Journal of Research in Biology - Volume 1 Issue 6

Journal of Research in Biology An International Scientific Research Journal
A new record of Caracal (Caracal caracal) in Melghat Tiger Reserve,

Maharashtra, Central India After decades
Authors: ERRATUM:
Narasimmarajan K, The authors of the article entitled A new record of Caracal (Caracal caracal)
Bidyut Bikas Barman in Melghat Tiger Reserve, Maharashtra, Central India - After decades here with
and Lalthan Puia. informing the errors prevailing in their article published in Vol 1: Iss 6; P 399-402 of
our journal. The paper deals about the new record of Caracal in Melghat Tiger
Reserve, but they recently found that the photograph which they have shown in the
article is a Jungle Cat. The corresponding author Mr. Narasimmarajan says, I am
herewith asking you to please accept my error and retract this article. He added
Institution: humbly to publish this error message too so that the reader could understand the
Post, Box No: 18, error that has happened in course of his article assignment.
Wildlife Institute of India, He said that the photo seemed to be like a caracal on writing this article but
Chandrabani, Dehradun now they have identified it as a jungle cat due to clear vision and the guidance of
248 001. experts. It has happened purely by human error and low pixel of the image. They
accept that this paper information is completely wrong because of the photograph.
The researchers are asked not to follow this article for their future research. The
publisher and editor-in-chief also apologize for not identifying the fact in the course of
Corresponding author: peer-review.
Narasimmarajan K
Note: The article has been removed from the website for not getting spread out
hereafter. The article will soon be removed from all the indexed servers. A note has
been placed to them for the removal.
Journal of Research in Biology
An International Online Open Access
Original Research Paper
Publication group
Fly as pollinator in Caralluma umbellata Haw. (Asclepiadaceae) found in

the Pachamalai hills, Eastern Ghats, Tamil Nadu.
Authors: ABSTRACT:
Anburaja V1,
Nandagopalan V2 and Pollination studies have been done in the Caralluma umbellata, a member of
Prakash S3. Asclepiadaceae found in the Pachamalai hills of Tamil Nadu. The hills lie between
latitudes 1109'00" to 1127'00" N and longitudes 7828'00" to 7849'00" E. The
studies were carried out between April 2006 and April 2008. The main scope of the
Institution: work was to investigate the pollination of the C. umbellata. Pollinator captures were
12
PG and Research carried out by (1) stalking near plants visually searching for arrival of probable
Department of Botany, pollinators and (2) random captures in the sampling area. The phenology of flowering
National College, is starting in the month of February and extended up to April. The fruit set is starting
Tiruchirappalli 620 001. in the month of June onwards. The pollination in the C. umbellata is mainly done by
3
Post Doctoral Scholar, house fly. A fleshy odour is produced during the peak flowering season. This odouring
VOLCANI Centre, Israel. attracts the pollinator towards the flowers. The activity of the pollinator is almost
peak during 14:15 hrs to 17:30 hrs. The time duration of the fly retaining in the flower
is varied from few minutes to 25 minutes. The average time of the fly visiting the
flower is 8.29 7.98 minutes.
Corresponding author:
Nandagopalan V
Email: Keywords:
tvn135@yahoo.co.in Caralluma umbellata, house fly, pollination.
Web Address: Article Citation:

http://jresearchbiology.com/
Anburaja V, Nandagopalan V and Prakash S.
Documents/RA0101.pdf.
Fly as pollinator in Caralluma umbellata Haw. (Asclepiadaceae) found in the
Pachamalai hills, Eastern Ghats, Tamil Nadu.
Journal of research in Biology (2011) 6: 403-407
Dates:
Received: 09 Sep 2011 /Accepted: 16 Sep 2011 /Published: 12 Oct 2011
Ficus Publishers.
This Open Access 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.
403-407 | JRB | 2011 | Vol 1 | No 6

