Journal of Research in Biology Volume 3 Issue 4

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Bharathiar University.

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TNAU, Coimbatore.

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Tarbiat Modares University (TMU), Iran.

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Al-Azhar university, Egypt

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Agharkar Research Institute, Pune, INDIA.

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Laboratorio de ecología y conservación de fauna Silvestre,

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Department of Pathology, Army Medical College, Rawalpindi, Pakistan.

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BCKV (Agri University), West Bengal, INDIA.

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Jawaharlal Technological University, Hyderabad.

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University of Kordofan, Elobeid-SUDAN.

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University of Mosul, Mosul,Iraq.

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Central Institute of Medicinal and Aromatic Plants, Lucknow, India.

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Hem. North Gujarat University, Patan.

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C.S.J.M. University, India.

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Federal University of Rondônia, Brazil.

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Stefan cel Mare University of Suceava, Romania.

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School of Life Sciences, Bharathidasan University, India.

Dr. Mrs Kavita Sharma [Botany]

Arts and commerce girl’s college Raipur (C.G.), India.

Suwattana Pruksasri [Enzyme technology, Biochemical Engineering]

Silpakorn University, Thailand.

Dr.Vishwas Balasaheb Sakhare [Reservoir Fisheries]

Yogeshwari Mahavidyalaya, Ambajogai, India.

Dr. Pankaj Sah [Environmental Science, Plant Ecology]

Higher College of Technology (HCT), Al-Khuwair.

Dr. Erkan Kalipci [Environmental Engineering]

Selcuk University, Turkey.

Dr Gajendra Pandurang Jagtap [Plant Pathology]

College of Agriculture, India.

Dr. Arun M. Chilke [Biochemistry, Enzymology, Histochemistry]

Shree Shivaji Arts, Commerce & Science College, India.

Dr. AC. Tangavelou [Biodiversity, Plant Taxonomy]

Bio-Science Research Foundation, India.

Nasroallah Moradi Kor [Animal Science]

Razi University of Agricultural Sciences and Natural Resources, Iran

T. Badal Singh [plant tissue culture]

Panjab University, India

Dr. Kalyan Chakraborti [Agriculture, Pomology, horticulture]

AICRP on Sub-Tropical Fruits, Bidhan Chandra Krishi Viswavidyalaya,

Kalyani, Nadia, West Bengal, India.

Dr. Monanjali Bandyopadhyay [Farmlore, Traditional and indigenous

practices, Ethno botany]

V. C., Vidyasagar University, Midnapore.

M.Sugumaran [Phytochemistry]

Adhiparasakthi College of Pharmacy, Melmaruvathur, Kancheepuram District.

Prashanth N S [Public health, Medicine]

Institute of Public Health, Bangalore.

Tariq Aftab

Department of Botany, Aligarh Muslim University, Aligarh, India.

Manzoor Ahmad Shah

Department of Botany, University of Kashmir, Srinagar, India.

Syampungani Stephen

School of Natural Resources, Copperbelt University, Kitwe, Zambia.

Iheanyi Omezuruike OKONKO

Department of Biochemistry & Microbiology, Lead City University,

Ibadan, Nigeria.

Sharangouda Patil

Toxicology Laboratory, Bioenergetics & Environmental Sciences Division,

National Institue of Animal Nutrition

and Physiology (NIANP, ICAR), Adugodi, Bangalore.

Jayapal

Nandyal, Kurnool, Andrapradesh, India.

T.S. Pathan [Aquatic toxicology and Fish biology]

Department of Zoology, Kalikadevi Senior College, Shirur, India.

Aparna Sarkar [Physiology and biochemistry] Amity Institute of Physiotherapy, Amity campus, Noida, INDIA.

Dr. Amit Bandyopadhyay [Sports & Exercise Physiology]

Department of Physiology, University of Calcutta, Kolkata, INDIA .

Maruthi [Plant Biotechnology]

Dept of Biotechnology, SDM College (Autonomous),

Ujire Dakshina Kannada, India.

Veeranna [Biotechnology]

Dept of Biotechnology, SDM College (Autonomous),

Ujire Dakshina Kannada, India.

RAVI [Biotechnology & Bioinformatics]

Department of Botany, Government Arts College, Coimbatore, India.

Sadanand Mallappa Yamakanamardi [Zoology]

Department of Zoology, University of Mysore, Mysore, India.

Anoop Das [Ornithologist]

Research Department of Zoology, MES Mampad College, Kerala, India.

Dr. Satish Ambadas Bhalerao [Environmental Botany]

Wilson College, Mumbai

Rafael Gomez Kosky [Plant Biotechnology]

Instituto de Biotecnología de las Plantas, Universidad Central de Las Villas

Eudriano Costa [Aquatic Bioecology]

IOUSP - Instituto Oceanográfico da Universidade de São Paulo, Brasil

M. Bubesh Guptha [Wildlife Biologist] Wildlife Management Circle (WLMC), India

Rajib Roychowdhury [Plant science]

Centre for biotechnology visva-bharati, India.

Dr. S.M.Gopinath [Environmental Biotechnology]

Acharya Institute of Technology, Bangalore.

Dr. U.S. Mahadeva Rao [Bio Chemistry]

Universiti Sultan Zainal Abidin, Malaysia.

Hérida Regina Nunes Salgado [Pharmacist]

Unesp - Universidade Estadual Paulista, Brazil

Mandava Venkata Basaveswara Rao [Chemistry]

Krishna University, India.

Dr. Mostafa Mohamed Rady [Agricultural Sciences]

Fayoum University, Egypt.

Dr. Hazim Jabbar Shah Ali [Poultry Science]

College of Agriculture, University of Baghdad , Iraq.

Danial Kahrizi [Plant Biotechnology, Plant Breeding,Genetics]

Agronomy and Plant Breeding Dept., Razi University, Iran

Dr. Houhun LI [Systematics of Microlepidoptera, Zoogeography, Coevolution,

Forest protection]

College of Life Sciences, Nankai University, China.

María de la Concepción García Aguilar [Biology] Center for Scientific Research and Higher Education of Ensenada, B. C., Mexico

Fernando Reboredo [Archaeobotany, Forestry, Ecophysiology]

New University of Lisbon, Caparica, Portugal

Dr. Pritam Chattopadhyay [Agricultural Biotech, Food Biotech, Plant Biotech]

Visva-Bharati (a Central University), India

Table of Contents (Volume 3 - Issue 4)

Serial No Accession No Title of the article Page No

1 RA0344 Influence of the growing area on oil palm (Elaeis guineensis) inflorescences insects population.

Koua Kouakou Hervé, Akpesse Apka Alexandre Moise, TUO Yalamoussa, and Hala N’klo.

940-946

2 RA0349 A chromosomal analysis of seven Cameroonian Acrididae species (Orthoptera: Acridinae, Oedipodinae and Spathosterninae) based on published data.

Seino Richard Akwanjoh and Dongmo Tonleu Ingrid.

947-953

3

RA0324

Impact of the residue of Deltamethrin and Endosulfan pesticides on biochemical toxicity and some neurotransmitter contents in different brain areas of male Albino mice.

Somaya M. Ismail, Azza A. Said and Samira M. El-Sayad.

954-966

4 RA0342 Prevalence and the effect of plant extracts on community associated methicillin resistant Staphylococcus aureus in Owerri, Imo State, Nigeria.

Amadi ES, Oguoma OI, Ibekwe VI, Abanobi SE, Chikwendu CI and Egbadon OE.

967-976

5 RA0346 Odonata diversity (Insecta: Arthropoda) in rice and vegetable fields in a north-eastern district of Tamil Nadu, India.

Veeramuthu Anbalagan, Michael Gabriel Paulraj and Savarimuthu Ignacimuthu.

977-983

6 RA0343 Hepatic enzyme markers and proteins in serum and some selected tissues in Clarias gariepinus from swamp around Kokori-Erhoike oil field, Nigeria.

Osioma E, Akanji MA and Arise RO.

984-992

http://jresearchbiology.com/documents/RA0344.pdf






Jou

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Biology

Influence of the growing area on oil palm (Elaeis guineensis) inflorescences

insects population

Keywords: culture area; pollinating insects; Lamé; Côte d’Ivoire.

ABSTRACT: Oil palm tree grows naturally on low ground and on plain. Seed production varies from one area to another on the same oil palm plantation. Pollination of oil palm is essentially entomophilous; it appeared useful to assess the influence of the growing area on the fluctuation of pollinating insects’ population. Samplings were performed each month on male and female inflorescences during two years on plots in lowland and plain. The insects showed no qualitative change from one area to another. Sixteen species of insects were observed on the male inflorescences against 10 species on female inflorescences. The inflorescences showed variation in the number of insects based on the growing area and the stage of flowering.

940-946 | JRB | 2013 | Vol 3 | No 4

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.

www.jresearchbiology.com Journal of Research in Biology

An International

Scientific Research Journal

Authors:

Koua Kouakou Hervé1,

Akpesse Apka Alexandre

Moise2, Tuo Yalamoussa3,

and Hala N’klo4.

Institution:

1. Félix Houphouet-Boigny

University of Cocody

(Abidjan, Côte d’Ivoire).

22 BP: 1611 Abidjan 22.




BP: 582 Abidjan 22.




BP: 582 Abidjan 22.

4. National Center of

Agronomic Research

(CNRA, Côte d’Ivoire)

BP: 1740 Abidjan 01.

Corresponding author:

Koua Kouakou Hervé.

Web Address: http://jresearchbiology.com/documents/RA0344.pdf.

Dates: Received: 25 Mar 2013 Accepted: 02 May 2013 Published: 16 May 2013

Article Citation: Koua Kouakou Hervé, Akpesse Apka Alexandre Moise, TUO Yalamoussa, and Hala N’klo. Influence of the growing area on oil palm (Elaeis guineensis) inflorescences insects population. Journal of Research in Biology (2013) 3(4): 940-946

Journal of Research in Biology An International Scientific Research Journal

Original Research

INTRODUCTION

Many problems are opposed to a good

production of oil palm. These include pests, pathogenic

fungi and especially the continuing decline of pollinating

insects. For several years many problems of fruit set

were observed in some regions of cultivation of oil palm

tree causing a gradual decline in seed production (Mariau

et al., 1991). The observation was made that the most

affected areas, spread over large areas of lowland.

There are many evidences that the pollinator

insects effectively contributes to the reproduction of

many cultivated plant species. Regarding the oil palm

tree, the discovery of the role of insects in pollination

was made by Chevalier in 1910. The works of Syed

(1979) and Syed et al. (1982) have confirmed this

finding. Pollination of oil palm is essentially

entomophilous (Corrado, 1985). Without pollination,

fruit set by wind is extremely low (Mariau et al., 1991).

Pollinating insects are thus an undeniable role. In Côte

d'Ivoire (West Africa), five species of Elaeidobius

(E. kamerunicus, E. plagiatus, E. subvitatus,

E. bilineatus, E. singularis), two species of Microporum

(M. congolense and M. dispar), two species of

Proseostus (P. minor, P. sculptilis), Atheta burgeoni,

Gabrius sp, Litargus sp, Thrips sp and Anthocoris sp

have been described by Desmier De Chenon, (1981) and

Hala et al., (2012) as pollinators of oil palm. It therefore

seemed appropriate to follow the dynamics of these

insects on two different ecological zones: lowland and

plain. The research question that we asked is whether the

growing area of oil palm has an influence on populations

of pollinating insects. Many factors can explain the

fluctuations of insect populations. The latest studies on

this subject have established outside the bioclimatic

factors which have a clear implication, that the use of

insecticides in the fight against pests do not spare

beneficial insects that are pollinators (Tuo et al., 2011).

This preliminary study established a quantitative

and qualitative comparison of oil palm inflorescences

insects in two agro ecosystems: lowland and plain.

MATERIAL AND METHODS

Study site

Our study site was the experimental station of La

Mé, located at 5°26, N, 3°50, W. The station is located to

about thirty kilometers north-east of Abidjan (Côte

d’Ivoire). This area is characterized by an ombrophilous

forest (Traoré and Mangara, 2009).

The study area has an equatorial climate

characterized by two distinct rainy seasons (March to

July and November). These two seasons are alternated by

two dry seasons: December to February and from August

to October (Pene and Assa, 2003). The monthly mean

temperature was about 27°C. The monthly average of the

highest temperature was recorded in March and the

lowest in August with respectively 28.55 and 25.5°C.

The average annual rainfall was about 1500 mm. The

average annual sunshine duration was about 1790 h; the

average monthly relative humidity was about 81%.

Insects of male inflorescences

Three operations were performed to assess the

male inflorescences of oil palm insect fauna: location,

sampling and identification of insects (Fataye, 1984).

Location

Each month a location was carried out in order to

count four inflorescences in the process of flowering.

This location has identified 192 male inflorescences at a

rate of eight per month during two years.

Sampling

As soon as the third florets of each inflorescence

were listed, with pair of secateurs four spikelets per

inflorescence were collected. This collection was done in

the beginning of anthesis (BA). The second (full

anthesis: FA) and third (end anthesis: EA) took place

respectively after the first three days and two days after

the second. Each batch of four spikelets collected was

placed in a bag and then the insects collected were

neutralized with an insecticide bomb. Before

Koua et al., 2013

941 Journal of Research in Biology (2013) 3(4): 940-946

identification, insects of each batch of spikelet were

collected in pillboxes containing alcohol 70%.

Identification

Using the collection of the insect fauna of oil

palm inflorescences of the National Agricultural

Research Centre of La ME and a binocular microscope,

insects of every month and each area were identified at

the species level.

Insects of female inflorescences

This study was conducted according to the

methods of N'goran (1982) and Fataye (1984).

Location

Two non-flowering inflorescences per plot were

identified and followed by month. The inflorescences

were cleared of husks and bulky leaves with machetes

and knives three days before flowering. Each

inflorescence thus revealed was covered with a muslin

cage and attached to the floral stem with a rubber. The

bagged inflorescences were controlled each afternoon to

follow the evolution of the inflorescence.

Sampling

All the insects that were attracted are placed on

the cage once flowering commences. Using a vacuum

cleaner, these insects were captured every hour for ten

minutes. This operation was performed at 6 AM to 6 PM

during the two days of the anthesis length. The collected

insects were immediately stored in pillboxes containing

70% alcohol. At the end of the day, insects collected

were sent to the laboratory. At each study site, sampling

was conducted on 48 inflorescences.

Identification

Insects collected were identified using the same

protocol as previously.

STATISTICAL ANALYSIS

Data processing was performed using Statistica

software version 7.1. An analysis of variance (ANOVA)

revealed significant differences between the data. The

test of Student-Newman-Keuls at 5% was used to

classify the means into homogeneous groups

RESULTS AND DISCUSSION

Variation in the number of insects on male

inflorescences

On male inflorescences of oil palm tree, the

insects mostly belong to Elaeidobius (E) genus. Five

species were observed: E. kamerunicus, E. plagiatus,

E. subvittatus, E. singularis and E. bilineatus.

Microporum (M) genus was represented by the species

M. dispar and M. congolense. Prosoestus genus was

present with two species P. sculptilis and P. minor.

Species, Atheta burgeoni, Lithargus sp., Anthocoride sp.,

Thrips sp, Gabrius sp. and bees (Nomia sp and

Apis mellifera) were also observed.

At the beginning of anthesis (BA)

The number of insects collected from the plot of

lowland (61%) is higher than that collected on the plain

(31%). At the species level, it was observed that

E. singularis, E. bilineatus, P. sculptilis, M congolense

and Anthocoris sp showed a significant difference

depending on the growing area with a higher effective in

lowland areas. The other species showed no preference

for one area (Figure 1A).

