Journal of Research in Biology Volume 4 Issue 1

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

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

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School of Life Sciences, Bharathidasan University, India.

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

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Silpakorn University, Thailand.

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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.

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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

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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.

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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

Dr. Preetham Elumalai [Biochemistry and Immunology] Institute for

Immunology Uniklinikum, Regensburg, Germany

Dr. Mrs. Sreeja Lakshmi PV [Biochemistry and Cell Biology] University of Regensburg, Germany

Dr. Alma Rus [Experimental Biology]

University of jaén, Spain.

Dr. Milan S. Stanković [Biology, Plant Science]

University of Kragujevac, Serbia.

Dr. Manoranjan chakraborty [Mycology and plant pathology]

Bishnupur ramananda college, India.

Table of Contents (Volume 4 - Issue 1)

Serial No Accession No Title of the article Page No

1 RA0410 Diversity of freshwater diatoms from few silica rich habitats of Assam,

India.

Dharitri Borgohain and Bhaben Tanti.

1162-1173

2 RA0395 Detection of biofilm formation in urinary isolates: need of the hour.

Saha R, Arora S, Das S, Gupta C, Maroof KA, Singh NP and Kaur IR.

1174-1181

3

RA0415

Foraging and pollination behavior of Apis mellifera adansonii Latreille

(Hymenoptera: Apidae) on Glycine max L. (Fabaceae) flowers at

Maroua.

Fernand-Nestor Tchuenguem Fohouo and Dounia.

1209-1219

4 RA0421 Determining the Natural Gypsophila L. (Coven) Taxa Growing in Tunceli

(Turkey).

Mustafa Korkmaz and Hasan Ozçelik.

1220-1227

5 RA0422 Distribution pattern of birds in Banni Grassland of Kachchh district,

Gujarat, India

Mukesh H. Koladiya, ArunKumar Roy Mahato, Nikunj B. Gajera and

Yatin S. Patel.

1228-1239

6 RA0414 Determination of age and growth by scale of a population of common

trout (Salmo trutta macrostigma, Dumeril, 1858) at the level of Sidi

Rachid River (Ifrane. Morocco).

Abba H, Belghity D, Benabid M and Chillasse L.

1240-1246

http://jresearchbiology.com/documents/RA0410.pdf






Article Citation: Dharitri Borgohain and Bhaben Tanti. Diversity of freshwater diatoms from few silica rich habitats of Assam, India. Journal of Research in Biology (2014) 4(1): 1162-1173

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Biology

Diversity of freshwater diatoms from few silica rich habitats of Assam, India

Keywords: Freshwater diatoms, silica rich soil, diatom diversity, Geological Survey of India.

ABSTRACT: Diatoms are a ubiquitous class of phytoplankton of extreme importance for the biogeochemical cycling of minerals such as silica. Few places of Nagaon district of Assam, India viz., Jiajuri, Borhola, Thanajuri and Chapanala have been recognized as the highest silica zones by Geological Survey of India. No any research has been conducted to explore the diatom diversity at this important silica rich habitat. In the present investigation, the morphology and diversity of freshwater diatom species were investigated during May 2012 to April 2013. The samples were subjected to acid wash treatment followed by microscopic observations. Altogether 103 species of diatoms belonging to 20 genera were recorded. Occurrence of diatom varied in all the four different study sites. The dominant genera includes: Stauroneis, Kobayasiella, Eunotia, Pinnularia, Nitzschia, Gomphonema, Frustulia, Surirella, Achnanthes, Rhopalodia, Navicula, Synendra, Encyonema, Achnanthidium, Cymbella, Hippodonta, Tabularia, Actinella, Encyonopsis and Luticola. Notably, all the diatom species belonged to pennate type.

1162-1173 | JRB | 2014 | Vol 4 | No 1

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:

Dharitri Borgohain and

Bhaben Tanti*.

Institution:

Department of Botany,

Gauhati University,

Guwahati - 781014, Assam,

India.

Corresponding author:

Bhaben Tanti.

Email Id:

Web Address: http://jresearchbiology.com/documents/RA0410.pdf.

Dates: Received: 07 Jan 2014 Accepted: 29 Jan 2014 Published: 15 Feb 2014

Journal of Research in Biology An International Scientific Research Journal

Original Research

INTRODUCTION

Diatoms belonging to the class Bacillariophyceae

are the major group of single-celled photosynthetic

eukaryotic algae which can be found in almost all

aqueous and humid environments. Diatoms are an

important component of phytoplankton in freshwaters.

There are over 250 genera of diatoms with more than

100,000 species (Gurung et al., 2012, Van Den Hoek

et al., 1997), which includes both marine and the

freshwater environments. These microscopic autotrophic

microalgae possess highly ornamented cell wall

composed of glass silica (SiO2) called frustules which

provide a variety of shapes from nano to micro-scale

structures. Diatoms can occur in large amounts, either

solitary or in colony and is cosmopolitan in distribution.

A major constituent of the plankton family, diatoms are

free floating, planktonic or attached to a substrate and

benthic forms (Werner, 1977). Diatoms are important

from the point of the biogeochemical cycling of silica.

Diatoms play a very significant ecological role by fixing

about 25% carbon globally. The diatoms of North East

region of India are still largely unexplored and

unexploited. Friable quartzite’s belonging to the Shillong

groups of rocks occur sporadically along eastern most

part of the Nagaon district. Borhola, Chapanala, Jiajuri

and Thanajuri are some of the important places where

friable quartzites are found abundantly. About 75% of

the glass sand may be recovered from this friable

quartzite by using different methods of beneficiation

(Goswami, 2006).

The Geological Survey of India (GSI) has found

significant reserves of silica deposits in the Jiajuri region

between the district of Nagaon and Karbi Anglong in

Assam (Borpuzari, 2012). The area is located about

30kms South-East from Nagaon and is adjacent to Jiajuri

Tea Estate. The deposit is bounded by latitude 26° 18′ 0″

to 26°19′ 0″ N and longitude 92°52′ 55″ to 92°54′ 15″ E.

Jiajuri hill covers an area of 2.9 km2 and the possible

friable quartzite is about 7.4 million tones. Chapanala

(26°20′10″ N latitude and 92°51′30″ E longitude) deposits

occur friable quartzite covering an area of 0.373 km2 and

possible reserve is 3.5 million tones. Thanajuri hill

(26°12′ 35’’ to 26°13′10″ N latitude 92°48′40″ to 92°50′35″

E longitude and) is situated in the northern part of Karbi-

Anglong plateau and southern part of Nagaon district.

The possible reserves of glass sand is about 1.788

million tones. Friable quartzite occurs in Borhola (26°26′

15″ N latitude and 92°56′45″ E longitude) covering an

area of 0.595 km2 and the possible reserve of glass sand

is about 1.25 million tones. Till date, there is no any

extensive work on the detailed investigation of diatom

diversity in these silica rich regions of Assam. Set in this

backdrop, the present investigation is assessed for the

exploration of diatom, having the genetic ability to

deposit natural silica over their cell surface in

characteristics nanoporous forms.

MATERIALS AND METHODS

Sample collection and growth conditions

Samples were collected from aquatic and semi-

aquatic habitats of the four study sites- Jiajuri, Borhola,

Thanajuri and Chapanala from May 2012 to April 2013

(Fig.1). The freshly collected samples were immediately

transferred to Diatom Medium (DM) proposed by

Beakes et al., (1988) which was standardized with slight

modifications and the composition of stock (per 200ml)

includes- Ca(NO3)2. 4H2O – 4g, KH2PO4– 2.48 g,

MgSO4.7H2O - 5 g, NaHCO3- 3.18 g, EDTAFeNa-

0.45g, EDTANa2 – 0.45g, H3BO3 – 0.496g, MnCl2.

4H2O –0.278g, (NH4) 6Mo7O24.4H2O – 0.20g,

Cyanocobalamine - 0.008g, Thiamine HCl – 0.008g,

Biotin – 0.008g and Na2SiO3.9H2O – 22.8g.

One ml of each stock solution was added to make

the final volume of 1L with distilled water, and adjusted

to pH 6.8. For solid medium, 1.5% agar was added. The

cultures were allowed to grow at 3K light at 18-20°C for

20-22 days. Repeated sub-cultures were done on the

solid medium to obtain pure cultures of diatom species.

Borgohain and Tanti, 2014

1163 Journal of Research in Biology (2014) 4(1): 1162-1173

Cleaning diatom frustules by acid wash method for

microscopic analysis

In order to analyze the diatom frustules for

microscopic studies, a cleaning procedure was needed

that removed the external organic matrix covering the

frustules. Plankton samples were subjected to acid wash

method according to the protocol of Hasle and Fryxell

(1970) before light microscopic observations. About

20ml of liquid cultures were transferred into a beaker and

treated with equal quantity of concentrated H2SO4 and

agitated gently. Freshly prepared KMnO4 was added to

the sample until the sample had a purple tint. Then

freshly prepared oxalic acid (COOH)2 was added to

obtain clear solution. The sample was centrifuged at

2500 rpm for 15 min and then rinsed with distilled water

until the cell suspension become less acidic. To confirm

the complete removal of organic matters, a drop of

cleaned samples was observed under the microscope.

For light microscopy (LM) observation, the

slides were prepared by evaporating drops of the cleaned

diatoms suspended in distilled water onto cover-slips and

the mounting was done by using Naphrax (a specific

diatom mountant with refractive index 1.74). The slides

were examined carefully under 1000x magnification and

the diatom images were documented in Nikon ECLIPSE

E200 with photo micrographic attachment.

Identification of diatoms

The diatoms obtained through laboratory pure

cultures were identified by consulting various literatures

and monographs (Gandhi, 1955; Husted, 1959; Hendey,

Journal of Research in Biology (2014) 4(1): 1162-1173 1164


Figure 1: Map showing the four study areas (source: www.mapsofindia.com).

1964; Patrick and Reimer 1966; Prescott, 1975;

Desikachary, 1989; Round et al., 1990; Nautiyal et al.,

1996; Anand, 1998; Gurung et al., 2013).

RESULTS AND DISCUSSION

During the present investigation, a total of 103

species of freshwater diatoms belonging to 20 genera of

class Bacillariophyceae were reported from the silica rich

soils of Nagaon district of Assam i.e. Jiajuri, Borhola,

Thanajuri and Chapanala. The prominent genera in

terms of its abundance and frequency were Nitzschia

(25), Gomphonema (17), Navicula (15), Pinnularia (14),

Eunotia (5), Stauroneis (4), Cymbella (4), Frustulia (3),

Synendra (3), Achnanthes (2), Achnanthidium (2) and

single species of the following diatoms: Actinella,

Luticola, Encyonema, Hippodonta, Surirella, Tabularia,

Encyonopsis, Kobayasiella and Rhopalodia. Pure

cultures of diatoms obtained in this study were identified

upto their genus level (Fig. 3-9). Morphological

descriptions of the diatom isolates obtained in pure

culture were enumerated.

Out of 103 diatoms species obtained in pure

cultures, 25 diatoms were found to be of different species

of Nitzschia representing 24.3% of the total diatom flora.

Further, there were 17 different species of Gomphonema,

15 different species of Navicula, 14 different species of

Pinnularia and 5 different species of Eunotia

representing 16.5%, 14.6%, 13.6% and 4.9%

respectively. There were four different species of

Stauroneis, Cymbella (3.9% each), followed by Frustulia

and Synendra (2.9% each) and Achnanthes and

Achnanthidium (1.9% each). The remaining diatoms viz.

Surirella, Tabularia, Encyonema, Actinella,

Encyonopsis, Rhopalodia, Luticola, Hippodonta and

Kobayasiella were represented by only one species

showing 8.7% out of the total diatoms identified in pure

cultures (Fig. 2).

Taxonomic account:

Taxonomic description of the 20 pennate

freshwater diatom genera obtained in the four silica rich


1165 Journal of Research in Biology (2014) 4(1):1162-1173

Diversity of diatom flora

Figure 2: Representation of diatom flora diversity.

sites during the study period are described below:

Class: Bacillariophyceae

Order: Bacillariales

Family: Naviculaceae

Genus: Navicula Bory 1822, Cleve 1894

Navicula sp. (Fig. 3 A-O)

Valves 36 µm long, 14 µm broad, broadly

elliptical with convex margins; ends slightly produced,

slightly capitate rounded; raphe thin, straight; central

nodules distinct; axial area narrow, linear; central area

somewhat obliquely rectangular; striae 23 in 10 µm, very

fine.


Order: Naviculales

Family: Pinnulariaceae

Genus: Pinnularia Ehrenberg 1843

Pinnularia sp. (Fig. 4 A-N)

Valves 53 µm long, 11 µm broad, linear, more or

less parallel margins with slightly tapering, broadly

rounded ends; raphe thick, straight, placed on one side

with distinct, unilaterally curved central nodules and

curved terminal fissures; axial area distinct, linear;

central area large reaching the sides; striae 7 in 10 µm,

coarse, 2-4 middle striae short and thick, radiate in the

middle, convergent towards apices.


Order: Cymbellales

Family: Gomphonemataceae

Genus: Gomphonema C.A. Agardh 1824

Gomphonema sp. (Fig. 5 A-L, 6 M-Q)

Valves 45 µm long and 8 µm broad, clavate with

capitate head pole and slightly capitate foot pole; axial

area linear, narrow, and widening into a small circular

central area with an isolated pore on the primary side of

the central nodule; raphe straight with distinct central

nodules; striae 10-11 in 10 µm, punctate and slightly

radiate, wider at the centre of the valve.


Order: Naviculales

Family: Amphipleuraceae

Genus: Frustulia Lange-Bertalot

Frustulia sp. (Fig. 6 A-C)

Valves 71-160 µm long and 15.3-30.2 µm

broad, rhombic-lanceolate, narrowing sharply to the

rounded apices. Axial and central areas narrow but

distinct. Transverse striae perpendicular to the raphe at



Figure 3(A-O): Navicula.

A

B

C D E

F G

H

I

J

K L

N M

O

the center of the valve, sometimes becoming slightly

convergent towards the ends of the valve, but radiate at

the apices, striae 20-30 in 10 µm.


Order: Cymbellales

Family: Cymbellaceae

Genus: Encyonema (Berkeley) Kutzing

Encyonema sp. (Fig. 6 D)

Valves 37-91 µm long and 15-30 µm broad,

robust and broadly dorsiventral and symmetrical to the

transapical axis. Dorsal margin normally arched, ventral

margin biarcuate to convex. Valve apices bluntly

rounded. Raphe straight with central endings deflected

dorsally and apical ends deflected ventrally, striae coarse

and 8-21 in 10 µm.


Order: Naviculales

Family: Diadesmidaceae

Genus: Luticola (Ehrenberg) D. G. Mann, 1990

Luticola sp. (Fig. 6 E)

Valves 12-24 µm long and 7-9 µm broad, linear

to linear-elliptical. Transapical striae radiate throughout,

composed of two to four rounded areolae. Largest

areolae near the valve margins. One isolated, circular

stigma present, striae 18-20 in 10 µm.


Order: Cymbellales


Genus: Encyonopsis (Grunow) Krammer, 1997

Encyonopsis sp. (Fig. 6 F)

Valves 21-25 µm long and 5.1-6.3 µm broad,

cymbelloid with dorsal margin strongly curved and

straight ventral margin. Axial area narrow, straight and

without a central area. Small central nodule. A stigmoid


Figure 4: (A-N) Pinnularia

1167 Journal of Research in Biology (2014) 4(1):1162-1173

Presented near the dorsal central striae, striae 14.2-16 in

10 µm.


Order: Rhopalodiales

Family: Rhopalodiaceae

Genus: Rhopalodia Otto Muller, 1895: 57

Rhopalodia sp. (Fig. 7 A)


isopolar and dorsiventral, lanceolate-elliptical in shape,

acute apices. The dorsal margin curved and straight at

the ventral margin. Striae composed of a single row of

puncta composes. Fibulae radiate, striae 14-20 in 10 µm.


Order: Naviculales


Genus: Kobayasiella Lange-Bertalot, 1999

Kobayasiella sp. (Fig. 7 B)

Valves 22-26 µm long and 5-7 µm broad, linear-

lanceolate with convex sides and short, capitate apices.

The axial area is narrow and nearly linear. The central

area is small and elliptical and bordered by alternately

long and short striae, striae 35-40 in 10 µm.


