

c 97.67± 0.33P

a 2.33± 0.33P

d Control

34.65± 2.41P

a 6.57± 0.28P

a 5.27± 0.18P

a 79.0± 0.58P

d 21.0± 0.58P

a D

19.25± 1.66P

b 4.27± 0.15P

b 4.5± 0.29P

b 82.33± 0.33P

c 17.67± 0.33P

b D+M

13.19± 1.88P

c 3.47± 0.26P

c 3.77± 0.28P

b 85.0± 0.58P

b 15.0± 0.58P

c D+M+Se

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163

3.4. DNA Fragmentation

The effects of nanoselenium and metformin drug on diabetic rats were investigated by DNA fragmentation assay (Figure 3). The result demonstrated that fragmented DNA, in the control group (8.33±0.33), was lower than that in the other treated groups. Also, fragmented DNA in diabetic animals (24.33±0.33) was significantly higher than that of the control group (P< 0.05). On the other hand, treated animals with metformin decreased the DNA fragmentation comparing with diabetic animals (P<0.05). In addition, the combined treatment with metformin /nanoselenium (12.17±0.47) decreased the fragmentation of DNA compared to the treatment with metformin alone (18.33±0.33) (P<0.05).

Figure 3. Percentages of DNA fragmentation in rat liver cells in all experimental groups.

4. Discussion

The present study indicated that the administration of STZ increased genetic alterations (micronuclei frequency, fragmented DNA, as well as chromosomal aberrations). The genotoxic effects of STZ, observed in the present study, are in agreement with the results obtained from previous studies (Vikram et al., 2007; Attia et al., 2009).0T 0T Also, 1TMartínez-Pérez0T1T et al. (2007)0T found that there were high levels of micronucleus frequency in type 2 diabetes with no microvascular or macrovascular complications. In addition, Corbi et al. (2014) indicated that there is an association between T2DM and DNA Damage by studying the frequency of micronucleated polychromatic erythrocytes. This genotoxicity may be due to the presence of hyperglycemia condition in T2DM that caused production of reactive oxygen species (ROS) (Pham-Huy et al., 2008; Robertson, 2004). Accumulation of ROS caused an oxidative stress, which plays a crucial role in cellular mechanism of tissue injury in a wide spectrum of disease states. It may lead to a constant threat to all living organisms and antioxidant defense system is employed by the body to eliminate it (Ceconi et al., 2003). In addition, stress causes increase resistance of insulin and disturbance in pancreatic β-cell function (Houstis et al., 2006).

Oxidative stress can attack all types of macromolecules including DNA by attacking the cell membrane, nucleus and then genetic materials, which leads to chromosomal aberrations (Attia, 2010; Otton et

al., 2004). DNA oxidative damaging occurs due to modifications in nucleotide bases or sugars, these modifications lead to mutations which may cause formation of tumors if they occur in somatic cells (Selvakumar et al., 2006), then early aging may occur which leads to death (Rehman et al., 1999).

Moreover, ROS damage the cell by different pathways, such as advanced formation of advanced glycation end production, hexosamine pathway, protein kinase and lipid peroxidation (Piconi et al., 2003), which is a result of attacking ROS to the residues of polyunsaturated fatty acid of cell membrane. The imbalance between production of ROS and the ability of antioxidant to detoxify their effect cause oxidative stress (Echtay, 2007; Roberts and Sindhu, 2009).

By using comet assay, the present study revealed that diabetes cause DNA damage. This result is similar to that obtained from the study of Sardas et al. (2001) who reported increase in oxidative DNA damage in type 1 and type 2 diabetes, and these damage was higher in type 2 diabetes compared to type 1 diabetes. Also, Marra et al. (2002) found that oxidative stress is increased in diabetes and this leads to cardiovascular disease.

Diabetic animals treated with Metformin showed decrease of most frequencies of structural and numerical chromosome aberrations and reduced the incidence of MN formation in PCEs in the diabetic animals. These may be due to the lowering of blood glucose by Metformin as a result of stimulating insulin releasing from functioning pancreatic beta cells (Chunying et al., 2006; Kirpichnikov et al., 2002) and hepatic glucose output as well as enhancing peripheral glucose uptake (Bailey and Turner, 1996), these to decrease fasting blood glucose by 20 to 4%, decrease body weight, decrease low density lipoprotein and increase high density lipoprotein (Howlett and Bailey, 1999). Therefore, a diabetes patient has three times normal rate of gluconeogenesis but metformin reduces this by one-third (Hundal et al., 2000). Metformin increased the insulin sensitivity and reduced the oxidative stress levels and the activity of catalase and superoxide dismutase when compared with diabetes rats (Vilela et al., 2016). So, the dual therapy with metformin drug promotes more benefits to oxidative stress control in rats.

The current data revealed that the treatment with SeNPs in addition to metformin drug resulted in a significant reduction in the genetic alterations (micronucleus formation, chromosomal aberrations) as well a comet assay. This may be due to the antioxidant property of a compound which could play a significant role in decreasing the nuclear injury caused by diabetes (Adler et al., 2009). These observations are consistent with those of Abdelaleem et al. (2016) who found that selenium nanoparticles possesses antioxidant and anti-diabetic activities by decreasing oxidative stress biomarkers as well as blood glucose level. Similarly, Ahmed et al. (2016) found that selenium nanoparticles have antidiabetic potency, repression of oxidative stress, potentiating of the antioxidant defense system, and inhibition of pancreatic inflammation. So, selenium plays a protective role against type 2 diabetes (Steinbrenner and Sies, 2009). The protective roles of selenium in mammalian cell are due to its function in the active site of many antioxidant enzymes, such as thioredoxin reductase, glutathione and GR (Flora

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 164

et al., 2002). Dietary selenium may cause an increase in glutathione peroxidase, which has the ability to detoxify ROS (Köhrle and Gärtner, 2009) that can interfere with insulin signaling and result in regulation of glucose levels and prevention of diabetes (Goldstein et al., 2005). Selenium is also a basic component of selenoprotein, an important enzyme in the body which protects from oxidative stress and inflammation (Rayman, 2012).

The nutritional deficiency of selenium causes muscular dystrophy, endemic fatal cardiomyopathy, and chronic degenerative diseases when used alone or in combination (Rayman, 2002).

5. Conclusion

In conclusion, the present study demonstrated the antidiabetic potential of SeNPs delivered in liposomes in the experimental model of T2DM. This effect appeared in decreasing the frequencies of chromosomal aberrations, in addition to decreasing the number of MNPCEs as well as repairing damaged DNA which is represent in DNA fragmentation assay and comet assay. Thus, SeNPs may benefit in clinical purposes, especially in enhancing the effect of T2DM.

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JJBS Volume 10, Number 3,September 2017

ISSN 1995-6673 Pages 167 - 176

Jordan Journal of Biological Sciences

Avifauna Diversity of Bahr Al-Najaf Wetlands and the Surrounding Areas, Iraq

Mudhafar A. Salim 1 and Salwan Ali Abed 2,*

1 Biodiversity and Natural Heritage Expert, The Arab Regional Center for the World Heritage, Kingdom of Bahrain;

2 Department of Environment, College of Science, University of Al-Qadisiyah, P.O.Box.1895, Iraq.

Received May 3, 2017 Revised June 27, 2017 Accepted July 31, 2017

Abstract

The Bahr Al-Najaf area is a natural depression located in the 'Middle Euphrates' area in the upper part of southern Iraq, 170km southwest of Baghdad. It represents a very important transitional area between the true desert regions to the west and the western limit of the alluvial plateau. The present study was conducted over the four seasons of 2015, and the area is located within latitudes (32° 09' 00" and 31° 48' 40"), and longitudes (43° 52' 30" and 44° 22' 00") where seven sampling locations were selected to represent the different environmental features and landscapes present. One hundred and sixty-eight bird species were observed during the surveys. Those bird species were resident breeding species or migrants (both wintering and passage). This illustrates the importance of the area in providing a suitable habitat for such a large spectrum of bird species that forms more than 40% of the entire checklist of the birds of Iraq. Based on the results of the current and previous studies, it seems that Bahr Al-Najaf wetlands and the surrounding areas deserve more protection and management towards better conservation of the natural heritage of the area.

Keywords: Bahr Al-Najaf, Avifauna, Diversity, Wetlands, Conservation, Iraq.

* Corresponding author. e-mail: Salwan.abed@qu.edu.iq; salwan_ali2000@yahoo.com.

1. Introduction

Iraq is located in the warm stripe between the cold, northern regions and the tropical, hot belt to the south, and that has resulted in enriching its avifaunal profile, both in quantity and diversity as represented by a relatively decent list of birds, most of which occur in considerable numbers (Allouse, 1960, 1961, 1962). Iraq is privileged in being located between the two hemispheres hosting key waterbirds migration routes between Eurasia and Africa (Boere and Stroud, 2006). The diversity of habitats and the variety in landscapes represent an added value in harboring large numbers of migrant birds each migration season, in addition to the resident breeding bird species (Salim et.al., 2006). One of the key habitat types that the migrant birds, especially the waterbirds, use are the wetlands and water bodies, both permanent and temporary, either during their passing over the country or using them as a wintering destination (Salim et al., 2009). Bahr Al-Najaf has a strong potential to be one of these key protected wetland areas in Iraq (Salman, 2015).

For these factors, the present study aims at highlighting the ecological importance of Bahr Al-Najaf area through shedding light on the biodiversity content of this area,

being poorly known wetlands along with the surrounding areas.

2. Methodology

The Study Area

Bahr Al-Najaf (Sea of Al-Najaf) is considered one of the key wetlands in Iraq that is located in the lower parts of Iraq, in a region called the Middle Euphrates to the west of the Euphrates River, and 2km west of Al-Najaf city (Figure 1). The marshes and the adjacent orchards are located to the south of the area that forms the edge of the Alluvial Plateau (Benni, 2001). The area to the north and west of Bahr Al-Najaf consists of the edges of the western desert that extends to the west and south towards the Saudi Arabian deserts. The location of Bahr Al-Najaf being between two different types of habitat regions (wetlands & desert) is reflected distinctively in the morphology of the area, and the rich biological diversity of the area (Mohammad et al., 2013b).

The sources of the water that feed the waterbody and the lower marshes are the water springs and oases from the northwest in addition to the local groundwater (Taleb, 2012). The Bahr also receives surface waters from the western side via the watercourses that bring water on a

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 168

seasonal basis from the upland desert to the west after any heavy rains (Al-Aboodi, 2008).

The study area consists of the following different habitat types: The open waterbody of the Bahr Al-Najaf (the lake); The marshes and reedbeds areas; The mudflats and 'Sabkha' (salt-crested soils) areas; The cliff-belt to the east, northeast, and north of the area; The desert and semi-deserted areas to the west; The natural springs that feed the area with permanent water, and The dense orchards and date-palm area to the southeast.

The diversity in topography that has resulted in the habitats and morphological landscapes has provided Bahr Al-Najaf and the surrounding areas with a variety of plant cover, and in turn in the biological diversity in the fauna species in the area (Benni, 2001).

The eastern and southern areas of the Bahr consist of a large variety of wetland habitats, of which the waterbody of the Bahr forms the main component of the study area, as shown in the map and the recent satellite image below:

Figure 1. The location of the study area: Bahr Al-Najaf and the surrounding areas (Source: courtesy Land-sat, March 2014)

This research project was conducted in 2015, and surveys were carried out in all the four seasons of that year. The study area spans around 1,000km2. It is located within the latitudes (32° 09' 00" and 31° 48' 40"), and the longitudes (43° 52' 30" and 44° 22' 00"). GPS coordinates for the seven sites in the study area are presented in (Table 1). The elevation of the study area ranges between 130m asl (above sea level) at the northwestern corner, and 9m asl. at the eastern parts.

An ‘Area Count’ methodology (Bibby et al., 2000) was followed Bahr Al-Najaf wetland in order to gain as thorough an idea as possible about the status of birds and to describe the environmental conditions and assess the different types of threats the area is facing and which affect the birds and their habitats. A Garmin GPS device was used to locate the site with the aid of 1:100,000 maps. 12X45mm binoculars and a 40x spotting scope were used during the observations, and photos were taken with a canon 7D camera with a 50-500mm lens. A 4X4 field vehicle was used to visit the seven observation sites within the study area. A unified field form was designed and used during the count in which the bird species and their numbers were noted. The work of Salim et al. (2006),

"Field Guide to the Birds of Iraq," and that of Porter and Aspinall (2010), "Birds of the Middle East," were used to assist in identifying the birds observed at each site. Several interviews with locals and hunters were made in order to gauge the hunting pressure on the bird species in the Bahr Al-Najaf wetland and to collect further information about the other fauna species in the area.

Table 1. Coordinates of the seven sampling sites in Bahr Al-Najaf and surrounding areas

Site Longitude Latitude

D M S D M S

BN1 32 6 0 43 54 17

BN2 32 5 39 44 8 0

BN3 32 7 15 44 1 2

BN4 32 1 19 43 57 27

BN5 31 58 25 44 17 54

BN6 31 53 40 44 17 20

BN7 31 54 19 44 3 13

3. Results

The diversity of the habitats within the study area (Figure 2) that was reflected in the density and distribution of plant cover in addition to the availability of permanent and seasonal water within the area, have influenced the occurrence and distribution of the bird species (both residents and migrants) in the study area in Bahr Al-Najaf.

Each of these habitats types includes 'sub-habitats' as follows: what is the reference of these classification – the different types of the habitats that were observed during the surveys were summarized in this list, so this is what the researchers have found, and was not taken from any references.

Terrestrial habitats: Desert and semi-desert landscape; Arid and salt-crest (Sabkha) landscape, and Cliff and alluvial stripes landscape.

Wetlands habitats: Natural springs and artificial wells; Open-lake landscape; Freshwater marshes with reedbeds; the wet farms and orchards, and Mudflats landscape.

A total of 168 bird species was observed during the different surveys in Bahr Al-Najaf and the surrounding areas.

It was noted that the majority of the avifaunal diversity in the Bahr Al-Najaf area is concentrated either in the wetlands within the study area, or close to those wetlands. The majority of the bird species were found at the edges and the mudflats of the lake and the marsh where large numbers of waders, terns, gulls, herons, and other waterbird species were observed. In comparison, the status of the birds in the desert and the relatively dryer areas was lower in terms of numbers of species and the populations of the birds.

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 169

A

B

C

D Figure 2. Different habitats within study area, A: Marsh, B: Waterbody, C: Sabkha, D: Semi-desert

The following seven sites (BN1-BN7) were observed

regularly, and the results of the field observations were as follows: 3.1. BN1

This site is located at the northwestern corner of the study area, 3 km south of the edge of the northern cliff.

The highest area in elevation within the study area is located to the north of this site. The site consists of open sandy/gravel desert with very few depressions. There is a small wetland body that seems seasonal to the south of this point. No human activities were observed in this site during the present study. The elevation at this point is 63m asl.

In comparison with the other sampling sites, it seems that this area is poor in bird species, and this might be due to the lack of diversity of habitats and plant cover within this area. 3.2. BN2

This site is located at the edge of the cliff, 5.5 km to the east of the paved road that divides the study area. It consists of a steep cliff that faces the northern part of the Bahr Al-Najaf depression. This site is a suitable habitat for owls and other cave-dwelling bird species. The elevation at this point is 86m asl.

In comparison with the other sampling sites, few bird species were spotted within this area, and this might be due to the lack of diversity of habitats and the lack to the water within this area. 3.3. BN3

This site is located in the upper part of the study area, between the waterbody and the desert. It is a Sabkha habitat (salty soil) and is close to a small wetland. Small-scale human activities were observed to the north of this site. The elevation at this point is 37m asl.

The number of bird species found in this area was more than that observed in the above-mentioned sites combined. 3.4. BN4

This site is located inside the waterbody of Bahr Al-Najaf. It is close to the paved road that runs north to south amid the waterbody and the study area. The habitat of this site consists of open water to the east which continues southwards as well, and a shallow waterbody at the western part. The elevation at this point is 9m asl.

The highest number of bird species was observed in this area. This might be due to the presence of the open-water lake and the muddy shores of the waterbody itself that provided good habitats for a wide spectrum of waders and other waterbirds to feed and rest in their typical habitat. 3.5. BN5

This site is located in the eastern part of the study area, in the middle of a shallow-water marsh habitat of reedbeds Phragmites and Typha. The area also includes some other aquatic and wetlands plant cover. It seems that this marsh provides a good habitat for marsh-dwelling bird species in addition to facilitating the movement of the buffalo and their keepers. No human activities were observed in this site except the presence of the buffalo-keepers. The elevation at this point is 13m asl.

The number of bird species observed in this site is considerable, compared with the rest of the surveyed sites. Most of the observed birds in this area were herons, terns, waders, and passerines, of which some are threatened species. It seems that this area harbors some endangered species where the Basra Reed Warbler Acrocephalus griseldis was recorded in the reedbeds of this freshwater marsh area.

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 170

3.6. BN6 This site is located in the southeastern part of the study

area, in a seasonal Sabkha marsh, poor with plant cover (salty soil flat area), 4.5 km to the east of the paved road. The elevation at this point is 14m asl.

In comparison with other surveyed sites, this location did not demonstrate good diversity in terms of bird species. Most of the birds that were observed within this site were land birds in addition to some passerines. 3.7. BN7

This site is located at the western part of the study area, 17km to the west of the paved road. This site represents the true desert habitat of which is located in one of the water courses that is common in the desert areas to the west of the study area. There are some scattered shrubs in the area especially at the bottoms of the seasonal watercourses. The elevation at this point is 57m asl.

Most of the bird species that were observed in this area were either desert dwellers or passage birds that use the shrubs of the watercourses almost frequently. Large numbers of passage Passerines were found in the shrubs of the watercourses especially during the migration season. The threatened Macqueen's Bustard Chlamydotis macqueenii (VU) was observed in the open, shrubby desert of this area. The locals have reported that the falconers used to collect some Falcons (mainly Falco cherrug) from or around this area.

The area is affected directly by the seasonal variation, and this was reflected obviously by the remarkable change in the occurrence of the bird species along the year. Table 2 below clearly shows the seasonal variation in the observed numbers of birds in the study area where the winter held the highest counts of the species number, while summer season was the lowest in numbers. The bird list in the Annex shows the entire bird species that were observed in Bahr Al-Najaf and the surrounding areas.

Table 2. Seasonal variation of bird’s occurrence in Bahr Al-Najaf

Survey Bird Spp. Numbers

1 Winter (Jan.) 126

2 Spring (Apr.) 82

3 Summer (June) 41

4 Autumn (Sept.) 98

4. Discussion

Based on the results of the present study, and the bird counts during the four seasons of the fieldwork, it seems that the diversity in the landscape and habitats in Bahr Al-Najaf area makes it one of the high-ranked areas in terms of biological diversity, especially in avifauna. Whilst the current study reported 168 bird species in the study area, in a previous survey of the same area, 73 species were recorded (Mohammed et al., 2013a), and this might illustrate the dynamic status of the avifauna in this area. Moreover, it illustrates the level of richness in the bird species in this area at more than 40% of the national checklist (Salim et al., 2012) (see the bird-list in the Annex.1).

