journal of research in biology volume 3 issue 3
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Journal of Research in Biology is an international scientific journal committed to the development and spread of research in Biological sciences. It accepts research articles with affiliation to biological science from all around the globe and publishes them in the journal. The submitted articles are peer-reviewed by experts in the field and editorial board members.TRANSCRIPT
Journal of Research in Biology
www.jresearchbiology.com
An International Scientific Research Journal for Biology Volume 3 Issue 3
ISSN: 2231 –6280 EISSN: 2231- 6299
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TNAU, Coimbatore.
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TNAU, Tirunelveli.
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Gorakhpur University, Gorakhpur
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S.S.(P.G.)College, Shahjahanpur, India.
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SS P.G.College, Shahjahanpur, India
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Al-Azhar university, Egypt
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Sri Ram Nallamani Yadava College of Arts & Sciences, Tenkasi, India.
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Sher-e-Bangla Agricultural University, Bangladesh
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Sheth M.N.Science College, Patan, India.
Kumudben Babulal Patel [Bird, Ecology]
Gujarat, India.
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College of Applied Medical Sciences, King Saud University.
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Agharkar Research Institute, Pune, INDIA.
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Aligarh Muslim university, Aligarh, india.
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Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India.
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University of Louisville, Kentucky.
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Laboratorio de ecología y conservación de fauna Silvestre,
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Department of Pathology, Army Medical College, Rawalpindi, Pakistan.
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Krishi Vigyan Kendra, Amritsar, Punjab, India.
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Universidade Federal de São João del-Rei, Brazil.
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BCKV (Agri University), West Bengal, INDIA.
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School of Chemical & Biotechnology, Sastra University, Tamilnadu, INDIA.
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Jawaharlal Technological University, Hyderabad.
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Bhagwan Mahavir Medical Research Centre, Hyderabad, India.
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University of Kordofan, Elobeid-SUDAN.
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University of Mosul, Mosul,Iraq.
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Central Institute of Medicinal and Aromatic Plants, Lucknow, India.
Dr. Sanjay M. Dave [Ornithology and Ecology]
Hem. North Gujarat University, Patan.
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C.S.J.M. University, India.
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Federal University of Rondônia, Brazil.
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Stefan cel Mare University of Suceava, Romania.
Dr. Meera Srivastava [Hematology , Entomology] Govt. Dungar College, Bikaner.
P. Gurusaravanan [Plant Biology ,Plant Biotechnology and Plant Science]
School of Life Sciences, Bharathidasan University, India.
Dr. Mrs Kavita Sharma [Botany]
Arts and commerce girl’s college Raipur (C.G.), India.
Suwattana Pruksasri [Enzyme technology, Biochemical Engineering]
Silpakorn University, Thailand.
Dr.Vishwas Balasaheb Sakhare [Reservoir Fisheries]
Yogeshwari Mahavidyalaya, Ambajogai, India.
Dr. Pankaj Sah [Environmental Science, Plant Ecology]
Higher College of Technology (HCT), Al-Khuwair.
Dr. Erkan Kalipci [Environmental Engineering]
Selcuk University, Turkey.
Dr Gajendra Pandurang Jagtap [Plant Pathology]
College of Agriculture, India.
Dr. Arun M. Chilke [Biochemistry, Enzymology, Histochemistry]
Shree Shivaji Arts, Commerce & Science College, India.
Dr. AC. Tangavelou [Biodiversity, Plant Taxonomy]
Bio-Science Research Foundation, India.
Nasroallah Moradi Kor [Animal Science]
Razi University of Agricultural Sciences and Natural Resources, Iran
T. Badal Singh [plant tissue culture]
Panjab University, India
Dr. Kalyan Chakraborti [Agriculture, Pomology, horticulture]
AICRP on Sub-Tropical Fruits, Bidhan Chandra Krishi Viswavidyalaya,
Kalyani, Nadia, West Bengal, India.
Dr. Monanjali Bandyopadhyay [Farmlore, Traditional and indigenous
practices, Ethno botany]
V. C., Vidyasagar University, Midnapore.
M.Sugumaran [Phytochemistry]
Adhiparasakthi College of Pharmacy, Melmaruvathur, Kancheepuram District.
Prashanth N S [Public health, Medicine]
Institute of Public Health, Bangalore.
Tariq Aftab
Department of Botany, Aligarh Muslim University, Aligarh, India.
Manzoor Ahmad Shah
Department of Botany, University of Kashmir, Srinagar, India.
Syampungani Stephen
School of Natural Resources, Copperbelt University, Kitwe, Zambia.
Iheanyi Omezuruike OKONKO
Department of Biochemistry & Microbiology, Lead City University,
Ibadan, Nigeria.
Sharangouda Patil
Toxicology Laboratory, Bioenergetics & Environmental Sciences Division,
National Institue of Animal Nutrition
and Physiology (NIANP, ICAR), Adugodi, Bangalore.
Jayapal
Nandyal, Kurnool, Andrapradesh, India.
T.S. Pathan [Aquatic toxicology and Fish biology]
Department of Zoology, Kalikadevi Senior College, Shirur, India.
Aparna Sarkar [Physiology and biochemistry] Amity Institute of Physiotherapy, Amity campus, Noida, INDIA.
Dr. Amit Bandyopadhyay [Sports & Exercise Physiology]
Department of Physiology, University of Calcutta, Kolkata, INDIA .
Maruthi [Plant Biotechnology]
Dept of Biotechnology, SDM College (Autonomous),
Ujire Dakshina Kannada, India.
Veeranna [Biotechnology]
Dept of Biotechnology, SDM College (Autonomous),
Ujire Dakshina Kannada, India.
RAVI [Biotechnology & Bioinformatics]
Department of Botany, Government Arts College, Coimbatore, India.
Sadanand Mallappa Yamakanamardi [Zoology]
Department of Zoology, University of Mysore, Mysore, India.
Anoop Das [Ornithologist]
Research Department of Zoology, MES Mampad College, Kerala, India.
Dr. Satish Ambadas Bhalerao [Environmental Botany]
Wilson College, Mumbai
Rafael Gomez Kosky [Plant Biotechnology]
Instituto de Biotecnología de las Plantas, Universidad Central de Las Villas
Eudriano Costa [Aquatic Bioecology]
IOUSP - Instituto Oceanográfico da Universidade de São Paulo, Brasil
M. Bubesh Guptha [Wildlife Biologist] Wildlife Management Circle (WLMC), India
Rajib Roychowdhury [Plant science]
Centre for biotechnology visva-bharati, India.
Dr. S.M.Gopinath [Environmental Biotechnology]
Acharya Institute of Technology, Bangalore.
Dr. U.S. Mahadeva Rao [Bio Chemistry]
Universiti Sultan Zainal Abidin, Malaysia.
Hérida Regina Nunes Salgado [Pharmacist]
Unesp - Universidade Estadual Paulista, Brazil
Mandava Venkata Basaveswara Rao [Chemistry]
Krishna University, India.
Dr. Mostafa Mohamed Rady [Agricultural Sciences]
Fayoum University, Egypt.
Dr. Hazim Jabbar Shah Ali [Poultry Science]
College of Agriculture, University of Baghdad , Iraq.
Danial Kahrizi [Plant Biotechnology, Plant Breeding,Genetics]
Agronomy and Plant Breeding Dept., Razi University, Iran
Dr. Houhun LI [Systematics of Microlepidoptera, Zoogeography, Coevolution,
Forest protection]
College of Life Sciences, Nankai University, China.
María de la Concepción García Aguilar [Biology] Center for Scientific Research and Higher Education of Ensenada, B. C., Mexico
Fernando Reboredo [Archaeobotany, Forestry, Ecophysiology]
New University of Lisbon, Caparica, Portugal
Dr. Pritam Chattopadhyay [Agricultural Biotech, Food Biotech, Plant Biotech]
Visva-Bharati (a Central University), India
Table of Contents (Volume 3 - Issue 3)
Serial No Accession No Title of the article Page No
1 RA0328 An ornithological survey in the vicinity of Agartala city of Tripura state, north-eastern India.
Partha Pratim Bhattacharjee, Rahul Lodh, Dipten Laskar, Joydeb Majumder and Basant Kumar Agarwala.
852-860
2 RA0327 Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms.
Ujowundu CO, Nwaogu LA, Igwe KO, Ujowundu FN, Belonwu DC
861-869
3 RA0167
Effect of age, sex and hemoglobin type on adaptive and blood biochemical characteristics in Red Sokoto Goats.
Akpa GN, Alphonsus C and Usman N.
870-875
4 RA0245 Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with special reference to its life table attributes in Tripura, India.
Samit Roy Choudhury and Basant Kumar Agarwala.
876-885
5 RA0329 Anti-inflammatory activity of lycopene isolated from Chlorella marina on carrageenan-induced rat paw edema.
Renju GL and Muraleedhara Kurup G.
886-894
6 RA0332 Identification of animal Pasteurellosis by PCR assay.
Venkatesan PS, Deecaraman M and Vijayalakshmi.
895-899
7 RA0345 Source of light emission in a luminous mycelium of the fungus Panellus stipticus.
Puzyr Alexey, Burov Andrey and Bondar Vladimir.
900-905
8 RA0274 Local people’s attitude towards conservation and development around Pichavaram mangrove ecosystem, Tamil Nadu, India.
Lakshmi Kodoth and Ramamoorthy D.
906-910
9 RA0318 Biodegradation of phenol at low and high doses by bacterial strains indigenous to Okrika River in the Niger Delta of Nigeria.
Nwanyanwu CE and Abu GO.
911-921
10 RA0317 Phenol and Heavy Metal Tolerance Among Petroleum Refinery Effluent Bacteria.
Nwanyanwu CE, Nweke CO, Orji JC and Opurum CC.
922-931
11 RA0337 Effect of Chromolaena odorata leaf extract on haematological profiles in Salmonellae typhi infested Wistar rats.
Nwankpa P, Ezekwe AS, Ibegbulem CO and Egwurugwu JN.
932-939
Jou
rn
al of R
esearch
in
Biology
An ornithological survey in the vicinity of Agartala city of Tripura state,
north-eastern India
Keywords: Avifauna, biodiversity hotspot, Agartala, Tripura, north-east India .
ABSTRACT:
North-east India is a part of Indo-Burma hotspot and among the richest bird zones in India. Tripura lies in the border of Indo-Burma global biodiversity hotspot area but is poorly covered by ornithological works. Avifauna of Tripura state is known by 277 species but there is lack of information about their distribution, particularly in and around Agartala city, which is the capital of Tripura state and is a tourist destination along the border of Bangladesh for its natural landscapes, inland water species, and strong presence of green flora. With a view to enhance its value for tourist attraction and naturalists, a study was conducted to record the species of birds that occur in and around the City. In the present study 73 bird species were recorded from Agartala city and its adjacent areas belonging to 41 families and 14 orders.
852-860 | JRB | 2013 | Vol 3 | No 3
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.
www.jresearchbiology.com
Journal of Research in Biology
An International Scientific
Research Journal
Authors: Partha Pratim Bhattacharjee,
Rahul Lodh, Dipten Laskar,
Joydeb Majumder and
Basant Kumar Agarwala.
Institution:
Ecology & Biodiversity
Laboratories, Department of
Zoology, Tripura University,
Suryamaninagar-799 022,
Tripura, India.
Corresponding author: Basant Kumar Agarwala.
Email: [email protected]
Web Address: http://jresearchbiology.com/documents/RA0328.pdf.
Dates: Received: 28 Jan 2013 Accepted: 15 Feb 2013 Published: 10 Apr 2013
Article Citation: Partha Pratim Bhattacharjee, Rahul Lodh, Dipten Laskar, Joydeb Majumder and Basant Kumar Agarwala. An ornithological survey in the vicinity of Agartala city of Tripura state, north-eastern India. Journal of Research in Biology (2013) 3(3): 852-860
Journal of Research in Biology An International Scientific Research Journal
Original Research
INTRODUCTION
Avifauna contributes most significantly to the
diversity of terrestrial vertebrates, which have a special
role in conservation of biodiversity of a particular area
(Daniels, 1994). Birds are very good indicator
of environmental changes as they respond in the minute
change in habitat structure and composition
(Robert et al., 2001). Indian subcontinent harbour nearly
1300 species of birds, which is more than 13% of total
bird species of the world (Grimmet et al., 2004), and
more than 60% of Indian birds are found in north-east
India (Choudhury, 2010). North-east India is one of the
most significant biodiversity hotspots of the world and
among the richest bird zones in India because of
convergence of the Indo-Malayan, Indo-Chinese and
Indian biogeographical realms. As a result, it is unique in
providing an abundance of habitats that harbour diverse
biota with a high degree of endemism (Chatterjee et al.,
2006; Narwade et al., 2011). Tripura (22°56´- 24°32´ N
and 91°10´- 92°21´ E, with an area of 10,490 km2) is a
small state of north-east India bounded by Bangladesh
on three sides and with Assam and Mizoram on the other
side. It lies in the border of Indo-Burma global
biodiversity hotspot area (Myers et al., 2000) but
very poorly covered by ornithological works
(Choudhury, 2010). Although avifaunal checklist for
Tripura state listed 277 species (Choudhury, 2010) but
little is known regarding the bird species found in the
vicinity of Agartala city, situated by international
boundary of Bangladesh.
STUDY SITES
Agartala city is situated in the western region of
Tripura state with the latitude of 23°45' North and
longitude of 91°45' East and an average elevation of
20.36 m above sea level. It is the capital town of Tripura
with a mix of urban and semi urban complex and a rich
green cover. Forests and farms adjoin the town on three
sides, and therefore, it is also called ‘Green City’. The
total city area is 62.02 km2 and is delimited on the west
side by international boundary with Bangladesh.
Climatic condition is of tropical monsoon type with an
average annual rainfall of 220 cm. Average minimum
and maximum temperature recorded in the region are
6.8°C in January and 37.70°C in June, respectively.
Present study was carried out in eleven
different sites (viz., College Tilla lake area, Golbazar,
Pratapgarh, Dashamighat, Arundhutinagar, Shanmura,
Bhubanban, Barjala, Jagannath Bari lake area, G B Bazar
and Nandannagar) (Table 1, Figure 1) covering different
sides of Agartala city and its adjacent areas.
METHODOLOGY
The study sites were visited fortnightly
throughout the study period from 2009-2011. Data on
Bhattacharjee et al., 2013
853 Journal of Research in Biology (2013) 3(3): 852-860
Sl. No Sites Coordinates Altitude (m)
1. College Tilla lake area 23°49'35.45" N; 91°17'42.28" E 17
2. Golbazar 23°49'38.30" N ; 91°16'57.15" E 16
3. Pratapgarh 23°49'08.98" N ; 91°17'17.10" E 16
4. Dashamighat 23°49'46.34" N ; 91°15'51.45" E 16
5. Arundhutinagar 23°49'01.44" N ; 91°16'21.68" E 31
6. Shanmura 23°50'51.98" N ; 91°16'07.20" E 15
7. Bhubanban 23°51'56.50" N; 91°15'73.70" E 21
8. Barjala 23°52'05.05" N ; 91°16'32.13" E 23
9. Jagannath Bari lake area 23°50'05.43" N; 91°16'53.70" E 14
10. G B Bazar 23°51'33.74" N ; 91°17'33.97" E 27
11. Nandannagar 23°51'43.68" N ; 91°17'57.00" E 28
Table 1: Geo-coordinate details of the study sites
present bird species were collected by direct observations
with the help of binoculars (VISTA LE 8 X 40). Almost
all the species mentioned in the checklist were
photographed. For this purpose, digital cameras of Canon
Power shot SX 200 IS (12 X Digital zoom), Cannon SRL
EOS 50D and Panasonic Lumix DMC FZ 40 were used.
In lake areas birds were observed from the bank,
peripheral areas, and urban areas were surveyed on foot
regularly. Farm and forested areas in the vicinity of the
city were surveyed to record the assemblages of different
bird species. Most of visits were made in morning and
afternoon time when birds are most active. Identification
of birds was based on the field guides produced by Ali
and Ripley (1995), Ali (1996 and 2002) and Grimmett et
al., (2003).
Status of the birds was classified as C-Common,
MC-Most common, NC-Not common, S-Singleton,
W- Winter visitor.
RESULTS AND DISCUSSION
In the present study 73 bird species were
recorded from Agartala city and its adjacent areas
belonging to 41 families and 14 orders (Table 2, Plate 1
and 2). There is no authentic information about the
avifauna of Tripura except that by Blyth (1845, 1846),
Ali and Ripley (1968-74) and International Waterfowl
and Wetlands Research Bureau on Asian Waterfowl
Census, 1989 (Scott and Rose 1989). Majumdar et al.,
(2002) recorded 259 species of birds, belonging to
56 families and 16 orders. Recently Choudhury (2010)
recorded 277 species of birds, in the annotated checklist
from Tripura, but avifaunal diversity of Agartala city is
not yet available. Out of 14 orders. Passeriformes was
found dominant with 22 families followed by
Coraciiformes with 3 families and Pelecaniformes and
Piciformes with 2 families each. Dominance of
Passeriformes was also recorded by Choudhury (2010)
and Majumdar et al., (2002) from the state and from
Nagpur district of central India (Chinchkhede and Kedar,
2012). The resident birds such as Pond heron, Cattle
egret, Lapwing, Blue rock pigeon, Spotted dove,
Parakeets, Asian koel, Kingfisher, Bee eater, Lineated
barbet, Woodpecker, Bush lark, Bulbul, Shrike, Robin,
Tailorbird, Cinereous tit, Sunbird, Sparrow, Starling,
Myna, Oriole, Black drongo and Crow etc were found
regularly throughout the study period. Little Cormorant,
Asian Openbill-Stork, Black headed Ibis, Lesser
Whistling Duck, Crested Serpent Eagle, Red Junglefowl,
Red Collared Dove, Yellow-footed Green Pigeon, Brown
Fish Owl, Asian Palm Swift, Indian Roller, Coppersmith
Barbet, Orange headed Thrush, Blue Rock Thrush,
White-rumped Shama, Scarlet-backed Flower pecker,
Tricoloured Munia etc were found less common in this
study. Common Sandpiper and Black headed Ibis were
observed during the winter season only in the paddy
fields of peripheral areas of the city. Common Hoopoe
Bhattacharjee et al., 2013
Journal of Research in Biology (2013) 3(3): 852-860 854
Figure 1. Showing the study sites in and around Agartala City.
Bhattacharjee et al., 2013
855 Journal of Research in Biology (2013) 3(3): 852-860
Sl.
No. Common name Scientific name
Status
IUCN Abundance
Cormorants [Phalacrocoracidae]
1. Little Cormorant Phalacrocorax niger Vieillot, 1817 LC NC
Herons & Egrets [Ardeidae]
2. Indian Pond Heron Ardeola grayii (Sykes, 1832) LC MC
3. Cattle Egret Bubulcus ibis (Linnaeus, 1758) LC MC
4. Median Egret Mesophoyx intermedia (Wagler, 1827) LC C
Storks [Ciconiidae]
5. Asian Openbill-Stork Anastomus oscitans Boddaert, 1783 LC NC
6. Black headed Ibis Threskiornis melanocephalus (Latham, 1790) NT W, NC
Ducks [Anatidae]
7. Lesser Whistling Duck Dendrocygna javanica (Horsfield, 1821) LC NC
Hawks & Eagles [Accipitridae]
8. Crested Serpent Eagle Spilornis cheela Latham, 1790 LC NC
9. Black Kite Milvus migrans (Boddaert, 1783) LC C
Pheasants [Phasianidae]
10. Red Junglefowl Gallus gallus (Linnaeus, 1758) LC NC
Rails & Coots [Rallidae]
11. White-breasted Waterhen Amaurornis phoenicurus Pennant, 1769 LC C
Lapwings [Charadriidae]
12. Red-wattled Lapwing Vanellus indicus (Boddaert, 1783) LC MC
Sandpipers [Scolopacidae]
13. Common Sandpiper Actitis hypoleucos (Linnaeus, 1758) LC W, C
Pigeons & Doves [Columbidae]
14. Blue Rock Pigeon Columba livia Gmelin, 1789 LC MC
15. Spotted Dove Streptopelia chinensis (Scopoli, 1768) LC MC
16. Red Collared Dove Streptopelia tranquebarica (Hermann, 1804) LC NC
17. Orange-breasted Green
Pigeon Treron bicinctus (Jerdon, 1840) LC C
18. Yellow-footed Green Pigeon Treron phoenicoptera (Latham, 1790) LC NC
Parakeets [Psittacidae]
19. Rose-ringed Parakeet Psittacula krameri (Scopoli, 1769) LC C
20. Red-breasted Parakeet Psittacula alexandri (Linnaeus, 1758) LC C
Cuckoos & Coucals [Cuculidae]
21. Asian Koel Eudynamys scolopaceus (Linnaeus, 1758) LC MC
22. Greater Coucal Centropus sinensis (Stephens, 1815) LC C
Owls [Strigidae]
23. Collared Scops Owl Otus lettia Hodgson, 1836 LC S
24. Spotted Owlet Athene brama (Temminck, 1821) LC C
25. Brown Fish Owl Bubo zeylonensis (Gmelin, 1788) LC NC
Table 2: List of birds in and around Agartala city during 2009-2011
Bhattacharjee et al., 2013
Journal of Research in Biology (2013) 3(3): 852-860 856
Swifts [Apodidae]
26. Asian Palm Swift Cypsiurus balasiensis Gray, 1829 LC NC
27. House Swift Apus affinis (J E Gray, 1830) LC C
Kingfishers [Alcedinidae]
28. Common Kingfisher Alcedo atthis (Linnaeus, 1758) LC MC
29. Stork-billed Kingfisher Halcyon capensis (Linnaeus, 1766) LC C
30. White-throated Kingfisher Halcyon smyrnensis (Linnaeus, 1758) LC MC
Bee-eaters [Meropidae]
31. Little Green Bee-eater Merops orientalis Latham, 1802 LC MC
Rollers [Coraciidae]
32. Indian Roller Coracias benghalensis (Linnaeus, 1758) LC NC
Hoopoe [Upupidae]
33. Common Hoopoe Upupa epops Linnaeus, 1758 LC S
Barbets [Capitonidae]
34. Lineated Barbet Megalaima lineata (Vieillot, 1816) LC MC
35. Coppersmith Barbet Megalaima haemacephala Muller, 1776 LC NC
Woodpeckers [Picidae]
36. Rufous Woodpecker Celeus brachyurus (Vieillot, 1818) LC C
37. Greater Flameback Chrysocolaptes lucidus (Scopoli, 1786) LC C
38. Fulvous-breasted Woodpecker Dendrocopos macei (Vieillot, 1818) LC C
Larks [Alaudidae]
39. Singing bush lark Mirafra cantillans Blyth, 1844 LC C
Pipits & Wagtails [Motacillidae]
40. Paddy field Pipit Anthus rufulus Vieillot, 1818 LC C
41. White Wagtail Motacilla alba Linnaeus, 1758 LC W,C
Bulbuls [Pycnonotidae]
42. Red-whiskered Bulbul Pycnonotus jocosus (Linnaeus, 1758) LC C
43. Red-vented Bulbul Pycnonotus cafer (Linnaeus, 1766) LC MC
Loras [Irenidae]
44. Common Lora Aegithina tiphia (Linnaeus, 1758) LC C
Shrikes [Laniidae]
45. Brown Shrike Lanius cristatus Linnaeus, 1758 LC W, MC
46. Grey-backed Shrike Lanius tephronotus (Vigors, 1831) LC W, C
Thrushes [Turdidae]
47. Orange headed Thrush Zoothera citrina (Latham, 1790) LC NC
Flycatchers [Muscicapidae]
48. Blue Rock Thrush Monticola solitarius (Linnaeus, 1758) LC NC
49. White-rumped Shama Copsychus malabaricus (Scopoli, 1786) LC NC
50. Oriental Magpie Robin Copsychus saularis (Linnaeus, 1758) LC MC
Bhattacharjee et al., 2013
857 Journal of Research in Biology (2013) 3(3): 852-860
Babblers [Timaliidae]
51. Rufous-necked Laughing-
thrush
Garrulax ruficollis (Jardine & Selby, 1838) LC C
Warblers [Sylviidae]
52. Common Tailorbird Orthotomus sutorius (Pennant, 1769) LC MC
Flycatchers [Stenostiridae]
53. Grey-headed Canary-
flycatcher
Culicicapa ceylonensis (Swainson, 1820) LC W, C
Tits [Paridae]
54. Cinereous Tit Parus cinereus Vieillot, 1818 LC MC
Flowerpeckers [Dicaeidae]
55. Scarlet-backed Flowerpecker Dicaeum cruentatum (Linnaeus, 1758) LC NC
Sunbirds [Nectariniidae]
56. Ruby-cheeked Sunbird Anthreptes singalensis (Gmelin, 1788) LC C
57. Purple-rumped Sunbird Nectarinia zeylonica (Linnaeus, 1766) LC C
58. Purple Sunbird Cinnyris asiaticus Latham, 1790 LC MC
White-eyes [Zosteropidae]
59. Oriental White-eye Zosterops palpebrosus (Temminck, 1824) LC C
Munias [Estrildidae]
60. Scaly-breasted Munia Lonchura punctulata (Linnaeus, 1758) LC C
61. Tricoloured Munia Lonchura malacca (Linnaeus, 1766) LC NC
Sparrows [Passerinae]
62. House Sparrow Passer domesticus (Linnaeus, 1758) LC MC
Weavers [Ploceidae]
63. Baya Weaver Ploceus philippinus (Linnaeus, 1766) LC C
Starlings & Mynas [Sturnidae]
64. Chestnut-tailed Starling Sturnus malabaricus (Gmelin, 1789) LC C
65. Asian Pied Starling Gracupica contra (Linnaeus, 1758) LC MC
66. Common Myna Acridotheres tristis (Linnaeus, 1766) LC MC
67. Jungle Myna Acridotheres fuscus (Wagler, 1827) LC MC
Orioles [Oriolidae]
68. Black-hooded Oriole Oriolus xanthornus (Linnaeus, 1758) LC MC
Drongos [Dicruridae]
69. Black Drongo Dicrurus macrocercus (Vieillot, 1817) LC MC
70. Greater racket-tailed Drongo Dicrurus paradiseus Linnaeus, 1766 LC NC
Crows & Treepie [Corvidae]
71. Rufous Treepie Dendrocitta vagabunda (Latham, 1790) LC C
72. House Crow Corvus splendens Vieillot, 1817 LC MC
73. Jungle Crow Corvus macrorhynchos Wagler, 1827 LC MC
Abbreviations:
Status: LC = least concern; NT = near threatened; C = common; MC = most common; NC = not common;
S = singleton; W = winter visitor.
