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INDEX ____________________________________________________________________________ Article Title Page No The insect pathogenic fungus Verticillium lecanii (Zimm.) Viegas and its use for pests control: a review Thiery B C ALAVO 337 – 345 Monitoring of Aflatoxin contamination at market food chain in East Java Agustina A. Rahmianna* and Eriyanto Yusnawan 346 – 352 Livelihood assessment of the fishermen community in the south west region of Bangladesh Mridula Rani Das, Sunuram Ray, Uttam Kumar* Salma Begum and Satya Ranjan Tarafdar 353 – 361 Effect of poultry manure and mineral fertilizer on the growth performance and quality of cucumber fruits OKOLI PSO and Nweke I A* 362 – 367 Plasmid stability and maintenance of copy number using natural marker Hamzah Basil Mohammed and Sudhakar Malla* 368 – 377 Ergastic crystals in identification of Costus pictus: a medicinal spiral ginger in herbal medicine Justin R Nayagam 378 – 383 Development of in vitro screening system for food habit related risk analysis Mandy Bruch* and Elmar Mohr 384 – 393

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Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

KEYWORDS

Bio-control

Entomopathogenic fungi

Verticillium lecanii

Crop Protection

ABSTRACT

Chemical insecticides play an important role in the control of plant damage and plant diseases.

However, extensive use of these products has led to the disruption of ecosystems because of several

reasons such as death of non-target species, accumulation of pesticide residues in the environment and

food, and buildup of pesticide resistance in the target species. Biological control is one of the

alternatives to chemical pesticides and it can be described as the limitation of the abundance of living

organisms and their products by other living organisms. Predators, parasitoids, fungi and other

beneficial organisms can be used for the biocontrol of insect pests. The fungus Verticillium lecanii is

one of the members of Deuteromycetes and it can be used for crop protection. This paper is a review of

the international literature related to V. lecanii for the bio-control of insects of agricultural importance.

Thiery B C ALAVO

Laboratoire d‟Entomologie Appliquée, Faculté des Sciences et Techniques, Université d‟Abomey Calavi; BP 215 Godomey (Bénin).

Received – May 05, 2015; Revision – May 16, 2015; Accepted – July 30, 2015

Available Online – August 20, 2015

DOI: http://dx.doi.org/10.18006/2015.3(4).337.345

THE INSECT PATHOGENIC FUNGUS Verticillium lecanii (Zimm.) Viegas AND ITS

USE FOR PESTS CONTROL: A REVIEW

E-mail: [email protected] (Thiery B C ALAVO)

Peer review under responsibility of Journal of Experimental Biology and

Agricultural Sciences.

* Corresponding author

Journal of Experimental Biology and Agricultural Sciences, August - 2015; Volume – 3(4)



ISSN No. 2320 – 8694

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1 Introduction

Chemical pest control occupies an important place among the

plant protection methods. Before World War II, chemical pest

control was mainly restricted to the application of nicotine and

some arsenical products (Schepers, 1989). Sprayed on the

crops, they kill the pests that come into contact with these

compounds, but they have neither systemic nor residual

effects. Since the war, chemical control of insects has made

rapid progress, starting with the development of DDT

formulations and other chlorinated hydrocarbons such as

lindane, which showed a residual effect but had no systemic

properties (Schepers, 1989).

The persistence of residues of the chlorinated hydrocarbons

causes accumulation in the food chain and since this

phenomenon has become known, unrestrained application of

those products no longer seems justified and is already

prohibited in many countries. The development of systemic

insecticides starting with the formulation of

organophosphorous compounds, opened new perspectives in

pests control (Schepers, 1989). Later, the carbamate

insecticides added more possibilities, as did the development

of synthetic pyrethroids. All these products played an

invaluable role in the control of plant damage and plant

diseases (Schepers, 1989). However, extensive use of chemical

insecticides has led to the disruption of ecosystems because of

the death of non-target species, the accumulation of pesticide

residues in the environment and food, and the build up of

pesticide resistance in the target species (FAO, 1989;

Devonshire, 1989).

Biological control can be described as the limitation of the

abundance of living organisms and their products by other

living organisms. The term encompasses both the biotic

component of natural control, which is, the naturally occurring

regulation of the numbers of a species by the action of its

natural enemies, and also, applied or manipulative biological

control, which is the use by man of biological means to control

a plant or animal (or its products) that has become a pest

(Carver, 1989).

Predators, parasitoids, fungi and other beneficial organisms can

be used for the bio-control of insect pests. The fungus V.

lecanii is one of several Deuteromycetes species that can be

used for crop protection. This work is a review of the

international literature related to V. lecanii for the bio-control

of insects of agricultural importance.

2 Biology and Ecology of Verticillium lecanii

V. lecanii lacks a sexual phase (perfect stage) and reproduces

by means of non-motile, asexual spores called conidia.

Germination of these conidia produces hyphae and after

subsequent growth, these hyphae produce conidiophores which

finally produce conidia (Alexopoulos & Mims, 1979). V.

lecanii is able to grow on both living and dead materials

(Schuler, 1991). It is non-fastidious and can grow on all

conventional mycological media so far tested, e.g. Czapeck-

Dox, Malt extract, Sabouraud and Potato dextrose agars,

including a media containing chitin as the sole source of

carbon and nitrogen. It has capability to produce conidia on

solid media; in contrast, V. lecanii assumes a semi-yeast

morphology in liquid media (Hall, 1981a).

V. lecanii is one of the most common and important

entomophagous Hyphomycetes fungi occurred on coccids,

aphids, thrips, Diptera, Homoptera, Hymenoptera, Lepidoptera

and mites and in all the climatic regions. Other important

substrates for V. lecanii are rusts and other fungi. It is a

consequence of this habit that the species is frequently isolated

from soil; it has also been isolated from leaf litter of oak, ash

and birch, tea leaves, barley seed, baker´s yeast, beet seed and

bursting corn kernels (Domsch et al., 1980; Sewify &

Mabrouk, 1990; Andreeva & Chternchis, 1995). During

greenhouse experiment, V. lecanii could control the cucumber

powdery mildew (Sphaerotheca fuliginea), keeping the mildew

severity with partially resistant cucumber variety, below 15%

of infected leaf area or under economic threshold (Verhaar et

al., 1993; Verhaar et al., 1996). Transmission electron

microscopy observations have provided evidence that V.

lecanii has also potential to colonize mycelial structures of

Sphearotheca fuliginea and reduced the pathogenicity of this

phytopathogenic fungus under laboratory conditions (Askary et

al., 1997; Askary et al., 1998). Isolates of V. lecanii readily

isolated from coffee rust lesions under laboratory conditions

and at high relative air humidity, showed hyperparasitic and

antibiotic properties. Other isolates of V. lecanii of insect

origin were also able to parasitise Hemileia vastatrix (Eskes et

al., 1991).

The parasitism of V. lecanii has been well demonstrated

against many other phytopathogenic fungi like Puccinia

graminis var. tritici (Hänssler et al., 1981), Puccinia

striiformis (Mendgen, 1981), Uromyces appendiculatus

(Grabski & Mendgen, 1985; Grabski & Mendgen, 1986),

Phaeoisariopsis personata and Puccinia arachidis (Ghewande,

1989; Ghewande, 1990; Zambettakis et al., 1985), Uncinula

necator (Heintz & Blaich, 1990), Puccinia horiana (Srivastava

et al., 1985; Whipps, 1993); Puccinia coronata (Leinhos &

Buchenauer, 1992) and Puccinia allii (Uma & Taylor, 1987).

This fungus also occurs on several species of Nematodes

(Schuler, 1991). The parasitism of V. lecanii on cysts or eggs

of several nematode species has been well reported (Hänssler,

1990; Uziel and Sikora, 1992; Meyer et al., 1990). The cyst

wall of Heterodera schachtii was penetrated by V. lecani

mycelium only after 60 hours of inoculation. Inside the cyst

cavity, the fungus passed through the eggshells and colonized

the larvae. V. lecanii secreted specific enzymes into the culture

medium which enable him to degrade constituents of the cyst

as well as the eggshell (Hänssler, 1990). Wild or mutant strains

of V. lecanii showed efficacy against nematodes under

greenhouse conditions (Meyer & Meyer, 1995; Meyer &

338 ALAVO

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Meyer, 1996; Meyer, 1994; Reddy et al., 1996; Meyer &

Huettel, 1996; Meyer et al., 1997). Like all the other

entomopathogenic fungi, V. lecanii infects its invertebrate

hosts through the external cuticle. Three phases have been

recognised in the development of insect mycosis: adhesion and

germination of the fungal spores on the host cuticle,

penetration of the insect integument by a germ tube, and

development of the fungus inside the insect body, generally

resulting in death of the infected host. Under good humidity

conditions, the dead host is covered by the fungal spores and

hyphae (Quinlan, 1988; Zimmermann, 1984).

Humidity and temperature are the most important climatic

factors which influence the growth of V. lecanii (Schuler,

1991). Virtually, all fungi require high humidity for spore

germination, growth and sporulation. V. lecanii conidia require

high humidity to germinate and possibly do so in water film

only (Hall, 1981a). The optimal temperature requirement for

spore germination and colony growth or infection may vary

from isolate to isolate, between 15°C and 25°C (Hall, 1981a;

Ekbom, 1979; Easwaramoorthy & Jayaraj, 1977; Barson,

1976; Hirte et al., 1989; Sermann et al., 1991; Hsiao et al.,

1992). At 5°C, spores of V. lecanii can germinate and grow

slowly and above 30°C its germination and growth may cease.

3 Insecticidal potential of V. lecanii

The fungus appears to have been first observed in Ceylon (Sri

Lanka) about 1861, on diseased Lecanium coffeae. It was

subsequentely found by Zimmermann on Lecanium viride on

coffee, in Java (Indonesia) and was briefly described by him

under the name Cephalosporium lecanii, in a short paper in

1898. Zimmermann stated that each dead scale was surrounded

by a white fungus. He cultivated the fungus on nutrient agar

and directed attention to the possibility that this fungus can be

utilised for controlling the scale-insect (Petch, 1925).

Guegnen published an exhaustive work in 1905 on fungus-

parasites of man and animals, and reported a new conidial

form, Acrostalagmus coccidicola, found by him on coccid in a

greenhouse in Paris (Parkin, 1906). Guegnen cultivated this

fungus on several media and described it. He attempted to

infect an undetermined coccid by applying the fungal spores to

the insect with a brush, but without success. According to the

description, Acrostalagmus coccidicola does not differ

morphologically from Cephalosporium lecanii (Petch, 1925).

In 1905, Dop published a paper on a new fungus parasite of

Aspidiotus from Martinique; he stated that the advent of the

fungus has practically saved the cocoanut palm cultivation in

this Island; the fungus was referred to the genus Hyalopus;

thus, it is probably near akin to Cephalosporium (Parkin,

1906).

Parkin (1906) reported fungus similar to Cephalosporium

lecanii from Ceylon on lecanium viride, L. hemisphaericum

and L. nigrum and stated that the ease whereby the fungi can

be artificially cultivated is a point in their favour for their

possible use for controlling the scale-insects. Successful

infection of the green-bug (Lecanium viride) on the Java coffee

by an artificial culture of Cephalosporium was then cited by

him. At that time, conditions for successful inoculation were

therefore somewhat obscure. In 1909, information with regard

to the distribution and effectiveness of insect-pathogenic

fungus in the West Indian Islands has been collected by

various researchers (South, 1910). The fungi which were

commonly reported by these researchers are: the white-headed

fungus (Ophionectria coccicola E. and E.), the black fungus

(Myriangium duriaci Mont.), the shield scale fungus (probably

Cephalosporium lecanii Zimm) (South, 1910).

In some districts of these islands where the general conditions

are favourable to their growth, the parasites of certain species

of insects exist naturally in large numbers and were responsible

for the comparative rarity of these species in those districts

(South, 1910). Work was carried out in order to introduce the

parasite into places in which the conditions are favourable to

its growth, but in which it has not previously been known to

occur and to produce it where possible by artificial means.

Methods of introducing these fungi include spraying the spores

and portions of the mycelium of the fungi onto trees which it is

intended to infect, tying infected material into trees which it is

desired to infect and finally, planting among the trees to be

infected, small trees whose foliage is well infected with

various parasitic scale fungi, so that the leaves of the small

trees come into contact with those of the larger ones. With

regard to the artificial formation of conditions suitable to these

fungi in localities where they are naturally unfavourable, two

methods were suggested: spraying trees with clean water, once

or twice a week and allowing the trees attacked by scale insects

to become covered with a fairly thick growth of Bengal beans

(Mucuna pruriens); the beans were supposed to create damp

conditions.

The results with Cephalosporium lecanii and another fungus

were said to be encouraging (South, 1910). Petch (1925)

collected specimens of insect pathogenic fungi from several

localities and cultivated them on nutritive media for further

study and reported that most of the insect pathogenic fungi

showed similarity with Cephalosporium lecanii.

V. lecanii has been employed in Brazil for controlling the

green scale of coffee by Viegas who gave him in 1939, its

current name (Schuler, 1991). Gams (1971), after cultural and

morphological examination of type specimens, regarded the

identifying characteristics of previously determined

Cephalosporium as well as other associated species insufficient

to justify their separation as distinct species; he, therefore,

proposed that all these species should be amalgamated as

synonyms of Verticillium lecanii. This fungus has never been

implicated, in temperate climates in epizootics, although it is

frequently isolated from individual insects (Barson, 1976; Hall,

1981a).

The Insect Pathogenic Fungus Verticillium lecanii (Zimm.) Viegas and its use for Pests Control: A Review 339

_________________________________________________________



Indoors, in the somewhat tropical environment of greenhouses,

in North Europe and USA, it frequently decimates populations

of scales and aphids, e. g. 100% mortality of several target

insects was reported in limited trials (Hall, 1981a).

The potential of the fungus has been realised in glasshouse

systems in Europe thanks to the extensive research in U. K., in

years 70. For that, laboratory methods for obtaining uniform

batches of conidiospores and stock insects have been

developed together with a bio-assay technique, using isolated

apterous insects on leaf discs, in order to quantify accurately

the pathogenicity of V. lecanii conidiospores against aphids. A

single spore isolate of V. lecanii obtained from a diseased

aphid Macrosiphoniella sanborni infesting chrysanthemums

was therefore cultured on Sabouraud dextrose agar and spore

concentration was determined using improved hemacytometer.

The bio-assay technique consists in treating artificially reared

aphids with the fungal spore on filter paper in Buchner funnel,

by pouring gently a known amount of the appropriate spore

suspension on them. After treatment, aphids were singly placed

on leaf discs in high humidity assay cells which were kept in

perplex cages at high humidity for the duration of the

experiment (Hall, 1976). This assay technique provides a good

measurement of the pathogenicity of batches of conidiospores

of V. lecanii.

Once the efficacy of the pathogen proved under laboratory, it

has been tested on aphid-infested Chrysantemum plants in

greenhouse. These plants were sprayed with conidial

suspension with pneumatic hand sprayer (Hall, 1979; Hall &

Burges, 1979). All these laboratory and greenhouse

experiments led to the development of two commercial

products “Vertalec” and "Mycotal" based on strains

specifically selected for use against aphids and whiteflies

(Gardner et al., 1984; Ramakers, 1989). V. lecanii has been

tested experimentally against a range of pests, in a number of

countries, with varying results which were said in general to be

encouraging (Kitazawa et al., 1984; Helyer & Wardlow, 1987;

Saito, 1988; Gour & Dabi, 1988; Gopalokrishnan, 1989;

Ravensberg et al., 1990a; Ravensberg et al., 1990 b; Byrne,

1991; Van der Schaaf et al., 1991; Meade & Byrne, 1991;

Masuda & Kikuchi, 1992; Pinna, 1992; Chandler et al., 1993;

Zukauskiene & Sirvinskas, 1993; Helyer, 1993; Ravensberg et

al. 1994; Miranpuri and Khachatourians, 1994; Fournier, 2000;

Gindin et al., 2000; Wang et al., 2004; Nirmala et al., 2006;

Liande et al., 2007; Jeong et al., 2007; Van et al., 2007; Goettel

et al., 2008; Jeong et al., 2008; Chavan et al., 2008; Shinya et

al., 2008).

The effectiveness of this bio-control agent has been proved

also in soil against the soilborne stages of the western flower

thrips (Frankliniella occidentalis) (Hirte et al., 1994; Sermann

et al., 1994; Sermann et al. 1996; Beyer et al., 1997a; Beyer et

al., 1997b). Nevertheless, despite the fact that greenhouses

offer an attractive environment for the exploitation of the

fungus, the effectiveness of V. lecanii still depends on high

humidity and selection of strains infecting the host rapidly or

investigation of methods able to favour the fungus action is

essential.

The effects of pesticides on spores germination, mycelial

growth and sporulation as well as on infection have been

investigated quantitatively and qualitatively with strains of V.

lecanii. Some chemicals were shown to be incompatible while

others proved relatively harmless to the fungus (Hall, 1981b;

Khalil et al., 1985; Saito & Yabuta, 1996). Synergistic

inhibitory action of innocuous chemicals on conidiospores

germination of the pathogen has been reported. On the basis of

all these studies, it is concluded that a careful selection of

pesticides and fungicides would permit the combined use of V.

lecanii and chemicals in integrated control programmes (Hall,

1983).

