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Publisher, Journal of Research in Biology.
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Bharathiar University.
SUGANTHY [Entomologist]
TNAU, Coimbatore.
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TNAU, Tirunelveli.
Syed Mohsen Hosseini [Forestry & Ecology]
Tarbiat Modares University (TMU), Iran.
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Norwegian University of Science and Technology (NTNU), Norway.
Dr. Ajay Singh [Zoology]
Gorakhpur University, Gorakhpur
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Kisan PG College, BAHRAICH
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S.S.(P.G.)College, Shahjahanpur, India.
Adarsh Pandey [Mycology and Plant Pathology]
SS P.G.College, Shahjahanpur, India
Hanan El-Sayed Mohamed Abd El-All Osman [Plant Ecology]
Al-Azhar university, Egypt
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Sri Ram Nallamani Yadava College of Arts & Sciences, Tenkasi, India.
T.P. Mall [Ethnobotany, Plant pathology]
Kisan PG College,BAHRAICH, India.
Mirza Hasanuzzaman [Agronomy, Weeds, Plant]
Sher-e-Bangla Agricultural University, Bangladesh
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Mahatma Gandhi Post Graduate College, Gorakhpur, India.
N.K. Patel [Plant physiology & Ethno Botany]
Sheth M.N.Science College, Patan, India.
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Gujarat, India.
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College of Applied Medical Sciences, King Saud University.
B.C. Behera [Natural product and their Bioprospecting]
Agharkar Research Institute, Pune, INDIA.
Kuvalekar Aniket Arun [Biotechnology]
Lecturer, Pune.
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Aligarh Muslim university, Aligarh, india.
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Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India.
Vaclav Vetvicka [Immunomodulators and Breast Cancer]
University of Louisville, Kentucky.
José F. González-Maya [Conservation Biology]
Laboratorio de ecología y conservación de fauna Silvestre,
Instituto de Ecología, UNAM, México.
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Department of Pathology, Army Medical College, Rawalpindi, Pakistan.
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Krishi Vigyan Kendra, Amritsar, Punjab, India.
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Universidade Federal de São João del-Rei, Brazil.
Dr.Amit Baran Sharangi [Horticulture]
BCKV (Agri University), West Bengal, INDIA.
Dr. Bhargava [Melittopalynology]
School of Chemical & Biotechnology, Sastra University, Tamilnadu, INDIA.
Dr. Sri Lakshmi Sunitha Merla [Plant Biotechnology]
Jawaharlal Technological University, Hyderabad.
Dr. Mrs. Kaiser Jamil [Biotechnology]
Bhagwan Mahavir Medical Research Centre, Hyderabad, India.
Ahmed Mohammed El Naim [Agronomy]
University of Kordofan, Elobeid-SUDAN.
Dr. Zohair Rahemo [Parasitology]
University of Mosul, Mosul,Iraq.
Dr. Birendra Kumar [Breeding and Genetic improvement]
Central Institute of Medicinal and Aromatic Plants, Lucknow, India.
Dr. Sanjay M. Dave [Ornithology and Ecology]
Hem. North Gujarat University, Patan.
Dr. Nand Lal [Micropropagation Technology Development]
C.S.J.M. University, India.
Fábio M. da Costa [Biotechnology: Integrated pest control, genetics]
Federal University of Rondônia, Brazil.
Marcel Avramiuc [Biologist]
Stefan cel Mare University of Suceava, Romania.
Dr. Meera Srivastava [Hematology , Entomology] Govt. Dungar College, Bikaner.
P. Gurusaravanan [Plant Biology ,Plant Biotechnology and Plant Science]
School of Life Sciences, Bharathidasan University, India.
Dr. Mrs Kavita Sharma [Botany]
Arts and commerce girl’s college Raipur (C.G.), India.
Suwattana Pruksasri [Enzyme technology, Biochemical Engineering]
Silpakorn University, Thailand.
Dr.Vishwas Balasaheb Sakhare [Reservoir Fisheries]
Yogeshwari Mahavidyalaya, Ambajogai, India.
Dr. Pankaj Sah [Environmental Science, Plant Ecology]
Higher College of Technology (HCT), Al-Khuwair.
Dr. Erkan Kalipci [Environmental Engineering]
Selcuk University, Turkey.
Dr Gajendra Pandurang Jagtap [Plant Pathology]
College of Agriculture, India.
Dr. Arun M. Chilke [Biochemistry, Enzymology, Histochemistry]
Shree Shivaji Arts, Commerce & Science College, India.