At full anthesis (FA)

The total number of insects collected was 42% in

the plain region and 58% in lowland areas. For the

species, E. plagiatus, E. kamerunicus, M. congolense,

M. dispar and A. Burgeoni, a significant difference was

found between their respective populations based on the

growing area. Only A. burgeoni presented a higher

effective in the lowland areas. The other species were

much more present in plain areas. Besides these species,

no differences were recorded between the number of

insects collected in lowland areas and those collected on

the plain (Figure 1B).

At the end of anthesis (EA)

The total number of insects differ from one area

to another. It was 75% in lowland areas against 25% on

Koua et al., 2013

Journal of Research in Biology (2013) 3(4): 940-946 942

the plain. With regard to species, only the number of

E. singularis, M. dispar, Lithargus sp and A. burgeoni

depends on the area. These species excepted Lithargus sp

were more abundant in the lowland than on the plain

(Figure 1C).

Variation in the number of insects on female

inflorescences

The species observed on female inflorescences

were: E. kamerunicus, E. plagiatus, E. subvittatus,

E. bilineatus, E. singularis, M. congolense, M. dispar,

P. minor, P. sculptilis and Atheta burgeoni.

First day of anthesis

The total population of insects was significantly

higher on the plain (78%) than in the lowland area

(22%). At the species level, only the species M. dispar,

M. congolense, E. plagiatus, E. kamerunicus,

E. subvittatus, E. singularis, A. burgeoni and P. minor,

were affected by the growing area. A. burgeoni attended

more inflorescences of the lowland area. Other species

were more present on the plain than in the lowlands

(Figure 2A).

Second day of anthesis

Insects were relatively influenced by the growing

area. Indeed, 78% of the insects were collected on

inflorescences of the plain region against 28% in the

lowlands. Regarding species, E. subvittatus, A. Burgeoni,

P. minor and P. sculptilis, had no preference for the

growing area. The species E. kamerunicus, E. plagiatus,

E. bilineatus, E. singularis, M. dispar and M. congolense

were receptive to the area of culture (Figure 2B).

Among the species, only E. kamerunicus,

E. plagiatus, E. singularis and M. dispar were affected

by the growing area both the first and second day of

anthesis.

The insects of oil palm inflorescences showed no

qualitative change from one area to another. These

species were always present on the oil palm tree and

colonize the inflorescences of this plant regardless of the

study area.

Sixteen insect species were observed on the male

inflorescences against only 10 species on female

inflorescences. The six species that were absent on the

Koua et al., 2013


A: Beginning of anthesis

B: Full anthesis

Lowland Plain

C: End of anthesis

Figure 1: Influence of the growing area on the

number of insects present on male inflorescences

E.su: Elaeidobius subvittatus; E.p: Elaeidobius

plagiatus; E.s: Elaeidobius singularis; E.b: Elaeidobius

balineatus; E.k: Elaiedobius kamerinucus;

P.m: Prosoestus minor; P.s: Prosoestus sculptilis;

M.c: Microporum congolense; M.d: Microporum

dispar; L.sp: Lithargus sp.; Ant.sp.: Anthocoris sp.;

A.bur: Atheta burgeoni: T.sp.: Thrips sp.; G.sp.:

Gabrius sp.

Histograms with the same letter are not

significantly different at the 5% level

female inflorescences (Lithargus sp., Anthocoride sp.,

Thrips sp., Gabrius sp., and bees (Nomia sp. and

Apis mellifera) would not intervene mainly in oil palm

pollination. According to Mariau et al., 1991, four

species provide the largest share of pollination of oil

palm tree: E. kamerinucus, E. plagiatus, E. subvittatus

and E. singularis.

The male inflorescences showed a variation in

the numbers of insects based on the growing area and the

stage of flowering. In general, the lowland areas showed

significantly more insects than upland areas (61% against

31% at the beginning of anthesis, 58% against 42% at

full anthesis and finally 75% against 25% at the end of

anthesis). Insects observed in the male inflorescences

live on them usually. For example, the male flowers are

the breeding sites of insects of the genus Elaeidobius

(Beaudoin-Ollivier et al., 2012). The differences could

be explained by changes in environmental factors. At the

beginning of anthesis, flowers began to appear on the

male inflorescences that induced an attractive factor

because of the strong smell of anise emitted by the male

flowers. To this, were added the environmental factors

including relative humidity and temperature. It was noted

that the number of insects on lowland inflorescences

were two times higher than those of insects collected on

plain region inflorescences. At the species level, if for

E. bilineatus, P. sculptilis and M. congolense, numbers

were highest in the lowland area than on the plain region.

These three species are unfortunately not effectively

intervening in the pollination of oil palm tree. The other

insect species showed no significant difference at the

beginning of anthesis.

In full anthesis, the number of insects on male

inflorescences has reached its maximum value. The

attractive factor of flowers took over on bioclimatic

factors. Thus, it has almost the same number of insects

from one region to another. In terms of species observed

in full anthesis, the number of E. plagiatus,

E. kamerunicus, A. Burgeoni, M. dispar and

M. congolense were significantly different from one area

to another. Indeed, apart A. burgeoni, all these species

were more prevalent in upland areas. This can be

explained by bioclimatic factors which were more

favorable to the activity of these insects.

We observed three times more insects on

lowland inflorescences than on those on plain at the end

of anthesis. This can be explained by the fact that on

plain, the flowers were fading and dry faster than the

inflorescences in lowland areas (lower temperature and

Koua et al., 2013


A: First day of anthesis

A: Second day of anthesis

Lowland Plain

Figure 2: Influence of the growing area on the

numbers of insects present on female inflorescences

E.su: Elaeidobius subvittatus; E.p: Elaeidobius

plagiatus; E.s: Elaeidobius singularis; E.b: Elaeidobius

balineatus; E.k: Elaeidobius kamerinucus; P.m:

Prosoestus minor; P.s: Prosoestus sculptilis; M.c:

Microporum congolense; M.d: Microporum dispar;

L.sp: Lithargus sp.; Ant.sp.: Anthocoris sp.; A.bur:

Atheta burgeoni: T.sp.: Thrips sp.; G.sp.: Gabrius sp.

Histograms with the same letter are not

significantly different at the 5% level.

higher relative humidity). Insects were removed quickly

on inflorescences located in plain region. At the species

level, only E. singularis, M. dispar, A. burgeoni and

Lithargus sp. showed significantly different numbers

from one area to another. Only bio-ecological

requirements of these species can explain this

distribution. According to Hussein et al., 1990, the

change in the population of pollinating insects in

plantations of oil palm is largely due to the influence of

intrinsic and extrinsic factors, in particular, biological

and chemical factors.

During the anthesis, the total number of insects is

higher on female inflorescences taken from the plain

region than in the lowland area. At the species level, only

P. sculptilis showed no preference zone during the two

days during anthesis. The other species except

A. burgeoni showed a preference for the plateau region.

The determining factor is mainly the higher temperature

which allows the presence and maximal activity of

insects found on female inflorescences (Mariau et al.,

1991). Thus, the first day of anthesis as the second, the

numbers of insects were higher in these areas.

CONCLUSION

The number of insects collected on the plain

region is significantly different from that harvested the

lowland area. The number of insect has been higher in

male inflorescences in lowland areas than on the plain.

This number was higher on the plain than in the lowland

area. The numbers of insects are influenced by the

culture area. This factor is to be taken into account in the

implementation and the entomological monitoring of oil

palm plantations.

REFERENCES

Beaudoin-Ollivier L, Frerot B, Coffi A, Flori A. 2012.

Duperie olfactive et pollinisation chez le palmier.

Journée Filière Palmier à Huile – juillet Cirad, Paris

Chevalier A. 1910. Documentation sur le palmier à

huile, végétaux utiles de l’Afrique Tropicale- VII - Paris,

Corrado F. 1985. La conformation des régimes de

palmier à huile (Elaeis guineensis Jacq.) dans quelques

plantations de Colombie. Oléagineux, 40 (4):173-187.

Desmier De Chenon R. 1981. Entomophil pollination of

oil palm in West Africa. Preliminary research. In: The oil

palm in agriculture in the eighties. Incorporated Society

of Planters ed., Malaysia, Vol. I, 239-291.

Fataye A. 1984. Rôle des principaux insectes dans la

pollinisation des palmiers à huile en Côte d’Ivoire.

Rapport de stage de fin de première année agronomique,

ENSA, Abidjan - Station palmier à huile IRHO-CIRAD

de La Mé, CI, 26.

Hala N, Tuo Y, Akpesse AAM, Koua HK and Tano

Y. 2012. Entomofauna of Oil Palm Tree Inflorescences

at La Mé Experimental Station (Côte d’Ivoire).

American Journal of Experimental Agriculture 2(3): 306-

319.

Hussein MY, Lajis NH and Ali JH. 1990. Biological

and chemical factors associated with the successful

introduction of Elaeidobius kamerunicus faust, the oil

palm pollinator in Malaisia. Acta Horticulturae, 288: 81-

87.

Mariau D, Houssou M, Lecoustre R and Ndigui B.

1991. Insectes pollinisateurs du palmier à huile et taux de

nouaison en Afrique de l’ouest ; Oléagineux., Vol. 46

(2) : 43-51.

N’goran DF. 1982. Etude du trafic des insectes sur les

fleurs femelles des palmiers à huile ; importance des

populations sur les fleurs mâles. Rapport de stage de fin

de première année agronomique, ENSA, Abidjan-

Koua et al., 2013


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Koua et al., 2013

Station palmier à huile IRHO-CIRAD de La Mé, CI.13p.

Syed RA. 1979. Studies on oil palm pollination by

insects. Bull. Ent. Res, 69(2): 213-224.

Syed RA, Law IH and Corley RH. 1982. VInsect

pollination of oil palm: introducing, establishment and

pollinating efficiency of E. kamerunicus FRAUSTY in

Malaysia. Incorporated Society of Planters ed. Vol. 58,

547-560.

Traoré K and Mangara, A. 2009. Etude

Phyto-écologique des Adventices dans les

Agroécosystèmes élaeicoles de la Mé et de Dabou. Eur.

J. Sci. Res., 65, 519-533.

Tuo Y, Akpesse AAM, Hala N and Koua HK. 2011.

Impact of terrestrial spraying of thiocyclam hydrogen

oxalate on oil palm pollinating insects. Journal of

Agriculture and Biological Sciences Vol. 2(7) pp.208 -

213.

Jou

rn

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esearch

in

Biology

A chromosomal analysis of seven Cameroonian Acrididae species (Orthoptera:

Acridinae, Oedipodinae and Spathosterninae) based on published data

Keywords: Acrididae, Acridinae, Oedipodinae, Spathosterninae, karyotype, relationships.

ABSTRACT: So far, the karyotypes of seven Acrididae species from Cameroon have been reported. These species included: Acrida turrita, Chirista compta, Coryphosima stenoptera producta, Oxycatantops spissus (Acridinae), Paracinema luculenta, Morphacris fasciata (Oedipodinae) and Spathosternum pygmaeum (Spathosterninae). Karyotype and meiosis relationships among these species were analysed from published data. The species had a common karyotype made up of 23 acrocentric chromosomes (males), the sex mechanism in all seven species was XX♀-XO♂ and meiosis was normal and chiasmate. The chromosomes in the species occurred in three size groups of long, medium and short. The number of chromosomes per size group however varied among the species (A. turrita = 4LL:5MM:2SS; C. compta =4LL:4MM:3SS; C. stenoptera product=2LL:6MM:3SS; O. spissus =5LL:3MM:3SS; P. luculenta = 6LL:2MM:3SS; M. fasciata = 6LL:2MM:3SS; and S. pygmaeum = 2LL:7MM:2SS). The X chromosome was long in the Oedipodinae, medium in the Acridinae and short in the Spathosterninae. Total length of chromosomal material was in the series C. compta > O. spissus > P. luculenta > S. pygmaeum > A. turrita > M. fasciata > C.s. producta.

947-953 | JRB | 2013 | Vol 3 | No 4



www.jresearchbiology.com

Authors: Seino Richard Akwanjoh1,2

and Dongmo Tonleu Ingrid1.

Institution:

1. Laboratory of Applied Ecology (LABEA),

Department of Animal

Biology, Faculty of Science,

University of Dschang,

P.O. Box 353, Dschang,

Cameroon.

2. Department of Biological

Science, Faculty of Science,

University of Bamenda,

P.O. Box 39, Bamenda, Cameroon.


Seino Richard Akwanjoh

Email:

[email protected]


Dates: Received: 15 Apr 2013 Accepted: 02 May 2013 Published: 20 May 2013

Article Citation: Seino Richard Akwanjoh and Dongmo Tonleu Ingrid. A chromosomal analysis of seven Cameroonian Acrididae species (Orthoptera: Acridinae, Oedipodinae and Spathosterninae) based on published data. Journal of Research in Biology (2013) 3(4): 947-953


Original Research

Journal of Research in Biology

An International Scientific

Research Journal

INTRODUCTION

The use of Orthoptera material for karyotype

studies dates from the inception of cytogenetics. This is

simply because Orthoptera material presents large

chromosomes and few chromosomes per karyotype.

Chromosome size and number are of important

cytotaxonomic value (Turkoglu and Koca, 2002). The

Orthoptera are also well known for their karyotypic

uniformity in chromosome number and morphology

(Ashwathanarayana and Ashwath, 2006; Chadha and

Mehta, 2011a).

It has been severally shown that analysis of

karyotype differentiation between species yields better

understanding of the evolutionary interrelationships and

divergence (Chadha and Mehta, 2011a; Sandhu and

Chadha, 2012). A survey of investigations on karyotype

evolution in different groups of animals has revealed that

several karyotypes are dynamic and are subject to

change. Therefore, the stable karyotypes of the Acrididae

are subject to change.

The cytogenetic diversity of Cameroonian

acridid grasshoppers has not been investigated. During

this study, published data on karyotypic characters were

analysed to determine similarities and differences as well

as interrelationships among seven Cameroonian

Acrididae species.

MATERIALS AND METHODS

The cytogenetics of only seven Cameroonian

Acrididae species have so far been described. The

species include Acrida turrita, Chirista compta,

Coryphosima stenoptera producta, Oxycatantops spissus

(Sub-family Acridinae) Paracinema luculenta,

Morphacris fasciata (Sub-family Oedipodinae) and

Spathosternum pygmaeum (Sub-family Spathosterninae).

The species and the sources from which karyotypic

information on them was obtained for this analysis are

shown in Table 1.

To analyse these karyotypes for similarities and

differences, the karyotypes of the seven species were

also arranged together (Figure. 1) and the morphometric

characters for the seven species were arranged in a

tabular form (Table 2).

RESULTS

Information on chromosome number,

morphology, size, and length of X chromosome obtained

for the seven species is summarised in Table 2. A perusal

of Table 2 revealed that among the seven species studied

A. turrita, C. compta, C. stenoptera producta and

O. spissus belonged to the sub-family Acridinae,

P. luculenta and M. faciata belonged to the subfamily

Oedipodinae and S. pygmaeum belonged to the

subfamily Spathosterninae (Mestre and Chiffaud, 2009).

Table 2 also revealed that the seven species investigated

had a common a diploid chromosome number of 2n=23

and the sex determining mechanism was XO in males.