Similarly, the combined use of V. lecanii with other biological

control agents such as the whitefly parasites Encarsia formosa,

Amblyseius spp. and other pests antagonists has been

investigated (Kanagaratnam et al., 1979; Bennison et al., 1990;

Van der Schaaf et al., 1991; Buxton & Wardlow, 1992).

Results showed that integration of V. lecanii with these bio-

control agents is also possible.

The virulence of two strains of V. lecanii originally isolated

respectively from the aphid Myzus persicae and whitefly

(Trialeurodes vaporariorum) was bioassayed against 3

different aphids‟ species. These strains (V24 and V18) were

capable of infecting all three aphids species, but their virulence

determined by LC50 and LT50 varied. The strain (V24) isolated

from M. persicae showed the highest virulence against the

homologous aphid species, but the whitefly derived strain

(V18) showed also the highest virulence against one of the

aphid species.

The variability of the bioassay results and the impossibility of

defining clearly traits associated with the virulence of a fungus

strain toward a specific insect species have been discussed and

spores improvement using adjuvants was suggested (Alavo et

al, 2002a). However, greenhouse trials aimed at controlling M.

persicae in chinese cabbage using V. lecanii blastospore

suspension either pure or with adjuvants such as soyflour and

rape oil were discouraging; the product failed to control the

aphid populations (Alavo et al, 2002b). Some other adjuvants

were said to improve germination and infection rate of V.

lecanii; nevertheless, effective pest control using V. lecanii

conidia suspension combined with such additives was not

demonstrated (Jin et al., 2006; Zhangyan et al. 2006).

Fluorescent microscopy investigations revealed almost 100%

spore loss from the cuticle of aphid larvae before infection.

Spore loss was attributed to the rapid moulting of aphids larvae

and the unreliable control of aphids using V. lecanii was

discussed (Alavo et al, 2002b; Alavo, 2000).

Nowadays, a formulation of V. lecanii is commercialized under

the name of „Mycotal®‟ only for use against Whitefly larvae.

This product efficacy is said to be improved if applied together

340 ALAVO

_________________________________________________________



with adjuvant based on emulsifiable vegetable oil (Koppert,

2015).

Conflict of interest

Authors would hereby like to declare that there is no conflict of

interests that could possibly arise.

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KEYWORDS

Peanut

Aflatoxin contamination

Aspergillus flavus

Seed moisture content

Damage seed

ABSTRACT

Peanut is a cheapest source of protein especially for developing countries communities and mostly it

obtained from traditional markets. Earlier studies showed that aflatoxin incidence was relatively less at

the farmer/trader levels while it is significantly higher at retail levels especially in traditional markets.

Present study was conducted to understand the factors leading to the post-harvest building up of

aflatoxin in peanuts sold in traditional market and in supermarket. This study was carried out at

Pasuruan regency, East Java Province, Indonesia from March 2005 to June 2006. During study period

peanut grains were collected from wholesalers, retailers and supermarkets at three months interval. In

each sampling point, 2kg of grains was obtained and then was divided into eight parts for the analysis of

parameters namely seed moisture content, physical quality, Aspergillus flavus infection and aflatoxin B1

contamination. The results showed that seed water contents at wholesalers, collectors, and retailers in

traditional wet markets were almost lower than 10%. They were thus ‘safe’ from aflatoxin B1

contamination as seed moisture contents were below the aflatoxin risk zone. Time of sampling did not

affect the level of aflatoxin B1 contamination. Under controlled condition generated from air-tight

container, the influence of seed moisture content and A. flavus infection on aflatoxin production was

significant.

Agustina A. Rahmianna* and Eriyanto Yusnawan

Indonesia Legumes and Tuber crops Research Institute (ILETRI), Jalan Raya Kendalpayak Km 8 Malang, PO Box 66 Malang 65101 East Java, Indonesia

Received – April 20, 2015; Revision – May 08, 2015; Accepted – July 31, 2015



MONITORING OF AFLATOXIN CONTAMINATION AT MARKET FOOD CHAIN IN

EAST JAVA

E-mail: [email protected] (Agustina A. Rahmianna)







ISSN No. 2320 – 8694















_________________________________________________________



1 Introduction

Community awareness related to the risk of aflatoxin

contamination is still low especially for people who have

limited access to education and information. Aflatoxin is a

carcinogen, immune suppressing and anti-nutritional natural

contaminant of peanuts and hence is a major food and feed

quality problem worldwide (Wotton & Strange, 1985;

Paramawati et al., 2006). This toxin produced by soil-borne

fungi Aspergillus flavus, A. paraciticus and A. nomius (Pitt &

Hocking, 1997) in peanut kernels. These fungi can colonized

and infect peanut even when the crops are on farm, which

represent that aflatoxin production in peanut seeds occur at

both pre and post harvesting stages. Pre-harvested aflatoxin

contamination occurs when peanut suffered from drought

stress especially during late generative growth phase (seed

maturation). Meanwhile post-harvest contamination usually

proceeds when the seeds are in warm and humid conditions

either during storage or transportation (Yameogo & Kassamba,

1999).

Extensive surveys on aflatoxin contamination at various levels

of the Indonesian peanut supply chain have found that

aflatoxin incidence was very minor at the farmer and collector

level where peanut is produced and sun-dried, respectively

(Dharmaputra et al 2003; Dharmaputra et al 2007; Rahmianna

et al 2007a). The toxin contamination increased as peanut

seeds went through the next level of supply chain i.e. the

marketing sectors especially at retail level in traditional

market; here the contamination was generally found to be the

highest. This indicates that aflatoxin contaminated peanuts

entered in the marketing chain, post-harvest contamination of

aflatoxin build up due to poor storage conditions (Dharmaputra

et al., 2003; Dharmaputra et al., 2004; Rahmianna et al., 2007;

Ndung’u 2013).

Early results of various studies confirmed that A. flavus is

highly prevalent in kernel samples stored at wholesaler and wet

markets with temperature (25 to 35oC) and relative humidity

(up to 60%) conditions being highly conducive for aflatoxin

production (Dharmaputra et al., 2003, Dharmaputra et al.,

2004; Rahmianna et al., 2007). Though Peanut is a cheapest

and important source of protein for population of developing

countries, and the current study is proposed to enhance our

understanding of factors leading to the post-harvest build up of

aflatoxin in peanuts during storage in three different selling

points (i.e. wholesaler, traditional ‘wet’ market and

supermarket) in Indonesia. The objective of this research was

to understand the incidence of aflatoxin contamination at three

market types during wet and dry seasons of study period.

2 Materials & Methods

A monitoring study was conducted by collecting peanut

kernels from the available points of market food chain in one

of the central production areas at East Java Province, i.e.

Pasuruan regency. In this regency, peanut is an important

secondary food crop and the grains are popularly consumed

either as food (peanut sauce, peanut cracker, etc) or in various

snacks. The monitoring study was conducted for 16 months

(from March 2005 to June 2006) with sampling scheduled in

every three months during the storage period (a total of 6

samplings: March, June, September, and December in year

2005, while in year 2006 was March and June). These six

samplings covered both dry and wet seasons, where from April

to October was dry season and November to March was wet

season. Peanut samples were collected from one wholesaler

(who distributes peanuts to traditional/wet market), three

shops/retailers in traditional market, and one supermarket.

At retailer, wholesaler and supermarket levels, three samples

were obtained in every sampling time as replication. An

analysis of variance was performed using a randomized block

design with two factors and three replications. Time of

sampling (six sampling times) was factor 1 and market type

(three market types) as factor 2.

The size of each sample was two kg of seeds. The air dried

seed were divided in eight parts by using a seed divider to

obtain working samples for analysing moisture content,

physical quality, percent of kernel infected by A. flavus and

aflatoxin B1 content (Dharmaputra et al., 2004). The procedure

for obtaining the working samples from the main peanut

samples described in the figure 1.

The moisture content of peanut kernels was measured by

gravimetric method. Physical quality of seed kernels was

quantified by manually segregating seed sample into three seed

qualities i.e. sound mature kernel (SMK), shriveled and

damaged kernels (only data on damaged kernels provided in

this paper). The damaged kernels included those cracked,

broken, discolored, and damaged by insects or fungi. The

percentage of seeds infected by A. flavus was determined by

plating 100 seeds per sample onto the Aspergillus flavus and

parasiticus agar (AFPA) media in 10 petri dishes (as

replications) or 10 seeds per each petri dish. The seeds were

incubated for 3 days.

The A. flavus infection was easily determined by the presence

of fungal colonies as shown by dark yellow or orange colour at

the bottom of the media. Aflatoxin B1 content in the seeds was

determined using Elisa method (Lee & Kennedy 2002).

Briefly, samples for aflatoxin B1 were extracted with methanol.

Supernatant was separated from the pellet. A competitive

ELISA was performed to measure aflatoxin B1 in the

supertanant. The absorbance values were determined using

spectrophotometer and plotted against a standard curve of

aflatoxin B1. The aflatoxin B1 contents of the samples were

expressed in part per billion (ppb).

347 Rahmianna and Yusnawan

_________________________________________________________



1) = samples for moisture content analysis

2) &5) = samples for physical quality of kernels analysis

7) = samples for A. flavus analysis

3), 4), 6) & 8) = samples for aflatoxin B1 analysis

Figure 1 The procedure for obtaining the working samples from the main peanut samples. [Source: Dharmaputra et al. (2004) ]

3 Results

3.1 Seed Moisture Contents

Entire samples obtained from three market types during the

various study periods, the seed water moisture contents were

found to be <10%, except from one sample that revealed

kernels with moisture content >10%. This high seed moisture

content was obtained from retailer at traditional market in

March 2006. Possible reason for this could be the usage of

opened-wooden box for the storage of seeds in the shops for at

least one week at ambient temperatures. The seeds obtained

from supermarket, however, had lowest moisture content

throughout the sampling times because peanut seeds were

properly packed in air-tight packaging and stored under air-

conditioned environment. Samples obtained from wholesalers

consistently contained 8-9 % moisture, as the seeds stayed for

very short period with maximum 7 days in this point, and

during storage, the seeds were packed in the gunny bag with

minimum contact to the surrounding air (Table 1).

Table 1 Seed moisture contents of peanuts collected from three different market types in Pasuruan Regency from March 2005 to June

2006.

Sampling times Market types

Traditional market Wholesaler Supermarket

March 2005 8.9 b 8.6 b 6.3 cd

June 2005 6.4 cd 8.2 bc 7.5 bcd

September 2005 7.9 bc 7.9 bc 6.3 cd

Januari 2006 8.0 bc 7.7 bcd 6.3 cd

March 2006 12.1 a 8.3 bc 7.2 bcd

June 2006 5.6 d 8.8 b 6.2 cd

Numbers followed by the same letter(s) indicated not significantly different based on DMRT 5%

Monitoring of Aflatoxin contamination at market food chain in east java. 348

_________________________________________________________



Table 2 Percent of damaged seeds of peanuts collected from three different market types in Pasuruan Regency from March 2005 to June

2006.



March 2005 29.6 ij 55.6 b 39.9 efgh

June 2005 50.0 bcde 24.6 j 52.1 bc

September 2005 35.0 hi 45.4 bcdefg 49.4 bcdef

December 2005 50.5 bcd 46.3 bcdef 65.9 a

March 2006 46.3 bcdef 35.6 ghi 40.2 defgh

June 2006 42.5 cdefgh 34.2 hi 39.5


Table 3 Percent of seeds infected by Aspergillus flavus on peanuts collected from three different market types in Pasuruan Regency from

March 2005 to June 2006.



March 2005 11.6 g 34.6 defg 42.3 bcde

June 2005 59.5 bc 33.8 defg 94.6 a

September 2005 34.3 defg 30.0 defg 16.0 fg

Januari 2006 25.6 efg 40.2 cde 21.0 efg

March 2006 52.5 bcd 63.2 b 36.5 def

June 2006 23.3 efg 41.6 bcde 30.5 defg


3.2 Physical quality

Despite three categories of physical quality observed, only data

of damaged seeds are presented in this paper since the damage

seeds had more risk in or were more prone to A. flavus

infection and aflatoxin B1 contamination based on our previous

studies (Rahmianna et al 2007a,. Rahmianna et al 2007b). In

general, the data indicated that percent of damage seeds were

high ranging from 24.8% to 65.9% in three market points.

Percent of damaged seeds at wholesaler was generally lower

than those at retailer level from six sampling times, except for

samples taken in March and September 2005 (Table 2).

Peanuts sold in the supermarket did not always have good

quality seeds in terms of seed coat color, intactness and

healthiness as pointed out by the sample obtained in June,

September, and ultimately December (Table 2).

3.3 Infection of A. flavus

Peanut seeds from all market chains were infected by A. flavus

at various levels, ranging from 11% to almost totally infected

(Table 3). The percentage of infection range was very wide,

especially for peanut from retailers in traditional market.

Among sampling time, samples taken in September 2005 had

low infection rate in all three market points.

3.4 Aflatoxin B1 contamination

The aflatoxin B1 (AFB1) contamination in most samples from

all three market types in Pasuruan regency was >10 ppb. In

addition, some samples had unacceptable levels of aflatoxin

since of its extremely high contamination (>3000 ppb) (Table

4). In general, the number of samples with aflatoxin content

>10 ppb from retailers was higher compared to those obtained

from wholesale and supermarket, except samples taken in

March and June 2006 (Table 5).

The higher contamination incidence at retailer level was due to

the longer period of storage under conditions that favorable for

the fungus to produce this secondary metabolite before the

seeds were processed for food.

4 Discussion and Conclusions

Any peanut kernel with moisture content ≥10% is stayed in

aflatoxin risk zone as the critical moisture for aflatoxin

production is in the range of 10-30% (Crop Link, 2000).

Having low aflatoxin contamination, the kernels should

contain 7-8% moisture and therefore the dry seeds had to be

kept under air-tight 0.05 mm poly propylene bag. This

condition was experienced by peanuts in wholesaler, where

they were closely packed. Dried condition of seeds together

with air-tight packed resulted in safe kernels with low aflatoxin

contamination even up to six months storage (Dharmaputra,

2002; Ginting, 2006).


_________________________________________________________



Table 4 Aflatoxin B1 content of peanuts collected from three different market types in Pasuruan regency from March 2005 to June 2006.

Market type Aflatoxin B1 content (ppb) of peanuts samples

March 2005 June 2005 Sept 2005 Dec 2005 March 2006 June 2006

Whole-sale 6.4 – 10.7 8.3 – 418.8 6.7 – 137.5 3.5 – 8.7 5.6 – 453.4 4.8 – 3583

Super-market* 6.5 11.5 9.1 7.7 6.3 265.8

Retailer in traditional market 1.7 – 18.1 8.1 – 3074.9 5.6 – 478.3 3.8 – 90.9 4.8 – 290.8 4.4 – 187.7

* one sample only

Table 5 Percentage of seeds with aflatoxin B1 content lower and higher than 10 ppb of peanuts collected from three different market

types in Pasuruan regency from March 2005 to June 2006.

Market type March 2005 June 2005 Sept 2005 Dec 2005 March 2006 June 2006

≤10

ppb

>10

ppb

≤10

ppb

>10

ppb

≤10

ppb

>10

ppb

≤10

ppb

>10

ppb

≤10

ppb

>10

ppb

≤10

ppb

>10

ppb

Whole-sale 72.7 27.3 70.0 30.0 76.9 23.1 100 0 20.0 80.0 60.0 40.0

Super-market* 100 0 100 0 100 0 100 0 100 0 0 100

Traditional market 81.3 18.7 38.1 61.9 64.7 35.3 89.0 11.0 89.0 11.0 85.7 14.3

*one sample only

High number of damaged seeds in traditional market was in

accordance to the results obtained from six retailers at

traditional markets in Banjarnegara region of Central Java,

where there was an average of 45% damaged seeds with the

range from 5 to 73.5% (Rahmianna et al., 2007a). The storage

system practiced by retailers in the traditional market at

Pasuruan (i.e. storing the grains in opened containers during

the trading hours under ambient temperature) favored high

incidence of damage seeds. Ginting (2006) concluded that

when seeds were stored in opened containers under ambient

temperature for 4 months generated huge damaged seeds by

80.9% compared to those kept in the 0.05 mm and 0.08 poly

propylene bags with only 1.3 and 1.9% of damaged seeds,

respectively. Seeds that were exposed to environment with free

air rich in oxygen in the traditional market was stimulate the

activity of microorganism both fungi and pest. Also, improper

post harvest handling such as shelling (both manually and

machinery) contributed to testa and/or cotyledons damage.

This physical damage did not only found in peanut seeds

obtained from in retailer level but that also found in wholesale

level.

High A. Flavus infected seeds obtained from traditional market

were also found in peanut seeds collected from traditional

markets at Banjarnegara (Rahmianna et al., 2007), Pati

(Dharmaputra et al., 2003), and Wonogiri regencies

(Dharmaputra et al., 2004). In this monitoring study, high

incidence of A. flavus infection in peanut obtained from

supermarket was surprisingly. Since A. flavus is a soil borne

fungus, the spores of A. flavus infected the pods during pre-

harvest. The physical condition of pods and the intactness of

testa plays an important role in entering the spores into the

seeds and the development of hyphae onto the seeds,

respectively (Mehan et al., 1983, Cole et al., 1985; Van Eden

et al., 1994). Generally the fungal infection was getting serious

when the seeds stayed in the marketing chains with poor

storage condition as pointed out by high number of seeds

infected by A. flavus. In contrast, the number of seeds infected

by A. flavus was very low (4.8-5.6% in average) at harvesting

time in the farmer’s field at Banjarnegara: (Rahmianna et al.,

2007). According to Lubulwa & Davis (1994) aflatoxin content

of 1000 ppb resulted in acute liver damage to human, livestock

and poultry. High contamination at retailer level also happened

in Cote-d’Ivoire of Africa (Pollet et al., 1992) as aflatoxin

contamination built up during storage and transportation

(Yameogo & Kassamba, 1999).