Dr. AC. Tangavelou [Biodiversity, Plant Taxonomy]
Bio-Science Research Foundation, India.
Nasroallah Moradi Kor [Animal Science]
Razi University of Agricultural Sciences and Natural Resources, Iran
T. Badal Singh [plant tissue culture]
Panjab University, India
Dr. Kalyan Chakraborti [Agriculture, Pomology, horticulture]
AICRP on Sub-Tropical Fruits, Bidhan Chandra Krishi Viswavidyalaya,
Kalyani, Nadia, West Bengal, India.
Dr. Monanjali Bandyopadhyay [Farmlore, Traditional and indigenous
practices, Ethno botany]
V. C., Vidyasagar University, Midnapore.
M.Sugumaran [Phytochemistry]
Adhiparasakthi College of Pharmacy, Melmaruvathur, Kancheepuram District.
Prashanth N S [Public health, Medicine]
Institute of Public Health, Bangalore.
Tariq Aftab
Department of Botany, Aligarh Muslim University, Aligarh, India.
Manzoor Ahmad Shah
Department of Botany, University of Kashmir, Srinagar, India.
Syampungani Stephen
School of Natural Resources, Copperbelt University, Kitwe, Zambia.
Iheanyi Omezuruike OKONKO
Department of Biochemistry & Microbiology, Lead City University,
Ibadan, Nigeria.
Sharangouda Patil
Toxicology Laboratory, Bioenergetics & Environmental Sciences Division,
National Institue of Animal Nutrition
and Physiology (NIANP, ICAR), Adugodi, Bangalore.
Jayapal
Nandyal, Kurnool, Andrapradesh, India.
T.S. Pathan [Aquatic toxicology and Fish biology]
Department of Zoology, Kalikadevi Senior College, Shirur, India.
Aparna Sarkar [Physiology and biochemistry] Amity Institute of Physiotherapy, Amity campus, Noida, INDIA.
Dr. Amit Bandyopadhyay [Sports & Exercise Physiology]
Department of Physiology, University of Calcutta, Kolkata, INDIA .
Maruthi [Plant Biotechnology]
Dept of Biotechnology, SDM College (Autonomous),
Ujire Dakshina Kannada, India.
Veeranna [Biotechnology]
Dept of Biotechnology, SDM College (Autonomous),
Ujire Dakshina Kannada, India.
RAVI [Biotechnology & Bioinformatics]
Department of Botany, Government Arts College, Coimbatore, India.
Sadanand Mallappa Yamakanamardi [Zoology]
Department of Zoology, University of Mysore, Mysore, India.
Anoop Das [Ornithologist]
Research Department of Zoology, MES Mampad College, Kerala, India.
Dr. Satish Ambadas Bhalerao [Environmental Botany]
Wilson College, Mumbai
Rafael Gomez Kosky [Plant Biotechnology]
Instituto de Biotecnología de las Plantas, Universidad Central de Las Villas
Eudriano Costa [Aquatic Bioecology]
IOUSP - Instituto Oceanográfico da Universidade de São Paulo, Brasil
M. Bubesh Guptha [Wildlife Biologist] Wildlife Management Circle (WLMC), India
Rajib Roychowdhury [Plant science]
Centre for biotechnology visva-bharati, India.
Dr. S.M.Gopinath [Environmental Biotechnology]
Acharya Institute of Technology, Bangalore.
Dr. U.S. Mahadeva Rao [Bio Chemistry]
Universiti Sultan Zainal Abidin, Malaysia.
Hérida Regina Nunes Salgado [Pharmacist]
Unesp - Universidade Estadual Paulista, Brazil
Mandava Venkata Basaveswara Rao [Chemistry]
Krishna University, India.
Dr. Mostafa Mohamed Rady [Agricultural Sciences]
Fayoum University, Egypt.
Dr. Hazim Jabbar Shah Ali [Poultry Science]
College of Agriculture, University of Baghdad , Iraq.
Danial Kahrizi [Plant Biotechnology, Plant Breeding,Genetics]
Agronomy and Plant Breeding Dept., Razi University, Iran
Dr. Houhun LI [Systematics of Microlepidoptera, Zoogeography, Coevolution,
Forest protection]
College of Life Sciences, Nankai University, China.
María de la Concepción García Aguilar [Biology] Center for Scientific Research and Higher Education of Ensenada, B. C., Mexico
Fernando Reboredo [Archaeobotany, Forestry, Ecophysiology]
New University of Lisbon, Caparica, Portugal
Dr. Pritam Chattopadhyay [Agricultural Biotech, Food Biotech, Plant Biotech]
Visva-Bharati (a Central University), India
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
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
Jou
rn
al of R
esearch
in
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
Borgohain and Tanti, 2014
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
Borgohain and Tanti, 2014
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.
