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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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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)
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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
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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
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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
Available Online – August 20, 2015
DOI: http://dx.doi.org/10.18006/2015.3(4).346.352
MONITORING OF AFLATOXIN CONTAMINATION AT MARKET FOOD CHAIN IN
EAST JAVA
E-mail: [email protected] (Agustina A. Rahmianna)
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)
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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
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Journal of Experimental Biology and Agricultural Sciences
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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
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Journal of Experimental Biology and Agricultural Sciences
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Table 2 Percent of damaged seeds 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 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
Numbers followed by the same letter(s) indicated not significantly different based on DMRT 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.
Sampling times Market types
Traditional market Wholesaler Supermarket
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
Numbers followed by the same letter(s) indicated not significantly different based on DMRT 5%
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).
349 Rahmianna and Yusnawan
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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.
Monitoring of Aflatoxin contamination at market food chain in east java. 350
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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.
Conflict of interest
Authors would hereby like to declare that there is no conflict of
interests that could possibly arise.
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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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
Available Online – August 20, 2015
DOI: http://dx.doi.org/10.18006/2015.3(4).353.361
LIVELIHOOD ASSESSMENT OF THE FISHERMEN COMMUNITY IN THE SOUTH
WEST REGION OF BANGLADESH
E-mail: [email protected] (Uttam Kumar)
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)
Journal of Experimental Biology and Agricultural Sciences
http://www.jebas.org
ISSN No. 2320 – 8694
Production and Hosting by Horizon Publisher (www.my-
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All rights reserved.
All the article published by Journal of Experimental
Biology and Agricultural Sciences is licensed under a
Creative Commons Attribution-NonCommercial 4.0
International License Based on a work at www.jebas.org.
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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
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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
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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
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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.
Livelihood assessment of the fishermen community in the south west region of Bangladesh 357
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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
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Journal of Experimental Biology and Agricultural Sciences
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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
Livelihood assessment of the fishermen community in the south west region of Bangladesh 359
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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.
Conflict of interest
Authors would hereby like to declare that there is no conflict of
interests that could possibly arise.
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.
Department of Fisheries (2010) Ministry of Fisheries and
Livestock, Dhaka, Bangladesh. National Fish Week
Compendium (in Bengali) Pp 144.
Department of Fisheries (2012) Ministry of Fisheries and
Livestock, Dhaka, Bangladesh. National Fish Week
Compendium (in Bengali) Pp 144.
Department of Fisheries (2013) 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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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
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Shahjahan M, Miah MI, Haque MM (2001) Present Status of
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Livelihood assessment of the fishermen community in the south west region of Bangladesh 361
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Journal of Experimental Biology and Agricultural Sciences
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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
Available Online – August 27, 2015
DOI: http://dx.doi.org/10.18006/2015.3(4).362.367
EFFECT OF POULTRY MANURE AND MINERAL FERTILIZER ON THE GROWTH
PERFORMANCE AND QUALITY OF CUCUMBER FRUITS
E-mail: [email protected] (Nweke I A)
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)
Journal of Experimental Biology and Agricultural Sciences
http://www.jebas.org
ISSN No. 2320 – 8694
Production and Hosting by Horizon Publisher (www.my-
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All rights reserved.
All the article published by (Journal of Experimental
Biology and Agricultural Sciences) / CC BY-NC 4.0
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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
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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
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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.
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
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
Available Online – August 27, 2015
DOI: http://dx.doi.org/10.18006/2015.3(4).368.377
PLASMID STABILITY AND MAINTENANCE OF COPY NUMBER USING
NATURAL MARKER
E-mail: [email protected] (Sudhakar Malla)
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)
Journal of Experimental Biology and Agricultural Sciences
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ISSN No. 2320 – 8694
Production and Hosting by Horizon Publisher India
(http://publizer.jebas.org/index.html).
All rights reserved.
All the article published by (Journal of Experimental
Biology and Agricultural Sciences) / CC BY-NC 4.0
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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 Materials and Methods
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
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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.
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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.
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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.
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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.
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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 7 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 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.
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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 9 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 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.
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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.
Plasmid Stability and Maintenance of Copy number using Natural marker. 376
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Journal of Experimental Biology and Agricultural Sciences
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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.
Conflict of interest
Authors would hereby like to declare that there is no conflict of
interests that could possibly arise.
References
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expression for therapeutic applications. Current Opinion in
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377 Hamzah and Sudhakar
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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
Available Online – August 27, 2015
DOI: http://dx.doi.org/10.18006/2015.3(4).378.383
ERGASTIC CRYSTALS IN IDENTIFICATION OF COSTUS PICTUS: A MEDICINAL
SPIRAL GINGER IN HERBAL MEDICINE
E-mail: [email protected] (Justin R Nayagam)
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)
Journal of Experimental Biology and Agricultural Sciences
http://www.jebas.org
ISSN No. 2320 – 8694
Production and Hosting by Horizon Publisher (www.my-
vision.webs.com/horizon.html).