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Anburaja et al.,2011
INTRODUCTION Methods
Plant-animal pollination relationships have Fieldwork was carried out from April 2006
held the interest of scholars since the late 18th to April 2008. Most of the observations were
century (Sprengel, 1793) and played an important diurnal, with limited nocturnal surveillance. Since
role in shaping early ideas about natural selection the main scope of the work was to investigate the
(Darwin, 1880). The dicotyledonous plant pollination of the C. umbellata. Pollinator captures
Caralluma umbellata belongs to the family were carried out by following two techniques: (1)
Asclepiadaceae. It is otherwise known as the stalking near plants visually searching for arrival of
milkweeds, arguably one of the most highly probable pollinators, (2) random captures in the
evolved of all dicotyledon families, rivaling the sampling area. Captures were done with an
monocotyledonous Orchidaceae in floral entomological net and specimens were kept
complexity. As in many orchids, asclepiad pollen is separately in plastic test tube with cork shaving
usually presented for a pollinator as a coherent imbued with ethyl acetate until they could be drying
mass known as pollinium. Specialized prepared for identification. The standard deviations
transportation devices called translators have were calculated by using Microsoft Excel Program.
evolved to provide attachment of pollen to the Photo documentation was done by using Canon
pollinator, whilst pollination is usually brought Power Shot A 550, with the 7.1 mega pixels and up
about by insertion of the pollinium into modified to 4x optical zoom.
anther organs. From this basic ground plan, a range
of floral forms has evolved. An overview of the RESULTS AND DISCUSSION:
morphological aspects of asclepiad pollination is The average height of C. umbellata shoot is
given by Kunze (1995) has proposed a model of 32.68 7.62 cm. A single individual plant can
asclepiad floral evolution. This paper emphasizes produces 2.91 1.16 branches. Thus the total
the ubiquity of the fly - pollination in C. umbellata. population of the plant contains 58 10.14 erect
branches. Among these branches only 4 2.19
MATERAILAS AND METHODS branches are producing the inflorescence. Even
Study area though, only 1.37 0.91 branches are getting
Pachamalai hill is situated in the two maturity. From a single inflorescence 59.42 9.10
districts of Salem and Trichy of Tamil Nadu. The individual flowers are produced. The opening of the
hills lie between latitudes 1109'00" to 1127'00" N flower is from the base to the centre (Figure 3).
and longitudes 7828'00" to 7849'00" E. The total Only 1.71 0.75 flowers are producing the pod in
geographical area is 14,122 sq. km. A Sub - tropical an inflorescence after pollination. The fully matured
climate prevails with a maximum temperature pod reaches the length up to 15 2.44 cm (Table).
ranging from 23C to 31C, and a minimum The phenology of flowering is starting in the
temperature ranging from 12C to 18C. The annual month of February and extended up to April. The
rainfall varies with years and the maximum rainfall fruit set is starting in the month of June onwards.
is 1250 mm. The total area of Pachamalai is 14, 121 The pollination in the C. umbellata is mainly done
ha. The forests occupy 3806.92 ha. (26.96%). by house fly (Figure 4). A fleshy odour is
Material produced during the peak flowering season. This
Caralluma umbellata is an erect succulent odouring attracts the pollinator towards the flowers.
herb. The stem and branches are angles and contain The complicated structure of the corona and rigid
watery latex. Leaves are caducous and leaving spine hairs on the corolla enable flies only a limited
like scars on the angles. It produces inflorescence in access to the nectar glands. They fumble with the
the terminal part of the branches. The peduncle is proboscis near the slots towards the mouth of the
stout and short (1 cm). Flowers are mostly solitary flower. Doing this, the hairs or bristles of the head
and sometimes paired. The length of the pedicel is or legs are often stuck in the guide rails and only
up to 3.5 cm. Calyx is 5 -7 lobed and sub equal. one way is possible upwards to the end of the
Calyx is lanceolate in shape and thick. Corolla is stamina lock, which is connected to the jag of the