Class: Bacillariophyceae
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.
Class: Bacillariophyceae
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.
Class: Bacillariophyceae
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
Borgohain and Tanti, 2014
Journal of Research in Biology (2014) 4(1): 1162-1173 1166
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.
Class: Bacillariophyceae
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.
Class: Bacillariophyceae
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.
Class: Bacillariophyceae
Order: Cymbellales
Family: Cymbellaceae
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
Borgohain and Tanti, 2014
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.
Class: Bacillariophyceae
Order: Rhopalodiales
Family: Rhopalodiaceae
Genus: Rhopalodia Otto Muller, 1895: 57
Rhopalodia sp. (Fig. 7 A)
Valves 21-30 µm long and 6-9 µm broad,
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.
Class: Bacillariophyceae
Order: Naviculales
Family: Naviculaceae
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.
Class: Bacillariophyceae
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.
Class: Bacillariophyceae
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.
Class: Bacillariophyceae
Order: Bacillariales
Borgohain and Tanti, 2014
Journal of Research in Biology (2014) 4(1): 1162-1173 1168
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.
Class: Bacillariophyceae
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.
Class: Bacillariophyceae
Order: Bacillariales
Family: Bacillariaceae
Genus: Nitzschia Hassall, 1845: 435
Nitzschia sp. (Fig. 8 A-Y)
Valves 27-30 µm long and 5.2-6.7 µm broad,
linear with concave sides and wedge shaped, constricted
produced ends, striae very fine, almost indistinct, striae
31-35 in 10 µm.
Class: Bacillariophyceae
Order: Naviculales
Family: Naviculaceae
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
Borgohain and Tanti, 2014
1169 Journal of Research in Biology (2014) 4(1): 1162-1173
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.
Class: Bacillariophyceae
Order: Surirellales
Family: Surirellaceae
Genus: Surirella Turpin 1828
Surirella sp. (Fig. 9 B)
Valves 55-65 µm long and 30-34 µm broad,
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.
Class: Bacillariophyceae
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.
Class: Bacillariophyceae
Order: Fragilariales
Family: Fragilariaceae
Genus: Tabularia (C. Agardh) D.M. Williams and
Round
Tabularia sp. (Fig. 9 E)
Valves 21-400 µm long and 3.1-5.3 µm broad,
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.
Class: Bacillariophyceae
Order: Cymbellales
Family: Cymbellaceae
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
Borgohain and Tanti, 2014
Figure 7 A: Rhopalodia, B- Kobayasiella, C- Actinella, D and E- Achnanthidium,
(F-J) Eunotia, (K-M) Synendra.
Journal of Research in Biology (2014) 4(1): 1162-1173 1170
stigmata at the ends of the middle ventral striae; striae
8-10 in 10 µm, punctate, radiate.
Class: Bacillariophyceae
Order: Naviculales
Family: Stauroneidaceae
Genus: Stauroneis Ehrenberg, 1843
Stauroneis sp. (Fig. 9 J-M)
Valves 62-66 µm long and 15-18 µm broad,
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
Borgohain and Tanti, 2014
Figure 8(A-R):Nitzschia
1171 Journal of Research in Biology (2014) 4(1): 1162-1173
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.
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Figure 9. A- Hippodonta, B- Surirella, C and D- Achnanthes, E- Tabularia, (F-I) Cymbella, (J-M) Stauroneis.
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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
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:
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.
Corresponding author:
Rumpa Saha.
Web Address: http://jresearchbiology.com/documents/RA0395.pdf.
Dates: Received: 01 Dec 2013 Accepted: 08 Feb 2014 Published: 17 Feb 2014
Journal of Research in Biology An International Scientific Research Journal
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.
MATERIALS AND METHODS
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
1175 Journal of Research in Biology (2014) 4(1): 1174-1181
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.
Journal of Research in Biology (2014) 4(1): 1174-1181 1176
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
1177 Journal of Research in Biology (2014) 4(1): 1174-1181
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
Journal of Research in Biology (2014) 4(1): 1174-1181 1178
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
1179 Journal of Research in Biology (2014) 4(1): 1174-1181
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.
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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
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www.jresearchbiology.com Journal of Research in Biology
An International
Scientific Research Journal
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.
Corresponding author:
Dounia.
Email Id:
Web Address: http://jresearchbiology.com/documents/RA0415.pdf.