Based on the data obtained in all market points during six

times of sampling undertaken both in dry and wet seasons,

aflatoxin contamination was directly governed mostly by seed

moisture content rather than by the number of seeds infected

by A. flavus and physical quality.(i.e. damaged seeds) as

pointed out by correlation coefficient values of 0.517, 0.384

and 0.194 respectively.

Theoretically, aflatoxin production was governed by the level

of A. flavus infection. Seed damage, among others, as a result

of A. flavus infection, also played a significant role in aflatoxin

production. The production of this secondary metabolite was

also influenced by seed moisture content. All these theories

were found significant in peanut seeds obtained from

supermarket. These phenomena did not find in peanut seeds

obtained from retailers and wholesalers. Air-tight container

absolutely prevents the seeds from the interference of

surrounding environment and air-conditioned storage room

warrants the stable air temperature and relative humidity.

Rahmianna et al. (2007) revealed the positive correlation

between seed moisture content, which was from 4.2 to 17.6%,

in line with the moisture content which risky to aflatoxin

contamination by 10-30% (Crop Link, 2000) with aflatoxin

contamination. The positive correlation was also significant

between A. flavus infection and aflatoxin, both for peanut

obtained from farmer’s level.


_________________________________________________________



Kernel moisture content most of peanut kernels sampled in

wholesale and retail outlets was lower than 10% at most of the

sampling times and therefore the incidence of aflatoxin

contamination was also low or under the safe level of

contamination. Despite a substantial proportion of damaged

kernels both caused by mechanical and biological damage in

seed lots obtained at each sampling time, these two elements

did not govern the level of aflatoxin contamination. Time of

sampling throughout the year did not affect the level of

aflatoxin contamination. Under controlled condition generated

from air-tight container and controlled condition, the influence

of A. flavus infection and seed moisture content on aflatoxin

production was significant.




Acknowledgment

The research was funded by Australian Center for International

Agricultural Research through project # PHT 97/017. Thanks

are due to Dr. Rachaputi Nageswara Rao for improving the

research proposal. We also thank to Miss Lina Kusumawati

and Mr. Langgeng Sutrisno, for their kindly help in the

laboratory and field activities.

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geocarphosphere temperatures that induce preharvest aflatoxin

contamination of peanuts under drought stress.

Mycopathologia 9: 41-46

Crop Link (2000) Aflatoxin in Peanuts. Tips to Reduce the

Risk. Queensland Department of Primary Industries. Farming

Systems Institute. 12 pp

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food and feedstuffs and their products. Biotropia 19: 26-46

Dharmaputra OS, Putri ASR, Retnowati I, Ambarwati S (2003)

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the delivery chains in Pati regency, Central Java Province.

Research Report. SEAMEO BIOTROP. 24 pp (in Indonesian)

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Aspergillus flavus and aflatoxin contamination in peanuts at

various stages of the delivery chains in Wonogiri regency,

Central Java Province. Biotropia 14 (2): 9-21.

Ginting E (2006) Quality and aflatoxin contamination in

peanut seeds cultivar Kancil and Mahesa under several

packaging materials. Jurnal Agrikultura 17(3):165-172 (in

Indonesian).

Lee AN, Kennedy IR (2002) Practical 1. University of Sydney

quick aflatoxin B1 ELISA Kit. Paper presented at ELISA

Workshop Analysis of Aflatoxin B1 in Peanuts, held in Bogor

on 12-13 February 2002. Organized by University of Sydney,

ACIAR and SEAMEO Biotrop Bogor. 8 pp.

Lubulwa ASG, Davis JS (1994) Estimating the social costs of

the impacts of fungi and aflatoxin in maize and peanuts. P.

1017-1042. In E. Highley et al (Eds). Stored Products

Protection. Proc. The 6th

Inter. Working Conf. On Stored-

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and potential for aflatoxin contamination in groundnuts and

peanut butter from farmers and traders in Nairobi and Nyanza

provinces of Kenya. Journal of Applied Biosciences 65: 4922 –

4934

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role of postharvest machineries and packaging in minimizing

aflatoxin contamination in peanut. Indonesian Journal

Agricultural Science 7: 16-20

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Academic & Profisional. London.

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E (1992) Three years’ studies on relationships between

traditional groundnut storage and aflatoxin problems in Cote-

d’Ivoire. Main results. Oleagineux 47: 71-85

Rahmianna AA, Taufiq A, Yusnawan E (2004) Evaluation of

ICRISAT groundnut genotypes for end-of-season drought

tolerance and aflatoxin contamination in Indonesia.

International Arachis Newsletter No 24: 14-17.

Rahmianna AA, Ginting E, Yusnawan E (2007a) Aflatoxin

contamination on peanut at various stages of the delivery chain

in Banjarnegara, Central Java. Jurnal Penelitian Pertanian

Tanaman Pangan 26(2): 137-144 (In Indonesian).

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harvest timing and post harvest storage conditions on aflatoxin

contamination in groundnuts harvested from the Wonogiri

regency in Indonesia. International Arachis Newsletter No 27:

43-45.

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(1994) The role of insect damage in the colonization of

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Wotton HR, Strange RN (1985) Circumstantial evidence for

phytoalexin involvement in the resistance of peanut to

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aflatoxin on tropical seed used for snacks: Arachis hypogaea,

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39: 46-49.

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39: 46-49.


_________________________________________________________



KEYWORDS

Fishermen

Livelihood

Batiaghata Upazila

Khulna

Bangladesh

ABSTRACT

This study was undertaken to update the livelihood status of fishermen community in Batiaghata upazila

of Khulna district from February to December, 2013. The data were collected through the questionnaire,

survey, group discussion and public interview. The results of the study revealed some interesting facts

and showed that most of involved fishermen are in 16-30 years age group (45%) whereas majority of

them were Hindu (62%). About 75% of fishing community was illiterate and 24% was literate.

Furthermore, it was reported that about 78% of the fishermen received treatment from Village doctors (

have little medical knowledge, mostly quack) and only 20% of the respondents concern the doctors from

upazila health complex and left over 2% got health service from MBBS doctors outside the upazila. It

was found that 61% house were Kacha (Made of mud and straw) while 37% house were Semi-pucca

(Tin shed building) and only 2% house were pucca. (Bricks Built house) About 23% of fishermen had

ability to access electricity and others 77% had no access to electricity. No Vulnerable Group Feeding

cards were provided by government for them in those areas. Lack of proper knowledge, illiteracy not as

much of governmental support was the major constraints.

Mridula Rani Das1, Sunuram Ray

2, Uttam Kumar

3,* Salma Begum

4 and Satya Ranjan Tarafdar

5

1,4

Environmental Science Discipline, Khulna University, Bangladesh 2Centre for Integrated Studies on the Sundarbans (CISS), Khulna University, Bangladesh

3World Vision International, Bangladesh

5Development Activist, Bangladesh

Received – June 03, 2015; Revision – June 18, 2015; Accepted – August 05, 2015



LIVELIHOOD ASSESSMENT OF THE FISHERMEN COMMUNITY IN THE SOUTH

WEST REGION OF BANGLADESH

E-mail: [email protected] (Uttam Kumar)







ISSN No. 2320 – 8694















_________________________________________________________



1 Introduction

Fish and fisheries products play a Significant socioeconomic

role of Bangladesh in terms of nutrition, income, employment

and foreign exchange earnings and depend on fish as the

principal source of animal protein. More than 80 percent of the

animal protein in the Bangladeshi diet comes from fish

products. Fisheries sector contribute about 90% of the nation’s

animal protein intake, nearly 6 to 8% GDP to national income

and 5.77% to the foreign earning of nation (DoF, 2012).

Annual fish production was 2,701,370MT in 2008-09 where

annual fish intake by an individual is 17.52kg and the annual

fish demand is 29.74 MT (DoF, 2010). It is estimated that

about 12 million people are directly or indirectly engaged in

fishing and other ancillary fishery activities and a large portion

of rural family members are engaged in part time fishing from

the beels (Hughes et al., 1994). About 12 million people of the

country directly or indirectly involved in fisheries and other

ancillary activities (DoF, 2013). Livelihood is sustainable

when it can cope with stresses & shocks and maintain or

enhance its capabilities to recover from it, while not

undermining the natural resource base (Chambers & Conway,

1992). For sustainable rural development and poverty

elimination, different approaches had been adopted and the

sustainable livelihood approach has been gradually expanded

with its own core and principles for poverty focused

development activities (DFID, 1999). The approach basically

based on the fundamental principle analysis of capital assets in

the context of the external environment. Scoones (1998)

addressed that a sustainable livelihood is a way of thinking

about the objectives, scope and priorities for development, in

order to enhance progress in poverty elimination.

Batiaghata upazila is adjacent to the Khulna town is one of the

most important ecosystem with much aquaculture impending.

This area is known as a fishery polli which play a very

important role in alleviation of rural poverty and supplying

food to the poor fishing community. Large scope of fish

culture system would be possible if fish farmers adopt improve

technology and most of the fishermen in this area uses

traditional fishing method. However, socioeconomic status of

this fishing community is not reasonable; production of fish is

also declining day by day. Considering the above fact, the

present study was conceded to assess the livelihood status and

constraint faced by the fishermen in the study. For this reason,

Batiaghata upazila was selected for the study to identify socio-

economic condition of the fish farmers.

2 Material and Methods

2.1 Study area and duration

This study was carried out in Batiaghata upazila of Khulna

district, Bangladesh. The study areas located between 22°34´

to 22°46´ Latitude and 89°24´ to 89°37´ Longitude. The study

period was from February to December, 2013 (figure 1).

2.2 Sampling frameworks

The study was composed of a livelihood systems assessment,

coastal regions of south-western Bangladesh. The primary data

were collected through the questionnaire, survey, group

discussion and interview. Weekly field survey was carried out

to collect the necessary information through random selection

method. A total of 100 fishermen households were surveyed in

ten villages, viz-Jharbanga, Machalia, Tengramari,

Katiadangla, Amtala, Titukhali, Andharia, Khejurtala, Persolua

and Brittisolua under Gangarampur union, Batiaghata upazila.

However, all the data were cross-checked for ensuring the

accuracy of data collected from the respondents. The Focus

Group Discussion were conducted to identify the problems and

to collect fishermen’s recommendations regarding the

problems identified so that effective solution to the problems

would develop. Necessary information on the socio- economic

condition of fishermen was collected from regional fisheries,

settlement, LGED and agricultural offices.

Figure 1 Location of study area in Batiaghata upazila under Khulna district, Bangladesh.

354 Mridula et al

_________________________________________________________



3 Results and Discussion

Total 100 fishermen were interviewed and it was reported that

the socio-economic condition of fishermen using the survey

indicators like, sex ratio, religion status, age distribution

marital status, educational status, house type, latrine, daily

intake of meal, diseases and treatment, land owner, occupation,

expenditure, monthly income, earn from per travel to the sea,

trends of fishing faced various levels problems. A detailed

analysis is made on these parameters and presented in this

section.

3.1 Sex Distribution

Results of the sex ratio and religion related study suggested

that total 82% male and 18% female respondents were actively

involved in the fishing (figure 2). DoF (1993) studied on

different coastal districts of Bangladesh and showed

involvement of sex ratio, male: female was 90:10 in fishing.

Figure 2 Sex distribution related to the fishing

3.2 Age distribution

Knowledge of age distribution of the fishermen is important in

estimating potentiality of the human resources. In the present

study, age classified into five groups as 0-15, 16-30, 31-45, 46-

60, and 61-75 years that presented in the figure 3. The

investigation showed majority of the fishing professional

belongs to the 16-30 age group (45%) while the 61-75 aged

class has been lowest involvement (4%).

Results of the study are controversial than the Minar (2012)

and revealed that 31-40 old people are highest (56%) and 41-

60 aged people are the lowest (14%) involved in fishing. In

another study Kostori (2012) revealed that majority (36%)

fishermen of chalan Beel were belonging to age group of 20-

30 years and these findings are in accordance with the findings

of present study.

Figure 3 Age distribution of the respondent fishermen

3.3 Family type and family size

Trends of joint family are continuously decreasing in the

fishermen society of Bangladesh. This may be due to the poor

economic condition and different natural calamities of the

fishermen. Result of the present study showed that maximum

numbers of studied fishermen leaved in single family (76%)

while very small number of the studied responded prefers

extended family (24%). The table (1) disclosed the number of

family people and respondent in the survey.

Table 1 Family size of the studied respondent

Family Size (Year) Respondents %

01-03 9

04-06 65

07-09 23

10-12 2

13-15 1

3.4 Educational status

Many of the fishermen were literate at various levels of

education. However, while responding it was found to just

class 1 to 10 as literature and such percentage was only 24%.

Among the studied responded only one fisherman had passed

S.S.C (Secondary School Certificate Exam). Above this level

no one had higher education while the maximum studied

respondent fishermen (75%) were illiterate. Most of the

sampled fishermen were compelled to enter into the fishing

profession in their early status of their parents and lack of

awareness about education. Another important factor is that,

there were hardly any educational institutions in this area. For

above reasons, most of the fishermen were illiterate of the

study area (figure 4).

0

20

40

60

80

100

Male Female

fish

erm

en %

Livelihood assessment of the fishermen community in the south west region of Bangladesh 355

_________________________________________________________



Figure 4 Educational qualification of the respondent fishermen

of the study area.

Literacy rate in the fishermen communities was found to be

25% being defined when a person found to just class-1 to 10

passed and S.S.C passed. Hossain & Pingali (1998) and

Shahjahan et al. (2001) studied socio-economic condition of

the fishermen and reported that majority of the fishermen were

illiterate (71.12% and 63.33% respectively) while 24.44%,

31.67% of the riverine fishermen had only primary level of

education and only 4.44%, 5% of them had only secondary

level of education respectively. In this aspect results of the

present study are in agreement with the findings of Hossain &

Pingali (1998) and Shahjahan et al. (2001).

According to the study of Hannan (1994), majority of the

fishermen (96.97%) were literate at the various stages of

education among the coastal fishing community of the

Kalapara Upazilla. Study of Ahamed (1999) in the polder

estuaries areas obtained literacy rates 25% and 23%

respectively. DoF (1993) in chanda beel and Ahamed (1996) in

Tangail found literacy rates 45% and 69% respectively.

3.5 Religion status

Results of the study revealed that the maximum number of

studied area fishermen (62%) belongs to Hindu community

while the rest 38%were belongs to Muslim community.

Figure 5 Religion status of the fishermen community.

Results of the present study are in accordance with the findings

of Shamima (2000) who had reported that 61.11% of the

fishermen of Gollamari Fishing Community, Khulna were

Hindus while 38.89% were belongs to Muslim community.

These results show similarities with the findings of present

study while the findings of Ahamed (1999) are controversial to

the present study and he reported that in coastal area showed

majority of the fishermen are Muslims (68.33%) (figure 5).

3.6 Ownership of house and house types

Present study showed that a great portion of fishermen were

either landless or nearly landless people. Among the studied

respondents, about 60% of fishermen were landless and they

had no arable land and about 30% of the fishermen owned only

1-40 decimal homestead land (figure 6). Ahamed (1996)

reported that 94% fishermen household live in their own

house. Furthermore, Mannu (1999) reported that 28% of

fishermen constructed house on the khal land outside of the

dam, 36% were owner land of the house, 8% in the father laws

house and 12% lived joint with father.

Figure 6 House types of fishermen in the study area.

From the figure, it is clear that 61% of the studied areas houses

were Kacha while the 37% houses are Semi-pucca and only

2% houses are pucca. So, economic condition of fishermen of

the study area was not well.

3.7 Use of Electricity

The study revealed that only 23% of the fishermen had

electricity access while majority (77%) of them had no

electricity access (figure 7). Results of the present study are in

accordance with the findings of Shamima (2000) who had

reported that 20% of fishermen had electricity in Gollamari

fishing community, Khulna. According to the DoF (2012) only

2% fishermen household used electricity and in this manner the

fishermen of the study areas had better condition with respect

to the electricity consumption.

Figure 7 Status of the electricity consumption by the study area

fishermen community

62%

38%

H…M…

356 Mridula et al

_________________________________________________________



3.8 Working pattern of the fishermen

In the study area, the fishermen caught white fish viz; Vola,

Taposhi, Khaira, shrimp (Chaka, Chapne, Horina) etc by using

small or medium sized boats. The research estimated that 57%

of fishermen have own-boat and only 18% of fishermen

worked as a day labour in Mohajon’s (Usurious) fishing boats

(figure 8). They have to go to the river 3-4 Km from their

residence for fishing.

Figure 8 Working patterns of the respondent fishermen

3.9 Travel based income for going to the river

The fishermen went to the river 3-4 times in a day for fishing.

The study showed that 17 % of the studied fishermen earn

monthly income ranged only 0-25 US$. In present study it was

reported that 35% and 39% of the study area respondents has

monthly income ranged 25-50$ and 50-76.8$ respectively.

About 5%, 3% and 1% of fishermen earned 77-102$, 102-128$

and >128$ respectively. So the graph (figure 9) revealed that

the fishing will not be main tools of fishermen livelihood.