Bhattacharjee et al., 2013
Journal of Research in Biology (2013) 3(3): 852-860 858
Plate 1. A-Lineated barbet, B-Cattle egret, C-Red-wattled Lapwing, D-Common Hoope, E-Little cormorant,
F-Stripe-breasted woodpecker, G-Rufous woodpecker, H-White throated kingfisher, I-Yellow footed green
pegion, J-Asian open bill stork, K-Chestnut-tailed starling, L-Collared scops owl.
Plate 2: M-Asian Koel, N-Crested serpent eagle, O-Common Tailorbird, P-Cinereous Tit, Q-Emerald dove,
R-Little green bee-eater, S-Grey-baked shrike, T-Indian pond heron, U-Red collared dove, V-White-rumped
shama, W-Singing bush lark, X-Black headed ibis.
and Collared Scops Owl were sighted only once in the
two years study. Brown Shrike, Grey-backed Shrike,
Grey-headed Canary-flycatcher were observed in the
winter season only (Table 2), which corroborates with
the findings of Choudhury (2010) and Majumdar et al.,
(2002).
CONCLUSION
The present avifaunal survey of Agartala city and
its adjacent areas revealed 73 bird species which is very
important as it is the first ornithological record of the city
and will give a baseline data for future study. Rich bird
diversity is influenced by the topographical location of
the city and adjacent areas of Bangladesh.
Expansion of the city by construction activities,
reducing forest and farm areas with population pressure,
filling of pond and lake areas, dumping of wastes and
garbage in the low lands, use of chemical pesticides in
agricultural fields and hunting of birds are the major
threats to the avifaunal diversity here which needs proper
conservation management practices.
ACKNOWLEDGEMENT
Authors are thankful to Mr. Dipankar Kishore
Sinha for his constant services, tireless field assistance
and in capturing photographs during the study.
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Jou
rn
al of R
esearch
in
Biology
Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of
Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms.
Keywords: Oil and gas, Pollution, Phytochemicals, Vitamins, Oha, Okra.
ABSTRACT: The phytochemical, proximate, mineral and vitamin contents of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms were investigated. Plant samples were harvested from Polluted Environment (PE) at Izombe in Oguta Local Government Area- an oil drilling and gas flaring environment. The results obtained were compared to identical vegetables harvested from Eziobodo in Owerri West Local Government Area, designated as Unpolluted Environment (UPE). Our result showed that A. esculentus and P. mildbraedii have excellent nutritional value, which can confer biochemical and physiological advantage to humans. The quantitative proximate composition showed that the carbohydrate and ash contents of samples harvested from PE differed significantly (P<0.05) from samples obtained from unpolluted environment. The protein, crude fibre, moisture and total fat contents of samples from PE differed non significantly (P<0.05) when compared with samples obtained from UPE. The phytochemical contents of A. esculentus and P. mildbraedii were significantly higher in samples from UPE than in samples from PE. The mineral and vitamin contents were also determined. The concentration of nutritionally important macro and micro elements indicates that the two vegetable samples studied are rich sources of minerals and, therefore, can be used to improve the diet of both humans and livestock. This study also showed that environmental pollutants emanating from the activities of oil and gas industries can impact negatively on some important chemical and nutritive compositions of edible vegetables.
861-869 | JRB | 2013 | Vol 3 | No 3
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.
www.jresearchbiology.com
Journal of Research in Biology
An International Scientific
Research Journal
Authors:
Ujowundu CO1,
Nwaogu LA1, Igwe KO1,
Ujowundu FN1,
Belonwu DC2.
Institution:
1. Department of
Biochemistry, Federal
University Technology
Owerri, Nigeria.
2. Departmentof
Biochemistry, University of
Portharcourt, Nigeria.
Corresponding author:
Ujowundu CO.
Email: [email protected]
Web Address: http://jresearchbiology.com/documents/RA0327.pdf.
Dates: Received: 16 Jan 2013 Accepted: 09 Feb 2013 Published: 11 Apr 2013
Article Citation: Ujowundu CO, Nwaogu LA, Igwe KO, Ujowundu FN, Belonwu DC. Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms. Journal of Research in Biology (2013) 3(3): 861-869
Journal of Research in Biology An International Scientific Research Journal
Original Research
INTRODUCTION
Nigeria, a major producer of crude oil, benefits
as well as suffers from the positive and negative effects
of crude oil drilling and gas flaring (Adeniye et al.,
1983). Gas flaring is the unscientific burning of excess
hydrocarbons gathered in an oil/gas production flow
station. Gas flaring is a major source of pollution in
Nigeria's Niger Delta because it is the preferred means of
disposing waste gas associated with oil exploitation in
that region by many multinational oil companies that
operate its fields. Gas flaring releases carbon monoxide,
oxides of sulphur and nitrogen, hydrocarbons, soot and
heavy metals (Coker, 2007; Ikoro, 2003). These
pollutants actually interfere with growth and survival of
living organisms in such environment especially in
plants. It pollutes seedlings and fruits of plants which in
turn have a devastating effect on humans who consume
them. Such effects include respiratory or cardiovascular
diseases. This study used Abelmoschus esculentus
Moench and leaves of Pterocarpus mildbraedii Harms,
two commonly consumed indigenous vegetables to
evaluate the biochemical effects of these environmental
pollutants.
Abelmoschus esculentus Moench (local name,
Okra) and leaves of Pterocarpus mildbraedii Harms
(local name, Oha) are vegetables commonly consumed
as a source of food and medication for their high content
of nutrients and phytochemicals and mainly used for
soup preparation. Consumption of vegetables provide
taste, palatability, increases appetite and provides fiber
for digestion and to prevent constipation. They play key
roles in neutralizing acids produced during digestion of
proteins and fatty foods and also provide valuable
roughages which helps in movement of food in the
intestine. Some of these vegetables possess the ability to
reduce or reverse so many disease conditions and
disorders such as those which require a reduced intake of
glucose (diabetes) (Mcdowell, 2001: Ogbonnia et al.,
2008). These vegetables can be deeply affected by
pollution.
The most glaring sight in gas production flow
station is the ten-meter-high flame that burns
continuously from vertical pipes at many facilities owned
by oil companies. These vertical pipes are fed with gas
given off during production. Gas flaring, for about four
decades has contributed to the high pollution level, and
the ecosystem of Izombe may have been impacted
negatively. A good example of such negative effect is
high soil acidity that creates chemical and biological
conditions which may be harmful to the soil and plants
(Nwaugo et al., 2006). One of these conditions is the
reduction in the capacity of plants to absorb cations
(Wild et al., 2005). The higher acidic nature of soil is
attributable to high concentrations of sulphur dioxide and
particulates from gases flared into the atmosphere which
is washed back to the soil as acid rain.
This study has two objectives, first to contribute
to the knowledge of nutritional and antinutritional
composition of A. esculentus Moench and P. mildbraedii
Harms. Secondly, to evaluate the effect of environmental
pollution resulting from crude oil exploration and
exploitation and other industrial processes within the
area of study. The arable nature and vast land mass of
Izombe, confers it the status of food basket of Imo State,
Nigeria.
MATERIALS AND METHODS
Collection and preparation of plant samples
Samples of A. esculentus Moench and
P. mildbraedii Harms were obtained at Izombe, in Oguta
Local Government Area and at Eziobodo, in Owerri
West Local Government Area both in Imo state, Nigeria
and identified by a plant taxonomist in the Federal
University of Technology, Owerri (FUTO). Izombe is a
rainforest ecosystem, which hosts multinational
industries specialized in crude oil exploration and
exploitation. Flaring of gases constitutes the major
method of waste gas disposal at these oil fields. Situate
Ujowundu et al., 2013
862 Journal of Research in Biology (2013) 3(3): 861-869
to these oil fields are communities of indigenous
inhabitants whose occupation are subsistence and semi-
commercial farming. Eziobodo is also within the
rainforest region of Nigeria, occupied by indigenous and
FUTO students population. It has no known industrial
activities, except few automobiles that convey
inhabitants in and out of the village. Samples were sorted
by removing extraneous materials, spoilt and unhealthy
ones. After washing, okro samples were carefully sliced.
The samples were oven dried, macerated, sieved and
properly stored.
Evaluation of proximate composition
The method described by James (1995) and
Onwuka (2005) were used to determine crude fiber. Fat
content was determined by the method of Min and Boft
(2003). Moisture content was determined by the method
of AOAC (1990). The sample’s total protein content was
determined by microkjeldhal method described by
James (1995). Protein concentration was obtained by
determining total nitrogen and multiplied by the
factor- 6.25. Carbohydrate contents was calculated using
the arithmetical difference method described by Pearson
(1976) and James (1995).
Evaluation of phytochemical content
Tannin content of samples were determined by
Folin-Denis colorimetric method (Kirk and Sawyer,
1998). Saponin, alkaloid and flavonoid were done by
method described by Harborne, (1973). The
spectrophotometric method as described by Griffiths and
Thomas (1981) was used for determining phytate
content. Determinations were done in triplicates and
results were expressed as averages of percent values on
dry weight basis.
Evaluation of vitamins content
Retinol, ascorbic acid and α-tocopherol contents
in the samples were determined using the method of
Association of Vitamin Chemist as described by Kirk
and Sawyer (1998).
Evaluation of mineral content
Some mineral contents were determined by
atomic absorption spectrophotometer (James, 1995). The
dry samples were burnt to ashes to remove all organic
materials leaving inorganic ash. The resulting ash was
dissolved in 10 ml of 2 M HCl solution and diluted to
100 ml with distilled water in a volumetric flask. The
mixture was filtered and the resulting extract was used
for the specific evaluation of copper, zinc and iron.
Sodium, potassium, calcium and magnesium were
determined with the aid of Jaway digital flame
photometer. Phosphorus was determined as phosphate by
the vanadomolybdate colorimetric method (Pearson,
1976)
Ujowundu et al., 2013
Journal of Research in Biology (2013) 3(3): 861-869 863
Environment of
sampling Carbo- hydrates
Crude
Protein Ash Crude Fibre Moisture Total Fat
Polluted 32.47±2.22a 14.28±0.30a 13.78±0.40a 22.13±1.40a 10.52±0.89a 6.82±0.90a
Unpolluted 37.94±1.78b 12.67±0.07a 7.99±0.16b 21.83±0.23a 12.71±1.71a 6.85±0.03a
Table 1: Proximate composition (%) of Abelmoschus esculentus Moench
Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.
Environment of
sampling Carbo- hydrates
Crude
Protein Ash Crude Fibre Moisture Total Fat
Polluted 29.38±1.24c 8.06±1.43c 19.40±0.57c 17.65±0.79c 22.37±1.18c 3.14±0.33c
Unpolluted 34.82±0.30d 9.67±0.07c 12.05±0.18d 16.58±0.21c 23.67±0.29c 3.21±0.06c
Table 2: Proximate Composition (%) of Pterocarpus mildbraedii Harms
Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.
Statistical analysis
Data obtained were expressed as
means± standard deviation. Statistical Package for the
Social Sciences (SPSS) was used for the Analysis of
Variance (ANOVA) for the test of significant difference
between means (P<0.05).
RESULTS
The proximate contents of A. esculentus and
P. mildbraedii are presented in Tables 1 and 2
respectively. Results obtained from unpolluted
environment (UPE) showed that A. esculentus have
higher content of carbohydrate, protein, fibre and total
fat compared to P. mildbraedii. However, higher ash
and moisture content were observed in P. mildbraedii
when compared to A. esculentus from UPE.
Carbohydrate content in samples obtained from polluted
environment (PE) were significantly (P<0.05) lower than
the UPE. But the ash contents were significantly higher
in samples from PE. The protein, crude fiber, moisture
and total fat contents of the samples showed no
significant difference.
Results of phytochemical analysis are presented
in tables 3 and 4. The concentrations of phytochemicals
were significantly higher (P<0.05) in samples from PE.
Also, the phytochemical contents of P. mildbraedii from
PE were higher than that of A. esculentus from PE. The
highest concentrations of phytochemicals were observed
in flavonoids (0.54±0.02%) and tannin (1.83±0.01%)
from A. esculentus and P. mildbraedii respectively from
PE. However, alkaloids and tannins contents were
highest in A. esculentus and P. mildbraedii respectively
from UPE.
Vitamin contents were presented in tables 5 and
6. Vitamin A concentration in A. esculentus and
P. mildbraedii were 627.59±0.47 mg/100g and
375.48±0.18 mg/100g respectively, indicating the
highest vitamin content in the samples. Also, vitamin C
and B5 contents in samples from UPE were significantly
higher (P<0.05) when compared to samples from PE.
Similarly P. mildbraedii have significantly higher value
of vitamin B5 (189.33±2.31 mg/100g) compared to
A. esculentus from UPE. In A. esculentus (table 5), all
the vitamins determined were significantly (P<0.05)
lower except vitamin B5, vitamin B9 and vitamin E in
samples from PE. Also, samples of P. mildbraedii from
PE when compared with samples from UPE showed
significantly lower content in all the vitamins (table 6)
except in vitamin B2, vitamin B3 and vitamin E.
The mineral contents are shown in tables 7 and 8.
The concentrations of copper, iron, zinc and lead in
A. esculentus from PE were significantly higher (P<0.05)
Ujowundu et al., 2013
864 Journal of Research in Biology (2013) 3(3): 861-869
Environment of
sampling Saponins Tannins Phytates Alkaloids Phenols Flavonoids
Polluted 0.33±0.03a 0.37±0.01a 0.23±0.01a 0.43±0.01a 0.26±0.04a 0.54±0.02a
Unpolluted 0.12±0.05b 0.20±0.01b 0.09±0.01b 0.27±0.09b 0.11±0.00b 0.24±0.02b
Table 3: Phytochemical Composition (%) of Abelmoschus esculentus Moench
Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.
Environment
of sampling Saponins Tannins Phytates Alkaloids Phenols Flavonoids
Polluted 0.41±0.02c 1.83±0.00c 0.37±0.01c 0.57±0.01c 0.44±0.02c 0.63±0.01c
Unpolluted 0.23±0.08d 1.56±0.14d 0.15±0.01d 0.28±0.12d 0.35±0.00d 0.45±0.08d
Table 4: Phytochemical Composition (%) of Pterocarpus mildbraedii Harms Vegetables
Values (mean ± SD of triplicate determinations) with different superscripts per column are significantly
(P<0.05) different.
Ujowundu et al., 2013
Journal of Research in Biology (2013) 3(3): 861-869 865
En
vir
on
men
t
of
sam
pli
ng
V
ita
min
A
Vit
am
in B
1
Vit
am
in B
2
Vit
am
in B
3
Vit
am
in B
5
Vit
am
in B
6
Vit
am
in B
9
Vit
am
in C
V
ita
min
E
Poll
ute
d
59
2.7
8±
19
.69
a 0
.02
±0.0
2a
0.0
5±
0.0
1a
0.8
3±
0.1
0a
21
.00
±2
.52
a 0
.58
±0.0
2a
0.6
7±
0.1
1a
68
.41
±2
.31
a 1
.52
±0.0
2a
Un
poll
ute
d
62
7.5
9±
0.4
7b
0.0
8±
0.0
1b
0.0
8±
0.0
1b
1.1
2±
0.0
1b
23
.33
±2
.31
a 0
.81
±0.1
0b
0.7
3±
0.0
8a
78
.03
±1
.02
b
1.8
8±
0.1
7a
Tab
le 5
: V
ita
min
Con
ten
t (m
g/1
00g
) of
Abelm
osc
hu
s esc
ule
ntu
s M
oen
ch
Va
lues
(mea
n +
SD
of
trip
lica
te d
ete
rm
ina
tion
s) w
ith
dif
fere
nt
sup
ersc
rip
ts p
er c
olu
mn
are s
ign
ific
an
tly (
P<
0.0
5)
dif
feren
t.
En
vir
on
men
t
of
sam
pli
ng
V
ita
min
A
Vit
am
in B
1
Vit
am
in B
2
Vit
am
in B
3
Vit
am
in B
5
Vit
am
in B
6
Vit
am
in B
9
Vit
am
in C
V
ita
min
E
Poll
ute
d
30
2.5
7±
7.0
1c
0.0
7±
0.0
2c
0.0
4±
0.0
1c
0.6
5±
0.0
9c
17
5.2
2±
6.9
7c
0.2
5±
0.0
8c
0.5
3±
0.1
7c
85
.29
±1
.79
c 1
.97
±0.1
8c
Un
poll
ute
d
37
5.4
8±
0.1
8d
0.1
2±
0.0
0d
0.0
6±
0.0
1c
0.5
7±
0.0
2c
18
9.3
3±
2.3
1d
0.5
9±
0.0
5d
0.8
9±
0.0
2d
99
.15
±2
.69
d
2.2
7±
0.1
7c
Tab
le 6
: V
ita
min
Con
ten
t (m
g/1
00g
) of
Pte
roca
rpu
s m
ild
bra
ed
ii H
arm
s V
egeta
ble
s
Va
lues
(mea
n +
SD
of
trip
lica
te d
ete
rm
ina
tion
s) w
ith
dif
fere
nt
sup
ersc
rip
ts p
er c
olu
mn
are s
ign
ific
an
tly (
P<
0.0
5)
dif
feren
t.
En
vir
on
men
t
of
sam
pli
ng
M
ag
nesi
um
C
alc
ium
P
ho
sph
oru
s S
od
ium
P
ota
ssiu
m
Cop
per
Iron
Z
inc
Lea
d
Poll
ute
d
22
.17
±1
.39
a 7
0.2
3±
2.0
2a
57
.35
±0
.59
a 6
.34
±0.4
6a
10
7.6
3±
4.4
5a
0.4
2±
0.1
5a
0.9
5±
0.2
6a
0.4
8±
0.1
4a
0.6
5±
0.0
7a
Un
poll
ute
d
39
.60
±1
.39
b
82
.83
±2
.32
b
68
.28
±0
.46
a 7
.47
±0.2
3a
13
0.4
0±
6.5
5b
0.0
6±
0.0
1b
0.7
4±
0.2
5b
0.3
2±
0.0
7b
0.3
5±
0.1
0b
Tab
le 7
: M
iner
al
Con
ten
t (m
g/1
00g
) of
Ab
elm
osc
hu
s esc
ule
ntu
s M
oen
ch
.
Va
lues
(mea
n +
SD
of
trip
lica
te d
ete
rm
ina
tion
s) w
ith
dif
fere
nt
sup
ersc
rip
ts p
er c
olu
mn
are s
ign
ific
an
tly (
P<
0.0
5)
dif
feren
t.
En
vir
on
men
t
of
sam
pli
ng
M
ag
nesi
um
C
alc
ium
P
ho
sph
oru
s S
od
ium
P
ota
ssiu
m
Cop
per
Iron
Z
inc
Lea
d
Poll
ute
d
48
.67
±4
.38
c 6
4.3
8±
3.9
5c
40
9.8
9±
0.4
3c
17
.16
±1
.71
c 2
50.7
3±
4.2
9c
0.3
4±
0.1
3c
0.7
7±
0.1
8c
0.3
4±
0.0
6c
0.5
0±
0.2
2c
Un
poll
ute
d
52
.80
±2
.40
c 7
7.4
8±
2.3
2d
41
3.8
9±
12
.48
c 2
1.1
3±
0.1
2d
28
4.2
7±
2.4
4d
0.0
4±
0.0
0d
0.5
8±
0.0
9d
0.1
6±
0.0
2d
0.1
9±
0.1
3d
Tab
le 8
: M
iner
al
Con
ten
t (m
g/1
00g
) of
Pte
roca
rpu
s m
ild
bra
ed
ii H
arm
s V
egeta
ble
s.
Va
lues
(mea
n +
SD
of
trip
lica
te d
ete
rm
ina
tion
s) w
ith
dif
fere
nt
sup
ersc
rip
ts p
er c
olu
mn
are s
ign
ific
an
tly (
P<
0.0
5)
dif
feren
t.
when compared to samples from UPE. Results presented
in table 8 showed that P. mildbraedii from UPE are
excellent source of phosphorus (413.89±12.48),
potassium (284.27±2.44), calcium (77.48±2.32) and
magnesium (52.80±2.40). Also, the concentration of
minerals in P. mildbraedii from PE and UPE were
significantly different in all except in magnesium and
phosphorus.
DISCUSSION
Gas flaring and other oil and gas activities for
about four decades have contributed to pollution in
Oguta, which have impacted on the ecosystem. Soots
were seen on vegetation within the communities around
the flaring site. Plants growing in such environment have
over the years taken in varying doses of pollutants which
invariably may affect the nutritional and chemical
contents.
Our result showed that A. esculentus had better
nutritional value than P. mildbraedii with respect to
protein and carbohydrate contents. Also, A. esculentus
and P. mildbraedii showed higher values in proximate
contents (except in protein) than A. hybridus as reported
by Nwaogu et al., (2006). Carbohydrates provide energy
to cells in the body, particularly to the brain, a
carbohydrate dependent organ in the body. (Nelson and
Cox, 2005). These vegetables can supplement the daily
energy intake of humans (Bingham, 1998; Effiong et al.,
2009). The crude fibre content, indicates that the
vegetables are good sources of fibre, thus making them
veritable source of roughage. The concentrations of
carbohydrate were significantly reduced while ash
contents were increased in plants from polluted
environment when compared to plants from unpolluted
environment (UPE). The reduced carbohydrate can be
attributed to the effect of air pollutants as reported by
Farzana (2005), in which he affirmed that it reduces
photosynthesis in chloroplasts. The contents of protein,
crude fiber and fat in samples from PE were lower than
those from UPE but were not significantly different,
which indicates that they were not adversely affected by
the pollution.
The phytochemical results indicate that
A. esculentus and P. mildbraedii are good sources of
these beneficial chemicals. They have antioxidative,
hypocholesterolemic, chemoprotective and antibacterial
properties (Price et al., 1987; Enechi and Odonwodo,
2003; Okwu, 2004). Both vegetables are rich in
alkaloids, flavonoids and tannins which indicates that
they have diuretic, antispasmodic, anti-inflammatory and
analgesic effects (Owoyele et al., 2002; Nobre-Junior,
2007 ; Alisi et al., 2011). Comparatively, P. mildbraedii
had higher content of the phytochemicals studied. Also,
significantly higher amount of phytochemicals were
observed in vegetables obtained from PE. The increase
can be linked to their role in oxidative stress in plants.
Phytochemicals are secondary metabolite of plants,
known to exhibit diverse pharmacological and
biochemical effects on living organisms. It has been
reported that certain phytochemicals play important role
in antioxidant defense systems of vegetative plants
(Ugochukwu and Babady, 2003). Pollution by gas flaring
is taught to generate free radicals in surrounding
environment. Thus, it is expected that plants may
increase synthesis of antioxidant defense compounds.
These vegetables showed significantly high
amount of vitamins especially vitamins A, B1, B2, B5, B6
and C in samples from UPE when compared
to samples from PE. These vitamins are involved in
intermediary metabolism of both plants and animals
acting as part or whole coenzyme to some specific
enzyme system and playing important role in both
enzyme and non enzyme oxidative stress defense
systems. The high concentrations of vitamins A and C
will contribute significantly to the daily requirements in
view of the reports of Murray (1998). Vitamin C
maintains blood vessel flexibility and improves
circulation in the arteries of smokers. The most important
Ujowundu et al., 2013
866 Journal of Research in Biology (2013) 3(3): 861-869
benefit of vitamins A and C is their involvement in free
radical scavenging processes (Trumbo et al., 2004;
Nwaogu et al., 2011). These chemically active radicals
are byproducts of many normal biochemical processes.
Their numbers are increased by environmental assaults
such as chemicals and toxins. The lower concentrations
of these vitamins in samples from PE suggest an inability
of the plants to synthesize these vitamins in sufficiently
large amount for their metabolic functions. Oxidative
stress caused by gas flaring in Oguta community can
interfere with the synthetic mechanisms of the plants in
the environment (Farzana, 2005).
Some of the mineral contents of
A. esculentus Moench and P. mildbraedii Harms are
comparable or higher than that reported for
Amaranthus hybridus (Nwaogu et al., 2006)
Mucuna utilis (Ujowundu et al., 2010),
Commelina nudiflora and Boerhavia diffusa (Ujowundu
et al., 2008). The values obtained for the minerals
indicates that the samples are good sources of mineral
and are of great nutritional importance. In animals,
potassium and sodium are important electrolytes.
Potassium is a major intracellular cation. Sodium is
involved in the regulation of acid-base equilibrium,
protection against dehydration and maintenance of
osmotic pressure in living system. It plays a role in the
normal irritability of muscles and cell permeability
(Schwart, 1975). Copper (Cu) is essential for
haemoglobin synthesis, normal bone formation and the
maintenance of myelin within the nervous system
(Passmore and Eastwood, 1986). In animals, the
manifestations of copper deficiency include; anaemia,
hypo-pigmentation, defective wool keritinization,
abnormal bone formation with spontaneous reproductive
and heart failure (Williams, 1982). In humans, it has
been established that occurrence of Cu absorption
disorder in after partial gastetomycin leads to severe
malnutrition just as when protein is severely deficient in
the diet; as in kwashiorkor (Davies, 1972). Calcium and
phosphorus are important and indispensable for the
synthesis of strong bones and teeth, kidney function and
cell growth (Uddoh, 1988; Brody, 1994). Phosphorus
and magnesium are also important in the regulation of
acid-alkaline balance in the body (Fallon, 2001).
The mineral contents, like Mg, Ca, P, S and K in
vegetables from PE have significantly (P<0.05) reduced
value compared to vegetables obtained from UPE. The
release of pollutants such as oxides of sulphur and
nitrogen, hydrocarbons and other volatile organic
carbons can create chemical and biological conditions
which may be harmful to plants and soil microorganisms.