All rights reserved.
All the article published by (Journal of Experimental
Biology and Agricultural Sciences) / CC BY-NC 4.0
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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.
2 Materials and Methods
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
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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
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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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Journal of Experimental Biology and Agricultural Sciences
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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
Authors would hereby like to declare that there is no conflict of
interests that could possibly arise.
References
Gothandam KM, Aishwarya R, Karthikeyan S (2010)
Preliminary screening of antimicrobial properties of few
medicinal plants. Journal of Phytology 2:1–6.
Hedge PK, Rao HA, Rao PN (2014) A review on insulin plant
(Costus igneus Nak). Pharmacognosy Reviews. 8: 67-72.
doi: 10.4103/0973-7847.125536.
Jayasri MA, Mathew L, Radha A (2009) A report on the
antioxidant activity of leaves and rhizomes of Costus pictus D.
Don. International Journal of Integrative Biology 5:20–26.
Jose B, Reddy LJ (2010) Analysis of the essential oils of the
stems, leaves and rhizomes of the Medicinal plant Costus
pictus from Southern India. International Journal Pharmacy
and Pharmaceutical Sciences 2:100–101.
Kokate CK, Purohit AP, Gokhale SB (2010) Pharmacognosy
Edition 46, Vol. I & II, Nirali Prakashan publishers, Shivaji
Nagar, Pune.
Kumudhavalli MV, Jaykar B (2012) Evaluation of antidiabetic
activity of Costus igneus (L) leaves on STZ induced diabetic
rats. Der Pharmacia Sinica Journal 3:1–4.
Mani P, Kumar AR, Bastin TM, Jenifer S, Arumugam M
(2010) Comparative evaluation of extracts of C. igneus (or C.
pictus) for hypoglycemic and hypolipidemic activity in alloxan
diabetic rats. International Journal for Pharmaceutical
Technology 2:183–95.
Ergastic crystals in identification of Costuspictus: a medicinal spiral ginger in herbal medicine 382
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Journal of Experimental Biology and Agricultural Sciences
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Manjula K, Pazhanichamy K, Kumaran S, Eevera T, Dale
Keefe C, Rajendran K (2012) Growth characterization of
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
male albino rats. International Journal of Integrative Biology
9:62–68.
Meléndez-Camargo ME, Castillo-Nájera R, Silva-Torres R,
Campos-Aldrete ME (2006) Evaluation of the diuretic effect of
the aqueous extract of Costus pictus D. Don in rat. Proceedings
of the Western Pharmacology Society 49:72–74.
Metcalfe CR (1960) Anatomy of Monocotyledons I
Gramineae. Clarendon Press, Oxford University Press, New
York.
Nadumane VK, Rajashekar S, Narayana P, AdinarayanaS,
Vijayan S, Prakash S (2011) Evaluation of the anticancer
potential of Costus pictus on fibrosarcoma (HT-1080) cell
line. Journal of Natural Pharmaceuticals 2:72–76.
Nakai T (1941) Notulae ad Plantas Asiae Orientalis XVI. The
Journal of Japanese Botany 17: 189-210.
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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Remya R, Daniel M (2012) Phytochemical and
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Sabu M (2006) Zingiberaceae and Costaceae of South India.
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383 Nayagam
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Journal of Experimental Biology and Agricultural Sciences
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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
DOI: http://dx.doi.org/10.18006/2015.3(4).384.393
DEVELOPMENT OF IN VITRO SCREENING SYSTEM FOR FOOD HABIT
RELATED RISK ANALYSIS
E-mail: [email protected] (Mandy Bruch)
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)
Journal of Experimental Biology and Agricultural Sciences
http://www.jebas.org
ISSN No. 2320 – 8694
Production and Hosting by Horizon Publisher
(http://publisher.jebas.org/index.html).
All rights reserved.
All the article published by Journal of Experimental
Biology and Agricultural Sciences is licensed under a
Creative Commons Attribution-NonCommercial 4.0
International License Based on a work at www.jebas.org.
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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.
2 Materials and Methods
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
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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.
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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)
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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
different at 5% DMRT.
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
significantly different at 5% DMRT.
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*
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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.
Figure 9 short circuit current (Isc) after the addition of sodium
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.
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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
different at 5% DMRT.
Figure 12 short circuit current (Isc) after the addition of sodium
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
significantly different at 5% DMRT.
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
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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.
Conflict of interest
Authors would hereby like to declare that there is no conflict of
interests that could possibly arise.
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