The diversity in the morphological features reflected in the plant-cover, in addition to the presence of the

freshwater sources (and the groundwater) (Al-Aboodi, 2008) and (Taleb, 2012), have created an active ecosystem that combines a spectrum of desert habitats that extends to intermediate/moist margins, to the actual wetlands in area of Bahr Al-Najaf and the surrounding areas. This has contributed to the richness of the area in fauna species (Mohammad et al., 2013a; Mohammed et al., 2015), taking into consideration the western extension of the desert landscape that are very important to the migrant birds over Iraq (Ctyroky, 1988) where new bird species are still being discovered, like the recently-recorded Thick-billed Lark from nearby areas (Mohammad et al., 2013b). It also seems that some of the recently-recorded bird species have found in Bahr Al-Najaf good hosting habitat where the Black-shouldered Kite Elanus caeruleus, Namaqua Dove Oena capensis, and Common Mynah Acredotheres tristis – all are new species to Iraq, frequently observed in the study area and around it (Salim, 1998, 2002, and 2008).

It was also notable that the highest numbers of the observed bird species and populations are located in or close to the wetlands. The Bird species numbers and population in the (BN4) site were the highest, and this might be due to the availability of water and mudflats that provide a wide range of micro-habitats for these species which attracts migrant waders and waterbirds (that are congregator species) in relatively large numbers (Boere and Stroud, 2006). It seems that the majority of these bird species that visit this wetland for resting and refueling belong to Gull, Tern, Duck, and other wader species are migrants between Eurasia and Africa (Cramp and Simmons, 1983).

The presence of some threatened bird species in Bahr Al-Najaf provides important insight into the significance of the area and why it is eligible for protection. Salim (2011) indicated the serious threat of falconry in the area and in the surrounding deserts, and pointed to the illegal but massive hunting that takes place in these habitats whereby large numbers of threatened birds, like the Macqueen's Bustard Chlamydotis macqueenii are shot or trapped. The wetland part of the area is also still a main target for hunters and bird collectors, exactly as is happening with the hunting and netting of threatened waterfowl, such as the Marbled Teal Marmaronetta angustirostris, in quite considerable numbers in different wetlands in Iraq (Abed, 2014) which also has a very low distribution around Iraq (Porter and Aspinall, 2010).

The desert parts of the study area seem to hold remarkable wildlife and a variety of bird species. However, further studies and more research with special methodologies are required in this regard. The desert areas seem to harbor some threatened species where the Macqueen's Bustard Chlamydotis macqueenii was observed in the open, shrubby desert to the west of the study area. This species is vulnerable according to the IUCN Red list and Bird Life International (Bird Life International, 2011), and is facing serious pressure of hunting and poaching (Salim, 2011). Moreover, the presence of the threatened Falco cherrug gives the study area and the surrounding areas special priority for conservation and management.

Based on the results of the present study, Bahr Al-Najaf is still one of the poorly known but biodiversely rich areas

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 171

in Iraq, and represents a very important habitat for fauna on the national level. The area has several first or second records for species in Iraq (Salim, 1998, 2008; Mohammad et al., 2013b). The number of these new bird species is an indicator of the virginity of the area and the necessity for more scientific and technical effort to better cover the flora and fauna of this relatively unknown area.

The present study supports the finding that two seasons (winter and autumn) are the highest in terms of bird species numbers, while spring and summer are relatively poorest in the occurrence of the bird species (Figure 3). For several reasons, including the severe hot temperature in this area during summer, and based on the results of the current fieldwork, only 41 bird species were observed in the area of which the majority was observed as breeding species that belong to either the resident or the summer-visitor populations (Mohammad et al., 2013a).

Figure 3. Migrant and resident bird species observed in Bahr Al-Najaf over the various seasons

The difference in the number of the species and the bird populations in this area over the different seasons might be due to different factors. Some key factors are the arrival of the migrant birds, in addition to the variety of the floral conditions in the area, and the diversity and availability of foraging areas and food that differ from season to season (Abbas et al., 2015; Mohammad et al., 2015.

The study area seems to hold a considerable number of threatened birds according to the IUCN Redlist (http://www.iucnredlist.org). Within the present study, eleven threatened bird species were observed in the area, of which two were endangered, four were vulnerable, and five species were near-threatened (Table 3). This makes the study area and the surrounding areas eligible for more protection; this is in agreement with and supports what Salman (2015) mentioned in her recommendations.

Table 3. Threatened and conservation concern bird species observed in Bahr Al-Najaf and surrounding areas

Bird Species Scientific Name Conservation Status *

Egyptian Vulture Neophron percnopterus

E

Basra Reed Warbler

Acrocephalus griseldis

E

Marbled Duck Marmaronetta angustirostris

V

Greater Spotted Eagle

Aquila clanga V

Saker Falcon Falco cherrug V

Macqueen's Bustard

Chlamydotis macqueenii

V

Ferruginous Duck Aythya nyroca NT

Pallid Harrier Circus macrourus NT

Black-tailed Godwit

Limosa limosa NT

European Roller Coracias garrulous NT

Semi-collared Flycatcher

Ficedula semitorquata

NT

5. Conclusion and Conservation Issues

Bahr Al-Najaf wetlands and the surrounding areas complex is an area of high diversity in terms of landscapes and habitats, which can be grouped into terrestrial and aquatic habitats.

This considerable diversity in habitats and landscapes is a major factor contributing to the richness of the area in terms of flora and fauna (especially avifauna), in turn. Both groups of birds, the resident and migrant species, use the different habitats that provide shelter and food. The area also harbors considerable congregations of waterfowl and large numbers of passerines during the migration and wintering seasons. A considerable proportion of the birds observed in this area were under some kind of threat, which makes it an area eligible for protection and environmental management, either as a Ramsar site or as another kind of protected areas.

A Notable but serious list of threats was observed after the intensive fieldwork at and around the area. The major threat that the birds in Bahr Al-Najaf area face is hunting and poaching, and these activities target even the threatened species, like the Macqueen's Bustard and falcons. The unsustainable use of the basic natural resources, like the harvesting of natural spring water for fish farming, is considered as an additional threatening factor. The intensive fish farming mostly occurs upstream at the upper-western parts of the area and by some of the nearby agricultural farms. The area faces other types of threats as well. The disposal of solid waste, disturbance, and unsustainable fishing, although on a small scale, were also documented within the area. The chemical pollution from agricultural drainage water might be an added threat. However, this requires further study to be confirmed. There might also be a potential threat from urban expansion, especially in the lower parts of the area.

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 172

Acknowledgement

The authors of the present study are grateful to the following institutions for their kind support and for providing useful logistical and funding support during the planning, fieldwork, data-analysis, writing of this paper, and their kind support in general: The Iraqi Organization for Conservation of Nature (IOCN), the University of Al-Qadisiyah / College of Science, and the Arab Regional Center for World Heritage (ARC-WH). Special thanks go to the Ministry of Health and Environment and the Local Government of Najaf. Thanks are also due to Mohamad Mahdi for his help in proofreading of the present paper.

References

Abbas R F, Nasrullah R P, Karamiani R and Mohammed G R. 2015.Taxonomic status of sand boas of the genus Eryx (Daudin, 1803) (Serpentes: Boidae) in Bahr Al-Najaf depression, Al-Najaf Province, Iraq. Ira. J Anim. Biosyst., 11(2):149-156. Abed S A. 2014. Bio-Ecological study of Marbled Teal Marmaronetta angustirostris in Hor Al-Dalmaj - Iraq. Ph.D. dissertation, University of Baghdad, Iraq. Al-Aboodi A H. 2008. Hydrochemical classification of groundwater in Bahr Al-Najaf, Western Desert, Iraq. J Basrah Res (Sciences) 34 (2): 23-32. Allouse B. 1960, this year not available in the main text – they were added to the text because we used the three volumes [Birds of Iraq]. Vols I, II, III. Al-Rabita Press, Baghdad. [In Arabic]. Benni TJ. 2001. Sedimentological and Palaeoclimatic Record of Bahr Al-Najaf Depression during Late Quaternary. Unpub. M.Sc. dissertation, University of Baghdad. Iraq. Bibby CJ. Burgess ND, Hill DA and Mustoe SH. 2000. Bird: Census Techniques. 2nd edition. Academic Press, London. Bird Life International. 2011. IUCN Red List for birds. www.birdlife.org. Boere GC and Stroud DA. 2006. The flyway concept: what it is and what it isn’t. In: Boere, GC, Galbraith, CA and StroudDA. (Eds), Waterbirds Around the World. The Stationery Office, Edinburgh, UK. pp. 40-47.

Cramp S and Simmons KEL. 1983. The Birds of the Western Palearctic. Vol 3. Waders to Gulls. Oxford University Press, UK. Ctyroky P. 1988. Autumn migration of birds in the western desert of Iraq. Beitrage Vogelkunde 34: 230–236. IUCN – the International Union for the Conservation of Nature: (http://www.iucnredlist.org). Mohammad K M, Ali B A and Hadi A M. 2013 a. The Biodiversity of Bahr Al-Najaf Depression, Al-Najaf Al-Ashrraf Province. Bull Iraq Nat Hist Mus., 12 (3): 21-30. Mohammad K M, Al-Rammahi H M and Lahony S R. 2013 b. The First Record Of The Thick-Billed Lark Rhamphocoris clotbey (Bonaparte, 1850) For Iraq. Bull Iraq Nat Hist Mus.,12 (3): 37-41. Mohammed RG, Nasrullah RP, Karamiani Rand Rhadi FA. 2015. The Lizard Fauna of AL-Najaf Province, Southern Iraq. Cumhuriyet University Faculty of Science, Cumhuriyet Sci J. (CSJ), 36( 6): Special Issue. Porter R and Aspinall S. 2010. Birds of the Middle East. Christopher Helm, London. Salim MA. 1998. The first Common Mynah Acredotheris tristis in Iraq. Sandgrouse 20: 148–149. Salim MA. 2002. The first records, including breeding, of the Black-winged Kite Elanus caeruleus in Iraq. Sandgrouse 24: 136–138. Salim, M.A. 2008. The first Namaqua Dove Oena capensis in Iraq. Sandgrouse 30: 100–101. Salim, M.A. 2011. The illegal hunting and trade increase the threat on Macqueen's Bustard Chlamydotis macqueenii in Iraq. NI Technical Publications NI-1011-02. [In Arabic]. Salim MA, Porter RF and Clayton R. 2009. A summary of the Birds Recorded in the Marshes of Southern Iraq, 2005–2008. Bio Risk 3: 205–219. Salim MA, Porter RE, Schiermacker-Hansen P, Christensen S and Al- Jbour. S. 2006. [Field guide to the birds of Iraq]. NI/Bird Life International, Baghdad. [In Arabic]. Salim MA, Al-Sheikhly OF, Majeed KA and Porter RE. 2012. An annotated checklist of the birds of Iraq. Sandgrouse 34: 4-43. Salman AH. 2015. The environmental features of Bahr Al-Najaf depression and the exploitation of their potentials as natural reserve. Thi-Qar J. 270. Taleb Z A. 2012. Measurement of some chemical characters for the water of Bahr Al-Najaf. Al-Qadisiyah J Agri Sci.,2(1). [In Arabic].

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 173

Annex (1)

Checklist of the Birds of Bahr Al-Najaf Wetlands and the Surrounding Areas

Bird Species Scientific Name

Black Francolin Francolinus francolinus

Eastern Greylag Goose Anser anser rubrirostris

Greater White-fronted Goose Anser albifrons

Common Shelduck Tadorna tadorna

Ruddy Shelduck Tadorna ferruginea

Gadwall Anas strepera

Mallard Anas platyrhynchos

Northern Shoveler Anas clypeata

Northern Pintail Anas acuta

Eurasian Teal Anas crecca

Marbled Duck Marmaronetta angustirostris

Ferruginous Duck Aythya nyroca

Little Grebe Tachybaptus ruficollis

Great Crested Grebe Podiceps cristatus

Greater Flamingo Phoenicopterus roseus

Western White Stork Ciconia ciconia

Glossy Ibis Plegadis falcinellus

Eurasian Spoonbill Platalea leucorodia

Little Bittern Ixobrychus minutus

Black-crowned Night Heron Nycticorax nycticorax

Squacco Heron Ardeola ralloides

Western Cattle Egret Bubulcus ibis

Grey Heron Ardea cinerea

Purple Heron Ardea purpurea

Little Egret Egretta garzetta

European Honey Buzzard Pernis apivorus

Black-shouldered Kite Elanus caeruleus

Black Kite Milvus migrans

Egyptian Vulture Neophron percnopterus

Short-toed Snake Eagle Circaetus gallicus

Western Marsh Harrier Circus aeruginosus

Hen Harrier Circus cyaneus Pallid Harrier Circus macrourus Eurasian Sparrowhawk Accipiter nisus Steppe Buzzard Buteo buteo vulpinus

Long-legged Buzzard Buteo rufinus

Greater Spotted Eagle Aquila clanga

Steppe Eagle Aquila nipalensis

Lesser Kestrel Falco naumanni

Common Kestrel Falco tinnunculus

Eurasian Hobby Falco subbuteo

Saker Falcon Falco cherrug

Peregrine Falcon Falco peregrinus

Macqueen's Bustard Chlamydotis macqueenii

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 174

Bird Species Scientific Name

Water Rail Rallus aquaticus

Little Crake Porzana parva

Purple Swamphen Porphyrio porphyrio

Common Moorhen Gallinula chloropus

Eurasian Coot Fulica atra

Common Crane Grus grus

Eurasian Stone-curlew Burhinus oedicnemus

Black-winged Stilt Himantopus himantopus

Pied Avocet Recurvirostra avosetta

Northern Lapwing Vanellus vanellus

Spur-winged Lapwing Vanellus spinosus

Red-wattled Lapwing Vanellus indicus

White-tailed Lapwing Vanellus leucurus

Common Ringed Plover Charadrius hiaticula

Little Ringed Plover Charadrius dubius

Kentish Plover Charadrius alexandrinus

Common Snipe Gallinago gallinago

Black-tailed Godwit Limosa limosa

Spotted Redshank Tringa erythropus

Common Redshank Tringa totanus

Marsh Sandpiper Tringa stagnatilis

Common Greenshank Tringa nebularia

Green Sandpiper Tringa ochropus

Wood Sandpiper Tringa glareola

Terek Sandpiper Xenus cinereus

Common Sandpiper Actitis hypoleucos

Ruddy Turnstone Arenaria interpres

Temminck's Stint Calidris temminckii

Curlew Sandpiper Calidris ferruginea

Ruff Philomachus pugnax

Cream-coloured Courser Cursorius cursor

Collared Pratincole Glareola pratincola

Slender-billed Gull Chroicocephalus genei

Common Black-headed Gull Chroicocephalus ridibundus

Great Black-headed Gull Larus ichthyaetus

Armenian Gull Larus armenicus

Gull-billed Tern Gelochelidon nilotica

Caspian Tern Hydroprogne caspia

Little Tern Sternula albifrons

Whiskered Tern Chlidonias hybrida

White-winged Tern Chlidonias leucopterus

Pin-tailed Sandgrouse Pterocles alchata

Spotted Sandgrouse Pterocles senegallus

Rock Dove Columba livia

Common Woodpigeon Columba palumbus

European Turtle Dove Streptopelia turtur

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 175

Bird Species Scientific Name

Eurasian Collared Dove Streptopelia decaocto

Laughing Dove Streptopelia senegalensis

Namaqua Dove Oena capensis

Rose-ringed Parakeet Psittacula krameri

Western Barn Owl Tyto alba

Pallid Scops Owl Otus brucei

Pharaoh Eagle Owl Bubo ascalaphus

European Nightjar Caprimulgus europaeus

Egyptian Nightjar Caprimulgus aegyptius

Common Swift Apus apus

Indian Roller Coracias benghalensis

European Roller Coracias garrulus

White-throated Kingfisher Halcyon smyrnensis

Common Kingfisher Alcedo atthis

Pied Kingfisher Ceryle rudis

Blue-cheeked Bee-eater Merops persicus

European Bee-eater Merops apiaster

Eurasian Hoopoe Upupa epops

Red-backed Shrike Lanius collurio

Daurian Isabelline Shrike Lanius isabellinus

Lesser Grey Shrike Lanius minor

Steppe Grey Shrike Lanius pallidirostris

Woodchat Shrike Lanius senator

Eurasian Magpie Pica pica

Rook Corvus frugilegus

Mesopotamian Crow Corvus capellanus

Brown-necked Raven Corvus ruficollis

Hypocolius Hypocolius ampelinus

Greater Hoopoe-Lark Alaemon alaudipes

Desert Lark Ammomanes deserti

Crested Lark Galerida cristata

Eurasian Skylark Alauda arvensis

Thick-billed lark Rhamphocoris clotbey

White-eared Bulbul Pycnonotus leucotis

Sand Martin Riparia riparia

Barn Swallow Hirundo rustica

Cetti’s Warbler Cettia cetti

Common Chiffchaff Phylloscopus collybita

Basra Reed Warbler Acrocephalus griseldis

Great Reed Warbler Acrocephalus arundinaceus

Eastern Olivaceous Warbler Iduna pallida

Upcher's Warbler Hippolais languida

Icterine Warbler Hippolais icterina

Zitting Cisticola Cisticola juncidis

Graceful Prinia Prinia gracilis

Iraq Babbler Turdoides altirostris

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 176

Bird Species Scientific Name

Afghan Babbler Turdoides huttoni

Eurasian Blackcap Sylvia atricapilla

Lesser Whitethroat Sylvia curruca

Eastern Orphean Warbler Sylvia crassirostris

Asian Desert Warbler Sylvia nana

Common Whitethroat Sylvia communis

Ménétries’s Warbler Sylvia mystacea

Common Myna Acridotheres tristis

Common Starling Sturnus vulgaris

European Robin Erithacus rubecula

Bluethroat Luscinia svecica

Common Nightingale Luscinia megarhynchos

Rufous-tailed Scrub Robin Cercotrichas galactotes

Black Redstart Phoenicurus ochruros

Common Redstart Phoenicurus phoenicurus

Whinchat Saxicola rubetra

Siberian Stonechat Saxicola maurus

Isabelline Wheatear Oenanthe isabellina

Northern Wheatear Oenanthe oenanthe

Eastern Black-eared Wheatear Oenanthe melanoleuca

Desert Wheatear Oenanthe deserti

Spotted Flycatcher Muscicapa striata

House Sparrow Passer domesticus

Spanish Sparrow Passer hispaniolensis

Dead Sea Sparrow Passer moabiticus

Yellow Wagtail Motacilla flava

Black-headed Wagtail Motacilla (flava) feldegg

Grey Wagtail Motacilla cinerea

White Wagtail Motacilla alba

Meadow Pipit Anthus pratensis

Water Pipit Anthus spinoletta

Corn Bunting Emberiza calandra

JJBS Volume 10, Number 3,September 2017

ISSN 1995-6673 Pages 177 - 183

Jordan Journal of Biological Sciences

Inhibitory Effect of Crude Ethanol and Water Extracts of Phytolacca dodecandra (L’ Herit) on Embryonic Development of

Anopheles gambiae (Diptera: Culicidae) Jared O. Yugi* and Joyce J. Kiplimo

School of Science and Technology, University of Kabianga, P. O. Box 2030-20200, Kericho Kenya.