Order: Eunotiales

Family: Eunotiaceae

Genus: Actinella Lewis, 1864

Actinella sp. (Fig. 7 C)

Valves 76-140 µm long and 5.7-8 µm broad,

arcuate, asymmetrical to both the apical and transapical

axes. External distal raphe ends extending slightly to the

valve face on both ends. Striae parallel, striae 13-19 in

10 µm.


Order: Achnanthales

Family: Achnanthaceae

Genus: Achnanthidium Kutzing, 1844

Achnanthidium sp. (Fig. 7 D and E)

Valves 6.2-14 µm long and 2-3.7 µm broad,

linear-elliptic, slightly or more elongated near the end,

and with bluntly rounded poles. Striae slightly radiate

and often a shortened striae near the small central area,

axial area narrow, striae 19-21 in 10 µm.





A B C

D

E F

G H

I J

K L

Figure 5 (A-L):Gomphonema.

Family: Eunotiaceae

Genus: Eunotia Ehrenberg 1837

Eunotia sp. (Fig.7 F-J)

Valves 68µm long, 12 µm broad, slightly arched,

dorsal margin convex with two wavy ridges at the

middle, gradually narrowing towards the ends, ventral

margin concave; ends slightly constricted on the dorsal

side, slightly produced, rounded; raphe thin; polar

nodules distinct, on the ventral side near the apices;

striae 13 in 10µm, coarse, lineate, parallel, somewhat

radiate and closely placed near apices.


Order: Fragilariales

Family: Fragilariaceae

Genus: Synendra Ehrenberg 1832: 87

Synendra sp. (Fig. 7 K-M)

Valves 44 µm long and 3.2- 3.8 µm broad, linear

with narrow and capitate ends. The central area reaches

the margins. Pseudo raphe linear and broad. Striae strong

and distantly placed, striae 13 in 10 µm.



Family: Bacillariaceae

Genus: Nitzschia Hassall, 1845: 435

Nitzschia sp. (Fig. 8 A-Y)


linear with concave sides and wedge shaped, constricted

produced ends, striae very fine, almost indistinct, striae

31-35 in 10 µm.


Order: Naviculales


Genus: Hippodonta (Ehrenberg)

Hippodonta sp. (Fig. 9 A)

Valves 20.2-29 µm long and 5.5-8 µm broad,

elliptic-lanceolate, ends subcapitate to capitate. Raphe

straight, filiform, central pores fairly close. Striae



Figure 6: (A-C) Frustulia, D– Encyonema, E-Luticola, F-Encyonopsis, (M-Q) Gomphonema.

noticeably broad, radiate in the middle, convergent at the

ends, striae 9-11 in 10 µm.


Order: Surirellales

Family: Surirellaceae

Genus: Surirella Turpin 1828

Surirella sp. (Fig. 9 B)


heteropolar, ovate with broad rounded ends. Middle line

absent. Middle field linear-lanceolate. Striae very thick,

widening towards the middle, set at unequal distances,

Striae 11-16 in 10 µm.


Order: Achnanthales

Family: Achnanthaceae

Genus: Achnanthes C.A. Agardh (1824)

Achnanthes sp. (Fig. 9 C & D)

Valves 12.5-16 µm long and 5-7 µm broad,

rectangular-elliptical to almost quadrate in the middle

portion, constricted at the ends which are rostrate. Axial

area narrow and central area linear reaching the margins.


Order: Fragilariales

Family: Fragilariaceae

Genus: Tabularia (C. Agardh) D.M. Williams and

Round

Tabularia sp. (Fig. 9 E)


elliptic or elongate and variable in outline, from narrowly

linear to linear- lanceolate or lanceolate valves with

rounded or capitate ends, striae 7.4-25 in 10 µm.


Order: Cymbellales


Genus: Cymbella, C.A. Agardh 1830

Cymbella sp. (Fig. 9 F-I)

Valves 118 µm long, 24 µm broad, ventricose,

curved, asymmetric, dorsal side convex, ventral side

slightly concave with middle inflation; ends slightly

constricted, produced rounded; raphe thick, arcuate,

excentric with ventrally curved central nodules; axial

area not narrow; central area elliptical with 3-4 isolated


Figure 7 A: Rhopalodia, B- Kobayasiella, C- Actinella, D and E- Achnanthidium,

(F-J) Eunotia, (K-M) Synendra.


stigmata at the ends of the middle ventral striae; striae

8-10 in 10 µm, punctate, radiate.


Order: Naviculales

Family: Stauroneidaceae

Genus: Stauroneis Ehrenberg, 1843

Stauroneis sp. (Fig. 9 J-M)


lanceolate with abruptly constricted, somewhat produced

capitate ends. Raphe thick with slightly unilaterally bent

central pores and curved terminal fissures. Axial area

moderate, linear or slightly widened between the middle

and ends: Striae radial, striae 20-22 in 10 µm.

It is interesting to note that all the diatom taxa

belonged to pennate type. No centric forms of diatom

were found in all the four sampling sites. Majority of the

forms were solitary and colonial forms were absent. The

dominant genera includes- Gomphonema, Nitzschia,

Stauroneis, Navicula, Frustulia, Eunotia and Pinnularia

which were common in all the sampling sites in all the


Figure 8(A-R):Nitzschia


Figure 8(S-Y):Nitzschia

seasons throughout the year. Kobayasiella, Cymbella,

Synendra, Achnanthidium and Tabularia were abundant

only in Chapanala while Luticola, Encyonema occurred

in Borhola. Pennate diatoms like Achnanthes,

Encyonopsis, Hippodonta, Actinella and Rhopalodia

were found only in Jiajuri. Only pennate diatom

Surirella was found in Thanajuri.

CONCLUSION

Silica rich soils Jiajuri, Borhola, Thanajuri and

Chapanala of Nagaon district of Assam harbours rich

assemblage of various forms of diatoms; many of which

are new to the region. As detailed taxonomic

investigations on the diatom flora of North- East India is

very limited, the present basic information of diversity

and distribution of diatoms would form a useful tool for

further monitoring and ecological assessment of these

silica rich soils of Assam. Further, the diversity of

freshwater diatoms could also be used as a resource

database for future applications.

ACKNOWLEDGEMENT

The author would like to acknowledge UGC-

SAP (Special Assistance Programme) for providing

Basic Scientific Research (BSR) fellowship in carrying

out the work.

REFERENCES

Anand N. 1998. Indian fresh water microalgae. Bishen

Singh Mahendra Pal Singh Publication, Dehradun, India.

p. 1-94.

Beakes GW, Canter HM and Jaworski GHM. 1988.

Zoospore ultrastructure of Zygorhizidium affluens and Z.

planktonicum, two chytrids parasitizing the diatom

Asterionella formosa. Canadian J Bot., 66(6):1054-1067.

Borpuzari P. 2012. Ministry to exploit silica reserves in

N-E India. The Financial Express 20 March.

Desikachary TV. 1989. Atlas of Diatoms: Marine

diatoms of the Indian Ocean region. Madras Science

Foundation. 6(1-13): 622-809.


Figure 9. A- Hippodonta, B- Surirella, C and D- Achnanthes, E- Tabularia, (F-I) Cymbella, (J-M) Stauroneis.


Gandhi HP. 1955. A contribution to our knowledge of

fresh-water diatoms of Pratapgarh, Rajasthan. J. Indian

Bot Soc., 34(4): 307-338.

Goswami ID. 2006. Mineral resources of Assam, In

Envis Assam, July-September, p. 2-4.

Gurung L, Tanti B, Buragohain AK and Borah SP.

2012. Studies on the freshwater diatom diversity in

Deepar Beel, Assam, India. J Assam Sci Soc., 53(2): 1-6.

Gurung L, Buragohain AK, Borah SP and Tanti B.

2013. Freshwater diatom diversity in Deepar Beel – a

Ramsar site. J. Res. Plant Sci., 2(2):182-191.

Hasle GR and Fryxell GA. 1970. Diatoms: cleaning

and mounting for light and electron microscopy.

Transactions of the Americans Microscopical Society. 89

(4): 469-474.

Hendey NI. 1964. An introductory account of the

smaller algae of British coastal water, Part V,

Bacillariophyceae (Diatoms). H.M.S.O., London. 317-

323.

Husted F. 1959. Die Kieselalgen Deutschlands,

Osterreichs Und Der Schweiz, Vol.2. Koeltz Scientific

Books, USA. p. 845.

Nautiyal R, Nautiyal P and Singh HR. 1996. Pennate

diatom flora of a cold water mountain river, Alaknanda

II suborder Araphideae. Phykos. 35(1-2): 57-63.

Patrick R and Reimer CW. 1966. The diatoms d

Hawaii.I. Monograph of the Acad. Nat. Sci. Philad. 13

(1): 668-672.

Prescott GW. 1975. Algae of the Western Great Lakes

Area. Michigan State University, USA. p. 998-1012.

Round FE, Crawford RM and Mann DG. 1990. The

diatoms: biology and morphology of the genera,

Cambridge University Press. 747.

Van Den Hoek C, Mann DG and Jahns HM. 1997.

Algae: An introduction to phycology, Cambridge

University Press, London.

Werner D. 1977. The Biology of Diatoms. University of

California Press. p. 498.

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Article Citation: Saha R, Arora S, Das S, Gupta C, Maroof KA, Singh NP and Kaur IR. Detection of biofilm formation in urinary isolates: need of the hour. Journal of Research in Biology (2014) 4(1): 1174-1181

Jou

rn

al of R

esearch

in

Biology

Detection of biofilm formation in urinary isolates: need of the hour

Keywords: Biofilm, biofilm detection, Congo Red Agar.

ABSTRACT: The purpose of the study was to estimate biofilm (BF) formation in urinary catheterized patients, by comparing three methods i.e. Tissue culture plate method (TCP), Congo Red Agar method (CRM) and Tube method (TM) and to study the antimicrobial resistance pattern in BF producing and non BF producing isolates. A total of 130 urinary catheterized patients were taken as the study group. From one milli litre of urine sample isolates > 102 colony forming units per milli litre were screened for the detection of BF by TCP, TM and CRM. Antibiotic sensitivity test for both BF producing and non BF producing bacterial and fungal isolates were done as per CLSI guidelines. From 130 urine samples in our study group, 55 samples grew microorganisms of significance, of which 11 samples were poly-microbial in nature. Of these biofilm production was seen in 49 microorganisms (89.09%) by any of the three methods used. TCP method picked up 69% of biofilm producers as compared to TM and CRM which picked up only 36% and 27% biofilm producers respectively. Our study reveals TCP method as the more dependable one as compared to TM and CRA methods for the quantitative biofilm detection, so it can be recommended as a screening method in laboratories.

1174-1181 | JRB | 2014 | Vol 4 | No 1




An International


Authors:

Saha R1*, Arora S1, Das S1,

Gupta C1, Maroof KA2,

Singh NP1 and Kaur IR1.

Institution:

1. Department of

Microbiology, University

College of Medical Sciences

and Guru Teg Bahadur

Hospital, Dilshad Garden,

Delhi – 110095, India.

2. Department of

Community Medicine,

University College of Medical Sciences and Guru

Teg Bahadur Hospital,

Dilshad Garden,

Delhi – 110095, India.


Rumpa Saha.


Dates: Received: 01 Dec 2013 Accepted: 08 Feb 2014 Published: 17 Feb 2014


Original Research

Abbreviations BF - Biofilms; TCP - Tissue Culture Plate; CRM - Congo Red Method; TM - Tube Method; CLSI - Clinical Laboratory Standard Institute; CAUTI - Catheter associated Urinary Tract Infection; CLED - Cysteine Lactose Electrolyte Deficient; BHIB - Brain Heart Infusion Broth; TSB - Trypticase soy broth; ELISA - Enzyme linked immunosorbent assay; MHA - Muller Hinton Agar; MIC -Minimum Inhibitory Concentration; ATCC - American type culture collection; GPC -Gram positive cocci; GNB - Gram negative bacilli.

INTRODUCTION

Indwelling urinary catheters play an essential

part in the management of disorders of the urinary tract,

especially in the elderly and disabled patients. These

urinary catheters serve as a portal of entry for

microorganisms leading to Catheter Associated Urinary

Tract Infections (CAUTI). Many of these microbes

colonize and adhere to the artificial surface of the

indwelling catheters, which then forms biofilms.

Biofilms are communities of microorganisms which are

embedded within a matrix of extracellular polymeric

material and display an altered phenotype. Based on the

type and length of the stay of a gadget, composition of

microorganism in a biofilm may vary from one to

numerous. The same is true for urinary catheter biofilms

where number of organisms is directly proportional to

length of exposure.

Microorganisms commonly isolated from

indwelling urinary catheters are Staphylococcus

epidermidis, Escherichia coli, Klebsiella pneumoniae,

Enterococcus faecalis, Proteus mirabilis and Candida sp

(Donlan, 2001).

Biofilms carry important clinical repercussions

as they provide a niche for survival of microbes, by

conferring protection to microbes from drying,

mechanical damage and other influences from external

environment, human immune system and antimicrobial

agents (Costerton et al., 1995; Mah and Toole, 2001).

High antimicrobial concentrations are required to

inactivate organisms growing in biofilms and resistance

may often increases thousand folds. (Stewart and

Costerton, 2001)

Moreover biofilms act as a persistent source of

infection or may provide reservoir for new infections.

The biofilms often leads to crystalline material blocking

the catheters and induce complications like painful

distension of the bladder, urolithiasis, reflux of infected

urine resulting in pyelonephritis and sometimes urinary

leakage around the outside of the catheter causing the

patient to become incontinent, thus leading to failure of

medical device.

There are different methods for the estimation of

biofilm formation including Tissue culture plate method,

Tube method, Congo Red agar method, bioluminescent

assay, light or fluorescence microscopic examination,

confocal laser scanning microscope and piezoelectric

sensor (Mathur et al., 2006).

There is paucity of data in Indian literature

regarding biofilm formation in urinary catheterized

patients. This study was undertaken with the aim to

estimate biofilm formation in urinary catheterized

patients, to compare three methods i.e. Tissue culture

plate method (TCP), Congo Red method (CRM) and

Tube method (TM) for biofilm production and to study

antimicrobial resistance pattern in biofilm producing

isolates.


The study was done over a period of one year

from April 2008 – March 2009 at department of

Microbiology, of our tertiary care hospital after obtaining

clearance from Institutional Ethical Committee. A total

of 130 urinary catheterized patients were taken as study

group who gave informed consent to the work. One ml of

urine samples were collected from catheter with aseptic

precautions and the samples were immediately sent to

the Microbiology laboratory. The samples were plated on

Cysteine Lactose Electrolyte Deficient (CLED) medium.

The age, sex, days of catheterization of the patients were

noted. Isolates were identified by standard

microbiological procedures. The presence of > 102 c.f.u./

ml in aseptically collected urine was taken as significant

bacteriuria (Winn et al., 2006). The cultures were

maintained on nutrient agar slopes, Enterococci were

maintained on brain heart infusion slopes and Candida

species were maintained on Sabouraud’s Dextrose Agar

(SDA) slopes. Control strains used for biofilm

production in the study were: S. epidermidis ATCC

Saha et al., 2014


35984 (strong biofilm producer), S. epidermidis ATCC

35983 (moderate biofilm producer) and S. epidermidis

ATCC 12228 (non biofilm producer), Acinetobacter

baumannii ATCC 19606 and Candida albicans ATCC

90028.

Biofilm formation was detected by the following

three methods:-

Tissue culture plate method (Christensen et al., 1995):

Isolates from freshly subcultured plates were

inoculated in trypticase soy broth (TSB) with 1% w/v

glucose and incubated for 18 hours at 37˚C in stationary

conditions and then diluted to 1:100 with fresh TSB.

Individual wells of sterile polystyrene 96 well flat

bottom microtitre plates were filled with 200μl aliquots

of diluted culture. Un-inoculated TSB served as a control

to check sterility and non specific binding of media.

Control strains were also inoculated in triplicate. The

microtitre plate was incubated for 24 hrs at 37˚C. After

incubation contents of each well was removed by tapping

the plates. After washing the wells for four times with

200μl of phosphate buffer saline (PBS pH 7.2), the

floating planktonic bacteria were removed. The biofilms

thus formed in plates were fixed using 2% w/v sodium

acetate for 10 minutes and tainted with 0.1% w/v crystal

violet for 30 minutes. After washing thoroughly with de-

ionized water to remove any excess stain, the plates were

dried. Micro-ELISA auto-reader at the wavelength of

540 nm was used to measure the Optical Density (OD) of

the stained adherent micro-organisms. The OD540 value

of sterile medium, fixative and dye were averaged and

subtracted from all test values. The mean OD540 value

from a control well was deducted from all test OD540

values. These OD540 values were considered as an index

of bacteria adhering to surface and forming biofilms.