One of such conditions is the reduction in the capacity of
plants to absorb cations (Wild et al., 2000). Crops grown
in soil with low mineral contents exhibit various forms of
mineral deficiency. In plants, potassium is an essential
nutrient and has an important role in the synthesis of
amino acids and proteins (Malik, 1982). Ca and Mg play
significant role in photosynthesis, carbohydrate and
nucleic acids metabolism (Russel, 1973). The reduced
content of these minerals will definitely affect these
important plant processes. Lead is yet to record any
physiological role in the biological system and are
known to be extremely toxic even at the slightest
concentration. Their presence in the samples calls for
serious concern
This study has shown that A. esculentus Moench
and P. mildbraedii Harms are good sources of nutrients
and their consumption should be encouraged. Improved
information on these plants will contribute to the
awareness of their nutritive value, especially in this time
of increased food insecurity. Also, gas flaring showed
negative effects on these plants, which could affect
animals that consume them. Similarly, the adverse health
consequences on the inhabitants around the gas flare
site are of great concern. Communities around such
environment should be enlightened on the inherent
dangers. Oil and gas industries should be compelled to
upgrade their waste disposal technologies, with emphasis
Ujowundu et al., 2013
Journal of Research in Biology (2013) 3(3): 861-869 867
in gas disposal. This will reduce the detrimental effects
on the health and well-being of inhabitants of Izombe in
Oguta Local Government Area of Imo State.
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Jou
rn
al of R
esearch
in
Biology
Effect of age, sex and hemoglobin type on adaptive and blood biochemical
characteristics in Red Sokoto Goats
Keywords: Adaptive coefficient, heart rate, rectal temperature, blood biochemical characteristics.
ABSTRACT: This study was conducted to evaluate the effect of haemoglobin (Hb) types, sex and age on adaptive and blood biochemical characteristics of Red Sokoto goats. Ninety four (94) goats were sampled from two locations: Dei-dei and Gwagwalada grazing reserved, Abuja. Data were collected on adaptive characteristics {heart rate(HR) and rectal temperature(RT) and adaptive coefficient (AC) was calculated from the HR and RT} and blood biochemical characteristics{ haemoglobin (Hb) types, Hb-concentration (Hb-conc), Potassium concentration (K-conc) and albumin concentration (alb-conc)}. The effects of haemoglobin type, sex and age on the adaptive and blood biochemical characteristics of the goats was analyzed by general linear model (GLM) procedure of SAS. The results showed that the mean RT of the sampled goats was 38.9°C with very minimal variations (CV=0.5). The mean HR of the goats was 76.1bpm, with min and max HR of 70 and 80bpm. The mean albumin, Hb and K concentration were 38.4g/l, 8.9g/dl and 4.0Mmol/l, respectively. The variation of Hb type with adaptive and blood biochemical characteristics was significant (P<0.05) except Hb concentration. Higher HR was observed in goats with Hb AA and AB. Age and sex had significant effect (P<0.05; P<0.01) on HR, AC and albumin concentration of the goats. Although there was no trend in the variation of HR and AC with age, but HR and AC were higher in the older goats than the younger, however the albumin concentration significantly decreased with progressive increase in age of the goats.
870-875 | JRB | 2013 | Vol 3 | No 3
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www.jresearchbiology.com
Authors:
Akpa GN, Alphonsus C
and Usman N.
Institution:
Animal Science
Department, Ahmadu Bello
University, Zaria, Nigeria.
Corresponding author:
Alphonsus C.
Email:
Web Address: http://jresearchbiology.com/documents/RA0167.pdf.
Dates: Received: 15 Dec 2011 Accepted: 01 Jan 2012 Published: 16 Apr 2013
Article Citation: Akpa GN, Alphonsus C and Usman N. Effect of age, sex and hemoglobin type on adaptive and blood biochemical characteristics in Red Sokoto Goats Journal of Research in Biology (2013) 3(3): 870-875
Journal of Research in Biology An International Scientific Research Journal
Original Research
Journal of Research in Biology
An International Scientific
Research Journal
INTRODUCTION
In recent years, advances in the field of
biotechnology have opened up a completely new area at
molecular levels with the introduction of techniques such
as routine electrophoresis employed for detection of
polymorphism at protein and enzyme loci as well as
other serological and immunogenetic procedures for the
measurement of variations (Salako et al., 2007). Data
obtain from this type of study could be useful as genetic
markers for important economic characteristics and
could aid significantly in selection of superior animals
for breeding purposes.
Haemoglobin typing is very important as
different Hb types may have selective advantage in
different geographical regions (Ndamukong, 1995).
Economic pressures of various kinds are forcing the
production of livestock into climatic environments that
are increasingly more remote from the considered ideal
for optimal production and feed utilization.
Thermal stress, which is one of the major factors
that affect the productivity of many farm animals can be
reflected in an easily observable changes in pulse rate,
respiration rate, and rectal temperature, although the
whole body reacts to thermal stress by an elaborate series
of chain reactions (Ahmed, 2004). The most obvious
index of thermal stress is body temperature response.
Deviation from normal rectal temperature indicates that
the animal is under stress, that its homeothermic
mechanisms are overtaxed Ahmed, 2004).
Adaptive characteristics of animals serve as a
key to managing any livestock operation. Adaptive traits
such as rectal temperature, heart rate, and flank
movement have been documented to have some
significant effect on genetic variations. Every normal
animal has a range of individual adaptive traits in
relation to a specific physiological pattern.
Because study of environmental physiology
involves so many variables and scientific discipline,
much is being published on this subject especially as
related to farm animals. Comprehensive reviews have
appeared under the authorship of Alderson, 1992,
Derman and Noakes, 1994, Tambuwal et al., (2002),
Otoikhian et al., 2009, chukwuka et al., 2010,
Opara et al., 2010, Gurcan et al., 2010 and a lot of
others. Research results reported in this paper is intended
to supplement data reviewed by authors listed.
Eventually, accumulated data will permit specific
recommendation on breeding, feeding and management
of farm animals.
This study therefore aimed at studying the effects
of Haemoglobin type, sex and age on adaptive and blood
biochemical characteristics of Red Sokoto goats.
MATERIALS AND METHODS
Location
The study was conducted at the Federal Capital
Territory Abuja, located within the Northern guinea
Savanna zone of Nigeria. It is laying between latitudes
8.25° and 9.0° N of the equator and Longitude 6.45° and
7.39° E of the Greenwich Meridian. (Presentation
Copyright@ falling Rain Genomics, 1996-2010).
Data collection
Ninety four (94) goats were sampled from two
locations: Dei-dei and Gwagwalada grazing reserve,
Abuja. Data were collected on the adaptive and blood
biochemical traits. The adaptive traits were Heart rate
(HR), Rectal temperature(RT) and Adaptive coefficient
(AC) while the blood biochemical characteristics were
haemoglobin (Hb) types, Hb-concentration (Hb-conc),
Potassium concentration (K-conc) and albumin
concentration (alb-conc).
METHODS OF MEASUREMENTS
Adaptive Traits
Heart Rate (HR)
Heart rate was taken by placing stethoscope on
femoral artery of the hind limb of the goat to count the
number of beat per minute.
871 Journal of Research in Biology (2013) 3(3): 870-875
Akpa et al.,2013
Rectal Temperature (RT)
This was taken using clinical thermometer which
was inserted into the rectum of the goat and left for
45-50 seconds. It was then removed and the temperature
level was read. The values read were recorded, and the
process was repeated for the other goats.
Blood Biochemical Characteristics
Blood samples were taken from each of the
experimental goats through the jugular vein. 5 mls of the
blood was taken from each goat, from which 2 mls was
put into heparinized vacutainer tubes containing
anticoagulant ethylene diamine tetra acetic acid (EDTA).
The remaining 3 mls of the blood was put into sterile
vacutainer tubes (without anticoagulant). The samples
were labeled accordingly. The blood samples in the
sterile vacutainer tube were centrifuged in order to have
a clear layer of serum. This serum was pipetted into
another sterile bottle and store in a refrigerator. The
blood samples were taken to the Haematological
laboratory of Ahmadu Bello Teaching Hospital from
where the analysis of blood biochemical characteristics
was carried out.
A spectrometer with wavelength capability of
600-650 nm (Zenway 5041 colorimeter) was used
to analyzed for the albumin concentration, while the
K concentration was analysed using Corning
flame photometer 410. Electrophoresis and
Cyanmethaemoglobin method was used to analyzed for
Hb-types and Hb-conc, respectively.
Data Analysis
The adaptability of the goats were measured by
determining the adaptive coefficient from the values of
the Rectal Temperature (RT) and Heart Rate (HR) as
thus
Adaptive coefficient (AC) = (RT/38.33) + (HR/23.00)
The effects of haemoglobin types, sex and age on
the adaptive and blood biochemical characteristics of the
goats were determined by general linear model (GLM)
procedure of SAS, (2005).
RESULTS AND DISCUSSION
Rectal Temperature (RT) is directly affected by
the surrounding and ambient temperature, and high
ambient temperature has a negative effect on
productivity of the animal. Chukwuka et al., (2010)
reported that negative effect of high ambient temperature
is direct in the form of stress suffered by the animal and
the diversion of energy from the purpose of production to
regulation of body temperature and indirectly by
affecting the availability of feed resources upon which
production is dependent. In this study, the mean RT of
the goats was 38.9°C with minimum and maximum body
temperature of 38.1 and 39.4 °C (Table 1). These values
were within the reference range of previous study
of goats in thermal neutral condition (Otoikhian et al.,
2009) and this indicate that the goats used for this
research showed no clinical signs of stress during the
research period. The body temperature of the goats
exhibited minimal variations (CV=0.5%), thus implying
that goats are homoeothermic animals, they can maintain
near constant body temperature under wide range of
environmental conditions.
The Heart Rate (HR) is the pulse that helps to
know the beating rate of the heart which is measured
Journal of Research in Biology (2013) 3(3): 870-875 872
Akpa et al.,2013
Characteristics N Mean±SE CV(%) Min Max
Rectal Temperature (°C) 94 38.9±0.02 0.5 38.1 39.4
Heart Rate (bpm) 94 76.1±0.39 3.8 70.0 81.0
Adaptive coefficient 94 4.3±0.02 5.0 4.1 4.6
Albumin concentration (g/l) 94 38.4±0.34 8.5 31.0 48.0
Hemoglobin concentration (g/dl) 94 8.9±0.16 17.1 4.0 12.7
Potassium concentration (Mmol/l) 94 4.0±0.06 14.7 3.0 5.8
Table 1: Summary Statistics of the measured characteristics in Red Sokoto Goats
in beats per minute (bpm) using stethoscope
(Otoikhian et al., 2009). The mean HR of the goats used
in this study was 76.1bpm, with the min and max HR of
70 and 80bpm. This is slightly higher than the range of
70-75bpm reported by Derman and Noaks (1994) in
goats. The minor difference observed in the values of the
HR may be explained by differences in geographical
conditions, season or climate and physiological
conditions of the sample goats.
Rectal temperature and heart rate have been
shown to be good indicators of the thermal stress and
may be used to assess thermal adversity of the
environment (Al- Haidary, 2004).
The Adaptive Coefficient (AC) (which is the
function of RT and HR) signifies the level of adaptability
of the goats to the environments varied significantly
(P<0.05) with Hb types, sex and age of the goats. The
goats with Hb AA and AB had higher AC than those
with BB and AC; likewise the bucks had higher AC than
the does.
Potassium is one of the intracellular elements
that regulate the intracellular density of the cell. The
amount of K-concentration is fairly high at intracellular
membranes (Gurcan et al., 2010). The values of
K-concentration reported by Opara et al., (2010) for
WAD bucks and does were 17.8 and 6.9mmol/l,
respectively. These values were higher than the mean
value of 4.0mmol/l observed in this study; this is
probably due to differences in breed and physiological
conditions of the sampled animals. Researchers had
identified the existence of different type of K in different
species of animals, and that in sheep for instance, there
are two types of K which is high and low K with the
low K type dominant over the high K type
(Soysal et al., 2003). Also Gurcan et al., (2010) reported
a range of 4.23 to 11.69mmol/l for low K type in
animals.
The concentration of albumin in this study
(38.4g/dl) was slightly higher than the 34.5g/dl reported
by Opara et al., (2010).
The variation of Hb type with adaptive and blood
biochemical characteristics was significant (P<0.05)
except Hb-concentration (Table 2). The relationship
between Hb types and HR can be linked to the different
Akpa et al.,2013
Characteristics Hemoglobin Type
AA BB AB AC SEM LOS
Rectal Temperature (°C) 39.0a 39.0a 39.0a 38.9b 0.02 *
Heart Rate (bpm) 76.6a 75.3b 76.2a 75.1b 0.40 *
Adaptive coefficient 4.4a 4.3b 4.4a 4.3b 0.02 *
Albumin concentration (g/l) 37.6b 39.2a 38.6a 38.8a 0.34 *
Hemoglobin concentration (g/dl) 9.1 8.6 8.8 8.6 0.16 ns
Potassium concentration (Mmol/l) 3.8b 3.9a 4.0ab 4.4a 0.06 *
Number of observations 30 11 41 12 94 ab: means within the same row with different superscripts differ significantly(P<0.05); ns:not significant;
Table 2: Effect of hemoglobin type on adaptive and blood biochemical characteristics
Characteristics Sex
Buck Doe SEM LOS
Rectal Temperature (°C) 39.0 39.0 0.03 ns
Heart Rate (bpm) 78.5a 75.0b 0.49 **
Adaptive coefficient 4.4a 4.3b 0.02 **
Albumin concentration (g/l) 39.8a 37.7b 0.49 **
Hemoglobin concentration (g/dl) 9.1 8.7 0.26 ns
Potassium concentration (Mmol/l) 3.9 4.0 0.08 ns
Number of observations 30 64 94 ab: means within the same row with different superscripts differ significantly(P<0.01); ns:not significant;
Table 3: Effect of Sex on adaptive and blood biochemical characteristics
873 Journal of Research in Biology (2013) 3(3): 870-875
levels of oxygen carrying capacity of the different
Hb types. In this study, higher HR was observed in goats
with Hb AA and AB, and Hb A is known to be the
haemoglobin allele with highest affinity for oxygen. This
is in line with the earlier report of Huisman et al., (1959)
who relates the preponderance of Hb A to it greater
affinity to oxygen. This could also explain the high
adaptive coefficient observed on goats with Hb types AA
and AB since adaptive coefficient is a function of HR
and RT.
The variation of HR, AC and Albumin
concentration with sex was highly significant (P<0.01;
Table 3). The RT of the buck and does were similar
however, the HR was higher in bucks (78.5bpm) than the
does (75.0bpm) this is probably due to the high sexual
activity of the bucks. There was no significant (P>0.05)
difference between the bucks and does in Hb and
K concentration. This is contrary to the study of
opera et al., (2010) who reported significant differences
between WAD bucks and does in there Hb and
K concentration. This is probably due to differences in
breeds and location of the animal, Hb type had been
reported to vary with breed and location (Ndamukong,
1995, Abdussamad et al., 2004 Essien et al., 2011)
Age significantly (P<0.05) influence HR, AC
and albumin concentration but had no significant
influence on the RT, Hb and K concentration (Table 4).
Although there was no trend in the variation of HR and
AC with age, but it was observed that the HR AC was
higher in the older goats than the younger, however the
albumin concentration significantly decreased with
progressive increase in age of the goats. The observed
significant influence of age on albumin concentration
is at variance with the earlier studies of
Piccione et al., (2009) and Opara et al., (2010) who
reported non-significant effect of age on albumin
concentration of WAD goats.
CONCLUSION
The mean body temperature (38.9°C) of the
goats used was within the reference normal range for
goats in thermal neutral condition and this indicates that
the goats showed no clinical signs of stress during the
research period.
The albumin concentration, heart rate and
adaptive coefficient of the goats had clear variation
based on differences in haemoglobin type, sex and age of
the animals.
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Characteristics Age of goat
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Rectal Temperature (oC) 39.0 39.0 39.0 39.0 0.02 ns
Heart Rate (bpm) 77.1b 74.4c 74.4c 78.5a 0.37 *
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Potassium concentration (Mmol/l) 4.0 4.0 4.0 4.0 0.06 ns
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Biology
Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with
special reference to its life table attributes in Tripura, India
Keywords: Catopsilia pomona butterfly, Pieridae, eco-biology, life table, Tripura, north east India.
ABSTRACT: Butterflies of the family Pieridae are common in tropical parts of the world. They are considered as major pollinators as well as pests of economically important plants. Catopsilia pomona is a dominant pierid butterfly found in association with wild plants of Tripura, northeast India. It is abundant throughout the year. Present study was conducted to document the eco-biology of Catopsilia pomona with special reference to its life table attributes in the state of Tripura. Survival rates of life cycle stages in the semi-natural as well as in the field were the maximum during the wet and hot season. Mortality (k value) of different life cycle stages as a proportion of individuals dying during development varied from 0.16 to 0.46 in different seasons. Results suggested that abiotic factors and mortality factors of egg significantly influenced the survival rate of C. pomona population. This butterfly depends on three species of Cassia plants, all shrubs, for their oviposition and larval development in the environment of Tripura.
876-885 | JRB | 2013 | Vol 3 | No 3
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licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.
www.jresearchbiology.com Journal of Research in biology
An International Scientific
Research Journal
Authors:
Samit Roy Choudhury and
Basant Kumar Agarwala*
Institution:
Ecology & Biodiversity
Laboratories, Department of
Zoology, Tripura University,
Suryamaninagar- 799022,
Tripura, India.
Corresponding author:
Basant Kumar Agarwala
Email:
Phone No:
0091 381 237 9083/9123
Web Address: http://jresearchbiology.com/documents/RA0245.pdf.
Dates: Received: 22 May 2012 Accepted: 28 May 2012 Published: 17 Apr 2013
Article Citation: Samit Roy Choudhury and Basant Kumar Agarwala. Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with special reference to its life table attributes in Tripura, India. Journal of Research in Biology (2013) 3(3): 876-885
Journal of Research in Biology An International Scientific Research Journal
Original Research
INTRODUCTION
Host selection for survival, development and
reproduction in majority of insects often vary in space
and time (van Nouhuys et al., 2003; Nylin et al., 2009)
which, in turn, depends on the availability (minimum
density per unit area) of closely related host plant species
(Thorsteinson, 1960), and trade off between host
preference by females for oviposition and larval
performance of insects (van Nouhuys et al., 2003).
However, adult butterflies and their caterpillars show
preference for certain host plants for tender shoots,
pollen and nectar as food source. Thus, butterfly
diversity of a particular habitat generally reflects the
overall plant diversity of that habitat. Butterflies are
essential component of any natural ecosystem. Their
value as indicators of biotope quality is being recognized
because of their sensitivity to minor changes in micro-
habitat, climatic conditions as well as seasonal changes
(Kremen, 1992; Murugesan and Muthusamy, 2011).
They are considered as ideal subject for ecological
studies of terrestrial landscapes (Thomas and Malorie,
1985).
North eastern region of India is blessed with
vegetation rich landscapes that support diverse butterfly
fauna and other insects (Alfred et al., 2002). The state of
Tripura, being a part of this region, also contains large
number of butterfly species which is evident from
infrequent records of these taxa (Mandal et al., 2002;
Agarwala et al., 2010; Majumder et al., 2011; Roy
Choudhury et al., 2011). Butterflies of the family
Pieridae are common in tropical parts of the world and
are considered as major pollinators of crop plants
(Borges et al., 2003), and a few of them are also
considered as pests of economically important plants
(Anonymous, 2007; Capinera, 2008). Despite their
common occurrence, there is a lack of substantial study
on the ecology, seasonal abundance, host preference and
life history of the most common pierid species
Catopsilia pomona F. found in association with wild
plants of north east India, including Tripura. However,
information on life table and host selections are available
on other pierid species that feed and oviposit on crop
plants (Chew, 1995). C. pomona, a dominant pierid
butterfly, is found throughout the year in the state of
Tripura (Agarwala et al., 2010; Majumder et al., 2011;
Roy Choudhury et al., 2011). It prefers green and moist
lands, pasture lands, farms, and edge of drains, moist
deciduous forests, hillocks, and semi-arid areas with high
abundance of grasses, small herbs and shrubs i.e.
secondary type of vegetation (Atluri et al., 2004).
Reported larval host plants of common emigrant
comprise of Cassia fistula L., C. sophera L.,
C. occidentalis L., C. tora L., C. siamea (Lam.) Irwin et
Barneby, Butea frondosa, and Bauhinia racemosa L.
(Kunte, 2000; Atluri et al., 2004). Among these plants
C. fistula, C. tora, C. occidentalis, C. sophera, and
B. racemosa are important as medicinal plants
(Anonymous, 2004; Danish et al., 2011; Harshal et al.,
2011; Singh and Dubey, 2012), and C. siamea is used in
social forestry (Atluri et al., 2004, Borikar et al., 2009).
Hence, it is very important to document the seasonal
occurrence and its host plant preference for oviposition
and larval development of C. Pomona. With this view,
the present study was conducted to know the eco-biology
of Catopsilia pomona with special reference to its life
history attributes in the state of Tripura.
Study site
Present study was conducted in Trishna Wildlife
Sanctuary of south Tripura district (23°26.137’ N,
91°28.184’ E: 51-82 m asl), having an area of about
194.7 sq. km. Study location is characterized by patches
of secondary moist deciduous forests and surrounded by
swamp areas. Forest patches are rich in sal trees, garjan
trees, bamboo bushes, herbs, shrubs and climbers.
Trishna sanctuary is known by 230 tree species, 110
species of shrubs, 400 species of herbs, and 150 species
of climbers (Economic review of Tripura, 2008-2009).
Among the known host plants of C. pomona, the study
Roy Choudhury and Agarwala, 2013
877 Journal of Research in Biology (2013) 3(3): 876-885
area contains three species of Cassia only viz.
Cassia sophera, C. tora and C. occidentalis which are
considered to be the preferred hosts of larvae. Some part
of the study area is used for rubber cultivation and paddy
cultivation (Figure 1). The area has a tropical climate,
with cold weather from November to February. Average
daily temperature varies from the minimum of 6.8°C in
January to the maximum of 37.7°C in June. The area
receives, on an average, 3353.4 mm rainfall annually.
MATERIALS AND METHODS
Field census of eggs, larvae and oviposition
preference of C. pomona
Prior to the study a reconnaissance survey was
made in the Trishna study area to locate the available
host plants distribution of C. pomona. Walk census for
leaves of host plants containing eggs and larvae were
held at an interval of 7-days from March 2007 to
February 2008. For this, two line transects (approx. 1 km
long and 5 m wide) were set up in the study area. Thirty
host plants, 10 plants each of C. sophera, C. tora and
C. occidentalis, were randomly selected for the study
along the length of transects and were marked with
plastic tags. Thus, sixty plants from three species were
selected from transects. Ovipositing females were
followed in the selected host plants for recording number
of eggs laid per female per leaf. Binoculars were used to
observe the females from a distance (about 2 m) without
disturbing them. The same host plant was also observed
for presence of larvae. All the females seen ovipositing
on the selected host plants was recorded during the
transect walk. Two transects were walked in two
consecutive days in a week. Ten apical leaves were
observed within a selected plant for egg and larval counts
which were made between 8.00 AM to 12.00 noon local
time. When a female was found to either laying eggs or
seen perching near a host plant, halt was made for
approx. 8 to10 minutes, and then move to the subsequent
host plants along the transect. Different host plants
selected by females for oviposition were recorded,
photographed, collected and later identified by
comparing with the herbarium deposited in the gallery of
Plant Taxonomy and Biodiversity Laboratories,
Department of Botany, Tripura University.
Roy Choudhury and Agarwala, 2013
Journal of Research in Biology (2013) 3(3): 876-885 878
Figure 1. Geographical map of Trishna and landscape of the Study area.
Larval host range and seasonal variation in
development
Leaves of the host plant species found to contain
freshly laid eggs of C. pomona in field were brought to
the field station (3 km from the study area), and
transferred individually to 10 cm diameter paired Petri
dishes lined with corrugated papers. These were fed with
surplus quantity of tender leaves of respective host plants
from which they were actually collected. Twenty
replicates were used for each host plant species. Food
was changed every 24 hrs intervals. Petri dishes were
cleaned at the time of food change. These were observed
twice in a day at 11 am and again at 5 pm to record the
incubation period of eggs, developmental time of larvae,
and pupae. Mortality in development, if any, was also
recorded. This was simultaneously done on each host
plant, once in five different seasons to record the
seasonal variation, if any. Experiments were set up at the
field station (Temp: 18°C ~ 27°C, RH: 45~75%, and
L: D: 16:8h) i.e. in the controlled environment.
Larval development in field
Selected plants with freshly laid eggs and
subsequent developmental stages were provided with
coloured tags and these were numbered for easy
identification. Individual eggs, larvae and pupae were
followed daily, and the disappearance of individuals or
those that failed to develop in to the next stage at
different life stages were recorded. Larvae were found to
be slightly sluggish and females laid solitary eggs,
usually one on each leaf. The study was repeated once in
different seasons.
Survival rate and K-factor analysis
An age-specific life table was constructed
following the method of Stiling (2002). To prepare the
life table, records were made on the larval durations and
survival rate at each developmental stage i.e. eggs to
emergence of adults from pupae. For this purpose,
409 eggs and 317 eggs of C. pomona were studied in
natural (in field) and in controlled conditions (ambient
condition of field station), respectively. Meteorological
data of Trishna study area were collected from the
records maintained by the Department of Agriculture,
Govt. of Tripura at Arundhuti Nagar, Agartala.
Data analysis
Field data on proportion of host plants used by
C. pomona for laying of eggs and distribution of eggs per
leaf of the different host species during a year were used
to draw population curves. For this purpose, weekly data
were pooled on monthly basis. Developmental time from
egg to the eclosion of pupae on different host plants and
between different seasons was subjected to one-way
analysis of variation (ANOVA). Mean values of
development time on different host plant species and
between different seasons were compared by Tukey’s
multiple comparison test. Differences in development
time recorded in field and in field station were compared
by Students t-test. A significance level of 0.05 was used
to reject the null hypothesis. Field data on distribution of
eggs on different host plant species were subjected to
regression analysis to reveal the relationship between
oviposition preference and host utilization. Based on the
life table data, survival rate and K factor value that
closely mirrors the overall population mortality was
Roy Choudhury and Agarwala, 2013
879 Journal of Research in Biology (2013) 3(3): 876-885
Host plant No. of leaves
observed
No. of larvae
counted
Mean (+SEM) no
of larvae/ leaf
ANOVA No. of eggs
counted
Mean (+SEM)
no of eggs/ leaf
ANOVA
C. sophera 4800 984 0.21 + 0.01 F = 6.909 ,
df = 2,14397,
P = 0.0001
1237 0.26+0.02 F = 5.26,
df = 2,14397,
P = 0.006 C.occidentalis 4800 563 0.12 + 0. 02 899 0.19+0.03
C. tora 4800 647 0.13 + 0.01 816 0.17+0.02
Table 1. Oviposition preference of C. pomona females on different host plants in the study area
determined. At each life stage, number of deaths
(k value) was calculated as under: k = log Nt - log Nt+1,
where Nt is the density of the population before it is
subjected to the mortality and Nt+1 is the density
afterward. Total generational mortality factor K is
determined as the sum of the individual mortality factors
k at egg, larval and pupal stage of the C. pomona species
(Stilling, 2002). For interpretation of colleted data, the
year was divided in to five seasons: spring (March,
April), summer (May, June), rain (July, September),
autumn (October, November), and winter (December-
February). To determine the relationship between
successful development (%) of C. pomona eggs and
climatic factors in the study area regression analysis was
carried out. Origin 7 software (www.originlab.com) was
used for the analysis of data.