Figure 9 Travel based income level of the respondent

fishermen

3.10 Alternative livelihood options

Present study estimated that the monthly income rose during

June to September but that is not handsome for fishermen.

Fishermen opine that tender system has increased their

investment costs and their dependency on middlemen. So, the

people involved in agriculture, day laboring, business, Van-

rickshaw pulling and so on as their secondary and tertiary

alternative options. Besides, male and female even child make

the net round year. Results of the survey showed that in the

year 2003 fishing was the primary occupation for most of the

respondent fishermen and with this secondary and other

occupational percentages were little bit. But at 2013 their

primary occupation was still fishing but their secondary and

other occupational percentages status were going to high. The

study (figure 10) ensures that fishermen are prone to

agriculture, scarcity of fish and bad weather.

Figure 10 Secondary occupations of the respondent fishermen

3.11 Changes of income sources

Maximum fisherman of the study area belongs to poor and

underprivileged class. They cannot improve their socio-

economic condition by fishing profession as the income from

fisheries sector is continuously reducing. So, they are shifted to

other sources.

Figure 11 Income percentages from different sources

In the study area, 73% fishermen were caught fish all the year

round dependent family and 27% were other sources

dependent family at 2003. But the graph showed that fishing

dependent families were decreasing and other sources

dependent families were increasing at the year 2013 (figure

11). Ahamed (1996) found similar result stating 81% carried

out fishing throughout the year. However, Mannu (1999)

reported that 72% were full time fishermen.

3.12 Problems faced by fishermen

The fishermen have to catch fishes in good and bad weather

condition. They have to go to the river and faces different

types of problems during fishing.


_________________________________________________________



Figure 12 Various problems faced by the fishermen during

fishing.

So, they faced different types of problems during fishing. Most

of them faced various types of disaster and the percentage of

these reached up to 65%. 12% fishermen faced problems by

diseases, 8% faced problems by pirates and 20% faced

problems by lack of capital (figure 12). Sometimes, they faced

a little bit transportation problems during fishing.

3.13 Present fish status in the river

Day after day, fish’s populations are decreased in the river and

new species of fish are not found in the river. (figure 13) The

graph indicates poor condition than previous status of fish.

Figure 13 Fishermen perception on status of fish into the river.

From the study it was found that 64% respondent opined that

the present status of fish into the river was low, 33% believed

that medium and only 3% said that high.

3.14 Process of fish preservation

Most of the fishermen caught fishes from nearby river of the

study area and try to come back early to the land and sold the

fish to the fish market or to the buyers. That’s why they did not

need any kind of preservation process. Among the studied

respondent’s fishermen, most of the fishermen did not preserve

fish (65%) but sometimes they may use ice (27%) or other

preservative methods (8%) for preserving their fish products

(figure 14).

Figure 14 Process of fish preservation

3.15 Fish selling process

The pattern of fish selling process was not satisfactory to the

fishermen of the study area. The fishermen of the study area

were poor. Most of them had no sufficient capital for fishing.

They had to take Dadon (High rated loan from usurious) for

fishing from moneylenders.

Figure 15 Fish selling process in the study area

Fishermen of the study area were not interested to take Dadon

from moneylender. But, they had to take Dadon as they had no

enough capital for fishing in the river. Fishermen lend money

from Mohajon’s for fishing purpose. But, they can’t back this

money directly. Under such condition, boat was also given by

Mohajon’s. When the fishermen come back from the river after

fishing the whole fishes are taken by the Mohajon’s.

Mohajon’s sold out all the collected fishes and received their

loan amount. At first Mohajon’s take this money that he gave

to the fishermen and then he distributes the rest of the money

as condition Mahajan’s: Fishermen at 10:6 ratios. From the

study (figure 15) it has been reported that the fish were selling

to the arot /storehouse (45%), market (15%), dealer (10%) and

wholesaler (30%).

3.16 Sources of drinking water

Drinking water have direct effect on the fishermen’s health but

among the total surveyed respondents only 89% fishermen

used tube-well water for drinking purposes, 3% used pond

water, 7% used river water and 1% used other sources of water

for drinking and other daily activities (cooking, bathing and

washing). In general, women produced in the study area were

using drinking water, this does not mean that all had tube-well

(figure 16).

Figure 16 Drinking water sources of fishermen

358 Mridula et al

_________________________________________________________



Half of the fishermen were found to have their own tube-wells

while others are using governmental tube-wells. A number of

schools had tube-wells in the survey area and also market place

contained one or two tube-wells in the study area. Mahbullah

(1986) noted that 41% fishermen use tube-well water for

drinking, cooking, bathing and washing. Rahman & Hassan

(1992) exhibited that 11% for drinking, cooking and bathing,

18% for drinking and cooking and 29% for only drinking in

the polder and estuarine fishing community in Bangladesh.

3.17 Effect of diseases

All members of the fishermen communities were affected by

various diseases viz. Diarrhoea, Fever, Jaundice, Typhoid,

Gastritis and so on.

Figure 17 Common diseases among the fishermen of the

studied area

From the survey it was found that most of the fishermen suffer

from water borne diseases such as Diarrhoea, Jaundice, and

Typhoid etc. Majority of them (34%) suffer from Jaundice

while 27% of them suffer from fever, 18% of them suffer from

Diarrhoea, 10% of them suffer from Fever and 9% of them

suffer from Typhoid (figure 17). Similar type of observation

was made by Samima (2000) who had reported that 52%

fishermen suffered from fever in Gollamari fishing

communities, Khulna District. Generally In Bangladesh, there

is a significant variation in temperature ranging from 10 to

38ºC; heat stress mortality may be observed among elders,

especially during mid-April and mid-August. Due to high

temperature and humidity, the elder and malnourished children

will face dehydration related problems. A temperature increase

of 1~2ºC would perhaps not cause a significant, but higher

intensity of extreme events may intensify heat stress and

associated health hazards.

3.18 Sanitation practice used by fishermen communities

Information was collected on the extent of sanitation practice

of the respondent fishermen. Majority of the fishermen (59%)

used katcha latrine for defection in the study area (figure 18).

Figure 18 Sanitation practice of the fishermen.

Second majority of the fishermen used sanitary latrine (40%)

for defection and 1% fishermen had no latrine.

3.19 Fishermen Comments about the Present Condition of the

river

Most of the fishermen go for fishing in the river 3-4 km away

from their residence. That’s why they go as a team of

partnership mostly. They go two times in a day for fishing.

But, at the time of day of the new moon and night of the full

moon they go 3-4 times for fishing because in this time the

amount of fishes increased. They have been done this work

since 20-30 years. So, they have vast experience about the

wave pattern, flow of water, high tide, low tide, the quantity of

fishes in the river and the extinction of the fish species in the

river. By the following table it is seen that the condition of

river is at now vulnerable condition in a great extent.

Fishermen said that the price of fish is not good enough to do

well their socio-economic condition. Fishing zones are altering

and 93% fishermen were saying that fishing zones are altering

and only 7% are responding negatively (table 2). They also

said about the decreases of water in river, pond, khal and beel

and amount of fish in river.

Table 2 Comments of the fishermen about the river.

Result Choice of Fish Price

(%)

Improper

Change (%)

Fishing Zone Alter

(%)

Are fishes Less Amount

in River? (%)

Decreases of

water (%)

Yes 3 99 93 100 100

No 97 1 7 0 0


_________________________________________________________



Conclusion

The socio-economic condition of the fishermen in the adjacent

area was not satisfactory. The fishermen were deprived of

many facilities. The education level of the fishermen was so

poor. Lack of awareness as well as the poor income, the

fishermen have to take loan from Mohajan at high interest. So

why, some educational institutes should be built up in the

adjacent area. The Govt. should take some important step by

providing some sorts of management policy as well as

providing of some extra providence during the ban season of

the fishing. That may be done within the providing of the

VGF card. Some forms of NGO’s activity must be ensured in

the adjacent area for the improvement of the life leading status

of the fishermen. The NGO’s must be helpful about the

providence of the loan which may be used for the up gradation

of the income procedure. As well as health facilities should be

ensured by the government assistance.

Acknowledgement

Authors are grateful to Environmental Science Discipline,

Khulna University, Bangladesh for the technical support to

conduct the study.




References

Ahamed N (1999) A study on socio-economic aspects of

coastal fishermen in Bangladesh, Bangladesh journal of

Zoology 24:20-26.

Ahamed NU (1996) Reports of the fishermen’s socio-

economic survey. Fisheries survey and monitoring program.

Department of fisheries, Bangail. Pp4.

Chambers R, Conway GR (1992) Sustainable Rural

Livelihoods: Practical Concepts for the 21st Century. IDS

Discussion Paper 296 available on

http://www.ids.ac.uk/publication/sustainable-rural-livelihoods-

practical-concepts-for-the-21st-century access on 24th March,

2015.

Department for International Development (1999). Sustainable

livelihoods guidance sheets, (DFID), London, UK.

Department of Fisheries (1993) Ministry of Fisheries and

Livestock, Dhaka, Bangladesh. National Fish Week

Compendium (in Bengali) Pp 144.










Hannan M (1994) Fisher folk organization in Bangladesh. In:

Socio-economic Issues in Coastal Fisheries Management.

Proceedings of the IPFC Symposium, Bangkok, Thailand, 23-

26 November 1993: Indo Pacific Fishery Commission (IPFC),

no. 8, pp. 216-222.

Hossain M, Pingali PL (1998) Rice research, technological

progress and impact on productivity and poverty: an

overview. In: Pingali P, Hossain M (Eds.) Impact of Rice

Research. Proceedings of the International Conference on the

Impact of Rice Research, 3-5 June 1996, Bangkok. Thailand:

Thailand Development Research Institute and Los Benos

(Philippines) and IRRI, Pp 1-2, 5.

Hughes R, Adnan S, Dalal-Clayton B (1994) Floodplains or

flood plans? A critical look at approaches to water

management in Bangladesh. International Institute for

Environment and Development (IIED), London and Research

and Advisory services, Dhaka.

Kostori MFA (2012) Scio- economic condition of fishermen of

the Chalan Beel under Talash thana of Sirajganj Disrtrict in

Bangladesh. Bangladesh Research Publication Journal 6: 393-

402.

Mahbullah M (1986) Case study of polder and estuarine

fishing community in Bangladesh. Socio-economic study of

tropical fishing community in Bangladesh, A report for the

food and agriculture organization (FAO), Rome. 1986(12-14).

Mannu MU (1999) Jeleder Sukh Dukh. The Daily Janakantha,

22 August 1999.

Minar MH, Arifur Rahman AFM, Anisuzzaman M (2012)

Livelihood status of the fisherman of the Kirtonkhola River

nearby to the Barisal town. Journal of Agroforestry and

Environment 6: 115-118.

Rahman MM, Hassan MR (1992) A study on fish and

fishermen of Kaptai Lake in Bangladesh. Report submitted in

Grants Commission, Dhaka, Pp. 49.

Shamima A (2000) Socio-economic conditions of fishing

community: Gallamary fish market, Khulna. A project thesis in

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http://www.ids.ac.uk/publication/sustainable-rural-livelihoods-practical-concepts-for-the-21st-century

http://www.ids.ac.uk/publication/sustainable-rural-livelihoods-practical-concepts-for-the-21st-century

_________________________________________________________



Fisheries and Marine Resource Technology Discipline, Khulna

University, Khulna, Bangladesh Pp 11-34.

Scoones I (1998) Sustainable rural livelihood: a framework for

analysis. IDS working paper No. 72.Institute of Development

studies,1998 Brighton.

Shahjahan M, Miah MI, Haque MM (2001) Present Status of

Fisheries in the Jamuna River. Pakistan Journal of Biological

Sciences, 4: 1173-1176 DOI: 10.3923/pjbs.2001.1173.1176.


http://dx.doi.org/10.3923/pjbs.2001.1173.1176

_________________________________________________________



KEYWORDS

Growth

Yield

Poultry

NPK 20:10:10

ABSTRACT

Effect of poultry manure and organic fertilizer (NPK 20:10:10) on the growth and quality of cucumber

fruits was studied at the experimental site of the Teaching and Research Farm of the Department of

Crop Science and Horticulture, Faculty of Agriculture, Chukwuemeka Odumegwu Ojukwu University,

Igbariam campus. The experiment was laid out in a randomized complete block design (RCBD) with

four levels of treatments consisting of 4tha-1

poultry manure (PM), 900 kgha-1

NPK in a ratio of

20:10:10 (NPK), 2 tha-1

poultry manure + 450 kgha-1

NPK fertilizer (PM + NPK) and 0 tha-1

control

(CO), where no treatment was applied. Data collected were tested using analysis of variance and

significant differences among treatment means were separated using LSD at 0.05 probability level. The

results obtained from the study indicated that the numbers of leaves of cucumber increased as weeks

after planting (WAP) increased. The highest leaves numbers was observed in the plots treated by PM.

The order of leaves increases from 2 to 6 weeks were PM > PM+NPK > NPK > CO. The length of

fruits, number of fruits, the quality of marketable fruits and weight of fruits increased proportionately in

PM treated plots and were significantly (P=0.05) different among the other treatments except for quality

of marketable fruits. The highest value of 171.25cm (length of fruits), 10.75 (number of fruits) and

2.38kgha- 1

(weight of fruits) were obtained in PM treated plots. Based on the results obtained it is

evident that poultry manure as organic manure and its mixture (PM + NPK) is a good source of soil

amendment, since it influenced the growth and yield components of cucumber.

OKOLI PSO1 and Nweke I A

2,*

1Department of Crop Science and Horticulture, Chukwuemeka Odumegwu Ojukwu University, Anambra State, Nigeria.

2Department of Soil Science, Chukwuemeka Odumegwu Ojukwu University, Anambra State, Nigeria.

Received – May 15, 2015; Revision – May 29, 2015; Accepted – August 25, 2015



EFFECT OF POULTRY MANURE AND MINERAL FERTILIZER ON THE GROWTH

PERFORMANCE AND QUALITY OF CUCUMBER FRUITS

E-mail: [email protected] (Nweke I A)







ISSN No. 2320 – 8694




All the article published by (Journal of Experimental

Biology and Agricultural Sciences) / CC BY-NC 4.0






_________________________________________________________



1 Introduction

Production of cucumber in Nigeria has increased probably due

to awareness being created by its market demand and

economic returns, short duration in maturity or due to its

nutritional and medicinal values. Hence it has become a

popular vegetable crop in Nigeria. Both older and young

people enjoy the cucumber fruit of which many in their leisure

time usually eat with fried groundnut in their offices, homes,

and market place or recreational areas. When given proper care

and protection, cucumber tends to produce well. It is a crop

that grows well in a well drained fertile soil with good

moisture retention ability. This crop required high amount of

soil nutrients from seedling stage to maturity and highly

sensitive to excessive water or waterlogs environment and

adequate soil tillage for easy fragile root penetration, is

required prior to sowing or planting (Nweke et al., 2014).

Water logging usually causes or trigger off leaf problem in

cucumber. Increase in cucumber production can be achieved

either by putting more land area under its cultivation or by

using improved varieties with appropriate cultural practices.

However, manure application either in form of organic or

mineral fertilizer is found to be the quickest and easiest ways

of increasing the yield of cucumber per unit area (Nweke et al.,

2014, Nweke & Nsoanya, 2015).

Mineral fertilizers were used to provide soil nutrients in order

to maintain optimum soil fertility conditions and healthy

growth of plants and quality yield. Chemical fertilizers help the

growing crops to withstand stress conditions and in some cases

these were used to correct plant nutrients deficiencies.

According to Leonard (1986), maximum net returns in crop

production can adequately be sustained with adequate fertilizer

program that will supply the amounts of plants nutrients

needed. Similar type of observation was reported by Akinrinde

(2006) in various crop production studies. There are also

various reports on the preferences of mineral fertilizer in the

growth and productivity of crops (Adediran & Banjoko, 2003;

Akinrinde, 2006; Nweke & Nsoanya, 2013a; Nweke & Emeh,

2013). NPK fertilizers are required greatly by crops for healthy

development and crop quality. Furthermore, Ologunda (1987)

reported that Nitrogen is the most limiting factor for

production of crops on soils around the world. Hence Djokoto

& Stephen (1961), argued that Nitrogen is the most important

element in the nutrition of compositing micro flora since it is

required for the simulation of carbon substrate in organic

waste. The next element after N that limits the crop production

in the tropical regions and indeed most regions of the world is

phosphorus ( Holford, 1997). According to HUE, (1995)

inadequate P supply will result in a decreased synthesis of

RNA, the protein maker, leading to decreased growth. Grain

yield is often severally reduced with P deficiency (Jones et al.,

2003). Potassium is required in least amount but in soil it is

required in large amount by many crops and it is important for

maintaining the osmotic potential and rigidity of plant cells;

hence it plays a vital role in water relations in the plant.

Marschner (1995) observed that K is involved in water uptake

from the soil, water retention in the plant tissue and long

distance transport of water in the xylem and of photosynthetic

in the phloem. With adequate K, cell walls are thicker, thereby

improving plant resistance to lodging, pests and diseases

(Bergmann, 1992).