Figure 1 also revealed that the in the seven species

investigated was acrocentric in morphology. The

chromosomes in all seven species occurred in three size

groups of long, medium and short. The number of

chromosome pairs per size group varied between species


Seino and Dongmo, 2013

S/No Species Subfamily Source of data

1 Acrida turrita

Acridinae

Seino et al, 2008

2 Chirista compta Seino et al, 2010

3 Coryphosima stenoptera producta Seino et al, 2010

4 Oxycatantops spissus Seino et al, 2010

5 Paracinema luculenta Oedipodinae

Seino et al, 2012

6 Morphacris fasciata Seino et al, 2012

7 Spathosternum pygmaeum Spathosterninae Seino et al, 2012

Table 1: The species analysed, their subfamilies and references from which karyotypic

information was obtained

and subfamilies (Table 2; Figure. 2). The Oedipodinae

showed most similarity since both of them revealed

6 long, 2 medium and 3 short chromosomes (6LL: 2MM:

3SS) in their karyotypes. The lengths of the

X chromosome was in the series P. luculenta >

C. compta > O. spissus > C.s. producta > M; fasciata >

A. turrita > S. pygmaeum. However, the X chromosome

was medium in the Acridinae, long in the Oedipodinae

and short in the Spathosterninae species (Figure. 2). The

total length of chromosomal material was in the series

C. compta> O. spissus> P. luculenta> S. pygmaeum>

A. turrita> M. fasciata > C.s. producta.

DISCUSSION

Every species has a unique karyotype which

provides an identity to the species (Channaveerappa and

Ranganath, 1997). Acridid grasshoppers are known to be

characterised by a basic karyotype of 23 chromosomes in

males. Due to this great cytogenetic uniformity Acridids

are considered as an example of ‘karyotypic

conservation’ (Aswathanarayana and Aswath, 2006).

In the present study, seven Acridids have been

investigated which belong to three different sub-families

that include the Acridinae, Oedopodinae and

Spathosterninae. The results of this study revealed that

the seven Acrididae have a chromosome number of

23 and a sex determining mechanism which is XO/XX.

Similar observations have been made for several other

Acrididae species. With respect to chromosome number,

chromosome morphology and sex determining

mechanism, Bugrov et al., (1994); Bugrov (1995),

Bugrov et al., (1999) Bugrov and Sergeev (1997)

observed similar results for Podisma and

Eyprepocnemidinae (Acrididae) grasshoppers in Russia

and Central Asia. Camacho and Cabrero (1983) also

reported similar results for European species of Acrotylus

(Oedopodinae). Yao (2006) and Chadha and

Mehta (2011a) reported similar results for

Spathosternum pransiniferum (Spathosterninae)



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respectively from Asia and India. So the Acridid

grasshoppers of different regions are showing

cytogenetic uniformity regarding chromosome number,

morphology and sex determining mechanism. The results

of this study confirmed that the basic Acrididae

karyotype is 23 acrocentric chromosomes and a sex

determining mechanism of the XX/XO type.

Metacentric chromosomes through fusions were not

observed in the seven species here investigated even

though they have been reported in several other

Acrididae species (White, 1973; Sharma and Gautam,

2002; Mayya et al., 2004; Chadha and Mehta, 2011a).

Turkoglu and Koca (2002) reported the presence of

metacentric, submetacentric and acrocentric

chromosomes in the karyotypes of Oedipoda schochi and

Acrotylus insbricus (Oedopodinae) from Turkey. The

aberrant chromosomes were the result of centric fissions.

X - autosome fusion resulting in the Neo - XY sex

mechanism have been reported in some acridid

grasshoppers (White, 1973). Bidau and Marti (2000)



Figure. 1: Mitotic Metaphase chromosomes in the seven species investigated.

a) Acrida turrita, b) Chirista compta, c) Coryphosima stenoptera producta, d) Oxycatantops spissus,

e) Paracinema luculenta, f) Mophacris fasciata, g) Spathosternum pygmaeum. Chromosomes are tapered

towards one end and centromeres were deemed to be towards the tapered ends of the chromosomes.

a b c

d e f

g



0

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4

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8

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Chromosome pair

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0

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6

8

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P. luculenta

0

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8

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Chromosome pair

Rela

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so

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(RC

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M. fasciata

0

2

4

6

8

10

12

14

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Chromosome pair

Rela

tive C

hro

mo

so

me L

en

gth

(RC

L)

S. pygmaeum

Figure. 2: Idiograms of the seven species investigated

r eported Neo-XY in Dichroplus v ittatus

(Acrididae: Melanoplinae). This type of sex

determination mechanism was absent in the seven

species investigated in this study.

The X-chromosome during this investigation was

found to be medium in the four Acridinae. However,

Chadha and Mehta (2011a), investigating Indian

Acridinae observed that the X chromosome in A. turrita

was the longest chromosome in the karyotype. There is

therefore disagreement of this report with that of the

present investigation. Chadha and Mehta (2011b)

reported the X chromosome in Oedaleus abruptus

(oedipodinae) to be the largest element in the karyotype.

During the present study, the X-chromosome in

P. luculenta and M. fasciata (Oedipodinae) were among

the large chromosomes of the karyotypes. There is no

doubt that the X chromosomes of different species of the

Oedipodinae is one of the largest elements in the

karyotype. Though this chromosome was acrocentric in

the two Oedipodinae investigated here, Turkoglu and

Koca (2002) found the same chromosome in Oedipodia

schochi schochi and Acrotylus insbricus (Oedipodinae)

from Turkey to be Metacentric in morphology.

ACKNOWLEDGEMENTS

The authors are thankful to Dr Watcho Pierre

(Associate Professor in the Department of Animal

Biology, University of Dschang - Cameroon) for reading

the manuscript and helpful suggestions. We are also

grateful to Professor Mpoame Mbida (Head of the

Laboratory of Applied Ecology (LABEA), University of

Dschang for laboratory space.

REFERENCES

Aswathanarayana NV and Ashwath SK. 2006.

Structural polymorphism and C-banding pattern in a few

Acridid grasshoppers. Cytology, 71(3):223 – 228.

Bidau CJ and Marti DA. 2000. Meiosis and the Neo-

XY system of Dichroplus vittatus (Melanoplinae:

Acrididae): a comparison between sexes.

Bugrov AG, Warchalowska-Sliwa E, Maryanska-

Nadachowska A. 1994. Karyotype evolution and

chromosome C-banding patterns in some Podisma

grasshoppers (Orthoptera: Acrididae) Caryologia, 47:

183-191.

Bugrov AG. 1995. Interpopulation sex chromosome

polymorphism in the grasshopper Podisma sapprorensis

Shir. From Sakhalin and Kurile Island. Folia Biologica

(Krakow), 43: 51 - 53.

Bugrov AG and Sergeev MG. 1997. A new

grasshopper species of the genus Podisma, Bertold

(Orthoptera-Acrididae) from the Southern Island and its

karyotypic features. Acta Zoologica Cracoviensia, 40: 47

-52.

Bugrov AG, Warchalowska E, Vysotskaya L. 1999.

Karyotypic features of Eyprepocnemidinae grasshoppers

from Russia and Central Asia with reference to the B

chromosomes in Eyprepocnemis plorans (Charp.). Folia

Biologica (Krakow), 47 (3 -4).

Camacho JPM and Cabrero J. 1983. karyological

differences between two species of grasshopper genus

Acrotylus (Acrididae: Oedipodinae). Caryologia, 36(2):

121-127.

Chadha P and Mehta A. 2011a. Chromosome study in

few species of Acridids (Acrididae:Tryxalinae):

karyotype analysis and distribution of constitutive

heterochromatin. Journal of Entomology and

Nematology, 3 (1): 14 – 19.

Chadha P and Mehta A. 2011b. Chromosome

complement and C-banding patterns in 6 species of

grasshoppers. International Journal of Genetics and

Molecular Biology, 3(1): 25-30.





Channaveerappa H and Ranganath H. 1997.

Karyology of few species of South Indian Acridids. II.

Male germ line karyotypic instability in Gastrimargus. J.

Biosci. 22 (3): 367 -374.

Mayya S, Screepada KS, Hegde MJ. 2004. Non-

banded and C-banded karyotypes of ten species of short-

horned grasshoppers (Acrididae) from South India.

Cytologia, 69(2): 167 - 174.

Mestre J and Chiffaud J. 2009. Acridien du Cameroun

et de Republique centrafricaine (Orthoptera Caelifera).

Supplément au catalogue et atlas des acridiens d’Afrique

de l’ouest. Edition numérique ISBN 978-2-9523632-1-1.

Sandhu KS and Chadha P. 2012. Karyological studies

of four species of grasshoppers from Gurdaspur district

of Punjab, India. Nucleus, online first article, Vo 55(3):1

-4.

Seino RA. 1989. Cytogenetic characterization of

seven Acridomorphoid grasshoppers. MSc Dissertation,

University of Lagos, Nigeria.

Sharma T and Gautam DC. 2002. Karyotypic studies

of eleven species of grasshoppers from north-western

Himalayas. Nucleus, 45(1-2): 27-35.

Turkoglu S and Koca S. 2002. Karyotype C- and

G-band patterns and DNA content of Callimenus

(=Bradyporus) macrogaster macrogaster. J. Insect Sci. 2

(24): 1-4.

White MJD. 1973. Animal cytology and evolution.

Third edition. Cambridge University Press.

Yao S. 2006. The karyotypes and C-band of five Locusta

species of Acridoidea in Guizhou. Journal of Guizhou

Normal University (Natural Sciences), 2: 3 - 14.

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Biology

Impact of the residue of Deltamethrin and Endosulfan pesticides on biochemical toxicity

and some neurotransmitter contents in different brain areas of male Albino mice

Keywords: Deltamethrin, Endosulfan pesticide, Laboratory-bred strain Swiss albino male mice, neurotransmitter contents (NE, DA and GABA).

ABSTRACT:

Evaluating the action of the residues of pesticides on non-target organisms has been of interest to many researchers. The present study aimed to evaluate the pesticides deltamethrin and endosulfan on biochemical toxicity and some neurotransmitter contents in different brain areas of male albino mice. The results showed that the daily oral administration of deltamethrin and endosulfan caused a significant decrease in neurotransmitter contents (NE, DA and GABA) in most of the tested brain areas (cerebellum, striatum, cerebral cortex, hypothalamus, brain stem and hippocampus). On the other hand a gradual significant reduction, ALT, AST and ALP enzyme activities, while the glucose level and acid phosphatase increase were observed in serum of mice treated with deltamethrin and endosulfan for two weeks. Also, this study has a significant inhibition in the activities of enzymes in liver tissues of treated mice including glutathione reductase. Meanwhile, the activity of lipid peroxide, glycolytic (PK, PFK and GPI) and gluconeogenic enzyme activities (F-1, 6-D-Pase) were significantly increased in liver tissues of treated mice in response to treatment. Additionally, total protein and glycogen content showed a significant reduction in liver tissues of mice treated with deltamethrin and endosulfan for two weeks. It was concluded that the pollution of the aquatic environment by deltamethrin and endosulfan pesticides, would adversely affect the metabolism of the mice.

954-966 | JRB | 2013 | Vol 3 | No 4






Research Journal

Authors:

Somaya M. Ismail1,

Azza A. Said2,

Samira M. El-Sayad2.

Institution:

1. Zoology department,

Faculty of Science,

Cairo university.

2. Zoology department,

Faculty of Science, Fayoum university.


Somaya M. Ismail

Email:

[email protected]

Tel: +201118244212


Dates: Received: 12 Jan 2013 Accepted: 22 Feb 2013 Published: 24 May 2013

Article Citation: Somaya M. Ismail, Azza A. Said and Samira M. El-Sayad. Impact of the residue of Deltamethrin and Endosulfan pesticides on biochemical toxicity and some neurotransmitter contents in different brain areas of male Albino mice. Journal of Research in Biology (2013) 3(4): 954-966


Original Research

An International Scientific Research Journal

INTRODUCTION

Using pesticides is an important procedure for

enhancing agriculture yield. However, the great

consciousness, brought back upon their deleterious

effects on human, animal and environmental health,

leading to the shortage of their use by imposing various

rules (Ahmsd et al., 2010; Botella et al., 2004).

Among pesticides, Deltamethrin, which is a type

II pyrethroids, has a wide acceptability, and is used in

agriculture and forestry because of its high

activity against a broad spectrum of insect pests

(Villarini et al., 1998). The oral route constitutes the

main sources of general population exposure to this

pesticide which is ingested within food and water

(Barlow et al., 2001).

It has been reported that deltamethrin caused

an oxidative damage in liver and intestine of

Carassius auratus gibelio explained by an increase of

LPO level and an enhancement of antioxidative defence

parameters (Dinu et al., 2010). Oral absorption of

deltamethrin is rapid and is metabolized with microsomal

enzyme system in liver and with tissue esterase present

in intestinal wall and liver in addition to plasma

carboxylesterases (Usmani et al., 2006). According

to Simsek et al., (2008), Deltamethrin applied at different

concentrations of 25, 50, 100, 200, 400, 800 and

1600 mg /L, for 1,24, 48, 72 and 96 h increased lipid

peroxidation which is accompanied by a decrease of

reduced glutathione and catalase activity in digestive

gland and gill of fresh water mussel.

Endosulfan (6, 7, 8, 9, 10-hexachloro -1,5,5a,6,

9a-hexahydro-6,9-methano -2, 4, 3 benzodioxathiepine-3

-oxide) is a broad-spectrum organochlorine pesticide

(insecticide and acaricide) first registered for use in the

United States in 1954 to control agricultural insect and

mite pests on a variety of fruits, vegetables, rice, grains,

tea, coffee, cotton and also in animal farm and houses

(US EPA). Results from a global monitoring network for

persistent organic pollutants revealed that endosulfan is

abundant in the environment and its use is increasing

(Pozo et al., 2006; Harner et al., 2006). It reaches aquatic

systems through direct application, as well as spray drift

and runoff from agricultural areas (Broomhall, 2002;

Jergentz et al., 2004 and Rand et al., 2010).

It is known that exposure to pesticides during

development may interfere with the normal development

of neurotransmitter systems and cause their direct

damage (Richardson et al., 2006). The central nervous

system (CNS) during development is particularly

susceptible to the toxic effects of xenobiotics (Tilson,

2000). The mechanism by which these effects occur is

not known but currently it is assumed that the

monoaminergic neurotransmitters play a role during

development, defined as “morphogenetic” (Buznikov

et al., 1996; Levitt et al., 1997; Nicotra and Schatten,

1990).

Organophosphate pesticide represent one of the

world’s most commonly used agrochemical.

Consequently, many of its residues are frequently found

in the environment. The aim of this study was to

determine the effects of the pesticides, deltamethrin and

endosulfan on biochemical toxicity and some

neurotransmitter contents in different brain areas of male

albino mice.


Pesticides

Deltamethrin

Deltamethrin is a synthetic pyrethroid pesticide

[ (S) -acyan o-3-ph en oxyben zyl (1R,3R) -3 -(2, 2-

dibromovinyl)-2,2-dimethylcyclopropanecarboxylate]

with molecular formula (C22H19 Br2NO3). Solubility in

water is <0.1 mg/L at 25oC. Relative molecular mass of

the compound is 505.2 g/mol, and the melting point is

100oC (Figure 1).

Endosulfan

Endosulfan is an off-patent organochlorine

pesticide and acaricide that is being phased out globally

Ismail et al., 2013


[6, 7, 8, 9, 10, 10-Hexachloro-1, 5,5a, 6, 9, 9a-hexahydro

-6, 9-methano-2,4,3-benzodioxathiepine-3-oxide]. With

the molecular formula of (C9H6Cl6O3S). Solubility in

water is 0.33 mg/L. Relative molecular mass is found to

be 406.93 g mol−1,and the melting point is 70-100°C,

343-373 K, 158-212 °F (Figure 1)

Animals

Swiss albino male mice of 10 weeks old with an

average weight of 28.5±2.5 g obtained from the National

Research Centre, Cairo, Egypt were used. They were

maintained in a well ventilated animal house. They were

housed in large polypropylene cages with free access to

food and water ad labium during the course of

the experiment. Animals were housed in groups

(5 animals/ group) and maintained under standard

conditions of temperature (23°C to 25°C), a relative

humidity of 65% to 86% and in a schedule of 12 hours of

light and 12 hours of dark.