purplish brown with yellow bands. Pollinial bags corpusculum of the pollinarium. Only one fly can
are minute. Receptacle found. Ovaries in two be accommodated at a time in the flower. But,
number but sometimes three. The shape of the seeds sometimes another fly comes and struggle with the
is oblong and obtuse in the ends with silky white already existing fly, the dominating fly is retaining
coma. in the flower. After getting enough nectar from the
404 Journal of Research in Biology (2011) 6: 403-407
flower, the fly start to go away from the flower and sapromyiophilous pollination syndrome. According
then only the fly comes to feel that their mouth to Dobson (Dobson. 2006) a single fly species may
parts are trapped in the receptacle. At this time, the visit distinct flowers for different purposes (i.e.
fly tries to escape from the receptacle with their full food versus oviposition) and therefore pollinate
force; finally it pulls out the pollinal bags and flowers. In particular flies prefer yellow in the
carries to the next flower. If the flies are too weak presence of sweet scents, which signal food
to remove the pollinarium, they remain trapped in sources, and brownpurple in the presence of odor of
the flower. At this time the weak flies are hunted by excrements, which indicate egg-laying sites.
the spiders (Figure 5). The pollinator is trapped Flowers of C. europaea are brown-purple with
again and if it is strong enough, it frees itself or yellow stripes, they contain compounds with both
tears off the remnant of the pollinarium at the sweet odors and compounds found in excrements,
translator arm. It can pollinate another flower with and in this way they may mimic both food
the remaining pollinium. The placed pollinium resources and oviposition sites thus augmenting the
remains in the receptive area of the style. spectrum of potential pollinators.
The activity of the pollinator is almost peak Flowers in taxa of the genus Caralluma
during 14:15 hrs to 17:30 hrs. The time duration of show decaying organic matter odours, like C.
the fly retaining in the flower is varied from few arachnoidea with scents of rotting fruits and are
minutes to 25 minutes. The average time of the fly pollinated by small Drosophilidae or Milichiidae,
visiting the flower is 8.29 7.98 minutes. while the floral odour of Desmidorchis flava can be
described as reminiscent of decaying urine or
TABLE: Vegetative and Flowering characteristics of pungent and ruinous and Coleoptera (Dermestidae)
C. umbellata. have been recorded as flower visitors, although it
S.
Parameter Observations
has not been determined whether they really act as
No pollinators (Jurgens et al., 2006).
1 Height of the Plant (cm) 32.68 7.62
Number of Branches in an CONCLUSION
2 2.91 1.16
Individual
The flowering of Caralluma umbellata is
Number of Branches Branch in
3 58 10.14 peak during the months February and April. The
a Population
Number of Branch initiating
pollination in the C. umbellata is mainly done by
4 4 2.19 house fly. A fleshy odour is produced during the
inflorescence
Number of inflorescence peak flowering season. This odouring attracts the
5 1.37 0.91 pollinator towards the flowers. After getting enough
Matured
Number of flowers in nectar from the flower, the fly tries to escape from
6 59.42 9.10
inflorescence the receptacle with their full force and at the same
Number of Pod Produced Per time it also pulls out the pollinal bags and carries to
7 1.71 0.75
Flower the next flower. If the flies are too weak to remove
8 Pod Length (cm) 15 2.44 the pollinarium, they remain trapped in the flower
and hunted by the spiders.
Similar results are obtained in some
Bulbophyllum species have flowers that are
pollinated by flies belonging to four dipteran
families, Calliphoridae, Lonchaeidae, Milichiidae,
as well as Tephritidae (Christensen, 1994). But
there is no information as to the actual chemical
component(s) responsible for fly attraction. The
foral structure of the Asclepiadaceae has lead to
highly specialized insect pollination that promotes
out breeding (Wyatt and Broyles, 1994).
It is interesting to note that all the Diptera
families involved in pollination have a biology
linked to decaying organic matters and therefore
Caralluma europaea falls within the
Journal of Research in Biology (2011) 6: 403-407 405