RESULTS
Egg abundance and oviposition preference
Females of C. pomona laid solitary eggs at edges
and on undersides of tender or young leaves (one egg/
leaf/female) of C. sophera, C. occidentalis and C. tora
plants throughout the year (Table 1, Figure. 2). In the
year-round census of 10000 m2 (1000 m long x 5 m wide
x 2 transects @ 1 ha) which represents less than 0.5% of
the study area (19.47 ha), 52.54% to 85.07% of
C. sophera plants, 21.31% to 69.47% of C. occidentalis
plants and 23.88% to 56.52% of C. tora plants were
found with one or more eggs. Between the three host
plant species, common emigrant females selected the
highest proportion of C. sophera for oviposition during
hot and wet months, and the maximum was recorded in
the month of August (Figure 2). In comparison,
distribution pattern of eggs on C. occidentalis plants
showed marked difference from the distribution of eggs
on C. sophera. Higher proportion of this host plant
species was recorded during dry and cooler months, and
the maximum was recorded in the month of January
(69.47%) (Figure. 2). In case of C. tora, the trend of egg
distribution was found to be nearly similar to that of
C. sophera but the proportion of host use was found to
be much lower than C. sophera (Figure. 2). Occurrence
of eggs showed that 4800 leaves each of C. sophera,
C. occidentalis and C. tora that were surveyed during the
year, contained 1237, 899 and 816 eggs, respectively
(mean + SEM: C. sophera: 0.26+0.02 eggs per leaf,
C. occidentalis: 0.19+0. 03 eggs per leaf and C. tora:
0.17+0.02 eggs per leaf, ANOVA: F = 5.26, df = 2,
14397, P = 0.006) (Table 1).
Larval host range
Larvae of C. pomona were found to feed on
tender leaves of the three host plant species, viz.
C. sophera, C. occidentalis and C. tora. Higher
proportion of C. sophera plants were used as food and
maximum was recorded in the hot and wet month of
August (26.70%). Incidence of larvae on C. occidentalis
Roy Choudhury and Agarwala, 2013
Journal of Research in Biology (2013) 3(3): 876-885 880
Month N Mean + SEM value (days)
C. sophera C. occidentalis C. tora
March 36 24.50 + 0.26 1 a 24.75 + 0.25 1 a 24.74 + 0.33 1 a
May 36 20.67 + 0.31 2 a 20.92 + 0.42 2 a 20.92 + 0.42 2 a
August 36 18.92 + 0.23 3 a 19.42 + 0.63 3 a 19.00 + 0.28 3 a
October 36 21.17 + 0.24 2 a 21.33 + 0.28 2 a 21.25 + 0.25 2 a
December 36 30.67 + 0.47 4 a 30.83 + 0.41 4 a 31.00 + 0.41 4 a
Dissimilar numbers following means in a column denote significant difference and similar letters accompanying
means show no difference between them by Tukey’s multiple comparison range test at 5% significant level.
Table 2. Development time (in days) of C. pomona on different host plant species
plants was recorded to be the highest in January
(20.61%) and lowest in August (1.64%), respectively. In
case of C. tora host, the highest proportion was recorded
in the month of June (17.24%) and the lowest in the
month of January (5.33%) (Figure. 3). Occurrence of
larvae showed that 4800 leaves each of C. sophera,
C. occidentalis and C. tora that were surveyed during the
year, contained 984, 563 and 647 larvae, respectively
(mean + SEM: C. sophera: 0.21 + 0.01 larva per leaf,
C. occidentalis: 0.12 + 0. 02 larva per leaf and C. tora:
0.13 + 0.01 larva per leaf, ANOVA: F = 6.909,
df = 2,14397, P = 0.0001) (Table 1).
Developmental time and seasonal variation
Developmental time of different immature stages
(egg to pupae) of C. pomona was found to vary in
different seasons but did not show difference in any one
season between different host species (Figure 4).
Development time was recorded to be the longest at
lower temperature and lower relative humidity
corresponding to the month of December (controlled
condition: average temperature=18°C, average relative
humidity=51.33%) and shortest at higher temperature
and higher relative humidity in August (average
temperature=27.91°C, average relative humidity
=77.07%) (Table 2).
Survival rate and K factor analysis
Results showed that in field about 30% of the
eggs deposited by C. pomona developed in to pupae
during the months of July and August (average
temperature 31.09°C, average humidity 70%, mean
rainfall 7.45 cm). Developmental success was limited to
13.04% in the month of December (average temperature
19.330C, average humidity 51%, rainfall 0 cm).
Regression analysis of survival rate showed positive
correlations with average temperature (y =1.08 + 0.87x,
Roy Choudhury and Agarwala, 2013
881 Journal of Research in Biology (2013) 3(3): 876-885
Figure 4. Development time (in days) of C. pomona on
different larval host plants in different months of a
year. Similar alphabets accompanying bars denote no
significant difference between the mean values in that
month.
Figure 3: Mean number of larvae of C. pomona
recorded on different host plants.
Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9P
rop
ort
ion
of h
ost p
lan
ts s
ele
cte
d fo
r o
vip
ositio
n
Months
C. occidentalis
C. sophera
C. tora
Figure 2. Proportion of host plants of three Cassia
species recorded with eggs of C. pomona in different
months of the year in the study area.
r2=0.74) (Figure 5a), average relative humidity
(y = 3.87 + 0.33x, r2 = 0.52) (Figure 5b), and with mean
rainfall (y =20.07 + 1.64x, r2 = 0.64) (Figure 4c).
Number of eggs that developed successfully in fields
(24.03+1.46; n=240) and in semi natural condition
(76.36+0.90; n=240) showed significant difference
(t =30.54, df =478, P =0.000).
K-value analysis showed maximum mortality in
development (0.46) in the month of December and
minimum (0.16) in the month of September. K - values
of the eggs recorded in different seasons were found to
be very high (0.21) and very low (0.09), respectively,
during these two months. Analyses showed that mortality
in the egg stage influenced the total K value the most
(Figures. 6a, b).
DISCUSSION
Natural populations of phytophagous insects
including butterflies frequently encounter wide choice of
host plants of differing suitability (Badeness et al., 2004;
Dennis et al., 2006). The dominant strategy among
herbivorous insects involves specialization on a set
of closely related plants that will maximize offspring
survival and fitness (Ward and Spalding, 1993; Gibbs et
al., 2006), and also to the phenological characteristics of
host plants. It is evident from the present study that
C. pomona butterflies utilize three species of Cassia for
oviposition and larval development in Trishna study
area. Among these host plants, maximum number of
C. pomona eggs were found in C. sophera with higher
proportion recorded during hot and wet months, and
lowest in dry and cooler months of the year. During dry
and cool months, females choose C. occidentalis in
higher proportion for oviposition followed by C. tora.
This might be due to the availability of more young
leaves in C. occidentalis and C. tora compared to
C. sophera in dry and cooler months of the year. Results
indicated that common emigrants preferred C. sophera
than the two other host plants but utilized three hosts
throughout the year depending on the host plant
phenology, and made the larval host range wider.
Patterns of host use have several effects on butterfly
Roy Choudhury and Agarwala, 2013
Journal of Research in Biology (2013) 3(3): 876-885 882
Figure 5. Regression analysis between successful
development (%) of C. pomona eggs and climatic
factors: (a) average temperature (oC), (b) average
relative humidity (%), and (c) mean rainfall (cm).
population dynamics (Hanski and Singer, 2001). Food
plant-insect herbivore association is based on resource
size and optimal synchronization of their respective
life-cycles. If resource size in time and given space is
large, insects will show monophagism. In comparison, if
resource size is short and patchy, then insect herbivores
are generally polyphagous or oligophagous (Price, 1997;
Dixon, 1998; Nylin et al., 2009). In this study,
availability of taxonomically closely related Cassia
plants in time and given area under study on which
C. pomona successfully completed their life history
attributes might widen their host range. This finding is in
conformity with the optimisation theory of species in
relation to host plants in time and space (Begon et al.,
1996; Scheirs and Bryn, 2002).
Population of C. pomona showed strong
relationships with climatic factors. They took longer time
for development in the dry and cooler months when the
suitable habitat for oviposition and larval development
were minimum than in wet and hot months. But,
developmental time on the different host plants did not
differ during a particular season that suggested possible
qualitative similarity between host plants. However,
several studies showed that ovipositing females of
phytophagous butterflies typically show a preference for
host plants that are capable of supporting fast larval
growth (Thompson, 1988a, b, c; Janz et al., 1994).
Climatic factors are well known for their
significant influence on population dynamics of animal
communities (Leonard et al. 1998). Analysis of K-value
in this study has revealed that the average temperature,
the average relative humidity and the mean rainfall
showed strong positive relationships with survival rate of
C. pomona. In the present study no biotic factors such as
parasites, predators were noticed which can also
influence the population dynamics of C. pomona
butterfly.
CONCLUSION
Results revealed that C. pomona females
occurred and laid eggs throughout the year on three host
plant species of Cassia. It preferred C. sophera host over
C. occidentalis and C. tora for oviposition and larval
development. Pattern of egg distribution i.e. oviposition
was found to be linked with host plant phenology. Egg
mortality was the major influencing factor in
determination of survival rate. The k-value of egg
mortality (k1) and total mortality factor (K) showed
strong positive relationship.
ACKNOWLEDGEMENT
Authors are thankful to the Head, Department of
Roy Choudhury and Agarwala, 2013
883 Journal of Research in Biology (2013) 3(3): 876-885
Figure 6. Key- factor analysis of development of
C. Pomona: (a) mortality in developmental stages
expressed as k values, (b) regression fit of mortality in
egg stage (k1) to the total K value.
Zoology, Tripura University for the laboratory facilities.
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Biology
Anti-inflammatory activity of lycopene isolated from Chlorella marina on
carrageenan-induced rat paw edema
Keywords: Microalgae, Chlorella marina, lycopene, anti-inflammation.
ABSTRACT:
Even though role of lycopene (all-trans) in controlling inflammation was reported, lycopene (cis and all-trans 40:60) isolated from green algae Chlorella marina was not reported so far. In this present study inflammation was induced in male Sprague dawley rats and edema was produced acutely by injecting 0.1 ml of carrageenan into the plantar region of the right hind paw of the rats subcutaneously. Intra peritoneal administration of algal lycopene (AL) at the dose of 10 mg/kg b.wt showed maximum (83%) inhibition on paw edema. The anti- inflammatory effect was significantly (P< 0.05) higher in rats fed with algal lycopene when compared to the standard drug voveran (71%) and all- trans tomato lycopene (TL) (63%). Carrageenan induced rats showed elevated levels of cyclooxygenase (COX) and lipoxygenase (LOX) activities in monocytes. Myeloperoxidase (MPO) in serum, C- reactive protein (CRP) and ceruloplasmin activity in plasma was also high in carrageenan induced rats when compared to normal. Lycopene from Chlorella marina showed significant effect in reducing the above parameters to that of the standard drug while tomato lycopene showed less effect when compared to algal lycopene. Therefore algal lycopene from Chlorella marina would be recommended for the treatment of anti-inflammatory disorders.
886-894 | JRB | 2013 | Vol 3 | No 3
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www.jresearchbiology.com
Journal of Research in Biology
An International Scientific
Research Journal
Authors:
Renju GL and
Muraleedhara Kurup G.
Institution:
Department of Biochemistry,
University of Kerala,
Trivandrum, India.
Corresponding author:
Muraleedhara Kurup G.
Email:
Phone:
+919447251408.
Fax:
91-471 2308078.
Web Address: http://jresearchbiology.com/documents/RA0329.pdf.
Dates: Received: 02 Feb 2013 Accepted: 22 Feb 2013 Published: 23 Apr 2013
Article Citation: Renju GL and Muraleedhara Kurup G. Anti-inflammatory activity of lycopene isolated from Chlorella marina on carrageenan-induced rat paw edema. Journal of Research in Biology (2013) 3(3): 886-894
Journal of Research in Biology An International Scientific Research Journal
Original Research
INTRODUCTION
Inflammation is a response which protects and
heals the host tissue after infection or injury. (Nathan,
2002). However, it is frequent that the inflammatory
response to several insults erroneously leads to the
damaging of normal tissues. Prostaglandin-E2 is
generated from arachidonic acid by the enzyme
cyclooxygenase (COX) at sites of inflammation in
substantial amounts and can mediate many of the
pathologic features of inflammation (Serhan and Levy,
2003). One of the early cellular events in inflammation is
the margination of leukocytes, primarily neutrophils and
this can be measured by myeloperoxidase activity
(Goulet et al., 1994).
Currently, non steroidal anti-inflammatory drugs
(NSAIDs) were used for inflammatory diseases. Even
though this drugs transiently suppresses inflammation,
but their long term use cause ulceration in the
gastrointestinal tract and renal morbidity (James and
Hawkey, 2003). However research focused on finding
newer drugs with pharmacological actions without side
effects.
Several antioxidants have been reported to
have anti-inflammatory and anti-arthritic activities
(Maxwell et al., 2006). In the present study a culturable
marine edible algae Chlorella marina was selected to
evaluate the anti-inflammatory activity of lycopene.
Generally tomatoes are the source of lycopene, but it has
many disadvantages (Shi and Le mague, 2000). The
content of lycopene in tomato is very less and the
configuration of lycopene is all-trans. Even though
lycopene from algae has been reported (Ishikawa and
Abe, 2004), no attempt has been made so far for the
commercialization of algal lycopene. It can be seen that
marine sources especially algae are the least exploited
for their bioactive molecules (Pinky and Goswai, 2012).
Work in our laboratory has shown that the lycopene
content in algae is comparatively high, when compared
to tomato lycopene. The most interesting observation
was that algal lycopene contain cis-configuration
(5-cis, 9-cis, 13- cis and 15-cis). Recently it has been
reported that the cis form of lycopene is more
biologically active than the trans form (Stahl and Sies,
1996).
MATERIALS AND METHODS
Chemicals
Lycopene, carrageenan, linoleic acid,
Histopaque, arachidonic acid other fine chemicals were
purchased from Sigma, St. Louis, MO, USA. Diclofenac
sodium (Voveran) was obtained from Novartis, India.
Salt and vitamin mixtures were purchased from Merck,
Germany. All other chemicals and reagents were
purchased from Sisco Research Laboratory Pvt.Ltd
(SRL), India, and were of analytical grade.
Algal source
Marine algae Chlorella marina Butcher was
collected from the Vizhinjam coast of Kerala, located at
Latitude 08° 22’ North Longitude. 76° 59’ East on the
south west coast of India and was cultured under
laboratory conditions. The microalgae were identified by
the botanist (Dr. G. Valsaladevi, Department of botany)
and a voucher specimen (No. KUBH 5812) has been
deposited in the Department of Botany, University of
Kerala, India.
Culture medium
Walne’s medium (1970) was used as a basal
medium for the cultivation of Chlorella marina. 5 g /L
glucose was added to the basal medium. Flasks were
incubated at 25°C with continuous illumination. The pH
was adjusted to 7.5. Nicotine (10 µM/ L) was sterilized
by autoclaving and was added to 5 days old cultures for
the production of lycopene.
Biomass harvest
Chlorella marina cells were grown in suspension
cultures up to 30 to 40 days. The cells were harvested at
stationary phase by withdrawing the cultures in 50 ml
polypropylene tubes and centrifuged at 5000 rpm for
Renju and Kurup., 2013
887 Journal of Research in Biology (2013) 3(3): 886-894
10 minutes. Removed the medium and the pellets were
freeze dried, weighed and stored under nitrogen at -20°C.
Isolation of lycopene from Chlorella marina (AL) and
analysis
Harvested biomass (5g dry weight) was
suspended with 5 ml of 80% cold acetone and kept
overnight under 4°C for better and easy recovery of
carotenoids. The mixtures were vortexed for 2 minutes
and centrifuged at 5000 rpm for 20 minutes. After
repeated extractions (4 times), the supernatants were
pooled and the colorless cell pellets were discarded. The
extracts were dried over anhydrous sodium sulphate and
reduced to a minimum volume by evaporating the
solvents using N2 stream. The crude extracts were kept
for further separation of carotenoids in amber colored
containers under nitrogen at -20°C. All operations were
done at subdued light under nitrogen atmosphere. The
absorbance in the solvent phase was quantified by
spectrophotometric method at 470 nm as described by
Lichtenthaler (1987).
Isolation of all-trans lycopene from tomato (TL)
Tomatoes obtained from the local market,
Trivandrum, India were used. The all-trans lycopene
from tomato was extracted and evaluated according to
the procedure of Fish et al., (2002).
Determination of lycopene by HPLC
Lycopene extracted from algal cells and tomatoes
were determined by HPLC method at 450 nm as
described by Shaish et al., (1992). HPLC analysis of
lycopenes were performed using a silia chrom® column
(250 x 4mm + 5 x 4, NCLIOSIL 100-5-C18 5.0µm),
K 1001 type pump and the UV detector type of K 2600,
Germany. Elution was performed isocratically with
methanol: acetonitrile (9:1) v/v at a flow rate of
1 ml min-1. A UV detector with a wavelength of 450 nm
was employed. Lycopene (95%) obtained from Sigma
chemicals were used as standard. The retention time was
recorded and peak areas of standards and tests were
noted on each run and used for calculation of
concentrations of different fractions. All samples were
injected in duplicate.
Experimental animals
Male Sprague Dawley rats with the average body
weight of 150- 200 g of the same breed were selected for
the study. These animals were housed in the department
animal house and provided standard pellet diet and water
ad libitum and maintained with temperature at 25 ± 1°C,
humidity (55-60%) and photoperiod (12:12 h) light and
dark cycle. Experimental procedures conducted on rats
were approved by the Animal Experiment Committee
(218/CPCSEA) for animal care of Kerala University
according to Government of Indian law on animal use
and care.
Induction of acute inflammation-Carrageenan
induced rat paw edema
Carrageenan-induced rat paw edema assay was
conducted according to the procedure as described by
Winter et al., (1962). Five groups of six rats were
treated as AL and TL with doses 10 mg/kg and reference
drug Voveran, a Diclofenac sodium preparation
(20 mg/kg) were given orally and intraperitoneally (i.p),
1 h before the injection of carrageenan. Control rats were
given 0.1 ml 1% carrageenan. Inflammation was
induced by 0.1 ml, 1% carrageenan suspension in
0.9% NaCl solution was injected into the right hind paw
after 1 hour. The volume of the right paw was measured
by paw edema meter before and after injection in
the third and fifth hour. The paw edema and inhibition
was calculated by the equation: Activity= 100 - (100 ×
average drug treated/average for control).
Treatment Protocol and Experimental Design in
Acute Inflammation
Edema was induced on rat right hind paw by
aponeurosis injection of 0.1ml of 1% carrageenan in
0.9% saline. The experimental groups consisted of 30
rats were divided in to five groups.
Group I: control (received saline only),
Group II: carrageenan alone
Renju and Kurup., 2013
Journal of Research in Biology (2013) 3(3): 886-894 888
Group III: carrageenan + algal lycopene (AL groups,
10 mg/kg i.p)
Group IV: carrageenan + tomato lycopene (TL groups,
10 mg/kg i.p)
Group V: carrageenan + Voveran (VOV groups,
20 mg/kg i.p.)
At the end of third hour, the animals were
sacrificed by euthanasia. Blood was removed to ice cold
containers for various biochemical analyses.
Activity of Cyclooxygenase (COX) and
Lipooxygenase (LOX) in Peripheral Blood
Mononuclear Cells (PBMC)
Mononuclear cells were isolated the procedure
described by Radhika et al., (2007). Cox activity was
measured by the method of Shimizu et al., (1984).
15-LOX activity was determined by the method of
Axelrod et al., (1981).
Biochemical analysis
Serum myeloperoxidase (MPO) activity was
measured by Mullane et al., (1985). CRP in plasma was
determined by using Immunoturbidometric kit (Diasys
Diagnostics, Germany). Ceruloplasmin was estimated by
the method of Ravin (1961). Protein was determined by
the methods of Lowry et al., (1951).
Statistical analysis
The Statistical package for social sciences
(SPSS/PC+), version 11.5 (SPSS Inc; Chicago. IL, USA)
was used to analyze the results for statistical significance
using one-way ANOVA followed by Duncan’s test.
P value < 0.05 was considered as significant.
RESULTS AND DISCUSSION
Sub plantar injection of carrageenan into the foot
of rats caused a time-dependent increase in paw volume.
The localized inflammatory response as evidenced
visually by the edema reached a maximum intensity at
third hour after carrageenan induction and this maximal
effect was seen until the fifth hour. Administration of AL
and TL has showed significant effects in decreasing
carrageenan-induced paw edema. Algal lycopene showed
maximum edema inhibition compared to all-trans tomato
lycopene and drug. AL exhibited 70% and 83% edema
inhibition at third/fifth hours, respectively. This effect
was comparable to the reference drug Voveran which
exerted 54% and 71% edema inhibition at third and fifth
hour, respectively. TL showed 51% and 63% edema
inhibition at third and fifth hour after carrageenan
induction (Figure. 1).
COX activity in PBMC was significantly
(p<0.05) increased in carrageenan treated rats when
compared to control rats (Figure. 2). Treatment with AL
showed significant (p<0.05) decrease in COX activity
when compared to carrageenan induced rats.
Prostaglandin is formed by the interaction of two distinct
but related enzymes, COX-1 and COX-2 and plays an
important role in promoting the signs and symptoms
of inflammation (Otterness and Bliven, 1985;
Ibegbulem et al., 2012). The activity of COX in PBMC
was decreased (p<0.05) in AL treated group when
compared to TL and voveran treated group. Reduction of
paw swelling and decreased activity of COX showed the
immunological protection rendered by the algal
lycopene. These results showed the anti-inflammatory
potential of the AL.
The activity of 5-LOX and 15-LOX in PBMC
was significantly (p<0.05) increased in carrageenan
induced rats when compared to normal rats
(Figure.3 and 4). Algal lycopene treatment significantly
reduced (p<0.05) in 5-LOX and 15-LOX activity, when
compared to CII rats. The effect was significantly higher
(p<0.05) than TL and drug treated groups.
Lipoxygenases are a family of key enzymes in the
biosynthesis of leukotrienes that are postulated to play an
important role in the pathophysiology of several
inflammatory diseases (Henderson, 1994; Yamamoto,
1992). In the normal situation, cellular leukotriene
production is suppressed by selenium dependent
peroxidases (Werz et al., 1997). On receiving
Renju and Kurup., 2013
889 Journal of Research in Biology (2013) 3(3): 886-894
inflammatory stimuli, leukotriene production is elicited
through the arachidonic acid cascade, causing micro
vascular injury, vasoconstriction and production of
pro-inflammatory cytokines (Peskar, 1991). Studies have
shown that LOX and leukotrienes have a profound role
in carrageenan-induced inflammation (Henderson, 1994;
Gamache et al., 1986). In the carrageenan-induced
inflammation model, AL significantly reduced
carrageenan-induced 5-LOX and 15-LOX activities in
mononuclear cells, indicating decreased leukotriene
production and hence a protective effect.
MPO activity in serum was significantly
increased (p<0.05) in carrageenan induced rats when
compared to normal group (Table 1). Treatment with AL
showed significant decrease (p<0.05) in MPO activity
when compared to carrageenan induced rats. The MPO
activity was significantly decreased when compared to
TL and drug treated groups. The activity of MPO is a
marker of neutrophil infiltration (Bradley, 1982), and
was found to be significantly increased in the paw tissue
of carrageenan-induced rats. AL significantly decreased
(p<0.05) the elevated MPO activity, an indicator of
neutrophil in inflamed paws, suggesting that inhibition of
neutrophil infiltration might be another mechanism by
which AL achieves its anti-inflammatory effect.
Table 1 also shows the variations in serum CRP
and ceruloplasmin level in the test animals compared to
control. Serum CRP and ceruloplasmin levels were
significantly increased (p<0.05) in carrageenan induced
rats when compared to normal rats. Supplementation
with AL significantly decreased (p<0.05) the serum
CRP and ceruloplasmin levels when compared to
carrageenan induced rats. The levels of CRP and
ceruloplasmin were decreased significantly (p<0.05),
when compared to TL and Voveran treated groups.
C-reactive protein is an acute phase protein that has been
identified as an important biomarker for various
inflammatory, degenerative, and neoplastic diseases.
Elevated levels of CRP have been found in the blood
during virtually all diseases associated with active
inflammation or tissue destruction, particularly in
patients with rheumatoid arthritis (Pepys and Hirschfield,
2003; Kushner, 1991). In our study the increased levels
Renju and Kurup., 2013
Journal of Research in Biology (2013) 3(3): 886-894 890
Figure 1: Effect of algal lycopene on carrageenan-
induced paw edema in normal and experimental rats.
Figure 2: Effect of algal lycopene on activity of COX in
PBMC of normal and experimental rats COX activity
is expressed as an optical density increase
(OD increase) per mg protein per minute. Val-
ues are expressed as mean ± SEM of six rats in each
group. a – Statistical difference of Control group with CII
group when p < 0.05. b – Statistical difference of CII group with group AL,
TL and VOV when p < 0.05. c – Statistical difference of VOV group with group AL
and group TL when p < 0.05. d –Statistical difference of TL group with
AL when p <0.05.
of CRP level was found to be significantly decreased in
algal lycopene treatment when compared to TL and
Voveran treatments.
The serum protein, ceruloplasmin is a powerful
free radical scavenger that oxidizes iron from the ferrous
to ferric state. Ceruloplasmin levels increase under
conditions leading to the generation of oxygen products
such as the superoxide radical and hydrogen peroxides
(Revnic, 1995). Serum ceruloplasmin level was
significantly increased in carrageenan induced rats when
compared to normal rats. Treatment with AL showed
significant decrease in the concentration of
ceruloplasmin. The increased levels of ceruloplasmin in
carrageenan induced rats could be decreased
significantly on treatment with algal lycopene when
compared to TL and standard drug Voveran might be
having a protective response against free radical
mediated lipidperoxidation.