It was based on the few highlighted qualities these three

elements are formulated into NPK – fertilizer with different

grade ratios. Some of the attendant problems of these elements

when applied as fertilizer in a tropical humid environment like

Nigeria is the development of soil acidity, fixation of P,

making it virtually unavailable for plant uptake, due to the

presence of large amount of iron and aluminum – oxides or

amorphous alumina silicate clays in tropical soils. Holford

(1997) estimated that as much as 90% added fertilizer

phosphorous in all its natural forms including organic solution

are fixed at any one time, while, Bergmann (1992), and Singh

& Prehan, (1997), observed that if K is not taken up by plants,

it might be lost by leaching as K is mobile and its retention by

the negative charges on Clay surface is temporal as the

application of Ca2t

or mg2t

containing dolomite or gypsum

displace it into the soil solution. Furthermore, Owen (2008)

reported that synthetic fertilizers do not have good

characteristics in aggregating soil particles. White (2006), in

his own study, reported that potassium fertilizers have

antagonistic effects on magnesium and directly, on the

phosphorus content in some plants, hence conventional crops

would contain toxic heavy metals such as cadmium.

Therefore, one of the major ways to reduce these problems is

to add organic matter inform of organic manure to the soil.

Organic manures are an excellent fertilizer containing nitrogen,

phosphorus, potassium and micro-nutrients for healthy growth

of plants. According to Magkos et al. (2003), poultry manure

supply macro and trace elements not contained in the organic

manure. It is a reservoir of nutrients, released during

humidification that is eventually made available to the growing

plants. Organic manure such as poultry manure can be used to

ameliorate the amount of toxic compound produced by the

chemical fertilizers. Poultry manure increase the organic

matter (OM) content of soil and in turn releases the plant

nutrients in available form for the use of the plants. Deskissa et

al. (2008) emphasized that manure enable a soil to hold more

water, improve the drainage, organic acids that help to dissolve

soil nutrients and then made available for the crops. Poultry

contained essential nutrient elements association with high

photosynthetic activities and thus promotes root and vegetable

growth (John et al., 2004).

The integration of inorganic fertilizer and organic manure has

also been reported to be more beneficial than the use of either

mineral fertilizer or organic manure alone especially in

intensive agricultural production. Nweke et al. (2013), Nweke

& Nsoanya (2013b) and Nweke & Nsoanya (2015) observed

that nutrient use efficiency are been increased through the

combination of organic manure and mineral fertilizer. It was

against this backdrop that this work was conceptualized to

363 OKOLI and Nweke

_________________________________________________________



evaluate the effect of poultry manure and mineral fertilize on

the growth performance and quality of cucumber fruits.

2 Materials and Methods

2.1 Experimental setup

The experiments were conducted at the Teaching and Research

Farm of the Department of Crop Science and Horticulture,

Chukwuemeka Odumegwu Ojukwu University (formerly

Anambra State University), Igbariam Campus, Anambra State,

Nigeria. The area lies between latitude 060 14 N and longitude

060 45 E. The rain fall pattern is bimodal and it occurred

between April and October with a mean annual rainfall of

about 1575mm. Annual temperature ranges from 25 – 35

degree centigrade and the relative humidity of the study area is

moderately high all year round with highest (85%) during the

wet season and lowest (64%) during the dry season.

A land area of size 176m2

was manually cleared and debris

removed. After cleaning, the plots were laid out as randomized

complete block design (RCBD) with four replicates. Plot size

was 3.25m x 2m and a pathway of 1m apart. The level of

treatment were no treatment or control (CO), 4tha-1

poultry

manure (PM), 900kgha-1

NPK in recommended ratio 20:10:10

(NPK), 2tha-1

poultry manure + 450kgha-1

at same ratio NPK

fertilizer (PM + NPK). The poultry manure treatment was

applied to their respective plots one week before planting in

order to enable proper decomposition and release of nutrients,

while the NPK fertilizer and the mixture (PM + NPK) were

applied two weeks after planting by ring method. Two seeds of

cucumber were planted per hole which was later thinned down

to one plant per hole, two weeks after germination. The seeds

were sown to the depth of about 4cm deep and planting

spacing of 50 cm x 50cm was used. Field borders were

properly cleared to avoid insects and rodents attacks. The field

was kept relatively weed free till harvest, while insects and

pests were controlled effectively using ZAP broad spectrum

insecticide. Data on growth parameter were collected at 2, 4

and 6 weeks after planting (WAP) on the number of leaves.

Yield parameters were collected on length of fruit (cm),

number of fruits, number of marketable fruits and fruit weight

(kg).

2.2 Statistical Analysis

The data were collected from various parameters subjected to

analysis of variance according to Steel & Torrie (1980).

Treatment means were separated using the least significant

difference at 5% probability level.

3 Results

Effect of the individual or combined application of the poultry

manure and organic fertilizers on the growth and yield of

Cucumber is represented in Table1. The result showed that the

effect of individual or combined application of poultry manure

and NPK fertilizer on the number of leaves were significantly

(P = 0.05) different at 4th

weeks of data collection. Results of

the study revealed that the values recorded for each treatment

increased as the weeks after plant increased from 2 weeks to 6

weeks and the highest value for each stage was obtained from

plots treated with poultry manure (PM) and the order of

increase from 2 weeks to 6 weeks were PM > PM + NPK >

NPK > CO. Data collected at the 4th week showed that values

recorded for PM, and PM + NPK are statistically similar but

significantly better than the control.

The results of fruit length, number of fruits, marketable fruits

and weight of fruits are presented in Table 2 and it showed that

the effect of poultry and mineral fertilizer were significantly (P

= 0.05) difference among the treatment except for marketable

fruits. The plots treated with PM gave the highest value in all

the selected parameter measured, this was followed by PM +

NPK and the least value were gotten from CO plots. The order

of increase in all the yield parameter measured were PM > PM

+ NPK > CO. The result equally revealed that the values

obtained from PM and PM +NPK plots were statistically

similar, but significantly better than the control plots in length

of fruits, number of fruits and weight of fruits.

Table 1 Effect of individual or combined application of poultry manure and mineral fertilizer on the number of leaves per cucumber

plant.

Treatment Weeks 2 After 4 Planting 6

PM 32.25 49.25 87.50

NPK 28.00 33.25 74.30

PM + NPK 29.25 48.25 83.50

CO 28.25 26.00 39.00

LSD0.05 Ns 17.99 Ns

PM = Poultry manure, NPK = NPK 20:10:10 Fertilizer, PM + NPK = Poultry manure + NPK 20; 10:10 Fertilizer mixture; CO = Contr ol

where no treatment was applied, LSD = Least Significant Difference, NS = Non – Significant.

Effect of poultry manure and mineral fertilizer on the growth performance and quality of cucumber fruits 364

_________________________________________________________



Table 2 Effect of individual or combined application of poultry manure and mineral fertilizer on yield components of cucumber.

Treatment Length of fruit cm Number of fruits Marketable fruits Weightof fruits kgha-1

PM 171.25a 10.75

a 9.0

a 2.38

a

NPK 100.00bc

6.75bc

3.8b 1.22

bc

PM + NPK 135.75ab

8.75ac

6.8ab

1.73ab

CO 64.25c 4.25

b 3.0

b 0.76

c

LSD0.05 57.22 3.63 4.79 0.84

Means on the same column with the same letter do not differ significantly (P=0.05). Note PM = Poultry manure; NPK = NPK 20:10 :10

Fertilizer; PM + NPK = Poultry manure + NPK 20:10:10 Fertilizer mixture; CO = control; LSD = Least significant difference.

4 Discussions

The result of the study revealed that growth and yield

components of cucumber plant were increased and the highest

values were observed in poultry manure (PM) treated plots and

it was followed by the treatment containing PM + NPK treated

plots. The higher values recorded in PM relative to PM + NPK

plots in all the parameter measured could be attributed to

higher level of nutrients especially nitrogen and phosphorous

in poultry manure available for plant growth and their release

as well as synchronization of nutrients released within the short

period of growth of the cucumber plant.

Ghanbarian et al. (2008) reported that poultry manure contains

higher nitrogen and phosphorous as compared to other

manures, while Garg & Bahl (2008) indicated that poultry

droppings readily supply phosphorous to plants than other

organic waste. The number of leaves of cucumber measured

increased as the week after planting increased from 2 weeks to

6 weeks. This indicated that organic manure has a profound

effect on the vegetative growth of the cucumber plant. Aliyu

(2000), Nweke & Obasi (2013) made similar observation in

garden egg and Okra Plant respectively. Dauda et al. (2005)

and Nweke et al. (2014) reported an increase in plant growth

following poultry manure application. The increase in growth

observed with poultry manure compared to the integration or

mixture (PM + NPK) may be mainly due to reasons of more

availability and release of nutrients by poultry manure through

the growing period of the cucumber plant.

The number and length of fruits increased with the application

of poultry manure which was significantly different among the

treatments applied. The results could be due to higher number

of leaves, flowers and fruiting buds which may have increased

fruit production (Nweke et al., (2014). Increase in the number

and length of fruits was equally reported by Nweke et al.

(2014) following poultry manure application in agricultural

crops. The marketable fruits result showed that sizable fruits of

good quality was observed mainly in PM treated plots, though

not significantly different from the other treatments, but the

result indicated that the yield of cucumber fruits size and

quality as well as price development and consumers demand

can be increased with the use of poultry manure in cucumber

production. Poultry manure replenishes soil N and other

elements and build up organic matter content of the soil that

support crop yield and greater abundance of soil invertebrates

and even reduced weeds growth (Hole et al., 2005; Herencia et

al., 2007). A positive effect of organic manure have been also

reported by Mc Robic (1998), and Adediran et al. (1999) to

produce higher and better crops that keeps longer and more

nutritious than organic manure. The weight of fruit result

indicated that the yield of cucumber increased significantly

with the application of treatments and was highest with poultry

manure. The significant high yields obtained in the present

study could be said to be caused by nutrient content of poultry

manure which was translated into high vegetative growth

giving rise to high photosynthetic activities which culminated

into high yield observed in PM. John et al., (2004) made

similar observation when they reported that poultry manure

contained essential nutrient elements that favour high

photosynthetic activities to promote plant roots and vegetative

growth. Increase in yield of agricultural crops following

organic manure application was reported in the works of

Nweke & Obasi (2013), Nweke & Nsoanya (2013c), Nweke

(2014), Okoli & Nweke (2015).

Conclusion

The result of the present study indicated that the application of

poultry manure had more effect on the vegetative growth and

yield components of cucumber compared to its mixture (PM +

NPK) as the highest values were recorded in PM treated plots

in all the parameters measured. Poultry manure released

enough nutrients which resulted in significant increase in

growth and yield of cucumber; also it serves as a good source

of soil amendment, and improvement of soil properties which

in turn resulted in improved growth and yield.




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367 OKOLI and Nweke

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KEYWORDS

Plasmid Stability

Sea squid

Transformation efficiency

Growth curve

ABSTRACT

Present study was conducted to study the plasmid stability with the help of natural plasmid isolated from

the bacteria which lodges the ink gland of the sea squid and emits bioluminescence. Isolated bacterial

strain was identified by using 16srRNA sequencing and its plasmid DNA was used for the experimental

studies. The plasmid is found to be responsible for the bioluminescence. The stability of this plasmid

was studied in shake flask method using the different sugar sources (Glucose, Sucrose, Lactose and

Maltose) as fermenting medium. The cultures were incubated at different time intervals in which the

plasmid stability was inferred by deducing the Bacterial Growth curve. The optical densities were read

out and the growth pattern was obtained. Along with the growth curve, the transformation efficiency and

CFUs (Colony forming Unit) was calculated for individual volumes of bacterial cultures. The

transformation efficiency was found to be increased after every 12 hrs of incubation which indicated the

improved plasmid stability. The effect of carbon sources was suggestively detected in the growth curve.

Among the four sources of carbon, addition of Glucose exhibited highest Optical density value and

inferred the enhancement in Plasmid stability.

Hamzah Basil Mohammed and Sudhakar Malla*

Department of Biotechnology, Indian Academy Degree College, Bangalore, India

Received – July 07, 2015; Revision – July 16, 2015; Accepted – August 25, 2015



PLASMID STABILITY AND MAINTENANCE OF COPY NUMBER USING

NATURAL MARKER

E-mail: [email protected] (Sudhakar Malla)







ISSN No. 2320 – 8694

Production and Hosting by Horizon Publisher India

(http://publizer.jebas.org/index.html).









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1 Introduction

DNA technology especially rDNA owes a great deal in

producing recombinant proteins, these protein can be produced

in bulk than many native proteins and moreover this, these can

be easily controlled in quality wise. These recombinant

proteins have heavy demand in the pharmaceutical sectors for

producing vaccines, therapeutic proteins and functional

enzymes (Andersen & Krummen, 2002). Over 150 human and

veterinary based biopharmaceuticals have received approval

and majority of these represent recombinant proteins in the

form of recombinant antibodies (Friehs, 2004).

Plasmid stability is a common term which frequently used in

recombinant protein industry. The success of the production of

the recombinant protein largely depends on the stability of the

plasmid. On successive generations there is a chance of the

reducing copy number and thereby affecting the production of

recombinants. Furthermore, the use of the antibiotic resistance

genes as selectable markers in the plasmid is also a major

concern, due to which the usage has been limited. Hence there

is a need to search an alternative mode of plasmid stability

selection. Here plasmid stability is a measure of how well a

plasmid is maintained in a bacterial population in the absence

of any selection for plasmid-encoded traits. Stability is

typically evaluated by growing bacterial cells with the plasmid

in a medium that does not select for the presence of the

plasmid. After a known number of cell divisions (generations)

the number of cells with and without the plasmid is counted.

Growth rate affects several parameters that determine

recombinant protein accumulation rate. Among them

percentage of substrate utilized for cellular maintenance, RNA

polymerase activity, ribosome number, plasmid stability,

plasmid copy number, plasmid multimerization, and the

distribution of cells in the cell-cycle phases are some common

one (Escriou et al., 2001). Thus, it is possible to control

recombinant protein production through growth rate and this

growth rate can be manipulated by the manipulating nutrient

availability. Namely, the main carbon or nitrogen source can

be maintained at a predetermined concentration to obtain the

desired growth rate. Such a manipulation can be achieved

through fed-batch or continuous cultures (Bloquel, 2004).

Gene therapy has more valuable interest and more than 1,700

clinical trials have already been approved worldwide. Plasmid

DNA production needs a specialized marker for screening after

the process of transformation and during the amplification

process (Colosimo et al., 2000). The plasmids once

transformed into the host for propagation or expression are

then checked for their stability. Stability refers to the ability of

the plasmid to remain stable segregationally and the daughter

cells should contain at least one copy of the plasmids. Plasmid

stability is always correlated with the number of copies of a

plasmid in the host bacteria. If plasmid cells are allowed to

grow under non-selective conditions, there will be a serious

threat to the plasmids of interest (Cooper, 1987). Infect

plasmid exerts an extra pressure on the metabolic activity of a

bacteria and also effecting its growth rate. Hence it is always

necessary to impose a selection pressure which favours the

selection of bacteria containing the gene of interest (Doig,

2001).

Antibiotic resistance genes are often used as selection markers

and in this plasmid containing cells are growing on the specific

medium containing the respective antibiotic. But use of

antibiotic resistance genes has been warned of serious threat to

the nature owing to the chance of recombining with the

homologous region in the hosts (El-Helow, 2000). If these

antibiotic genes are used they might be spread into the wild

pool contaminating the gene pool and making them resistant to

the antibiotics. This might lead to future complications in

relation to clinical significance. One major reason for

considering this sort of gene markers is they might lead to an

immune response and create allergy when used in the

production of recombinant vaccines. Henceforth it will be

importance for the rDNA sector if new innovations are made in

the vectors will reduce the effect of cross contaminating the

gene pool (Gardlik, 2011).

Limited information’s are available on the stability of

plasmids. According to Maskos, (2000) media composition

plays a potential role in cell growth rate and it also ahelps in

the maintaining plasmid copy number (Jones, 1980).

Therefore, the possible effects of medium and its components

should be evaluated for their role on the cell growth and

plasmid productivity. Acetate accumulation has also been

proved to be a major problem in medium design and also

fermentation of recombinant E. coli at high density levels

might inhibit the cell growth and protein expression (Summers,

1998). Among the different carbon sources used, glucose was

preferably used for the production of plasmid pCMV-AP in E.

coli. Glucose was found to be more useful for high plasmid

yield both on the shake flask cultivation and pilot plant.

Glycerol was also used as supplement to increase the yield of

plasmid up to 70.6% (Summers, 1998).

In present study the plasmid used as a natural marker unlike

the other available artificial markers. This proves to be an

added advantage in the Therapeutics. The plasmid stability

studies were done by shake flask method. The plasmids were

also used for optimization by growing in various carbon

sources such as Glucose, Lactose, Maltose, Sucrose and the

growth rate was observed. The study provides an insight on the

plasmid stability.


2.1 Dissection of sea squid

Defrosted squid was used for obtaining the ink gland. The

squid was laid along the dorsal side down on a piece of wax

paper in a dissecting tray. The squid was laid to a position in

such a way that with its head to the left and its siphon up. The

pen from the animal was removed from the dorsal side by

369 Hamzah and Sudhakar

_________________________________________________________



grasping it firmly with the fingers and pulled it free from the

mantle. The mantle is now cut using a scalpel or dissecting

scissors. The chromatophores which are of small freckle-like

spots on the outer layer of the mantle were observed and

peeled off a small piece of the skin that contains the spot.