Animal treatment

The animals were divided into three groups (n=6)

of equal number, The control group (1) was orally and

daily administered with equivalent amount of the vehicle

(distilled water) for two weeks, the second group was

given drinking water with 1.28 mg/kg BW of

deltamethrin (Yousef et al., 2006) during two weeks of

oral and daily administration and the third group was

orally and daily administered with endosulfan

(1.5 mg /kg BW). At the ends of the experimental period

(2 weeks), the mice were sacrificed under diethyl ether

anesthesia at fasting state.

Effect of deltamethrin and endosulfan (pesticide) on

some neurotransmitter contents in different brain

areas of male albino mice

During the experiment six mice of each group

were decapitated each week till the end of the 2-week

duration times. The mice were killed by sudden

decapitation at the designed times. The brain was rapidly

and carefully excised and then dissected on dry ice glass

plate according to the method of Glowinski and Lversen

(1966) into the following regions; cerebellum, striatum

cerebral cortex, hypothalamus, brain stem and

hippocampus. Brain tissues were wiped dry with filter

paper, weighed, wrapped in plastic films and then in

aluminum foil and quickly frozen in dry ice. NE and DA

were extracted and estimated in the brain tissues

Ismail et al., 2013


Fig. 1. Chemical structure of the pesticides

Deltamethrin and Endosulfan

Fig. 2. Changes (%) of activities of glucose level (GL),

some enzymes (alanine aminotransferase (ALT),

aspartate aminotransferase (AST), Alkaline

phosphatase (ALP), acid phosphatase (ACP) in serum

of male mice,. Lipid peroxide (LP) glutathione

(GSH) , pyruvate kinase (PK), phosphofructokinase

(PFK), glucose phosphate isomerase (GPI), Fructose -

1, 6-diphosphatase (F-1, 6-ase) enzymes, Total protein

(TP), glycogen content in tissues of male mice liver

exposed to LC25 of Deltamethrin and Endosulfan

pesticides for 2 weeks.

according to the method of Chang (1964) modified by

Ciarlone (1978). GABA were extracted and estimated in

the brain tissues according to the method of Sutton and

Simmonds (1973). The fluorescence was measured in

Jenway 6200 fluorometer.

Effect of deltamethrin and endosulfan (pesticide) on

biochemical toxicity of male albino mice

Serum samples were obtained by the

centrifugation of blood of six rats of each group at

4000 rpm for 15 min at 4°C, and were then divided in to

Eppendorf tubes. Isolated sera from each group were

stored at -20°C until they were used for the analyses. For

preparation of tissue homogenates of mouse liver tissue

of six mice of each group, one gram of liver tissues of

mouse from each group was homogenized in 5 ml

distilled water at pH 7.5.A glass homogenizer was used

and the homogenate was centrifuged for 10 minutes at

3000 rpm, fresh supernatant was used.

The levels of serum alanine aminotransferase

(ALT), aspartate aminotransferase (AST) were measured

according to Reitman and Frankel (1957). Alkaline

phosphatase (ALP) was measured according to Belfield

and Goldberg (1971) and acid phosphatase (ACP) was

measured according to Wattiaux and De Duve (1956)

and sera glucose concentrations (GL) were determined

according to the glucose oxides method of Trinder

(1969). Total protein (TP) content was determined

according to Bradford (1976) Determination of tissues

glycogen was evaluated according to Nicholas et al.,

(1956). Lipid peroxide ( LP) was measured according to

Buege and Aust (1978). Glutathione (GSH) was

measured according to Moron et al., (1979) Pyruvate

kinase (PK) relative activity was measured

spectrophometrically by the method of Bucher and

pfleiderer (1975). phosphofructokinase (PFK) was

measured according to Zammit et al., (1978) Glucose

phosphate isomerase (GPI) was measured according to

King (1965). Fructose -1, 6-diphosphatase (F- 1, 6-ase)

was measured according to Sand et al., (1980). All

biochemical l parameters determined in this study were

determined spectrophotometrically, using reagent kits

purchased from BioMerieux Company, France. Kits

purchased from BioMerieux Company, France.

Statistical analysis

The results obtained in the present work are

represented as means ± standard deviation (SD), and

were analyzed using analysis of variance (ANOVA). The

significance of difference between means were

calculated using the Duncan Multiple Range Test (Steel

and Torrie, 1980).

RESULTS

Results in Table 1 showed that the daily oral

administration of deltamethrin and endosulfan resulted in

Ismail et al., 2013


Pesticides

Cerebellum mean ± S.E.

Striatum

mean ± S.E.

Cerebral cortex

mean ± S.E.

Hypothalamus mean ± S.E.

Brain stem mean ± S.E.

Hippocampus mean ± S.E.

Deltamethrin

C 122.7± 0.72 280.5±0.64 52.3±0.084 433 ± 4.2 310.2±0.45 222.1±0.6

T 82.3±1.2* 186.6±0.6* 36.5±0.21* 146.3±2.1** 254.5±1.4 160.2±0.62

% 32.79% 33.69% 30.21% 66.21% 17.69% -27.87%

Endosulfan

C 122.7± 0.72 280.5±0.64 52.3±0.084 433±4.2 310.2±0.45 222.1±0.6

T 48.2±2.42** 145.12±2.3** 21.4±0.73** 116.4±2.5*** 192.63± 1.5** 112.6±1.6**

% 60.72% 48.26% 59.1% 73.12% 38.11% 49.30%

Table (1): Effect of oral administration of Deltamethrin and Endosulfan on dopamine (DA) content in the

different brain areas of male albino rat.

- Statistical analyses were performed between control (C=6) and treated (T=6) animals by using paired t' test

% : Percentage of change from control *p< 0.05,**p< 0.01 & ***p< 0.001

a significant decrease in DA content in all brain area.

The maximal decrease (p<0.001) in DA content was

found in the hypothalamus of mice treated with

deltamethrin and endosulfan at the concentrations of

66.21% and 73.1%, respectively. Also, Table 2 showed

that the daily oral administration of deltamethrin and

endosulfan caused a significant (p<0.001) decrease in

GABA content in all the brain area, the maximal

decrease (p< 0.001) in GABA content was found in

brain stem of mice treated with deltamethrin and

endosulfan at the concentration of 72.52% and 80.52%,

respectively.

The results obtained from Table 3 showed that

the maximal decrease (p<0.001) in NE content was

found in the hypothalamus of mice treated with

deltamethrin and endosulfan at the concentrations of

54.1% and 62.89%, respectively.

The results in Table 4 showed that a clear

reduction (P<0.001) in liver enzyme activities in serum

of mice treated with deltamethrin and endosulfan as

compared to the control mice. On the other hand, the

glucose concentration and Acid phosphatase in serum of

treated mice showed a marked increase (P<0.001) in

comparison with the control group. Glycogen content in

tissues of treated mice showed a significant (p>0.001)

decrease in comparison with the control group. The

reduction rates were 36.32% and 58.24% for mice

treated with deltamethrin and endosulfan, respectively

(Table 7).

Ismail et al., 2013


Pesticide Cerebellum

mean ± S.E.

Striatum

mean ± S.E.

Cerebral cortex

mean ± S.E.

Hypothalamus

mean ± S.E.

Brain stem

mean ± S.E.

Hippocampus

mean ± S.E.

Deltamethrin

C 165.7±0.65 154.21±0.8 44.2±0.62 321.6±0.82 121.2±0.197 204.3±1.6

T 102.6±1.3** 92.6±0.428** 35.2±0.8 2925±0.43* 33.3±0.764** 98.8±0.577**

% -61.97% 39.82% 18.1% 9% 72.52% 51.64%

Endosulfan

C 165.7±0.65 154.21±0.8 44.2±0.62 321.6±0.82 121.2±0.197 204.3±1.6

T 60.6±1.2*** 72.4±0.87** 28.6±0.83*** 252±1.6 23.6±0.82* 88.4±1.6***

% 63.43% 53.1% 35.29% 21.64% 80.52% 56.73%

Table (2):Effect of oral administration of Deltamethrin and Endosulfan on gama-butyric acid (GABA) content

in the different brain areas of male albino rat.

- Statistical analyses were performed between control (C=6) and treated (T=6) animals by using paired t' test.

% : Percentage of change from control. *p< 0.05,**p< 0.01 & ***p< 0.001

Pesticide Cerebellum

mean ± S.E.

Striatum

mean ± S.E.

Cerebral cortex

mean ± S.E.

Hypothalamus

mean ± S.E.

Brain stem

mean ± S.E.

Hippocampus

mean ± S.E.

Deltamethrin

C 102.6±1.4 434.2±1.6 64.6±1.54 462.2±2.11 342±0.53 233.1±1.4

T 77.5±0.56** 344.23±1.4** 35.2±0.54* 212.2±052** 243.1±0.45** 106.2±0.62***

% 24.64% 20.72% 48.57% 54.1% 28.95% 45.44%

Endosulfan

C 102.6±1.4 434.2±1.6 64.6±1.54 462.2±2.11 342±0.53 233.1±1.4

T 48.2±0.62** 223.3±0.61** 22.3±1.4*** 210.8±1.1*** 168.5±1.4*** 86.5±0.83***

% 53% 48.57% 65.48% 99.45% 50.73% 62.89%

Table (3): Effect of oral administration of Deltamethrin and Endosulfan on norepinephrine (NE) content

in the different brain areas of male albino rat.

- Statistical analyses were performed between control (C=6) and treated (T=6) animals by using paired t' test.

% : Percentage of change from control. *p< 0.05,**p< 0.01 & ***p< 0.001

The present result in Table 5 indicated that a

significant increase in lipid peroxide accompanied with a

significant reduction in glutathione and total protein in

liver enzyme activities of mice treated with deltamethrin

and endosulfan as compared to the control mice.

The present results in tables (6, 7) demonstrate a

significant elevated level of glycolytic (PK, PFK and

GPI) and gluconeogenic enzyme activities (F-1,6-D-

Pase) in tissue of mice treated with deltamethrin and

endosulfan as compared to the control. The elevation

rates in the activities of PK, PFK, GPI and F-1-6, D-Pase

enzymes were 97.36%, 76.1%, 74.84% and 69.1%,

respectively for mice treated with endosulfan.

DISCUSSION

Many monoamine neurotransmitters, including

DA , NE and GABA are important in the regulation of

brain development prior to assuming their roles as

transmitters in the mature brain (Whitaker-Azmitia,

1992; Di Pino, 2004; Ansorge, 2008), thus any

circumstance that affects these neurotransmitters in the

developing brain can alter the final structure and function

of the brain. Developmental neurotoxicity involves

alterations in behavior, neurophysiology.

From the present results, it is clear that the daily

oral administration of deltamethrin and endosulfan

caused reducing side effect in some neurotransmitter

tissue in the brain and a significant decrease in

neurotransmitter contents (NE, DA and GABA) in most

of the tested brain areas. Cerebellum which is

responsible for the voluntary movement; pons and

medulla oblongata which is responsible of essential

reflexive acts; striatum which is a brain region

responsible for motor activity; cerebral cortex is

responsible for sensation including visual, auditory and

olfactory as well as motor coordination and association,

also is responsible for higher mental function such as

thinking, planning, reasoning, memory and

consciousness and hippocampus, this is the key area

Ismail et al., 2013


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Del

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concerned with learning (Ansorge, 2008). Brain stem is

responsible for integration of coordination of essential

reflexive acts such as swallowing, vomiting and

respiration (Bloom, 1983).

Our findings support the idea that deltamethrin

and endosulfan is neurotoxic in the developing brain.

The present result found that these pesticides induced a

decrease in DA levels in cerebellum, striatum, cerebral

cortex, hypothalamus, brain stem and hippocampus of

treated mice. The loss of hippocampus DA levels was

higher in treated mice. DA is an important component of

the neuroendocrine mechanism that regulates the

activation of male sexual behavior in mammalian species

(Castagna and Ball, 1997). Moreover, steroidogenesis in

the brain may play a critical role in mammalian brain

developmental of both sexes (Konkle and McCarthy,

2011). Steroids play a role in the development of

catecholamines systems (Leret, 2009; Muneoka et al.,

2010; Pappas et al., 2010).

It is known that DA is the major compound

involved in the control of the motor system. Bernardi and

Palermo-Neto, (1983) showed that locomotion and

rearing frequencies observed in an open field might be

used to detect drug-induced dopaminergic interference.

Locomotors activity as measured in the open field

appears to be associated with the dopaminergic system

(Chiavegatto et al., 1998). Also, in the present study, we

similarly found a loss of the NE and gamma-butyric acid

(GABA) content in the cerebellum, striatum, cerebral

cortex, hypothalamus, brain stem and hippocampus. The

loss of brain stem DA levels and the loss of hippocampus

GABA levels were higher in treated mice.

These effects may represent a large number of

actions involved in the development of synaptic

dysfunction in these neurotransmitter systems that

ultimately contribute to behavioral anomalies.

Nevertheless further behavioral testing is needed to

confirm this suggestion. Moreover, the present findings

might indicate that prenatal and postnatal exposure to

pesticide altered the program for developmental of DA,

NE, and GABA synaptic functions. Given that, the

dysfunction in serotonin and dopamine systems is

involved such as appetite, affective, locomotion,

learning, neurological and neuropsychiatric disorders

(Insel et al., 1990; Kaye, 2008), further testing of this

function is needed to confirm that alteration of these

neurotransmitter systems is the cause of some of these

dysfunctions. In general, our results support the

Ismail et al., 2013


Lipid peroxide(LP)

(ug/g tissue)

Glutathione(GR)

(ug/g tissue)

Total protein(TP)

(mg/ml)

% change % Change % change

Control 0.65 ±0.01 30.22 ±1.22 52.44 ±2.11

Deltamethrin 0.95 ±0.57 -46.15% 20.20 ±1.12 33.16% 41.11 ±1.15 21.61%

Endosulfan 1.22±0.06 -87.69% 15.16 ±0.85 49.83% 31.22 ±1.65 40.46%

Table 5: Effect of Deltamethrin and Endosulfan on lipid peroxide, glutathione and total protein in male mice liver.

Glycolytic enzmes (umole/mg protein/min.)

PK PFK GPI

% change % change % change

Control 4.16 ±0.26 7.44 ±1.16 77.34 ±2.43

Deltamethrin 7.18 ±1.44 -72.6% 10. 1 ±1.22 -35.75% 113.50 ±3.2 -46.81%

Endosulfan 8.23 ±1.64 -97.36% 13.1 ±1.23 -76.1% 135.22 ±6.4 -74.84%

Table 6: Effect of Deltamethrin and Endosulfan on some glycolytic enzymes in male mice liver.

Data represent mean values of five replicates. Within columns for dose, time and (dose x time), mean values followed by

different letters are statistically significantly different based on LSD at P = 0.05.

suggestion that at least some of the effects of these

disorders that are increasing in humans can be caused by

exposure to neurotoxin environmental contaminants

(Slikker W and Schwetz, 2003).

In conclusion, the results observed in this study

reinforce the idea of the use of neurochemical measures,

such as the DA, NE and GABA content and its

metabolites in brain regions as indicators of

neurotoxicity, including developmental neurotoxicity,

induced by chemical agents. Because of serotonergic

dysfunction is involved in appetite and affective

disorders, and the catecholamine DA and NE have been

most often linked to the behavioral pathology of a

number of neurological and psychiatric disorders, studies

of pesticide on DA, NE- and GABA. Related behaviors

in animal models will be needed to clarify the outcomes

of long-term alterations in noradrenergic, serotonergic

and dopaminergic systems identified here.