Figure 1 Figure 2
Figure 3 Figure 4
Figure 5 Figure 6
Figure 1: The habit of C. umbellata.

Figure 2: Formation of the inflorescence at the apex of the fleshy shoot.
Figure 3: Base to the Centre Opening of the developed flowers.
Figure 4: Fly busy in pollination.
Figure 5: Fly hunted by the spider.
Figure 6: After pollination

REFERENCES
Christensen DE. 1994. Fly pollination in the
Orchidaceae. In: Arditti J, ed. Orchid biology:
Reviews and perspectives VI. London: John Wiley
& Sons 415-454.
Darwin C. 1880. The different forms of flowers on

plants of the same species. London: Murray.
Dobson HEM. 2006. Relationship between floral

fragrance composition and type of pollinator. In
Biology of Floral Scent; Dudareva NA, Pichersky
E., Eds.; CRC Press Taylor & Francis Group:
London, UK, 147-198.
Jurgens A, Dotterl S, Meve U. 2006. The

chemical nature of fetid floral odours in stapeliads
(Apocynaceae-Asclepiadoideae-Ceropegieae). New
Phytol., 172:452-468.
Kunze H. 1995. Bau und Funktion der

Asclepiadaceenblute. Phyton (Austria) 35:1-24.
Sprengel CK. 1793. Das entdeckte Geheimnis der

Natur im Bau in der Befruchtung der Blumen. (The
secret of nature in the form and fertilisation of
flowers discovered). Berlin: F. Vieweg.
Wyatt R, Broyles SB. 1994. Ecology and evolution

of reproduction in milkweeds. Annual Review of
Ecology and Systematics 25:423-441.

Publication group
First report of anthracnose disease of Aloe vera caused by Colletotrichum

gloeosporioides
Authors: ABSTRACT:
Shubhi Avasthi1, Ajay
Kumar Gautam2 and
Rekha Bhadauria1.
A new disease of Aloe vera was found in Madhya Pradesh, India having
Institution:
1 reddish brown to brown colour lesions on leaf surface. The disease was found to be
School of Studies in
Botany, Jiwaji University, caused by a fungus. The fungus was exclusively isolated from the disease spots, and
Gwalior- 474011 (M.P.) typical symptoms were reproduced after inoculation with the isolate. The causal
India. fungus was identified as Colletotrichum gloeosporioides Penz. & Sacc.
2
Department of Botany,
Abhilashi Institute of Life
Sciences, Mandi-175008,
(H. P.), India.
Corresponding author: Keywords:

Rekha Bhadauria Aloe vera, Anthracnose, Colletotrichum gloeosporioides.
Email: Article Citation:

rekhabhadauria@yahoo.com Shubhi Avasthi, Ajay Kumar Gautam and Rekha Bhadauria.
First report of anthracnose disease of Aloe vera caused by Colletotrichum
gloeosporioides.
Web Address: Dates:

http://jresearchbiology.com/ Received: 16 Sep 2011 /Accepted: 28 Sep 2011 /Published: 12 Oct 2011
Ficus Publishers.
cited.
408-410 | JRB | 2011 | Vol 1 | No 6