Received January 2, 2017 Revised May 24, 2017 Accepted May 29, 2017

Abstract

Plant extracts have been demonstrated to interfere with activities of juvenile hormones that facilitate egg hatching by the embryo. In the present study we demonstrate ovicidal effect of crude ethanol and water extracts of mature green fruits and leaves of Phytolacca dodecandra against Anopheles gambiae eggs under ambient laboratory condition. Freshly laid An. gambiae eggs were exposed to 33.33mls of water and ethanol extracts of mature green fruits and leaves of shoot and midsections of P. dodecandra. The solutions were a serial dilution of 40, 20, 10, 5 and 5mg/100mls of the crude extracts dispensed in plastic containers measuring 6cm top × 5.7cm bottom × 3.5cm height. Ovicidal activity was assessed 48 hours post exposure under dissecting microscope (Leica Zoom 2000) at ×10 magnification. Unhatched eggs were judged dead when non-hatched and with unopened opercula and alive when hatched or with open operculum. WHO threshold of > 80 % mortality was used to assess ovicidal effectiveness. Crude water extracts of leaves of the shoots killed more than 80% while ethanol extracts of the same parts killed 60% of exposed eggs. Leaf extracts of the shoots were more effective compared to extracts of leaves of midsection and mature green fruits. Egg mortality was higher for P. dodecandra extracts compared to Neem or deltamethrin. It was concluded that water extracts were more potent as ovicides than ethanol extracts irrespective of part of P. dodecandra used. Extracts of P. dodecandra can be used as an alternative to chemical insecticide in malaria vector control.

Keywords: Anopheles gambiae, Deltamethrin, Ethanol, Neem, Phytolacca dodecandra

* Corresponding author. e-mail: yugijared@gmail.com; jowiti@kabianga.ac.ke.

1. Introduction

Mosquitoes are vectors of medically important viruses (Polwiang, 2015; Ghimire and Dhakal, 2015: Kindhauser et al., 2016) and malaria (Beck-Johnson et al., 2013; Kumar et al., 2013) and as such are a threat to public health. However, the war against mosquitoes using chemical insecticides has failed due to development of resistance strains (Aizoun et al., 2013; Jones et al., 2013; Stenhouse et al., 2013; Nardini et al., 2013; Vannini et al., 2014) . In addition, the pesticides have been found to negatively impact human health and environment as they are non-biodegradable and some even bioaccumulate (Cartilla and De la Cruz, 2012). For these reasons, research for new control tools is focused on biopesticides and botanical formulations, which are considered eco-friendly, pest specific, biodegradable (Bowers, 1992), and composed of novel compounds with a wide range of activities (Ghosh et al., 2008). The plant-borne compounds are also effective in small doses (Benelli, 2015; Pavela, 2015) and can be employed for rapid synthesis in nanoformulations (Benelli, 2016).

In recent years, plant products and phytochemicals have been evaluated against different developmental stages

of mosquitoes, either on individual mosquito stage or a combination (Shaalam et al., 2005; Ivoke, 2005; Chansang et al., 2005; Mehlhorn et al., 2005; Amer and Mehlhorn, 2006; Gleiser and Zygadlo, 2007; Govindarajan et al., 2008; Muthu et al., 2012; Kaliyamoorthy et al., 2012; Ramar et al., 2014; Valentina et al., 2015). Plant extracts have also been found efficacious as growth inhibitors against eggs of different mosquito strains. For instance; Boswellia dalzielii (Younoussa et al., 2016) and Ocimum basilicum (Foko et al., 2016) against An. gambiae, Spathodea campanulata (Pravin et al., 2015), Argemone Mexicana (Warikoo and Kumar, 2015) against Ae. aegypti, Boswellia dalzielii (Younoussa et al., 2016) against Culex quinquefasciatus (Say) and Melanochyla fasciculiflora leaf and Gluta renghas leaf (Zuharah et al., 2015) against Aedes albopictus.

Phytolacca dodecandra a biopesticide, has demonstrated potency against snail vectors of bilharzias (Erko et al., 2002; Abebe et al., 2005), filarial vector Culex quinquefasciatus Say (Misganaw et al., 2012), An. gambiae larvae (Yugi et al., 2015) as well as adults (Yugi et al., 2014; 2016) with impressive outcome. Nonetheless, despite these positive results, there is a dearth of literature report on its inhibitory effect on An. gambiae embryo

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development. It was with respect to this that the present study was designed to evaluate and report on the activity of ethanol and water extracts of P. dodecandra on An. gambiae eggs under laboratory condition.

2. Materials and Methods

2.1. Study Area, Experimental Mosquitoes and Study Design

The experiments were conducted in the Entomology laboratory at the Centre for Global Health Research / Kenya Medical Research Institute (CGHR/KEMRI). An. gambiae mosquitoes, maintained in the laboratories and reared following standard procedures (Das et al., 2007), were used in the experiments. A randomized informal ‘after-only with control’ experimental design (Kothari, 2004) was used to investigate the embryonic inhibitory effect of crude ethanol and water extracts of P. dodecandra on the eggs. The climatic conditions within the insectary and the laboratory were 28 – 30°C temperatures 70 - 80% relative humidity and 12:12, L:D photoperiod (Yugi et al., 2014). 2.2. Deltamethrin (KOTab 1-2-3®) and Plant Materials Acquisition and Preparation

Deltamethrin (KOTab 1-2-3®) was acquired and prepared as described (Yugi et al., 2015). Fresh leaves (shoot and midsection) and mature green fruits of P. dodecandra and fresh leaves of Azadirachta indica (Neem), were acquired, identified and voucher specimen deposited as earlier described (Yugi et al., 2015). The plant parts were shade dried at room temperature, grounded and extracts obtained using ethanol and water following Tilahun et al. (2003); Parekh et al. (2005) and Das et al. (2010) procedures. 2.3. Ovicidal Assays

A modified method of Elango et al. (2009) was used to determine the embryonic inhibitory activity of crude ethanol and water extracts of green mature fruits and leaves (shoot and midsection) of P. dodecandra on An. gambiae eggs. Freshly laid eggs were collected from the entomology laboratory at CGHR/KEMRI, counted in batches of 20 eggs each from filter papers under a dissecting microscope (Leica Zoom 2000) at ×10 magnification using fine tipped painting brushes and placed in smaller Whitman No. 1 filter papers. Each of such filter paper was then placed in plastic containers (Figure 1) containing approximately 33ml of a given treatment.

Figure 1. Plastic containers used in exposing eggs of An. gambiae

The treatments were prepared by weighing 80 milligrams of stock’s crude extracts of P. dodecandra and dissolving this in 100 millilitres of rain water. The resulting solution was then serially diluted to different concentrations of 40, 20, 10, 5 and 2.5 mg/100ml. A solution of a particular concentration was later distributed among a set of three plastic containers. Each plastic container held an approximate volume of 33mls. The sets of containers were arranged in ascending order with respect to concentration of solutions therein. The arrangement was repeated for similar concentrations of Neem and deltamethrin. Extracts of Neem and solutions of deltamethrin were used as positive control while exposure to rain water alone as negative control. The experimental set ups were replicated five times for each treatment with appropriate controls.

Embryonic inhibitory activity was assessed 48 hours post treatment by observing the eggs under dissecting microscope (Leica Zoom 2000) at ×10 magnification. Egg hatchability was scored as a function of hatched larvae or unhatched but live egg. Unhatched egg was judged as live and capable of hatching or dead and incapable of hatching from observation. A dead eggs appeared non-hatched and with unopened opercula while live egg was one that appeared unhatched but with open operculum. Dead eggs were counted for each treatment, and the percent mortality calculated using a modified version of Abdullahi et al. (2011) formula:

Bioassay tests showing more than 20% control

mortality were discarded and repeated. However, when control mortality ranged from 5% to 20%, the corrected mortality was calculated using Abbott's formula (Abbott, 1925):

Standard WHO procedure was used to assess

effectiveness of the extracts as an ovicide at a mortality rate of > 80% (WHO, 2005). 2.4. Data Analysis

Data obtained from the bioassays were analysed using one way analysis of variance (ANOVA). Regression (probit) analysis was used to calculate the lethal concentration (LC50) and χ2 statistics of the extracts used. The means were separated using LSD and differences between them as well as that of χ2 statistics considered significant at P < 0.05. All statistical analysis was performed using SAS statistical package version 20. .

3. Results

This experiment was conducted for a period of two months using 10, 800 freshly laid An. gambiae eggs. It was found that crude water and ethanol extracts of leaves and shoot of P. dodecandra inhibited >80% and <60% hatching of the exposed eggs respectively. Extracts of P. dodecandra leaves from shoots recorded a higher percentage of unhatched eggs than extracts of mature

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green fruits or leaves of the midsection. Solvent used in extraction did not influence embryonic inhibitory effect of P. dodecandra extracts as numbers of unhatched eggs from exposures were close. Extracts of P. dodecandra were more potent as embryonic inhibitors compared to statistics on extracts of Neem leaves or deltamethrin (Figure 2).

Activity of the extracts was dose dependent with activity reducing with reduced concentration irrespective of source or solvent used. Water extracts of P. dodecandra were more potent than ethanol extracts regardless of concentration or source of P. dodecandra plant (Figure 3). Extracts of P. dodecandra sourced from the Highlands (Eldoret) were more potent than those sourced from the lowlands (Nyando), though the influence of extracts of P. dodecandra from the two sources on embryonic inhibitory

activity was similar (Table 1). Overall, extracts of P. dodecandra were more potent as embryonic inhibitors compared to extracts from Neem and deltamethrin (Table 2). Calculated estimates of the lethal concentration (LC50) of P. dodecandra extracts were relatively low irrespective of parts or solvent used. Estimated values for water extracts were lower than those of ethanol extracts and even that of deltamethrin. All calculated χ2 values were less than the χ2 critical value of 22.362 (df = 13; p < 0.05). This means that the H0 hypothesis of no relationship between dose and mortality of An. gambiae eggs was retained. The influence of the lethal concentrations (LC50) of P. dodecandra on the inability of the exposed An. gambiae egg to hatch did not however, significantly differ (Table 3).

Figure 2. Ovicidal effects of ethanol and water extracts of different parts of P. dodecandra sourced from Eldoret and Nyando against An. gambiae eggs

Figure 3. Ovicidal efficacies of different concentrations of ethanol and water extracts of P. dodecandra from Eldoret and Nyando against An. gambiae eggs

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Table 1. Comparative of regional effectiveness of ethanol and water extracts of P. dodecandra against An. gambiae eggs.

P. dodecandra source

df Solvent used in extraction

Water Ethanol

F P F P

Nyando 4 167.687 0.000 7.919 0.000

Eldoret 4 285.249 0.000 47.838 0.000

Notes: df stands for degree of freedom; F stands for the F statistical factor; P stands for probability for the level of significance. P was taken as significant at p < 0.05

Table 2: Duncan’s Statistics on ovicidal effectiveness of different treatments against An. gambiae eggs.

Treatments df F P F P

P. dodecandra (Nyando) 4 0.636 0.640 8.044 0.000

P. dodecandra (Eldoret) 4 0.397 0.810 7.729 0.000

Neem 4 2.869 0.080 2.869 0.080

Deltamethrin 4 6.000 0.010 6.000 0.010

Control 4 5.859 0.011 5.859 0.011

Notes: 1. df stands for degree of freedom; F stands for the F statistical factor; P stands for probability for the level of significance. P was taken as significant at p < 0.05

Table 3: Estimated lethal concentration (LC50) of ethanol and water extracts of P. dodecandra used against An. gambiae eggs. The estimated are reported together with standard errors (SE).

Phytolacca dodecandra df

Solvent of extraction

Ethanol Water

Source Parts used LC50±

SE χ2 p

LC50± SE χ2 p

Eldoret Fruit 13 8.11± 0.18

5.879 0.950 1.45± 4.19

18.005 0.157

Leaves of mid-section

13 10.54± 0.18

4.588 0.983 2.30± 0.18

3.152 0.997

Leaves of shoot

13 13.50± 0.18

4.921 0.977 1.70± 0.28

10.221 0.676

Nyando Fruit 13 8.97± 0.25

15.976 0.250 1.74± 0.29

6.072 0.943

Leaves of mid-section 13 1.74±

0.29 6.072 0.943 24.09±

0.20 3.191 0.997

Leaves of shoot 13 11.26±

0.24 12.89 0.546 1.62±

0.25 10.029 0.692

Control Neem 13 19.31± 0.19

4.505 0.985 5.39± 0.21

9.466 0.737

Deltamethrin 13 2.52± 0.19

7.562 0.871 2.52± 0.19

7.562 0.871

Notes: 1. df = degrees of freedom (n-2); 2. χ2 = chi-square test statistics of relationship between the considered factors; p = level of significance. The relationships were considered significant at p<0.05; SE = standard error

4. Discussion

The present study demonstrates the following on effectiveness of ethanol and water extracts of P. dodecandra plant parts as ovicides. First, it has an inhibitory effect on An. gambiae mosquito embryo growth

and development. Second, the effectiveness of the extract is dose dependent and that potency reduces with reduced concentration irrespective of plant part or solvent used. Third, water is the best solvent for extracting bioactives of P. dodecandra as water extract were more potent than ethanol extract irrespective of plant part used. Lastly, extract of P. dodecandra leaves are more potent than that of mature green fruits.

Plant extracts such as Pemphis acidula (Samidurai et al., 2009), C. celata (Reegan et al., 2013) on Cu. quinquefasciatus and An. aegypti eggs, Exacum pedunculatum on An. stephensi (Elangovan et al., 2012) to mention but a few have been evaluated against mosquito eggs and found potent. However, no report on embryonic inhibitory activities for any part of P. dodecandra is available against any mosquito species. The present study reports and demonstrates that concentrations of 20 mgs/100mls and higher of ethanol and water extracts of mature green fruits and leaves of P. dodecandra are potent against An. gambiae eggs as they hindered more than 80% of the exposed An. gambiae eggs irrespective of solvent used or geographical source of P. dodecandra plants from hatching. The effectiveness however, reduced with reduced concentration. This showed that effectivity of P. dodecandra extracts as ovicides were dose dependent an observations that correlated with those of Elumalai et al. (2010); Ramar et al. (2014); Younoussa et al. (2016) and Foko et al. (2016) who reported the efficacy of various essential oils and leaf extracts against Culex quinquefasciatus eggs, Spodoptera litura and Anopheles gambiae, respectively

These findings place P. dodecandra extracts among such products as essential oils from three spontaneous plants against Aedes aegypti and Anopheles gambiae complex (Bassolé et al., 2003), Hyptis suaveolens leaf extracts against Anopheles gambiae (Ivoke et al., 2009) and Cardiospermum halicacabum leaf extract against Culex quinquefasciatus and Aedes aegypti (Govindarajan, 2011) among others. It also places P. dodecandra extracts in the same rating as methoxyfenozide that has been demonstrated not only to disrupt growth but also to delay development in Culex pipiens L. (Hamaidia and Soltani, 2016).

In the present study, solvent played a significant role in shaping activity of the extracts. Water extracts were more potent than ethanol extracts. P. dodecandra bioactives also seemed to distribute differentially along the vertical stretch of the plant with the leaves having more potent bioactives than the green fruits. These findings were consistent with earlier observation that reported a correlation of levels of potency of plant extracts with type of plant part and solvent used in the extraction of bioactives (Jeyabalan et al., 2003; Mgbemena, 2010; Anupam et al., 2012).

P. dodecandra extracts from the highlands (Eldoret) were more potent than those from the lowlands (Nyando) irrespective of the parts used. This observation demonstrates that spatial disposition of a plant influences the concentration of bioactives within and that plants from different geographical zones have bioactives of different potency. The differential potency of bioactives of P. dodecandra from different ecological regions reported in this study is consistent with that of Anupam et al. (2012)

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and Mgbemena, (2010) that demonstrated that geographical source influences level of potency of bioactives in plant samples.

The fact that higher concentrations of P. dodecandra extracts killed >80% of exposed An. gambiae eggs, demonstrates that the extracts are effective as ovicides (WHO, 2005). These observations demonstrate without doubt that P. dodecandra extracts are potent inhibitors of mosquito embryo development and are in the same rank as insect growth disruptor as methoxyfenozide (Hamaidia and Soltani, 2016). The mechanism through which the embryos died may not be known as this was not investigated by the present study but it is speculated it could have been due to blockage of the micropyle thereby interfering with gaseous exchange or by interfering with purity of the gases within the operculum preventing the embryo from accessing air and thus ultimately dying (Bhatnagar and Sharma, 1994).

Eggs play a crucial role in the life cycle of insects as they are the starting point of new generation and their correct development and timely hatching is important. Studies have shown that egg hatchability in insects and most importantly An. gambiae is influenced by several factors among which are temperature, salinity and humidity (Lyons et al., 2013), organic substances and bacteria (Lindh et al., 2008; Sumba et al., 2008). Delay or reduction in the rate of growth and development or even death to the eggs definitely affects individual fitness, population structure and dynamics (Spencer et al., 2002). It is therefore understandably that anything that interferes with the correct development of this stage (eggs) of malaria vector greatly impact malaria transmission and morbidity.

In conclusion, the present study report demonstrates two things. First, that water extracts of P. dodecandra are more potent as ovicides than ethanol extract irrespective of part used and second, that extracts of P. dodecandra interferes with embryo development in An. gambiae leading to inability of the eggs to hatch. It is recommended that extracts of P. dodecandra be used as an alternative to chemical insecticide in malaria vector (An. gambiae mosquito) control.

Authors’ Contributions

YJO conceived the concept, designed, sourced for funds and wild mosquito larvae and wrote the manuscript. YJO and KJJ, conducted the experiments, read and corrected the manuscript.

Competing Interests

The authors declare that they have no competing interests and that Neem and Deltamethrin were used purely for experimental purpose.

Acknowledgements

We thank Richard Amito, Charles Owaga, Patience Akoth and Trevor Omondi for processing and culturing the experimental mosquitoes, Centre for Global Health Research/Kenya Medical Research Institute (CGHR/KEMRI) for laboratory space, mosquitoes and

equipments for conducting the experiments and National Commission for Science Technology and Innovation (NACOSTI) for funding the project (Grant contract # NCST/5/003/3rd Call PhD/056 to YJO).

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JJBS Volume 10, Number 3,September 2017

ISSN 1995-6673 Pages 185-191

Jordan Journal of Biological Sciences

Comparative Anatomy of Stem, Petiole and Flower Stalks and its significance in the Taxonomy of Some Members of Cucurbits

Chimezie Ekeke*, Ikechukwu O. Agbagwa and Alozie C. Ogazie

Department of Plant Science and Biotechnology, Faculty of Science, University of Port Harcourt, P.M.B. 5323, Port Harcourt, Rivers State,

Nigeria.

Received, January 26, 2017; Revised May 10, 2017; Accepted May 27, 2017

Abstract

We compared the anatomy of the flower stalk (male and female) anatomy, stem, petiole, tendrils and median vein shape of three representative members of Cucurbitaceae (Citrullus lanatus, Cucumeropsis mannii and Citrullus colocynthis). These materials (flower stalks of the male, female, midrib, petiole and the tendril) were fixed in FAA, wax embedded, sectioned, stained and photographed with Leitz Diaplan photomicroscope fitted with Leica WILD MPS 52 camera. The midrid and lamina anatomy of these species showed clear variations in the layers of mesophyll, number and arrangement of vascular bunbles which are of taxonomic significance and are reported for the first time. These anatomical features suggest taxonomic affinity amongst the species and enhance the delimitation of the species. It also strengthens the fact that the species maintained as separate species and confirm the fact that the anatomical characteristics of the stem, petiole, flower stalks, midrib and leaf lamina are an important line of evidence in classification of these species.

Keywords: Anatomy, Cucurbitaceae, Cucurbit, Flower Stalk, Midrib, Vascular Bundles.

* Corresponding author. e-mail: ekeke.uche@uniport.edu.ng.