Lycopene from edible marine microalgae
C. marina showed higher anti-inflammatory activity than
all-trans tomato lycopene and standard drug Voveran.
These effects might be due to the presence of two
isomeric form of lycopene (cis and all-trans) in the
microalgae. Reports available indicate that the
cis-lycopene has a high antioxidant potential when
compared to all-trans lycopene (Stahl and Sies 1992;
Clinton et al., 1996). Algal lycopene isolated from
C. marina could reduce cell influx, oedema formation
Renju and Kurup., 2013
891 Journal of Research in Biology (2013) 3(3): 886-894
Figure 3: Effect of algal lycopene on activity of
5- LOX in PBMC of normal and experimental
rats
5-LOX activity is expressed as an optical density
increase (OD increase) per mg protein per min-
ute. Values are expressed as mean ± SEM of six
rats in each group. a – Statistical difference of Control group with CII
group when p < 0.05. b – Statistical difference of CII group with group
AL, TL and VOV when p < 0.05. c – Statistical difference of VOV group with group
AL and group TL when p < 0.05. d –Statistical difference of TL group with AL
when p <0.05.
Figure4: Effect of algal lycopene on activity of
15- LOX in PBMC of normal and experimental
rats
15-LOX activity is expressed as an optical den-
sity increase (OD increase) per mg protein per
minute. Values are expressed as mean ± SEM of
six rats in each group. a – Statistical difference of Control group with
CII group when p < 0.05. b – Statistical difference of CII group with group
AL, TL and VOV when p < 0.05. c – Statistical difference of VOV group with
group AL and group TL when p < 0.05. d –Statistical difference of TL group with AL
when p <0.05.
and release of mediators associated with inflammatory
condition, and therefore has the potential to be used as
an anti-inflammatory agent. Further studies are in
progress to evaluate the molecular mechanism of its
anti-inflammatory activity.
ACKNOWLEDGEMENT
We express gratitude to Dr. Anantha Lekshmi,
Veterinary Doctor, Department of Biochemistry,
University of Kerala, Kariavattom, India for helping us
with the animal experiments.
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Renju and Kurup., 2013
Journal of Research in Biology (2013) 3(3): 886-894 892
Groups MPO
(µm/min/mg)
CRP
(mg/ml)
Ceruloplasmin
(mg/dl)
Control 5.85 ± 0.37 22.0 ± 1.24 0.10 ± 0.006
CII 20.52 ± 1.11a 97.71 ± 3.80a 0.34 ± 0.014a
AL 7.45 ± 0.37bc 45.94 ± 2.01bc 0.19 ± 0.007bc
TL 13.75 ±0.48bcd 80.64 ±3.18bcd 0.28 ± 0.016bcd
VOV 9.74± 0.39b 56.89 ± 2.42b 0.22 ± 0.010b
Table 1: Levels of CRP, Ceruloplasmin in plasma and
MPO in serum of experimental animals.
Values are expressed as mean ± SEM of six rats in
each group.
a – Statistical difference of Control group with CII
group when p < 0.05.
b – Statistical difference of CII group with group AL,
TL and VOV when p < 0.05.
c – Statistical difference of VOV group with group AL
and group TL when p < 0.05.
d –Statistical difference of TL group with AL when
p <0.05.
Table 2: Statistical table of Myeloperoxidase in one
way ANOVA followed by Duncan’s test
MPO
Sum of
Squares df
Mean
Square F
Between
Groups 827.642 4 206.911 91.002
Within
Groups 56.842 25 2.274
Total 884.485 29
Where df is degrees of freedom, F is F- ratio.
Table 3: Statistical table of CRP in one way ANOVA
followed by Duncan’s test
Where df is degrees of freedom, F is F- ratio.
CRP
Sum of
Squares df
Mean
Square F
Between
Groups 20984.623 4 5246.15
6 121.093
Within
Groups 1083.082 25 43.323
Total 22067.705 29
Table 4: Statistical table of CRP in one way ANOVA
followed by Duncan’s test
Ceruloplas-
min
Sum of
Squares df
Mean
Square F
Between
Groups .196 4 .049 59.148
Within
Groups .021 25 .001
Total .217 29
Where df is degrees of freedom, F is F- ratio.
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Renju and Kurup., 2013
Journal of Research in Biology (2013) 3(3): 886-894 894
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Biology
Identification of Animal Pasteurellosis by PCR Assay
Keywords: Culture lysate, genomic DNA, Pasteurella multocida, PCR .
ABSTRACT:
Diagnosis of pasteurellosis has become difficult, as there are five different capsular types and 16 somatic types. Molecular techniques like PCR are adapted nowadays for rapid and accurate diagnosis in early stage of the disease and also it provides useful information for epidemiological studies. The present study was conducted to study the efficiency of polymerase chain reaction (PCR) in the identification of P. multocida isolates and evaluation of different PCR methods viz., (i) PCR using genomic DNA (ii) PCR using culture lysate and (iii) PCR by colony touch method. In the present study P. multocida specific PCR was performed by using KMT1SP6 and KMT1T7 oligos. These oligos amplified the genomic DNA from P. multocida isolates only. All the three methods produced PCR amplified product at 460 bp and colony touch method was found to be the best method.
895-899 | JRB | 2013 | Vol 3 | No 3
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.
www.jresearchbiology.com
Journal of Research in Biology
An International Scientific
Research Journal
Authors:
Venkatesan PS,
Deecaraman M and
Vijayalakshmi M.
Institution:
Department of IBT,
Dr. M.G.R. Educational &
Research Institute,
Department of IBT,
Maduravoyal,
Chennai - 600095.
Corresponding author:
Venkatesan PS.
Email:
Web Address: http://jresearchbiology.com/documents/RA0332.pdf.
Dates: Received: 04 Feb 2013 Accepted: 05 Mar 2013 Published: 30 Apr 2013
Article Citation: Venkatesan PS, Deecaraman M and Vijayalakshmi. Identification of animal Pasteurellosis by PCR assay. Journal of Research in Biology (2013) 3(3): 895-899
Journal of Research in Biology An International Scientific Research Journal
Original Research
INTRODUCTION
The various forms of pasteurellosis caused by
Pasteurella multocida are the major health problem for
livestock population worldwide. Diagnosis of
pasteurellosis has become difficult, as there are five
different capsular types and 16 somatic types. Molecular
techniques like PCR are adapted nowadays for rapid and
accurate diagnosis in early stage of the disease and also it
provides useful information for epidemiological studies.
Pasteurellosis has high impact on economic status of
Indian farmers. The overall incidence rate of
haemorrhagic septicaemia (HS) was reported as
6.4 per lakh population during 1974-86, resulting in
losses exceeding ten million rupees annually
(Dutta et al., 1990; singh et al., 1996).
Isolation and identification of P. multocida from
specimens like fresh tissues or heart blood followed by
the performance of various biochemical and serological
methods have been used to study P. multocida. These
include catalase, indole, oxidase and sugar fermentation
tests. Due to time consuming procedure and limitations
of these methods, molecular techniques like polymerase
chain reaction (PCR) were adapted nowadays. PCR has
advantages over the conventional techniques in rapidity,
sensitivity and specificity to identify the P. multocida.
The present study was conducted to assess the efficiency
of PCR in the identification of P. multocida from poultry
and ruminants and to evaluate the different methods in
PCR assay viz. PCR using genomic DNA, PCR using
culture lysate and PCR by colony touch method.
MATERIALS AND METHODS
Isolation and Identification of P. multocida
Fifty two samples were collected from various
geographical areas of Tamil Nadu, India. Specimens
such as heart blood swab, liver, spleen and long bones
collected from various animals, were streaked directly
onto 5% sheep blood agar and Pasteurella multocida
selective agar as reported earlier (Moore et al., 1974) and
incubated at 37°C with 5-10 % CO2 for 24-48 h. Plates
were examined for colonies, the suspected colonies were
subjected to grams staining, and biochemical test as
per standard techniques. Standard vaccine strain of
P. multocida P52 (B:2) was taken as reference strain.
Pathogenicity test in mice were carried out for all the
fifteen isolates and PCR was performed for all the
isolates.
Isolation and Purification of Genomic DNA
A 900 µl cell suspension of each sample were
resuspended in 100 µl of 10x Tris-EDTA (TE) buffer
(pH 8.3) with 10 mg of lysozyme and were incubated at
37°C for 1.5 h. Bacterial cultures were treated with 10 µl
of proteinase K (10 mg/ml) and incubated at 50°C
for 1 h. The nucleic acid was extracted with
phenol-chloroform-isoamyl alcohol followed by ethanol
precipitation as per the method of Sambrook et al.,
(1989) and Sachithanandam et al., (2011).
PCR Using Culture Lysate
One Milliliter of 18 h broth culture or take few
freshly grown pure colonies from blood agar plate and
suspend in 500 µl sterile distilled water and centrifuge at
4000 g for 1 minute and collect the pellet. The pellet was
washed with sterile distilled water, resuspended in 100 µl
sterile distilled water and boiled for 10 min. The samples
were centrifuged to sediment cell debris and 10 µl of the
supernatant was used in the PCR reaction.
PCR Using Colony Touch Method
A single pure colony grown on agar plates was
used to perform PCR. A pipette tip was lightly touched
onto a colony and then suspend in PCR amplification
mixture.
PCR Technique
The species-specific primers KMT1SP6 and
KMT1T7 designed by Townsend et al., (1998) were used
in this study to amplify the gene sequences in
P. multocida.
Primers 1 KMT1SP6 5’-GCT GTA AAC GAA CTC
GCC AC- 3’
Venkatesan et al., 2013
896 Journal of Research in Biology (2013) 3(3): 895-899
Primers 2 KMT1T7 5’- ATC CGC TAT TTA CCC AGT
GG-3’
PCR mixture was prepared using PCR kit
obtained from FINNZYME, Finland. The 50 µl of
reaction mixture was prepared with 10 µl template DNA,
10ng of each primers, 200 µM concentration of each
dNTPs, 10x PCR buffer and 1 unit Taq DNA
polymerase. PCR amplification was carried out in an
automated thermal cycler (Perkin Elmer Gene AMP PCR
system 2400) with the following thermal programme.
Initial denaturising at 95°C for 4 min followed by
30 cycles of denaturising at 95°C for 1 min., annealing at
55°C for 1 min., extension at 72°C for 1 min. and final
extension at 72°C for 9 min, were carried out. After
amplification, PCR products were checked in
1.5% agarose gel electrophoresis along with the standard
molecular weight marker (Lambda DNA Hind III digest
and ϕ X 174 DNA Hae III digest; FINNZYME,
Finland).
The biochemical tests were carried out as per the
standard procedure followed in Arun kumar et al., (2012)
RESULTS
Out of total collection of 52 suspected samples,
procured from cattle sheep, goat and poultry, 15 samples
were confirmed as P. multocida based on biochemical
tests (Table 1) and PCR. All the P. multocida isolates
were pathogenic to mice and dies within 24 h. PCR was
performed for all the 15 isolates by 3 methods viz.,
colony touch method, culture lysate and with genomic
DNA. P52 strain of P. multocida, obtained from the
Institute of veterinary preventive medicine (IVPM)
Ranipet, Tamil Nadu, taken as a positive control
and the following bacteria Escherichia coli,
Clostridium chauvoei, Salmonella enteritidis,
Salmonella typhimurium, Bacillus anthracis,
Venkatesan et al., 2013
Journal of Research in Biology (2013) 3(3): 895-899 897
Tests Name of the Isolates
D1P D2P FP GP HP KP LP NP OP AS CS TS YS BG MC
Hemolysis on Blood agar
- - - - - - - - - - - - - - -
Growth on MacConkey agar
- - - - - - - - - - - - - - -
Motility - - - - - - - - - - - - - - - Gelatin Liquefaction - - - - - - - - - - - - - - - Methyl Red Test - - - - - - - - - - - - - - - H2S (Hydrogen sulphide)
+ + - - + + + - + + + + + + -
Catalase + + + + + + + + + + + + + + + Oxidase + + + + + + + + + + + + + + +
Nitrate Reduction + + + + + + + + + + + + + + + Indole + + + + + + + + + + + + + + + Lysine Decarboxylase - - - - - - - - - - - - - - - Ornithine Decarboxylase
- + + + + + - + + + + + + + +
Urease - - - - - - - - - - - - - - - Pyrase - - - - - - - - - - - - - - - Esculin Hydrolysis - - - - - - - - - - - - - - -
VT (Voges Proskaeur - - - - - - - - - - - - - - - Phenylalanine - - - - - - - - - - - - - - - β-Galactosidase (ONPG)
- - - - - - - - - - - - - - -
β-Glucuronidase + + + + + + + + + + + + + + + α-Galactosidase + + + + + + + + + + + + + + + β-Xylosidase - - - - - - - - - - - - - - - N-acetyl β-D-glucosaminedase
- - + + + + - + - - + + + + +
Table 1. Biochemical Profiles for the Identification of Pasteurella multocida Isolates
+ : Positive, - : Negative,
Venkatesan et al., 2013
898 Journal of Research in Biology (2013) 3(3): 895-899
Staphylococcus aureus and Klebsiella spp. used as
negative controls. The expected amplification size of
460 bp was obtained in all the 15 isolates. PCR
amplification was noticed at approximately 460 bp by all
the three methods and in all the 15 isolates as like that of
positive control (figure 1). No amplification product was
observed in negative controls (figure 1). Molecular
weight of PCR product was estimated based on the
standard molecular weight marker.
DISCUSSION
The 15 isolates of P.multocida collected from
different places and sources of origin produced
approximately 460 bp amplified product as that of
reference strain P52, but no amplified product was
noticed among the negative controls. It is concluded that
the primers were highly specific to P. multocida isolated
from various sources. The above result agrees with the
previous reports of earlier workers (Townsend et al.,
1998; Hunt et al., 2000; Miflin and Blackall, 2000; OIE
manual, 2000; Dutta et al., 2001). In this study the
amplified product of approximately 460 bp was observed
using three different methods viz. colony touch method,
culture lysate method and purified genomic DNA
method (figure 1). The intensity of the amplified PCR
product varies (figure 1), due to the variation in DNA
concentrations. Townsend et al., (1998) reported that
PCR using colony touch method produced amplification
Figure 1: Pasteurella multocida – specific PCR (PM-PCR) assay
CS TS YS BG MC PC NC M
460bp→
D1P D2P FP GP HP PC NC M
460bp→
KP LP NP OP AS PC NC M
These figures illustrate fragments specifically amplified by PCR in all the P. multocida isolates by means of the
primers KMT1SP6 and KMT1T7. Variation in the intensity of the amplified product was observed, due to variation in
DNA concentration of each sample.
D1P, D2P, FP, GP, HP, KP, LP, NP, OP, AS, CS, TS, YS, BG, and MC are the names of P. multocida isolates.
product approximately at 460 bp and the intensity of the
amplified product varied due to inconsistency of the
DNA concentration. Dabo et al., (2000) reported that the
boiled cell extract method has the advantages of
simplicity and rapidity in the identification of
P. multocida isolates. Since the PCR amplified product
of 460 bp was noticed in all samples of poultry and
ruminants, using oligos KMT1SP6 and KMT1T7, the
oligos are considered as specific to P. multocida
affecting all species of poultry and ruminants.
Considering the cost and time involved in the preparation
and purification of genomic DNA, the colony touch
method has advantages of simplicity and rapidity for
epidemiological surveys involving large number of
P. multocida isolates. PCR using colony touch method
would be an adaptable easy to perform method in
regional laboratories for rapid diagnosis of HS and FC
from field cases without the need to obtain pure culture
and extensive biochemical and serological tests.
ACKNOWLEDGEMENT
The authors thank the Head, department of
microbiology, Madras Veterinary College, Chennai, for
providing the facilities to carry out this work.
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Sharma GC. 1990. Epidemiological studies on
occurrence of haemorrhagic septicaemia in India. Indian
Veterinary Journal 67(10): 893-899.
Dutta TK, singh VP and Kumar AA. 2001. Rapid and
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assay. Indian Journal of Comparative Microbiology,
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Hunt ML, Alder B and Townsend KM. 2000. The
molecular biology of Pasteurella multocida. Veterinary
Microbiology 72(1-2):3-5.
Manual of Standards for diagnostics tests and
vaccines. 2000. Office International Des Epizootics
Manual, France. 446-456.
Sachithanandam V, Mohan PM, Dhivya P,
Muruganandam N, Baskaran R, Chaaithanya IK and
Vijayachari P. 2011. DNA barcoding, phylogenetic
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179-183.
Sambrook J, Fritisch EF and Mamiatis T. 1989.
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BS. 1996. Significance of Haemorrhagic Septicaemia in
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May 28-30.
Townsend KM, Frost AJ, Lee CW, Papadimitrion
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Microbiolgy 36(4):1096- 1100.
Venkatesan et al., 2013
Journal of Research in Biology (2013) 3(3): 895-899 899
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Biology
Source of light emission in a luminous mycelium of the fungus
Panellus stipticus
Keywords: Bioluminescence, Panellus stipticus, luminous mycelium, confocal microscopy.
ABSTRACT:
Mechanism of bioluminescence and light-emitting sources in higher fungi remain as an open question for a long time. We investigated the mycelium of cultivated luminous Panellus stipticus using confocal microscopy. No excitation light was imposed on the sample. Two types of sources of bioluminescence and their location were determined in the substrate mycelium. One were small 0.1-3 µm local formations disposed on the surface of hyphae, the other - relatively vast areas in bulk of the nutrient medium. No luminescence signal was recorded inside the hyphae. This may mean that the components of luminescent reaction are spatially separated within the cells, or the intracellular conditions block the reaction. The origin and formation of the light-emitting structures are discussed.
900-905 | JRB | 2013 | Vol 3 | No 3
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.
www.jresearchbiology.com
Journal of Research in Biology
An International Scientific
Research Journal
Authors:
Puzyr Alexey,
Burov Andrey and
Bondar Vladimir.
Institution:
1. Institute of Biophysics SB
RAS, Krasnoyarsk.
2. Special Design-
Technology Bureau "Nauka"
KSC SB RAS, Krasnoyarsk.
3. Institute of Biophysics SB
RAS, Siberian Federal
University Krasnoyarsk.
Corresponding author:
Burov Andrey.
Email:
Web Address: http://jresearchbiology.com/
documents/RA0345.pdf.
Dates: Received: 02 Apr 2013 Accepted: 27 Apr 2013 Published: 06 May 2013
Article Citation: Puzyr Alexey, Burov Andrey and Bondar Vladimir. Source of light emission in a luminous mycelium of the fungus Panellus stipticus. Journal of Research in Biology (2013) 3(3): 900-905
Journal of Research in Biology An International Scientific Research Journal
Original Research
INTRODUCTION
Bioluminescence in fungal cells, which involves
the emission of light generated by a chemical reaction,
has long attracted attention of scientists (Harvey, 1952;
Shimomura, 2006; Desjardin et al., 2008). Researchers
studying bioluminescence of fungi focus their efforts on
three key areas: (i) methods of cultivation under
laboratory conditions and characteristics of the light
emission (Weitz et al., 2001; Prasher et al., 2012; Dao,
2009; Mori et al., 2011), (ii) the molecular organization
of luminescence system and light emission mechanism
(Shimomura, 2006; Airth and McElroy, 1959;
Kamzolkina et al., 1983; Oliveira and Stevani, 2009;
Bondar et al., 2011), (iii) - application of fungal
luminescence in analytical techniques (Weitz et al.,
2002; Mendes and Stevani, 2010).
There has been little research conducted to
determine sources of luminescent light in the fungal
structures. To the best of our knowledge, only the
mycelium of Panus stipticus and Armillaria fusipes,
growing on agar were investigated for light source
detection (Berliner and Hovnanian, 1963). The used
photographic process allowed to record light from a
single hypha.
However, a low resolution of the technique
limited by the emulsion grain size denied localizing the
source of light. The authors of this, obviously, pioneer
work, suggested that the light was emitted over the entire
cell. Given the size of the objects under study, such
research should employ methods of microscopic
investigations. Calleja and Reynolds, who studied
Panus stipticus and Armillaria mellea by optical
microscope with EMI 4-stage image intensifier tube,
came to the conclusion that light emission in an
individual hypha was limited to a segment removed from
the apical point (Calleja and Reynolds, 1970). Absence
of later works related to structural and morphological
studies of mycelium of luminous fungi with microscopy
is astonishing as all known microscopic methods are
widely used to investigate non-luminous fungi
(Riquelme and Bartnicki-Garcia, 2008; Roberson et al.,
2011; Steinberg and Schuster, 2011).
In this report the mycelium of luminous
Panellus stipticus was studied using confocal
microscopy to determine and localize the source of light
emission. In our opinion it is important to find in
luminous fungi structures (or formations), which are the
light-emitting sources, and their location. On the one
hand, this can provide additional knowledge about
morphology of luminous fungi, on the other - might give
insight into molecular-cellular organization of fungal
luminescent system and mechanism of light emission.
MATERIALS AND METHODS
In this work we studied the culture of
Panellus stipticus luminous fungus (Bull:Fr.) Karst.,
IBSO 2301 (Figure 1). The mycelium was grown in
plastic Petri dishes at temperature 22°С on a commercial
nutrient medium Potato Dextrose Agar (HiMedia
Laboratories Pvt., India), or on richer medium containing
in 1 liter: 10 g of glucose, 5 g of peptone, 3 g of yeast
extract, 3 g of malt extract, 20 g of agar-agar. The
specimens exhibiting the highest light intensity were
selected for the experiments.
For confocal microscopy, a confocal laser
scanning microscope (LSM-780 NLO, Carl Zeiss,
Gottingen, Germany) equipped with a high sensitivity
GaAsP was used. Bioluminescence was recorded in the
accumulation mode with the 491–631 nm filter. The laser
was turned off (laser power = 0.0%) so that no excitation
light was imposed on the sample. This was done to avoid
fungal autofluorescence - emission of light by biological
substances such as flavins, lipofuscins and porphyrins
when excited by ultraviolet, violet, or blue light (Zizka
and Gabriel, 2008).
Images were processed using ZEN 2010 software
(version 6.0; Carl Zeiss). To prepare a specimen for
microscopy a fragment of agar with mycelium was cut
Puzyr et al., 2013
901 Journal of Research in Biology (2013) 3(3): 900-905
out and transferred to the cover glass.
RESULTS AND DISCUSSION
Figure 2 shows a 3D projection of the mycelium
by producing a Z-stack with 82 sections, 0.208 μm thick
each. No bioluminescence was detected from the aerial
mycelium. The light emission was recorded from the
surface of specimen to a depth of ~ 16 μm with
maximum intensity localized at the depth of Z= 6-8 μm
where the main body of mycelium was located. Only
isolated signals were detected at Z=8-16 μm that
confirmed that the agar did not contribute to the observed
bioluminescence.
Two types of sources emitting luminescent
signals could be distinguished. One light source were
small 0.1-3 µm local formations, associated with the
substrate hyphae, the other – vast areas in bulk of agar
(Figure 3). Light intensity recorded in the agar was much
higher than that of the local sites in the area of hyphae.
The use of the larger magnification (Figure 4) and bright
field microscopy (Figure 5a) suggests that the local
luminous sites are cellular excretions located on the
hyphae surface while vast luminescent areas are formed
by their aggregation in agar.
While presence of luminous sites on the surface
of hyphae could be assumed, finding of luminescent
areas in the agar came as a surprise. It is uncontroversial
that the recorded bioluminescent signals result from the
interaction of mixing light components synthesized by
the fungal cells. Luminescent signals were recorded by
the confocal microscope only when these components
were outside the cells. No bioluminescence inside
hyphae may mean that inside the cells the components of
luminescent reaction are spatially separated and do not
interact with each other, or the intracellular conditions
(pH, oxygen concentration, presence of inhibitors, etc.)
block the reaction.
One could argue that the surface of glowing
structures should be either hydrophobic or they have a
membrane enclosing the internal volume. Only under
these conditions components necessary for the
luminescent reaction do not mix with the water phase
contained within the nutrient medium. This suggestion is
based on the sharp boundaries exhibiting by both small
local formations on the walls of hyphae and vast areas in
the nutrient agar (Figure 5b).
So far it is not clear whether the luminous
structures containing components necessary for the
emission are formed within the fungal hypha or on/in
their surface. In the first case it requires a transport
system providing for the mechanism excreting the
Puzyr et al., 2013
Journal of Research in Biology (2013) 3(3): 900-905 902
Figure 1 View of culture of Panellus stipticus (IBSO 2301) growing on agar in natural light (A) and in the dark (B).
forming structures outside the cell. This is plausible
because the Golgi apparatus, that synthesizes secretory
vesicles containing products of vital functions and
excretes them from the cell, is well known. In the second
case on/in the wall cell there should exist structural
elements performing specialized secretory function.
On the basis of the results above we hypothesize
the following. Cells of P. stipticus synthesize and
localize the components required for bioluminescence in
structures which can originate within the cell and then
are moved on the outside surface of the hyphae by
a mechanism analogous to the mechanism of transport
via the Golgi complex. They can be also assumed to
form directly on/in outside surface of the hyphae by
structural elements of the cell possessing secretory
function. Such enclosed structures make possible to
concentrate the necessary components within a small
volume. Separation of luminous structures from the
surface of hyphae and their subsequent diffusion into the
bulk of the nutrient medium produce the vast
Puzyr et al., 2013
903 Journal of Research in Biology (2013) 3(3): 900-905
Figure 2 Fragment of 3D pattern of bioluminescence produced by P. stipticus.
Figure 3 Confocal luminescence image of the
P. stipticus mycelium. Figure 4 Confocal luminescence image of an
individual hyphae.
20µm
5µm
areas of luminescence in the agar.
CONCLUSION
Confocal microscopy due to its high resolution
and ability to record low light signals offers new
opportunities in investigation of fungal bioluminescence
system. Using this technique the sources of light
emission were identified for the first time in the
mycelium of P. stipticus (IBSO 2301) cultivated on agar
medium. One source were local formations disposed on
the surface of the substrate hyphae, the other – vast areas
in bulk of agar formed by aggregation of these luminous
structures. Further study is required for a detail
understanding whether the discovered structures are
specific for this fungus or they are common among other
luminous fungi.
ACKNOWLEDGEMENTS
The authors thank Mr. Barinov A.A. (OPTEC,
Novosibirsk) and Dr. Baiborodin S.I. (TsKP for
microscopic analysis of biological objects, SB RAS,
Novosibirsk) for technical assistance with confocal
microscopy. We are grateful to Dr. Medvedeva S.E.