Experiments were performed in triplicate. Interpretation

of biofilm production was done according to the criteria

of Stepanovie et al., (2007). (Table 1)

Tube method:

A quantitative method was used as described by

Christensen et al., (1982). Ten milli litre of BHI broth

with 1% w/v glucose was taken in test tubes and was

inoculated with loop full of microorganism from

overnight culture plates and incubated at 37˚C for 24 hrs.

The tubes were washed with PBS (pH 7.3) after

decanting the culture. The dried tubes were then stained

with crystal violet (0.1% w/v) for 30 minutes after fixing

with sodium acetate (2% w/v) for 10 minutes. Through

washing was again done with de-ionized water to remove

excess stain. Tubes were then kept in inverted position

for complete drying. Biofilm formation was detected by

the presence of visible film on the wall and bottom of the

tube. Ring formation at the liquid culture interface was

taken as negative. The amount of biofilm formation was

scored according to the results of control strains and

graded as 0, 1, 2 and 3 denoting absent, weak, moderate

and strong biofilm formation respectively. Experiments

were performed in triplicate.

Congo red agar method (Freeman et al., 1989):

Congo red media was prepared as a concentrated

aqueous solution of 0.8 g/l of Congo red and autoclaved

separately from other medium constituents [brain heart

infusion broth (37 g/l), sucrose (50 g/l), agar (10 g/l)];

then added when agar gets cooled to 55˚C. The required

microbial strains were inoculated on the prepared media

and incubated aerobically for 24 hrs at 37˚C. Growth of

black colonies with a dry crystalline consistency was

taken as positive biofilm production; pink colonies with

occasional darkening at the centre of the colonies were

non biofilm producers. Black colonies without dry

crystalline colonial morphology indicated indefinite

results. The experiment was performed in triplicate and

repeated for three times.


Saha et al., 2014

Average OD value Biofilm production

≤ OD540C/ OD540C < ~ ≤ 2x OD540C Non/weak

2x OD540C < ~ ≤ 4x OD540C Moderate

> 4x OD540C Strong

Table 1. Interpretation of biofilm production

Antimicrobial susceptibility testing was done

on Muller-Hinton agar (MHA) for both biofilm

producing and non biofilm producing bacterial isolates

by Kirby Bauer disk diffusion method as per Clinical and

Laboratory Standards Institute guidelines (CLSI, 2006).

The antibiotic panels used were 25μg Cotrimoxazole,

30μg Cefotaxime, 30μg Vancomycin, 300 units

Nitrofurantoin, 10μg Norfloxacin, 120μg High level

gentamicin, 30μg Tetracycline, 30μg Amikacin, 10μg

Gentamicin, 10μg Imipenam, 100μg Piperacillin; 10μg

Tazobactam and 300 units Polymyxin B . Antibiotics

discs were procured from HiMedia Laboratories Pvt. Ltd,

India.

Antifungal susceptibility profile of BF forming

and non biofilms forming Candida isolates was done by

determining MIC for Amphotericin B, Itraconazole and

Fluconazole by microdilution method as described by

CLSI guidelines (CLSI, 2008). Candida albicans ATCC

90028 were used as control.

Statistical Analysis:

Data entered in MS Excel and SSPS 17.0 were

used for data analysis. Chi square test was used to

compare proportions between various groups.

Sensitivity, Specificity and predictive values were

calculated using the standard formulae.

RESULTS

Among 130 urine samples from our study group,

55 samples grew microorganisms of significance of

which 11 samples were polymicrobial in nature. Of these

biofilm production was seen in 49 microorganisms

(89.09%) by any of the three methods used. All sets of

polymicrobial organisms were biofilm producers. All

comparisons were done keeping TCP as gold standard.

The different organism isolated and their biofilm

producing capacity is compared in Table 2.

TCP method picked up 69% (38) of biofilm

producers as compared to TM and CRM which picked up

only 36% (20) and 27% (15) of biofilm producers

respectively. This difference was found to be highly

significant (x2 = 17.55, P < 0.001). Table 3 shows

sensitivity and specificity of TM and CRM. By TCP

method, the number of strong biofilm producers were 20

Saha et al., 2014


Isolate TCP (%) TM (%) CRM (%) No BF producer (%)

Gram positive organism n-12 11(91.66) 2(16.66) 2 (16.66) 1 (8.33)

Staphylococcus aureus n = 8 7 1 2 1

Enterococcus sp n = 4 4 1 0 0

Gram negative organism n-37 24(64.86) 17(45.94) 12 (32.43) 4 (10.81)

Escherichia coli n = 20 13 11 5 3

Klebsiella sp n = 7 4 3 3 1

Citobacter sp n = 2 1 0 1 0

Proteus sp n = 2 1 1 2 0

Acinetobacter sp n = 2 2 1 0 0

Pseudomonas sp n = 4 3 1 1 0

Candida sp n-6 3 (50) 1(16.66) 1 (16.66) 1 (16.66)

Candida albicans n = 2 1 0 0 1

Candida tropicalis n = 4 2 1 1 0

Total n = 55 38(69.09) 20(36.36) 15 (27.27) 6 (10.90)

Table 2. Comparison of biofilm production by three methods – TCP, TM and CRM

Parameters TM CRM

Sensitivity 34.21% 21.05%

Specificity 58.82% 58.82%

Positive Predictive Value 65.00% 53.33%

Negative Predictive Value 28.57% 25.00%

Table 3. Diagnostic parameters TM and CRM for

Biofilm detection

and the same by TM and CRM was 3 and 14 respectively

and this difference was found to be highly significant

(x2 = 21.4, P < 0.001, d.f = 2). (Figure1). When degree

of biofilm production was compared, TM showed similar

detection rate with TCP for moderate biofilm producers,

but the same is not true for strong biofilm producers.

This difference was also highly significant.(x2 = 21.06,

P < 0.001, d.f = 1). Figure 2 shows colonies of biofilm

and non biofilm producers on Congo Red medium.

The antimicrobial resistance pattern of the

biofilm producing isolates is given in Table 4. Among

the gram negative organism, the resistance was more for

biofilm producers as compared to non biofilm producers

however it was not significant except for Cotrimoxazole

(x2 = 4.911, P = 0.0266).

Biofilm production has also increased

significantly with the days of catheterization (x2 = 16.88,

P < 0.001) (Figure 3).

DISCUSSION

More than 40% of all healthcare associated

infections are due to CAUTI. Eradication of biofilm

based catheter related infection is often challenging

because they exhibit increased resistance to antimicrobial

therapies by various mechanisms (Douglas, 2003).

Saha et al., 2014


Table 4. Comparison of antimicrobial resistance pattern of BF producer with

non BFproducers

Antimicrobial agents BF producer (%) Non BF producer (%)

Staphylococcus aureus n =8 n= 7 n=1

Cotrimoxazole 6(85.71) 1 (100)

Cefotaxime 5(71.42) 1 (100)

Vancomycin 0 0

Nitrofurantoin 3(42.86) 0

Norfloxacin 6(85.71) 0

Enterococcus n-4 n=4 n = 0

Vancomycin 1 (25) -

High level Gentamicin 4(100) -

Nitrofurantoin 2 (50) -

Norfloxacin 4(100) -

Tetracycline 4(100) -

Gram negative organism n=33 n= 21 n = 12

Amikacin 15 (71.43) 6 (50)

Gentamicin 15 (71.43) 6 (50)

Cotrimoxazole 18 (85.71) 6 (50)

Imipenam 7 (33.33) 1 (8.33)

Piperacillin-Tazobactam 15 (71.43) 5 (41.67)

Norfloxacin 17 (80.95) 8 (66.67)

Nitrofurantoin 13 (61.90) 4 (33.33)

Pseudomonas n = 5 n = 4 n = 1

Amikacin 3 (75) 1 (100)

Gentamicin 3 (75) 1 (100)

Imipenam 3 (75) 0

Piperacillin-Tazobactam 2 (50) 0

Polymyxin B 0 0

Norfloxacin 3 (75) 0

Candida spp n = 6 n=3 n=3

Fluconazole 2 (66.67) 1 (33.33)

Itraconazole 3 (100) 2 (66.67)

Amphotericin B 0 0

In this study we evaluated 55 isolates by three

different screening methods for their ability to form

biofilms. In our study we have found that TCP method

detected biofilm formation in 69% of isolates. We have

used 1% sucrose in BHI for growing biofilms in

microtitre plate. Addition of sugar increases the biofilm

production; as reported by other authors (Mathur

et al.,2006; Bose et al., 2009 ; Hassan et al., 2011).

Overall TCP method detected maximum biofilm

producers. The ability to detect biofilm production of

Gram Positive Cocci (GPC) was less for TM and CRM

method as compared to TCP method whereas TM and

CRM picked up greater number of biofilm producers

among the Gram negative bacilli (GNB). This difference

was however not significant (x2 = 197, P = 0.1226,

d.f = 2).

TM detected 36% of isolates as biofilm

producers while 63% isolates were identified as non

biofilm producers. TM is only 34.21% sensitive, 58.82%

specific for biofilm detection. This is not consistent with

the findings of Mathur et al., 2006; Bose et al., 2009

from India, who reported higher sensitivity and

specificity for Tube method. In our study, this method

correlated well with TCP for identifying moderate

biofilm producers (30.90% i.e. 17 / 55), but detection

rate for high biofilm producer was very low (5.45% i.e.

3/55). This difference may be due to the inter-observer

variability in the reading of results, resulting in low

sensitivity and specificity in our study.

Only 27% isolates were identified as biofilm

producers by CRM similar to Ruzicka et al., 2004 who

detected 43.5% of biofilm producers by this method.

This was higher in comparison to the 3-6% detection rate

by other workers from India and Pakistan (Mathur et al.,

2006; Bose et al., 2009; Hassan et al., 2011). The

sensitivity and specificity, however, remained low

(21.05% and 58.82% respectively). Surprisingly, in this

study CRM outscores TM in the detection of high

Saha et al., 2014


Figure 1 Degree of biofilm formation by TCP, TM and CRM

Figure 2. Colonies of biofilm and non biofilm

producers on Congo Red agar medium

biofilm producers. CRM detected 25.45% (14/55) while

TM detected 5.45% isolates as high biofilm producers

and this difference was highly significant. CRM is a

comparatively easier method and also over-rules

inconsistency by observation which could possibly

explain such finding.

The antimicrobial susceptibility pattern of

microbes isolated from clinical samples has important

implications especially in clinical settings as it helps

clinicians to decide treatment protocol for patients and

also help hospital infection control team to formulate

hospital antibiotic policies. As biofilms form significant

reservoir of infection, it is essential to find antibiogram

for biofilm producing isolates. In our study, we found

that biofilm producing gram negative isolates were more

resistant to antimicrobial agents as compared to non

biofilm producing isolates. However comparison could

not be done in case of Enterococci sp as all the isolates

produced biofilm and in case of Staphylococcus aureus

and Pseudomonas sp there were unequal distribution of

biofilm producing and non biofilm producing isolates.

More antimicrobial resistance among biofilm producers

has also been seen in other studies (Hassan et al., 2011;

Ruzicka et al., 2004). Some of the non biofilm producing

strains were also resistant to antimicrobial drugs. The

enhanced survival of drug resistant pathogens may be

due to the widespread injudicious use of broad spectrum

antibiotics in our setup, which is a tertiary care hospital.

CONCLUSIONS

The ability of microorganisms to form biofilms

on the medical devices is a challenge for the clinicians

because biofilm associated microorganisms are much

more resistant to antimicrobial agents, which may result

in treatment failure. Therefore effective treatment

strategies should be explored to deal such infections. Our

findings indicate that TCP is a suitable and reproducible

method for the screening of biofilm producers in health

care setups.

REFERENCES:

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Article Citation: Fernand-Nestor Tchuenguem Fohouo and Dounia. Foraging and pollination behavior of Apis mellifera adansonii Latreille (Hymenoptera: Apidae) on Glycine max L. (Fabaceae) flowers at Maroua. Journal of Research in Biology (2014) 4(1): 1209-1219

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Biology

Foraging and pollination behavior of Apis mellifera adansonii Latreille

(Hymenoptera: Apidae) on Glycine max L. (Fabaceae) flowers at Maroua

Keywords: Apis mellifera adansonii, Glycine max, flower, visit, nectar, pollination.

ABSTRACT: To assess the impact of Apis mellifera adansonii on pod and seed yields of Glycine max, its foraging and pollinating activities were studied in Maroua, during the two season seasons (August-September 2010 and 2011). Observations were made on 51 to 17866 flowers per treatment. Treatment 1 represented by free flowers; treatment 2 bagged flowers and treatment 3 flowers visited only by A. m. adansonii. In addition, all flower visitors were recorded. The abundance of bee, duration of visits, impact of activity of A. m. adansonii on fruiting percentage, the influence of this bee on formation of pods, number of seeds in each pods and average of normal seeds (well developed) were recorded. Individuals from 28 species of insects were recorded on the flowers of G. max, after two years of observations. Apis mellifera adansonii with 23.18% of 954 visits was the most frequent, followed by Polyrachis sp. 1 (14.77%), Macronomia vulpina (14.22%), Lipotriches collaris (11.07%). This honey bee intensely and exclusively foraged for nectar. The mean foraging speed was 12.56 ± 5.79 flowers per minute. Flowers visited by insects had higher fruiting rate compared with the others while those bagged had the lowest. Apis mellifera adansonii foraging resulted to a significant increment in fruiting rate by 14.14 and 11.98%, as well as the number of seeds per pod by 36.95 and 35.65%, and the percentage of normal seeds by 32.61 and 29.26% respectively in 2010 and 2011. The installation of A. m. adansonii colonies in G. max plantations is recommended to improve pod and seeds production of this species.

1209-1219 | JRB | 2014 | Vol 4 | No 1




An International


Authors:

Fernand-Nestor

Tchuenguem Fohouo1 and

Dounia1-2*.

Institution:

1. Laboratory of Zoology,

Faculty of Science,

University of Ngaoundéré,

Ngaoundéré, Cameroon.

2. Laboratory of Zoology, Higher Teacher Training

College, University of

Yaoundé I, Yaoundé,

Cameroon.


Dounia.

Email Id:


Dates: Received: 15 Jan 2014 Accepted: 04 Feb 2014 Published: 11 April 2014


Original Research

INTRODUCTION

Glycine max is an annual plant originated from

Northern and Central regions of China (Hymowitz, 1970).

The plant is an annual, herbaceous, erect, and can reach a

height of 1.5m; there are cultivars of soybean

indeterminate, determinate and semi-determinate growth

(Gallais and Bannerot, 1992). The first leaves are

simple, opposite and swallowed, while the following are

trifoliate and alternate; the pod is straight or slightly

curved, with a length of two to seven cm; the seed is

generally oval, but may vary depending on the cultivar,

almost spherical, elongated and flattened (Hymowitz and

Harlan, 1983). Flowers are grouped by two to eight on a

short racemes inserted on the stem axile sheets and are

purple or white (Boyeldieu, 1991). Each flower has a

tubular calyx of five sepals, a corolla of five petals, a

single carpel and ten stamens, nine of which being

welded and the tenth is free (Hymowitz and Harlan,

1983). Each flowers Produce nectar and pollen which

attract insects (Milfont et al., 2013). The reproduction

system is autogam/allogam (Ibarra-Perez et al., 1999).

Soybean is grown primarily for its seeds, which have

many uses in the food and industrial sectors (USDA,

2002). It is a major edible oil and vegetable sources of

protein (38-40%) for the feed of men and other animals

(Boyeldieu, 1991; Tien et al., 2002; USDA, 2002).

Currently the production of G. max in Cameroon is low

whereas the demand for seeds is high (MINADER,

2010). Therefore, it is important to investigate on the

possibilities of increasing the production of this plant

in the country. This can be done if flowering insects of

G. max in each region are well known and exploited

(Milfont et al., 2013). Unfortunately no research has

been reported on the relationships between G. max and

its anthophilous insects in Cameroon. In Maroua A. m.

adansonii visit flowers of G. Max (unpublished data), and

this study is carried out to assess the effects of foraging

activities of A. m. adansonii on yields of G. max.