Dates: Received: 15 Jan 2014 Accepted: 04 Feb 2014 Published: 11 April 2014
Journal of Research in Biology An International Scientific Research Journal
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.
MATERIALS AND METHODS
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
1210 Journal of Research in Biology (2014) 4(1): 1209-1219
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)
Journal of Research in Biology (2014) 4(1): 1209-1219 1211
Fohouo and Dounia, 2014
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.
Fohouo and Dounia, 2014
1212 Journal of Research in Biology (2014) 4(1): 1209-1219
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
Fohouo and Dounia, 2014
Journal of Research in Biology (2014) 4(1): 1209-1219 1213
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
Fohouo and Dounia, 2014
1214 Journal of Research in Biology (2014) 4(1): 1209-1219
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
Journal of Research in Biology (2014) 4(1): 1209-1219 1215
Fohouo and Dounia, 2014
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
Fohouo and Dounia, 2014
1216 Journal of Research in Biology (2014) 4(1): 1209-1219
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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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
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www.jresearchbiology.com
Journal of Research in Biology
An International
Scientific Research Journal
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.
Corresponding author:
Mustafa Korkmaz.
Email Id:
Web Address: http://jresearchbiology.com/documents/RA0421.pdf.
Dates: Received: 04 Feb 2014 Accepted: 05 Mar 2014 Published: 16 April 2014
Journal of Research in Biology An International Scientific Research Journal
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
1221 Journal of Research in Biology (2014) 4(1): 1220-1227
This study was aimed to determine the Gypsophila taxa
naturally distribute in the province of Tunceli city of
Turkey.
MATERIALS AND METHODS
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.
Journal of Research in Biology (2014) 4(1): 1220-1227 1222
Korkmaz and Ozçelik, 2014
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
RESULTS AND DISCUSSION
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
Korkmaz and Ozçelik, 2014
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
1223 Journal of Research in Biology (2014) 4(1): 1220-1227
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
Korkmaz and Ozçelik, 2014
Journal of Research in Biology (2014) 4(1): 1220-1227 1224
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.
www.jresearchbiology.com Journal of Research in Biology
An International
Scientific Research Journal
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.
Corresponding author:
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
Journal of Research in Biology An International Scientific Research Journal
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
1229 Journal of Research in Biology (2014) 4(1): 1228-1239
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
Journal of Research in Biology (2014) 4(1): 1228-1239 1230
Koladiya et al., 2014
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
Koladiya et al., 2014
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
1231 Journal of Research in Biology (2014) 4(1): 1228-1239
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
Koladiya et al., 2014
Figure. 3. Bird species diversity in various habitats of Banni grassland, Kachchh
Journal of Research in Biology (2014) 4(1): 1228-1239 1232
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
Koladiya et al., 2014
Figure 4. Seasonal abundance (%) of birds in Banni grassland of Kachchh, Gujarat
1233 Journal of Research in Biology (2014) 4(1): 1228-1239
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
Koladiya et al., 2014
Figure 5. Population density of birds in various habitats of Banni grassland, Kachchh
Journal of Research in Biology (2014) 4(1): 1228-1239 1234
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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Koladiya et al., 2014
1235 Journal of Research in Biology (2014) 4(1): 1228-1239
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
Koladiya et al., 2014
Journal of Research in Biology (2014) 4(1): 1228-1239 1236
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
Koladiya et al., 2014
1237 Journal of Research in Biology (2014) 4(1): 1228-1239
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
Koladiya et al., 2014
Journal of Research in Biology (2014) 4(1): 1228-1239 1238
Koladiya et al., 2014
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1239 Journal of Research in Biology (2014) 4(1): 1228-1239
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.
www.jresearchbiology.com Journal of Research in Biology
An International
Scientific Research Journal
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.
Corresponding author:
Abba H
Email Id:
Web Address: http://jresearchbiology.com/documents/RA0414.pdf.
Dates: Received: 19 Dec 2013 Accepted: 15 Jan 2014 Published: 16 April 2014
Journal of Research in Biology An International Scientific Research Journal
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.
MATERIALS AND METHODS
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
1241 Journal of Research in Biology (2014) 4(1): 1240-1246
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
Journal of Research in Biology (2014) 4(1): 1240-1246 1242
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.
RESULTS AND DISCUSSION
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
1243 Journal of Research in Biology (2014) 4(1): 1240-1246
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.
Journal of Research in Biology (2014) 4(1): 1240-1246 1244
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.
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Abba et al., 2014
Age
group
Age
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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)
1245 Journal of Research in Biology (2014) 4(1): 1240-1246
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