(IBP SB RAS, Krasnoyarsk) for the cultivation of
luminescent fungi.
This work was supported: by the Federal Agency
for Science and Innovation within the Federal Special
Purpose Program (contract No 02.740.11.0766); by the
Program of the Government of Russian Federation
«Measures to Attract Leading Scientists to Russian
Educational Institutions» (grant No 11. G34.31.058); by
the Program of SB RAS (project No 71).
REFERENCES
Airth RL and McElroy WD. 1959. Light emission from
extracts of luminous fungi. J Bacteriol.;77(2):249-250.
Berliner MD and Hovnanian HP. 1963.
Autophotography of luminescent fungi. J Bacteriol. 86
(2):339-341.
Bondar VS, Puzyr AP, Purtov KV, Medvedeva SYe,
Rodicheva EK, Gitelson JI. 2011. The luminescent
system of the luminous fungus Neonothopanus nambi.
Doklady Biochem Biophys.;438(1):138-140.
Calleja GB, Reynolds GT. 1970. The oscillatory nature
of fungal bioluminescence. Trans Br Mycol Soc. 55:149-
154.
Dao TV. 2009. Pilot culturing of a luminous mushroom
Omphalotus af. illudent (Neonothropanus namibi).
Biotechnology in Russia. 6:29-37.
Desjardin DE, Oliveira AG, Stevani CV. 2008. Fungi
bioluminescence revisited. Photochem Photobiol Sci.;7
(2):170-182.
Harvey EN. Bioluminescence. New York: Academic
Press. 1952.
Puzyr et al., 2013
Journal of Research in Biology (2013) 3(3): 900-905 904
Figure 5 Confocal luminescence (A), bright field (B) and overlay (C) images of the substrate. Scale bar = 20 μm.
Kamzolkina OV, Danilov VS, Egorov NS. 1983.
Nature of luciferase from the bioluminescent fungus
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Mendes LF and Stevani CV. 2010. Evaluation of metal
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medium. Environ Toxicol Chem. ;29:320-326.
Mori K, Kojima S, Maki S, Hirano T, Niwa H. 2011.
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Mycena chlorophos. Luminescence. 26(6): 604-10.
Oliveira AG and Stevani CV. 2009. The enzymatic
nature of fungal bioluminescence. Photochem Photobiol
Sci. 8(10):1416-21.
Prasher IB, Chandel VC, Ahluwalia AS. 2012.
Influence of culture conditions on mycelial growth and
luminescence of Panellus stipticus (bull.) P. Karst. J Res
Biol. 2(3):152-9.
Riquelme M and Bartnicki-Garcia S. 2008. Advances
in understanding hyphal morphogenesis: ontogeny,
phylogeny and cellular localization of chitin synthases.
Fungal Biol. Rev.;22(2):56-70.
Roberson RW, Saucedo E, Maclean D, Propster J,
Unger B, Oneil TA, Parvanehgohar K, Cavanaugh C,
Steinberg G, Schuster M. 2011. The dynamic fungal
cell. Fungal Biol. Rev.;25(1):14–37.
Shimomura O. Bioluminescence: chemical principles
and methods. Singapore: World Scientific, 2006.
Weitz HJ, Ballard AL, Campbell CD, Killham K.
2001. The effect of culture conditions on the mycelial
growth and luminescence of naturally bioluminescent
fungi. FEMS Microbiol Lett. 202(2):165-170.
Weitz HJ, Colin D, Campbell CD, Killham K. 2002.
Development of a novel, bioluminescence-based, fungal
bioassay for toxicity testing. Environ Microbiol. 4(7):
422-429.
Puzyr et al., 2013
905 Journal of Research in Biology (2013) 3(3): 900-905
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Local people’s attitude towards conservation and development around
Pichavaram mangrove ecosystem, Tamil Nadu, India.
Keywords: Mangrove ecosystem, Livelihood, Attitudes, Conservation, Development.
ABSTRACT Studies in mangrove ecosystem are often focused on biological or ecological criteria and interdependency between mangroves and people is normally neglected. The situation is similar in Tamil Nadu; India which has a coastline of about 950 km. One of the major mangrove forests in Tamil Nadu is situated in Pichavaram, Cuddalore district. The present study was carried out in the seventeen hamlets, which are directly or indirectly dependent on the Pichavaram mangrove wetlands for their livelihood and survival. These seventeen hamlets consist of over 2600 households many of whom derive their principal income from fishing and related activities. Individual surveys were carried out for 10% of the households in each of the selected hamlets. Semi-structured questionnaires were used for surveys to study the attitude and perception of the community on the conservation and importance of mangrove wetlands and resources. The study was conducted to assess the awareness, attitudes and views of people dependent on the mangrove ecosystem towards conservation issues and development options. It was observed that a large percentage of the sampled population showed a positive inclination towards conservation of the ecosystem and were well aware of their responsibility towards it.
906-910 | JRB | 2013 | Vol 3 | No 3
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www.jresearchbiology.com Journal of Research in Biology
An International Scientific
Research Journal
Authors:
Lakshmi Kodoth and
Ramamoorthy D.
Institution:
Department of Ecology &
Environmental Sciences,
Pondicherry University,
Puducherry.
Corresponding author:
Lakshmi Kodoth.
Email:
Web Address: http://jresearchbiology.com/documents/RA0274.pdf.
Dates: Received: 08 Aug 2012 Accepted: 26 Aug 2012 Published: 06 May 2013
Article Citation: Lakshmi Kodoth and Ramamoorthy D. Local people’s attitude towards conservation and development around Pichavaram mangrove ecosystem, Tamil Nadu, India. Journal of Research in Biology (2013) 3(3): 906-910
Journal of Research in Biology
Original Research
An International Scientific Research Journal
INTRODUCTION
The Mangrove ecosystem has been studied
extensively by scientists more in the ecological and
biological sense. During the 1980s and early 1990s, more
attention was given to research involving the human
interactions with the forested wetlands (FAO, 1985;
Hamilton et al., 1989; FAO, 1994; Cormier-Salem,
1999). Mangrove wetlands are a dominant feature of the
intertidal areas of the tropical and subtropical regions in
between 25°N and 25°S latitudes. The mangrove
ecosystem provides a number of ecological services:
provision of plant and animal products (Macnae, 1974;
Rasolofo, 1997; Spaninks and Beukering, 1997),
sediment trapping and nutrient uptake and transformation
(Furukawa et al., 1997; Hussain and Badola, 2008), they
provide detritus food for the aquatic fauna, harbour
migratory and aquatic birds, serve as spawning ground
for fishes, mussels and prawns. They also act as a natural
shield against storms and tidal waves (Kathiresan and
Rajendran, 2005).
The coastal communities are largely dependent on
the mangrove forests for firewood, timber, honey, fodder
and for its fishery resources. Most coastal communities
in the tropics are significantly dependent on the harvest
of marine and coastal resources for sustaining their
livelihoods (Kunstadter et al., 1986). The majority of
people living near the mangrove areas derive their
income predominantly from fishing and related activities.
Hence, the present study was carried out as it is essential
to understand people’s attitude and perception towards
the mangrove ecosystem as they derive their livelihood
from it; it helps us in formulating better policies and
enhances the developmental plan for the ecosystem.
Study Area
India has a coastline of 7,516 km of which Tamil
Nadu has about 950 km. Extensive mangrove wetlands
are located in two places namely, i) in Pichavaram,
Cuddalore district and ii) Muthupet in Thivarur and
Tanjore districts.
The Pichavaram mangrove wetland is located in
the northern extreme of Cauvery delta between the
Vellar and Coleroon estuaries (figure 1). Geographically,
it is located between 79°47’E longitude and 11°27’N
latitude. The Pichavaram mangrove forests have an area
of about 1,350 ha, which are colonised by 13 true
mangrove species. Rhizophora Sp and Avicennia Sp are
the predominant mangrove species present in the
Pichavaram mangrove forests. Pichavaram mangrove
wetland is also rich in its fishery resources (figure 2).
Annually about 245 tons of fishery resources are
harvested from this mangrove wetland, of which prawns
alone contribute 85% of the catch (Selvam et al., 2003).
Methods
People belonging to 17 hamlets surrounding the
Kodoth and Ramamoorthy, 2013
907 Journal of Research in Biology (2013) 3(3): 906-910
Figure 1 Glimpse of the Pichavaram mangrove forest
Figure 2 Fishing in the mangrove backwaters
Pichavaram mangroves wetland were selected for survey.
For each selected hamlet 10% of the households were
picked up randomly for the household survey. Using
semi-structured questionnaires, information on the
demography, land use, income and occupational pattern
as well as local dependence on the mangrove resources
were gathered (Badola and Hussain, 2003; Glaser,2003) .
Few open ended questions were also included to
determine the attitude and perception of villagers
towards development and conservation issues. A total of
324 households were surveyed. The responses we got
were mostly in terms of yes, no and we don’t know.
RESULTS AND DISCUSSION
Assess the awareness and views towards conservation
The results (Table 1) showed that majority of the
respondents i.e. 91% (n=324) were aware that
Pichavaram mangrove was as declared protected area
and this awareness was gained largely because of,
NGO’s working in that area and the forest department.
An overwhelming percentage (84%) of the local
population felt responsible towards the protection of the
mangrove ecosystem and another 76.7% are in favour of
eco-development projects in the area. Out of the 324
respondents, 67% of the people are willing to cooperate
with the forest department for the same. Only a small
percentage of people feel their rights being violated
because of the protected area status if the ecosystem.
When questioned regarding their views on
eco-development initiatives and its implementation, a
majority of the respondents (44.7%) were in favour of
the community led initiatives. 32% felt that NGO’s
should take lead in eco-development and the rest
23% felt that the government should take up
eco-developmental projects by itself ( Table 2).
The importance of the mangrove forests to the
local population was emphasized when a majority of
people were against cutting down of the forests. A
majority of the respondents (71%) felt that more
mangrove plantations need to be carried out, while
28.4% felt that the present conditions of the mangrove
forests were good (Table 3).
Kodoth and Ramamoorthy, 2013
Journal of Research in Biology (2013) 3(3): 906-910 908
Table 1: Attitude of people towards Pichavaram Mangrove Ecosystem and conservation (n= 324)
Questions Yes (%) No (%) Don’t Know (%)
Are you aware that Pichavaram Mangrove Ecosystem is declared
as Protected area? 91 9 -
Do you feel any sense of responsibility for the protection of the
ecosystem? 84 13.8 2.2
Do you feel your rights have been violated after the declaration of
PA? 11.9 80.5 7.6
Do you face any problem because of PA? 15.8 78.6 5.6
Are you in favour of the implementation of an ecodevelopment
project? 76.7 15.3 8
Would you like to co-operate with the forest department with regard
to the ecodevelopment project? 67 23 10
Table 2: View of respondents towards
Eco-Development itiatives (n = 324)
Views Frequency Percentage
Want through govt.
initiative 75 23.1
Want through
Community initiative 145 44.7
Want through NGO
initiative 104 32
Table 3: View of local people towards various
management alternatives (n = 324)
Management Alternatives Responses (%)
Forests should be cut and land used
for other purposes 0.6
Current situation of protecting the
forests is good 28.4
Increase in mangrove plantations
needed 71
The findings in this study are similar to that of
the study in Bitarkanika mangrove ecosystem in Orissa
(Badola and Hussain, 2003) which shows that the
villagers are well aware of their responsibility to the
ecosystem and willing to participate in the conservation
efforts of both the government and NGO’s.
Developmental Options
Recently, eco tourism has been promoted to a
large extent in Pichavaram mangrove forests. Majority of
the respondents (76%) were in favour of developing eco
tourism as it will improve job opportunities for the local
population. Shrimp farms are not favoured in the area as
83% of the responses were against setting up of such
farms. This is primarily due to the fact that shrimp farms
in the area are the reason for increase in salinity of the
canal water (Table 4).
Ecological functions and values identified by local
community
The respondents were given a list of ecological
functions to find out how much they were aware of the
functions and its direct or indirect importance in their
livelihoods.
Table 5 shows ranking of use values, 76% gave
highest ranking to contribution of mangroves towards
fishing. 63% gave agriculture as their second preference.
Incase of ranking ecological functions performed by
the Pichavaram mangrove ecosystem, 77.8% of the
responses favoured Tsunami/cyclone mitigation. 67.2%
gave second preference to nutrient cycling (Table 6).
The results show that the respondents were aware
of both the direct and indirect benefits of the mangrove
ecosystem. It is evident from the results that people value
the uses or function which more beneficial to them in
their day today lives.
CONCLUSION
The results showed that in general people have a
positive attitude towards conservation and are aware of
their responsibility in sustaining these mangrove forests.
The socio economic and market conditions influence the
people’s attitude towards the resources. Eco
developmental plans were in favour with the local
population since it will be helpful in formulating
sustainable policies for ecosystem. The promotion of eco
tourism in the area had a largely positive response hence
it should be capitalised on to improve local economy.
Inclusion of the local people in decision making process
can lead to successful management of the Pichavaram
mangrove ecosystem.
REFERENCE
Badola R and Hussain SA. 2003. Valuation of the
Bhitarkanika mangrove ecosystem for ecological security
and sustainable resource use. Study report. Wildlife
Institute of India, Dehra Dun.
Kodoth and Ramamoorthy, 2013
909 Journal of Research in Biology (2013) 3(3): 906-910
Table 5: Ranking of the use values in Percentage
(n=324)
Use values Rank 1 (%) Rank 2 (%) Rank 3 (%)
Fishing 76 18 6
Agriculture 26 63 11
Tourism 35 56 9
Table 6: Percentage ranking of various functions
(n=324)
Ecological
functions
Rank 1
(%)
Rank 2
(%)
Rank 3
(%)
Fish 59.4 34.3 6.3
Aesthetic 38 59 3
Tsunami/cyclone
mitigation 77.8 22.2 0
Nutrient 32.2 67.2 0.6
Table 4: View of local people towards various developmental options (n = 324)
Queries Yes (%) No (%) Don’t know(%)
Are you in favour of developing eco tourism in the area 76 16 8
Are you in favour of shrimp farms 8 83 9
Has Shrimp farms been useful to you? (n=14) 47 46 7
Cormier-Salem MC. 1999. The Mangrove: an area to
be cleared…for social scientists. Hydrobiologia. 413:
135-142.
FAO. 1985. Mangrove management in Thailand,
Malaysia and Indonesia. FAO Environment Paper 4,
Food and Agriculture Organization of the United
Nations, Rome.
FAO. 1994. Mangrove forest management guidelines.
FAO Forestry Paper 117, Food and Agriculture
Organization of the United Nations, Rome.
Glaser M. 2003. Interrelations between mangrove
ecosystem, local economy and social sustainability in
Caete Estuary, North Brazil. Wetland Ecology and
Management. 11:265-272.
Furukawa K, Wolanski E and Mueller H. 1997.
Currents and sediment transport in mangrove forests.
Estuarine, Coastal and Shelf Science. 44:301-310.
Hamilton LS, Dixon JA and Miller GO. 1989.
Mangrove forests: an undervalued resource of the land
and of the sea. In: Borgese EM, Ginsburg N, Morgan JR.
(Eds.), Ocean Yearbook 8. University of Chicago Press,
Chicago. 254-288.
Hussain SA and Badola R. 2008. Valuing mangrove
ecosystem services: linking nutrient retention function of
mangrove forests to enhanced agroecosystem production.
Wetlands Ecology and Management. 16:441-450.
Kathiresan K and Rajendran N. 2005. Coastal
mangrove forests mitigated tsunami. Estuarine, Coastal
and Shelf Science. 65:601-606.
Kunstadter P, Bird ECF and Sabhasri S. (Eds.). 1986.
Man in the Mangroves. United Nations University,
Tokyo.
Macnae W. 1974. Mangrove forest and fisheries. FAO/
UNDP Indian Ocean Fishery Programme. Indian Ocean
Fishery Commission. Publication IOFCDev. 74:34-35.
Rasolofo MV. 1997. Use of mangroves by traditional
fishermen in Madagascar. Mangroves Salt Marshes.
1:243-253.
Selvam V, Ravichandran KK, Gnanappazham L and
Navamuniyammal M. 2003. Assessment of
community-based restoration of Pichavaram mangrove
wetland using remote sensing data. Current Science.
85:6,794-798.
Spaninks F and Beukering PV. 1997. Economic
Valuation of Mangrove Ecosystems: Potential and
Limitations. CREED Working 14.
Kodoth and Ramamoorthy, 2013
Journal of Research in Biology (2013) 3(3): 906-910 910
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Biodegradation of phenol at low and high doses by bacterial strains indigenous to
Okrika River in the Niger Delta of Nigeria
Keywords: Biodegradation, phenol, bacteria, Okrika River.
ABSTRACT: Assessments on biodegradation at low and high doses of phenol by bacterial strains indigenous to Okrika River in Niger Delta of Nigeria were carried out. Growth at low dose of 0.01 µg/l phenol showed that highest and lowest cell density values of OD540nm of 0.15 and 0.09 in Pseudomonas sp. SD1 and Citrobacter sp. RW1 while at 1.0 µg/l phenol concentration the highest cell density values of OD540nm of 0.28 was observed in Staphylococcus sp. RW2. The highest specific growth rate of 0.019 h-1 at 500 mg/l of phenol was obtained for Pseudomonas sp. SD1 while Citrobacter sp. RW1 had the lowest specific growth rate of 0.014 h-1 at 500 mg/l of phenol. The specific phenol degradation rate ranges from 55.35 to 130.98 mg/(L.h.OD). The order of specific phenol consumption rate at 1000 mg/l by the organisms is: Bacillus sp. SD3>Pseudomonas sp. SD1>Citrobacter sp. RW1>Staphylococcus sp. RW2. Citrobacter sp. RW1 exhibited highest growth yield in 250 mg/l of phenol with the growth yield of 6.24 (x 10-4 A540 unit.l/mg). The results showed that the test organisms might be the most suitable bacterial strains for removal of phenols at low and high doses in phenolic polluted media.
911-921| JRB | 2013 | Vol 3 | No 3
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.
www.jresearchbiology.com Journal of Research in biology
An International Scientific
Research Journal
Authors:
Nwanyanwu CE 1*
Abu GO2.
Institution:
1.Department of
Microbiology, Federal
university of Technology,
P.M.B.1526, Owerri,
Nigeria.
2.Department of
Microbiology, University of
Port Harcourt, P.M.B. 5323, Port Harcourt, Nigeria.
Corresponding author:
Nwanyanwu CE.
Email: [email protected]
Web Address: http://jresearchbiology.com/documents/RA0318.pdf.
Dates: Received: 26 Dec 2012 Accepted: 17 Jan 2013 Published: 06 May 2013
Article Citation: Nwanyanwu CE and Abu GO.
Biodegradation of phenol at low and high doses by bacterial strains indigenous to Okrika River in the Niger Delta of Nigeria. Journal of Research in Biology (2013) 3(3): 911-921
Journal of Research in Biology An International Scientific Research Journal
Original Research
INTRODUCTION
Contamination of aquatic environment brought
about by the discharge of wastewater resulting from
anthropogenic activities clearly continues to be a major
environmental issue. Effluents are very important
sources of chemicals entering aquatic ecosystems. They
may contain hundreds, or even thousands of chemicals,
but only a few of them are responsible for effluent
toxicity (Tisler et al., 1999). High strength wastewaters
have been reported to be associated with chemical
processing industries. Wastewaters generated from these
processing industries such as petrochemical, oil
refineries, coke-processing plants, etc contain a large
number of organic and inorganic pollutants at high
concentrations that exhibit adverse effect on the
environments when released (Papadimitriou et al., 2009).
The presence of high level of these contaminants formed
the major pollutant in the water body as a result of
continuous discharge of effluents by industries into the
ecosystem. In water these pollutants of the discharged
effluent sorbs onto particulate materials and if not
degraded eventually end up in sediments. As an ultimate
respiratory of most xenobiotic contaminants that enter
water bodies, sediments act as both carrier and sources of
contaminants in aquatic environment (Akan et al., 2010).
Thus, the contaminated sediments may represent a
continual threat of recontamination of the aquatic
environment as the adsorbed pollutants if not degraded,
in turn lead to the exposure of aquatic life to organic
pollutants such as phenol (Mort and Dean-Ross, 1994).
On the other hand, the release of contaminants from
sediments could increase the amount of toxic compounds
in the waters making them more available to organisms
and affecting their life cycles, reproduction, metabolism
and physiology. Microorganisms being ubiquitous in
nature exploit many carbon and energy sources in its
niche for growth. Several species of micro-organisms
inhabiting hostile ecological niche have been reported by
Colwell and Walker (1977), Atlas (1981), Heinaru et al.
(2000) and Polymenakou and Stephanou (2005).
Microorganisms indigenous to aquatic environment are
crucial for the biodegradation of organic matter and the
cycling of nutrients, while these microorganisms are
susceptible to toxic pollutants from industrial effluent
discharges, especially petroleum refinery. Therefore,
perturbations of aquatic microbial communities could
have consequences for the higher trophic levels and for
the overall aquatic environment.
The composition of effluents from petroleum
refineries varies according to their origin, storage and
treatments as these wastewaters are enriched with
different pollutants. Phenol and its derivatives along with
other organic and inorganic compounds is one of the
most common contaminants present in refinery effluents
(Jena et al., 2005) which renders refinery effluents its
toxic nature. Phenols as constituents of industrial
effluents may remain in water body for much longer
period if it is continually or consistently released into the
aquatic environments from sources thereby increasing its
elevation in the environment. The toxic nature of phenol
and its derivatives to microbial cells is well documented
(Kahru et al., 2002; Keweloh et al., 1990). Owing to
toxic nature of phenol, its contact with microorganisms
always results in the decrease of microbial enzyme
activity (Nwanyanwu and Abu, 2011) as well as leading
to death of organisms at higher concentration.
A large number of microbial genera possess the
capability to degrade organic pollutants. Among the
bacterial genera implicated in the degradation of phenol
include Pseudomonas, Bacillus, Corynebacterium
species etc. The ability of organisms to degradation
phenol and other toxicants is related to adaptation of the
microorganisms to the compound of concern and
adaptation is associated with synthesis of new enzymes
capable of transformation of the toxicant to harmless
substances (Jaromir and Wirgiliusz, 2007). The resultant
effect of biodegradation of phenol and other organic
compounds is growth as the organic pollutants are used
Nwanyanwu and Abu, 2013
912 Journal of Research in Biology (2013) 3(3): 911-921
as the source of carbon and energy.
This research assessed the growth and utilization
of phenol at low and high doses by bacterial strains
indigenous to Okrika River in the Niger Delta of Nigeria.
MATERIALS AND METHODS
Chemical reagents
All chemical reagents used in the study were of
analytical grade and were obtained from sigma chemical
company, St Louis Missouri, USA, BDH chemicals,
Poole, England and HACH chemical company.
Sample collection and analysis
The Okrika River is a small tidal river that
empties into Bonny estuary in Niger Delta of Nigeria.
The River is highly polluted as a result of effluent
discharges from Port Harcourt petroleum refinery
industry sited along its bank (IAIA09 Conference
Proceeding, 2009). Sediment and water samples were
collected from the river as described by Nweke et al.,
(2007) and the samples analyzed within few hours of
collection. The results of the physicochemical analysis of
the samples are as shown in Table 1.
Isolation and identification of bacterial strains
The bacterial strains used in this work were
isolated from the samples by spreading one tenth of
decimally diluted sediment suspension and water
samples on mineral salt agar-phenol (2.5 mM) medium
and the isolated organisms identified as described
elsewhere (Nwanyanwu et al., 2012). The isolates were
designated according to their sources (RW for River
water, SD for sediment) and were then maintained on
nutrient agar slants.
Preparation of inoculum
The bacterial strains used for the assay were
grown in 100 ml of sterile nutrient broth media for 48 h.
The turbid culture medium were harvested, washed
and suspended in deionized distilled water then
followed by standardization of the suspensions
spectrophotometrically to an optical density of
0.4 at 540 nm and used as inocula.
Assay for isolates growth in very low concentrations
of phenol
The ability of the isolates to grow and utilize
phenol at low concentrations (0-1.0 µg/l) was assessed in
sterile Bushnell Haas (BH) mineral salt broth medium.
The assay was carried out as described by
Nwanyanwu et al., (2012) with little modification. The
medium without agar was used instead for the assay.
After inoculation of the flasks, growth profile of the
organisms was monitored by the optical density
(OD540nm) on daily basis.
Growth and biodegradation of phenol at high
concentration
Degradation of phenol at high concentration by
the organisms was carried out in sterile BH medium
contained Erlenmeyer flasks. The flasks were
supplemented with aliquot of sterile phenol (2000 mg/l)
to bring the final phenol concentrations in the flasks to
250, 500, 750 and 1000 mg/l. The flasks after inoculation
with the test organisms were incubated at 30oC in an
incubator. At predetermined time, samples were
withdrawn to determine cell growth and phenol
concentration. Controls, one without phenol and another
without cells in BH medium were set up. At
predetermined time, samples were removed and used to
measure for cell growth (optical density, OD540nm) and
Nwanyanwu and Abu, 2013
Journal of Research in Biology (2013) 3(3): 911-921 913
Table 1: Physicochemical characteristics
of Okrika River
Parameter/unit
Sample source
Water Sediment
pH 8.90 6.90
Elect. conduc. (µscm-1) 364 615
Oil and grease (mg/l) 16.0 103.0
BOD (mg/l) 8.16 -
COD (mg/l) 84.0 -
PO4 (mg/l) 0.15 0.90
SO4 (mg/l) 118 117
Phenol (mg/l) 6.1 15.5
Zn (mg/l) 0.03 3.48
Cu (mg/l) <0.01 0.06
Pb (mg/l) <0.01 <0.01
phenol residue (4-amino antipyrine) in cell free samples.
Analytical methods
C e l l g r o w t h w a s d e t e r m i n e d
spectrophotometrically while phenol was analyzed by
photometric method using 4-aminoantipyrine as the
colouring agent and measuring the absorbance at 500 nm
(Folsom et al., 1990).
Data Analysis
Specific growth rate
The specific growth rate (µ) for each
concentration of phenol was calculated from the slope of
linear logarithmic plots of optical density against time as
expressed in equation 1 (Gokulakrishnan and Gummadi,
2006):
Specific degradation rate
The specific degradation rate (Qs) was
determined through the relationship of equation 2 (Loh
and Wang, 1998):
Where: [Ph] denotes phenol concentration
(mg/l), t denotes incubation time (h) and X denotes cell
concentration (optical density, OD540 nm).