1. Introduction

Cucurbitaceae is a group of flowering plants with 100 genera and 750 species (Yamaguchi, 1983). These plants are widely distributed in the tropical part of the world. They are herbs or rarely undershrubs with watery juice, often scabrid; stems scandent or prostrate; tendrils mostly present, spirally coiled. Flowers are monoecious or dioecious, very rarely hermaphrodite and actinomorphic. Male flower: calyx tubular, lobes imbricate or open; corolla polypetalous or gamopetalous, lobes imbricate or induplicate-valvate; stamens free or variously united, mostly 3, rarely 1–5, one anther always 1-celled, the others 2-celled, cells straight or often curved, flexuous or conduplicate, connective often produced. Female flower: calyx-tube adnate to the ovary and often produced beyond it; staminodes usually not present; ovary inferior or very rarely free; placentas often 3, parietal but often meeting in the middle; ovules numerous, rarely few, arranged towards the walls of the ovary; style simple or rarely 3 free styles; stigmas thick. Seeds various, often flattened, without endosperm (Hutchinson and Dalziel, 1952).

Leaves, roots and fruits of members of this family are used in Nigeria and other African countries for different purposes (food and medicine) depending on the ethnic group (Ajuru and Okoli, 2013; Aguoru and Okoli, 2008; Burkill, 1985; Okoli, 1984; Aguoru and Ogaba, 2010;

Jansen van Rensburg et al., 2004). Among other plant species and cucurbits, anatomical characteristics of the petiole, leaf lamina, midrid, flower stalks and the number of vascular bundles in the different parts of the plants have been demonstrated to be valuable in delimitating the plants of the same genus or family (Ekeke and Agbagwa, 2016; Ekeke, et. al., 2016; Ekeke and Mensah, 2015; Agogbua et al., 2015; Ekeke et al., 2015; Ajuru and Okoli, 2013; Aguoru and Okoli, 2012). Although a number of recent studies have been carried out on the phylogenetic relationship among cucurbits, the significance of fruit stalks, petiole, midrib and tendril anatomy in the taxonomy of these species from Nigeria is lacking. The present work, therefore, investigates the comparative anatomy of these plant parts to enhance the delimitation of these species.

2. Materials and Methods

2.1. Source of Material

Seeds of properly identified plants (Citrullus colocynthis (L.) Schard, Cucumeropsis mannii Naud and Citrullus lanatus (Thunb.) Matsum and Nakai were harvested in September 2015, processed and stored. In February 2016, the seeds were germinated in pots and the vegetative parts, used for the present study, were harvested at maturity (when the plants had started flowering and fruiting).

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 2 186

2.2. Anatomical Studies

The flower stalks of the male and female flowers, petioles (distal – base of the leaf, median – middle of the petiole and proximal – petiole portion close to the stem), median portion of the midrib and stems of plants were excised and fixed in FAA for 24hrs, dehydrated in 30%, 50% and 70% ethanol for 1hr each and preserved in absolute ethanol till when needed. Thereafter, the specimens were wax embedded, trimmed, and hand-sectioned. Thin sections selected, stained with 1% safranin or alcian blue, mounted on slide, observed under microscope and micro-photographed using Leica WILD MPS 52 microscope camera on Leitz Diaplan microscope (Ndukwu and Okoli, 1992; Ajuru and Okoli, 2012).

3. Results

The comparative anatomical characteristics of the stem, flower stalk (female and male), tendril, petiole and midrib of three species of cucurbits (Citrullus lanatus, Cucumeropsis mannii and Citrullus colocynthis) are summarized in Tables 1 and 2 and Figures 1-3). 3.1. Petiole

The anatomy of the petiole showed that the number of vascular bundles in the distal, median and proximal portions varied 7 to 9 (Figure 1 and Table 1). Citrullus lanatus had 9 vascular bundles at the distal part, 7 at the median portion and 7 at the proximal portion; Cucumeropsis mannii had 9 vascular bundles at the distal part, 9 at the median portion and 7 at the proximal portion while Citrullus colocynthis had 9 vascular bundles each at the distal, median and proximal portion (Figure 1). 3.2. Midrib

In the present study, we noted variations in the midribs of the species studied which are of taxonomic importance. The number and arrangement of the vascular are discriminating among the species studied. C. colocynthis had 7-vascular bundles (open crescent of 5 separate bundles with 2-accessory bundles at the adaxial surface) Figure 2A. In C. lanatus, the midrib had 3-vascular bundles in which 2 are parallel or superimposed and 1-accessory bundle at the adaxial surface (Table 1 and Figure 2D) and C. mannii had 5-vacular bundles (3 adxial separate vascular bundles in a crescent with abaxial strand between the ends of the crescent and 1-accessory bundle at the adaxial portion) (Figure 2G and Table 1). 3.3. Leaf Lamina

The adaxial and abaxial epidermal layers are made up of single layer of cells (Table 1, Figure 2 and Appendix II). The adaxial epidermal cells of C. colocynthis and C. mannii are periclinally elongated. The palisade mesophyll layers comprised 1-layer of cells in C. mannii and C. lanatus and 2-layers of cells in C. colocynthis. The mesophyll cells are shortened in C. colocynthis and C. mannii while in C. lanatus, they are anticlinally elongated (Figures 2B, E and H). The layers of spongy mesophyll varied from one species to another. These include C. colocynthis (3-4 layers) C. mannii (6 layers) and C. lanatus (5 layers).

3.4. Stem

The stem of C. lanatus had 11-vascular bundles, C. colocynthis and C. mannii 10 vascular bundles each (Figure 2). The thickness of the parenchymatous cells are 4-6 layers, 3-8 layers and 2-7 layers for C. lanatus, C. colocynthis and C. mannii respectively. The sclerenchy-matous cell formed continuous/partly interrupted layer of cells in all the species while their thickness include 3-4 layers in C. lanatus and C. mannii, and 2-4 layers in C. colocynthis (Table 1). 3.5. Female Flower Stalk

The number of vascular bundles in the female flower stalk of all the species studied is ten. However, the nature and thickness of the sclerenchymatous and parenchy-matous cells varied slightly among them (Figures 3A, D and G). For instance, in C. lanatus and C. colocynthis, the sclerenchymatous cells are continuous but are interrupted in C. mannii. In contrast, the thickness/layer of cells include 3-4 layers in C. lanatus, 2-7 layers in C. colocynthis and 3-5 layers in C. mannii (Figure 3 and Table 2). Also, the thickness of parenchymatous cells include 11-14 layers in C. lanatus, 8-12 layers in C. colocynthis and 6-9 layers in C. mannii (Figures 3, Table 2 and Appendix III). 3.6. Male Flower Stalk

In the male flower stalk, the number and nature of vascular bundles were found to be diagnostic. The number of vascular bundles in C. lanatus is 9, C. colocynthis 10 and C. mannii 11 (Figure 3). The sclerenchymatous cells are continuous in all the species but slightly differed in thickness. In C. lanatus (3-4 layers), C. colocynthis (2-7 layers) and C. mannii (4-7 layers). In the same vein the thickness/layers of parenchymatous include C. lanatus (1-3 layers), C. colocynthis (5-7 layers) and C. mannii (2-4 layers) Appendix III. 3.7. Tendril

The anatomical characters of the tendrils are fairly similar. For example, the sclerenchymatous cells are continuous in all the species studied. Also, the thickness of the sclerenchymatous and parenchymatous are as follows; C. lanatus (3-4 layers and 3-6 layers), C. colocynthis (3-6 layers and 4-7 layers) and C. mannii (4-5 layers and 5-8 layers), respectively (Figures 3C, F and I; Table 2 and Appendix III).

Figure 1. Sketch (Drawing) for transverse section of distal (A, D, G), median (B, E, H) and proximal (C, F, I) of the petiole. (A-C) Citrullus lanatus; (D-F) Cucumeropsis mannii and (G-I) Citrullus colocynthis

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 2

187

Figure 2. Sketch for anatomical sections of midrib (A, D, G), leaf lamina (B, E, H) and stem (C, F, I). (A-C) Citrullus colocynthis; (D-F) Citrulus lanatus and (G-I) Cucumeropsis (Avb = accessory vascular bundle, Sp = spongy mesophyll, Vb = vacular bundle, Ep = epidermal cell)

Figure 3. Sketch for transverse section of the female flower stalks (A, C, E), male flower stalks (B, D, F) and tendril (C, F, I). (A-C) Citrullus lanatus; (D-F) Cucumeropsis mannii and (G-I) Citrullus colocynthis

Table 1. Summary of the anatomical features of the petiole, midrib and stem of the cucurbits studied

Plant part Features

Species name

Citrullus lanatus (Thunb.) Matsum. & Nakai.

Citrullus colocynthis (L.) Schard.

Cucumeropsis mannii Naud.

Petiole Number of vascular bundles

9 at the distal part, 7 at the median portion and 7 at the proximal portion

9 at the distal part, 9 at the median portion and 7 at the proximal portion

9 at the distal part, 9 at the median portion and 9 at the proximal portion

Midrib Number of vascular bundles

3 7 5

Nature/arrangement of vascular bundle

Exhibits 2 parallel or superimposed vascular bundles with 1-accessory bundle at the adaxial surface

Exhibits open crescent of 5 separate bundles with 2-accessory bundles at the adaxial surface

Exhibits adaxial crescent of 3 separate vascular bundles with abaxial strand between the ends of the crescent and 1-accessory bundle at the adaxial portion

Leaf lamina

Nature of leaf lamina 1-layer of enlongated palisade mesophyll and 3-4 layers of spongy mesophyll

2-layer of short palisade layer and 5-layers of spongy mesophyll

1-layer of short palisade layer and 5-layers of spongy mesophyll

Nature of adaxial epidermis

Single layer Single layer but periclinally elongated

Single layer but periclinally elongated

Stem Number of vascular bundles

11 10 10

Parenchymatous cell thickness

4-6 layers 3-8 layers 2-7 layers

Nature and thickness of sclerechymatous cells

Continuous and partly interrupted, 3-4 layers

Continuous and partly interrupted, 2-4 layers

Continuous and partly interrupted, 3-4 layers

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 2 188

Table 2. Summary of the anatomical features of the flower stalk and tendril of the cucurbits studied

Plant part Features Species name

Citrullus lanatus Citrullus colocynthis Cucumeropsis mannii

Female flower stalk

Number of vascular bundles 10 10 10 Nature and thickness of sclerenchymatous cells Continuous with 3-4

layers Continuous with 2-7 layers

Interrupted with 3-5 layers

Thickness of parenchymatous cells 11-14 layers 8-12 layers 6-9 layers Male flower stalk

Number of vascular bundles 9 10 11 Nature and thickness of sclerenchymatous cells Continuous with 3-4

layers Continuous with 1-3 layers

Continuous with 2-4 layers

Thickness of parenchymatous cells 1-3 layers 5-7 layers 4-7 layers Tendril Number of vascular bundles 9 9 8

Nature and thickness of sclerenchymatous cells Continuous with 3-4 layers

Continuous with 3-6 layers

Continuous with 4-5 layers

Thickness of parenchymatous cells 3-6 layers 4-7 layers 5-8 layers

4. Discussion

Anatomical line of evidence has been used in classifying different plant taxa (Metcalfe and Chalk, 1979; Aguoru and Okoli, 2008; Ekeke and Agbagwa, 2016; Ekeke, et al., 2016; Ekeke and Mensah, 2015; Agogbua et al., 2015; Ekeke et al., 2015; Ajuru and Okoli, 2013; Aguoru and Okoli, 2012). The species investigated showed close affinity based on the anatomical characteristics of their stems. In addition, the vascular bundles in all the species are of the same type (bicollateral vascular bundles). The thickness of the sclerenchymatous cells in the stems varied from 2-4 layers and was continuous or partly interrupted in all the species studied. The thickness of parechymatous cells and the number of vascular bundles varied slightly and included C. lanatus (4-6 layers and 11), C. colocynthis (3-8 layers and 10) and C. mannii (2-7 layers and 10), respectively. The variation in the number of vascular bundles at the distal, median and proximal portions of the petiole could be a fairly diagnostic feature among the species studied. For instance, all the species had 9-vascular bundles at the distal portion of the petiole. This indicates that they are closely related and supports their placement in one family (Hutchinson and Dalziel, 1954; Jerffery, 1990, 2005). At the median portion, C. mannii had 9-vascular bundles while C. lanatus and C. colocynthis had 7-vascular bundles. On the other hand, at the proximal portion, C. colocynthis had 7-vascular bundles while C. mannii and C. lanatus had 9-vascular bundles each. The number of vascular at these points confirmed the placement of C. lanatus and C. colocynthis in this same genus. The midrib and lamina anatomy of these species showed clear variations in the layers of mesophyll, number and arrangement of vascular bundles which are of taxonomic significance and are reported for the first time (Table 1). In the midrib, C. lanatun had 3-vascular (2 parallel or superimposed vascular bundles with 1-accessory bundle at the adaxial surface), C. colocynthis had 7-vascular bundle (open crescent of 5 separate bundles with 2-accessory bundles at the adaxial surface) while C. mannii had 5-vascular bundles (adaxial crescent of 3 separate vascular bundles with abaxial strand between the ends of the crescent and 1-accessory bundle at the adaxial

portion). The thickness or the layers of lamina among the species were distinct. For instance, C. lanatus had 3-4 layers of cells, C. colocynthis had 6-layer of cells and C. mannii 5-layer of cells.

A dichomotous taxonomic key based on the anatomical characters of the species studied in the present work is presented below:

1. Petiole with 9-vascular bundles at the distal portion and 7-vascular bundles at the proximal portion; sclerechymatous cells in female flower continuous; tendril with 9-vacular bundles --------------------------------- 2

1' Petiole with 9-vascular bundles at both the distal, median and proximal portions; sclerechymatous cells in female flower interupted; tendril with 8-vacular bundles; midrib with 5-vascular bundles; male flower stalk with 11-vascular bundles …………………….Cucumeropsis mannii

2. Midrib with 3-vascular bundles (2-parallel or superimposed vascular bundles and 1-accessory bundle at the adaxial surface); stem with 11-vascular bundles; 1-layer of palisade mesophyll; male flower stalk with 9-vascular bundles …………………… Citrullus lanatus

2' Midrib with 3-vascular bundles (5 separate or open crescent bundles and 2-accessory bundles at the adaxial surface); stem with 10-vascular bundles; 2-layers of palisade mesophyll; male flower stalk with 10-vascular bundles , …………………… Citrullus colocynthis

5. Conclusion

These anatomical features suggest taxonomic affinity amongst these species and enhance the delimitation of the species. They also strengthen the fact that the species should be maintained as separate species (Hutchison and Dalziel, 1954; Jeffrey, 1964 and 1980) and confirm the fact that the anatomical characteristics of the stem, petiole, flower stalks, midrib and leaf lamina is an important line of evidence in classification of these species.

References

Agogbua J, Chimezie, E and Bosa B E. 2015. Morpho-anatomical characters of Zehneria capillacea (Schumach) C. Jeffrey and Zehneria scabra (L.F.) Sond Cucurbitaceae. African J Plant Sci., 9(12): 457-465.

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189

Aguoru C U and Ogaba J O. 2010. Ethonobotanical survey of anti-typhoid plants amongst the Idoma people of Nigeria. Int. Sci. Res. J. 2: 34-40.

Aguoru, CU and Okoli BE. 2008. Seed coat anatomy of Momordica L. (Cucurbitaceae) in parts of tropical western Africa. Int. J. Trop. Agric. Food Syst. 2(1): 29-33.

Aguoru C U and Okoli B E. 2012. Comparative stem and petiole anatomy of West African species of Momordica L (Cucurbitaceae). African J Plant Sci., 6(15), 403-409, DOI: 10.5897/AJPS11.309.

Ajuru M G and Okoli B E. 2013. The morphological characterization of the melon species in the Family Cucurbitaceae Juss. and their utilization in Nigeria. Inter J Modern Bot., 3(2): 15-19

Burkill H M. 1985. The Useful Plants of West Tropical Africa. 2nd Ed. Vol. 1. Families A-D. Kew, UK: Royal Botanical Gardens, p.960.

Ekeke C and Agbagwa I O. 2016. Anatomical characteristics of Nigerian variants of Caladium bicolor (Aiton) Vent. (Araceae). African J Plant Sci., 10(7): 121-129.

Ekeke C and Mensah S I. 2016. Comparative anatomy of midrib and its significance in the taxonomy of the Family Asteraceae from Nigeria. J Plant Sci., 10(5): 200-205.

Ekeke C, Agogbua J and Okoli B E. 2015. Comparative anatomy of tendril and fruit stalk in Curcubitaceae Juss. from Nigeria. Int. J. Biol. Chem. Sci. 9(4): 1875-1887.

Ekeke C, Ogazie C A and Mensah S I. 2016. Importance of leaf, ltem and flower stalk anatomical characters in the identification of Emilia Cass. Inter J Plant & Soil Sci., 12(6): 1-12.

Hutchinson J and Dalziel J M. 1954. Flora of West Tropical Africa. Crown Agents, London UK.

Hutchinson, J and Dalziel J M. 1952. Flora of West Tropical Africa. Vol.1. Second Edition revised by Keay, R. W. J.

Jansen van Rensburg W S, Venter S L, Netshiluvhi T R, van den Heever E, Vorster H J and de Ronde J A. 2004. Role of indigenous leafy vegetables in combating hunger and malnutrition. South Afr. J. Bot. 70(1):52-59.

Jeffrey C. 1964. Key to the Cucurbitaceae of west tropical Africa with a guide to localities and little know species. J W Afr Sci Assoc., 9: 79- 97.

Jeffrey C. 1980. A review of Cucurbitaceae. Bot. J. Linn. Soc. 81: 233-2479.

Jeffrey C. 1990. An outline classification of the Cucurbitaceae. In: Bates DM, Robinson RW, Jeffrey C (Eds.), Biology and Utilization of the Cucurbitaceae. Cornell University Press Ithaca, NY. pp. 449-463.

Jeffrey C. 2005. A new system of Cucurbitaceae. Bot. Zhurn 90: 3332-3335.

Metcalfe C R. and Chalk L. 1979. Anatomy of the Dicotyledons. 2nd Ed., Clarendon Press, Oxford, UK. P 276.

Ndukwu B C and Okoli B E. 1992. Studies on Nigerian Curcurbita moschata. Nigerian J of Bot., 5:18-26.

Okoli B E. 1984. Wild and cultivated cucurbits in Nigeria. Economic Bot., 38: 350-357.

Yamaguchi M. 1983.World Vegetables: Principles, Production and Nutritive values: West port, Conn.

Appendix I

Figure 1. Anatomical sections of midrib (A, D, G), leaf lamina (B, E, H) and stem (C, F, I). (A-C) Citrullus colocynthis; (D-F) Citrulus lanatus and (G-I) Cucumeropsis.

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 2 190

Appendix II

Figure 2. Transverse section of the female flower stalks (A, C, E), male flower stalks (B, D, F) and tendril (C, F, I). (A-C) Citrullus lanatus; (D-F) Cucumeropsis mannii and (G-I) Citrullus colocynthis. It would be better to rearrange the section photos for each species in a sequence manner for the three studied species.

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191

Appendix III

Figure 3: Transverse section of distal (A, D, G), median (B, E, H) and proximal (C, F, I) of the petiole. (A-C) Citrullus lanatus; (D-F) Cucumeropsis mannii and (G-I) Citrullus colocyhthis.