2.2 Isolation of bacteria

The sea water complete (SWC) media (0.38M NaCl, 0.02M

MgCl2. 6H2O, 0.25M Mg SO4. 7H2O, 8mM KCl, Peptone:

0.5g, Yeast extract: 0.3g, Glycerol: 0.3ml, Agar: 1.5g, Distilled

water: 100ml) was prepared under sterile conditions. The ink

squeezed from the ink gland was spread plated on SWC media.

The plates were kept for incubation at 21° C for 24-48 hrs.

Following the plates were observed under the UV illuminator.

The colonies showing the presence of luminescence were

further subcultured and stored at 40C. The colonies were

confirmed using the gram staining procedure.

2.3 16s rRNA sequencing

The colonies with peculiar variations in the morphology were

made of pure culture and then sent for sequencing. The

sequencing protocol followed was to be capillary based

(Macrogen, Seoul, Korea).

2.4 Genomic DNA isolation

2ml of O/N culture was taken in a centrifuge tube and

centrifuged at 5000rpm for 10 minutes. The supernatant

obtained was discarded and the pellet was re-suspended in

300µl of lysis buffer (1M Tris HCl, 0.5M EDTA, 1% SDS)

and gently vortexed. To the contents, 500µl of TE buffer (1M

Tris HCl, 0.5M EDTA-2ml) and 500µl of chloroform were

added and the tube is inverted gently upside down. The

contents centrifuged at 9000rpm for 10 minutes. The upper

aqueous layer was collected in a fresh eppendorf tube and

added with 1/10th

volume of 3M sodium acetate. After gentle

mixing of the contents, 2 volumes of chilled ethanol were

added along the walls of the tube. The precipitated DNA is

now pelleted and washed with 70% ethanol for purity and

finally re-suspend in 30-50µl of TE buffer (Sambrook et

al., 1989).

2.5 Plasmid DNA isolation

Plasmid DNA was isolated by the method described by

Sambrook et al., 1989. For this 2ml overnight luminescent

culture was centrifuged at 14000rpm for 10 minutes and the

pellet obtained was re-suspended in 200µl of freshly prepared

solution I (50mM Tris, pH-8, 10mM EDTA,100µg/ml RNase

A, 50mM Glucose). The contents are mixed well and added

with 200µl of solution II (200mM NaOH, 1% SDS) and 200µl

of solution III (Pottassium Acetate 3M). The contents were

mixed by inverting the tube gently. The tubes were then

centrifuged at 14,000rpm for 10 minutes and the supernatant

obtained was transferred to a fresh set of tubes and the DNA

was precipitated using 900 µl of 100% ethanol. The DNA

obtained was resuspended in 50µl of TE buffer with 5µl of

freshly prepared RNAse and the tubes were stored at -20° C

until further use.

2.6 Transformation

Competent cells of E.coli were prepared using the calcium

chloride method (50mM CaCl2). The prepared cells were used

for the transformation experiments. Briefly 100µl of competent

cells were taken in two eppendorf tube and added with 5µl of

plasmid DNA in one tube labelled as positive and the other

was kept negative (no plasmid added). Both the tubes, positive

and negative were incubated on ice for 20 minutes and later

kept in hot water bath at 42° C for 2 min. following which the

tubes were removed immediately and placed on ice for 10

minutes. 0.5ml of LB broth was added to both the tubes and

was incubated at 37 °C for 1 hr. Meanwhile 2 plates (positive

and negative) of LB agar were prepared. About 20µl of the

samples was spread plated on their respective plates. The

plates were then incubated at 37°C for 48 hrs in an inverted

position and observed for the transformed colonies.

2.7 Shake flask cultivation

The culture obtained was further used for the stability studies

by shake flask culture. The experiment was first carried out in

20ml of LB medium, this was followed by 100ml and 2 litres

medium containing flask culture. Sugars were used as minimal

medium to check the level of stability with different sugars

(Glucose, lactose, Maltose and Sucrose). A single colony of

isolated & identified bacterium was inoculated into 20ml LB

medium contained in 250ml conical flask. The culture was

incubated for 12hrs at 37 °C at 250rpm on rotatory shaker.

After 12hrs of incubation 10ml of culture was diluted by

inoculating into another 20ml LB medium. 0.5ml of 12hrs old

culture was diluted with 4.5ml of LB broth and the absorbance

was recorded at 600nm. Bacterial growth curve was analysed

after every 12 hrs culture. Repeated inoculations were carried

out into 20ml LB broth in a 250ml conical flask after every 12

hrs for growth curve. Meanwhile 4 plates containing LB agar

along with the individual carbon source sucrose (1gm/L),

glucose (1gm/l), lactose (1gm/l), maltose (1gm/l)) were

prepared. 100µl of culture was spread plated on their

respective plate. The plates were incubated at 37°C for 24-48

hrs and the numbers of colonies are counted by colony forming

unit (cfu). The same procedure was repeated for 100ml and 2

litre flasks culture also.

The transformed colonies were counted using coulter counter

(Digital colony counter (Optopercision services, model: DCC-

110) and the colonies were then checked for the transformation

efficiency. Plates with more than 300 colonies cannot be

counted and are designated too many to count (TMTC). Plates

with fewer than 30 colonies are designated too few to count

(TFTC). The number of bacteria (CFU) per millilitre or gram

of sample was calculated by dividing the number of colonies

by the dilution factor multiplied by the amount of specimen

added to liquefied agar.

Plasmid Stability and Maintenance of Copy number using Natural marker. 370

http://www.srcosmos.gr/srcosmos/generic_pinakas.aspx?pinakas=cited_refs&alpharef=Sambrook%20J


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Transformation efficiency is the efficiency by which cells can

take up extracellular DNA and express genes coded by it. This

is based on the competence of the cells. It is calculated by

dividing the no of successful transformants by the amount of

DNA used during a transformation procedure. Transformants

are cells that have taken up the DNA and which can express

genes on the introduced DNA.

3 Results and Discussion

3.1 Isolation and identification of bacteria from squid

The bacteria were isolated from the ink gland of squid and

cultured on the SWC media (Figure 1). The colonies exhibiting

fluorescence under UV transilluminator were picked up and

subcultured on separate SWC agar medium. Isolated bacteria

were initially identified by the Gram staining and selected

strains were taken for phylogenetic analysis by 16s rRNA

sequencing (Figure 2). The sequence was found to be about

990bp. These sequences were taken for the sequence similarity

with the identited organisms by using blast. The query was

found to have 99% identity with Bacterium Vibrio harveyii

strain IS01 16S ribosomal RNA gene partial sequence (Figure

3 & Figure 4).

Figure 1 Left: Figure showing the giant Sea squid. Right: Ink gland dissected from the squid.

Figure 2 A: Pure colonies of isolated bacteria on SWC Agar medium; B: Pure colonies of isolated Bacteria exhibiting fluorescence under

UV transilluminator; C: Transformed colonies on LB agar; D: Transformed colonies exhibiting fluorescence under UV transilluminator;

E: Transformed colonies without plasmid DNA (negative control) LB agar; F: Transformed colonies without plasmid DNA (negative

control) LB agar; G: More colonies grown on LB agar with the sole carbon source Glucose comparative than all the carbon sources; H:

Very few colonies grown on LB agar with the sole carbon source Maltose; I: Fewer colonies grown on LB agar with the sole carbon

source Sucrose; J: Few colonies grown on LB agar with the sole carbon source Lactose.


_________________________________________________________



Figure 3 Figure showing the 990bp sequence obtained by 16srRNA sequence of the bacterium isolated from the ink gland of the sea

squid.

Figure 4 Left: Figure showing the blastn results of the sequence obtained by 16srRNA sequencing. Right: Figure showing the blastn

score results showing the percent similarity to the nearest identical bacteria.


_________________________________________________________



3.2 Isolation of genomic and plasmid DNA

Genomic and plasmid DNA was isolated and quantified by

using UV spectrophotometer. The samples were then run on

0.8% Agarose gel together with 1kb ladder DNA for reference

and checked for the purity. The concentration of the genomic

and plasmid DNA obtained was of 2.4325 & 2.123µg/ml

respectively.

3.3 Transformation

Transformation efficiency was calculated by dividing the

number of successful transformants by the amount of DNA

used during a transformation procedure. The concentration of

the plasmid DNA is 20ng/µlitre. The amount of Plasmid DNA

used for the transformation is 5 µlitre. So the amount of DNA

transformed is 100ng (20x5). The Volume of the culture plated

for every 1ml is 25µl. Therefore the amount of DNA for 100µl

is 10ng. The numbers of colonies observed on the plate were

65colony/plate. Plasmid stability was also reported Colosimo

et al., 2004; Bloquel et al 2004 and found similar to the present

study.

3.4 Shake flask for 20ml of culture

The shake flask method was carried out for 20ml of the culture

and the growth was calculated by photometric assay by using

calorimeter. The colony forming unit was calculated by

counting the number of colonies grown on the individual

plates.

Figure 5 Graph showing the number of colonies and transformed colonies of the transformed culture. All the values are the averages o f

triplicates. The dilution of the transformed culture was 1:10.

Figure 6 Graph showing the number of colonies and transformed colonies of the transformed culture for different incubation hours. The

dilution of the transformed culture was 1:10.


_________________________________________________________



A two way ANOVA between the different incubation period

and carbon sources was conducted to compare the effect of

carbon sources on plasmid stability. There was a significant

effect of different carbon sources and incubation period

remembered at the p<0.05 level. The significance effect of the

carbon source and incubation period was reported on the

colony forming unit [F (3,12) = 88.4545, p = 1.89E-08] and

[F(4,12)=23.8, p=1.24512E-05]. Furthermore, a significance

effect of the carbon source and incubation on the

Transformation Efficiency was also reported [F(3, 12) =

140.144, p = 1.32E-09] and [F(4, 12) = 20.8011, p =

2.50374E-05 ]. A significant amplification was reported in the

colony forming units as well as in the transformation efficiency

with increasing the time for incubation period. Also among the

different carbon sources added to the growth medium, glucose

was reported best in raising the transformation efficiency

(Figure 5 & Figure 6)

Similarly the experiment was carried out in 100ml culture and

the growth was calculated by photometric assay by using

calorimeter while the colony forming unit was calculated by

counting the number of colonies grown on the individual

plates. Result of the study are presented graphically against

incubation time v/s Cfu (Colony forming Units) &

Transformation efficiency (Figure 7 & 8).



Figure 8 Graph showing Colony forming unit and transformation efficiency. All the values are the averages of triplicates. The dilution of

the transformed culture was 1:10.


_________________________________________________________



A two way ANOVA between the incubation period and

different carbon sources was conducted to compare the effect

of carbon sources on plasmid stability. There was a significant

effect of different carbon sources and incubation period

remembered at p<0.05 level. The significance effect on the

colony forming unit between carbon source and incubation

period [F(3, 12) = 27.9444, p = 1.07E-05] and [F(4,12)=

7.02381, p=0.003740167].

Further, it also showed a significance effect on the

Transformation Efficiency between carbon source and

incubation period [F(3, 12) =96, p =1.18E-08 ] and [F(4, 12)

=17.6914 , p =5.69455E-05 ].

Furthermore, a substantial increase in the number of colonies

as the incubation period was increased. The CFU/ml was

raised and the transformation efficiency was improved as the

incubation hours were elevated. Addition of Glucose in the

medium again showed an increase in the transformation

efficiency.

3.5 Shake flask for 2 litres of culture

Like 20 and 100 ml culturing, shake flask method was also

carried out for 2 litres culture and a graph was plotted against

incubation time v/s Cfu (Colony forming Units) &

Transformation efficiency (Figure 9 & Figure 10).



Figure 10 Graph showing Colony forming unit and transformation efficiency. All the values are the averages of triplicates. The dilution

of the transformed culture was 1:10.


_________________________________________________________



A two way ANOVA between the incubation hours and

different carbon sources to compare the effect of carbon

sources on plasmid stability and a significant effect of different

carbon sources and incubation period was reported at the

p<0.05 level. The significance effect on the colony forming

unit between carbon source and incubation period [F(3, 12) =

30.5285, p = 6.73E-06] and [F(4,12)= 8.56694 , p=0.00166].

And also showed a significance effect on the Transformation

Efficiency between carbon source and incubation period [F(3,

12) =61.1344, p = 1.53E-07] and [F(4, 12) = 13.9355, p

=0.00018. There was a substantial increase in the number of

colonies as the incubation period was increased. The CFU/ml

was raised and the transformation efficiency was improved as

the incubation hours were elevated. Addition of Glucose in the

medium again showed an increase in the transformation

efficiency. Similar type of plasmid study was conducted by

Cooper (1987) & Friehs (2004) they reported similar type of

results.

3.6 Growth Curve

After every 12 hrs of incubation, the sample culture was

observed for the turbidity. It was than diluted and was read at

620nm calorimetrically. The optical density values were noted

down and a graph was plotted against the incubation period

(Figure 11).

The growth was accelerated after every 12 hrs with 2m, 100ml

and 2 litre of culture. The carbon sources added into the

growth medium. Among all, addition of glucose exhibited a

rise in the Optical densities. Both the treatments are at par to

each other and are statistically differ than the sucrose and

glucose.

Conclusion

Markers used in Production of recombinant Proteins are

crucial. Another factor which influences the production of

genetically engineered proteins is the stability of the Plasmid.

Nowadays, many artificial markers are used in the

biotechnology industry. The marker helps in detecting the

production of desired protein in the respective cell. It helps in

detecting the success rate of the recombinant protein. Without

a marker, it becomes difficult for a biotechnologist to detect

the production of Recombinant protein involved. Use of

Natural Marker in production of Recombinant protein has

always proven to be an excellent choice. The Bacterial Plasmid

isolated in the current study can be used as a marker as it

exhibits fluorescence and being natural, it has potential

applications in the field of Biotechnology. For further

identification, the culture was sent for 16srRNA sequencing.

The bacterium was found sharing 99% similarity with

Bacterium 4D902 deposited in the website.

The shortcoming with the artificial Marker is the possibility of

its integration into the host chromosome. This leads to the

ethical tissues and limits the use of such Markers in the

Biotechnology industry. Natural Marker proves to be of an

advantage here. The Plasmid stability is another limitation for

the production of recombinant proteins on a large scale. The

protein expression decreases after every generation and this

limits the use of recombinant protein in therapeutics.

Figure 11 Graph showing the rise in the Optical density values of the bacterial culture incubated at different time intervals and ca rbon

sources.


_________________________________________________________



In this project, the plasmid stability was improved by the

addition of different carbon sources in a shake flask method.

The effect of carbon sources was suggestively detected in the

growth curve. Among the four sources of carbon, addition of

Glucose exhibited highest Optical density value and inferred

the enhancement in Plasmid stability. Furthermore,

transformation efficiency increased after every 12 hrs of

incubation which indicated the improved plasmid stability.

The extracted Plasmid has an additional advantage being

natural and can be used as a marker. It will aid in dodging the

ethical issues related to the implementation of Recombinant

protein in therapeutics and Biotechnology industry. The

fluorescence exhibited by Plasmid is easily detectable under

UV transilluminator. By studying the effect of different carbon

sources on the plasmid stability, it was shown that glucose

addition enhances the Plasmid stability in the successive

generations.




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http://www.srcosmos.gr/srcosmos/generic_pinakas.aspx?pinakas=cited_refs&alpharef=Fritsch%20EF

http://www.srcosmos.gr/srcosmos/generic_pinakas.aspx?pinakas=cited_refs&alpharef=Maniatis%20T



_________________________________________________________



KEYWORDS

Costus pictus

Costus speciosus

Ergastic crystals

Raw drug

ABSTRACT

Present study was conducted to compare the morphological, anatomical and histochemical features of

exotic species Costus pictus with its related C. speciosus, introduced in Peninsular India during the

recent past. Identifying features of C. pictus is well documented through the present study with samples

collected from different agro climatic regions along with its closely related species C. speciosus, which

is available from the past even in wild without any characteristically reported medicinal property. The

comparative study of fresh specimen shows distinctive features for identification with regard to

morphological and anatomical characters at its flowering condition. The dried raw drug can be

distinguished with the analysis for the presence of cuboidal solid crystal in the leaf mesophyll. Result of

comparative study of leaf suggests that C. pictus leaves do not show the presence of cuboidal ergastic

crystal in their leaf mesophyll where as C. speciosus leaves possess the same.

Justin R Nayagam

Department of Botany, Union Christian College, Aluva, (Affiliated to Mahatma Gandhi University,Kottayam) Kerala, India

Received – May 02, 2015; Revision – May 23, 2015; Accepted – July 26, 2015



ERGASTIC CRYSTALS IN IDENTIFICATION OF COSTUS PICTUS: A MEDICINAL

SPIRAL GINGER IN HERBAL MEDICINE

E-mail: [email protected] (Justin R Nayagam)







ISSN No. 2320 – 8694











_________________________________________________________



1 Introduction

Costus pictus D. Don, (Costus mexicanus Liebm. Costus

igneus Nak. Costus mexicanus Liebm ex Petersen or Costus

congenitus Rowle) commonly known as fiery costus or step

ladder or spiral flag or insulin plant (Jose & Reddy, 2010;

Hedge et al., 2014). C. pictus is an introduced medicinal spiral

ginger to Peninsular India and it belongs to family Costaceae.

This plant is distributed along the coast from Mexico to Costa

Rica and is locally known as canaagria or cana de jabaliin

Mexico (Remya & Daniel, 2012).