Yield factor
Yield factor (Y) of the biomass was calculated
using equation 3 (Bajaj et al., 2009):
Where dX is the change in cell biomass related to
the change in substrate concentration dS. X was replaced
with the OD at 540 nm.
RESULTS AND DISCUSSION
The phenol content of Okrika River water and
sediment were 6.1 and 15.5 mg/l while oil and grease of
the River water and sediment were 16.0 and 103.0 mg/l
respectively (Table 1). This level of oil and grease as
well as phenol in the River water and sediment were
much higher than the previously reported levels of
10.56 and 15.23 mg/l (oil and grease) and 5.13 and
16.0 mg/l (phenol) (Otokunefor and Obiukwu, 2005).
This indicated that these compounds have accumulated
in Okrika River over time and pose the major pollutants
of the river.
Figure 1 shows the growth of the test organisms
in low concentration of phenol amended mineral salt
medium. All the organisms showed progressive growth
in low phenol concentration medium. Highest growth of
the organisms was observed in phenol concentration of
1.0 µg/l followed by 0.1 µg/l. The least growth was
observed in 0.01 µg/l. Among the test organisms,
Staphylococcus sp. RW2 showed the highest growth in
0.1 and 1.0 µg/l of phenol with optical density (OD)
values of 0.23 and 0.28 respectively while Citrobacter
sp. RW1 showed the least growth in all the low
concentrations (0.01, 0.1 and 1.0 µg/l) of phenol
amended medium with OD values of 0.09, 0.11 and 0.13
respectively. Growth of microorganisms especially
bacterial species at phenol concentration as low as
microgram per litre have been reported by many authors.
Chesney et al., (1985) have reported growth of water
microorganism in water sample supplemented with 0.001
to 1.0 µg/ml of phenol. Also Goldstein et al., (1985)
have reported the growth of Pseudomonas sp. in a
112
12
tt
XXIn
2/
X
dtPhdQs
3dS
dXY
Nwanyanwu and Abu, 2013
914 Journal of Research in Biology (2013) 3(3): 911-921
Table 2: Yield factor (Y) of biomass after growth at
different initial phenol concentrations
Bacteria
Yield factor, Y(x 10-4A540
unitsa. l/mg)
Phenol concentration (mg/l)
250 500 750 1000
Citrobacter sp. RW1 6.24 4.46 2.69 3.11
Staphylococcus sp.RW2 4.96 3.80 3.28 3.00
Pseudomonas sp. SD1 4.96 3.80 3.28 3.00
Bacillus sp. SD3 3.28 4.46 2.69 3.11 a A540 units = optical density at 540 nm
medium amended with 1.0 and 10.0 µg/l concentration of
2, 4-dichlorophenol. Pahm and Alexander (1993) found
that Pseudomonas sp. K, Flavobacterium sp. M4,
Flavobacterium sp. M1 and Pseudomonas sp. SP3 grown
in p-nitrophenol (PNP) of concentration of 0.1 µg/l
reached a total viable count of 105 and 106 cells/ml.
Figures 2 and 3 showed typical profiles of cell
growth and biodegradation of phenol at high
concentrations by bacterial strains of Okrika River
ranging from 250 to 1000 mg/l. The lag phase of the
organisms in phenol fortified medium was short. The
short in lag phase period depends on the pre-exposure of
the organism. Phenol was completely utilized by the
isolates within 180 h of incubation. Phenol
concentrations of 500, 750 and 1000 mg/l was degraded
completely within 96, 132 and 156 h by Pseudomonas
sp. SD1 while same concentrations of phenol was
degraded completely within 108, 144 and 180 h by other
test organisms. Time-dependent degradation of organic
compounds has been reported to be linked with
concentration of the organic compound as observed by
many authors (Colwell and Walker, 1977; Kotresha and
Vidyasagar, 2008). This may be due to changes in the
transport mechanism of the substrate across the cell
membrane in response to high phenol concentration
hence diminished capacity to catabolize phenol. This is
in line with the reports of Gilbert and Brown (1978),
Keweloh et al., (1990), Collins and Daugulis (1997) and
Nwanyanwu and Abu, 2013
Journal of Research in Biology (2013) 3(3): 911-921 915
Figure 1: Growth profile of the bacteria in mineral salt medium fortified
with phenol concentrations
Time (h)
Ab
sorb
an
ce (
A54
0 n
m)
Nwanyanwu and Abu (2011) who observed the toxic
effect of phenol at the membrane level, thereby
disrupting the activity of enzymes in phenol-utilizing
bacteria. Also, Joseph and Joseph (1999) and Ye and
Shen (2004) reported that phenol toxicity depends on the
sensitivity as well as source of organism.
The growth profiles of the pure cultures expressed
as optical density and phenol residues at different initial
concentrations are shown in figures 2 and 3. The cells
gradually increase in number as the phenol residues of
the medium progressively decreased. This may be due to
high phenol concentration made available more carbon to
the organism for growth. Pseudomonas sp. SD1
degraded 1000 mg/l of phenol in 160 h with a cell
biomass (OD540nm) of 0.363.
The dependence of specific growth rate on
phenol concentration is shown in Figure 4. From this
plot, the specific growth rate increased with increase in
the initial phenol concentration upto 250 mg/l and then a
progress decrease started with increase in phenol
Nwanyanwu and Abu, 2013
916 Journal of Research in Biology (2013) 3(3): 911-921
Ab
sorb
an
ce (
A54
0 n
m)
Figure 2: Biodegradation and cell growth profile of planktonic bacteria of Okrika River in high
phenol concentrations
Time (h)
Ph
en
ol
(mg
/l)
concentration. In the present study, at 500 mg/l of phenol
concentration, the specific growth rate of Pseudomonas
sp. SD1 is increased (highest µ =0.017 h-1). For
concentration higher than 500 mg/l, the specific growth
rate of Pseudomonas sp. SD1 decreases and became
almost constant at 750 mg/l (µ = 0.011 h-1) and
1000 mg/l (µ = 0.011 h-1) of phenol. This is quite similar
to the result obtained by Dey and Mukherjee (2010) who
observed increase in specific growth rate (0.093 h-1) of
mixed microbial culture up to 300 mg/l of initial phenol
concentration and then started decreasing to a constant
(0.057 h-1) at 600 and 700 mg/l of phenol. This trend
suggested that the phenol is an inhibitory substrate. Thus
the parameter has been found to be a strong function of
initial phenol concentration. At 250 and 500 mg/l, the
highest specific growth rate values of 0.026 and 0.017 h-1
were observed in Citrobacter sp. RW1 and Pseudomonas
sp. SD1 respectively while the lowest specific growth
rate of 0.016 and 0.014 h-1 at the same concentration of
phenol was observed in Pseudomonas sp. SD1 and
Nwanyanwu and Abu, 2013
Journal of Research in Biology (2013) 3(3): 911-921 917
Figure 3: Biodegradation and cell growth profile of sediment bacteria of Okrika River in high
phenol concentrations
Time (h)
Ph
en
ol
(mg
/l)
Ab
sorb
an
ce (
A54
0 n
m)
Citrobacter sp. RW1 respectively. However, the
growth rates of the test organisms are similar
to that of Pseudomonas aeruginosa and
Pseudomonas pseudomallei degrading phenol in saline
solutions (Afzal et al., 2007).
The specific rate of phenol degradation of the
organisms is depicted in figure 5. The specific
degradation rate (Qs), was estimated by correlating
phenol concentration versus culture time using
regression technique in Microsoft Excel to obtain the
equation of best fit of the degradation curve. The
correlation were differentiated with respect to time and
then divided by the cell mass (Loh and Wang, 1998).
The specific degradation (consumption) rate of a
compound was suggested to be a measure of microbe
performance. The highest specific consumption rate of
phenol was observed in Bacillus sp. SD3 with specific
degradation rate value of 130.98 mg/(L.h.OD) at
1000 mg/l while Staphylococcus sp. RW2 showed the
least specific consumption rate of phenol with a specific
degradation rate value of 99.83 mg/(L.h.OD) at the same
concentration. The organisms in this work showed a
robust decrease in specific degradation rate as the phenol
concentration decreases. This is in line with the work of
Cho et al., (2000) who observed an increase in specific
degradation rate as phenol concentration increases in
their assessment of influence of phenol on
biodegradation of p-nitrophenol by freely suspended and
immobilized Nocardioides sp. NSP41. Agarry and
Solomon (2008) also made similar reports in their work
on kinetics of batch microbial degradation of phenols by
indigenous Pseudomonas fluorescence.
Table 2 shows the growth yield of the test
organisms expressed as absorbance, A at 540nm unit litre
of cells produced per mg of phenol substrate utilized.
The growth yield varied among the test organisms
ranging from 2. 69 to 6.24 (x 10-4A540 units. l/mg). High
growth yield were obtained at low concentration of
toxicant (phenol) while low values of growth yield were
obtained at high phenol concentration. At 250 mg/l
highest and lowest growth yield were observed in
Citrobacter sp. RW1 and Bacilllus sp. SD3 with cell
yield coefficients of 6.24 and 3.28 (x 10-4A540 units.l/mg)
respectively. The higher value of Y observed in
Citrobacter sp. RW1 indicate that phenol was degraded
very efficiently by the organism. All the growth yields
Nwanyanwu and Abu, 2013
918 Journal of Research in Biology (2013) 3(3): 911-921
Phenol, So (mg/l)
Figure 4: Specific growth rate of the organisms
at different initial phenol concentrations
Sp
ecif
ic g
row
th r
ate
(h
-1)
Figure 5: Specific degradation rate at different
initial phenol concentrations by the bacterial strains
Phenol, So (mg/l)
Sp
ecif
ic d
egra
dati
on
rate
(m
g/(
L.h
.OD
))
reported here were lower than those reported by other
authors. Yield coefficients of 0.14 and 0.16 have been
reported (Bajaj et al., 2009). The yield coefficients
reported by Yoong et al., (1997) are 0.16 and 0.27.
As Citrobacter sp. RW1, Staphylococcus sp. RW2,
Pseudomonas sp. SD1and Bacillus sp. SD3 shown high
specific phenol consumption rate, they have
demonstrated strong potential to utilize and grow in
phenol of low and high phenol concentrations of upto
1000 mg/l. This indicated that these strains have great
potential for application in the treatment of phenolic
wastewater and in the bioremediation of phenol impacted
media.
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Journal of Research in Biology (2013) 3(3): 911-921 921
Nwanyanwu and Abu, 2013
Jou
rn
al of R
esearch
in
Biology
Phenol and Heavy Metal Tolerance Among
Petroleum Refinery Effluent Bacteria
Keywords: Phenol, heavy metals, refinery effluent bacteria.
ABSTRACT: Bacterial isolates from petroleum refinery effluent were evaluated for growth in increasing doses of phenol and heavy metal ions. All the test organisms were able to grow in mineral salt medium with phenol concentration of 15.0 mM (≈ 1412.0 mg/l) except Pseudomonas sp. RBD3. Aeromonas sp. RBD4, Staphylococcus sp. RBD5 and Pseudomonas sp. RBD10 showed the highest tolerance to 15.0 mM of phenol followed by Corynebacterium sp. RBD7 while Escherichia coli RBD2 and Citrobacter sp. RBD8 showed the least tolerance to 15.0 mM of phenol. The minimum inhibitory concentrations (MICs) ranged from 1.0 mM for mercury and 4.5 mM for chromium, nickel, lead and copper. The bacterial strains were most susceptible to mercury toxicity. Viable counts of the organism on mineral salt-phenol agar showed a typical growth pattern for inhibitory substrate. The threshold concentration is 0.5 mM for Bacillus sp. RBD1, Escherichia coli RBD2, Bacillus sp. RBD6, Citrobacter sp. RBD8, Streptococcus sp. RBD9, Pseudomonas sp. RBD11 and Escherichia coli RBD12 and 1.0 mM for Pseudomonas sp. RBD3, Aeromonas sp. RBD4, Staphylococcus sp. RBD5, Corynebacterium sp. RBD7 and Corynebacterium sp. RBD10. The results suggest that microorganisms isolated from petroleum refinery effluent are potentially useful for detoxification of phenol impacted systems in the presence of heavy metals.
922-931 | JRB | 2013 | Vol 3 | No 3
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.
www.jresearchbiology.com Journal of Research in Biology
An International Scientific
Research Journal
Authors:
Nwanyanwu CE,
Nweke CO, Orji JC,
Opurum CC.
Institution:
Department of
Microbiology, Federal
University of Technology,
P.M.B. 1526, Owerri,
Nigeria.
Corresponding author:
Nwanyanwu CE.
Email: [email protected]
Web Address:
http://jresearchbiology.com/
documents/RA0317.pdf.
Dates: Received: 24 Dec 2012 Accepted: 09 Jan 2013 Published: 10 May 2013
Article Citation: Nwanyanwu CE, Nweke CO, Orji JC, Opurum CC. Phenol and Heavy Metal Tolerance Among Petroleum Refinery Effluent Bacteria. Journal of Research in Biology (2013) 3(3): 922-931
Journal of Research in Biology An International Scientific Research Journal
Original Research
INTRODUCTION
Petroleum refinery effluents are wastes liquids
that resulted from the refining of crude oil in petroleum
refinery. The effluents are composed of oil and grease
along with many other toxic organic and inorganic
compounds (Diya’uddeen et al., 2011). Among the toxic
components of these effluents are heavy metals. Heavy
metals include cobalt, chromium, nickel, iron,
manganese, zinc, etc. They usually form complexes with
different non metal donor atoms which account for their
participation in various microbial metabolisms in the
environment (Kamnev, 2003). Some of these heavy
metals such as cobalt, chromium, nickel, iron
manganese, zinc, etc. are required in trace amount by
microorganisms at low concentration as nutrients, since
they provide vital co-factors for metalloproteins and
enzymes and are known as essential metals while others
such as cadmium, mercury, lead, etc have no
physiological functions and are known as nonessential
metals (Sevgi et al., 2010). At high concentration both
essential and nonessential heavy metals exert an
inhibitory action on microorganisms by impairing the
essential functional groups as well as modifying the
active conformation of biological molecules. This results
in reduction of microbial activity leading to increased lag
phase as well as slow growth rate (Aleem et al., 2003).
It is expected that petroleum refinery effluents
will contain some of these metals in reasonable quantity
as well as aromatic compounds such as phenols. Organic
and inorganic mixed pollutants are known to be
commonly present in industrial effluents and also other
contaminated sites. In this case, apart from affecting the
viability of the microbiota, the metal activity may have
synergistic effect on biodegradation processes of the
aromatic compounds. Thus studies related to the
association of the bacterial tolerance properties to metals
and degradation of phenolic compounds may be relevant
to applications in bioremediation processes (Silva et al.,
2007).
Discharge of these metals into natural waters at
increased concentration in refining operations can have
severe toxicological effects on aquatic environment and
humans. Heavy metals as well as phenol are known to
be harmful pollutants emanating from industrial
wastewaters that have negative effects on
microorganisms. These metals are in the form of
inorganic and metallo-organic compounds while phenol
appears to be a soluble component of the industrial
effluents (Nwanyanwu and Abu, 2010; Hernandez et al.,
1998). These environmental pollutants which are
environmentally mobile tend to accumulate in organisms,
and become persistent because of their chemical stability
or poor biodegradability (Emoyan et al., 2005).
Contamination of wastewater with high concentration of
heavy metals caused a significant decrease in
the numbers of bacteria in biological system
(Otokunefor and Obiukwu, 2005). It is obvious that
heavy metals are one of the toxic contaminants in
wastewaters and causes disorder in biological wastewater
treatment (Sa’idi, 2010). Microorganisms being
ubiquitous in nature have been reported to be found in
inhospitable habitats such as petroleum refinery
effluents, coke effluents, etc (El-Sayed et al., 2003;
Hidalgo et al., 2002) as the effluents are characterized by
the presence of phenols, metal derivatives, surface active
substances and other chemicals (Suleimanov,1995).
Bruins et al., (2000) in their work reported that
organisms in such inhospitable environment must have
developed metal resistance systems in an attempt to
protect sensitive cellular components. On the other hand,
utilization of phenol and other pollutant is enhanced by
adaptation and production of appropriate enzymes by
organisms for the removal of the toxicants
(Nwanyanwu et al., 2012).
This study investigated the tolerance to heavy
metals and phenol by bacterial population in petroleum
refinery effluent.
Nwanyanwu et al., 2013
923 Journal of Research in Biology (2013) 3(3): 922-931
MATERIALS AND METHODS
Sample collection
Petroleum oil refinery effluent was collected
from Biological treatment plant unit (Rotary biodisk,
RBD) of Port Harcourt oil refinery complex and
transported to the laboratory for physicochemical
analysis which includes pH, total dissolved solids,
biological oxygen demand (BOD), chemical oxygen
demand (COD), phosphate (PO4), nitrate (NO3), oil and
grease, phenol, electrical conductivity and heavy metals
content. The methods used for the analysis were as
shown elsewhere (Nwanyanwu et al., 2012).
Microbiological analysis
Microbiological counts were estimated by plating
0.1 ml of the 102 - 106 decimally diluted effluent samples
in physiological saline on appropriate agar plates. Total
heterotrophic bacterial count was done on nutrient agar
plates while phenol-utilizing bacterial count was done on
phenol-agar medium of Hill and Robinson (1975). The
inoculated plates were incubated for 24 h at 30oC for the
heterotrophic bacterial count and 72 h for phenol-
utilizing bacteria count.
Isolation and identification of bacterial strains
The discrete bacterial colonies that developed on
phenol-agar medium were purified, characterized
biochemically and identified as described by
Nwanyanwu et al., (2012).
Preparation of inoculum
The organisms were grown in nutrient broth
medium contained in Erlenmeyer flasks (100 ml) at
28±2oC for 48 h. Thereafter, the cells were harvested and
washed in sterile deionized distilled water. The cell
suspensions were standardized by adjusting the turbidity
to an optical density of 0.1 at A540.
Screening of isolates for phenol tolerance
Into 5.0 ml mineral salt broth medium contained
in 15.0 ml screw capped glass culture tubes were added
aliquots of phenol stock solution (200 mM). The tubes
were sterilized by autoclaving at 121oC for 15min and
allowed to cool at room temperature (28±2oC).
Thereafter, 0.1 ml aliquot of cell suspensions were
seeded into the tubes and incubated at 30oC for 96 h. The
final concentrations of phenol in the tubes ranged from
0.1-100 mM. Controls included cells in mineral salt
medium without phenol and mineral salt medium
supplemented with phenol but without cells.
Development of turbid culture depicted tolerance to
phenol stress. Isolates that exhibited phenol tolerance
from 5.0 mM and above were used for further phenol and
heavy metal toxicity assay.
Nwanyanwu et al., 2013
Journal of Research in Biology (2013) 3(3): 922-931 924
Table1: Physicochemical and microbiological
analyses of biological treatment unit of petroleum
refinery wastewater
Parameter/ unit Value
pH 8.18
Elect. conduct (µs/cm) 485
Oil and grease (mg/l) 15.0
TDS (mg/l) 250
BOD (mg/l) 8.0
COD (mg/l) 76.0
Phenol (mg/l) 13.6
PO42- (mg/l) 0.14
NO3- (mg/l) 1.20
Metal concentration
Zn2+ (mg/l) 0.02
Cu2+ (mg/l) <0.02
Cr2+ (mg/l) 0.05
Pb3+ (mg/l) <0.01
Ni2+ (mg/l) 0.02
Cd2+ (mg/l) <0.01
Microbial load
THBC (CFU/ml) 2.52 x 108
TPUBC (CFU/ml) 1.14 x 108
% TPUBC (%) 45.24
THBC = Total Heterotrophic bacterial count; TPUBC = Total phenol-utilizing bacterial count
Growth on phenol-mineral salt agar
The isolates were tested for their ability to grow
on mineral salt agar medium (MSM) amended with
increasing phenol concentrations. An aliquot (100 µl) of
decimally diluted standardized inoculum of each isolate
in physiological saline was spread plated onto surface
of MSM plates with 2.0-20 mM of phenol
concentrations. Control included cells in MSM plates
without phenol. The culture was incubated at 30oC for
72 h (Kahru et al, 2002). The number of the colony that
developed was enumerated as colony forming unit per ml
(CFU/ml).
Minimum inhibitory concentration (MIC)
determination
Stock solutions of Cd, Zn, Hg, Cu, Pb, Ni, Co
and Cr as salts of CdCl2, ZnSO4, HgCl2, CuSO4, PbCl2,
Ni(NO3)2, CoCl2.6H2O and K2Cr2O7 were prepared in
deionized distilled water. All the chemicals used were
analytical reagent grade.
The minimum inhibitory concentrations (MIC) of
eight heavy metal ions at which no growth was observed
were determined at pH 7.2 against each bacterial isolate
using tube dilution method (Hassen et al., 1998) with
little modifications. Graded concentrations of each heavy
metal ranging from 0.05 mM to 10.0 mM were prepared
in tryptone soy broth (TSB) contained in screw capped
culture tubes. The supplemented TSB-heavy metal
medium was sterilized by autoclaving at 121oC for
15 min. On cooling to room temperature (28±2oC), the
tubes were seeded with 100 µl of the bacterial
suspension and incubated at 30oC for 72 h. Inoculated
medium free of heavy metal ions and uninoculated
medium with metal ions served as positive and negative
controls respectively. The MIC of the metal to the test
isolates is the lowest concentration that totally inhibited
growth of the organisms.
RESULTS AND DISCUSSION
The physicochemical and microbiological
properties of the petroleum refinery effluent are shown in
Table 1. Phenol-utilizing bacteria represented 45.24% of
the microbial load of biodisk effluent. The high
population of phenol-utilizing bacteria obtained could be
related to natural selection and adaptation to phenol at
the unit. The concentration of heavy metals in the
effluent present in the effluent may be as a result of
physicochemical treatment (oxidation and reduction,
chemical precipitation, etc) given to the raw wastewater
before been channeled into the biological treatment unit.
The result of screen test for phenol tolerance is
shown in Table 2. With the exception of Pseudomonas
sp. RBD3 that tolerated phenol up to 10 mM, all the
organisms are able to tolerate phenol stress up to
15.0 mM. The growth of the isolates in the medium with
phenol concentrations above 10.0 mM may be attributed
to previous exposure to phenolic raw wastewater influent
into the biological treatment unit (RBD). This is in line
with the report of Santos et al., (2001) in which they
related the growth of Trichosporom sp. in phenolic
amended medium of 10.0 mM concentration to previous
phenolic wastewater shock load from stainless steel
industry. Moreso, the tolerance of the organisms to high
concentration of phenol (15.0 mM) may be the ease with
which the isolates open the phenol ring for its subsequent
uptake as carbon and energy source (Ajaz et al., 2004).
Gurujeyalakshmi and Oriel (1989) in their work have
reported that Bacillus stearothermophilus strain BR219
could grow on phenol at levels up to 15 mM. In contrast,
growth inhibition of Bacillus, Pseudomonas and
Citrobacter species at phenol concentration above
1.0 mM has been reported by many authors
(Obiukwu and Abu, 2011). Janke et al., (1981) reported
inhibition of phenol hydroxylase activity in
Pseudomonas species at 0.25 mM phenol concentration.
Yang and Humphrey (1975) found that the growth
of Pseudomonas putida was strongly inhibited
above phenol concentration of 0.5 mM. Buswell and
Twomey (1975) reported that growth of
Nwanyanwu et al., 2013
925 Journal of Research in Biology (2013) 3(3): 922-931
Bacillus stearothemophilus was inhibited at phenol
concentration above 5.0 mM.
The effect of increasing doses of phenol
(0.05 - 15.0 mM) on the population of the test organisms
are shown in Figure 1. Generally, the viable counts
increased with the concentration of phenol until a certain
concentration when the growth of the organisms was
inhibited. The growth of the organisms on phenol
followed a substrate inhibition pattern. Increasing phenol
concentration resulted in decrease in microbial growth
and eventually very minimal growth was detected at the
highest phenol concentration (15.0 mM) in all the test
organisms. The growth of Bacillus sp. RBD1,
Escherichia coli RBD2, Bacillus sp. RBD6, Citrobacter
sp. RBD8, Streptococcus sp. RBD9, Pseudomonas sp.
RBD11 and Escherichia coli RBD12 with a total viable
count of 7.1 x 106, 8.0 x 106, 7.2 x 106, 7.8 x106,
7.5 x 106, 8.8 x 106, 7.4 x 106 and 7.4 x 106 CFU/ml
respectively were stimulated at phenol concentrations up
to 0.5 mM (≈ 47.06 mg/l). Similarly, at phenol
concentration up to 1.0 mM (≈ 94.11 mg/l), the growth
of Pseudomonas sp. RBD3, Aeromonas sp. RBD4,
Staphylococcus sp. RBD5, Corynebacterium sp. RBD7
and Corynebacterium sp. RBD10 with a total viable
count of 8.4 x 106, 7.5 x 106, 7.2 x 106 and
8.4 x 106 CFU/ml respectively, were stimulated.
Thereafter, the total viable counts progressively
decreased as the phenol concentration increases. This
growth pattern is typical of in an inhibitory substrate like
phenol. The inhibition of bacterial growth by phenol is
well-documented. However, some bacteria are more
tolerant to phenol than others. For instance, the growth
inhibition constant (Ki) for bacteria degrading phenol
have been reported as 54.1mg/l (0.57 mM)
(Monteiro et al., 2000), 129.79 mg/l (1.379 mM) (Kumar
et al., 2005), 2434.7 mg/l (25.87 mM) (Arutchelvan et
al., 2006) and 7.818 mM (Wei et al., 2008). In this study,
all the test organisms tolerated phenol up to 10.0 mM
(≈ 941 mg/l) and with the exception of Pseudomonas sp.