Concerning, ALT, AST and ALP enzyme

activities, gradual significant reduction was observed in

serum of mice treated with deltamethrin and endosulfan

for two week. The reduction observed in AST and ALT

attributed to the hepatocellular damage resulting from

chemical-toxicity, where the transaminases levels

showed an intimate relationship to cell necrosis and /or

increased cell membrane permeability which led to the

discharge of enzyme to blood stream. The decrease in

transaminase levels providing additional support for the

side effect of the deltamethrin and endosulfan on

mitochondria of the hepatic cells as it is the subcellular

localization of transaminases (El –Shazly et al., 2001).

This was attributed to the irritation of liver cells by

toxins or due to increase loss of intracellular enzyme by

diffusion through cell membrane. In the present study,

acid phosphatase show significant elevation in serum

of treated mice. Higher levels of acid phosphatase in

tissue was observed by El-Aasar et al., (1989) and

Abdel-Rahman et al., (1993), which was attributed to the

irritation of liver cells by toxins or metabolic products of

growing schistosomula of adult worms and eggs or due

to increase loss of intracellular enzyme by diffusion

through cell membrane which appear to act as a stimulus

to the synthesis of more enzyme.

Regarding the sources of energy for mice,

deltamethrin and endosulfan significantly decreased the

glycogen content in liver tissues of treated mice, while

the glucose level increased in the serum of treated mice.

This may be attributed to the activity of the pesticides

that impedes oxygen consumption of mice, thus inducing

anaerobic respiration. Under hypoxic conditions, animals

derive their energy from anaerobic breakdown of

glucose, which is available to the cells by increased

glycogenolysis (Vincent et al., 1995; Sambasiva, 1999).

Nakano and Tomlinson (1967) have suggested that

catecholamine levels rise under stressful environmental

conditions, enabling the increased utilization of glycogen

for energy production. To restore its energy

requirements, the mouse has to increase the rate of

glycolysis thus bringing about a reduction of the

glycogen content and increase glucose level in the blood

(Baskaran and Palanichamy, 1990; Vasanthi and

Baskaran, 1990).

Ismail et al., 2013


Glycogen( mg/g tissue ) Fructose-1,6-diphos-phatase

(umole/mg protein/min.)

% change % Change

Control 6.8 ±0.64 12.6±1.22

Deltamethrin 4.33±0.64 36.32% 15.4 ±1.11 22.22%

Endosulfan 2.84±1.02 58.24% 21.3 ±1.43 69.1%

Table 7: Effect of Deltamethrin and Endosulfan on Glycogen and some Gluconeogenic enzymes in male mice liver.

The data obtained in the present study showed

that lipid peroxide was elevated in the liver of mice

treated with deltamethrin and endosulfan. Lipid

peroxidation is known to require the participation of

highly reactive oxygen and other reactive metabolites in

the chain of biochemical reaction (Botros et al., 2007).

At the same time, liver GSH was drastically depleted in

the liver. Such depletion is critical, as shown by the

increased cytotoxicity of H2O2 in endothelial cells, as a

result of inhibition of glutathione reductase, which keeps

glutathione in its reduced state (El-Rigal et al., 2006).

In a good agreement with the present results

Mittelstaedt et al., (2004) suggested that nuclei and

mitochondria act as major targets of MG toxic action,

probably by increasing the generation of free radicals,

lipid peroxidation and DNA adducts formation.

Total protein content showed significant

reduction in liver tissue of mice treated with deltamethrin

and endosulfan for 2 weeks. This could be attributed to

cellular damage caused by toxin (Parasad et al., 1991).

The significant decrease in total protein content is mainly

due to increase in messenger RNA degradation which is

the possible cause for the hypoalbuminemia (Metwally

et al., 1990).The depletion of the protein fraction in the

liver tissue of mice in this experiment may have been

due to interference of active substance of pesticides in

protein metabolism by inhibiting protein synthesis (Volpi

et al., 1997).

The current investigation revealed significant

enhancement of PK, PFK and GPI the rate limiting

glycolytic enzymes, in liver tissue of treated mice

with two pesticides compared to control group

accompanied by a marked increase in the

gluconeogensis; F,1-6-diphosphtase. The increase in

gluconeogenic enzymes may be also responsible for the

production of glucose during treatment (Klover and

Mooney, 2004). Stimulation of PK in pesticide toxicity

ascertained the enhancement of glycolytic flux

previously reported by Ahmed and Gad (1995).

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Jou

rn

al of R

esearch

in

Biology

Prevalence and the effect of plant extracts on community associated

methicillin resistant Staphylococcus aureus in Owerri, Imo State, Nigeria

Keywords: Antibiotic resistance, Staphylococcus aureus, Methicillin, Plant extracts, Isolates.

ABSTRACT: The prevalence of Methicillin resistant Staphylococcus aureus (MRSA) among apparently healthy inhabitants of Eziobodo Community and Students of Federal University of Technology Owerri (FUTO), Imo State, Nigeria was studied. The work further ascertained the antibacterial activities of medicinal plants including Azadirachta indica, Pterocarpus mildbraedii, Garcinia kola, Phyllanthus amarus and Vernonia amygdalina against the MRSA isolates. A total of two hundred nasal swab specimens were randomly collected from the participants. The Kirby-Bauer technique was used to determine the susceptibility pattern of the isolates to Vancomycin (5µg), Ciprofloxacin (5µg), Ceftriaxone (30µg), Oxacillin (5µg), Methicillin (10µg) and Erythromycin (15µg). The antibacterial properties of the ethanolic plant extracts were determined using the agar well diffusion technique. A total of 181 (90.5%) and 141 (70.5%) of the nasal swab samples, yielded Staphylococcus species and Staphylococcus aureus respectively. The antibiotic sensitivity screening revealed that 38 (27%) of the S. aureus isolates were methicillin resistant. The MRSA isolates also exhibited the highest resistance to vancomycin and the least to ceftriaxone. Furthermore, the result showed that crude ethanolic extracts of all tested plant extracts except Pterocarpus mildbraedii exhibited antibacterial activities against the MRSA isolates. Phytochemical components such as Alkaloids, Tannins, Glycosides, Saponins, Flavonoids, Terpenoids, Phlobatannins, Steroids and Anthraquinones were detected in the plant materials in varying proportions. This study unveils a relatively high occurrence of MRSA among the study population which could be a risk factor for infection with MRSA. These plant extracts could also serve as potential sources of therapy for the treatment of MRSA infections.

967-976 | JRB | 2013 | Vol 3 | No 4






Research Journal

Authors:

Amadi ES1, Oguoma OI1,

Ibekwe VI1, Abanobi SE2,

Chikwendu CI1 and

Egbadon OE1.

Institution:

1. Department of

Microbiology, School of

Science, Federal University

of Technology, P.M.B. 1526,

Owerri Imo State, Nigeria.

2. Department of Biochemistry, School of

Science, Federal University

of Technology, P.M.B. 1526,

Owerri Imo State, Nigeria.


Chikwendu CI.

Email:


Dates: Received: 20 Mar 2013 Accepted: 09 May 2013 Published: 05 June 2013

Article Citation: Amadi ES, Oguoma OI, Ibekwe VI, Abanobi SE, Chikwendu CI and Egbadon OE. Prevalence and the effect of plant extracts on community associated methicillin resistant Staphylococcus aureus in Owerri, Imo State, Nigeria. Journal of Research in Biology (2013) 3(4): 967-976


Original Research

INTRODUCTION

Staphylococcus aureus is a coagulase positive,

gram positive cocci, which apart from being a normal

flora of the anterior nares, skin and large intestine, is also

capable of causing a wide range of diseases varying from

minor skin infections to life threatening septicemia,

pneumonia, endocarditis, deep-seated abscess among

others (Willey et al., 2008; Lowy 2003; Kuehnert et al.,

2006; Tenover and Gaynes, 2000; Holmes et al., 2005;

Nester et al., 2007). Penicillin and later methicillin were

very efficacious in the management of Staphylococcus

infections in the early 1960s. However, over the years,

most strains have acquired resistance to these drugs due

to acquisition of gene encoding the enzyme penicillinase.

In recent times, strains of S. aureus have emerged that

not only produce penicillinase, but also have Penicillin

binding proteins (PBPs) with low affinity for all β-lactam

drugs. These strains referred to as methicillin resistant

Staphylococcus aureus (MRSA) are resistant to

meth icil l in and other β -lactam drugs

(Nester et al., 2007; Willey et al., 2008). Nearly all

MRSA have additional genetic material known as mec A

gene not found in methicillin sensitive strains, which

encodes PBP 2a, a cell wall transpeptidase, having

reduced affinity for β -lactam antibiotics. The mec A

gene is found as a part of a mobile genetic element found

in MRSA strains known as Staphylococcal cassette

chromosome mec (SCC mec) (Jeshina and Surekha,

2009; Pinho et al., 2001).

In addition to β lactam drugs, MRSA isolates

have become resistant to a number of antimicrobial

agents such as, fluoroquinolones, aminoglycosides and

macrolides (Shittu et al., 2009). MRSA could be

categorized as either hospital acquired (HA-MRSA) or

community acquired (CA-MRSA), depending on the

source of acquisition. While the former occur in

individuals who are/have recently been in a hospital or

other healthcare facility, the latter are acquired by

persons not recently hospitalized. According to David

and Daum (2010), all infections occurring among

outpatient or among inpatients with an MRSA obtained

earlier than 48 hours after hospitalization could be

regarded as CA-MRSA. In addition, livestock associated

MRSA (LA-MRSA) have been reported to pose a

challenge particularly in countries with low level of

MRSA (Stefani et al., 2012). Morris et al., (2012)

reported the potential for pet animals to harbour MRSA

when residing with human MRSA patients.

The fact that MRSAs are becoming more

prevalent worldwide and also resistant to a wide range of

antibiotic groups, underlines the need for alternate

strategies to stem the immense public health challenge

posed by these organisms. Natural products from local

medicinal plants are increasingly being used in the

treatment of many hard to treat diseases and the search

for more potential compounds from plants has continued

(Lai et al., 2010; Newman and Cragg, 2007). According

to WHO, 65-80% of the world population rely on

traditional medication for their ailments (Gurinder and

Daljit, 2009). A number of works has highlighted the

efficacy of local indigenous plants against a wide range

of pathogens (Ugbogu et al., 2010; Lai et al., 2010;

Aliyu et al., 2008; Aliyu et al., 2011; Ajibade et al.,

2010). The present study was aimed at ascertaining the

occurrence of MRSA among apparently healthy

Eziobodo community inhabitants and FUTO students as

well as their susceptibility to different antibiotic groups.

It also determined the antibacterial effects of some local

plant extracts on the MRSA isolates.


Collection of nasal specimens

Two hundred (200) nasal specimens were

collected, 100 each from the anterior nares of apparently

healthy individuals of Eziobodo community (one of the

communities hosting FUTO) and students of Federal

University of Technology (FUTO), all in Owerri West

LGA, Imo State, Nigeria. They were aseptically

Amadi et al., 2013


collected using sterile swab sticks between August and

November 2010.

Cultivation and isolation of Staphylococcus aureus

The respective nasal specimens were cultivated

within one hour of collection in Mannitol salt agar and

nutrient agar using standard techniques to obtain discreet

colonies. The plates were incubated at 37oC for 24 hours.

The axenic cultures of the isolates were subsequently

identified using colony morphology, microscopy and

biochemical tests including catalase and coagulase tests

(Cheesbrough, 2002).

Antibiotic susceptibility test

The antibiotic susceptibility screening of the

S. aureus isolates was conducted using the Kirby-Bauer

disc diffusion method (Cheesbrough, 2002). Standard

inoculum, equivalent of 0.5 McFarland standards of the

isolates was evenly spread on Mueller Hinton agar

plates. Antibiotic discs including Vancomycin (5µg),

Ciprofloxacin (5µg), Ceftriaxone (30µg), Oxacillin

(5µg), Methicillin (10µg) and Erythromycin (15µg)

(Oxoid) were aseptically placed on the plates. The plates

were then incubated at 37oC for 24 hours and the

inhibition zones recorded in millimeters using a meter

rule.

Subsequently, all the isolates identified as

Methicillin resistant Staphylococcus aureus (MRSA)

were subjected to antibiotic screening test using the same

disc diffusion technique as above. The following

antibiotics were used; Vancomycin (5µg), Ciprofloxacin

(5µg), Ceftriaxone (30µg) and Erythromycin (15µg)

(Oxoid).

Collection of plant materials

The leaf and bark of Pterocarpus mildbraedii,

Azadirachta indica, leaves of Vernonia amygdalina, and

whole plant of Phyllanthus amarus were obtained from

the premises of FUTO. The seeds of Garcinia kola

however, were purchased from Ekeukwu Owerri market,

Imo State. The plant materials were subsequently

authenticated by a taxonomist.

Preparation of plant extracts

The leaves, barks and seeds of the plants were

washed and dried at room temperature and later

pulverized. 20gm of each plant powder was separately

mixed with 250ml of ethanol and the extraction was done

using the soxhlet extraction procedure.

Phytochemical screening

The phytochemical screening of each plant

extract was carried out to determine the presence or

absence of Alkaloids, Tannin, Saponins, Glycosides,

Anthraquinone, Steroids, Flavonoids, Terpenoids, and

phlobatannins (Harbone, 1973; Sofowora, 1993).

Susceptibility screening of MRSA isolate to plant

extracts

The antibacterial effects of each plant extract on

MRSA were determined using the agar well diffusion

technique (Perez et al., 1990). Standard inoculum,

equivalent of 0.5 McFarland standards of the isolates was

evenly spread on Mueller Hinton agar plates. Sterile cork

borer was used to make wells on the agar. The

reconstituted extracts (25mg/ml, 50mg/ml, 100mg/ml

and 200mg/ml) were respectively introduced into wells

and labeled accordingly. Following the incubation of the

plates at 37oC for 24 hours, the inhibition zone diameters

were recorded using meter rule.

Amadi et al., 2013


Figure 1: Antimicrobial resistance rates (%) of

MRSA isolates to different antibiotics

RESULTS AND DISCUSSION

Out of the total of 200 specimens collected, 181

isolates were identified as Staphylococcus species, while

141 isolates were identified as S. aureus, representing a

prevalence rate of 90.5% and 70.5% respectively

(Table 1). Also, the antibiotic resistance screening of the

isolates showed that 38 (27%) of the S. aureus isolates

were MRSA. The MRSA isolates exhibited their highest

sensitivity to Ceftriaxone and the least to Vancomycin

antibiotic (Table 2 and Figure 1).

The Phytochemical screening of the plant

extracts revealed the presence of Phytochemical

components such as alkaloids, saponins, flavonoids and

others in varying quantities (Table 3).

The antibacterial screening of the ethanolic

extracts of the medicinal plants used in this study

indicated that all except Pterocarpus mildbraedii,

exhibited inhibitory activity against MRSA isolate.

(Table 4).

The result of this study showed that 90.5% and

70.5% of the isolates from the anterior nares of Eziobodo

inhabitants and FUTO students were respectively

Staphylococcus species and Staphylococcus aureus. This

is consistent with the findings of Ugbogu et al., (2010)

and Chigbu and Ezeronye (2003), but higher than the

33.3% prevalence reported from Amassoma community

in Niger Delta, Nigeria (Onanuga and Temedie,

2011).This high occurrence in our present work is not

unexpected, since S. aureus is a normal microflora of the

human body, particularly the upper respiratory tract

(Willey et al., 2008; Cheesbrough 2002).