JJBS Volume 10, Number 3,September 2017

ISSN 1995-6673 Pages 193 - 197

Jordan Journal of Biological Sciences

Estimation of Median Lethal Dose of Commercial Formulations of Some Type II Pyrethroids

Prabhu N. Saxena and Brijender Bhushan*

Toxicology Laboratory, Department of Zoology, School of Life Sciences, Dr. B.R. Ambedkar University, Khandari Campus, Agra – 282002 ,

India

Received February 14, 2017 Revised May 3, 2017 Accepted June 4, 2017

Abstract

The present study aims to investigate the median lethal dose (LD50) of three commercially formulated type II pyrethroids, namely Fenvalerate (Hafen), Deltamethrin (Decis) and Cypermethrin (Cybil) against female albino rats. Acclimatised experimental animals were divided into three sets and respective doses of pyrethroids under investigation were administrated orally to them. Survival and mortality number was observed following 96 hours of intoxication. LD50 was calculated statistically by log-dose/probit regression line method from the numerical data obtained and came out to be 1532.5, 656.2 and 618.2 mg/kg b.wt. for Hafen, Decis and Cybil respectively. Structural changes in type II pyrethroid vide supra have been considered responsible for the differences in LD50.

Key words: LD50, Hafen, Decis, Cybil, Female albino rat.

* Corresponding author. e-mail: bantu.sls@gmail.com

1. Introduction

Synthetic pyrethroids have emerged as a group of multipurpose pesticides, considered comparatively safer than other available counterparts, such as Organochlorines and Organophosphates. These pesticides presently account for about 30% of the total insecticide market worldwide by virtue of comparatively higher LD50, easy biodegrability and less non-target toxicity. These pesticides are synthetically modified analogues of basic pyrethrins contained in the flowers of genus Chrysanthemums. On routine basis, many new pyrethroids are synthesised and added up in the market to meet enhanced global food demands, vector borne diseases and genetically modified pesticide resistant pest species. On the same line, Fenvalerate, Deltamethrin and Cypermethrin in the formulated state have been added up in the market and broadly used throughout the world (Saxena and Tomar, 2003; Saxena and Doneriya, 2004; Bhushan et al., 2010; Pande et al., 2014; Moustafa and Hussein. 2016.

A consequence of their indiscriminate and excessive use can also be in the form of toxicity to various non-target species (Doneriya and Saxena, 2004; Bhushan et al., 2013; Bhushan et al., 2013; Rajawat et al., 2015). Considering the same concept, comparative median lethal dose (LD50) evaluation has been undertaken in the present

study, so as to assess the effect of structural modifications on non-target toxicity.

2. Materials and Methods

2.1. Experimental Animal

Ninety female albino rats, Rattus norvegicus, eight weeks old, selected from an inbred colony weighing 110 ± 20 gm were used in the present study. These experimental animals were well acclimatized for two weeks to laboratory conditions and provided standard rat pellet feed and water ad libitum. The rats were then divided into three main sets each of which was further sub-divided in five sub-sets comprised of seven rats corresponding to different doses of Hafen (Fenvalerate), Decis (Deltamethrin) and Cybil (Cypermethrin), respectively. The experimentation was approved by the Ethical Committee of Dr. B. R. Ambedkar University, Agra, India. 2.2. Experimental Compounds

Commercial formulations of Fenvalerate, Deltamethrin and Cypermethrin were obtained from Bayer India Ltd., Mumbai. Concentrations of experimental pyrethroids in these commercial formulations was 92, 92.6 and 91%, respectively. The LD50 was calculated (Finney, 1971).

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 194

2.3. Dose Administration and Determination of LD50

2.3.1. Hafen (Fenvalerate) Experimental albino rats were divided into five groups,

each consisting of seven individuals. Standard solution of experimental test compound Hafen (Fenvalerate) was prepared by dissolving in distilled water. Different doses 1200, 1600, 2000, 2400 and 2800 mg/kg b.wt. were administrated orally. The mortality and survival numbers of the rats were recorded for each dose after 96 hours. The data were analyzed statistically by log dose/probit regression line method (Finey, 1971). Regression line was drawn on the basis of two variables, log dose and empirical probit on a simple graph paper and used to determine the expected probit necessary for LD50 determination (Table 1). 2.3.2. Decis (Deltamethrin)

Experimental albino rats were divided into five groups, each consisting of seven individuals. Standard solution of experimental test compound Decis was prepared by dissolving in distilled water. Different doses 640, 800, 960, 1200 and 1280 mg/kg b.wt. were administrated orally. The mortality and survival numbers of rats were recorded for each dose after 96 hours. The data were analyzed statistically by log dose / probit regression line method (Finey, 1971). Regression line was drawn on the basis of two variables, log dose and empirical probit on a simple graph paper and used to determine the expected probit necessary for LD50 determination (Table 1). 2.3.3. Cybil (Cypermethrin)

Experimental albino rats were divided into five groups, each consisting of seven individuals. Standard solution of experimental test compound Cybil was prepared by dissolving in distilled water. Different doses 315, 650, 1260 and 2520 and 5040 mg/kg b.wt. were administrated orally. The mortality and survival numbers of rats were recorded for each dose after 96 hours. The data were analyzed statistically by log dose/probit regression line method (Finey, 1971). Regression line was drawn on the basis of two variables, log dose and empirical probit on a simple graph paper and used to determine the expected probit necessary for LD50 determination (Table 1). 2.4. Statistical Analysis

Results obtained were statistically analysed using log-dose/probit regression analysis (Finey 1971).

3. Results

In the different experimental sets, experimental rats were treated with different doses of different concentrations of Hafen, Decis and Cybil for estimation of LD50. Survival number and percentage for each dose have been noted after 96 hours which decreased with increasing dose of Hafen, Decis and Cybil (Table 1). LD50 has been calculated (Tables-2-4, Figure 1-3) by log dose/probit regression line method (Finney, 1971). The test doses were then converted to their logarithm. The empirical probit values, equivalent to percentage mortality were then obtained from the table (Finney, 1971) and plotted against log dose on graph paper. The provisional lines, drawn fitting to the expected values, were read for the values of log dose (X).

Working probits Y = Y0 + kp where, p = percentage mortality Y0 and k are two factors. The weighting coefficients for each point were also

obtained from the table. The weight has been calculated by multiplying each coefficient by number of rats used (Table 2-3). The value of ‘b’ is obtained by the formula:

−

−= ∑ ∑∑ ∑ wxXwxwyXwxyb 2/

The values of Y can be obtained by regression equation (Figures1-3)

XXbYY −+= ( ) From the equation values of X, equivalent to Y and Y were estimated, and the calculated values of LD50 thus been obtained (Tables-2-4, Figures 1-3). Variance V was calculated followed by 95% confidence fiducial limits as below:

Variance (V) =

−Σ

−+

∑∑∑

wwx

wx

XXwb 2

2

2

2 )()(11

By the following formula, 95% confidence fiducial limits have been obtained (Table 5): m1 = m + 196 V m2 = m-1.696 V

Applying all these calculation, LD50 for Hafen, Decis and Cybil came out to be 1532.5, 656.2 and 618.2 mg/Kg b.wt., respectively.

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 195

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 196

Figure 1.Graph between probit mortality and log-dose showing the regression line for the calculation of mortality index (LD50) of Hafen(Fenvalerate)

Figure 2. Graph between probit mortality and log-dose showing the regression line for the calculation of mortality index (LD50) of Decis(Deltamethrin)

Figure 3. Graph between probit mortality and log-dose showing the regression line for the calculation of mortality index (LD50) of Cybil(Cypermethrin)

4. Discussion

Hafen, Decis and Cybil, are all type II pyrethroids, which are commonly and extensively used all over the world, therefore reflect risk to non-target species (Tomar and Saxena, 2003; Saxena and Doneriya, 2004; Doneriya and Saxena, 2004; Bhushan et al., 2013; Bhushan et al., 2013; Amir et al., 2015; Abdul-Hamid et al., 2017).

Median lethal dose (LD50) determination provides an overview of the toxic potential of a compound and is helpful in minimizing the accidental acute exposures such as in case of domestic intoxications. In the present era of competition, research and development of new more effective pesticides and further their indiscriminate use makes it necessary to compare the toxicity of new compounds with formerly present counterparts. However, limited guidelines for toxicological analysis in pyrethroid poisoning cases have been cited; evaluation of LD50

becomes essential for better evaluation of toxic characteristic of pyrethroids (Saxena and Yadav, 2011; Raj et al., 2013; Chandra et al., 2014; Deasi et al., 2015; Elblehi et al., 2015; Rajawat et al., 2015).

In the present investigation, the order of toxicity in increasing order based on LD50 values has been observed as Cybil followed by Decis and lastly Hafen. Structural differences may be kept the sole factor responsible for this difference in the toxicity. Fenvalerate contains two halogenated groups, which in turn are chlorine, where as two bromine atoms attached to a tri cyclic structure in case of deltamethrin, where as in case of Cypermethrin, a chlorine atom attached to a benzene ring (Figures. 4-6). Chlorine is more electronegative than bromine, but bromine atoms attached in case of Deltamethrin are attached to a cyclic group which provides the compound an additional stability, which in turn increases many folds in case of Cypermethrin where chlorine atom is attached to a benzene ring, providing an additional stability and therefore the present order of toxicity (Key et al., 1997; Finar, 1998; Bhushan et al., 2013; Bhushan et al., 2013; Yadav et al., 2014).

Figure 4. Structure of Fenvalerate

Figure 5. Structure of Deltamethrin

Figure 6. Structure of Cypermethrin

5. Conclusion

A preliminary idea of the comparative toxicity evaluations of newer pyrethroids with the former representatives has been provided in the present study, which revealed enhanced toxicity with advancement, thus also is a suggestive of proper precautions and appropriate use so as to minimize risk to non-target organisms.

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Saxena PN and Tomar V. 2003. Assessment of comparative heamatoxicity of Cybil and fenvalerate in Rattus norvegicus. Bull Environ Contam Toxicol., 70: 839-846.

Saxena PN and Yadav E. 2011. Differential susceptibility of Rattus norvegicus (Berkenhout) to α-cyano type II pyrethroids: An assessment based on serum cholinesterase activity. Proc Nat Acad Sci India Sect. B, 81(11): 180-184.

Yadav E, Singh M and Saxena PN. 2014.Structure activity relationship of some type II pyrethroids: A study based on atomic charges, molecular electrostatic potential surfaces and molecular orbitals analysis. Nat Acad Sci Lett., 37 (3):245-251.

JJBS Volume 10, Number 3,September 2017

ISSN 1995-6673 Pages 199 - 203

Jordan Journal of Biological Sciences

Morphometric Relationships of the Endangered Ticto barb Pethia ticto (Hamilton, 1822) in the Ganges River (NW Bangladesh)

through Multi-Linear Dimensions

Fairuz Nawer1, Md. Yeamin Hossain1*, Md. Alomgir Hossen1, Dalia Khatun1, Most. Farida Parvin1, Jun Ohtomi2 and Md. Ariful Islam3

1Department of Fisheries, Faculty of Agriculture, University of Rajshahi, Rajshahi 6205;

2 Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima 890-0056, Japan; 3Bangladesh Fisheries Research Institute, Shrimp Research Station, Bagerhat 9300, Bangladesh

Received February 23, 2017 Revised June 7, 2017 Accepted June 12, 2017

Abstract

The present study illustrates the morphometric relationships including Length-Weight Relationships (LWRs) and Length-Length Relationships (LLRs) using a total of 13 linear dimensions and meristic characters of the threatened ticto barb Pethia ticto (Hamilton, 1822) in the Ganges River through multi-linear dimensions. A sum of 100 specimen of P. ticto was sampled occasionally from the Ganges River during July 2014 to October 2015, using various traditional fishing gears (e.g., cast, gill, and square lift net). Counts of fin rays and scales (including lateral line scale) were done by a magnifying glass. Additionally, here total 14 different morphometric lengths were assessed to 0.1 cm, and Body Weight (BW) was measured to the nearest 0.1 g for each individual. The fin formula of P. ticto is: dorsal, D. 10–11 (ii/8-9); pectoral, P1. 12 (ii/10); pelvic, P2. 8- 9 (ii/6-7); anal, A. 7-8 (ii/5-6); and caudal, C. 20–24 (iv /16–20), respectively. Total Length (TL) was recorded as minimum (4.9 cm) and maximum (9.5 cm), whereas Body Weight (BW) was varied from 1.5-12.9 g. All LWRs were highly significant (p < 0.001) with r2 values ≥ 0.987. Based on r2 value, LWR by BW vs. TL was the best-fitted model among 14 equations. In addition, the LLRs had also significance with r2 values ≥ 0.986. According to r2 values, LLR by TL vs. FL was the best-fitted model among 13 equations. The present study is very effective for species recognition in the Ganges River, NW Bangladesh and in other river systems in its vicinity.

Keywords: Bangladesh; Pethia ticto; morphometric and meristic characters, Ganges River.

* Corresponding author. e-mail: hossainyeamin@gmail.com.

1. Introduction

The ticto barb, Pethia ticto (Hamilton 1822), is a fresh- and brackish-water subtropical, small-indigenous fish belonging to the family Cyprinidae. It is found in still, shallow waters and rivers, frequently with muddy bottoms (Bisht & Das 1981). P. ticto is a native species of the Asian countries, i.e., Bangladesh, India, Myanmar, Nepal, Pakistan, Sri-Lanka and Thailand (Talwar & Jhingran 1991). This species is a commercially important target species for small-scale fishers in Bangladesh, and elsewhere in Asia, who apply a variety of traditional fishing gears (Rahman 1989). It is also the most favored aquarium fish among barb species in Asian countries (Froese & Pauly 2016). P. ticto is a considerable source

of animal protein and micro-nutrients in the diet of rural small-scale farmers (Roos et al., 2007). It is vulnerable in Bangladesh (IUCN Bangladesh, 2015); lower risk but near threatened in India (Balasundaram et al., 2000; Sarkar et al., 2010); threatened in Sri-Lanka (IUCN Sri-Lanka 2000).

Morphometric and meristic characteristics are helpful for species recognition and classification (Begenal & Tesch 1978; Jayaram 1999, Hossen et al., 2016). Furthermore, morphometric characters play a vital role in fisheries research, as it is used for comparing life history and morphological trends of populations across regions (Hossain et al., 2017a, 2016a; Elahi et al., 2017). Morphometric and meristic characteristics, including Length-Weight Relationships (LWRs) (Hossain et al.,

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 2 200

2016b, 2017b), and length-length relationships (Hossain et al., 2006, 2016c) of many threatened species of Bangladesh, are well documented, but none covers a large quantity of linear dimensions. However, only a few conservation studies including LLR and LWR have been done on the ticto barb (Hossain et al., 2009a, 2009b, 2014, 2015a; Hossain, 2010, 2014; Kaushik & Bordoloi, 2015). Therefore, the present study illustrates the morphometric and meristic traits of P. ticto from the Ganges River, northwestern (NW) Bangladesh using a number of specimens with a wide variety of body sizes.

2. Material and Methods

A total of 100 individuals of P. ticto (see Figure 1) was collected occasionally from the Ganges River (24°22′ N; 88°35′ E; known as the Padma River in Bangladesh) during July 2014 to October 2015 using various types of traditional fishing gears that include cast net (mesh size ranges: 1.5 - 2.5 cm), gill net (mesh size ranges: 1.5–2.0 cm), and square lift net (mesh size: ~2.0 cm). The fresh samples were instantly chilled in ice on site and fixed with 10% buffered formalin upon arrival in the laboratory, where all morphometric and meristic features were computed according to Froese & Pauly (2016).

Figure 1. Photo of Pethia ticto, collected from the Ganges River, northwestern Bangladesh on 5 June 2015

The total number of the fin rays and scales in different body parts (including the lateral line) was counted by using magnifying glass. Body Weight (BW) of each individual was weighed using an electronic balance and different linear dimensions, i.e., lengths (see Table 1 and Figure 2) were measured with digital slide calipers to the nearest 0.1 g and 0.1 cm accuracy, respectively. The LWR was calculated with the equation: W= a× Lb, where W is the Body Weight (BW) and L is the 14 different lengths in cm. The parameters a and b were calculated through linear regression analyses depends on natural logarithms: ln (W) = ln (a) + b ln(L). Moreover, 95% confidence limit (CL) of a and b and the co-efficient of determination (r2) were estimated. Extreme outliers were excluded from the regression analyses. A t-test was used to verify whether b values obtained in the linear regressions were significantly dissimilar from the isometric value (b = 3), (Sokal & Rohlf 1987). In the present study, 13 LLRs were analyzed by linear regression analysis (Hossain et al., 2006). The best model for both LWRs and LLRs was selected depending on the highest value of determination r2. Statistical analyses were carried out with Graph Pad Prism 6.5 software. All statistical analyses were significant at 5% (p < 0.05).

Table 1. Morphometric measurements of the Pethia ticto (Hamilton, 1822) (n = 100) captured from the Ganges River, northwestern Bangladesh.

Measurements Min (cm)

Max (cm)

Mean ± SD

Mode 95% CL

%TL

TL (Total length)

4.9 9.5 7.69 ± 1.08

8.3 7.48-7.90

BW (Body weight)

1.5 12.9 7.06 ± 2.88

4.8 6.49-7.63

91.9

FL (Fork length)

4.2 8.5 6.82 ± 1.03

7.7 6.61-7.02

88.6

SL (Standard length)

3.6 7.5 5.88± 0.93

6.5 5.69-6.06

76.5

HL (Head length)

0.8 1.9 1.48± 0.26

1.7 1.43-1.53

19.3

PcL (Pectoral length)

0.9 1.8 1.42± 0.22

1.6 1.38-1.46

18.5

PvL (Pelvic length)

1.7 3.5 2.76± 0.41

3.1 2.68-2.84

35.9

PrDL (Pre Dorsal length)

1.7 3.6 2.89± 0.40

3.2 2.81-2.97

37.6

PoDL(Post Dorsal length)

2.4 4.9 3.90± 0.57

4.3 3.79-4.01

50.7

DFBL (Dorsal fin base length)

0.7 1.3 1.01± 0.18

1.1 0.97-1.04

13.1

PrAnL (Pre Anal length)

2.7 5.2 4.20± 0.58

4.7 4.08-4.31

54.6

PoAnL (Post Anal Length)

3.1 6 4.80± 0.70

5.4 4.67-4.94

62.5

AFBL (Anal fin base length)

0.4 0.8 0.61± 0.12

0.7 0.58-0.63

7.9

AnsL (Anus length)

2.5 5.1 4.02± 0.63

4.5 3.89-4.14

52.3

BD (Body depth)

1.4 3.2 2.4± 0.41

2.7 2.32-2.48

31.2

Min, minimum; Max, maximum; SD, standard deviation; CL, confidence limit for mean values; TL, total length

Figure 2. Showing the morphometric measurements of Pethia ticto from the Ganges River, Northwestern Bangladesh

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 2

201

3. Results

The body of P. ticto is elongated, strongly compressed, and silvery with two black spots (one above the pectoral fin and the other on the central point of the caudal peduncle). The mouth is small and terminal, with no barbel.

There are about 25-29 scales in the lateral line, 4.5-6.5 scales above the lateral line, and 6.5-7.5 scales beneath the lateral line. In addition, 9-10 pre-dorsal scales were found. All morphometric and meristic measurements are given in Table 1 & 2. The fin formula of P. ticto is dorsal, D. 10–11 (ii/8-9); pectoral, P1. 12 (ii/10); pelvic, P2. 8- 9 (ii/6-7); anal, A. 7-8 (ii/5-6); and caudal, C. 20–24 (iv /16–20), respectively. In the present study, TL was varied from 4.9-9.5 cm (mean ± SD = 7.69 ±1.08) and BW was ranged from 1.5-12.9 g (mean ± SD = 7.06 ±2.88). Fork length (88.6%) and standard length (76.5%) showed higher proportion of TL (Table 1).