Study site, experimental plot and biological material

The experimental is carried, from June to October,

in 2010 and 2011 at Mayel - Ibbé (Latitude 10° 62' N,

Longitude 14°33' E and altitude 400 m), Maroua, Far

North Region of Cameroon. This Region belongs to the

Savanna zone, with unimodal rainfall (Letouzey, 1985).

It has a Sahel-Sudanian climate type, characterized by

two seasons: a more extended dry season (November to

May) and a brief rainy season (June to October) (Kuete

et al., 1993). The maximum rainfall and

temperature are 1100mm and 38°C respectively

(Kuete et al., 1993). The experimental plot was 28m x

5m. The biological material was represented by Apis

mellifera adansonii Latreille (Hymenoptera: Apidae),

and others insects present in the environment. Seed of

G. max was provided by the Institute of Agricultural

Research for Development (IARD).

Sowing and weeding

On th e June 12, 2010 and June 15, 2011, the

experimental plot was cleaned and divided into 24

subplots, each measuring 1m × 1m. Sowing and weeding

was done as described by Douka and Tchuenguem

(2013).

Determination of the reproduction system of Glycine

max

On July 22, 2010, eight subplots carrying 106

plants with 34395 flowers at the bud stage were labeled.

Four subplots carrying 80 plants with 17187 flowers

were left to be open pollinated (treatment 1) (figure 1)

and four subplots carrying 17208 flowers were

protected with gauze mesh prevent to insect or other

pollinating animals vi s i t s (treatment 2) (figure 2).

On July 28, 2011, the experiment was repeated, for

treatment 1 four subplots carrying 80 plants with 17866

flowers and four treatment 2 four subplots carrying 80

plants with 15875 flowers.

Twenty days after shading of the last flower,

the number of pods was assessed in each treatment.

Fohouo and Dounia, 2014


The podding index (Pi) was then calculated as described

by Tchuenguem et al., (2004): Pi = F2/F1, where F2 is

the number of pods formed and F1 the number of

viable flowers initially set.

The allogamy rate (Alr) from which derives the

autogamy rate (Atr) was expressed as the difference in

podding indexes between treatment 1 (unprotected

flowers) and treatment 2 ( bagged flowers) as follows

(Demarly, 1977):

Alr = [(Pi1 - Pi2) / Pi1] × 100, Where Pi1 and Pi2

are respectively the podding average indexes of

treatments I and II. Atr = 100 – Alr.

Study of the foraging activity of Apis mellifera

adansonii on Glycine max flowers

The frequency of A. m. adansonii in the flowers

of G. max was determined based observations on

flowers of treatments 1 in 2010 and 2011. Experience

were made on 17187 individual opened pollinated

flowers (treatment 1) each day, from July 26 to August

20, 2010 and from August 2, to August 24 , 2011 at

7 – 8 h, 9 – 10 h, 11 – 12 h, 13 – 14 h, 15 – 16 h and 17

– 18 h. Capture and determination of insects that visited

G. max flowers was realize as described by Borror and

White (1991).

The determination of the relative frequency of all

insects visit the G. max flowers was calculated

(Tchuenguem, 2005).

During the same time that A. m. adansonii

encountered on flowers were registered, the types of

floral products collected by this bee were noted. This

parameter was measured to determine if A. m. adansonii

is strictly a pollenivore, nectarivore or pollenivore and

nectarivore. This could give an idea on its implication as

a cross pollinator of G. max.

In the morning of each day, the number of

opened flowers was counted. The determination of

frequency of visits, the duration of A. m. adansonii on

the flower of G. max was recorded according to

Tchuenguem (2005). The number of pollinated visits,

the abundance of foragers, the number of flowers

visited by A. m. adansonii per minute was recording

every day of observation. The method of observation was

followed as given by Tchuenguem et al., (2004).

The foraging speed was calculated according to

Jacob – Renacle (1989) by this formula: Vb = (Fi/di) x

60 where di is the time (s) given by a stopwatch and Fi is

the number of flowers visited during di. The

interaction between A. m. adansonii and the

competitors and the attractiveness exerted by the flower

of other plant species around the experimental plot on

A. m. adansonii were recorded (Tchuenguem et al.,

2004). The climatic factor (temperature and humidity)



Figure 1. Glycine max plot showing unprotected

plants in bloom.

Figure 2. Glycine max plot showing isolated

plants in bloom.

was registered as described by Douka and Tchuenguem

(2013).

Evaluation of the impact of Apis mellifera adansonii

and other insects on Glycine max yields

This evaluation was based on the impact of

visiting flowers on pollination, the impact of pollination

on fructification of G. max, and the comparison of

yields [fruiting rate, mean number of seeds per pod and

percentage of normal (well developed) seeds] of

treatments 1 and 2. The fruiting rate due to the activity

of insects (Fri) was calculated as follows by Tchuenguem

et al., (2004): Fri = {[(Fr1– Fr2) / Fr1] × 100}

Where Fr1 and Fr2 are the fruiting rate in treatments

1 and 2.

The fruiting rate (Fr) is: Fr = [(F2/F1) × 100]

Where F2 is the number of pods formed and F1 the

number of flowers initially set.

At maturity, pods were harvested from each

treatment. The mean number of seeds per pod and the

percentage of normal seeds were then calculated for

each treatment.

Evaluation of the pollination efficiency of

Apis mellifera adansonii on Glycine max

In 2010, along with the development of

treatment 1 and 2, 11 plants belonging to four subplots

and carrying 47 flowers were protected using gauze mesh

(treatment 3). In 2011 the same experience was repeated

but on 16 plants carrying 51 flowers. Between 7 and

9am, of each observation date, the evaluation or the

efficiency pollination of A. m. adansonii on G. max

was realized as according of Douka and Tchuenguem

(2013). The impact (Frx) of A. m. adansonii to fruiting

rate was calculated as follows by Tchuenguem et al.,

(2004) the formula:

Frx = {[(Fr3– Fr2) / Fr3] x 100}

Where Fr3 and Fr2 are the fruiting rates in treatment

3 (protected flowers visited exclusively by

A. m. adansonii) and treatment 2 (protected flowers).

The number of seeds per pod, the percentage of normal

seeds (well developed) was then calculated for each

treatment 3.

Data analysis

Data were analyzed using descriptive statistics,

student’s t-test for the comparison of means of the two

samples, correlation coefficient (r) for the study of the

association between two variables, chi-square (χ2) for

the comparison of two percentages using SPSS statistical

software and Microsoft Excel.

RESULTS

Reproduction system of Glycine max

According to table 2 : the allogamy rate was

6.59% and 5.38% respectively in 2010 and 2011 and

autogamy rate was 93.41% and 94.62% respectively in

2010 and 2011. Glycine max (used in our experiments)

has a mixed reproduction system autogamous -

allogamous, with the predominance of autogamy.

Frequency of A. m. adansonii in the floral entomofauna

of Glycine max

Among the 532 and 422 visits of 24 and 24

insect species counted on G. max flower in 2010 and

2011, respectively, A. m. adansonii was the most

r ep r e s en t ed insect with 132 visits (24.81 %) and 91

visits (21.56 %), in 2010 and 2011, respectively. The

difference between these two percentages is not

significant (χ2 = 1.39‚ df = 1‚ p > 0.05) (Table 1). In

2010, the highest mean number of A. m. adansonii

simultaneously in activity was one per flower (n = 50; s

= 0) and 2.88 per 1000 flowers (n = 60; s = 3.53; maxi

= 19). In 2011, the corresponding figures were one per

flower (n = 50; s = 0) and 1.97 per 1000 fl owers (n

= 60; s = 2.59; maxi = 12). The difference between the

mean number of foragers per 1000 flowers in 2010 and

2011 was highly significant (t = 9.19; df = 118, p <

0.001).

Activity of Apis mellifera adansonii on Glycine max

Floral reward harvested

During each of the two flowering periods, A. m.



adansonii was found to collect nectar intensively and

exclusively (Figure 3).

Relationship between visits and flowering stages

Visits were most numerous when the number of

open flowers was highest (Figure 4) Furthermore a

positive and significant correlation was found between

the number of G. max opened flowers and the number

of A. m. adansonii visits in 2010, as well as 2011

(r2010 = 0.90; df = 8; p < 0.05; r2011 = 0.85; df = 8; p <

0.05). Apis mellifera adansonii foraged on G. max

flowers throughout the blooming period, with a peak of

activity situated between 10 and 11am (Figure 5).

Duration of visits per flower

In 2010 and 2011, the mean duration of A. m.

adansonii visit is 2.50 sec (n = 132; s = 1.34; maxi = 6

sec) and 2.61 sec (n = 91; s = 1.40; maxi = 6 sec)

respectively. The difference between the duration of the

visit in 2010 and 2011 is higher significant (t = 22.25;

df = 221, p < 0.001). For the two cumulated years‚ the

mean duration of a flower visit were 2.55 sec.

Foraging speed of Apis mellifera adansonii on Glycine

max flowers

On the pot of G. max, A. m. adansonii visited

between 4 and 24 flowers/min in 2010 and between five

and 25 flowers/min in 2011. The mean foraging speed

was 11.65 flowers/min (n = 50; s = 5.77) in 2010 and

13.48 flowers/min (n = 50; s = 5.82) in 2011. The

difference between these means is highly significant (t =

- 7.95; df = 98, p < 0.001). For the two cumulated years‚

the mean foraging speed was 12.56 flowers /min.

Effect of climate on foraging activity of Apis mellifera

adansonii on Glycine max flowers

Climatic condition seem not to influence the

activity of A. m. adansonii. T he correlation was

negative and not significant (r2010 = - 0.34; df = 11; p

> 0.05 and r2011 = 0.28; df = 11; p > 0.05) between the

number of A. m. adansonii visits on G. max flowers

and the temperature. It was positive and not significant

(r 2010= 0.48; df = 11; p > 0.05 and r2011 = 0.07; df = 11;

p > 0.05) between the number of A. m. adansonii visits

and relative humidity (Figure 6).

Impact of anthophilous insects on pod formation and

seed yields of Glycine max

During nectar harvest on G. max, foraging

insects always shook flowers and are regularly in contact



Figure 3. Apis mellifera adansonii collecting nectar in

a flower of Glycine max

Figure 4: Variation of number of flowers and

number of visits of Apis mellifera adansonii on the

flowers of Glycine max in 2010 and 2011.

Figure 5. Variation of number of flowers and visits of

Apis mellifera adansonii on the flowers of Glycine

max according to daily time in 2010, 2011.

with the anthers and stigma (Figure 3), increasing cross

pollination possibility of G. max fruiting rate, number of

seeds per pod and percentage of normal seeds in different

treatments (Table 2).

a - The difference observed was highly

significant between fruiting rate of free opened flowers

(treatment 1) and that of bagged flowers (treatment 2),

the first year (χ2 = 248.73, df = 1, p < 0.001) and the

second year (χ2 = 299.84, df = 1, p < 0.001). T he

fruiting rate of t r e a t m e n t 1 ( unprotected flowers)

was higher than treatment 2 (protected flowers) in 2010

and in 2011. The fruiting rate due to the action of insects

was 5.92 and 5.81% in 2010 and 2011 respectively.

For the two cumulated years, the fructification rate due to

the influence of insects was 5.86%.

b - For the mean number of seeds per pod,

there was a highly significant difference between

treatments 1 and 2 (t2010 = 4315.78; df = 30462; p <

0.001; t2011 = 5958.33; df = 30670; p < 0.001).

Consequently, a high mean number of seeds per pod in

treatment 1 (opened flowers) were noticed compared to

treatments 2 (bagged flowers). The number of seeds per

pod attributed to the activity of insects was 26.11% in

2010 and 36.47% in 2011, giving an overall mean of

31.29%.

c - There was a highly significant difference

between the percentage of normal seed of treatment 1

and that of treatment 2 in the first year (χ2 = 4329.98; df

= 1; p < 0.0001) as well as the second year (χ2 =

6094.38; df = 1; p <0.0001). Thus, the percentage of

normal seeds in opened flowers was higher than that of

protected flowers in 2010 and 2011. The percentage of

the normal seeds due to the action of insects was 24.81%

in 2010 and 20.90% in 2011. For all the flowers studied,

the percentage of the normal seeds due to flowering

insects was 22.85%.

Pollination efficiency of Apis mellifera adansonii on

Glycine max

Apis mellifera adansonii foragers were always

in contact with the stigma and the anthers of G. max

(contacts with anthers and stigma was 100% for all

visits). C o n s e q u e n t l y t his bee increased

possibilities of the pollination of G. max flowers.

a - the difference observed between the fruiting

rate of treatments 2 and that of treatment 3 was highly

significant in 2010 (χ2 = 7.73; df = 1; p < 0.001) as

well as 2011 (χ2 = 6.93; df = 1; p < 0.001). The

fruiting rate of flowers exclusively visited by A. m.

adansonii (treatment 3) was higher than those of bagged

flowers (treatment 2). The fruiting rate due to A. m.

adansonii activity was 14.14% and 11.98% respectively

in 2010 and 2011. The percentage of the fruiting rate

due to A. m. adansonii activity was 13.06 %

b - There was a highly significant difference

between treatments 2 and 3 (t = 64.76; df = 14821; p <

0.001) the first year and the second year (t = 49.28; df =

14023; p < 0.001). High mean number of seeds per pod

of flowers of treatment 3 was noticed compared to

flowers of treatment 2. The augmentation of the number

of seeds per pod due to A. m. adansonii was 36.95% and



Figure 6. Daily distribution of A. m. adansonii visits on 17187 and 17866 G. max flowers over 10 days in 2010

(A) and 10 days in 2011 (B) respectively, mean temperature and mean humidity of the study site.

B



Insects 2010 2011

Order Family Genus, species, sub-species n1 p1% n2 p2%

Hymenoptera Apidae Apis mellifera adansonii n 132 24.81 91 21.56

Amegilla sp. 1 n 4 0.75 0 0

Xylocopa sp. 1 n 3 0.56 1 0.24

Halictidae Macronomia vulpina n 87 16.35 51 12.09

Lipotriches collaris n 56 10.53 49 11.61

Megachilidae Chalicodoma sp.1 n 13 2.44 2 0.47

Megachile sp. 1 n 3 0.56 1 0.24

Megachile sp. 2 n 0 0 4 0.95

Formicidae Polyrachis sp. 1 sh 79 14.85 62 14,69

Vespidae Synagris cornuta n 11 2.07 4 0.95

(1 sp.) n 1 0.19 0 0

Sphecidae Philanthus triangulum pr 6 1.13 2 0.47

(1 sp.) pr 1 0.19 0 0

Lepidoptera Pieridae Catopsilia florella n 28 5.26 29 6.87

(sp. 1) n 17 3.20 8 1.90

(sp. 2) n 12 2.26 3 0.71

Nymphalidae (1 sp.) n 19 3.57 23 5.45

Acraeidae Acraea acerata n 13 2.44 17 4.03

Diptera Muscidae Musca domestica n 26 4.89 49 11.61

Drosophilidae Drosophila sp. 1 n 12 2.26 8 1.90

Syrphidae (1 sp.) n 2 0.38 3 0.71

Calliphoridae (1.sp.) n 3 0.56 0 0

Hemiptera Coreidae Anoplocnemis curvipes n 1 0.19 1 0.24

Pyrrhocoridae Dysdercus voelkeri n 1 0.19 2 0.47

Orthroptera (sp.1) lv 0 0 5 1.18

(sp.2) lv 0 0 2 0.47

Nevroptera (sp.1) pr 2 0.38 1 0.24

(sp.2) pr 0 0 4 0.95

Total 28 species 532 100 422 100

Table 1. Diversity of floral insects on Glycine max in 2010 and 2011, number and

percentage of visits of different insects

Comparison of percentages of Apis mellifera adansonii visits for two years: χ2 = 1.39 ([df = 1; P > 0.05]).

n1: number of visits on 17187 flowers in 10 days.

n2: number of visits on 17866 flowers in 10 days.

p1 and p2: percentages of visits.

p1 = (n1 / 532) x 100. p2= (n2 / 422) x 100.

n: Visitor collected nectar.

lv: Visitor eating leaves.

sh: visitor shelter

pr: Predation.

sp.: Undetermined species.

35.65% respectively in 2010 in 2011. The percentage of

the mean number of seeds per pod attributed to the

activity of A. m. adansonii was 36.30%.

c - There was highly significant difference

between the percentage of normal seed of treatment 3

and that of treatment 2 in first year (χ2 = 67.76; df = 1;

p < 0.001) as well as the second year (χ2 = 58.58; df

= 1; p < 0.001). The percentage of normal seeds in

treatment 3 was higher than in treatment 2. The

percentage of the normal seeds due to A. m. adansonii

was 32.61% in 2010 and 29.26% in 2011. T he

percentage of the number of seeds per pod attributed to

the activity of A. m. adansonii was 30.93%.