Recently this plant gains more medicinal interest due to its

medicinal properties, leaves of the plant have anti-diabetic

activity in humans (Mani et al., 2010; Kumudhavalli & Jaykar,

2012).The plant has very high medicinal potential and shows

various pharmacological activities such as hypolipidemic

(Mani et al., 2010;), diuretic effect (Meléndez-Camargo et al.,

2006), anti-oxidant (Jayasri et al., 2009; Maruthappan &

Sakthisree, 2010), antimicrobial (Gothandam et al., 2010;

Saraswathi et al., 2010) anti-cancers (Nadumane et al., 2011)

and putative activity (Manjula et al., 2012) apart from its anti

diabetic activity.

C. speciosus is a native plant to India but form the beginning

onward it confused with C. pictus. Rama Rao (1914) listed C.

speciosus (Koenig) Smith under the family Zingiberaceae, later

on many earlier Indian floras placed this genera (Costus)

under family Zingiberaceae. Recently, Costaceae were

classified as Costoideae, a subfamily or as tribe (Costeae)

within Zingiberaceae. Now, genus Costus along with genera

Dimerocostus, Monocostus and Tapeinochilus were transferred

to the family Costaceae form the family Zingiberaceae (Nakai,

1941).

Presence of the ergastic crystals in dicotyledons and

monocotyledons families was reported (generally of calcium

oxalate) by many researcher in many. Forms and distribution

of these crystals have taxonomic importance (Metcalfe, 1960).

Present study was aimed to resolve confusion between much

confused species C. pictus and C. speciosus. Differentiation

between these two species was purely based on the presence

and form of ergastic crystal. In addition to this, conventional

taxonomic and anatomic comparison was also carried out

because published data on these aspects are in scanty for these

species.


Samples were collected from five locations of south India i.e.

Botanic garden, University of Kerala (Thiruvananthapuram),

Kidangoor, Edayar, Palakkad and from the foot hills of

Kodaikkanal. Collected plant samples were identified and

authenticated by Botanical Survey of India (MH accession no:

173772), Southern Circle Herbarium, Coimbatore, Tamil

Nadu, South India. Fresh as well as herbarium preserved

vegetative and reproductive specimens were used for

morphologic studies whereas fresh and preserved specimens

were used for anatomic studies. Plant parts used for anatomic

studies include adventitious roots, aerial shoot, underground

rhizome and foliar leaves. Differentiations between these two

species are basically based on the ergastic crystals by

anatomical study.

3 Results

Various morphological characters observed from fresh

vegetative and reproductive structures of C. pictus and C.

speciosus are summarized in table 1.

Table 1 Comparative morphological feature of C. pictus and C. speciosus

Character C. pictus* C. speciosus

*

Plant height at flowering stage 35 – 240 cm 25 – 95cm

Spiraling nature of stem Minimum towards tip Prominent towards tip

Adventitious buds on aerial stem Present (3 – 18) Absent

Number of leaves/branch 18 – 36 7 – 18

Length of longest leaf 18 – 23 cm 12.5 – 27 cm

Width of largest leaf 6.8 – 8.3 cm 3.89 – 10.6 cm

Size of Mature Rhizome Length 42cm & 3.5cm thick Length 33cm & 2.8cm thick

Flower bud Yellow with rounded tip Flesh colored with pointed tip

Flower color (Figure 1 & 2) Lemon yellow Creamy white

Flower size 5.1 cm – 6.9 cm 5.1 cm – 8.1 cm

Labellum Lemon yellow with reddish markings Creamy white

Labellum size 5.5 cm x 4.5 cm 7.6 cm x 6.8 cm

Fruits Not reported in Peninsular India Loculicidal capsule with persistent calyx

Seeds Not reported in Peninsular India Black with white arils and angular end

Means of vegetative propagation Aerial shoot cuttings, adventitious buds and rhizomes Rhizomes *All the values given in tables are means of sample collected from all study sites

379 Nayagam

_________________________________________________________



Table 2 Comparative anatomic features of C. pictus and C. speciosus

Character C. pictus*

C. speciosus*

Calcium oxalate crystals Present in all parts except leaves Present in all parts

Metaxylem in root Rounded Bigger with trigonous outline

Crystals in leaf Absent Cuboidal in outline *All the values given in tables are means of sample collected from all study sites

Table 3 Comparative herbarium features of C. pictus and C. speciosus

Character C. pictus C. speciosus

Leaf texture Smooth surface Coarse surface

Appearance Smooth upper and lower surface Distinct upper and lower surface

Sections cutting of fresh and preserved specimens of leaves,

aerial shoot, rhizomes and adventitious roots were examined

microscopically and all the reported differences are

summarized in table 2. The presence of calcium oxalate

(ergastic) crystals is more consistent in the aerial shoot and

rhizomes (Figure 3-10).

Herbarium specimens of both the samples were examined to

find the differences and the results obtained are presented in

table 3. The herbarium specimens of C pictus prepared was

authenticated by Botanical Survey of India and deposited with

accession no 173772 Southern Circle Herbarium, Coimbatore,

Tamil Nadu, South India. Voucher specimens of herbarium are

maintained at the research center.

Figure 1 Inflorescence – Costus pictus Figure 2 Inflorescence – Costus speciosus

Figure 3 T.S. of aerial stem C.pictus Figure 4 Portion of Rhizome C. pictus

Ergastic crystals in identification of Costuspictus: a medicinal spiral ginger in herbal medicine 380

_________________________________________________________



4 Discussions and Conclusion

Tropics are blessed with numerable plants which are of

multifarious use. The combined effect of plant introduction and

cultivation has largely accelerated the interest of scientists and

industrialists to focus on herbal medicine and other economic

products (Nayagam, 2015B). Published evidence for the

presence of C. speciosus in South Indian land is available since

17th

century (Rheede, 1692) but, C. pictus is a recently

introduced species (Jose & Reddy 2010; Hedge et al., 2014).

Correct taxonomic identification is most important before

proceeding to any analytical procedure and utilization.

Comparative approach on morphological and anatomical

features provides distinguishable features (Sabu, 2006;

Nayagam, 2015A) but most of the reliable distinguishing

characters are with respect to reproductive morphology (Figure

1 and 2)(Table 1). Morphological features of vegetative parts

with qualitative value are varies with the localities with respect

to growing regions and cultivars are considered. Since the

flowers of the studied species are produced seasonally and the

economically important part is leaves, so identification based

on reliable anatomical characteristic may be useful for making

differentiation in these two species and help in the

identification of raw drug. Ergastic crystals can be an

important diagnostic tool for the identification of such

economically important species (Metcalfe, 1960). Presence of

characteristic cuboidal ergastic crystal in the leaves of several

plant species including C. speciosus has been well reported

(Wallis, 2005; Kokate et al., 2010).

Figure 5 T.S. of C. speciosus Rhizome – outer cortex Figure 6 T.S. of C. speciosus Rhizome

Figure 7 T.S. of Adventitious root C. pictus Figure 8 T.S. of C. speciosus root

381 Nayagam

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Figure 9 T.S. of Costus pictus lamina

Figure 10 T.S. of Costus speciosus Leaf with Characteristically

shaped Crystals

Since the cuboidal crystals of calcium oxalate are present in

the mesophyll cells of C. speciosus and these are not reported

from the mesophyll cell of C. pictus leaves, it can be become a

consistent and easily identifiable characteristic between these

two species. The presence of oxalate crystals is characteristic

in aerial shoot and rhizome, where size of crystals found

smaller towards the tip of shoot whereas size of crystals

towards is bigger in size. Rhizome possesses crystals with

comparatively larger size in C. pictus (Figure 4). The cortex of

rhizome in C. speciosus is characterized with several layers of

compressed cells (Figure 5) followed by cells with numerous

ovoid crystals (Figure 4). So the presence of ergastic crystals

from various plant parts, its size and structure is an important

taxonomic key for the making difference between medicinally

important exotic species C. pictus and native C. speciosus.

Acknowledgements

The author expresses his heartfelt gratitude towards Mr.

Kunjachan, Managing Director, Dr. Benny, Director -

Technical, Dr. Merrina Benny, General Manager and Dr, Binu

T. Kuruvilla Assistant General Manager (R & D) of Arjuna

Natural Extracts, Aluva, for providing specimens from

cultivation plots. Sincere thanks to Dr. Thomas Philip,

Principal, and Dr. Thara K. Simon, Head of the Botany

Department, Union Christian College, Aluva for providing all

facilities to carry out this research work, Thanks to Mr.

Thomachen, Gardener, Dr. T.C Joseph Memorial Botanical

Garden, Department of Botany, Union Christian College,

Aluva, for maintaining the field specimens throughout the

study period. Extending a word of thanks to Dr. Suharabeevy

and Dr. Helen, University of Kerala for providing plant

specimens from their conservation plot. Sincere thanks to Dr.

Murthy, Dr. Sudhagar and Dr. Mohanan, Scientists, Botanical

Survey of India, Southern Circle, Coimbatore, Tamil Nadu for

helping in species identification and herbarium deposition

during the research work.

Conflict of Interest



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Ergastic crystals in identification of Costuspictus: a medicinal spiral ginger in herbal medicine 382

_________________________________________________________



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calcium oxalate monohydrate crystals influenced by Costus

igneus aqueous stem extract. International Journal of Pharmacy

and Pharmaceutical Sciences 4:261–70.

Maruthappan V, Sakthisree K (2010) Ameliorative effect

of Costus pictus D. Don rhizome on mitochondrial enzymes in

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9:62–68.

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Nayagam JR (2015 A) Compendium on Costus pictus: A

medicinal spiral ginger. Lambert Academic Publishers,

Germany.

Nayagam JR (2015 B) Plantation technology for seven tropical

tree species. Lambert Academic Publishers, Germany.

Rama Rao M (1914) Flowering Plants of Travancore.

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publishers, New Delhi, India Pp. 566-570.

383 Nayagam

_________________________________________________________



KEYWORDS

Using chamber

IPEC-J2 cells

Porcine intestine

Papain

Risk assessment

ABSTRACT

In order to assess the health risk that associated with the consumption of unknown feed or food

ingredients, there is a strong need of developing an in vitro screening system. The test system should be

fast, reliable, inexpensive and without the necessity of performing animal tests. Furthermore, it should

also provide important clues to the potential danger of unknown substances. The present study examines

the extent to which cell and tissue cultures can be used for such studies. It should be ascertained whether

the cell cultures can replace the native intestinal epithelium in terms of their sensitivity and provide

accurate results as a quick “screening system”. As a model for intestinal operations ex vivo tissue

cultures from the native intestinal epithelium of the pig and the permanent cell line IPEC-J2 were used.

The cell culture was characterized in terms of their morphological and functional properties (TEER,

tight-junction proteins).Various studies (short-circuit measurements, translocation of [3H]-mannitol)

were performed to IPEC-J2 cells and the native intestinal epithelium in order to compare the functional

properties of both systems. Finally, the response of the addition of "unknown” test substances (papain

and wheat extract) were investigated to determine whether the functional parameters of both systems are

affected by these test substances or not. The IPEC-J2 cells show a more significant influence in their

functionality by “unknown” substances than the control variant. Results of study revealed that the in

vitro system reacts rapidly in response of unknown test substances and it is more sensitive. Therefore, it

is possible to operate a “risk assessment” for “unknown” substances with the help of this developed

screening system.

Mandy Bruch* and Elmar Mohr

University of Rostock, Faculty of Agricultural and Environmental Sciences, Chair of Animal Health and Animal Welfare, Justus-von-Liebig-Weg 6b, 18059

Rostock

Received – April 24, 2015; Revision – May 13, 2015; Accepted – August 31, 2015

Available Online – September 02, 2015


DEVELOPMENT OF IN VITRO SCREENING SYSTEM FOR FOOD HABIT

RELATED RISK ANALYSIS

E-mail: [email protected] (Mandy Bruch)







ISSN No. 2320 – 8694

Production and Hosting by Horizon Publisher

(http://publisher.jebas.org/index.html).












_________________________________________________________



1 Introduction

Unknown ingredients of food and feed stuffs may pose a health

risk for human and animal when consuming (Krul et al., 2000;

Schmidt et al., 2011). Studies which should elucidate

constructive the possible effect of these substances on an

organism, often are based on animal experiments (Glaeser &

Fromm, 2008; Cehak et al., 2013; Yan et al., 2013). Feeding

trials with genetically modified plants (Reuter & Aulrich,

2003) or food additives (Lodemann et al., 2013) are expensive

and potentially under ethically controversial agenda, because

the animals are killed after food intake in order to examine the

tissue and the blood. Problem related to the comparability of

the results in potentially inhomogeneous test material like

animals are also associated with these types of studied. For

these reasons, it is feasible to support or partially replace these

studies by in vitro systems? Cell and tissue cultures have long

been used in medical and pharmaceutical research. Effect of

drugs, macromolecules and other substances on cell absorption

and transportation properties are already being studied in cell

cultures (Artursson & Karlsson, 1991; Uil et al., 1997;

Neumann et al., 2004; Weng et al., 2005; Grunwald et al.,

2006; Cardinali et al., 2013; Jarmolowska et al., 2013).

The intestinal epithelium has been used especially for the

absorption of ions (Frömter & Diamond, 1972; Frings et al.,

1999), macromolecules (Warshaw et al., 1971; Bruch et al.,

2008) and probiotics (Johnson et al., 2010; Lodemann et al.,

2006). The ussing technique is an accepted procedure for the

use of tissues and cell cultures for transport studies (Bajka et

al., 2003; Rozehnal et al., 2012; Song et al., 2013). The native

porcine intestinal epithelium is an established model for

studies of the transport of ions and high molecular weight

substances (Herrmann et al., 2012; Miyake et al., 2013). The

permanent cell line IPEC-J2 is characterized and considered a

suitable model for the jejunum of pig (Schierack et al., 2006;

Mariani et al., 2009; Geens & Niewold, 2011; Brosnahan &

Brown 2012). The combination of these already known

methods of investigation can be used to estimate the possible

occurring risk of an unknown substance. A rapid and easily

performing screening system could be based on a cell or tissue

culture giving first indications of a potential hazard. With the

development of such an in vitro test system the normally

required animal testing’s could be reduced and complemented

by a low-cost alternative method. The present study examined

the extent to which appropriate cell and tissue cultures can be

used for the detection of potentially hazardous substances.

Furthermore, suitable parameters in order to detect the effect of

harmful substances in an in vitro system, reactivity of cell

culture and ex vivo tissue cultures (native intestinal

epithelium), differences in sensitivity between cell and tissue-

culture, suitability of cell and tissue-cultures, substance

dependent differences between these two and the accuracy of

cell culture screening system were also tested in the present

study.


The studies used as a model for intestinal operations ex vivo

tissue cultures of native intestinal epithelium of the pig

(slaughterhouse material) and IPEC-J2 cell cultures. For these

two systems morphological and functional parameters were

determined before study. The morphological characteristics

were studied by histological and intravital-microscopic

analysis. Additionally the molecular biological detection of

tight-junction proteins was occurred. The passive functional

properties were characterized by the diffusion of tritium-

labeled Mannitol (H³). As a measure of active transport

properties of cell culture monolayers and the native epithelium,

the response to the addition of sodium and the transport of

essential amino acids was used (Rhoads et al. 1994; Zhang et

al. 2014).

In order to investigate to extend at which these system have the

ability to transporting gradients with high molecular mass,

GFP (green fluorescent protein) was used (Bruch et al. 2008).

Papain and wheat extract, which consisted of the variety

Greina, in the wild-type variant (isogenic) and in the

genetically modified variant (transgenic) were used to

determine the effect of an “unknown” substance on the above

parameters. The statistical analysis was performed using Excel

2010 and Sigma Plot 11th

. Statistical analysis of data was

carried out using standard analysis of variance. The

significance was determined using the F-test, least significant

difference (LSD) was computed at the 5% probability level.

Significant differences have been represented by different

letters.

2.1 Native epithelium

The native epithelium (tissue culture) was recovered from the

ileum of pigs of the breed German Landrace (male, castrated,

age on average 180 days). After commercial slaughter, the

tissue was removed, placed in ice-cold buffer containing

indomethacin (2.8 nM) and gassed with carbogen (a mixture of

5% carbon gas and 95% oxygen gas). This part of the gut was

chosen because of the occurrence of jejunal transporters in the

ileum as well as M-cells mediated transport mechanisms in

Peyer’s patches.

2.2 Cell line and Culture Conditions

To compare the results obtained by native epithelium to cell

line, commercially available IPI-21 cells from the ileum of the

pig would be the best choice. Unfortunately, they do not grow

on transwell-plates so transport studies in Ussing chamber

experiments are not possible. To work around this problem

IPEC-J2 cells were used. The IPEC-J2 cell line is a non-

transformed intestinal cell line originally derived from jejunal

epithelia isolated from a neonatal, unsuckled piglet and

maintained as a continuous culture (Berschneider, 1989). Cells

were purchased from FLI (Federal Research Institute for

Animal Health, Germany). Unless otherwise indicated, cells

385 Bruch and Mohr

_________________________________________________________



were cultured on Dulbecco’s modified eagle medium

(DMEM)/HAM’s F-12 (1:1) supplemented with 10% fetal calf

serum (Biochrom, Germany) and maintained in an atmosphere

of 5% CO2 at 37°C. Cells reached confluence after 3-4 days

and were cultivated on polyester membranes with 0.4 µm pore

size (Corning Costar). The investigations were carried out in

the period of 6-8 days after sowing (Schierack et al., 2006), in

a passage of 20-40.