The regression parameters (a and b), their 95% confidence intervals, coefficients of determination (r2) for LWRs, of P. ticto are given in Table 3. All LWRs were highly significant (p < 0.001) with r2 values ≥ 0.904. Based on r2 value, LWR by BW vs. TL was the best fitted model among 14 equations. LLRs, which are shown in Table 4, were also highly correlated with r2 values ≥ 0.902. Based on maximum values of r2, LLR by TL vs. FL was the best-fitted model among 13 equations.

Table 2. Meristic characters of Pethia ticto (Hamilton, 1822) (n = 100) captured from the Ganges River, northwestern Bangladesh.

Meristic data Numbers (Unbranched /Branched)

Dorsal fin rays 10-11 (ii/8-9)

Pectoral fin rays 12 (ii/10)

Pelvic fin rays 8-9 (ii/6-7)

Anal fin rays 7-8 (ii/5-6)

Caudal fin rays 20-24 (iv/16-20)

Lateral line scale 25-29

Scale upper the lateral line to base of dorsal fin

4.5-6.5

Scale below the lateral line to pelvic fin

6.5-7.5

Pre-dorsal scale 9-10

Unbranched, single fin ray; Branched, upper portion of fin is divided into several rays

Table 3. Descriptive statistics and estimated parameters of the length-weight relationships of Pethia ticto (Hamilton, 1822) (n = 100) from the Ganges River, northwestern Bangladesh.

Equation a b 95% CI of a

95% CI of b r2

BW= a × TLb

0.0086 3.26 0.0074-0.0101 3.18-3.33 0.987

BW= a × FLb

0.0208 3.00 0.0179-0.0241 2.95-3.08 0.984

BW= a × SLb

0.0416 2.86 0.0352-0.0492 2.77-2.96 0.973

BW= a × HLb

2.4798 2.51 2.3319-2.6371 2.37-2.66 0.922

BW= a × PcLb

2.3879 2.90 2.2879-2.4922 2.79-3.02 0.963

BW= a × PvLb

0.2838 3.10 0.2540-0.3172 2.99-3.21 0.970

BW= a ×PrDLb

0.0219 3.24 0.1860-0.2413 3.12-3.36 0.966

BW=a × PoDFLb

0.0941 3.13 0.0835-0.1060 3.04-3.21 0.981

BW= a × DFBLb

6.5258 2.52 6.3417-6.7153 2.37-2.68 0.912

BW= a × PrAnLb

0.0574 3.31 0.0512-0.0644 3.23-3.39 0.985

BW=a × PoAnLb

0.0469 3.15 0.0416-0.0530 3.07-3.23 0.985

BW=a × AnFBLb

19.7283 2.18 18.2198-21.3618 2.03-2.32 0.904

BW= a × AnsLb

0.1148 2.92 0.1025-0.1285 2.83-3.00 0.981

BW= a × BDb

0.6669 2.63 0.6074-0.7323 2.52-2.73 0.961

See Table 1 for abbreviations; n, sample size; a and b are LWR parameters; CI, confidence intervals; r2, coefficient of determination

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 2 202

Table 4. The estimated parameters of the length-length relationships (y = a +b × x) Pethia ticto of (Hamilton 1822), (n = 100) from the Ganges River, northwestern Bangladesh.

Equation Regression parameters

95% CI of a

95% CI of b

r2

a b

TL = a + b ×

FL

0.6124 1.039 0.4413-0.7835

1.014-1.063

0.986

TL = a + b ×

SL

0.9867 1.140 0.7480-1.2253

1.100-1.180

0.970

TL = a + b ×

HL

1.6581 4.076 1.3392-1.9770

3.863-4.288

0.937

TL = a + b ×

PcL

0.8854 4.789 0.6469-1.1238

4.623-4.954

0.971

TL = a + b ×

PvL

0.4491 2.620 0.1990-0.6992

2.530-2.710

0.972

TL = a + b ×

PrDL

0.0040 2.659 -0.2601-0.2680

2.568-2.750

0.972

TL = a + b × PoDL

0.3716 1.877 0.1426-0.6010

1.819-1.936

0.977

TL = a + b × DFBL

1.8145 5.835 1.4208-2.2083

5.450-6.220

0.902

TL = a + b × PrAnL

-0.0704 1.850 -0.2503-0.1094

1.807-1.892

0.985

TL = a + b × PoAnL

0.3108 1.536 0.1520-0.4695

1.503-1.569

0.984

TL = a + b × AnFBL

2.6094 8.356 2.3025-2.9162

7.861-8.851

0.920

TL = a + b ×

AnsL

0.8704 1.697 0.6826-1.0582

1.651-1.743

0.982

TL = a + b ×

BD

1.5067 2.576 1.2787-1.7348

2.483-2.670

0.985

See Table 1 for abbreviations; n, sample size; a, intercept; b, slope; CI, confidence intervals; r2, co-efficient of determination

4. Discussion

There are very few studies on morphometric and meristic characters of P.ticto (Talwar & Jhingran 1991; Hossain et al., 2009a; Kaushik & Bordoloi, 2015). In the present study, we found 25-29 scales in the lateral line, 10–11 dorsal fin rays, 12 pectoral fin rays, 7-8 anal fin rays and 8-9 pelvic fin rays were observed, which was dissimilar to the studies done by Talwar & Jhingran (1991).

A total of 100 individuals collected for the present study comprised various body sizes (4.9 – 9.5). However, during this study, absence of P. ticto smaller than 4.9 cm TL may be attributed either to the fishermen who did not catch the smaller size due to low market price or to the selectivity of fishing gears (Hossain et al., 2012a, 2016d, 2017c). In the present study, maximum length recorded (9.5 cm) is lower than the maximum length (TL= 10.0 cm) reported by Talwar & Jhingran (1991). Maximum length helps to estimate the growth parameters, i.e., asymptotic length, growth coefficient, vital for fisheries resource planning and management (Ahmed et al., 2012).

The allometric co-efficient (b) values of LWRs ranging from 2.5 to 3.5 are more common (Froese, 2006). In the present study, most of the b values were in the limit of 2.5–3.5, which is similar to Hossain et al. (2009a) who estimated the b value as 2.92 for P. ticto, though some were within the range of 2.0–4.0. However, b values may vary in the same species due to one or more factors, these include growth differences among organs, genders, and preservation methods, additionally differences in the physiology and the examined length series of the specimens collected (Tesch, 1971; Hossain et al., 2012b, 2015b), which were not considered in this study. In addition, all the LLRs were highly correlated. Due to lack of literature data, it restrains to compare our results with other findings except the study by Kaushik and Bordoloi (2015). However, the present study found the best model among equations using different lengths based on coefficient of determination, which will offer baseline for comparisons with future studies using any linear dimension.

The present study should be a valuable tool for fishery managers to identify P. ticto and initiate stock assessment of the existing stocks of this threatened species in the Ganges River, NW Bangladesh and other subtropical countries. Also, these results will impart valuable information for the FishBase and providing an important baseline for future studies within the Ganges River and surrounding ecosystems.

Acknowledgements

The authors would like to extend their sincere appreciation to the (i) IDRSBFRI, Bangladesh Fisheries Research Institute, Shrimp Research Station, Bagerhat, Bangladesh (Serial No. 04, Session: 2013-2014) and (ii) Ministry of Education, Bangladesh (No. 37.200000.004.003.005.2014-1309/1 (42); Date: 10-08-2014) for funding to carry out this research and (iii) R. L. Vadas Jr. (WDFW, WA, U.S.A) for critically reviewed the draft manuscript.

Conflicts of interest

The authors declare that there is no conflict of interest regarding the publication of the present paper.

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JJBS Volume 10, Number 3,September 2017

ISSN 1995-6673 Pages 205-212

Jordan Journal of Biological Sciences

Antibacterial Activities (Bacitracin A and Polymyxin B) of Lyophilized Extracts from Indigenous Bacillus subtilis Against

Staphylococcus aureus

Marwan Y. Hussain*, Adnan A. Ali-Nizam and Samir M. Abou-Isba

Department of plant Biology, Faculty of Science, Damascus University, Syria

Received February 28, 2017 Revised June 4, 2017 Accepted June 20, 2017

Abstract

The antibacterial compounds, which are produced from Bacillus species, have a strong inhibition ability, and the composition of these compounds have not been elucidated. The aim of the present study is to explore the antibacterial activity of crude lyophilized extracts, then isolate and characterize these antibacterial. Bacillus subtilis strains were isolated from Damascus zone and identified by two methods which are the API CHB 50 and 16S DNA. Staphylococcus aureus strains were isolated from clinical samples, and identified by API Staph 20 and coagulase test. The crude antibacterial was extracted by flasks content LB where incubated for 72 h. in shaker incubator at 37°C at 150 rev/min, then centrifugated 12000 rev/minor 15 min. at 4 °C and filtrated the supernatants throw 0.45 µm, next frozen and lyophilized. The crude extracts were run on HPLC system to separate the antibiotics. Some strains of B. subtilis gave high activity against strains of S. aureus and others were not. Peptides families of antibiotics were separated from lyophilized extracts of B. subtilis, and then they were identified as bacitracin A and polymyxin B. This crude extract could function as antibacterial activity against S. aureus pathogens.

Keywords: Antibacterial, Staphylococci, Well diffusion, Crude, Lyophilized, Peptide.

* Corresponding author. e-mail: mrgoldeny@gmail.com.

1. Introduction

Staphylococcus aureus is responsible for a variety of infections, like carbuncles, impetigo, pneumonia, osteomyelitis, acute endocarditis, conjunctivitis, wounds, and urinary tract infections in man, women, and children (Brooks et al., 2007; Wolff et al., 2013).

S. aureus, especially methicillin resistance S. aureus (MRSA), is still one of the five most common causes of nosocomial infections; in addition, the infections are associated with surgical sites, wound infections, catheters and prosthetic implant. The increase of infections by these organisms has been correlated with the wide medical uses of prosthetic and indwelling devices and the growing number of immune compromised patients in hospitals (Cal et al., 2005; Dockrell et al., 2010; Owens and Fowler, 2012). More medical and pharmaceutical applications were benefited from the antibacterial which were produced from Bacillus subtilis (Pag and Sahl, 2002; Stein, 2005).

Many studies focused on the ability of Bacillus species to produce antibiotics and antifungals. In a study by Chen et al. (2008), the lipopeptide antibiotics were isolated and characterized from Bacillus subtilis, and, according to Chen et al., these compounds could play as a

biocontrol agent against a large spectrum of pathogens. There are many different support factors, like pH, time incubation, that have an effect on growth microbial cells, and production metabolites, like enzymes and antibiotics (Awais et al., 2010). Bacillus subtilis was isolated from the soil, and showed activity against S. aureus, Pseudomonas aeruginosa, and Escherichia coli (Sethi et al., 2013). The effects of the endophyte B. subtilis were investigated on the growth of cacao seedlings in which the root length and the leaf were increased up to 30% and 14%, respectively while in the stem height was increased up to 7.6% only. Besides, it also showed the strongest inhibition against the fungi Colletotrichum sp. and C. gossypii (Falcäo et al., 2014). The antiviral activity of subtilosin from Bacillus amyloliquefaciens was studied and showed strong inhibition against the extracellular and total virus production of herpes simplex virus type 2 (Quintana et al., 2014). Several antimicrobial substances and antibiotic macrolactin E were produced by B. amyloliquefaciens (Yuan et al., 2014). Due to the prevalence of antimicrobial resistance among key microbial pathogens is increasing in worldwide. However, there are no specific reports available on the lyophilized extracts from Bacillus spp. against S. aureus. Therefore, the present study presents and characterizes some new strains of B. subtilis that can

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 206

produce antibiotics like bacitracin with their evaluation the antibacterial activity potential and characterizes the partially purified antibacterial compounds from these new strains.

2. Material and Methods

2.1. Collection the Samples of Soils and Water

Three different soil samples were taken from every site from the 15 -20 cm layer, then mixed to form a composite sample for each site. The samples were collected in the sterile polypropylene bags from cultivated and barren lands in, and around Damascus: Al-Tall, Doma, Khan-Alsheh, Meliha, Adawee, Jedaideh, and Yafour. Three different water samples were taken from three sites from Bab-Sharqi, the wastewater and one canal of Barada river in Damascus, had been collected in the sterile glass bottle. Finally, the samples were prepared for identification. 2.2. Isolation and Identification of B. subtilis

From soil, each 10 g of sample was suspended in 90 mL of sterile distilled water and shaken vigorously for 2 min, while from water; 10 mL of sample was suspended in 90 mL of sterile distilled water and shaken vigorously for seconds. Both of the samples were heated at 80 °C for 15 min in a water bath. Then the soil suspensions were diluted in sterile distilled water and 1 mL of dilutions from 10-1 to 10-6 were plated on Nutrient Agar (NA) (Abtec, England). The plates were incubated at 37 °C for 24 h. The identification of bacterial strains was performed both by microbiological, and biochemical methods, according to the Bergey’s Manual 2009, by using API 50 CHB (BioMérieux, France) and results of tests were read by API 50 CHB web, in addition, the spore morphology, gram characteristics and motility tests were carried out (Benson, 2001; Logan and De vos, 2009). 2.3. Molecular Identification of B. subtilis

Bacteria was grown in 50 mL Lactose Broth (LB) (Merck, Germany) for 24 h then DNA extraction was done according to (Japoni et al., 2004; Kalia et al., 1999) with simple modifications. All Bacillus isolates were identified by 16SrDNA sequence and genomic DNA amplified using Gene Pro thermal cycler (Model TC-E-96G), with forward and reverse primers BacF (5՝-GTGCCTAATACATGCAAGTC-3՝) and BacR (5՝-CTTTACGCCCAATAATTCC-3՝) from Alpha DNA Company (Canada), were flanked a highly variable sequence region of 545 bp (Nair et al., 2002). The reaction conditions consisted of one initial denaturation cycle at 94 °C for 3 min, 45 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min and a final extension step at 72 °C for 10 min then cold 4 °C. The amplified products were run on a 1.25% agarose gel and visualized on a U.V. transilluminator. The nucleotide sequence analysis of the 16S rDNA of strains was identified at NCBI server using BLAST-n (www.ncbi.ncm.gov/blast), and the sequences compared. The sequences were submitted to Genbank and accession number obtained.

2.4. Isolation and Identification of S. aureus

Staphylococci strains were taken from patients hospitalized in three hospitals from different departments: Al-Mouwasat, University children hospital, and Dermal Venereal Diseases hospital (ear, eye, urine, cerebrospinal fluid (CSF), bronchial lavages, peritoneal fluid, pleural effusion, abscess, furuncle, bullous, pus, wound) and healthy people (human skin) in Damascus city and plated on NA and blood agar (BA) (Abtec, England) and mannitol salt agar (MSA) (Biolife, Italy). The plates were incubated at 37 °C for 24 h then morphology of cultures, Gram stain, production of catalase and coagulase slide test (Sigma, Germany) in addition, the API Staph was carried out for identification (Garcia and Isenberg, 2007; Colaninno, 2009). 2.5. Microbial Strains and Culture Conditions.

All strains were isolated between June 2013 to August 2014, seven strains of B. subtilis (six from soil, and one from wastewater), and 52 strains of S. aureus from different sources (patients and healthy skin) were used in the experiments. Bacterial strains were maintained on NA slants at 4°C for a short time, and glycerin 20% in LB at −30°C freezer for a long-time. 2.6. Growth and Production of Antimicrobial by Lyophilization

Production of antimicrobial lyophilized crude carried out according to Haddad et al. (2009) and Bhatta and Kapadnis (2010) with some modifications. After preparing 1000 mL of LB (Merck, Germany), the media were allocated in separated Erlenmeyer flasks 500 mL, each one containing 100 mL of LB and autoclaved, the flasks were inoculated with a fresh culture of B. subtilis by using the sterilized loop and incubated for 72 h at 37 °C ± 1 °C in shaker incubator (JSR, Korea) at 150 rev/min until cell numbers reach 15 ×10P

7P CFU/mL by optical density OD

(ODR600 R0.5 = 5 ×10P

7 PCFU/mL) (WPA CO8000, Biochrome

England). After growth, culture media were centrifuged (Hittch, Germany) at 12000 rev/min for 15 min at 4 °C and the supernatants were filtered through 0.45 μm membranes (Sartorius, Germany). The resulting filtrates (pH 7.0–8.0) were collected in special vials and put in freezer −30 °C between 6 - 8 h then put in lyophilizer (Lyotrap, England) at −40 °C and less than 1 millibar for 48-72 h until complete lyophilization, finally the crude lyophilized extract was weighed and stored at 2 - 8 °C and these crudes were used to evaluate antimicrobial activity. 2.7. Antimicrobial Activity

The antimicrobial activity of the crude lyophilized extract of B. subtilis was checked by agar well diffusion method. Mueller-Hinton Agar MHA surface (Merck, Germany) plates were swab-inoculated with S. aureus were grown in Nutrient Broth (NB) (SRL, India) and the turbidity of the cultures of S. aureus were adjusted by OD (ODR600R 0.5 = 1 ×10P

8P CFU/mL). Wells were made in the

inoculated plates using sterile stainless still borer (diameter 6 mm), then 20 μL of the crude lyophilized extract was added into wells (0.5 mg/µL) and after the plates were incubated at 37 °C for 24 h, the diameter of the inhibition zone was measured in mm with well size of 4 mm, in addition, the standard antibiotic disc was used as positive control (Kumar et al., 2009; Bhatta and Kapadnis, 2010).

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2.8. Determination the Minimum Inhibition Concentration (MIC) of Lyophilized Extracts

MIC of the crude lyophilized extract against S. aureus were determined by broth dilution method, according to the Clinical and Laboratory Standards Institute (CLSI, 2007). The crude was dissolved in distilled water (0.5 mg/µL) and double dilution of the compound was made in NB. Then 1 mL inoculum (108 CFU/mL) of S. aureus was added separately to an equal volume of two-fold dilutions of respective antimicrobial solution. For positive control, 1 mL inoculum (108 CFU/mL) of target organisms was added to 1 mL growth medium without antimicrobial. All the tubes were incubated for 24 h at 37 °C.MICs were determined as the lowest concentration of a compound that showed no visible growth of the organism (Bhatta and Kapadnis, 2010). 2.9. Statistical Analysis

Statistical analysis (One Way ANOVA) was performed using SPSS program software, version 17 to validate the signification of the results. The data are presented as means (± SD) of three replicates. 2.10. Partial Purification of Extracted Antimicrobial Substance by HPLC

Antibiotics in the crude lyophilized extract (water solution, 0.5 mg/µL), were detected and quantified by reversed-phase HPLC as follows: A 50µL sample was injected into the HPLC column [PREP-ODS C18, 250×4.6 mm]. The temperature of the column was maintained at 25°C throughout the experiment. The mobile phase components were solution (A) 0.1% trifluoroacetic acid (TFA) in water and solution (B) 0.1% TFA in acetonitrile. The compounds were eluted at a flow rate of 1 mL/min with a linear gradient of solvent B increasing from 0 to 50%. The elution pattern was monitored at 268 nm, pooled fractions were collected and concentrated (Chen et al., 2008; Snyder et al., 2010; Yuan et al., 2014). 2.11. Determination of Functional Groups of Crude Antibacterial Extracted by FTIR

The FTIR (Fourier transform infrared) spectrum was recorded using FTIR 4200 (Jascoo, Japan). The spectra were scanned in the range of 400 – 4000 cm-1. The spectra were obtained using potassium bromide pellet technique. Potassium bromide (AR grade) was dried under vacuum at 100°C for 48 h. Thus, 100 mg KBr was mixed with 5 mg of sample. The spectra of intensity versus wave number were plotted.