DISCUSSION

Honey bee was the main floral visitor of

G. max during the observation period. This bee has

been reported as the main floral visitor of this Fabaceae

in USA (Rortais et al., 2005) and Brazil (Milfont et al.,

2013). Apis mellifera adansonii was also shown to be the

most abundant floral visitors of other Fabaceae members

such as Phaseolus coccineus in Yaoundé, Cameroon

(Pando et al., 2011a), and Phaseolus vulgaris in

Ngaoundéré, Cameroon (Kingha et al., 2012) and in

Maroua by Douka and Tchuenguem (2013). The

significant difference between the percentages of A. m.

adansonii visits for the two studied years could be

attributed to the variation of the number of colonies of

this honey bee around the experimental site. The peak of

activity of A. m. adansonii on G. max flowers was at

between 10 and 11am, which correlated to the period

of highest availability of nectar on G. max flowers. The

positive and highly significant correlation between the

number of G. max flowers and the number of A. m.

adansonii visits indicates the attractiveness of G. max

nectar with respect to this bee. The significant

difference observed between the duration of visits in

2010 and 2011 could be attributed to the availability of

nectar, the floral morphology of this crop or the variation

in the diversity of flowering insects from one year to

another. At Maroua in 2010 and 2011 (in the rainy

season), A. m. adansonii intensely and regularly

harvested nectar on the flowers of G. max during

flowering periods. This could be attributed to the needs

of colonies during the flowering period. During our

investigations, the interruption of visits by other insects

or the same honey bee reduced the duration of A. m.

adansonii visits. Similar results were found in

Cameroun by Tchuenguem et al., (2009b) and Douka

and Tchuenguem (2013) on flowers of Vigna

unguiculata (L.) (Fabaceae) and Phaseolus vulgaris

(Fabaceae) respectively. I t indicates that

A. m. adansonii can increased the possibility of

pollination of G. max flowers. During the collection of



Treatment Year Flowers Pods Fruiting rate Seeds / Pod Total

seeds

Normal

seed

% normal

seed Mean sd

Unlimited visits 2010 17187 15688 91.28% 3.14 1.42 48853 42609 87.22

Protected plot 2010 17208 14776 85.87% 2.32 1.01 34162 22415 65.61

Protected plot 2011 17866 16697 93.46% 3.92 2.06 66020 57137 86.54

Bagged flowers 2011 15875 13974 88.03% 2.49 1.52 63176 43250 68.46

A. m. adansonii 2010 47 47 100.00% 3.68 1.84 152 148 97.37

A. m. adansonii 2011 51 51 100.00% 3.87 1.88 217 201 92.63

Table 2. Glycine max yields under pollination treatments.

nectar, A. m. adansonii foragers regularly come into

contact with the stigma and carry the pollen to the anthers

for stigma. The weight of A. m. adansonii shoot the

fl owers of G. max dur ing nectar collection and this

movement played a positive role in liberation of pollen

by anthers for the optimal occupation of the stigma.

This phenomenon was also reported by Ahrent and

Caviness (1994) and Rortais et al., (2005) on G. max.

Thus in addition to their direct pollination role,

A. m. adansonii foragers also indirectly effected self-

pollination and cross-pollination of G. max flowers. The

positive and significant contribution of A. m. adansonii

in pods, seed yields and percentage of normal seeds of

G. max is justified by the action of this bee on

pollination. The similar have been obtain in Britain

(Kendall and Smith, 1976) on Phaseolus coccineus

(Fabaceae), USA (Ibarra-Perez et al., 1999) on

Phaseolus coccineus (Fabaceae), Ngaoundéré

(Cameroon) (Kingha et al., 2012) on Phaseolus vulgaris

(Fabaceae), Maroua (Cameroon) (Douka and

Tchuenguem, 2013) on Phaseolus vulgaris (Fabaceae)

and Brazil (Milfont et al., 2013) on G. max who showed

that these plants species produce fewer seeds per pod in

the absence of efficient pollinators. The higher

percentage of pods, seeds and normal seeds in the

treatment with unlimited visits or treatment visiting

exclusively by A. m. adansonii compared to treatment

with protected, showing that insect visits were effective

in increasing cross-pollination or self- pollination.

Our results confirmed those of Caviness (1970), Ahrent

and Caviness (1994), Rortais et al., (2005) and Milfont

et al., (2013) who revealed that G. max flowers set little

pods in the absence of insect pollinators. Similar

experiments on cr op sp eci e s r ea l i z ed in England

(Free, 1966) and in Brazil (Free, 1993) have shown that

pollination by insects was not always needed.

Woodworth (1922) showed that self-pollination of

G. max flowers produced as many pods and seeds as

exposed plants. Thus, pollination requirements may

differ between plant varieties and /or region.

CONCLUSION

This study reveals that t h e va r i e t y o f G. max

studi ed is a nectariferous bee plant that obtained

benefits from the pollination by insects among which A.

m. adansonii is the must important. The comparison of

pods and seeds set of unprotected flowers with that of

flowers visited exclusively by A. m. adansonii

underscores the value of this bee in increasing pods and

seed yields as well as seed quality. The installation of

A. m. adansonii co l on i e s to G. max fields should

be recommended for the increase of pod and seeds

yields of this valuable crop.

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bodies of its pollinators, Apis mellifera and Centris

tarsata. J. Apicul. Res. 36 (1): 15-22.

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végétales cultivées. INRA, Paris.p.768.

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soybean. Econ. Bot. 24 (4): 408-421.

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to North America by Samuel Bowen in 1765. Econ. Bot.

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Waines JG. 1999. Effects of insect tripping on seed

yield of common bean. Crop Sci. 39 (2): 428-433.

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l’abeille domestique et des abeilles sauvages dans des

vergers de pommiers en Belgique. Apidologie. 20 (4):

271-285.

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of honey bee and Bumblebee visits to flowers of the

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13 (3): 749-752.

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Nouvelle géographie du Cameroun. EDICEF, Paris. p.

207.

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Brückner D. 2012. Foraging and pollination activities

of Xylocopa olivacea (Hymenoptera, Apidae) on

Phaseolus vulgaris (Fabaceae) flowers at Dang

(Ngaoundere-Cameroon) J. Agric. Extension and Rural

Development. 4 (10): 330-339.

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phytogéographique du Cameroun au 1/500000. Inst.

Carto. Intern. Végétation, Toulouse et Inst. Rech.

Agron., Yaoundé.

Milfont MO, Rocha EEM, Lima AON, Freitas BM.

2013. Higher soybean production using honeybee and

wild pollinators, a sustainable alternative to pesticides

and autopollination. Environ. Chem. Lett. 11(4) :335-

341. Doi 10.1007/s10311-013-0312 .8.

Minader. 2010. Annuaire des Statistiques du Secteur

Agricole, Campagnes 2007 & 2008. Direction des

Enquêtes et des Statistiques, Agricoles. AGRI-STAT

Cameroun n° 16, p.98.

Pando JB, Tchuenguem FF-N, Tamesse JL. 2011a.

Foraging and pollination behaviour of Xylocopa calens

Lepeletier (Hymenoptera: Apidae) on Phaseolus

coccineus L. (Fabaceae) flowers at Yaoundé

(Cameroon). Entomol. Res. 41(5): 185-193.

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2005. Modes of honeybees exposure to systemic

insecticides: estimated amounts of contaminated pollen

and nectar consumed by different categories of bees.

Apidologie., 36 (1): 71-83).

Tchuenguem FF-N, Messi J, Brückner D, Bouba B,

Mbofung G, Hentchoya HJ. 2004. Foraging and

pollination behaviour of the African honey bee (Apis

mellifera adansonii) on Callistemon rigidus flowers in

Ngaoundéré (Cameroon). J. Cam. Acad. Sci.

4(2): 133-140.

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unguiculata L. Walp.) to the foraging activity of Apis



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8 (9): 1988-1996.

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inoculants and nitrogen Fixation of legumes in

Vietnam”. Edited by D. Herridge. ACIAR proceedings

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Article Citation: Mustafa Korkmaz and Hasan Ozçelik. Determining the Natural Gypsophila L. (Coven) Taxa Growing in Tunceli (Turkey). Journal of Research in Biology (2014) 4(1): 1220-1227

Jou

rn

al of R

esearch

in

Biology

Determining the Natural Gypsophila L. (Coven) Taxa Growing

in Tunceli (Turkey)

Keywords: Coven, Gypsophila, Habitat, Biodiversity, Tunceli, Turkey.

ABSTRACT: 56 species belonging to 60 taxa (out of 126 species in the World) of Caryophyllaceae family grows naturally in Turkey with Gypsophila sps L. as the third largest genus. The endemism ratio of the genus is 60% in Turkey. Because Turkey is the gene center of Gypsophila and economically very valuable; determining the geographic distribution and biological characteristics of the taxa is very necessary. They have well-developed roots, that prevent soil erosion. Because of containing saponin (10-25 %) in their root, its extract is used as fire extinguisher, gold polisher, cleaner and softener of delicate fabrics and crispness giving substance for halva. It is also used for making liqueur, herbal cheese, ice cream and some other foods. Some taxa are boron hyper acumulators and vegetative mining can be conducted by hyper accumulation. They are also thought to be the cleaning tools for toxid areas by fitoremediation. In this study, 12 records from eight Gypsophila taxa were collected around Tunceli. These are G. aucheri Boiss. (1), G. elegans Bieb. (1), G. pallida Stapf. (2), G. perfoliata L. var. perfoliata (1), G. ruscifolia Boiss. (3), G. sphaerocephala Fenzl ex Tchihat var. cappadocica Boiss. (1), G. venusta Fenzl (1) and G. viscosa Murray (2). With addition of G. briquetiana Schischk. and G. hispida Boiss. the total number is reaching to 10 and it shows that the city is an important diversity center of the genus. G. aucheri, G. briquetiana and G. sphaerocephala var. cappadocica are endemic to Turkey and G. pallida, G. perfoliata L. var. perfoliata, G. venusta and G. viscosa are determined to be new records for Tunceli.

1220-1227 | JRB | 2014 | Vol 4 No 1



www.jresearchbiology.com

Journal of Research in Biology

An International


Authors:

Mustafa Korkmaz1* and

Hasan Ozçelik2.

Institution:

1. Erzincan Üniversity,

Science and Arts Faculty,

Department of Biology,

Erzincan-Turkey.

2. Süleyman Demirel

Üniversity, Science and Arts Faculty, Department of

Biology, Isparta-Turkey.


Mustafa Korkmaz.

Email Id:


Dates: Received: 04 Feb 2014 Accepted: 05 Mar 2014 Published: 16 April 2014


Original Research

INTRODUCTION

Caryophyllaceae family distributes mostly in

Mediterranean region of southern hemisphere. It has a

large diversity with over 2000 species. Gypsophila L.

genus, which has 126 species on the World, has natural

distribution in the Irano-Turanian and Mediterranean

phytogeographic regions (Williams, 1989; Sumaira et al.,

2008). There are about 500 species of Caryophyllaceae

family in Turkey. More than half of totally 126

Gypsophila species in the world are found in Caucasian,

the North Iraq and the North Iran regions. There are

about 56 Gypsophila species found in Turkey. Many of

them are known from the type collection. G. heteropoda

Freyn & Sint. subsp. minutiflora Bark. is a rare endemic

taxon peculiar to Cappadocica sub region in Inner

Anatolia of Turkey and an endangered taxa on global

scale (Ekim et al., 2000; Ozhatay et al., 2005).

Gypsophila L. is the third biggest genus of

Caryophyllaceae family after Silene L. and Dianthus L.

(Davis, 1967; Davis et al., 1988; Güner et al., 2000;

Çelik et al., 2008; Korkmaz and Özçelik, 2011b).The

most important factor for the distribution of this genus is

the soil structure which contains gypsum, lime and

calcium; these are important for these plants to grow.

There are gypsum habitats around Sivas, Çankırı,

Çorum, Ankara, Eskişehir, Niğde and Erzincan. Because

of that, Gypsophila taxa are rich in these areas.

Soap root has been exported from Anatolia for a

long time. The collection of coven from natural habitats

and extraction have been increasing rapidly especially in

the Eastern and South-east Anatolia for nearly 40 years

(Kılıç, et al., 2008). In Turkey Gypsophila taxa are

generally known by the name “Çöven Otu” and they are

mostly used by the public for different purposes. The

word “Soaproot” or “Soapworth” terms are used for

Gypsophila species; in Europe the members of the genus

are widely known as “Baby’s Breath”. In Turkey the

plants are also called “Dişi Çöven, Tarla Çöveni, Helva

Çöveni, Şark Çöveni” by the local people (Kılıç, et al.,

2008; Korkmaz et al., 2010; Korkmaz and Özçelik,

2011a).

Turkish Covens are commonly obtained from

Gypsophila graminifolia Bark. G. arrostii Guss.var.

nebulosa (Boiss. and Heldr.) Bark., G. eriocalyx Boiss.,

G. bicolor (Freyn&Sint.) Grossh., G. perfoliata L.,

G. venusta Fenzl subsp. venusta and Ankyropetalum

gypsophiloides Fenzl. (İnan, 2006; Kılıç, et al., 2008).

G. ruscifolia Boiss. and G. bitlisensis Bark. are the least

preferred species. The most preferred species are

G. bicolor, G. arrostii and A. Gypsophiloides (Baytop,

1984; Özçelik, and, Özgökçe, 1999; Korkmaz and

Özçelik, 2011a).

Saponin chemical was first produced from the

roots of Saponaria officinalis (Baytop, 1984). The

amount of saponin in the roots of Gypsophila taxa differs

from 4 % to 25 % (Sezik, 1982). Gypsophila bicolor

(Van Çöveni), G. arrostii var. Nebulosa (Beyşehir,

Isparta Çöveni), G. perfoliata (Niğde Çöveni),

G. venusta subsp. Venusta and G. eriocalyx

(Çorum-Yozgat Çöveni) are most preferred taxa for

obtaining coven extract in Turkey (H´eroldand Henry,

2001; Battal, 2002).

Soap root extract is composed of sugar, resin and

saponin. It protects the plant from germ and fungal

infection, increases the nutritive value and facilitates the

digestion. The production phases of the extract starts

with cutting the roots in the form of chips and continuous

with boiling them for two times. After second boiling

stage the extract can be obtained. (Korkmaz et al., 2010;

Korkmaz and Özçelik, 2011a).

The main areas of the use of them are in the food

industry, the chemistry, in hygiene industry, in

horticulture, in mining, in whitening gold and in fire

extinguishers. They have antimicrobial effect and used in

medicines. Every year the average export of soap root

from Turkey is about 90 tones by gaining approximately

66 000 US Dollars (Baytop, 1984; Korkmaz and Özçelik,

2011a; Özçelik and Özgökçe, 1996).

Korkmaz and Ozçelik, 2014


This study was aimed to determine the Gypsophila taxa

naturally distribute in the province of Tunceli city of

Turkey.


Material of this study contains Gypsophila taxa

growing around Tunceli. With regard to this aim we have

collected eight taxa of the genus from 13 different

localities in the area. Collection date, record number,

habitat types and some other properties of the identified

taxa were determined (and given in Table 1). For the

identification of taxa Flora of Turkey and the East

Aegean Islands (Davis, 1967) has been used extensively.

Identifications were done with the help of stereo-zoom

microscope. Identified samples were converted to

herbarium specimen. Economic importance of the taxa is

given according to our early papers (Özçelik and

Özgökçe, 1999; Korkmaz et al., 2010; Korkmaz and

Özçelik, 2011a,b).

As it is given in the Table-2, endemic taxa and

the risk categories, phytogeographic regions, altitudes,

life forms and new records have been determined.

Turkish names of Gypsophila taxa grows around Tunceli

have been determined from Türkiye Bitkileri Listesi

(Güner et al., 2012) as they were given in Table 2.

Endemic taxa of the genus and their threat categories

have been determined from Ekim et al. (2000) and given

in the same table.