The Methicillin resistant Staphylococcus aureus

(MRSA) prevalence rate of 27% among apparently

healthy individuals as reported in the present work is

considerably low compared to a report by Ugbogu et al.,

(2010) who isolated 83.5% of MRSA from healthy

individuals in Abia State, South East Nigeria and

Onanuga et al., (2005) that recorded 69% isolation from

healthy women in Zaria, Nigeria. Similarly, our current

finding is also lower than the report of Olowe et al.,

(2007) and Onanuga and Temedie (2011), in which

47.8% and 47.6% MRSA were isolated in South West

and Niger Delta regions of Nigeria respectively. The

prevalence rate of 47.15% and 43% has also been

reported from Ibadan and Jos, Nigeria (Ghebremedhin

et al., 2009; Ekeh, 2003). However, the current result is

consistent with the report of Nwankwo et al., (2010) in

which 28.6% was recorded. The difference in the

prevalence of MRSA obtained in the present study and

those of previous works could be attributable to strain

variation in different geographical regions and locations

(Ikeagwu et al., 2008).

It is important to note that the recovery of MRSA

from apparently healthy community inhabitants in the

present study is very significant particularly at this time

when infiltrations of Community acquired MRSA

(CA-MRSA) to healthcare facilities has been reported in

some parts of the world (Stefani et al., 2012). According

to Creech et al., (2005), Farley et al., (2008), and

Hidron et al., (2005) enormous reservoirs of MRSA now

exist outside health care settings and this implies that the

current methods of MRSA control in health facilities are

not likely to succeed. In this regard, preventive measures

Amadi et al., 2013


Table 1: Prevalence (%) of Staphylococcus aureus and MRSA

isolates from Eziobodo and FUTO inhabitants

Target population No of samples Staphylococcus sp. Staphylococcus aureus MRSA

Eziobodo 100 95(95) 66(66) 20(30.3)

FUTO students 100 86(86) 75(75) 18(24)

Total 200 181(90.5) 141(70.5) 38(26.9)

to stop the possible transmission in the communities is a

viable approach ( Charlebois et al., 2004; Cooper et al.,

2004; David et al., 2008; Liu et al., 2008).

The antibiotic susceptibility test revealed that all

the S. aureus isolates exhibited the least resistance to

ceftriaxone antibiotic in the present study. This finding is

consistent with the report of Masood and Aslam (2010)

in which 96.1% susceptibility of S. aureus isolates to

ceftriaxone was highlighted. Ceftriaxone was apparently

recommended by these workers as a drug of choice for

infections caused by S. aureus, Escherichia coli,

Pseudomonas aeruginosa, Klebsiella pnuemoniae and

Salmonella typhi. On the other hand, the S. aureus

isolates were more resistant to Ciprofloxacin and

Erythromycin (Tables 2 and 3). The resistant rates are in

line with the reports of Shanhraz et al., (2012), and

Onanuga and Temedie (2011), but quite low compared to

the over 70% resistance recorded by Ojulong et al.,

(2009) in Kampala, Uganda. Azeez-Akande et al.,

(2008), however reported a susceptibility rate of 93.9%

of MRSA isolates to ciprofloxacin.

Furthermore, vancomycin has been described as

a reliable alternative for the treatment of MRSA

infections. Elhamzaoui et al., (2009) and Nwankwo and

Nasiru (2011) reported 100% sensitivity of S. aureus

isolates from a University hospital in Rabat Morocco and

a tertiary health institution in Kano, Nigeria, to

Vancomycin respectively. Nevertheless, this antibiotic,

vancomycin, which was initially a drug of choice in the

treatment of MRSA infections, is witnessing resistance

in recent times (Von-Eiff et al., 2001). In the present

work, over 50% of the MRSA isolates were resistant to

Vancomycin. This is worrisome because Vancomycin

has been described by various workers as very effective

Amadi et al., 2013


Table 2: Frequency (%) of antibiotic resistance S. aureus isolates from nasal

samples of Eziobodo and FUTO inhabitants

No (%) of resistant isolates

Eziobodo FUTO

Antibiotics No of

isolates

No of resistant

isolates

No of

isolates

No of resistant

isolates

Total no of

isolates

Total no of

resistant

isolates

Oxacillin 66 21(31.8) 75 20(26.7) 141 41(29.1)

Methicillin 66 18(27.3) 75 20(26.7) 141 38(27)

Ciprofloxacin 66 16(24.2) 75 10(13.3) 141 36(25.5)

Vancomycin 66 15(22.7) 75 7(9.3) 141 22(15.6)

Erythromycin 66 10(15.1) 75 9(12) 141 19(13.5)

Ceftriaxone 66 3(4.5) 75 0(0) 141 3(2.1)

Table 3: Phytochemical components of plant extracts

Phytochemical components Plant

extracts Alkaloids Tannins Glycosides Saponinss Flavonoids Phlobatannins Steroids Anthraquinones Terpenoids

AIL + + - + + - + + -

AIB - + + + + + - + +

PML + + + + + - - - -

PMB - + + - - + - + +

PA - + - - - - + + -

VA + + + + + + -

GA - + + + - + - + +

Key: AIL – Azadirachta indica Leaf, AIB – Azadirachta indica Bark, PA – Phyllanthus amarus, PML – Pterocarpus mildbraedii Leaf, PMB – Pterocarpus mildbraedii Bark, VA – Vernonia amygdalina, GK – Garcinia kola.

against MRSA and in fact a drug of choice in the

treatment of multidrug resistant S. aureus infections

(Ojulong et al., 2009; Elhamzaoui et al., 2009). The

vancomycin resistance rate as recorded in the current

study is however contrary to the report of Onanuga and

Temedie (2011) in Niger Delta Nigeria and

Shanhraz et al., (2012) in which over 70% susceptibility

was recorded. The present finding thus suggests that

vancomycin may be inefficient in the treatment of

infections caused by MRSA in the near future among our

target population. The present study therefore

recommends ceftriaxone as a drug of choice for the

treatment of MRSA infections in our study area.

The increasing resistance of MRSA to β-lactam

and other broad spectrum antibiotics has stimulated

recent investigations on plant parts for naturally

occurring active compounds as alternatives to treatment

of MRSA caused infections. The phytochemical

screening of the plant extracts used in this study revealed

the presence of alkaloids, Tannins, saponins, flavonoids,

terpenoids, anthraquinones, glycosides and steroids

(Table 4). The antibacterial screening of the plant

extracts showed that all the plant materials used except

Pterocarpus mildbraedii exhibited inhibitory activity

against MRSA. This effect could be attributed to the

concomitant effect of the active compounds contained by

these plants on MRSA. However, none of the extracts

were active against the MRSA isolates at the lowest

concentration of 25mg/ml (Table 4).

The inhibitory effect of Garcinia kola extract on

MRSA as observed in the present study is in agreement

with the work of Ugbogu et al., (2010) and

Adeleke et al., (2006), in which Garcinia kola extracts

exhibited antibacterial activities against MRSA isolates

in Nigeria. Also, Taiwo et al., (1999) reported that

Garcinia kola exhibited strong activity against MRSA.

Similarly, the inhibitory properties of Azadirachta indica

and Vernonia amygdalina against MRSA as recorded in

the present work is consistent with the reports of

Skariyachan et al., (2011), Aliyu et al., (2011)

and Aliyu et al., (2008). Furthermore, that

Phyllanthus amarus extract had antibacterial activity

against MRSA is in line with the findings of

Aliyu et al., (2008). Ajibade et al., (2010) also

highlighted the antimicrobial activity of Phyllanthus

species against MRSA. Undoubtedly, the findings of this

study support the local use of these plant materials in the

treatment of most hard to treat infections.

In conclusion, the recovery of CA-MRSA from

the external nare of apparently healthy individuals in this

study underscores the significance of the nasal region as

a reservoir of S. aureus, and by implication MRSA. In

fact, MRSA colonization of the nares is believed to be a

risk factor for a clinically apparent infection with MRSA

(Croft et al., 2009; Huang et al., 2006; Lu et al., 2007;

Muder et al., 1991). It is therefore very imperative that

strategies should be designed to halt the further spread of

MRSA in communities and most especially to

immunodeficient individuals. According to

Stefani et al., (2012), CA-MRSA clones spreading in the

community could also infiltrate healthcare facility in

many parts of the world. This certainly would exacerbate

the challenges already posed by MRSA. Interestingly

however, the therapeutic activities of the plant materials

Amadi et al., 2013


Table 4: Inhibitory activities of plant extracts

against MRSA isolates

Mean zone of inhibition (mm)/

Concentration of plant extracts (mg/ml)

Plant

extract 25 50 100 200

AIL - - - 9

AIB - 7 8 12

PML - - - -

PMB - - - -

PA - - - 8

VA - - 9 10

GK - - - 11

Key: AIL – Azadirachta indica Leaf, AIB – Azadirachta indica Bark, PA – Phyllanthus amarus, PML – Pterocarpus mildbraedii Leaf, PMB – Pterocarpus mildbraedii Bark, VA – Vernonia amygdalina, GK – Garcinia kola.

used in this study could hold a great promise as a

potential precursor in the development of therapies for

the management of MRSA infections, if properly

harnessed.

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Article Citation: Veeramuthu Anbalagan, Michael Gabriel Paulraj and Savarimuthu Ignacimuthu. Odonata diversity (Insecta: Arthropoda) in rice and vegetable fields in a north-eastern district of Tamil Nadu, India. Journal of Research in Biology (2013) 3(4): 977-983

Jou

rn

al of R

esearch

in

Biology

Odonata diversity (Insecta: Arthropoda) in rice and vegetable fields in a

north-eastern district of Tamil Nadu, India

Keywords: Dragonflies, Damselflies, Libellulidae, Pantala flavescens

ABSTRACT: Odonata diversity in vegetable fields (brinjal and okra) and rice fields was studied from January 2005 to December 2008 in Tiruvallur district of Tamil Nadu. Totally 23 species of Anisoptera (dragonflies) and 12 species of Zygoptera (damselflies) were recorded and all these species were grouped into eight families. In vegetable fields 31 species of dragonflies and damselflies were recorded under 22 genera. In rice fields the species richness (21 species) and total genera (16) were less than vegetable fields during the entire study period. Libellulidae was the large family in both vegetable and rice fields which comprised maximum number of species. Pantala flavescens (Fabricius), a migratory species, was the most dominant in numbers throughout the year. Diversity indices clearly showed that odonata diversity was higher in vegetable fields than in rice fields.

977-983 | JRB | 2013 | Vol 3 | No 4



www.jresearchbiology.com Journal of Research in Biology

An International

Scientific Research Journal

Authors:

Veeramuthu Anbalagan,

Michael Gabriel Paulraj

and Savarimuthu

Ignacimuthu*

Institution:

Entomology Research

Institute, Loyola College,

Chennai-34.


Savarimuthu Ignacimuthu

Email Id:


Dates: Received: 06 Apr 2013 Accepted: 23 May 2013 Published: 05 June 2013


Original Research

INTRODUCTION

Dragonflies and damselflies in the order Odonata

are important group of insects in agroecosystems, forest

ecosystems and aquatic ecosystems. They are potential

biocontrol agents of agricultural, horticultural and forest

pests. Many studies have shown that the larval stages of

Odonata are important biological control agents of

mosquito larvae (Mandal et al., 2008; Spencer et al.,

1999). According to Corbet (1999), dragonflies and

damselflies are excellent ecological indicators. Around

6,000 species and subspecies of Odonata have been

described under 630 genera in 28 families throughout the

world (Tsuda, 1991). In India, 499 species, 139 genera

and 17 families of dragonflies and damselflies have been

documented (Prasad and Varshney, 1995; Sharma,

2010). Odonata diversity has been extensively studied in

different forest areas. Emiliyamma (2005) has recorded

31 species of dragonflies and damselflies from southern

Western Ghats in the Kottayam district of Kerala. Very

few investigators have studied the Odonata diversity in

agricultural fields (Gunathilagaraj et al., 1999;

Kandibane et al., 2005). A knowledge on Odonata

diversity in different agro ecosystems is very essential to

understand the influence of crop type on species

richness, abundance and evenness of dragonflies and

damselflies. Hence the present work was undertaken to

assess the Odonata diversity in two different agricultural

fields, i.e. rice and vegetable fields in Tiruvallur district

of Tamil Nadu.

MATERIAL AND METHODS

Study site:

Dragonflies and damselflies were sampled from

vegetable fields, viz. brinjal and okra in Kolappancheri

village and rice fields in Vayalanallur village of

Tiruvallur district. The geocordination of Tiruvallur

district is 12° 15 and 13° 5`N Latitude and 99° 15` and

80° 20` E Longitude.

Sampling of Odonates:

In each village, dragonflies and damselflies were

sampled in three different locations by quadrate method.

Quadrates of 25 m x 10 m size were laid down with

threads inside rice, brinjal and okra fields separately.

Totally three quadrates were put in each rice and

vegetable fields. Perched dragonflies and damselflies

found inside the quadrates were collected by sweeping

net (25 cm in diameter) during day times (between 10.00

AM to 15.00 PM). Flying Odonates inside quadrate area

were also caught with sweeping net. Sampling was done

twice in a month from January 2005 to December 2008.

Specimens from replications were pooled together.

Identification:

The specimens were identified using

identification keys provided by Fraser (1933, 1934 and

1936) and Subramanian (2009). After identification and

counting the total number of specimens, few specimens

from each taxa were retained and others were left behind

alive in the field. Specimens which were not identified in

the field were brought to the laboratory for identification.

The identified specimens were deposited at the

Entomology Research Institute, Loyola College,

Chennai.

Meteorological Data:

Data on atmospheric temperature, relative

humidity, mean total rainfall and total number of rainy

days from 2005 to 2008 were obtained from Regional

Meteorological Centre, Chennai.

Diversity indices:

Total number of dragonflies and damselflies

collected during the study period was recorded. Total

abundance, Simpson‟s index of diversity (1-D), Shannon

-Wiener Diversity Index (H), Shannon entropy, species

richness and species evenness were calculated by using

the software „Past.exe‟ (ver. 2.14). Jaccard‟s similarity

index was calculated to find out the similarity in Odonata

diversity between vegetable and rice fields.

Anbalagan et al., 2013


The formulae for the diversity indices are as follows:

Simpson‟s index (D) = Σ ni (ni-1) / N (N-1)

i = 1

Where ni = number of individual for each species

N = total number of individuals

Shannon index of general diversity ( )

log

Where ni = number of individual for each species

N = total number of individuals

Evenness (e)

Where = Shannon index

S = number of species

The similarity in odonata diversity between

vegetable fields and rice fields was assessed by using the

formula of Jaccard‟s similarity index as follows:

Jaccard‟s Index = A/ (A+B+C)

Where A= total number of species present in both

communities

B= the number of species present in community 1 but not 2

C= the number of species present in community 2 but not 1

RESULTS

Totally 35 species of dragonflies and damselflies

were recorded collectively from vegetable and rice fields

in Tiruvallur district from January 2005 to December

2008 (Figure1). The species composition, richness,

evenness and other diversity indices showed variations

between vegetable and rice fields.

Species composition and diversity in vegetable fields

Three families viz., Aeshnidae, Gomphidae and

Libellulidae were recorded under Anisoptera

(dragonflies) and five families viz., Calopterygidae,

Coenagrionidae, Euphaeidae, Lestidae and

Platycnemididae were recorded under Zygoptera

(damselflies) (Table 1). Totally 31 species of dragonflies

and damselflies were recorded under 22 genera, of which

15 genera and 22 species were dragonflies and 7 genera

and 9 species were damselflies. Libellulidae was found

to be the largest family, which has the highest number of

species (18 species) throughout the study period. Species

richness was 31 throughout the study. Total abundance

was maximum (4167) in 2008. Maximum evenness of

0.899 was recorded in vegetable fields in 2007 and this

was correlated with the maximum Shannon-Wiener

diversity index of 3.328 during the same study year

(Table 2). The similarity index (Jaccard‟s similarity

index) was calculated as 0.660 for each study

year (Table 2).

Species composition and diversity in rice fields

Five different families namely Aeshnidae,

Gomphidae, Libellulidae, Coenagrionidae and Lestidae

were recorded in rice field. All the species collected from

rice fields were grouped under 16 genera (12 Anisoptera

and 4 Zygoptera). Total number of species recorded in

rice field was 21 (15 Anisoptera and 6 Zygoptera).