3. Results

3.1. Isolation and Identification of B. subtilis

Table 1 shows the number of strains of B. subtilis and characteristics of soil: color, humidity, and sources, B. subtilis was able to isolated from the soil of Al-Tall, Doma, Khan-Alsheh, Meliha, Adawee, Jedaideh except from Yafour and be isolated from wastewater of Bab-Sharqi. The strains of S. aureus, which were isolated from pathogen samples and healthy human skin were showed in Table 2 and Table 3.

3.2. Identification of B. subtilis by API 50 CHB and Molecular Method

The microscopic study of bacterial cultures showed these strains to be Gram-positive rods, less than 1 μm in diameter. All strains sporulated aerobically without swelling of the cell and produced catalase. These data indicated that tested strains belong to Bacillus genus. Additional testing with API 50CHB kit identification resulted that all strains were B. subtilis. Partial sequence of 16S rDNA of B. subtilis (BS1A, BS2D, BS3K, BS4M, BS5W, BS6J, BS7SH) were submitted to the Genbank database under accession numbers KM052377, KM192148, KF792061, KM251459, KF792060, KM189125 and KF792061, respectively. In addition, the results of 16S rDNA gene sequence confirmed with the obtained results of biochemical tests. Table 1. Characteristics of soils and the strains of B. subtilis from soil and water. Sample Source’s

sample Color Humidity B. subtilis

Al-Tall soil black little wet BS1A

Doma soil black wet BS2D

Khan-Alsheh

soil white dry BS3K

Meliha soil black wet BS4M

Adawee soil black wet BS5W

Jedaideh soil black dry BS6J

Yafour soil red little wet 0

Bab-Sharqi (canal of Barada)

waste water

- - BS7SH

3.3. Antimicrobial activity Table 2 shows that two crude extracts (BS1A, BS2D)

from strains of B. subtilis, which had been isolated from the soil and BS7SH, which had been isolated from wastewater, have an intermediate to a high antibacterial activity in which they gave a bigger zone against most of S. aureus strains (8-40 mm), whereas another crude extracts from strains of B. subtilis, which were isolated from the soil (BS3K, BS4M, BS5W, BS6J), did not give antibacterial activity against most of these strains of S. aureus.

As shown in Table 3, most of S. aureus strains have between intermediate to good sensitivity with diameter inhibition zone (8-25 mm) facing the crude extracts from strains of B. subtilis (BS1A, BS2D) and (BS7SH), while the crude extracts from strains of B. subtilis (BS3K, BS4M, BS5W, BS6J) did not have antibacterial activity against S. aureus strains except S31, S36, S43, S47. 3.4. MIC of crude lyophilized extract

The minimum inhibitory concentration of the antimicrobial compound in the crude lyophilized extract was found to be 5-7.5 mg/mL for S. aureus. 3.5. Statistical Results

According to the statistical results, the differences between the antibacterial activity against S. aureus were subjected to One Way ANOVA statistical test and it

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showed that there is a significant difference between some strains of B. subtilis towards S. aureus (P<0.05) and there

is no significant difference between others (P>0.05) (Table 2 and Table 3).

Table 2. The mean, ±SD, and results of Anova(a,b,c..) of the diameters of inhibition zone (mm) of crude lyophilized extract against S. aureus strains which isolated from patients as test organisms.

S. aureus No.

Source’s sample

BS1A BS2D BS3K BS4M BS5W BS6J BS7SH

S1 CSF 40.66±1.15a* 23.33±1.15b 8±0c 10±0c 8±0c 12±2.0c 12±0c

S2 Ear 29.33±1.15a 12.66±1.15b - - - - 8.66±1.15b

S3 pus 30.33±0.57a 15.33±1.15b - - - - 8.66±1.15b

S4 Bronchial lavages

- - - - - - 8±0

S5 Pus 28.66±1.15a 14.33±0.57b - - - - 8.66±1.15c

S6 Ear 13.33±2.88a 14±0a - - - - 9.33±1.15a

S7 bronchial lavages

- 16.66±1.15a - - - - 10±0b

S8 Abscess - - - - - - -

S9 Ear - 18.66±1.15a - - - - 10.66±1.15b

S10 Pus - 18.66±1.15a - - - - 12.66±1.15a

S11 Pus 14±0a 10.66±1.15a - - - - 10.66±1.15a

S12 Pus - 15.33±1.15a - - - - 11.33±1.15a

S13 Furuncle 11.33±1.15a 12±2.0a - - - - 8.66±1.15a

S14 Pus - - - - - - 10±0

S15 Abscess 21.33±1.15a 10.66±1.15b - - - - 10.66±1.15b

S16 Abscess 13.33±1.15a 14.66±0.57a - - - - 11.33±1.15a

S17 Urine 15.33±1.15a `16±5.29a 9.33±1.15b 10.66±1.15b 10.66±1.15b 9.33±1.15 13.33±1.15a

S18 Urine 11.66±2.88a 12.66±1.15a 8.66±1.15a 8±0a - - 8.66±1.15a

S19 Abscess 11.33±1.15a 14±0a - - - - 8.66±1.15a

S20 CSF 17±2.64a 10±0b - - - - 10.66±1.15b

S21 Ear - 10±0a - - - - 10±0a

S22 Eye - 10±0a - - - - 10.66±1.15a

S23 pleural effusion - - - - - - 10±0

S24 Urine - 15.33±1.15P

a - - - - 10.66±1.15P

b

S25 Urine 28±2P

a 15.33±1.15P

b - - 8±0P

c - 10±0P

d

S26 Peritoneal fluid 12.66±1.15P

a - - - - - 8.66±1.15P

a

S27 Pus - 11.33±1.15P

a 6±0P

b - - - 10±0P

a

S28 Ear 12.66±1.15P

a 10.66±1.15P

a - - - - 10±0P

a

S29 Wound - 11.33±1.15P

a - - - - 10±2.0P

a

S30 CSF - 14.66±1.15P

a - - - - 11.33±1.15P

a

Sensitive: ≥16 mm, intermediate sensitive: 10 – 15 mm, non-sensitive: 4 - 6 mm, (-) no inhibition zone.

*Different letters in the same line indicate significant differences among the samples

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Table 3. The mean and ±SD and results of Anova(a,b,c..) of the diameters of inhibition zone (mm) of crude lyophilized extract against S. aureus strains which isolated from (skin’s infections, healthy skin) as test organisms.

S. aureus Source’s sample

BS1A BS2D BS3K BS4M BS5W BS6J BS7SH

S31 Eczema 12.66±1.15a* 8.66±1.15a 11.33±1.15a 9.33±1.15a - - 12.66±1.15a S32 common

Bullous 9.33±1.15a - - - - - -

S33 Bullous 11.33±1.15a 13.33±1.15a - - - - - S34 Furuncle 8.66±1.15a 11.33±1.15a 10±0a 9.33±1.15a - - 10.66±1.15a

S35 Bullous 18.66±1.15a - - - - - 8.66±1.15b S36 Ulcers 25±0a 22±2.0a 10.66±1.15b 10.66±1.15b 11.33±1.15b 10.66±1.15b 14±0b

S37 Bullous - 12±0a - - - - 8.66±1.15a S38 Bullous 10±0a 12±2.0a - - - - 14.66±1.15a S39 Bullous - 10.66±1.15 - - - - 11.33±1.15a S40 Furuncle 10.66±1.15a 14±0b - - - - 8.66±1.15a S41 Face 12.66±1.15a 12±0a - - - - 10.66±1.15a S42 Face 13.33±1.15a 12.66±1.15a 8±0b 8±0b - - 13.33±1.15a

S43 Hand 15.66±0.57a 10±0b 14±0a 10.66±1.15b 10.330.57b 8.66±1.15b 16.33±0.57a

S44 Face - - - - - - - S45 Face 16.66±1.15a 14.66±1.15a - - - - 12±0a

S46 Hand - - - - - - 10.66±1.15 S47 Face 17.33±2.30a 15.33±1.15a 8.66±1.15b 7.33±1.15b 8.66±1.15b 11.33±1.15b 13.33±1.15c

S48 Face - 10.66±1.15a 12±0a 10.66±1.15a - - 10.66±1.15a

S49 Around mouth 16.66±1.15a 14.66±1.15a - - - - 10±0b

S50 Around mouth 12.66±1.15a 10±0b 9.33±1.15b 8±0b - - 12.66±1.15a

S51 Hand 10.66±1.15a 16.66±1.15b 8.66±1.15a 9.33±1.15a - - 10±0a

S52 Face - - - - - - 11.33±1.15 Sensitive: ≥16 mm, intermediate sensitive: 8 – 15 mm, non-sensitive: 4 - 7 mm, (-) no inhibition zone.

*Different letters in the same line indicate significant differences among the sample

3.6. Partial Purification by FTIR

The FTIR spectrum exhibited characteristic absorption valley at 3361 cm-1 (amide bonds) and valley at 1649 cm-1 (carbonyl bonds C=O) and valley at 3300 cm-1 (carboxyl group) and valley at 3500 cm-1 (hydrogen bonded OH groups) and valley at 1456 cm-1( carbon-carbon bond C=C) and it perhaps indicates the existence of a cyclical peptides. The valley at 2965 cm-1 shows C-H stretching, and valley at 1338 cm-1 shows C-O bond and valley at 2360 cm-1 shows S-H hydrogen sulfide bond. All indicated that substance contains peptide bonds (Figure 1 and Figure 2). So the characterizations of crude lyophilized extracts were showed in Table 4. 3.7. Partial Purification of Extracted Antimicrobial Substance by HPLC

The standard of bacitracin A and polymyxin B was run on HPLC. Bacitracin A gave peak with retention time 7.88 min while polymyxin B gave peak with retention time 28.36 min, then crude lyophilized extracts were purified by HPLC and were attributed to the peptides, like Bacitracin A. About 4 to 6 peaks were appeared in each extract, then, eluted fractions were collected and concentrated. The peak of bacitracin A purified from the strains BS1A, BS2D, BS7SH with retention time 8.65 and 9.57 min. (Figure 3 and Figure 4) and concentration 50, 50.20, 50.25 µg,

respectively, and correlates the antibacterial activity of its crudes extracts against S. aureus strains. However, there is no peak of bacitracin A appeared for the strains BS3K, BS6J, BS5W, and BS4M which correlates with antibacterial activity of its crudes extracts. The peaks of polymyxin B were appeared in these extracts BS1A, BS2D, BS7SH with retention time 28.36 and 28.72 min. (Figure 3 and Figure 4), then the fractions of peaks were collected and concentrated which were 114, 112 and 114.7 µg, respectively, but the other peaks were unknown because the standards were not available. Thus, these strains of B. subtilis which isolated from soil and wastewater can be added to the limited number of B. subtilis strains were reported to co-produce peptides previously. Table 4. Physical and chemical properties of crude lyophilized extract from B. subtilis.

Properties Results

Color light yellow to brownish when lyophilized and brown after soluble in water.

Nature light, smooth, brittle, special scent, porosity, morphous

Solubility water soluble, methanol, and soluble in 50% methanol-ethanol, but insoluble in chloroform, and Absolute ethanol

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 210

Figure 1. FTIR spectra of the Antibacterial compounds of crude lyophilized extract from B. subtilis was isolated from soil’s Doma.

Figure 2. The FTIR spectrum of the Antibacterial compounds of crude lyophilized extract from B. subtilis was isolated from wastewater Bab-Sharqi

Figure 3. Reversed-phase HPLC chromatograms of antibiotics produced by Bacillus subtilisBS1A, BS2D. Peaks marked 1 is Bacitracin A, and 2 is Polymyxin B.

Figure 4. Reversed-phase HPLC chromatograms of antibiotics produced by Bacillus subtilis BS7SH. Peaks marked 1 is Bacitracin A, and 2 is Polymyxin B.

4. Discussion

To the best of our knowledge, the reports on antibacterial activity from B. subtilis against S. aureus pathogens especially are few. In our study, as shown in as Table 2 and Table 3, the crude lyophilized extract from B. subtilis BS1A, BS2D and BS7SH showed a high antibacterial activity against S. aureus and the mean of diameters of inhibition zone was 8 - 40 mm, with 62.5% percent for BS1A, and 76.78% for BS2D, while percentage for BS7SH was 89.28%. ,whereas the crude extracts from BS1A and BS2D showed good antibacterial activity against S. aureus which isolated from skin’s infections, like bullous, ulcers, furuncles, and isolated from microflora of skin, like face and hand, with 74.07% percent for BS1A and 77.77% for BS2D (Table 2 and Table 3). On the other hand, the crude extracts from strains BS3K, BS4M, BS5W andBS6J did not give antibacterial activity against most of these strains of S. aureus except S1, S17, S36, S43, S47, in addition, the crude extracts from BS3K and BS4M were better than BS5Wand BS6J, which gave intermediate activity against S1, S17, S18, S31, S34, S36, S42, S43, S47, S48, S50, S51 (Table 2 and Table 3). At the same time, there were some strains of S. aureus resistant to the crude extracts, even the crudes extracts of species BS1A, BS2D and BS7SH, like S8, S44and resistant to the crudes extracts of BS1A and BS2D) but sensitive to the crude extracts of BS7SH, such as S4, S14, S23, S46, S52.

Some previous research studies used the liquid extracts, and S. aureus targets were isolated from clinical and food, while in the present study lyophilized extracts were used against clinical strains of S. aureus. It was found in a study by Aslim et al. (2002) and a study by Kim et al. (2003) that no effect of B. subtilis on S. aureus; in another study by Ouoba et al. (2007), it is showed that the S. aureus seemed resistance toward the antibiotic compounds which produced by B. subtilis. However, the studies of Motta et al. (2004), Fernandes et al. (2007), Sethi et al. (2013) and Ramachandran et al. (2014) reported that B. subtilis gave moderate activity against S. aureus and the diameters of inhibition zone varied from 8 to 16 mm. But Faheem et al. (2007) showed that Brevicin, which is isolated from B. brevis, gave a low activity against S. aureus. Whereas, Devi et al. (2008) showed that the antimicrobial activity from B. clausii was moderate against S. aureus. Also, the studies of Kuta et al. (2009), Mohammad et al. (2009), Bhatta and Kapadnis (2010), Moshafi et al. (2011) and Moore et al. (2013) showed that the antimicrobial compounds were produced from B.subtilis showed higher activity against S. aureus and the diameters of inhibition zone varied from 19 to 28 mm. On the other hand, Tabbene et al. (2010) characterized and identified a new antibacterial and antioxidant produced from B. subtilis and it has an activity against S. aureus, so Fuchs et al. (2011) isolated Entianin, which is a novel Subtilin from B. subtilis and gave a strong activity against S. aureus (MRSA).

These differences between the antibacterial activities of Bacillus species may be due to the production of antibiotics of each strain, or due to the environment in which they live, or may be due to the genetic differences which are important factors for the production of the antibiotics, and the biophysical and chemical factors in each soil.

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By the results of FTIR, presence the amide bonds, carbonyl bonds (C=O), carboxyl group, carbon-carbon bond C=C and hydrogen bonded OH groups indicate to a polypeptide, where these results were similar to the results of Kumar et al. (2009) and Bhatta and Kapadnis (2010).

Few peptides antibiotics, produced by B. subtilis, are active against Gram-positive bacteria, as staphylococci; however, using HPLC, the antibiotics were isolated and purified from the crude lyophilized extracts of strains of B. subtilis. As mentioned above, these strains BS1A, BS2D, and BS7SH showed strong antibacterial activity against S. aureus strains. About 4-6 peaks appeared from every extract, two important peaks were showed, the first peak is for bacitracin A with retention time 8.65, 9.57 (Figure 3 and Figure 4) and the latter is for polymyxin B with retention time 28.36, 28.72 (Figure 3 and Figure 4) and the activity of second peak against S. aureus was lower when compared to peak of bacitracin, that is because polymyxin B is used for Gram-negative Bacteria while bacitracin is used for Gram-positive Bacteria. So, related to these results are some of studies that purified and identified the antibiotics, like subtilin, and antifungal, like iturin from B. subtilis, and other species (Smitha and Bhat, 2012; Ouoba et al., 2007).

The strains, which showed a high antibacterial activity, were the BSIA, BS2D, and BS7SH. These strains will be suitable to undertake further studies on the antimicrobial activity of the B. subtilis isolates in order to isolate, characterize and identify the antimicrobial compounds produced against the pathogens.

5. Conclusions

This crude extract could function as an antibacterial activity against S. aureus pathogens. A new approach for structural characterization of antibiotics from B. subtilis were provided in the present study which uses the crude lyophilized extract against S. aureus.

Acknowledgment

The authors thank Prof. Duran Kala who is a lecturer in Ishik University and Editor-in-Chief of Eurasian Journal of Science and Engineering for reading this research to be best in publishing.

No conflict of interest

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JJBS Volume 10, Number 3,September 2017

ISSN 1995-6673 Pages 213 - 217

Jordan Journal of Biological Sciences

Antibacterial and Cytotoxic Activity of the Bark of Phoenix paludosa in Different Solvents

Saikat Ranjan Paul1, Md. Raad Sayeed1, Md. Lukman Hakim2*

1Department of Pharmacy, Southeast University; 2Department of Pharmaceutical Sciences, North South University, Dhaka, Bangladesh

Received March 12, 2017; Revised June 12, 2017; Accepted June 20, 2017

Abstract

Plant origin is an important source of drugs. In this experiment, the ethanolic extract of the bark of Phoenix paludosa fractioned with three different solvents (Petroleum ether, Chloroform and Ethyl acetate), was investigated to evaluate the antibacterial and cytotoxic activity. Most popular disc diffusion method was applied to determine the antibacterial activity of 500 µg/disc crude extract of the plant against 12 pathogenic Gram positive and Gram negative bacteria. Cytotoxic test was performed by following the brine shrimp lethality bioassay at different concentrations (100, 50, 25, 12.5, 6.25 µg/ml) where Vincristine sulphate served as positive control. Though the antibacterial activity is not so promising compared to the standard Kanamycin, only the chloroform fraction of ethanolic extract exhibited activity against all experimental bacteria (zone of inhibition ranging from 7 to 15 mm), the plant has been proven to possess good cytotoxic activity, having LC50 value of 13.03, 9.24 and 7.47 µg/ml for petroleum ether, chloroform and ethyl acetate fraction of ethanolic extract of the bark of Phoenix paludosa, respectively.

Keywords: antibacterial activity, brine shrimp bioassay, cytotoxicity, disc diffusion method, Phoenix paludosa.

* Corresponding author. e-mail: mlhshohag1221@gmail.com.

1. Introduction

Infectious diseases represent 41% of total health problems of the world along with non-infectious (43%) and injuries (16%) (Noumedem et al., 2013). Although there are many available promising antimicrobial drugs, pathogenic microorganisms are constantly developing resistance to these agents (Khan et al., 2008). In previous decades, a great number of antimicrobial agents have been launched by pharmaceutical companies, but even then the drug resistance has increased and most of these drugs failed to cope with multi-drug resistant bacteria (Ishaq et al., 2014). Bacterial resistance to antimicrobial agents is not only a medical problem with public health, but it has socioeconomic and even political implications (Massih et al., 2010). Multi-Drug Resistant (MDR) bacteria have become a global concern and scientists are trying heart and soul to overcome this problem.

Cancer, a disease of abnormal cell growth, is a leading cause of death in human. The death rate due to cancer is continuously increasing and it has been estimated that by 2030, 17 million deaths per year due to cancer will occur (Thun et al., 2010). Due to side effects and prolong

duration, the patients do not find the conventional cancer treatment (therapy, medicine) suitable. So there is still a search for anticancer drugs that can satisfy patient’s demand.