No Taxon Record

number Date Locality Habitat

1 G. aucheri Boiss. K: 1769 03.07.2009 Tunceli: Tunceli-Pertek, 10 km

to Pertek

Rocky places

2 G. elegans Bieb. K: 1741 02.07.2009 Tunceli: Erzincan- Pülümür,

near to Pülümür

Rocky places

3 G. pallida Stapf.

K: 1740 02.07.2009 Tunceli: Erzincan- Pülümür,

near to Pülümür

Rocky places

K: 1748 02.07.2009 Tunceli: Tunceli- Ovacık, 40

km to Ovacık

Inclined slopes

4 G. perfoliata L. var.

perfoliata

K: 1745 02.07.2009 Tunceli: Pülümür-Tunceli, near

to Pülümür

Rocky slopes

5 G. ruscifolia Boiss.

K: 1746 02.07.2009 Tunceli: Pülümür-Tunceli, 30

km to Tunceli

Rocky slopes

K: 1760 02.07.2009 Tunceli: Tunceli-Ovacık, 10 km

to Ovacık

Flowing slopes

K: 1761 02.07.2009 Tunceli: Ovacık, Munzur

Çayı Gözeleri

Rocky places

6 G. sphaerocephala

Fenzl ex Tchihat var.

cappadocica Boiss.

K: 2588 12.06.2011 Tunceli-Erzincan, Munzur

Mountain

Rocky slopes

K: 2638 11.07.2011 Tunceli-Erzincan Munzur

Mountain

Slopes

7 G. venusta Fenzl K: 1749 02.07.2009 Tunceli: Tunceli- Ovacık, 25

km to Ovacık

Rocky slopes

8 G. viscosa Murray K: 1750 02.07.2009 Tunceli: Tunceli Ovacıkarası,

25 km to Ovacık

Rocky slopes

K: 1752 02.07.2009 Tunceli: Tunceli-Ovacık, 10 km

to Ovacık

Rocky places

Table 1. Locality and habitat informationof Gypsophila taxa collected around Tunceli

K: Korkmaz


The results of the study are summarized in Table

-1 and Table-2. As seen in Table-1, 8 Gypsophila taxa

were collected from the area in 2009 and 2011. All of the

plant samples were collected from Pülümür, Tunceli,

Ovacık and Munzur Mountains. Generally, the collected

plants are naturally grown in rocky and slopy places.

Photograph of all collections were taken during the field

work. Totally 8 Gypsophila taxa were collected from 13

different localities. As seen in Table-2 there are 10

Gypsophila taxa determined in the flora of Tunceli.

G. aucheri, G. briquetianaand G. sphaerocephala var.

cappadocica are endemic taxa available in the vicinity.

Threat (risk) category of G. aucheri is Vulnerable (VU)

and the other two taxa is at the category of Low Risk

(LR). Flowering periods of the taxa changes from April

to August. All of the determined taxa are Irano-Turanian

phytogeographic region elements and distributes from

800 to 2500 m altitudes in the area. G. elegans

and G. viscose are annual life forms and the others are

perennial life forms. G. aucheri, G. briquetiana,

G. elegans, G. hispida, G. ruscifolia and


Table 2. Taxonomic information of Gypsophila taxa growing around Tunceli

No Taxon name

(Turkish name) Endemic Fl.

P.G.

region

Altitude

(m)

Life

form

New record or

recorded before

1 G. aucheri Boiss.

(Taş Çöveni)

Endemic

(VU)

6-7 Ir.-Tur. 1200-1600 P Tunceli, Pertek

2 *G. briquetiana Schischk.

(Gül Çevgeni)

Endemic

(LR)

7-8 Ir.-Tur. 1700-2500 P Tunceli, Ovacık,

Munzur Mountain

3 G. elegans Bieb.

(Hoş Çöven)

- 6-7 Ir.-Tur. 650-2600 A New record to

Tunceli

4 *G. hispida Boiss.

(Kıllı Çöven)

- 6-7 Ir.-Tur. 1100-2150 P Tunceli, between

Tunceli and Ovacık

5 G. pallida Stapf.

(Şark Çöveni)

- 6-8 Ir.-Tur. 850-2000 P New record to

Tunceli

6 G. perfoliata L. var.

Perfoliata (Helvacı Çöveni)

- 6-8 - 1000-1500 P New record to

Tunceli

7 G. ruscifolia Boiss.

(Acem Çöveni)

- 6-7 Ir.-Tur. 300-1800 P Tunceli, Ovacık

8 G. sphaerocephala Fenzl ex

Tchihat var. cappadocica Boiss.

Endemic

(LR)

7-8 Ir.-Tur. 800-1900 P Tunceli, Munzur

Mountain

9 G. venusta Fenzl

(Kara Çöven)

- 5-7 Ir.-Tur. 300-1600 P New record to

Tunceli

10 G. viscosa Murray

(Sadırlı Çöven)

- 4-6 Ir.-Tur. 350-1400 A New record to

Tunceli

* :Gypsophila taxa not available in the area, P: Perennial, A: Annual, P.G.: Phyto-geographic, Fl.: Flowering period


G. sphaerocephala var. cappadocica are early recorded

in Tunceli but, G. pallida, G. perfoliata var. perfoliata,

G. venusta and G. viscose (4 taxa) are new records.

Habitat types of Gypsophila taxa growing naturally in

the province are rocky places, in clined or flowing slopes

and slopes of mountains. Their flowering period starts in

July. The general vegetation type of the plants are arid or

semiarid steppes.

Soap roots have economic value in medicine,

food, decoration and cleaning and chemistry to produce

saponin. It is used as fire extinguisher, gold polisher,

fabric, cleaner and for purification of contaminated soil

such as by removing the boron. In addition, it is possible

to perform vegetative mining by boron

hyper-acumulation from soil to the upper parts of the

plant (Babaoğlu et al., 2004; Korkmaz and Özçelik,

2011a). Turkish soaproot is mostly obtained from

G. graminifolia, G. bicolor, G. arrostii var. nebulosa,

G. eriocalyx, G. perfoliata var. anatolica, G. venusta and

Ankyropetalum gypsophiloides species and the gene

center of both of the species is Turkey (Korkmaz and

Özçelik, 2011a,b). The harvest time of these plants is

from March to June. Because the roots of these plants are

generally used, the plants don’t produce seeds for the

next years. So, the plants are increasingly disappearing

from the nature and under the threat of extinction. This

problem becomes more important when the plants are

rare or endemic. Because of unemployment soap roots

have been collected for a long time in the rural parts of

the country. For preservation of Gypsophila species they

should not only be collected from nature but also its

cultivation should be planned and other soap root

yielding plant species should be identified.

The most important floristic study related with

Tunceli in the area is Flora of Munzur Dağları

(Yıldırımlı, 1995). The mountains are situated between

Erzincan and Tunceli in B7 grid square and in

Irano-Turanian phytogeographic region. It starts from

Kemaliye and reach to Pülümür at the west-east direction

as forming a natural border between Erzincan and

Tunceli. The width of the mountain is 25-30 km and the

length of it is 100-130 km. Altitude of the area changes

from nearly 850 to 3462 m. The climate of the area is hot

and dry summers and long and snowy winters.

According to the study there are 1407 vascular plant

species. The number of endemic species is 275 and some

of them were described as new to science. In this study

G. briquetiana Schischk., G. sphaerocephala,

G. ruscifolia, G. elegans Bieb, G. bitlisensis Bark. and

G. hispida Boiss. are given in the list of the plants.

Munzur Dağları is one of the most important ÖBA

(Önemli Bitki Alanı) of Turkey with its very rich floristic

diversity. Munzur Valley is also an important national

park of the country. There are 43 plant species peculiarto

Munzur Dağları. In addition to the study of Yıldırımlı

(1995) Özhatay et al. (2005), this is another important

study on biological diversity of the mountains.

Gypsophila briquetiana Schischk., Gypsophila elegans

Bieb. and Gypsophila ruscifolia Boiss. are three species

of the genus growing in the area of Munzur mountains

(Koyuncu and Arslan, 2009). Polat et al. (2012)

evaluated ethno botanical studies performed in the

Eastern Anatolian region including Tunceli. According

to this study there are only five ethnobotanical study

(Tuzlacı ve Doğan, 2010; Yıldırımlı, 1985; 1991; 1994

a;b) conducted in Tunceli. Also in another study

performed by Karlıdağ in (2009) related with both of

Elazığ and Tunceli, they determined local names and

medicinal uses of 53 plants.The least studied cities in

East Anatolian region are Ağrı, Ardahan, Bingöl, Bitlis,

Erzincan, Kars, Muş, Hakkari and Tunceli. So, it is

necessary to record and prevent ethnobotanical culture in

these cities by conducting news tudies (Polat et al.,

2012).

CONCLUSION:

There are 60 naturally growing Gypsophila taxa

in the Turkey. Many species of the genus are highly



potential to be used in economy. G. sphaerocephala and

G. perfoliata are known as boron hyper accumulators

and they are very important for boron mining. Because

of their well- developed root stock they can be used for

soil erosion. They easily regulate themselves to the

drought in summer by storing water in their leaves and

enlarged roots (Sameh et al., 2011).

As İnan (2006) said, collecting plants in an

uncontrollable way from natural environments,

industrialization, urbanization, enlargement of fields for

agricultural goal, overgrazing, tourism, environmental

pollution, deforestation, forest fires are main factors

threatening the diversity of plants in Turkey. Because of

these factors many endemic, economic and traditionally

used medicinal plants are increasingly disappearing.

There are at least 10 Gypsophila taxa growing around

Tunceli. G. aucheri, G. briquetiana and

G. sphaerocephala var. cappadocica are endemic taxa

available in the province. Threat category of G. aucheri

is Vulnerable (VU) and it needs protection studies. The

most important factor that threat these taxa in the area is

animal husbandry.

The richness of the area with regard to

Gypsophila (coven) taxa is very necessary to use these

species in economic development of the city. Because of

that reason construction of a saponin factory in the

region or in East Anatolian region have huge importance

for the people living in the region. Instead of collecting

these plants from the nature culturing these species to

produce saponin is another necessity for preserving the

threatened species. Moreover, ecological, chemical,

genetic, ethno-botanical, culturing and conservation

studies on these taxa should be planned and performed in

the near future immediately.

ACKNOWLEDGE:

Some of the plant samples in this study were

collected with the support of the project numbered as

TÜBİTAK (TBAG-107T147). We are much obliged to

the support provided by the institution

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Article Citation: Mukesh H. Koladiya, ArunKumar Roy Mahato, Nikunj B. Gajera and Yatin S. Patel. Distribution pattern of birds in Banni Grassland of Kachchh district, Gujarat, India. Journal of Research in Biology (2014) 4(1):1228-1239

Jou

rn

al of R

esearch

in

Biology

Distribution pattern of birds in Banni Grassland of

Kachchh district, Gujarat, India

Keywords: Bird, distribution, density, habitat, Banni grassland, Kachchh

ABSTRACT: Birds are interesting group of animals which are distributed in all major types habitat. Banni is one of the large grassland of India invaded by Prosopis juliflora, an alien plant species. Invasion of this species and some other natural and anthropogenic factor leads the grassland converted into a mixture of heterogeneous habitats. A study was attempted to understand the distribution of birds in this heterogeneous grassland. The habitats were identified based on dominant species of plants. The population estimates of birds were surveyed using line transects method and point count census method. A total of 91 species were recorded during the survey in the various habitats of this grassland. Among the seven habitats, sparse Prosopis was the most diverse habitat for bird species whereas Prosopis-Capparis was the least diverse habitat for bird species. The highest mean population density of birds were recorded in Prosopis-Capparis (15.9 individuals/km2), while lowest recorded in sparse Prosopis habitat (9 individuals/km2). It was found that, Prosopis-Salvadora (23.10±9.47) was the most dense and Prosopis-Capperis (8.84±5.26) was the least dense habitat for common birds of Banni grassland. In conclusion, bird species diversity and their population density estimates were varied among the various heterogeneous habitats of Banni grassland both in time and space gradients.

1228-1239 | JRB | 2014 | Vol 4 | No 1

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.


An International


Authors:

Mukesh H. Koladiya1,

ArunKumar Roy Mahato2,

Nikunj B. Gajera3 and

Yatin S. Patel4.

Institution:

1,2,3. Gujarat Institute of

Desert Ecology, Bhuj,

Kachchh-370001, Gujarat.

4. Samarth Organization

Trust, Vijapur, Mehsana-

382870, Gujarat.


Mukesh H. Koladiya.

Email Id:

Web Address: http://jresearchbiology.com/

documents/RA0422.pdf.

Dates: Received: 10 Feb 2014 Accepted: 24 Feb 2014 Published: 16 April 2014


Original Research

INTRODUCTION:

Various group of animals varied from survival

strategies in a landscape which are evolved in long

course of evolution. The distribution patterns of animals

in various habitats are preferred in response to various

uses and selective processes (Clark and Shutler, 1999).

The distributions of life forms are not typically random

in the habitat and it is generally assumed that non-

random distribution of life forms is due to natural

selection (Southwood, 1977). The distribution range

across a heterogeneous landscape may depend on the

habitat selected by the species, and animal which favors

their distribution in a greater proportion of the habitat

(Veech et al., 2011).

Banni grassland is one of the largest remnant

grassland of India. The landscape of this grassland is flat

and most part of it is filled with water during monsoon

which makes the grassland as a wetland. The soil

salinity is normally high in most of the part due to its

connection with Great Rann of Kachchh (GRK), a salt

inflated marshy land. To protect the grassland from salt

intrusion from GRK, Prosopis juliflora was introduced

in fringe areas of GRK to check desertification in Banni

grasslands. In present, P. juliflora is proved to be an

invasive species for the grassland and now major part of

the grassland is invaded by the species.

Birds are very important animal for this

ecosystem as they are good indicators of biodiversity.

Birds are one of the typical groups of animal distributed

in large landscape and even some species prefer to live in

heterogeneous environment distributed over continents.

To understand the processes of habitat selection and

preference by birds is dependent on an accurate

representation of the patterns of habitat occupancy

(Wiens et al., 1987). Birds generally colonize in an area

having presence of suitable habitat for their survival

needs (Veech et al., 2011). The distribution pattern of

birds might also influence by distribution patterns of bird

species richness (Shiu and Lee, 2003). The above

understanding on the distribution pattern and habitat

preference of bird communities over heterogeneous

environment is very much essential for conservation and

management of birds in regional as well as in local

environment (Kattan and Franco, 2004).

Banni grassland is one of the rich areas of birds

due to its varied micro-habitat and act as a seasonal

wetland. The distribution pattern of birds across the

grassland is very less understood due to the lack of study

in the area. Therefore, the present study was conducted

to understand the pattern of distribution of birds in time

and space gradient in the grassland for their conservation

and management.

MATERIALS AND METHODS:

Study Area:

Banni, the largest remnant grassland in India,

situated on the south-west portion of the Kachchh

Biosphere Reserve (KBR) and attached to the fringes of

greater Runn of Kachchh (23°19' to 23°52' N latitude and

68°56' to 70°32' E longitude), encompassing an area of

over 2,600 km2 is taken into consideration for our study

(Fig-1). A large tract of the southern part of Banni

grassland is marshy land and salty waste remains a

wetland in the monsoon season, known as Little Rann of

Banni, which separates the Banni grassland from the

mainland of Kachchh district (Shah and Somusundaram,

2010). The climate of the Banni is arid and semi-arid

type therefore, the temperature is high during most of the

time and it reaches a maximum of 48°-49°C during May-

June and low during winter season (8°-10°C) in the

month of January and February. The average yearly

rainfall of this grassland is 317 mm with scanty rainfall

and droughts are the common phenomenon of this area.

The grassland is situated in the semi-arid bio-

climatic zone of India. The major part of grassland is

now invaded by Prosopis juliflora, an invasive alien

species. The grassland has varied types of habitat patches

that attract large number of birds. Further, the seasonal

Koladiya et al., 2014


water bodies (locally known as Dhandh) inside the Banni

region serve as the wintering ground for many migratory

species of birds.

METHODOLOGY:

A preliminary survey was made to whole of the

Banni grassland for identifying transect location and

number of transect location required for the survey.

Based on this survey various micro-habitats were

identified. A total of 60 km distance was covered by

walking through various transects. The field data were

collected by two observers during the whole study period

between the months of June 2009 to May 2011. The

birds were identified using the field guide produced by

Ali (1996) and survey was conducted by using standard

data sheet, GPS-Garmin, binocular (8X40) and camera.