Maximum total abundance (1703) was recorded in 2008.

Maximum Shannon-Wiener diversity index (2.871) and

H

H N

ni

N

ni

S

H

log

H



Figure 1. Total number of genera and species

collected under different families of Odonata

collectively from vegetable and rice fields

e =

= -

evenness (0.8409) in rice fields were recorded during

2007. Odonata diversity in rice fields was lower than

vegetable fields. The similarity index (Jaccard‟s

similarity index) was calculated as 0.660 for each study

year.



Table 1. Taxonomic composition and total number of individuals collected under different species of Odonata

from North-Eastern Tamilnadu during 2005-2008

Sl.No. Species Number of individuals collected

Vegetable fields Rice fields

2005 2006 2007 2008 2005 2006 2007 2008

Anisoptera

Family: Aeshnidae

1 Anax guttatus (Burmeister) 0 0 0 0 9 11 7 6

2 Anax immaculifrons (Rambur) 25 52 67 72 12 6 14 11

Family: Gomphidae

3 Heliogomphus selysi (Fraser) 179 158 124 186 38 88 76 54

4 Ictinogomphus distinctus (Rambur) 128 94 108 134 0 0 0 0

5 Ictinogomphus rapax (Rambur) 112 75 82 92 29 42 37 32 Family: Libellulidae

6 Brachythemis chalybea (Brauer) 128 142 108 129 0 0 0 0

7 Brachythemis contaminata (Fabricius) 106 85 122 148 78 55 86 73

8 Bradinopyga geminata (Rambur) 27 35 42 33 0 0 0 0

9 Crocothemis servilia (Drury) 220 145 189 238 36 42 46 58

10 Diplocodes trivialis (Rambur) 175 205 218 232 125 163 158 182

11 Neurothemis tullia (Drury) 98 112 147 121 58 82 117 93

12 Orthetrum glaucum (Brauer) 116 105 98 165 78 67 63 85

13 Orthetrum sabina (Drury) 125 145 102 148 51 25 48 60

14 Orthetrum testaceum (Burmeister) 114 108 122 148 0 0 0 0

15 Pantala falvescens (Fabricius) 480 306 318 372 185 211 203 197 16 Rhyothemis variegata (Linn.) 219 184 225 236 89 58 62 71

17 Sympetrum vulgatum flavum (Bartenef) 90 109 128 114 0 0 0 0

18 Tholymis tillarga (Fabricius) 30 18 45 55 0 0 0 0

19 Tramea basilaris (Palisot de Beauvois) 170 165 138 145 31 27 41 29

20 Tramea limbata (Desjardins) 150 120 111 165 0 0 0 0

21 Trithemis aurora (Burmeister) 112 78 65 92 26 34 31 40

22 Trithemis festiva (Rambur) 107 118 128 108 0 0 0 0

23 Trithemis pallidinervis (Kirby) 72 110 95 108 52 45 69 42

Zygoptera

Family:Calopterygidae

24 Caliphaea sp 27 35 42 33 0 0 0 0

Family: Coenagrionidae 25 Agriocnemis femina femina (Brauer) 0 0 0 0 110 74 101 122

26 Agriocnemis pygmaea (Rambur) 0 0 0 0 92 68 81 105

27 Ceriagrion coromandelianum(Fabricius) 190 78 158 212 140 61 125 156

28 Ischnura aurora (Brauer) 70 78 65 128 43 59 78 88

29 Ischnura delicata (Hagen) 0 0 0 0 121 82 88 106

30 Ischnura inarmata (Calvert) 71 65 108 108 0 0 0 0

31 Ischnura senegalensis (Rambur) 92 84 149 132 0 0 0 0

Family: Euphaeidae

32 Euphaea sp 30 45 68 73 0 0 0 0

Family: Lestidae

33 Lestes viridulus (Rambur) 69 80 118 120 61 68 91 93 Family: Platycnemididae

34 Copera marginipes (Rambur) 70 78 92 55 0 0 0 0

35 Platycnemis sp 54 65 88 65 0 0 0 0

Total 3656 3277 3670 4167 1464 1368 1622 1703

Meteorological data:

The meteorological data is given in the table 3.

Mean maximum and minimum yearly temperatures were

low in 2007 compared to other three years. Also the

relative humidity was high in the year 2007.

DISCUSSION

Present study reports the odonata diversity in

vegetable and rice agroecosystems. Odonates are

predaceous insects and they are important biocontrol

agents of agricultural pests and vector mosquitoes.

In the present study families Libellulidae in Anisoptera

and Coenagrionidae in Zygoptera were found to be more

diverse families in terms of the number of species.

Similar findings were already reported by some

investigators. Ghahari et al.,(2009) have reported that

families Libellulidae and Coenagrionidae were dominant

in terms of number of species in rice fields in Iran.

Kumar and Mitra (1998) reported that family

Libellulidae was represented by high number of species

(18 species) among a total collection of 42 species from

Sahstradhara, Dehra Dun. Similar reports were

published by Prasad (2002), Kumar (2002) and

Vashishth et al., (2002).

Several investigators have reported that

dragonflies and damselflies are very common in rice

agroecosystems. Kandibane et al., (2003) have recorded

12 species of Odonata under three families in rice fields

of Madura. In the present work the number of species

and families recorded in rice fields were high compared

to the results of Kandibane et al., (2003, 2005). Among

the various species, Pantala flavescens, a migratory

species, was abundant in numbers. The damselfly

Ceriagrion coromandelianum was abundant in both

vegetable fields and rice fields. In rice field,

Agriocnemis femina femina was also found to be

abundant. Kandibane et al., (2003) have reported that

A. femina was more abundant in rice ecosystems.

The diversity and distribution of insects may be

influenced by type of ecosystems and climate. In the

present study the species richness, total abundance and

diversity of Odonata were high in vegetable ecosystems

compared to rice ecosystem. Higher evenness values

were recorded in vegetable fields than rice fields during

2006, 2007 and 2008. When the richness and the

evenness of a community increases, the Shannon index

also increases. In the present study the Shannon index

was higher in vegetable fields than rice fields. This was



Table 3. Mateorological data for the years from 2005 to 2008

Year Mean Maximum

temperature (oC)

Mean minimum

temperature (oC)

Mean Relative

Humidity (%) @

0830/1730 hrs IST

Mean Total

Rainfall (mm)

Total number of

rainy days (2.5mm

and above)

2005 33.6 24.8 66.8-75.6 199.8 73

2006 33.8 24.6 64.3-75.9 123.9 67

2007 33.3 24.5 67.1-75.7 106.9 68

2008 33.7 24.8 64.3-75.3 150.2 63

Table 2. Diversity indices for Odonata in vegetable and rice fields from 2005 to 2008

Sl.

No. Diversity Indices

2005 2006 2007 2008

Rice Fields Vegetable

Fields Rice

Fields Vegetable

Fields Rice

Fields Vegetable

Fields Rice

Fields Vegetable

Fields

1 Species richness (S) 21 31 21 31 21 31 21 31

2 Total no. of individuals

1464 3656 1368 3277 1622 3670 1703 4167

3 Shannon-Wiener Diversity Index (H)

2.84 3.221 2.828 3.3 2.871 3.328 2.847 3.308

4 Simpson 1-D 0.9326 0.9508 0.9288 0.9584 0.9358 0.9601 0.934 0.9591

5 Evenness 0.815 0.8082 0.8056 0.8749 0.8409 0.8991 0.8208 0.8817

6 Jaccard Similarity Index

0.660 0.660 0.660 0.660

due to the higher species richness and evenness in

vegetable fields. The dominance of species was found to

be lower in vegetable crops compared to rice fields.

Hence the Simpson„s index of diversity (1-D) was higher

in vegetable crops and it clearly explained that species

distribution in vegetable crops was equal.

Besides the type of crop, the climatic factors

such as rainfall, atmospheric temperature and humidity

also affect the insect diversity. The average annual

temperature was the lowest in the year 2007. This lowest

average temperature in 2007 coincided with the

maximum insect diversity in both rice and vegetable

crops. Brinjal and okra plants grow taller with branches

and provide suitable microclimate and resting place for

perching adult Odonata. Vegetable fields also harbour

variety of small insects, which are the main prey of

Odonates. Latif et al., (2009) have reported 20 species

of pest insects and 10 families of predaceous insects in

brinjal field. Hence the presence of variety of prey

insects might be the reason for higher odonata diversity

in vegetable fields.

CONCLUSION

It is concluded that dragonfly and damselfly

diversity was influenced by type of crop because

vegetable ecosystem supported more taxa of Odonates

than rice field.

ACKNOWLEDGMENTS

The authors thank Entomology Research Institute

for financial support

REFERENCES

Corbet PS. 1999. Dragonflies: Behavior and Ecology,

Cornell University Press, Ithaca, New York, 829.

Emiliyamma KG. 2005. “On the odonata (Insects)

fauna of Kottayam District, Kerala, India”. Zoos Print J.,

20(12): 2108-2110.

Fraser FC. 1933. The Fauna of British-India Including

Ceylon and Burma, Odonata.vol.1, Taylor and Francis

Ltd., London.


Ceylon and Burma, Odonata. vol. II. Taylor and Francis

Ltd., London.


Ceylon and Burma, Odonata. vol. III. Taylor and Francis

Ltd., London.

Ghahari H, Tabari M, Sakenin H, Ostovan H and

Imani S. 2009. “Odonata (Insecta) from Northern Iran,

with comments on their presence in rice fields,” Mun.

Ent. Zool, 4(1): 148-154.

Gunathilagaraj K, Soundarajan RP, Chitra N and

Swamiappan M. 1999. “Odonata in the rice fields of

Coimbatore”, Zoo`s Print J., 14(6): 43-44.

Kandibane M, Mahadevan NR and Gunathilagaraj

K. 2003. “Odonata of irrigated rice ecosystem of

Madurai, Tamil Nadu,” Zoo’s Print J., 18: 1155-1156.

Kandibane M, Raguraman S and Ganapathy N.

2005. “Relative abundance and diversity of Odonata in

an irrigated rice field of Madurai, Tamilnadu,” Zoo’s

Print J., 20(11): 2051-2052.

Kumar A. 2002. “Odonata Diversity in Jharkhand State

with Special Reference to Niche Specialization in their

Larva Forms,” In: Current Trends in Odonatology,

Kumar, A., (Ed.). Daya Publishing House, New Delhi,

297-314.

Kumar A and Mitra A. 1998. “Odonata diversity at

Sahastredhara (Sulphur springs), Dehra Dun, India,” with

notes on their habitat ecology. Fraseria, 5(1/2): 37-45.

Latif MA, Rahman MM, Islam MR and Nuruddin

MM. 2009. “Survey of arthropod biodiversity in the

brinjal field,” J. Entomol., 6(1): 28-34.




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Mandal SK, Ghosh A, Bhattacharjee I and Chandra

G. 2008.“Biocontrol efficiency of odonate nymphs

against larvae of the mosquito,” Culex

quinquefasciatus Say, 1823. Acta Tropica,106(2):109-

114.

Prasad M. 2002. “Odonata Diversity in Western

Himalaya, India,” In: Current Trends in Odonatology, A.

Kumar, (Ed.). Daya Publishing House, Delhi, 221-254.

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odonata of india including data on larval studies,”

Oriental Insects, 29(1): 385-428.

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Sharma G. 2010. “Studies on odonata and lepidoptera

(Insecta: Arthropoda) fauna of Mount abu, Rajasthan,

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Subramanian KA. 2009. Dragonflies of India: A Field

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Jou

rn

al of R

esearch

in

Biology

Hepatic enzyme markers and proteins in serum and some selected tissues

in Clarias gariepinus from swamp around Kokori-Erhoike oil field, Nigeria

Keywords: Fish, Kokori-Erhoike, Clarias gariepinus, Albumin, Alkaline phosphatase, Haemoglobin.

ABSTRACT: This study determines changes in some biochemical parameters in serum and tissues of Clarias gariepinus obtained from fish natural habitat in the oil exploration environs of Kokori-Erhoike in Delta State, Nigeria. Sampling sites include Ethiope River (Eku axis, reference Site A); Erhoike swamp (Site B) and Erhoike fish pond (Site C). However, Sites B and C are located in the oil exploration region of Erhoike. Clarias gariepinus (n=8) were collected from each site and used for the study. Levels of total proteins, albumin, haemoglobin as well as the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase were determined in serum, gill, liver, brain and muscle tissues. Results showed that total protein concentrations were comparable (p>0.05) in serum. Albumin concentrations of fish from Site B and C were lower (p<0.05) as compared with that of site A in gill, muscle and brain tissues. Haemoglobin concentration was significantly lower (p<0.05) in fish from Site A as compared with that of Sites B and C. Results also indicated that total protein and albumin concentrations were significantly (p<0.05) higher in gill, muscle and brain tissues of Clarias gariepinus from Site A as compared with fish tissues from Sites B and C. Hepatic enzymes (ALT and AST) and ALP activities were elevated (p<0.05) in serum, gill, brain and liver of fish from Sites B and C as compared with that of Site A. The observed biochemical changes in fish from Sites B and C could have resulted from contaminants arising from the oil exploration activities in Site B and the presence of organic/inorganic contaminants in Site C due to the presence of fish feeds. These biochemical alterations show that the fish were under stress in their natural habitat. These biomarkers could be employed in the environmental monitoring of crude oil pollution as well as early warning signs of the adverse effects of environmental pollution.

984-992 | JRB | 2013 | Vol 3 | No 4






Research Journal

Authors:

Osioma E1*, Akanji MA1

and Arise RO1.

Institution:

1. Department of

Biochemistry, Faculty of

Science, University of Ilorin,

Nigeria.


Osioma E.

Email:


Dates: Received: 23 Mar 2013 Accepted: 23 May 2013 Published: 13 June 2013

Article Citation: Osioma E, Akanji MA and Arise RO. Hepatic enzyme markers and proteins in serum and some selected tissues in Clarias gariepinus from swamp around Kokori-Erhoike oil field, Nigeria. Journal of Research in Biology (2013) 3(4): 984-992


Original Research

INTRODUCTION

Over the years, the extent of oil exploration

activities and its related environmental effects has been

on the increase, (Tolulope, 2004). Some quantities of

petroleum and its products may be released into the

environment during oil exploration activities due to

operational, accidental, transportation or other means.

Apart from contaminating the flesh of commercially

valuable fish, crude oil compromise fish hatcheries in

coastal waters (Leighton, 1991) and its products are the

most relevant to aquatic ecotoxicology (Pacheco and

Santos, 2001). In Nigeria, crude oil was discovered at

Oloibiri in 1959 (Akpofure et al., 2000) and exploration

activities has been carried out in Kokori- Erhoike

environment for over 30 years (Emoyan, 2009).

The cause-effect relationship and result of

xenobiotic pollution in an ecosystem can be assessed

through the analysis of biochemical alterations on

organisms inhabiting that environment. These

biochemical alterations may be sensitive and specific as

early indicators of aquatic pollution (Norris et al., 2000;

Strinac and Braunbeck, 2000)

Protein plays a vital role in the physiology of

living organisms and its metabolism according to Adams

et al., (1990) provides information on the general energy

mobilization of an animal and show relationship with

effects of contaminants in these organisms. The

concentration of plasma albumin is a useful index of the

state of protein repletion and it makes the major

contribution to plasma sulphydryl groups which can

function as a chain breaking antioxidant (Halliwell,

1988). Haemoglobin contained in the red blood cells

which serve as the oxygen carrier in blood has been

employed in assessing the health of fish and monitoring

stress response of several environmental contaminants

including petroleum hydrocarbons (Soivio and Oikari.,

1976; Gabriel et al., 2007).