Since the appearance of human on the planet, plants are affording us with food, shelter, medicine and the most indispensable oxygen. According to the information provided by the World Health Organization (WHO) 80% of the world population is dependent on the traditional medicine (Lilybeth et al., 2013). 87% of all human diseases are treated with natural products and related drugs (Nasrin et al., 2015). Lot of medicinal plants have been identified to have natural antimicrobial and cytotoxic compounds (Naik et al., 2014). About half of the present clinically used anticancer drugs are of natural product origin and it is estimated that about 60% of New Chemical Entities (NCEs) commenced as anticancer drugs in the period 1980-2002 were natural products or were derived from a natural lead compound (Apu et al., 2013). Over 400 compounds, including prominent anticancer drugs such as vincristine, vinblastine, topotecan, etoposide, lapachol have been isolated from plants (Amirghofran et al., 2010). Therefore the search of natural drugs of plant origin becomes an effective way to discover new medicines.

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 214

Phoenix paludosa (Family: Arecaceae) is a mangrove plant found normally in Bangladesh, India, Sri Lanka, Thailand, Mayanmar and Malaysia. In Bangladesh, the local name of the plant is Hantal palm, a thorny 6-7 m high plant with 2-3 m long leaves naturally found in Sundarban (Alam et al., 2009). Previous studies revealed the analgesic, antidiarrhoeal (Saha et al., 2012), antioxidant and cytotoxic (Samarakoon et al., 2016) activities of the leaf. Twig of the plant is known to have antioxidant as well as lipid peroxidation inhibition properties (Bunyapraphatsara et al., 2003). The whole plant is a rich source of antioxidant (Patra et al., 2014a). The present study is dedicated to finding antibacterial and cytotoxic potential of the bark of Phoenix paludosa.

2. Materials and Methods

2.1. Collection of Plant Material

The bark of the plant was collected from Sundarban (Koromjol region of Satkhira district) located in Khulna division of Bangladesh in the month of June, 2016. The plant was identified by the Bangladesh National Herbarium, Dhaka and a voucher specimen (43205) was preserved. 2.2. Extraction and Fractionation of Plant Material

The collected barks of plants were made free from unwanted materials by washing with running water and then were subject to dry at room temperature. Barks were cut into small pieces, sun dried for seven days. The dried small pieces of barks were ground into coarse powders. The powdered materials (400 g) were extracted with ethanol (2 L) in a flat bottle glass container through occasional shaking and stirring for 7 days. The extract was filtered through cotton at first and then through the Whatman No.1 filter paper. Then, it was concentrated by the help of a rotary evaporator. Solvent-solvent partitioning was performed by slightly modifying the model of Kupchan and Tsou and modified version of Wagenen et al. (Kupchan and Tsou, 1973), (Muhit et al., 2010). 5 g of the crude extract was triturated with 90% ethanol. The solution was next fractioned with petroleum ether (820 mg), ethyl acetate (665 mg) and chloroform (550 mg). The fractions were evaporated by means of a rotary evaporator at a temperature of 390C and preserved. 2.3. List of Bacteria

Both Gram positive (Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus, Sarcina lutea) and Gram negative (Salmonella paratyphi, Vibrio parahaemolyticus, Vibrio mimicus, Shigella boydii, Shigella dysenteriae, Escherichia coli, Pseudomonas aeruginosa) bacteria were used as test organisms to observe the antibacterial activity of the test sample. The organisms were collected from the Microbiology Laboratory, Southeast University.

2.4. Antibacterial Assay

The most popular disc diffusion method (Bauer et al., 1966) was used to carry out the present study. Sterile paper discs were fecundated with 500 µg bark extract of the plant. After drying, the discs were placed on nutrient agar medium seeded with Gram (+)ve and Gram (-)ve sample bacteria. For maximum diffusion, the plates were kept at 4oC for 24 hours. Then the plates were placed in an incubator at 37oC for 24 hours to permit the growth of bacteria. After 24 hours of incubation, the antibacterial activity of the test materials was determined by measuring the diameter of the zones of inhibition in millimeter. In the experiment, standard antibiotic kanamycin 30µg/disc (Oxoid Ltd, UK) served as positive control, whereas blank discs containing only solvent were considered negative control. 2.5. Cytotoxic Assay

In the brine shrimp lethality bioassay (Meyer et al., 1982), the brine shrimp (Artemia salina) should be grown in sea water. So 38 gm NaCl was dissolved in 1000 ml filtered distill water to gain artificial sea water. Sea water was kept in a small tank that was divided into two parts, one was covered with black cardboard before adding the eggs and the other was illuminated with 100 watt bulb to attract brine shrimp. Oxygen was supplied continuously for the next 48 hours besides the maintenance of 25-30oC temperature and a pH of 8.5. Two days were allowed for the shrimp to hatch and mature as nauplii. Then these nauplii were considered for bioassay. Test samples were prepared by dissolving them into DMSO (not greater than 50 µl in 5 ml solution) and adding sea water to get the concentration of 100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml, 6.25 µg/ml. 50µl DMSO diluted to 5 ml used as control, whereas Vincristine sulfate served as positive control (Hamid et al., 2011). 10 brine shrimp nauplii were applied to each vial individually and allowed for next 24 hours. After 24 hours the vials were observed, the number of surviving nauplii in each vial was counted and the LC50 value was determined.

3. Results

3.1. The Result of Antibacterial Assay

Among the three fractions (petroleum ether, chloroform and ethyl acetate) of ethanolic bark extract (500 µg/disc) the highest zone of inhibition (15 mm) is found in chloroform fraction against Staphylococcus aureus with a zone of inhibition of 7 to 15 mm. In case of petroleum ether fraction the greatest zone of inhibition (7mm) is determined against Staphylococcus aureus and Salmonella paratyphi. For ethyl acetate fraction maximum antibacterial activity (zone of inhibition 9mm) is recorded against both Gram positive Bacillus cereus and Gram negative Escherichia coli. Both the petroleum and ethyl acetate fractions of the extract failed to exhibit antibacterial activity (no zone of inhibition) against many experimental bacteria (Table 1).

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3

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Table 1. Zone of Inhibition of different fractions of ethanolic bark extracts of Phoenix paludosa and the standard antibiotic Kanamycin against test bacteria.

Group

Bacteria

Zone of Inhibition (mm)

Petroleum ether fraction (500µg/disc)

Chloroform fraction (500µg/disc)

Ethyl acetate fraction (500µg/disc)

Kanamycin

(30µg/disc)

Gram positive

Bacillus subtilis - 11 - 32

Bacillus megaterium - 12 - 33

Bacillus cereus - 7 9 30

Staphylococcus aureus 7 15 6 31

Sarcina lutea 6 8 - 28

Gram negative

Salmonella paratyphi 7 10 8 30

Vibrio parahaemolyticus - 12 5 33

Vibrio mimicus - 10 - 30

Shigella boydii 4 9 5 31

Shigella dysenteriae 6 8 - 33

Escherichia coli - 10 9 31

Pseudomonas aeruginosa

- 7 - 30

3.2. The Result of Cytotoxic Assay

In comparison to the positive control; Vincristin sulphate (Table 2 and Figure 1), all the fractions of the extract at different concentrations yielded distinguished cytotoxic potency. The mortality rate of nauplii is dependent on the concentration. In most cases, higher mortality rate was recorded in greater concentration. The best cytotoxic property was observed in ethyl acetate fraction (LC50 7.47 µg/ml), other two fractions also exhibited prominent cytotoxic activity, LC50 value of 9.24 µg/ml and 13.03 µg/ml of chloroform and petroleum ether, respectively (Table 3 and Figure 2). Table 2. Effect of vincristine sulphate on brine shrimp

Concentration (µg/ml)

Number of nauplii

Number of dead nauplii

% of mortality

LC50

(µg/ml)

10 10 10 100 5 10 9 90 2.5 10 8 80 1.45 1.25 10 6 60 0.63 10 5 50

Table 3. Effect of ethanol bark extract of different fractions of Phoenix paludosa on brine shrimp.

Type of fractions

Concentration (µg/ml)

Number of nauplii

Number of dead nauplii

% of mortality

LC50 (µg/ml)

Petroleum ether

100 10 9 90 13.03 50 10 7 80 25 10

10 6 70

12.50 5 50 6.25 10 4 30

Chloroform

100 10 10 100 9.24 50 10 8 70 25 10 7 60

12.50 10 6 50 6.25 10 4 50

Ethyl acetate

100 10 9 90 7.47 50 10 8 70 25 10 6 60

12.50 10 6 60 6.25 10 5 40

Figure 1. Effect of Vincristine sulphate on brine shrimp lethality bioassay.

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 216

Figure 2. Brine shrimp lethality bioassay of different fractions of the ethanolic extracts of the bark Phoenix paludosa

4. Discussion

From the ancient history, numerous medicines have been developed from plants. The dependence on medicinal plants can be easily understood by considering the prominent usage of natural products by patients even in this modern technological era. Based on the vast unrevealed source of natural plant drug origin, the present study is dedicated to finding out antibacterial and cytotoxic activities of bark extracts of natural plant Phoenix paludosa.

To carry out the antibacterial activity of ethanolic bark extracts of different fractions of Phoenix paludosa both Gram (+)ve and Gram(-)ve bacteria were used. The bark extracts were applied at concentrations of 500µg/disc besides the antibiotic Kanamycin (30µg/disc). Kanamycin was considered as a standard to compare the antibacterial activities of three different fractions of the ethanolic extract of the experimental bark. Though due to complicated cell wall structure Gram (-)ve bacteria are considered less vulnerable than Gram (+)ve bacteria, the chloroform fraction exerted similar effects against both classes. But in most cases the zone of inhibitions achieved by the chloroform fraction was half or one third or even more less compared to the zone of inhibition counted for standard Kanamycin. The performance of the petroleum ether and ethyl acetate fractions was not up to the mark. Both of these fractions could not score a single zone of inhibition against Bacillus subtilis, Bacillus megaterium, Vibrio mimicus and Pseudomonas aeruginosa.

An agent having potent cytotoxic activity may use as an anticancer drug. Among the procedures to conduct the cytotoxicity test, the brine shrimp (Artemia salina) lethality bioassay is rapid (24 hours), simple and inexpensive, besides these no aseptic technique is necessary to perform the test. Moreover, this bioassay has been proved to have a good correlation with cytotoxic activity in some human solid tumours (Kumar et al., 2011). In cytotoxicity test Vincristin sulphate was considered as control. Concentrations of 100µg/ml, 50µg/ml, 25µg/ml, 12.5µg/ml, and 6.25µg/ml were tested to determine cytotoxic property. A relation between death rate and concentration was observed, the higher the concentration the higher the number of death nauplii. So the death rate was directly proportional to the concentrations. To determine LC50, percent (%) of

mortality was plotted against concentration values, respectively. The LC50 of the ethanolic bark extract of petroleum ether, chloroform and ethyl acetate are promising which indicates a potent cytotoxic activity of the plant. So, the present study evidenced the cytotoxic property of bark extracts of Phoenix paludosa and raise the demand for further sophisticated investigations.

The investigated plant Phoenix paludosa is abound with numerous phytochemicals like flavonoids, polyphenols, glycosides, gum, steroids, tannins (Lima et al., 2010; Saha et al., 2012; Samarakoon et al., 2016), phytosterols, triterpinoids (Alam et al., 2009) and so on. These ingredients are responsible for different pharmacological properties including antimicrobial and cytotoxic potency. Especially flavonoids, polyphenols, tannins, terpinoids are considered as common antimicro-bial plant chemicals (Cowan, 1999). The better antibacterial activity of the chloroform fraction may be due to the high extraction capacity of flavonoids, terpinoids, polyphenols and other antimicrobial phytochemicals (Cowan, 1999; Dhawan and Gupta, 2017; Samarakoon et al., 2016). The larvicidial toxicity of the above mentioned phytochemicals is responsible for the cytotoxic response of the plant and it may become clinically useful anticancer as well as pesticide agents as the brine shrimp assay is a considerable preliminary assessment of toxicity (Rahman and Islam, 2013). The exhibited cytotoxic property of the bark of Phoenix paludosa in this study supports the previous studies on cytotoxic potency of the leaf of the plant in different solvents (Alam et al., 2009; Lima et al., 2010; Samarakoon et al., 2016). Previous studies on the plant showed unreliable antimicrobial activity (Patra et al., 2014b). A study of 400µg/disc of leaf extract reported an insensitive to an antimicrobial growth (Alam et al., 2009). Here, in the present study, a low to a moderate antibacterial activity of the bark of phoenix paludosa is observed at a concentration of 500µg/disc.

5. Conclusion

The present study identifies the antibacterial and cytotoxic activities of bark extracts of Phoenix paludosa. The results support low to moderate antibacterial property besides good cytotoxic potency of the bark of the plant. Further investigation should be carried out to identify the selective compounds responsible for medicinal properties.

Acknowledgement

The authors are thankful to the authority of Southeast University for providing all the facilities for conducting the present work.

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JJBS Volume 10, Number 3,September 2017

ISSN 1995-6673 Pages 219 - 220

Jordan Journal of Biological Sciences Short Communication

Eragrostis nairii Kalidass C. (Poaceae): New Record for Southern Western Ghats, India

Moorthy Pavithra , Manogaran Parthipan, Iwar Kanivalan and Arumugam

Rajendran*

Phytodiversity Research Laboratory, Department of Botany, School of Life Sciences,Bharathiar University,

Coimbatore- 641 046. Tamil Nadu, India.

Received April 2, 2017 Revised June 12, 2017 Accepted June 20, 2017

Abstract

The present paper confirms the occurrence of Eragrostis nairii Kalidass C., a newly described taxon from Similipal Biosphere Reserve in Odisha in the Southern Western Ghats of Tamil Nadu. In the present study, a detailed description and ecology of this newly recorded species is provided.

Keywords: Eragrostis nairii, Poaceae, Nilgiri, Southern Western Ghats, Tamil Nadu, New record.

* Corresponding author. e-mail: arajendran222@yahoo.com.

1. Introduction

Eragrostis Wolf, the largest Chloridoid grass genus, is morphologically diverse and taxonomically complicated (Veldkamp 2002; Ingram 2010) with an estimated total of 423 species, occurring in tropical, subtropical and warm temperature regions of the world (Ingram & Doyle, 2007). In India, the genus Eragrostis is represented by 36 species (Karthikeyan et al., 1989). Various workers have added eight additional species for India either as new species or as new records (Ravichandran et al., 1996; Matthew, 1999; Vivek et al., 2012; 2013a, & 2013b). Eragrostis nairii Kalidass C. (2015) was described as a new species much later from Sal forests in Similipal Biosphere Reserve, Mayurbhanj.

During the floristic studies of Nilgiri hills of Southern Western Ghats in Tamil Nadu, a few species of monocots were collected. On critical examination of literature concerning the genus Eragrostis it is identified as Eragrostis nairii Kalidass Chitherto unknown/unreported species in the Southern Western Ghats forms a new distributional record.

Notes

The newly described Eragrostis nairii is an annual herb; it is very allied to E. pilosa (L.) P. Beauv. but differs from by having culms 15-25 cm in high, panicles lanceolate or elliptic, to 15 cm long, spikelets linear-oblong or elliptic, florets ca. 8-florets, stamens 2 and caryopsis ovoid, to 0.3 mm long (Kalidass, 2015).

Systematic Treatment

Eragrostis nairii Kalidass C. in J. Econ. Taxon. Bot. Vol. 39(1): 126-129. 2015.

Herb, densely tufted, Culms densed, branched at base, up to 25 cm high; 1.0-1.5 mm in wide; nodes glabrous. Leaves sheaths 1.0-4.5 cm, ligulate; ligule a membranous rim, vilous; leaf blade linear, 3-8 x 0.1 – 0.15 cm, base truncate & apex filiform. Panicles lanceolate or elliptic, 10-15 cm long, purple to violet, shade from base, breaking up from the base, c. 8-florest, rachilla branched, persistent, opposite florets slightly overlapping. Lower glume, ovate to lanceolate, to 0.3 mm, acute at apex, cuneate at base, glabrous, chartaceous, minutely ciliated, 1-nerved. Upper glume, ovate-lanceolate, up to 1.0 mm, tips acuminate, obtuse at base, charatceous, 1-nerved, Lemma broadly ovate, to 1.1 mm, prominent, membranous, tips acuminate, 3-nerved. Palea, obovate, to 0.7 mm long, 2 keeled, minutely ciliate along the keels. Lodicules 2, oblong, c. 0.05 mm. stamens 2, anthers elliptic, purple, c. 0.1 mm long, versatile, glabrous, filaments c. 0.03 mm long. Ovary ovate to obovate; c.0.04 mm; style c. 0.04 mm long, divided into two filaments with ovary, stigmas plumose, c.0.2 mm long. Caryopsis globose, 0.005 mm.

Flowering and Fruiting: July - December

Distribution and Ecology: It is described based on the collections from the

riparian banks of semi-evergreen forest of Similipal Biosphere Reserve, Patbil and Thakurmundha forest, Mayurbhanj district, Odisha. The species E. nairii is

© 2017 Jordan Journal of Biological Sciences. All rights reserved - Volume 10, Number 3 220

growing along the forest, infrequently found in some cool areas. Sometimes this species living associates with some grasses. Hence, it is first time report from Nilgiri hills in Southern Western Ghats of Tamil Nadu.

Acknowledgements

We are grateful to Professor and Head, Department of Botany, Bharathiar University, Coimbatore, Tamil Nadu, India, for providing necessary facilities to carry out the present study.

References

Ingram AL. 2010. Evolution of leaf blade anatomy in Eragrostis (Poaceae) – Syst. Bot. 35: 755-765.

Ingram AL and Doyle JJ. 2007.Eragrostis (Poaceae); monophyly and infrageneric classification. Aliso, 23: 595-604.

Kalidass C. 2015.Eragrostis nairii (Poaceae: Eragrostideae) – A new species from Odisha, India. J. Econ. Tax. Bot. 39(1): 2050-9768.

Karthikeyan S, Jain SK, Nayar MP and Sanjappa M. 1989. Florae Indicae Enumeratio; Monocotyledonea. Botanical Survey of India, Calcutta.

Matthew KM. 1999. Flora of the Palni Hills, South India. Rapinat Herbarium Tiruchirapalli, India.

Ravichandran P, Krishnan S, Samson NP, Subbiah VR, Azakanandam K and Narasimhan D. 1996. Eragrostis dayanandani: A New Grass from Tamil Nadu, India. Kew Bull. J. 51(1): 155-157.

Veldkamp JF. 2002. Revision of Eragrostis (Gramineae, Chloridoideae) in Malesia. Blumea, 47: 157- 204.

Vivek CP, Murthy GVS and Nair VJ. 2012. A new species Eragrostis henryi (Poaceae: Eragrostideae) from Tamil Nadu, India. Nelumbo, 54: 9-12.

Vivek CP, Murthy GVS and Nair VJ. 2013a. Eragrostis collinensis (Poaceae: Eragrostideae) a new species from the Hills of Kerala and Tamil Nadu, India. Ind. J. Forest, 36(3): 401-404.

Vivek CP, Murthy GVS and Nair VJ. 2013b. Eragrostis nilgiriensis sp. nov. (Poaceae: Eragrostideae) from Nilgiri District, Tamil Nadu, India. Nord. J. Bot. 31: 700-703.

Figure 1.Eragrostis nairiiKalidass C.:AHabit, B.Glume, C.Palea, D.Anther,E. lodicules,F.Caryopsis

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