Habitat classification:

Banni was earlier divided by 10 habitat types by

Koladiya et al. (2012). In the present study, the Banni

grassland was divided into 7 major habitat types based

on the dominant plant species. It includes; Dense

Prosopis, Moderate Prosopis (medium Prosopis

density), Sparse Prosopis, Prosopis-Capparis Mixed,

Prosopis-Suaeda-Calotropis Mixed, Prosopis-Salvadora

Mixed and Suaeda Dominant. The vegetation of the

study area was also recorded by making quadrate on the

line transect and calculated the density of vegetation by

using Misra (1968).

Avi-faunal Survey:

The population and distribution of birds were

recorded using line transect method and point count

census method (Bibby et al., 1992; Bhupathy, 1991). A

total of 51 transects were laid down in the whole



Figure 1. A map of Banni grassland, and its location in the Kachchh district of Gujarat.

surveyed area. The presence of individual and group of

birds within 25 m radius of circular plot was made in

every 200 m distance along the line transect. The species

of bird was identified using binoculars and with the help

of Ali and Ripley (1983) and Grimmett et al.(2006).

Generally, the surveys were made during the morning

(7.30 am to 11.30 am) and afternoon (4.00 pm to 6.30

pm) hours of each season during 2009 and 2011.

The data recorded during the study was used to

calculate vegetation density, bird’s population density

(Gaston, 1973; Burnham et al., 1980) and tested by

ANOVA between micro-habitat using Microsoft Excel

2007.

RESULTS AND DISCUSSION:

Habitat category & Vegetation density:

Among the seven identified habitats of Banni

grassland Prosopis juliflora is the most dominant species

and found in all habitats except Suaeda dominant habitat.

The flag ship and dominant species of plants in the seven

identified habitat were Prosopis juliflora, Capparis

decidua, Suaeda spp., Calotropis spp. and Salvadora

spp. The density of major plant species calculated in

each habitat type is given in table-1.

Species Richness and diversity:

A total of 91 Species of avi-fauna belonging to

62 genera under 35 families and 11 orders were observed

during the whole study period (given in Annexure-I).

Among the total observed bird species, 59 were resident

and 32 were migratory in nature. The number of bird

species recorded in Banni grassland based on their

feeding guilds included; granivorous (32 species),

insectivorous (30 species), frutivorous (12 species),

piscivorous (10 species) and others (7 species). Based

on the transect survey in various seasons, the maximum

bird species recorded during monsoon (83 species), next


Habitat class Vegetation Mean individuals of bird/Km2

Dominant species Density/ Ha Winter Summer Monsoon

Dense Prosopis (DP) Prosopis juliflora 1200.00 12.4 4.50 20.5

Moderate Prosopis (MP) Prosopis juliflora 833.33 12.3 4.30 17.4

Sparse Prosopis (SP) Prosopis juliflora 483.33 8.9 2.80 15.3

Prosopis-Capparis mixed (PC)

Prosopis juliflora 733.33

15.5 3.00 29.1

Capparis decidua 1400.00

Prosopis-Suaeda-Calotropis

mixed (PSC)

Prosopis juliflora. 1050.00

7.8 4.40 16.6 Suaeda sps. 2133.30

Calotropis sps. 8933.30

Prosopis-Salvadora mixed (PS) Prosopis juliflora 433.33

21.4 5.70 17.2

Salvadora sps. 366.67

Suaeda dominant (SD) Suaeda sps. 10000.00 13.0 4.20 20.4

Mean±SD - - 13.1±4.50 4.12±0.98 19.5±4.64

Table 1. Major plant species density and birds population density in various

micro-habitats of Banni grassland


to that in winter (67 species) and minimum during

summer (32 species).

The total number of avian species was recorded

lower than number of species (163) recorded by Gajera

et al. (2012, 2013a, 2013b) in wetland, arid grasslands

and mining areas respectively distributed in western part

of Kachchh district. It is also noted that 56 species of

birds recorded alone from the Pena thattah, a seasonal

wetland located in the western part of Banni grassland by

Koladiya et al. (2013).

The species diversity (Shannon_H) was recorded

to found highest in Sparse Prosopis (H=2.20) habitat and

lowest in Prosopis-Capparis mixed (H= 0.91) habitat

(fig-2). The above result highlighted that avian species

diversity was also lower in comparison to the species

diversity recorded by Gajera et al. (2012, 2013a, 2013b)

in wetland, grassland and mining areas distributed in

western parts of Kachchh district.

Distribution of birds in various micro-habitat:

Out of the total species recorded during the

whole study period, the number of bird species recorded

in 7 identified habitats were as follows; dense Prosopis

(45 species), moderate Prosopis means Prosopis density

between more than 500 and less than 1000 individuals/

Figure 2. Seasonal Avian species richness in various habitat of Banni grassland


Figure. 3. Bird species diversity in various habitats of Banni grassland, Kachchh


ha. (56 species), sparse Prosopis (60 species), Prosopis-

Capparis mixed (28 species), Prosopis-Suaeda-

Calotropis mixed (50 species), Prosopis-Salvadora

mixed (30 species) and Suaeda dominant (40 species)

respectively. The above result highlighted that sparse

Prosopis was the rich habitat for bird species diversity

and Prosopis-Capparis mixed was the least supportive

habitat for bird species diversity in Banni grassland. The

number of species diversity between three season

(summer, monsoon and winter) was significantly varied

(F=14.40, df=2, p<0.001) while species diversity

between various habitat were significantly not varied.

On analysis of seasonal distribution of bird

species in 7 identified habitats of Banni grassland, it was

found that sparse Prosopis, Prosopis-Suaeda-Calotropis

and dense Prosopis were the preferred habitat during

monsoon season; moderate Prosopis, dense Prosopis and

Suaeda dominant are the preferred habitat during winter

season; moderate Prosopis and Prosopis-Suaeda-

Calotropis are the most preferred habitat during the

month of summer (Fig-3). The percent of species

recorded in each type of habitat in seasonal basis is

shown in Figure-4.

We found that the mean population density

(Mean ± SD) of birds was highest during monsoon

season (19.5±4.64) and least density during summer

season (4.12±0.98). The seasonal population density of

birds in various habitats of Banni grassland is given in

table-1. It was found that the highest population density

of birds was found in Prosopis-Capparis mixed habitat

(29.1 individuals/km2) during monsoon and least density

was recorded in sparse Prosopis habitat (2.8 individuals/

km2) during summer season. The mean population

density of birds recorded in various habitats of Banni

grassland is shown in fig-5. Among the various habitat,

the highest mean population density of birds were


Figure 4. Seasonal abundance (%) of birds in Banni grassland of Kachchh, Gujarat


recorded in Prosopis-Capparis (15.9 individuals/km2)

and Prosopis-Salvadora habitats (14.8 individuals/km2)

while lowest mean population density was recorded in

sparse Prosopis habitat (9 individuals/km2). The result

revealed that the density of birds in Banni grassland was

higher in relation to the density of birds recorded by

Gajera et. al (2013b) in western part of Kachchh.

Distribution pattern of common birds:

We analyse the population density estimates of

commonnly occuring 10 species of birds in identified

seven habitat types of Banni grassland (Table-1). It was

found that, Prosopis-Salvadora was the most dense

habitat of six common species of birds viz. house crow,

lark, babblar, dove, bee eater and bul bul; sparse

Prosopis was the most dense habitat of pegion and

drongo; dense Prosopis for sand groose and Prosopis-

Suaeda-Capparis was the most dense habitat for

francolin. Similarly, Suaeda dominent was the least

dense habitat of four species viz. babblar, dove, bee eater

and bul bul; Prosopis-Capparis and Prosopis-Suaeda-

Capparis were the least dense habitat for three species of

common birds viz. house crow, francolin, dansgroose

and lark, pigeon, drongo respectively. On estimating the

overall mean density (Mean±SD) of common birds, it

was found that, Prosopis-Salvadora (23.10±9.47) was

the most dense habitat and Prosopis-Capparis

(8.84±5.26) was the least dense habitat for the common

birds of Banni grassland.

CONCLUSION:

In conclusion, the diversity of birds in banni

grassland is rich with sparse Prosopis is the richest

habitat compare to other habitat in relation to species

diversity. Prosopis juliflora, an invasive alien species of

plant in the grassland is playing major role in the

distribution of avi-fauna in this region. Prosopis juliflora

is the dominant species of plant of this grassland which

provide habitat for nesting of birds and greater visibility

of birds for preying. Based on the results of the study, it

was found that monsoon season attracts more number of

species of birds in the grassland because large portion of

the grassland is converted into seasonal wetland during

the season. However, habitats with dominance of mixed

vegetation are the dense in habitat for birds compared to

other habitats of the grassland.

ACKNOWLEDGEMENTS:

We would like to thank Dr. R. V. Asari, Director,

Gujarat Institute of Desert Ecology (GUIDE) for

providing logistic supports and his encouragement. We


Figure 5. Population density of birds in various habitats of Banni grassland, Kachchh


are thankful to Mr. Yatin Patel for his help in Plant data

analysis for the manuscript. We are also thankful to all

scientist and scholars of Terrestrial Ecology Division of

GUIDE for their help and valuable comments. We are

grateful to State Forest Department, Gujarat for

providing funds for conducting this study.

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S. No Family Scientific Name Common Name MS Habitat

1 Phasianidae Francolinus pondicerianus Grey Francolin R DP, MP, SP, PSC,SD

2 Upupidae Upupa epops Common Hoopoe R MP, SP, SD

3 Coraciidae Coracias garrulus European Roller RM MP, SP, SD

4 Coracias benghalensis Indian Roller R SP, PC

5 Meropidae Merops orientalis Green Bee-eater R DP, MP, SP, PC, PS

6 Merops leschenaulti Chestnut-Headed

Bee-Eater

R DP, MP, SP, PC, PS

7 Cuculidae Eudynamys scolopacea Asian Koel R SP, PC, PS

8 Centropodidae Centropus sinensis Greater Coucal R MP, SP, SD

9 Psittacidae Psittacula krameri Rose-Ringed Parakeet R PC, PS, SD

10 Apodidae Apus affinis House Swift R MP, SP, SD

11 Strigidae Bubo bubo Eurasian Eagle-Owl R DP, MP, SP

12 Columbidae Columba livia Blue Rock Pigeon R DP, MP, SP, PC

13 Streptopelia decaocto Eurasian Collared Dove R DP, MP, SP, PC

14 Streptopelia tranquebarica Red Collared Dove R DP, MP, SP, PC

15 Streptopelia chinensis Spotted Dove R DP, MP, SP, PC

16 Streptopelia senegalensis Little Brown Dove R DP, MP, SP

17 Pteroclididae Pterocles exustus Chestnut-bellied

Sandgrouse

R DP, MP, SP, PC

18 Pterocles indicus Painted Sandgrouse R DP, MP, SP, PC

19 Accipitridae Circus pygargus Montagu's Harrier RM MP, PSC

20 Circus aeruginosus Eurasian Marsh Harrier WV DP, MP, PSC

21 Circus cyaneus Hen Harrier WV DP, MP, PSC

22 Circus macrourus Pallid Harrier R DP, MP, PSC

23 Accipiter badius Shikra R MP, SP, PSC

24 Elanus caerulus Black-Shouldered Kite R MP, SP, PSC

25 Milvus migrans Black Kite R MP, SP

26 Pandion haliaetus Osprey RM SP, SD

27 Aquila pomarina Lesser Spotted Eagle R DP, MP, PSC

28 Aquila nipalensis Steppe Eagle WV DP, MP, PSC

29 Falconidae Falco tinnunculus Common Kestrel WV DP, MP, PSC

30 Lanidae Lanius excubitor Grey Shrike RM DP, MP, PSC, PS

Annexure I

List of bird species recorded in various habitat of Banni grassland



31 Lanius cristatus Brown Shrike M DP, MP, PS, SD

32 Lanius vittatus Bay-backed Shrike R DP, MP, PS, SD

33 Lanius schach Rufous-tailed Shrike R DP, MP, PS, SD

34 Lanius meridionalis Southern Grey Shrike RM DP, MP, PS, SD

35 Corvidae Corvus splendens House Crow R DP, MP, SP, SD

36 Corvus macrorhynchos Jungle Crow R DP, MP, SP, SD

37 Dicrurus macrocerus Black Drongo R DP, MP, PS, SD

38 Muscicapidae Saxicola jerdoni Jerdon's Bushchat R MP, SP, PS, SD

39 Saxicola caprata Pied Bush Chat R MP, SP, PS, SD

40 Oenanthe deserti Desert Wheatear RM MP, SP, PSC, SD

41 Oenanthe picata Variable Wheatear M SP, PSC, SD

42 Oenanthe isabellina Isabelline Wheatear M SP, PSC, SD

43 Copsychus saularis Oriental Magpie Robin R DP, MP, SP, PC, SD

44 Saxicoloides fulicata Indian Robin R DP, MP, SP, PC, SD

45 Sturnidae Sternus roseus Rosy Starling WV DP, MP, PS

46 Acridotheres tristis Common Myna R DP, MP, PS

47 Acridotheres ginginias Bank Myna R DP, MP, PS

48 Paridae Parus nuchalis Pied Tit R MP, SP, SD

49 Hirundinidae Hirundo rustica Barn Swallow WV SP, SD

50 Hirundo smithii Wire-tailed Swallow R SP, SD

51 Hirundo daurica Red-Rumped Swallow R SP, SD

52 Delichon urbica Northern House-Martin RM SP, SD

53 Pycnonotidae Pycnonotus cafer Red-Vented Bulbul R DP, MP, PC, PSC,PS,SD

54 Pycnonotus leucotis White-eared Bulbul R DP, MP, PC, PSC,PS,SD

55 Cisticolidae Prinia buchanani Rufous-fronted Prinia R DP, SP, PSC, PS

56 Prinia inornata Plain Prinia R DP, SP, PSC, PS

57 Prinia sylvatica Jungle Prinia R DP, SP, PSC, PS

58 Prinia socialis Ashy Prinia R DP, SP, PSC, PS

59 Sylvidae Orthotomus sutorius Common Tailorbird R DP, MP, PC, PSC, PS

60 Hippolais caligata Booted Warbler R DP, SP, PC, PSC, PS

61 Turdoides caudatus Common Babbler R DP, MP, PC, PSC, PS

62 Turdoides malcolmi Large Grey Babbler R DP, MP, PC, PSC, PS

63 Turdoides striatus Jungle Babbler R DP, MP, PC, PSC, PS

64 Alaudidae Galerida cristata Crested Lark R SP, PC, PSC

65 Eremopterix grisea Ashy-crowned, Sparrow-Lark R SP, PC, PSC



66 Mirafra erythroptera Indian Bushlark R DP, MP, PC, PSC

67 Mirafra cantillans Singing Bushlark R MP, SP, PC, PSC

68 Calandrella raytal Short-toed lark M MP, SP

69 Galerida deva Sykes's Crested Lark R MP, SP, PSC

70 Nectarinidae Nectarinia asiatica Purple Sunbird R DP, SP, PC, PSC, PS

71 Passeridae Passer domesticus House Sparrow R SP, PSC, PS

72 Anthus rufulus Paddyfield Pipit RM DP, PSC, PS

73 Lonchura malabarica Indian Silverbill R DP, PC, PSC, PS

74 Motacilla alba White Wagtail WV SP, PSC

75 Motacilla flava Yellow Wagtail WV SP, PSC

76 Motacilla cinerea Grey Wagtail WV SP, PSC

77 Ploceus philippinus Baya Weaver R SP, PC

78 Alcedinidae Alcedo atthis Common Kingfisher R MP, PSC

79 Dacelonidae Halcyon smyrnensis White-breasted Kingfisher R SP, PSC

80 Cerylidae Ceryle rudis Pied Kingfisher R SP,PC, SD

81 Gruidae Grus grus Common Crane WV SP, PSC, SD

82 Grus virgo Demoiselle Crane WV SP, PSC, SD

83 Charadridae Vanellus indicus Red-Wattled Lapwing R MP, PSC, SD

84 Anhingidae Anhinga melanogaster Darter R PSC, SD

85 Ardeidae Bubulcus ibis Cattle Egret R MP, PSC, SD

86 Casmerodius albus Great Egret R SP, PSC, SD

87 Egretta garzetta Little Egret R SP, PSC, SD

88 Mesophoyx intermedia Intermediate Egret R SP, PSC, SD

89 Threskiornithidae Pseudibis papillosa Black Ibis R MP, PC, SD

90 Platalea leucorodia Eurasian Spoonbill R SP, PC, SD

91 Ciconidae Mycteria leucocephala Painted Stork R SP, PC, SD

MS: Migratory Status, R: Resident, RM: Resident Migratory, WV: Winter visitor, DP: Dense Prosopis, MP:

Moderate Prosopis, SP: Sparse Prosopis, PC: Prosopis-Capparis mixed PSC: Prosopis-Suaeda-Calotropis mixed,

PS: Prosopis-Salvadora mixed, SD: Suaeda dominant





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Galerida deva

Aquila nipalensis

Accipiter badius Grus grus

Banni grassland

Upupa epops

Annexure II. Photographs showing Banni grassland and a few birds sited

Article Citation: Abba H, Belghity D, Benabid M and Chillasse L. Determination of age and growth by scale of a population of common trout (Salmo trutta macrostigma, Dumeril, 1858) at the level of Sidi Rachid River (Ifrane. Morocco)

Journal of Research in Biology (2014) 4(1): 1240-1246

Jou

rn

al of R

esearch

in

Biology

Determination of age and growth by scale of a population of common trout

(Salmo trutta macrostigma, Dumeril, 1858) at the level of Sidi Rachid River

(Ifrane. Morocco)

Keywords: River trout, age, growth, scales, Sidi Rachid River. Morocco

ABSTRACT: The determination of age and growth from the scales of trout river (Salmo trutta macrostigma, Dumeril, 1858) at Sidi Rachid River; was employed out of 438 specimens used the size varies between 6.3 cm and 37.5 cm, the relation linking the growth in length of the fish and the growth of the scale. Varied according to the equation Log Lt = 0.8674 ×Log Rt + 0.5349, with a coefficient of correlation( r) = 0.86592138. The period of the end of growth to this population of trout is between December and January, this period is characterized in the middle of the atlas by important reductions in temperature on one hand, the decrease of the network trophique on the other hand which gets coincided with the period of reproduction of the trout. The resumption of the growth is made in a important way from March. The age of the trout's determined by scales varies between 0 + to 5 +. The retro measures are lower than those observed and the equation of theoretical growth of Van Bertalanffy is of the following type: Lt = 34, 96 (1-exp-0,309 (t-0, 27)).