2.3 Morphological Characterization

The IPEC-J2 cells were stained immunohistochemically after 3

days growth on “cover slips". The cell nucleus, F-actin and

tubulin structures of the cells were stained with antibodies

(Dapi, Bodipy and anti-tubulin, Sigma Aldrich, Germany) and

fluorescent secondary antibodies (Alexa 594, Sigma Aldrich,

Germany). Immunofluorescence microscopy was performed

with a Zeiss LSM 510 META confocal laser scanning

microscope (CLSM, Zeiss, Germany)

2.4 Functional parameters

2.4.1 Transepithelial electrical resistance (TEER)

The cells were cultured as described above and seeded with a

density of 6*105 cells/cm². Transwell filters or snapwell filter

made by polycarbonate with 0.4µm pore size (Corning, The

Netherlands) was used for this culturing. The TEER was

measured daily with a volt-ohm-meter (WPI, Germany). The

transepithelial resistance of the native epithelium was

determined during the Ussing chamber experiments.

2.4.2 Ussing chamber experiments

The retrieved samples of intestine were prepared by removing

the muscularis and serosa and fixed in Ussing chambers

(Scientific Instruments, Germany) with an area of 1 cm². On

both sides, 5 ml buffer (115 mM NaCl; 25 mM NaHCO3; 0,4

mM NaH2PO4; 2,4 mM Na2HPO4; 5 mM KCl, 5 mM glucose;

1,2 mM CaCl2; 1,2 mM MgCl2 add 1,4 µM Indomethacin,

pH7.4) was added. The chambers were gassed with carbogen at

a temperature of 37°C. The initial resistance of the epithelium

was in the range of 90-150 Ώ. Confluent cell culture

monolayers on snapwell filters have been investigated in

Ussing chambers under the same conditions described above.

During the experiments, test substances were added to the

mucosal or serosal side of the Ussing chamber.

2.4.3 Short circuit (Isc) measurements

As a functional parameter the current after mucosal addition of

NaCl (115 mmol) was used. After an equilibration period of 15

min (constant value of Isc) NaCl was added and the reaction of

tissue or cell culture monolayer in the absence or presence of

the test substance was registered. After completion of the

experiment, Theophylline (10-2

mol*l-1

) was added to prove the

viability. Only in case of a reaction to Theophylline data were

used for further investigation.

2.4.4 Diffusion of [3H]-mannitol

Tightness of tissue or monolayer was investigated with tritium-

labeled mannitol (11.7 Ci/mM, conc. 3.85*10-6

mM,

Amersham, Great Britian). It added to the mucosal side of

native epithelium and cell culture in the Ussing chamber.

Hourly a sample from the serosal compartment was taken. The

samples were analyzed by liquid scintillations chromatography

(Liquid Scintillation Analyser, Tri-Carb 2900TR, Perkin

Elmer, USA) and the diffusion in relation to the initial amount

of [3H] mannitol was calculated in %/h*cm² or as standard

permeability coefficient (Papp).

2.4.5 Transport of essential amino acids

The tritium-labeled amino acids methionine, leucine, lysine

and tyrosine were added to the mucosal side of the Ussing

chamber (79.7 Ci/mM, 0.57*10-6

mM). Hourly a sample was

taken from the serosal side of the Ussing chamber. The

samples were analyzed by liquid scintillations chromatography

(Liquid Scintillation Analyser, Tri-Carb 2900TR, Perkin

Elmer, USA) and the transportin relation to the initial amount

of [3H]-amino acids was calculated in %/h*cm².

2.4.6 Transport of high molecular weight substances

To investigate a potentially available transport of high

molecular substances, in buffer dissolved GFP (green

fluorescent protein, about 4 mg/ml contain in genetically

modified tobacco plants) was added to the mucosal side of the

native epithelium or the IPEC-J2 cells. Hourly a sample was

taken from the serosal side of the Ussing chamber. The

samples were analysed by ELISA (sandwich-ELISA, Bioserv

GmbH, Germany) and the transport/diffusion in relation to the

initial amount of GFP was calculated in %/h*cm².

2.4.7 Effect of complex test substances

As a model for the effect of „unknown” complex test

substances on cell function, papain and wheat extract were

selected. Papain (Sigma Aldrich, Germany) is a proteolytic

enzyme from papaya. It serves the plant to repel insects and

affects the fibrin structures (Wittmack & Tomaschek 1978,

Konno & Barber 2014). In addition, it is considered to be

allergenic and therefore it was added in the concentration of 1

mg/ml to the mucosal side of the native epithelium or to

mucosal and serosal side of the cell monolayer.

Wheat extract containing gluten, known to be an agent

responsible for potential incompatibilities (Smecuol et al.,

1999; Menard et al. 2012) was chosen as an additional test

substance. A solution prepared from wheat grains of the

variety Greina (isogene (wild type variant) and transgenic

(genetically modified) variant), conc. 1mg/ml buffer, was

added to the mucosal side of the native epithelium and the

IPEC-J2 cells.

Development of in vitro screening system for food habit related risk analysis. 386

_________________________________________________________



3 Results

3.1 Morphological characterization

Results demonstrated by Figure.1 and proofed by the

occurrence of Occludin (Figure. 2), IPEC-J2 cells formed a

dense monolayer with formation of tight-junctions. The TEER

ranged between 4000-4500 Ώ/cm² on day 7 of cultivation. The

standard permeability coefficient (Papp) of [3H]-mannitol was

1.3386E-06 on IPEC-J2 monolayer and 3.572-E-07 on native

epithelium at a resistance of 110 Ώ (Figure. 3).

3.2 Functional characterization

To characterize electrophysiological properties the response to

addition of sodium was examined. Both the IPEC-J2 cells, as

well as the native epithelium showed an increase in short-

circuit current Isc: about 16 µA/cm² in cell culture monolayer

and about 7 µA/cm² in native epithelium (Figure. 4).

3.3 Measurement of amino acid transport

Both in native epithelium and IPEC-J2 monolayer a transport

of the amino acids leucine, lysine, methionine and tyrosine was

measured. The transport rates for the native epithelium have

been 0.007 (Lys), 0.013 (Met), 0.017 (Leu), 0.024 (Tyr)

pmol/s*cm² and for the IPEC-J2 cells are 37.80 (Met), 58.56

(Leu), 41.55 (Lys), 104.13 (Tyr) pmol/s*cm² (Figure. 5).

3.4 Transport of high molecular substances

To investigate the occurrence of unspecific transport

mechanisms for high molecular substances (e.g. ABC-

transporter), GFP was used. A transport of a high molecular

substance such as GFP could be detected. In native intestinal

epithelium a transport of GFP of 0.133 %/h*cm² of the initial

concentration was measurable, in the IPEC-J2 monolayer the

transport of GFP was only 0.0306 %/h*cm² (Figure. 6).

3.5 Influencing the morphological and electrophysiological

parameters by complex test substances

3.5.1 Papain

An influence on the functional and morphological parameters

of the test systems, by papain is detectable especially on IPEC-

J2 monolayer. Native epithelium did not response to papain

given on the mucosal side. In contrast, IPEC-J2 monolayers are

affected in most cases, only by papain donation to the serosal

side. Figure 7 shows the influence of serosal addition of papain

to the IPEC-J2 monolayers. It is visible already after 2 hours

and further strengthened over the test period. In the same way

lysine transport is affected (Figure. 8).

Figure 1 IPEC-J2 cells, 7 days cultured on polycarbonate

membrane

Figure 2 IPEC-J2 cells, nucleus (blue, Dapi) and Occludin

(green, Alexa 488)

387 Bruch and Mohr

_________________________________________________________



Figure 3 standard permeability coefficient (Papp) of [3H]-

mannitol on native intestinal epithelium and on IPEC-J2-

monolayer (n=8).Values fallowed by the same letter are not

significantly different at 5% DMRT.

Figure 4 short circuit current (Isc) after the addition of sodium

on the apical side of IPEC-J2 cells and native epithelium

(n=8).

Figure 5 Transport of amino acids (methionine, leucine, lysine,

tyrosine) by IPEC-J2 cells and native tissue (Ileum,

n=10).Values fallowed by the same letter are not significantly

different at 5% DMRT.

Figure 6 Transport of intact GFP (green fluorescent protein) by

IPEC-J2 cells and native tissue (n=8). Values fallowed by the

same letter are not significantly different at 5% DMRT.

Figure 7 Diffusion of [3H]-mannitol by IPEC-J2 monolayer

and native epithelium after mucosal addition of papain

(n=8).Values fallowed by the same letter are not significantly


Figure 8 Influence of mucosal or serosal addition of papain on

the translocation of lysine on the IPEC-J2 monolayer (n=8)

during 6 hours.Values fallowed by the same letter are not


time [min]

00:39:00 00:44:00 00:49:00

I [µ

A/c

m2]

-6

-4

-2

0

2

4

6

8

10

12

14

IPEG-cells

native epithel

Na*


_________________________________________________________



Furthermore no reaction was reported on the mucosal addition

and a significant increase in the presence of papain on the

serosal side. An effect of papain is also detectable in the

measurement of short circuit current caused by sodium. The

sodium-induced Isc in IPEC-J2 monolayer is 4µA/cm² and was

significantly reduced by the addition of papain (Figure. 9).

Interestingly, there is no significant difference in the reaction

between additions of papain to the mucosal orserosalside. The

Isc was always reduced by about 5µA/cm² compared to the

control.

3.5.2 Wheat extract

Presence of wheat gluten with the resultant potential

incompatibilities (Smecuol et al., 1999; Menard et al., 2012)

could have an influence on cellular transport mechanisms. In

addition, because wheat was available as isogenic and

transgenic version, the possible influence of non-identical

genetic material was of some interest and studied in this

experiment. To evaluate the potential influence on lysine

transport of IPEC-J2 cells wheat extract was admit to the

mucosal side. The lysine transport showed a significant

increase compared to the control variant without wheat extract

(Figure. 10).

There were no significant differences between the different

wheat varieties. The diffusion of [3H]-mannitol am IPEC-J2-

monolayer increased significantly after mucosal addition

(Figure.11). In contrast, a similar effect in native epithelium

could not be observed. In the next step, the influence of wheat

extract on the sodium-induced short circuit current (Isc) was

investigated. The results indicate a possible influence of Isc by

wheat extract: the addition of isogenic and transgenic wheat

extract was made to the mucosal side. The Isc increased

immediately and was significantly higher than in the control

variant over the entire time course (Figure. 12).

4 Discussion and conclusion

4.1 Morphological and functional characterization

The IPEC-J2 cells are capable due their origin to serves as a

model for the porcine intestine. Due the high compliance of

height, weight, anatomy and physiology of the gastrointestinal

tract of pigs and humans (Wernersson et al., 2005), this model

seems also to be suitable for human (Schierack et al., 2006,

Brosnahan et al., 2012). This hypothesis is strengthened by the

observation of tight junctions in cells like it is detectable in

human beings. The measured TEER values are consistent with

the data from literature (Schierack et al., 2006). Furthermore,

the diffusion of [3H]-mannitol under control conditions in

IPEC-J2 monolayer compared to the native intestinal

epithelium is not significantly different. This result indicates

that a dense intact epithelium was formed. From these facts it

was derive that this in vitro system is morphologically

comparable with other described in vitro systems (Geens &

Niewold, 2011).

In the reaction of cells and native epithelium to the addition of

sodium a significantly higher change in the short circuit current

(Isc) in the cell culture was reported. This effect can be

explained by the higher resistance of the complete intestine.

There are several cell layers are present, causing an overall

higher resistance. This shows that the cell culture system

should be preferred when investigating the influence on

sodium dependent transport processes. In the study of the

transport of essential amino acids in the native epithelium and

the IPEC-J2 monolayer a significant difference in the rate of

transport was observed by various researchers (Rhoads et al.,

1994; Zhang et al. 2014). The measured rate of transportation

of amino acids methionine, tyrosine, lysine and leucine is in

IPEC-J2 cells was reported significantly higher than in native

porcine intestine.


on the apical side of IPEC-J2 cells under the influence of

mucosal or serosal addition of papain (curve fit by Sigma Plot

11.0).

Figure 10 Diffusion of [3H]-mannitol by IPEC-J2 monolayer

and native epithelium after addition of wheat extract (n=8,

isogen (cereal-isogen) and transgenic (cerea-KP4). Values

fallowed by the same letter are not significantly different at 5%

DMRT.

389 Bruch and Mohr

_________________________________________________________



Figure 11 Translocation of [3H]-lysine on the IPEC-J2

monolayer (n=8) by addition of wheat extract during 6 hours.

Values fallowed by the same letter are not significantly



on the apical side of IPEC-J2 cells under the influence of

addition of wheat extract (isogenic or transgenic, curve fit by

Sigma Plot 11.0).Values fallowed by the same letter are not


Investigations with the aid of IPEC-J2 cells are able to show

the transport of these essential amino acids faster and more

effectively than it can be done using native epithelium.

In contrast, in the study about transport of high molecular

substances, e.g. GFP, a significantly higher transport by the

native intestinal epithelium can be detected. Cell culture

systems therefore, appear to be less suitable for studies about

the absorption and transport of high molecular substances.

Possibly, the mechanisms responsible for the transport of high

molecular substances are formed only to a very limited extent

in cell culture monolayers. In intestinal transport other cell

types, such as M-cells may be involved, which may be present

only in the native epithelium of the intestine. This observation

confirms the tests described in the literature, for example, that

Caco-2 cells alone are not in a position to carry GFP (Heppner

et al., 2001). Therefore it is recommended to investigate the

transport of high molecular substances only in native

epithelium, especially when transport routes are not yet known.

4.2 Use of in vitro systems for risk assessment

The suitability of the in vitro system for a risk assessment was

checked using the enzyme papain and an unknown mixture of

wheat extract. Papain is a protein-splitting enzyme and it

serves insects repellent and affects the fibrin structures

(Wittmack & Tomaschek 1978; Huby et al., 2000; Konno &

Barber, 2014). The applied wheat extract was a sample from

genetically modified wheat, the potential ability to influence

physiological processes in the gastrointestinal tract should be

investigated. Due to the higher sensitivity of cell cultures, the

reaction of these is faster and more sensitive as the ex-vivo

systems to a possible influence of paracellular transport routes.

As the results show in figure 5, the influence of the diffusion of

[3H]-mannitol under test conditions, such the addition of

papain (Figure. 8) and „wheat“(Figure. 10) is significantly

more pronounced in cell cultures.

4.3 Response to the addition of papain

Both the diffusion of [3H]-mannitol, the sodium –induced Isc

and the transfer of the amino acid lysine influenced by the

addition of papain. However, this effect could be detected only

in serosal addition to the native intestinal epithelium and on the

cell monolayer. The increased diffusion of mannitol on IPEC-

J2 monolayer in serosal addition of papain indicates a possible

change in terms of diffusion properties and in the integrity of

the monolayer. May be the damage of the epithelium on the

basolateral side by the addition of papain is the trigger for the

significantly higher mannitolflux. Papain also showed an effect

on the transport of lysine. Again, mucosal addition did not

significantly influence the transport of the amino acid. Butif

added serosal, the transport of lysine was significantly higher.

Probably proteolysis action of the enzyme causes possibly a

damaging effect on the basolateral side of the monolayer.

Because no blocker experiments were performed, it could not

be distinguished between an increase in transport and/or

diffusion. By the addition of papain the Na-Isc is significantly

reduced. The cell monolayer has been no longer able to

respond appropriately to the addition of sodium. Interestingly,

this effect occurs in serosal and in mucosal addition of papain.

Possibly, the epithelium was damaged or the

electrophysiological properties were affected by the addition of

papain. This will be investigated in further studies.

4.4 Response to the addition of wheat extract


_________________________________________________________



In addition, wheat extract was used as a test substance. The

diffusion of mannitol was significantly increased by mucosal

addition to the IPEC-J2 monolayer. Also the transport of lysine

exhibits an increase under the influence of wheat extract. The

Na-Isc was also affected by the addition of wheat extract.

Serosal addition of wheat extract was omitted because the test

system was mainly developed to simulate the processes in the

intact intestine. One possible explanation for the effect of

wheat extract on mucosal addition to the cell culture might be

bases on the effect of the wheat ingredients, because they

contained gluten, which has an effect on the tight junction

proteins (Smecuol et al., 1999).

Based on the response of the two test systems on the various

test substances a stronger influence always shown on the

morphological and functional properties of the cells culture

monolayer compared to the control variant (complete ex vivo

portion of the porcine intestine). The constant resistance of

IPEC-J2 cells and the native epithelium during the experiment

proof the stability of both test systems. The significantly higher

response in the cell monolayer is therefore no artifact but

inherent to the system.

The in vitro system reacts sensitively to any test substances.

The observed changes in diffusion of mannitol, as wells as the

electrophysiological (Na-Isc) and transport properties (transport

of lysine) are due to the influence of unknown components. In

a risk assessment setup, the presented test systems should be

implemented because the observed different responses make it

possible to characterize the various possible influences of

morphological and functional properties by the addition of

various test substances.

Test systems that are based on in vitro experiments, are an

effective alternative to animal experiments. The existing

technical possibilities should be used to develop further in vitro

test systems and apply this. The higher the accuracy and

comparability with in vivo data, the number of applications

would be developed. Research should invest in in-vitro

systems to replace animal tests largely.

Acknowledgment

The authors thank Angelika Hauth for her excellent technical

assistance.




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