Alanine aminotransferase (ALT) and aspartate

aminotransferase (AST) catalyze the transfer of α-amino

group from α-amino acid to α-keto acid. AST and ALT

are biological responses of severe hepatic injury and

their bioassay can serve as a diagnostic tool for

estimating necrosis of the liver cells. (Cappo et al.,

2002). The determination of ALT and AST activities has

been applied in fish research to indicate bacteria, viral

and parasitic infection, intoxications and water pollution

(Bucher and Hofer, 1990). Alkaline phosphatase

comprises group of enzymes which is responsible for

hydrolyzing phosphoric ester bonds present in organic

compounds at an alkaline pH (Akcakaya et al., 2007).

The enzyme (ALP) has been reported to be a marker

enzyme for the plasma membrane and endoplasmic

recticulum (Akanji et al., 1993).

Fish species are excellent subjects for the study

of various effects of contaminants (El-Shehami et al.,

2007) and African catfish (Clarias gariepinus) has been

used in fundamental research and toxicological studies

(Nguyen and Janssen, 2002)

A significant body of research has investigated

the effects of crude oil (or its derivatives) and refinery

effluents on fish health. The investigators include:

Yarbrough et al., 1976; Kuehn et al., 1995; Sunmonu

and Oloyede, 2006; Wegwu and Omeodu, 2010;

Mahmoud et al., 2011 and Nwaogu et al., 2011.

However, there is dearth of information on the effect of

crude oil exploration activities on African catfish

(Clarias gariepinus) obtained from swamps (fish natural

habitat) around Kokori- Erhoike oil field located in Delta

State, Nigeria. This information deficiency prompted this

study. Therefore, the aim of this research is to determine

changes of some biochemical parameters in serum and

tissues (gill, liver, brain and muscle) of

Clarias gariepinus obtained from swamps around Kokori

- Erhoike petroleum flow station in Delta State, Nigeria.


Sampling sites:

Osioma et al., 2013


This study was carried out in Ethiope East Local

Government Area of Delta State, Nigeria. Experimental

areas have been previously described by Aries et al.,

2013 and are represented in Figure 1 below.

Site A (reference site) is the Eku axis of the

Ethiope River, Delta State. There is no presence of oil

facilities/operations or any industry located along the

Ethiope River from its source, Umuaja, about 22km to

the Eku axis. The upper axis of the Ethiope River has

been reported to be relatively unpolluted (Ikomi et al.,

2005; Agbaire and Obi, 2009; Aries et al., 2013). This

qualifies the Eku axis of Ethiope River as a reference site

for this study. Site B is the swampy environment of

Kokori-Erhoike petroleum flow station where oil

exploration activities have been on for more than 35

years. This area has a number of oil wells and flow

stations. The aquatic ecosystem in the area is constituted

by non-tidal freshwater swampy forest characteristics of

those found within the freshwater survey zone of the

Niger Delta. Site C is a natural fish pond located within

Kokori-Erhoike environment. The main uses of water in

the catchments include domestic, recreational (e.g.

swimming) and fishing. Their major occupation includes

farming (cassava, yam, Okro etc.), fishing and petty

trading on food stuff.

Osioma et al., 2013


Fig 1. Map of Ethiope East showing the location sampling sites

Source: Ministry of lands, Surveys and urban Development, Asaba. (2008)

Fish

Eight African catfish (Clarias gariepinus) were

collected from each site in October, 2011. At site A (Eku

River) and site B (Erhoike swamp), fish were caught

with the help of professional local fishermen and at site

C (Erhoike Fish pond) fish net was used to catch the fish.

Preparation of Serum

About 1.5ml blood was taken by caudal arterial

puncture from each fish into a sterilized plain tube.

Blood was allowed to clot for about 5min dislodged and

centrifuged at x15,000g for 15min to obtain the serum,

which was stored frozen at – 20°C until analyzed.

Preparation of Tissue Homogenate

The fish were dissected and the gill, liver, brain

and muscle tissues were quickly removed. The tissues

(gill, liver, brain and muscles) were washed in cold

saline (0.9% NaCl) solution several times and then 1g of

wet tissue was homogenized in 9 ml of the physiological

solution (normal saline). The resulting homogenate was

centrifuged at 5000g for 20min. The supernatant was

decanted and used for further biochemical analysis.

Biochemical Investigations

The concentrations of total protein and albumin

were determined in serum and tissues (gill, liver, muscle

and brain) employing the methods of Doumas et al.,

1981 and Doumas et al., 1971 respectively.

Haemoglobin level was estimated by the method of Tietz

(1976), while the method of Roy (1970) was used to

determine the activities of alkaline phosphatase. Alanine

aminotransferase and aspartate aminotransferase were

analyzed using the method of Reitman and Frankel

(1957). All assays were carried out with the aid of

commercially available kits supplied by TECO

Diagnostics, Anahem, USA and Randox Laboratories,

Ardmore, United Kingdom.

Statistics

Analysis of Variance (ANOVA) was used to

analyse data obtained from the various biochemical

investigations. Group means were compared by the

Duncan’s Multiple Range Test (DMRT) at 5%

probability level. All statistical analysis was performed

using SPSS version 16.

RESULTS

Results (Table 1) show that gill, muscle and

brain total protein concentrations were significantly

higher (p<0.05) in fish from Site A as compared with

that of Site B and C. Total protein concentration in serum

and liver of Clarias gariepinus from all sites were

comparable at p>0.05. The data also indicated significant

reduction (p<0.05) in gill and muscle albumin

concentration of Clarias gariepinus from Sites B and C

as compared with that of Site A. Levels of serum and

liver albumin were comparable in fish from all sites.

Results in Table 1 also revealed that fish from Sites B

and C has elevated (p<0.05) haemoglobin concentration

compared with that of Site A.

Osioma et al., 2013


Table 1: Levels of total protein, albumin and

haemoglobin concentrations in serum and tissues (gill,

liver, muscle and brain) of Clarias gariepinus from

swamps around Kokori-Erhoike Petroleum Flow

Station in Delta State, Nigeria

Values are given as Mean ± SD. Means not sharing a

common superscript letter on a given row differ

significantly at p<0.05. A= Ethiope River (Eku axis);

B= Erhoike swamp; C= Erhoike fish pond.

SAMPLING SITES

A B C

Total protein concentration (g/dl) Serum (n=8) Gill (n=8)

9.31±0.53a 2.82±0.05a

9.30±0.31a 2.46±0.24b

9.29±0.33a 2.43±0.03b

Liver (n=8) Muscle (n=8) Brain (n=8)

2.81±0.37a 5.28±0.32a 4.17±0.17a

2.91±0.15a 4.29±0.10b 3.72±0.13b

2.98±0.40a 4.08±0.40b 3.96±0.12c

Albumin concentration (g/dl)

Serum (n=8) Gill (n=8)

7.25±0.32a 2.48±0.36a

7.01±0.01a 2.03±0.16b

6.96±0.13a 2.08±0.24b

Liver (n=8) Muscle (n=8) Brain (n=8)

2.52±0.43a 4.09±0.17a 3.78±0.22a

2.51±0.26a 2.09±0.42b 2.25±0.11b

2.69±0.18a 2.55±0.12b 3.68±0.10a

Haemoglobin concentration (g/dl)

Blood (n=8) 22.24±1.57a 27.03±1.65b 27.29±1.65b

Increased (p<0.05) activities of alanine

aminotransferase in serum and liver of

Clarias gariepinus from Sites B and C were observed as

compared with that of Site A (Table 2). Similar trend

was observed for the activity of liver aspartate

aminotransferase. The table also showed that the activity

of aspartate aminotransferase in serum of fish from all

sites (A, B and C) were comparable (p>0.05), although,

fish from Site A had relatively lower aspartate

aminotransferase activity. Results (Table 2) also showed

that the activity of alkaline phosphatase in (serum, liver,

gill and brain) of Clarias gariepinus from Sites (B and

C) were significantly (p<0.05) higher as compared with

that of Site A.

DISCUSSION

Biochemical markers of pollution are considered

indicators employed in fish toxicity tests and for field

monitoring of aquatic contamination. They established

contact of the sample with definite groups of chemical

compounds and clarify their metabolic fate. Biochemical

investigations allow cause-effect relationship to be

established at an early stage of pollution and these

sensitive and predictive diagnostic tools (Biomarkers) for

assessing animal exposure and toxic effects of chemical

contaminants are needed as aquatic environmental

contamination assessment indicators.

The total protein concentration in serum, gill,

liver, muscle and brain of Clarias gariepinus from Eku

River is higher than the values obtained from fish

samples in Erhoike swamp and Erhoike fish pond. The

differences were significant (p<0.05) in the gill, brain

and muscle tissues.

Proteins play a vital role in the physiology of

living organisms and provide information on the general

energy mobilization of an animal and show relationship

with the effect of contamination in these organisms

(Adams et al., 1990). The over 30 years of petroleum

exploration activities in Erhoike vicinity could have

contaminated the aquatic environment and petroleum

hydrocarbon can act as a mediator in free radical

generation in fish (Achuba and Osakwe, 2003). During

stress conditions, fishes need more energy to detoxify the

toxicants and to overcome stress, thus carbohydrate

reserve is depleted to meet energy demand (Nelson and

Cox, 2005; Sudhanshu and Ajay, 2009). Since fish have

a very little amount of carbohydrate, the next alternative

source of energy is protein to meet the increased energy

demand occasioned by a pollutant.

The decrease in total protein level observed in

the gill, brain and muscle tissue could be to meet the

higher energy demands for metabolic purposes due to the

presence of petroleum hydrocarbon in Erhoike

environment and could also be related to impaired food

intake, increased energy cost of homeostasis, tissue

repair and detoxification mechanism during stress.

Albumin is the most soluble and most electrically

mobile of all the major serum protein components and it

is synthesized entirely by the hepatic parenchymal cells.

Lower albumin concentration was observed in the serum,

Osioma et al., 2013


Table 2: Changes in the activities of alanine

aminotransferase (ALT), aspartate aminotransferase

(AST) and alkaline phosphatase (ALP) in serum and

tissues (liver, gill and brain) of Clarias gariepinus

collected from swamps around Kokori-Erhoike

Petroleum Flow Station in Delta State, Nigeria

SAMPLING SITES

A (n=8) B (n=8) C (n=8)

Alanine aminotransferase (IU/L)

Serum

Liver

43.86±2.79a

25.10±0.80a

48.28±0.34b

45.90±1.63b

49.19±0.65 b

42.38±1.82c

Aspartate aminotransferase (IU/L)

Serum

Liver

53.75±8.84 a

52.31±0.79a

54.34±2.88 a

69.70±1.63b

54.25±3.15 a

65.94±1.82c

Alkaline phosphatase (IU/L)

Serum

Liver

Gill

Brain

32.86±3.14 a

43.01±1.48a

29.73±3.73a

52.94±2.36a

46.22±3.05 b

52.67±1.79b

36.79±1.39b

57.34±1.21b

45.76±3.49 b

53.83±2.91b

32.74±5.36a

60.37±2.25c

Values are expressed as Mean± SD. Means not sharing a

common superscript letter on a given row differ

significantly at p<0.05. A= Ethiope River (Eku axis);

B= Erhoike swamp; C= Erhoike fish pond.

gill, liver, muscle and brain tissues of Clarias gariepinus

from Erhoike swamp and Erhoike fish pond compared

with corresponding albumin level of Clarias gariepinus

from Eku River (control site). The utilization of proteins

as an alternative source of energy by fish in stress

condition could have accounted for the reduced albumin

level in fish from Erhoike swamp and Erhoike fish pond.

Albumin has also been regarded as an antioxidant

molecule. It reacts with and neutralizes peroxyl radicals

(Stocker and Frei, 1991) and it is considered as a

sacrificial molecule that prevents damage when it acts as

an antioxidant because albumin is destroyed in the

process (Halliwell, 1988). Therefore, the observed

reduction of albumin concentration in African Catfish

from the oil exploration areas may be linked to its

participation as an antioxidant molecule to quench free

radical reactions in other to mitigate the impact of

oxidative stress or its utilization as a source of energy by

the fish in stress condition.

This study showed that Clarias gariepinus from

Erhoike swamp and Erhoike fish pond have higher levels

(p<0.05) of haemoglobin as compared with haemoglobin

concentration of Clarias gariepinus from the control site

(Eku River). Elevated levels of haemoglobin observed in

African catfish from Erhoike swamp and fish pond could

be as a result of stress induced by the presence of crude

oil and other contaminants (as in the case of the fish

pond) that leads to environmental hypoxia as a result of

chronic exposure to the contaminants and anaerobic

condition which lead to increase haemoglobin

concentration as a compensatory mechanism for

increased oxygen demand. This result corroborates with

the findings of Mdegela et al., (2010) who reported

significant elevation of haemoglobin concentration in

fish from Mzumbe sewage water. Zaki et al., (2010) also

reported significant increase in haemoglobin levels in

Tilapia zilli exposed to acute lethal concentration dose of

lead (Pb).

Alanine aminotransferase and aspartate

aminotransferase (ALT and AST) are enzymes directly

associated with the conversion of amino acids to keto

acids. Apart from being considered to be important in

assessing the state of the liver and some other organs

Verma et al., (1981), transamination of the same

represents one of the main pathways for synthesis and

deamination of amino acid, thereby allowing interplay

between carbohydrate and protein metabolism during the

fluctuating energy demands of the organisms in various

adaptive situations. ALT and AST activities are direct

indicators of intense hepatic damage, thus their bioassay

can assist as a diagnostic tool for determining necrosis of

the liver cells (Whitehead et al., 1999; Cappo et al.,

2002). Ugwu et al. (2008) concluded that AST enzyme

activity in Heterobranchus bidorsalis adults could be

used as biomarker for monitoring crude oil pollution in

Nigeria.

Compar ed wi th th e con tr ol ( i . e .

Clarias gariepinus from Ethiope River, Eku axis) the

activities of ALT and AST in serum and liver of

Clarias gariepinus from Erhioke swamp and Erhoike

fish pond were higher. Such increase of ALT and AST

may be partly due to hepatic damage resulting from

petroleum pollution (in case of Erhoike swamp) or

organic/inorganic contaminants (present in Erhoike fish

pond, Arise et al., 2013) –induced oxidative insults on

the hepatocytes. In addition, increased protein catabolism

might be responsible for the elevation of these

transaminases. These results agree with the findings of

Ayalogu et al., (2001); Orisakwe et al., (2005).

In this study, serum, liver, gill and brain alkaline

phosphatase act i vi t y were measured in

Clarias gariepinus from the three sampling sites. Marked

increase in ALP activity was recorded in the serum, liver,

gill and brain tissues of Clarias gariepinus from Erhoike

swamp and Erhoike fish pond as compared with ALP

activity of Clarias gariepinus from the control site (Eku

River). ALP together with ALT and AST provide an

Osioma et al., 2013


indication of the degree of inflammation as well as

possible causes of hepatocellular damage as well as

distortion of the plasma membrane and endoplasmic

recticulum. Results of the research agree with the

findings of Yarbrough et al., (1976) and Ayalogu et al.,

(2001).

CONCLUSION

The study demonstrates that the level of

contamination is enough to cause changes in the protein,

albumin and haemoglobin level with attendant effect on

the integrity of hepatocytes as evidence in the elevated

activities as seen in liver marker enzymes and alkaline

phosphatase activity of Clarias gariepinus from Erhoike

swamp and Erhoike fish pond. The above biochemical

changes showed that the fish were under stress in their

natural habitat (Erhoike swamp) thus; these markers

could be employed in the environmental monitoring of

crude oil pollutant and their associated metabolic

changes as early warning signs of adverse effects of

environmental pollution.

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