1240-1246 | JRB | 2014 | Vol 4 | No 1

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.


An International


Authors: Abba H 1, Belghity D1,

Benabid M2 and Chillasse L3.

Institution:

1. Biology and Health

Laboratory. Environmental

and Parasitology Team /

UFR Doctoral Parasitology

compared: Medical and

Veterinary Applications."

Sciences Faculty. Ibn Tofail

University. Kénitra B.P.

133, 14000. Morocco.

2. National Center of

Hydrobiology and

Pisciculture (NCHP) Azrou

Morocco.

3. Laboratory of biodiversity

and wet zones .Uni My

Ismail. Faculty of Science.

Meknes.


Abba H

Email Id:


Dates: Received: 19 Dec 2013 Accepted: 15 Jan 2014 Published: 16 April 2014


Original Research

INTRODUCTION

The fishing of salmonids constitutes one of the

main concerns of the members of fishing associations in

the nation. Both the common trout (Salmo trutta

macrostigma, Dumeril, 1858) and the rainbow trout

(Oncorhynchus mykiss) are appreciated in the fishing

sport. This activity plays an important role in the

socioeconomic development of the region. To alleviate

the disappearance of the endemic common trout, the

administrators in Morocco resort to the repopulation of

rivers with vesicle alevins stemming from artificial

reproduction which is carried out at the salmon farming

station of Ras El Ma.

For a long time, numerous studies were

conducted in the determination and knowledge of the

lines of fish the populations of in various aquatic circles.

Besides the parameters size and mass, we also quote the

age of the fish. These various biological lines once

determined, can be exploited in the perspectives of

management of the various types of peaches

professionally. The estimation of the age of a fish is of a

big importance to understand the dynamics of a

population. This determination of the age can be made

either in a direct way, or in an indirect way. In this study,

we limited ourselves to one of the direct methods by

means of the osseous structures (Spillmann, 1961;

Bagliniere et Maisse, 1990). Although the use of scales

for certain species are questioned (Pikitch et Demory,

1988), the scales are used for a majority of families with

species dulçaquicoles and amphihalines temperate or

cold regions to be known, almonds, cyprinids and

precedes (Bagliniere et Lelouarn, 1987; Meunier, 1987;

Bouhbouh, 2002). During this study, method used for the

determination of age and growth of brown trout (Salmo

trutta macrostigma, Duerile 1858) is by the number, size

and pattern of scales. Indeed, the growth of the

structures mineralized as the scales is proportional to the

length of the fish (Lea, 1910; Hattour et al., 2005). In

temperate zones, the growth of the fish presents a

seasonal rhythm with fast growth at the spring and

summer and a stops its growth during winter period. This

annual growth rate is marked on the various osseous

structures among which scales are present. The study of

these osseous structures will allow determining the

period of the stop of growth and consequently the age

and its relation with the size of the specimens of the

population of trout in the Sidi Rachid River.


Presentation of the environment of study

The environment of study (Figure-1) is Sidi

Rachid River, present in the geographical coordinates of

5°9'N N and 33°28'W W. It is at a height of 1620m and

belongs to the rural district of Ait Ali Ouikoub (province

of Ifrane). The brook is fed by the sources of Sidi Rachid

of which it takes its name with a maximum debit of 172

L/S (Abba, 2011) for a main source as well as the waters

from the station of salmon farming of Ras El Ma (Abba

et al., 2011). From the morphométric point view, the

River presents a low width which can vary from 2m to 6

m, and a depth which does not exceed 1m generally.

Biological material

Sampling of fishes

The method used in our case is the electric

fishing realized by technicians' team of the National

Center of Hydrobiology and Fish farming of Azrou. The

number of fish every month varies between 30 and 50

specimens. For every sinned fish, we have proceed to the

measure of its total length (Lt (cm)) with an ichtyometer,

and before putting it back in the housing environment,

scales in number from 6 to 20 were removed in the zone

recommended for salmonids according to Ombredane

and Richard(1990). Scales are then tidied up in

envelopes and numbered for further study in the

laboratory with a microfiche (×40).

Determination of the structure of the population

The determination of the number of classes of

size of the population of trout at the level of the Sidi

Abba et al., 2014


Rachid River during the period of study was made by

applying the ruler of Sturge. Number of class = 1 + (3, 3

log N), were N is the sample Size.

Preparation and reading of scales

The preserved scales dried on the referenced

envelopes were taken and rubbed between fingers and

cleaned by the water to eliminate any sorts of residues

(Jearld, 1983). The examination of scales can be made by

several ways. The reading chosen in this work as the

determination of the age of the fish was made by a reader

of microfiche. The criteria used for the determination of

rings for the stop of growth vary according to the

species. For the salmon kind, the criteria are generally as

follows:

Contraction of several circuli in the form of a

concentric band making the tour (ballot) of the scale

(Bagliniere and Lelouarn , 1987);

Discontinuity of circuli or absence of discontinuity

of the circuli in which the thickness decreases;

Stepping of the circuli of the annulus on those

previously trained in the side fields either

Measures made on scales

The rings of ruling of growth allow making

measurements on the scale to calculate the marginal

extension (AM). The latter is used to determining of the

period of stop of growth. The front of the scale generally

held to salmonids (Bagliniere et al., 1991) is used for the

determination of the total shelf R and other shelves r

corresponding to the various annuli, r1, r2, r3 to rn. The

measure was made by means of a graduated ruler on a

device microfiche for the same swelling (×42). To work

always in the same condition, the measure of the beam

was made on the main line, which corresponds to the

previous field of the scale. The Extension Margin (EM)

was calculated according to, Benabid (1990).

Determination of the retro calculation on growth

The relation binding the size of the fish and the

shelf of its scale is linear and is determined by the

following formula (Bryuzgin, 1970): L = b Ra (or Log L

= a Log R + Log b), with, , ‘L’:: length of the fish (cm)

in the capture, ‘R’: the previous shelf of the scale of the

fish (cm) ie., distance between the center of the scale and

its outside edge according to a direction strictly constant,

‘a’: and , ‘b’: are constants.

The formula of Le Cren (1947) and Philippart,

(1975) allows then the retro calculation of the size of the

fish every age. Log Ln = Log L + a (Log Rn - Log R).

With, ‘Ln’ length calculated at the time of the training of

the nème ring of the stop of growth in mm; ‘L’: length


Abba et al., 2014

Figure 1: Situation of area of study (Extracted from the map of Azrou. E: 1/50. OOO;

division of the map, 1974)

observed by some fish in mm; , ‘R’:: length observed by

the previous beam of the scale in mm; , ‘Rn’: length of

the previous beam of the scale up to the nème ring in

mm; and , ‘a’: constant. The theoretical model of growth

used is the one of Von Bertalanffy (1938): (Lt = L∞ [1-

exp (-K (t-t0))]). (Benabid, 1990; Bouhbouh, 2002).

With K (years- 1): growth rate; L∞ (cm): cut that the fish

in time infinite should have; t0 (years): the age in the

worthless length.


The histogram of the structure of population of

the trout (Figure-2) shows a good representation of the

individuals and the size of which is between 14 (the

Middle = 13.8) and 17 cm (the Middle of 16.8).This type

of structure is a characteristic of young populations. This

structure is explained by the fact that the adults are

generally fished by farmers in the station of fish farming

as a source of gametes during the period of artificial

reproduction which comes true in the station of Ras El

Ma.

Among 438 individuals sampled during the

period of study, the number of river trout presenting

scales of regeneration is 50 specimens, this constitutes a

number raised with regard the size of the sample; it is 11,

44 % (Ombredane and Richard, 1990).

The determination of the period of appearance of

the rings to the stop of growth was made by monthly

analysis of the variations of average Marginal Extension

(AM) on 387 trout's which presents normal scales.

During this study, some scales do not present rings on

the stop of growth; it is about scales of truitelles

stemming from on-the-spot cross-posted or born alevins

from March, 2007. The (figure-3) shows the results

obtained for all the scales of fishes representing stops of

growths.

The analysis of variations of the results showed

that Marginal Extension presents the minimum only one

marked well for December and January. This minimum

translates not only shows the ring of wintry stop of

growth but also it corresponds to the period of

heavyweight at the river trout. Indeed this stop of growth

is not only due to the period of reproduction which slows

down the growth of the fish but also on the severe

conditions which exist during this period of year as the

important decrease of temperature and trophiques

resources (Pourriot and Meybeck, 1995) which are

generally due to the snow coverage which knows in this

region. The resumption of the growth begins gradually

from February and reaches its maximum during August.

Abba et al., 2014

Figure 2: Representation schedules of various classes of common trout and their staff

at the level of the Sidi Rachid River during the period of study


This important growth is due to the favorable conditions

of the housing environment as the temperature and the

abundance of the food reserves, on 438 scales examined

(51, scales of regeneration), the age is between 0 + and

4+ for sizes going from 6.3 cm to 37.5 cm. The

determination of the size of the trout's at the various

moments of their life is based on the principle of

proportionality of the growth of the scale with that of its

body. For this end, the equation connecting the previous

beam R of the scale and the total length (Lt) used in this

study was determined as continuation. Log Lt = 0, 8674

×Log Rt + 0, 5349. The relation between the total length

of the body of the trout (Lt) and the length of the

previous shelf of its scale (R) (Figure-4) can be

allometrique (Giles and Giguere, 1992).

The introduction of the coefficient of regression

of the relation length (Lt) and length (R) of the scale

gives the following equation: Log Ln = Log L + 0.8674

× (Log Rn - Log R) (Le Cren, 1947; Benabid, 1990;

Bouhbouh, 2002). The total retro measure lengths from

the equation above are listed in the table -1.

The results obtained for the total retromeasures

lengthes are used for the determination of the annual

average linear increase ( C ) as well as the specific speed

of growth noted VSC established by Ricker ( 1958 ):

C = Ln-Ln-1. (Ln and Ln-1: annual lengthes retro

measures in time n and n-1 expressed in years. VCS = Ln

-Ln-1 × 100/Ln-1. The obtained results showed that, the

calculated total retro measures lengths are quite lower

than the observed annual average lengths. This

Abba et al., 2014

Figure 3: Monthly evolution of Average Marginal Extension

(AME) of the river trout

Figure 4: Relation between the length of the fish and the previous shelf of

its scale at the common trout of the Sidi Rachid River.


difference of length can give some explanation by the

fact that the observed average lengths correspond to the

lengths of fish at various moments of the year or the

growth is made. On the other hand the total retro

measures lengths correspond to the lengths of fish at the

time of the training of annuli stag of stop of growth

during December generally. The average lengths

observed to both sexes and individuals of the indefinite

sex do not present notable difference for age groups I (1

+) and II (2 +). Beyond this age, we notice a variation in

favour of females (age groups III (3 +) and IV (4 +)), to

become slightly raised to the males of age group V (5 +).

These variations can be due to the sexual maturity which

influences the growth and which is premature in a

general way at males. Also, the retro measure averages

are slightly superior at the females than at the males of

the same age group. As for the specific speed of the

growth, it is very important for the class II (2 +) and it

exceeded 40 % (combined sexes and various sexes). The

decrease is in a very remarkable way as the age of the

fish increases and achieves approximately 10 % for

fishes of age group V (5 +). For the theoretical model of

the growth of Von Bertalaffy (the obtained results watch

that the theoretical maximal size of the fish is of L8 =

34.96cm. The theoretical equation becomes then for the

population of trout of the Sidi Rachid River is Lt = 34.96

(1-exp-0.309 (t-0.27)). The theoretical length (34.96cm)

is lower than the maximal length of the biggest trout

(37.5cm) scales of which are used for the determination

of the age. The use of reliable software can give even

more reliable results for this equation because the sizes

sinned in other circles sometimes exceed 40cm.

CONCLUSION

The use of scales and other osseous structures

allow determining particularly the aspects of age and the

analysis of dynamics of a fish population growth. With

salmonids, the most recommended method is the scale,

despite some disadvantages such as the difficulty of

scales reading or the high number of scales of

regeneration that we obtain. Similarly, the use of another

method can be very beneficial and will allow having

more information.

ACKNOWLEDGEMENTS

I thank the persons in charge of the station of fish

farming of Ras El Ma/ Azrou/ Morocco.

REFERENCES

Abba H. 2011. Etude écologique et biologique de la

truite commune (Salmo trutta macrostigma, Dumeril

1858) dans une rivière de Moyen Atlas (Oued Sidi

Rachid) Maroc. Thèse de Doctorat Es Science. Univ.

Ibn Tofail. Fac .Sc. Kenitra. Maroc. pp.194.

Abba H, Belgghyti D Benabid M, ELIbaoui H, Fadli

M and Eby Ould Mohamadou A. 2011.

Physicochemical Typology of water of a Middle Atlas

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macrstigma, Duméril, 1858) live: Oued Sidi Rachid.

Abba et al., 2014

Age

group

Age

group

Observed average

length (mm)

Length averages retro measures (mm)

I II III IV V

2008 I 134.60 91.20

103.27

141.25

2007 II 152.40

2006 III 190.09 104.71 147.90 177.07 - -

2005 IV 263.35 112.20 165.95 213.79 245.47 -

2004 V 318.11 128.82 190.54 234.42 275.42 309.02

Number of fish retro measures 358.00 285.00 194.00 83.00 9.00

Annual average length retro measures 108.63 161.41 208.42 260.44 309.03

Standard deviation 13.84 20.04 29.04 21.17 -

Increase in annual average length (mm) 91.20 37.98 29.17 31.68 33.61

Specific speed of growth 44.39 28.33 14.85 12.20

Table 1: Linear retro measures at the Growth of common trout (combined Sexes)


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Materials and Methods

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Results and Discussion

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Reference Style

References should be cited in the text in the form (Author et al, 1987) and listed in alphabetical order at the end of the article as follows:

Schernewski G, Neumann T. The trophic state of the Baltic Sea a century ago: a model simulation study. J Mar Sys., 2005;53:109–

124.

Kaufman PD, Cseke LJ, Warber S, Duke JA and Brielman HL. Natural Products from plants. CRC press, Bocaralon, Florida. 1999;

15-16.

Kala CP. Ecology and Conservation of alphine meadows in the valley of flowers national park, Garhwal Himalaya. Ph.D Thesis,

Dehradun: Forest Research Institute, 1998; 75-76.

http://www.ethnobiomed.com/content/pdf/1746-4269-1-11.pdf.

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Journal of Research in Biology Volume 4 Issue 1

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