RBD 3, all the bacterial strains tolerated 15 mM
(≈ 1412 mg/l). This is in line with the report of Worden
et al., (1991) that Bacillus stearothermophilus BR219
Nwanyanwu et al., 2013
Journal of Research in Biology (2013) 3(3): 922-931 926
Table 2: Phenol tolerance of the test isolates in different concentrations of phenol
Bacteria
Growth in mineral salt broth with added phenol
Phenol concentration (mM)
0.1 0.2 0.5 1 2 5 10 15 20 50 100
Bacillus sp. RBD1 + + + + + + + + - - -
Escherichia coli RBD 2 + + + + + + + + - - -
Pseudomonas sp. RBD 3 + + + + + + + - - - -
Aeromonas sp. RBD 4 + + + + + + + + - - -
Staphylococcus sp. RBD 5 + + + + + + + + - - -
Bacillus sp. RBD 6 + + + + + + + + - - -
Corynebacterium sp. RBD7 + + + + + + + + - - -
Citrobacter sp. RBD8 + + + + + + + + - - -
Streptococcus sp. RBD9 + + + + + + + + - - -
Pseudomonas sp. RBD10 + + + + + + + + - - -
Corynebacterium sp. RBD11 + + + + + + + + - - -
Escherichia coli RBD12 + + + + + + + + - - -
+ = growth ; - = no growth
tolerated phenol concentration of 15.0 mM. Similarly,
Corynebacterium species was reported to resist 15 mM
phenol while Staphylococcus, Corynebacterium, Bacillus
and Proteus were found to resist 10 mM of phenol
(Ajaz et al., 2004). However, many authors have
reported inhibition of microorganisms at such high
phenol concentration (Hossein and Hill, 2006; Kotturi et
al, 1991). Li and Humphrey (1989) as well as
Gurujeyalakshmi and Oriel (1989) have reported
microbial growth inhibition at relatively low
concentrations of 2.0 mM and 0.25 mM respectively.
927 Journal of Research in Biology (2013) 3(3): 922-931
Nwanyanwu et al., 2013
Phenol (mM)
Tota
l V
iab
le C
ou
nt
(x 1
06 C
FU
/ml)
Figure 1: Growth of bacteria on mineral salt agar medium supplemented with increasing doses of phenol.
0
2
4
6
8
10
Pseudomonas sp. RBD3
0
2
4
6
8
10
0 4 8 12 16
Citrobacter sp. RBD8
The tolerance levels of refinery wastewater
phenol-utilizing bacteria to heavy metals expressed as
minimal inhibitory concentrations (MIC) are shown in
Table 3. The test isolates in this study showed similar
trend of susceptibilities to heavy metal ions based on
minimal inhibitory assay. The high MIC values obtained
in the study may be as a result of long term exposure of
the organisms to metal ions in the refinery effluent.
Highest MIC values were exhibited in Chromium,
Copper and Nickel while the least MIC was shown in
mercury among the isolates with a maximum value of
>3.0 mM and minimum value of <2.0 mM.
Pseudomonas sp. RBD3 showed maximum MICs value
range of 1.5 - 4.5 mM whilst Escherichia coli RBD12
showed minimum MICs value range of 1.0 - 3.5 mM in
all the metals tested. The MICs are higher than that
reported by El-Deeb (2009) for some phenol-degrading
bacteria. However, the MIC values are similar to the
values reported elsewhere (Nieto et al., 1989,
Nweke et al., 2006a, Akinbowale et al., 2007). The MIC
of metal ranging from 0.5 - 2.5 mM, 1.25 - 2.5 mM,
5.0 - 12.0 mM, 1.0 - 1.25 mM, 0.25 - 1.0 mM and
1.25 - 5.0 mM against hydrocarbon-utilizing bacteria was
reported for cadmium, chromium, lead, cobalt, mercury
and copper respectively (Nweke et al., 2006a). These
reported MICs in most cases corroborates the values
observed in this study. The MIC in growth inhibition
assay is analogous to the concentration of metal ion
that exhibited 100 % inhibition in dehydrogenase
activity assay. Thus, the MIC of zinc against river
water planktonic bacteria have been reported as
1.558 ± 0.037 mM, 1.283 ± 0.068 mM,
2.469 ± 0.045 mM and 1.328 ± 0.094 mM for
Escherichia, Proteus, Micrococcus and Pseudomonas
species respectively (Nweke et al., 2006b). Likewise, the
concentration of zinc that gave 100% inhibition of
dehydrogenase activity in sediment Bacillus and
Arthrobacter species are 1.442 ± 0.062 mM and
1.199 ± 0.042 mM respectively (Nweke et al., 2007).
Also, Hassen et al., (1998) have reported MIC values of
0.1, 0.8, 1.5, 1.6 and 1.8 mM for Mercury, Cobalt, Zinc
and Cadmium, Copper and Chromium respectively on
Pseudomonas aeruginosa, Citrobacter freundii,
Staphylococcus aureus, Streptococcus sp. and
Bacillus thurieniensis. Hassen et al., (1998) in their work
reported 3.0 mM chromium as the MIC for
Nwanyanwuet al., 2013
Journal of Research in Biology (2013) 3(3): 922-931 928
Organism
MIC of metal (mM)
Cd Zn Hg Cu Pb Ni Co Cr
Bacillus sp. RBD1 3.5 2.0 1.5 4.0 4.5 3.5 2.0 4.0
Escherichia coli RBD2 3.5 2.5 1.0 4.0 3.0 4.0 2.5 3.5
Pseudomonas sp. RBD3 4.0 3.0 1.5 4.5 3.0 4.5 3.0 4.5
Aeromonas sp. RBD4 3.5 3.0 1.0 3.0 4.0 4.0 2.0 4.0
Staphylococcus sp. RBD5 4.0 2.0 1.0 3.5 3.0 4.0 3.0 4.0
Bacillus sp. RBD6 3.0 2.5 1.5 3.5 4.0 4.0 3.0 4.0
Corynebacterium sp. RBD7 3.0 2.0 1.5 3.0 3.5 3.5 2.5 3.5
Citrobacter sp. RBD8 3.5 1.5 1.0 2.5 3.0 3.0 2.0 2.5
Streptococcus sp. RBD9 4.0 2.0 1.0 3.5 2.5 4.0 3.0 2.5
Pseudomonas sp. RBD10 3.5 3.0 1.5 4.5 4.0 4.5 3.5 4.0
Corynebacterium sp. RBD11 2.5 2.0 1.0 4.0 4.0 3.0 4.0 4.5
Escherichia coli RBD12 3.0 1.5 1.0 3.0 3.5 3.5 2.5 3.0
Table 3: Minimal inhibitory concentrations of heavy metals
Pseudomonas aeruginosa S8 and Citrobacter freundii
S24. The variation in the tolerance of heavy metal could
be attributed to the bacterial strain involved, assay
technique or culture conditions. However, the study has
proved that heavy metals such as mercury, zinc and lead
do indeed have toxic effect on bacteria. Although it may
vary from one species to another, there is no doubt that
heavy metals do inhibit bacterial growth.
Metals as toxic contaminants of various
environmental sites have been reported to have adversely
affected potential biodegradation processes occurring in
the environment (Said and Lewis, 1991). Amor et al.,
(2001) reported that the level of metal inhibition of
microbial growth depends on concentration as well as
nature of the metal and the type of microbial species.
Sandrin and Maier (2003) reported that metals such as
copper, zinc, cadmium, chromium, nickel, mercury and
lead are known to inhibit biodegradation of organic
pollutants by microorganisms. Phenol biodegradation
have also been reported to be inhibited by metals
(Nakamura and Sawada, 2000; Alves de Lima
et al., 2007, El-Deeb, 2009). Due to accumulative
behaviour of heavy metals, the effluents from petroleum
refinery industries could constitute enriched media to
propagate and spread microbial populations which are
resistant to metallic ions. Thus, microorganisms isolated
from petroleum refinery effluent having combined
abilities to grow in high concentration of phenol medium
and resistance to metals is potentially useful for
detoxification of phenolic wastewater co-contaminated
with heavy metals.
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Jou
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al of R
esearch
in
Biology
Effect of Chromolaena odorata leaf extract on haematological profiles in
Salmonellae typhi infested Wistar rats
Keywords: Salmonellae typhi, Chromolaena odorata, Blood cells, Anti-haematotoxic, Rats.
ABSTRACT: Haematological indices provide an insight about the internal environment of a given organism. In this present study, the possible anti-haemototxic effect of Chromolaena odorata on Salmonellae typhi – induced haematotoxicity in rats were investigated. The experimental animals were divided into three groups. Group A received only food and water (control). Group B and C received in addition to food and water, single dose of stock Salmonellae typhi at a dose of 106cfu/ml. The animals in group B and C were allowed to be infected with Salmonellae typhi for 7 days and confirmed by widal test, after which group C was treated with 750mg/kg body weight/day ethanolic extract of Chromolaena odorata for 10 days. The result showed a significant (p < 0.05) decrease in Red Blood Cells (RBC) count, packed cell volume (PCV), haemoglobin (Hb), mean corpuscular haemoglobin (MCH), Mean Corpuscular haemoglobin Concentration (MCHC), neutrophil and increase in platelet, total White Blood Cell (WBC) and lymphocytes in animals infected with Salmonellae typhi when compared to the control non-infected group. Treatment of animals in group C with ethanolic extract of Chromolaena odorata showed a significant (P < 0.05) increase in mean values of RBC count, PCV, Hb, MCH, MCV, MCHC and decrease in platelets, WBC and lymphocytes when compared to the group infested with Salmonellae typhi only. The results above suggest the anti-haematotoxic potential of ethanolic extract of Chromolaena odorata in Salmonellae typhi infested rats.
932-939 | JRB | 2013 | Vol 3 | No 3
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www.jresearchbiology.com
Journal of Research in Biology
An International
Scientific Research Journal
Authors:
Nwankpa P1, Ezekwe AS1,
Ibegbulem CO3 and
Egwurugwu JN2.
Institution:
1. Department of Medical
Biochemistry Imo State
University, Owerri, Nigeria
2. Department of
Physiology, Imo State
University, Owerri, Nigeria
3. Department of
Biochemistry, Federal
University of Technology
Owerri, Nigeria.
Corresponding author:
Promise Nwankpa
Web Address: http://jresearchbiology.com/documents/RA0337.pdf.
Dates: Received: 15 Feb 2013 Accepted: 05 Mar 2013 Published: 11 May 2013
Article Citation: Nwankpa P, Ezekwe AS, Ibegbulem CO and Egwurugwu JN. Effect of Chromolaena odorata leaf extract on haematological profiles in Salmonellae typhi infested Wistar rats. Journal of Research in Biology (2013) 3(3): 932-939
Journal of Research in Biology An International Scientific Research Journal
Original Research
INTRODUCTION
Enteric fever, also called typhoid fever caused by
the bacterium Salmonellae typhi, is an acute life
threatening febrile ailment (Kotton, 2007). Typhoid fever
is distributed worldwide and prevalent throughout the
tropics where it is the commonest cause of fever
(Wilcocks and Manson-Bahr, 1972). Literature reports
have shown that two million cases of typhoid and 200
thousand related deaths occur worldwide each year
(Steinberg et al., 2004). One challenge of development in
developing countries, is the provision of portable water
for the populace as poor sanitary condition and hygiene
has been reported to increase the prevalence of
Salmonellae typhi infection with reduced incidence in
developed countries (Kotton, 2007). Available reports
indicate that typhoid infection is the leading cause of
morbidity and mortality in a developing country like
Nigeria where water carriage method of sewage disposal
is inefficient (Crump et al., 2004). Salmonellae typhi
infection causes gastroenteritis which symptoms include
nausea, vomiting and diarrhea (Parry et al., 2002). The
affected organs include spleen, liver and other tissues
which habor the bacterium before entering the blood
(Jones and Falkow, 1996). During metabolism, bacterial
cells, release chemical toxins which interactions damage
the tissue of the host organism. This tends to disrupt the
blood components or blood forming tissues.
Blood is one of the specialized body fluid
responsible for the transportation of nutrients, oxygen,
hormones and other metabolites to the body’s cell and
metabolic waste products away from those cells to sites
of elimination. It is known to be the most important body
fluid that regulates various vital functions of the body
such as excretion, respiration, circulation, osmotic and
temperature balance etc. Mammalian circulation of blood
transports specific nutrients, gases, metabolic products
and hormones between different tissues and organs
(Baynes and Dominiczak, 2005). Literature reports
indicated that haematological profiles of different species
of animals may be influenced adversely by diabetic
condition (Edet et al., 2011), phenylhydrazine (Sanni
et al., 2005), some anti-retroviral drugs (Kayode et al.,
2011) and aqueous leaf extract of Ocimum gratissimum
(Obianime et al., 2011).
Chromolaena odorata (known as siam weed,
independent weed, killer weed) is a perennial shrub
which grow in rainforest, grassland and arid bushvelds
(Timbilla and Braimah, 2002). The leaves of the plant
has been reported to be widely used as herbal remedy for
the treatment of various ailments. Available reports have
shown a decotion of the leaf extract effective in the
treatment of malaria and cough (Suksamran et al., 2004).
Akah (1990) has reported the haemostatic and
anti-inflammatory property of the leaf extract while
Thang et al., (2005) has shown the stimulation of
granular tissue and re-epithelization of the epithelial
tissue during wound healing. Recently Nwankpa et al.,
(2012) reported the antioxidative effect of ethanolic leaf
extract of Chromolaena odorata in rats. Other medicinal
uses including anti-hypertensive, anti-diarrhoeal and
diuretic has been reported (Iwu, 1993).
In rural communities in Nigeria, the use of
Chromolaena odorata for treating Salmonellae typhi
infection is common but the effect of the plant on
haematological indices in typhoid fever is not known.
This study was therefore designed to assess the effect of
Chromolaena odorata on haematological profiles in
Salmonellae typhi infested rats.
MATERIALS AND METHODS
Plant Material: The Chromolaena odorata leaves were
collected from a natural habitat in Owerri and
authenticated by professor S.C. Okeke, a taxonomist at
the department of Plant Science and Biotechnology, Imo
State University Owerri, Nigeria. The voucher specimen
was kept in the university herbarium for references.
Preparation of Extract: Large quantities of fresh leaves
of Chromolaena odorata, washed free of sand and
Nwankpa et al., 2013
933 Journal of Research in Biology (2013) 3(3): 932-939
debris, were dried under shade at room temperature at
27°C for 3 weeks. Electric blender was used to
homogenize the dried leaves to a powder form. A 700g
of the powder macerated in 1.1 litres of 80% (v/v)
ethanol were allowed to stand for 24 hours. A chess clot
was used to filter the mixture and the filtrate
concentrated in vacuo at 37-40°C to 10% its original
volume using a rotary evaporator. The concentrate was
evaporated in a water bath at 40°C to a solid residue, the
extract. The extract was dissolved in 100ml of 10%
ethanol to an approximate concentration used for the
experiment.
Salmonellae typhi: The stock Salmonellae typhi was
procured from Federal College of Veterinary and
Medical Laboratory Technology of the National
Veterinary Research Institute Vom, Jos, Plateau State,
Nigeria. Nutrient agar plate, cesteine lactose electrolyte
deficient plate (DCA) was used to sub-culture the micro-
organism which was incubated at 37°C for 24 hours and
examined for growth. The stock sample used for the
experiment was prepared as culture slants using
McCartney bottle and nutrient agar. Salmonellae typhi
from the sub-cultured medium was aseptically incubated
for 18 hours at 37°C.
Animals: Albino Wistar rats of both sexes weighing
between 150-200g were obtained from the animal house
of Faculty of Medicine, Imo State University Owerri,
Nigeria. They were maintained at room temperature and
acclimatized for 12 days to daily handling. They were
fed ad-libitum with commercial rat chow (Product of
Pfizer Nigeria Ltd) and had free access to water.
Induction of typhoid: Each rat was orally administered
with 1ml of Salmonellae typhi at a dose of 106cfu/ml to
induce typhoid (Kirby, 1960).
Experimental design: Twenty - four albino Wistar rats
were used for the study. They were randomly assigned
into 3 groups. Each group has 8 rats.
Group A: The rats in this group were fed with rat chow
and had free access to water. They were not administered
with Salmonellae typhi and serve to monitor successful
induction of typhoid.
Group B: The rats in this group served as control. They
were fed with rat chow and had free access to water.
Single dose of Salmonellae typhi at106cfu/ml was orally
administered to rats in this group but were not treated
with the plant extract.
Group C: The rats in this group were fed with
rat chow and had access to water. Single dose
of Salmonellae tysphi at 106cfu/ml were orally
administered to the rats in this group. After 7 days
of infection, 750 mg/kg ethanolic leaf extract of
Chromolaena odorata were orally administered to the
animals daily for 10 days.
Collection and preparation of blood samples for
analysis
At the end of the treatment, the animals were
fasted for 24 hours, re-weighed and sacrificed under
chloroform anesthesia. By cardiac puncture, blood
sample was collected from each animal with a sterile
syringe and needle, in EDTA anti coagulated bottle. The
anti-coagulated blood samples were used for
haematological analyses which were carried out within
24 hours of sample collection.
Haematological analysis
Full blood counts such as packed cell volume
(PCV), Haemoglobin (Hb), Red Blood Cell (RBC), Total
White Blood Cells (TWBC), Platelet count, differential
white blood cell (like lymphocytes, monocytes,
eosinophils, neutrophils) and red cell indices including
Mean Corpuscular Haemoglobin (MCH), Mean
Corpuscular Volume (MCV), Mean Cell Haemoglobin
Concentration (MCHC) were estimated using the
Sysmex® Automated Haematology Analyzer KX-2IN,
Sysmex Corporation, Kobe, Japan.
Statistical analysis
Data generated were statistically analysed by
one-way analysis of variance (ANOVA) of the SPSS
statistical programme of Microsoft Excel. Values were
Nwankpa et al., 2013
Journal of Research in Biology (2013) 3(3): 932-939 934
declared significantly different at p<0.05.
RESULTS AND DISCUSSION
Table 1 and 2 shows the effect of
Salmonellae typhi infection and subsequent treatment
with ethanolic leaf extract of Chromolaena odorata on
haematological parameters in rats. The results showed a
significant (P < 0.05) decrease in Red Blood Cells (RBC)
count, haemoglobin (Hb), Packed Cell Volume (PCV),
Mean Corpuscular Haemoglobin (MCH), Mean
Corpuscular Volume (MCV), Mean Corpuscular
Haemoglobin Concentration (MCHC) and percentage
nuetrophil levels in Salmonellae typhi infested rats
compared to the non-infested group (Table 1 and 2).
On the contrary, the total White Blood Cell (WBC),
platelets and lymphocyte levels in rats infested with
Salmonellae typhi showed a significant (P < 0.05)
increase compared to the non-infested group (Table 2).
Treatment of the rats in group C with ethanolic leaf
extract of Chromolaena odorata showed a significant
(P < 0.05) increase in RBC count, Hb, PCV, MCH,
MCV, MCHC and percentage neutrophil levels
compared to the Salmonellae typhi infested non-treated
group (Table 1 and 2) while treatment of rats in group C
with ethanolic leaf extract of Chromolaena odorata
showed a significant (P < 0.05) decrease in platelets,
WBC and lymphocyte levels compared to the non-treated
Salmonellae typhi infested group (Table 2). However the
results of this study showed no significant (P > 0.05)
difference in RBC, Hb, PCV, MCV, MCH, MCHC,
platelets, WBC, and lymphocytes in Salmonellae typhi
infested rats treated with Chromolaena odorata
compared to the non-infested rats (Table 1 and 2).
Haematological indices provide relevant
information regarding the internal milieu of an organism.
Nutritional, environmental and microbial infection are
among several other factors which have been reported to
have adverse effects on the haematological profiles of
most organisms. Vitamin B12 and folic acid deficiency
(Jee et al., 2005, Murray et al., 2007) and exposure to
environmental pollutants such as carbondisulphide,
insecticide, hexane, gasoline vapour, nitrocellulose
thinner has been reported (Dhembara and Pandhe, 2000;
Uboh et al., 2007; 2009; 2012 and Savithri et al., 2010).
Bacterial infection in living cells release toxins which
metabolism results to increase in release of free radical
species with attendant damage to the cells (Stipanuk,
2000). In this study, Salmonellae typhi infection
significantly decreases the level of RBC, PCV, Hb,
MCH, MCV, MCHC, neutrophils and increases the level
of WBC and lymphocytes. The observation made in this
study agrees with the report of Wilcocks and Manson-
Bahr (1972) in Salmonellae typhi infection and Kumar
and Kuttan (2005) on cyclophosphamide induced
Nwankpa et al., 2013
935 Journal of Research in Biology (2013) 3(3): 932-939
Group Treatment RBC
X1012/L Hb
(g/dL) PCV (%)
MCV (fL)
MCH (pg)
MCHC (g/dL)
A Negative control/water
3.69 ± 0.21 14.43 ± 0.65 44.33 ± 2.13 63.12 ± 1.60 17.19 ± 1.12 31.27 ± 1.20
B Salmonellae typhi (Positive control)
1.62 ± 0.03a 10.09 ± 0.71a 33.26 ± 2.14a 54.85 ± 1.55a 12.52 ± 1.30a 24.12 ± 1.23a
C
Salmonellae typhi + Chromolaena odorata
3.49 ± 0.05bc 14.15 ± 0.79bc 43.40 ± 2.34bc 61.95 ± 1.32bc 16.55 ± 1.02bc 30.12 ± 1.33bc
Table 1: Effect of Chromolaena odorata on mean values of red blood cells, packed cell volume, hemoglobin and
red cell indices in both experimental and control groups.
Mean ± SD (n = 8) a Significantly different compared with negative control (P < 0.05). b Significantly different compared with Salmonellae typhi (positive control) (P < 0.05). c No significant difference compared with negative control (P > 0.05).
toxicity. The haematotoxic effect of Salmonellae typhi
infection may be explained by the interaction of the
bacteria or its toxins with the blood forming tissues/
organs which may inhibit the rate at which some specific
or generalized haemopoeitic committed stem cells are
synthesized by the tissues. Some reports have shown that
hexane, cyclophosphamide and benzene induced
haematotoxic effect is associated with the interaction of
their metabolites with the haematopoeitic tissues and
cause depression in their haematopoeitic activities
(Synder and Hedli, 1996; Kumar and Kuttan, 2005).
Increase in total white blood cells and lymphocytes as
well as decrease in neutrophils seen in this study is
consistent with the reports on effect of insecticides and
pesticides such as fenvalerate, lindane, aldrin among
others, on total white blood cells and the differential
counts in experimental animals (Synder and Hedli, 1996;
Kumar et al., 1996; Savithri et al., 2010). This may be
explained by increased lymphopoeisis and/or enhanced
release of lymphocytes from lymph myeloid tissue (Das
and Mukherjee, 2003). This response may be a direct
stimulatory effect of toxic substance on lymphoid tissue/
pollutant induces tissue damage and disturbance of the
non-specific immune system leading to increase in
production of leukocytes. Neutrophils are known to be
involved in the phagocytosis of foreign substances in the
body during which some of them are ruptured. This may
explain the decrease in neutrophil count on infection
with Salmonellae typhi.
Ethanolic extract of Chromolaena odorata
significantly increased the level of RBC, Hb, PCV,
MCV, MCH and MCHC thereby reducing and
ameliorating the anaemic condition induced by
Salmonellae typhi infection. The observed increase in
RBC, Hb, and PCV may be explained by the role of
Chromolaena odorata extract in reversing bone marrow
depression with attendant improvement in erythrocyte
membrane stability through the antioxidant potential of
the plant extract, thus reducing haemolysis (Krause and
Mahan, 1984; Naaz et al., 2007, Nwankpa et al., 2012).
The improvement on the haematopoetic activities of the
tissues and/or maintenance of red blood cell membrane
integrity relieves the anaemic condition observed in
Salmonellae typhi infection.
Consequently, increase in RBC count on
administration of Chromolaena odorata leaf extract
translates to an increase in MCV while increase in Hb
translates, to an increase in MCH and MCHC.
Furthermore, inhibition of microbial growth by the plant
extract has been reported. Okigbo and Ajalie (2005) and
Alisi et al., (2011) showed that Chromolaena odorata
leaf extract possess antibacterial activity which inhibit
the growth of Salmonellae typhi in cells. Decrease in
total white blood cell, lymphocytes and attendant
increase in neutrophils on administration of the plant
extract may be explained by the inhibition of growth of
Nwankpa et al., 2013
Journal of Research in Biology (2013) 3(3): 932-939 936
Group Treatment Platelets X103μL-1
TWBC
X103μL-1 Lymphocytes
(%) Neutrophils
(%)
Eosinophils
(%) Monocytes
(%)
A Negative control/water
855.18 ± 2.11 16.24 ± 0.78 70.11 ± 2.01 20.19 ± 1.15 1.98 ± 0.6 2.51 ± 0.11
B Salmonellae typhi (Positive control)
880.13 ± 1.5a 25.85 ± 1.16a 82.14 ± 2.11a 11.56 ± 0.87a 3.20 ± 1.10 2.90 ± 0.55
C Salmonellae typhi + Chromolaena odorata
858.82 ± 1.46bc 17.14 ± 1.21bc 72.18 ± 1.88bc 19.26 ± 1.11bc 2.10 ± 0.80 2.6 ± 0.52
Table 2: Effect of CO on mean values of platelets, total white blood cells and differential cell counts in both
experimental and control groups
Mean ± SD (n = 8) a Significantly different compared with negative control (P < 0.05). b Significantly different compared with Salmonellae typhi (positive control) (P < 0.05). c No significant difference compared with negative control (P > 0.05).
Salmonellae typhi in the cell. The inhibition of growth of
the microorganism lead to the destruction of excess
WBC and lymphyocytes released by the cell in response
to bacterial infection (Nancy et al., 2005). Conversely,
increase in neutrophil count on administration of the
plant extract may be explained by reduced phagocytosis
of the microbial cell consequent upon drastic reduction
in the growth of microbial cell.
CONCLUSION
This study has established the anti-haematotoxic
pot en t ia l of ethanol i c lea f extract of
Chromolaena odorata against Salmonellae typhi induced
haematotoxicity in rats.
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or statements cited in your conclusion must have been stated previously in the article. Do not introduce new information in the conclusion.
Acknowledgement
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References
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Accuracy of citation is the author's responsibility.
Reference Style
References should be cited in the text in the form (Author et al, 1987) and listed in alphabetical order at the end of the article as follows:
Schernewski G, Neumann T. The trophic state of the Baltic Sea a century ago: a model simulation study. J Mar Sys., 2005;53:109–
124.
Kaufman PD, Cseke LJ, Warber S, Duke JA and Brielman HL. Natural Products from plants. CRC press, Bocaralon, Florida. 1999;
15-16.
Kala CP. Ecology and Conservation of alphine meadows in the valley of flowers national park, Garhwal Himalaya. Ph.D Thesis,
Dehradun: Forest Research Institute, 1998; 75-76.
http://www.ethnobiomed.com/content/pdf/1746-4269-1-11.pdf.
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