Avasthi et al.,2011
INTRODUCTION with 1% sodium hypochlorite (NaOCl) for 2 min

Aloe barbadensis (L.) Burm. Fil. popularly and washed 3-4 times in sterile distilled water. The
known as Aloe vera, is a perennial, drought surface sterilized leaf pieces were then aseptically
resistance, succulent medicinal herb belongs to the transferred to petriplates containing Potato Dextrose
family Aloeaceae (Barcroft and Myskja, 2003). It Agar media. Plates were incubated at 252 for 4 to
has a long, fleshy sword- shaped leaves, with sharp 5 days and the isolate was purified by single spore
points arranged in a rosette pattern. Aloe vera is isolation and maintained on PDA medium to keep
believed to have originated in African continent the cultures viable.
specifically in Egypt (Daudu, 2000).The central The fungal colony appeared on inoculated
bulk of the leaf contains colourless mucilaginous tissue were white to grey with puffy mycelium and
pulp (A. vera gel), made up of large, thin walled dark orange colour, surrounded by white zone on
mesophyll cells. The plant contains 95 96% water the reverse side (Figure 1D). Acervuli were formed
and over 75 other constituents which include after15-20 days of inoculation. Conidiophores were
vitamins, minerals, enzymes, sugars, phenolic simple, short and erect. The conidia were hyaline,
compounds, saponins and amino acids (Boudreau one celled, ovoid to oblong and dumbbell shaped.
and Beland, 2006). It is highly appreciated due to The size of conidia varied from 12.5-183-5 m
its short growth period and highly economic value. (Fig.1F). Setae were 1 -4 septate, brown and ranged
In India the plant is mainly cultivated in Rajasthan, in size from 42- 150 4-5 m (Figure1E). Based
Andhra Pradesh, Gujarat, Madhya Pradesh and on the symptoms, mycelia and conidial characters,
Maharashtra. Total production of A. vera in India the fungus was identified as Colletotrichum
has been estimated to be 1, 00,000 tonnes (Dubey gloeosporioides (Penz.) Penz. & Sacc., which was
and Pandey, 2009). It is used for its laxative, anti- further confirmed at Indian Agricultural Research
inflammatory, immunostimulant and antiseptic Institute, New Delhi (#ITCC- 7800.10).
effect (Capasso et al, 1998). It is very much Pathogenicity of the fungus
effective for the treatment of sore and wounds, skin Pathogenicity of the fungus was carried out
diseases, reduce blood sugar in diabetes, arthritic by pin prick method on detached leaves. Small 5-6
swelling, constipation and pile (Rajendran et al, month old plants of Aloe vera were planted in pots
2007). filled with fertilized soil and cultivated for 6-8
Occurrence and symptoms weeks in a glasshouse. Healthy leaves were selected
During the survey of various nurseries of and a suspension (105 conidia/ml) of 7-8 days old
Gwalior city, India, a typical anthracnose symptom culture of C. gloeosporioides was sprayed onto
on the leaf surface of A. vera was observed in pinpricked leaves in petridishes lined with moist
August, 2010. The symptoms of anthracnose were sterile blotting paper. Detached pinpricked leaves
began as a small round to oval, water-soaked dark sprayed with sterile distilled water were served as
green area about 1-2 mm in diameter. These area control. The petridishes were incubated at 252C
increase into circular spots with tan to light brown for 6-7 days under laboratory conditions. The
centre bordered by water soaked tissue. As these pathogen was appeared on the inoculated leaves and
spots expand, centre of the lesion become reddish the fungus was consistently re-isolated from
brown to brown color. The average diameter of the infected leaf onto Potato Dextrose Agar media. No
spots was 3-30 mm and the size of the necrotic symptoms were observed in control leaves.
areas increases as spots coalesce (Figure 1A & B). Colletotrichum gloeosporioides has
The acervuli on infected leaves produced black previously been reported as an anthracnose
coloured spore mass under high humid condition pathogen of olives (Olea europaea) in Australia
(Figure1C). In the advance stage of infection, spots (Sergeeva et al, 2008), on onion (Allium cepa) in
appeared on both the surfaces of leaf, affected area Benin (Sikirou et al, 2011) and in Kokum (Garcinia
lost the mucilaginous gel and leads the death of indicia) (Jadhav et al, 2009) and clove (Syzygium
infected leaves. aromaticum) (Jadhav et al, 2008) from India. To the
Isolation and Identification of the fungus best of our knowledge; this is the first report of
Isolation of the pathogen was done by anthracnose disease of Aloe vera in India.
collecting diseased plant samples from various
nurseries. Leaves showing the typical symptoms ACKNOWLEDGEMENT
were thoroughly washed in tap water and cut into The authors are thankful to Head, School of
small pieces. These pieces were surface sterilized Studies in Botany, Jiwaji University, Gwalior,
Avasthi et al.,2011
Fig.1. (A): Leaf showing initiation of anthracnose and coalesce symptom (B) Typical anthracnose symptom (C)
Acervuli of C. gloeosporioides on leaf (D) Seven-day old Culture of C. gloeosporioides (E) Acervuli with dark
brown setae (F) Conidia of C. gloeosporioides.
Madhya Pradesh, India, for providing necessary Daodu T. 2000. Aloe vera, the miracle healing
laboratory facilities and constant support. plant. Health feild Corporation, Lagos, 36.
REFERENCES Boudreau MD, Beland FA. 2006. An evaluation

Sikirou R, Beed F, Hotgni F, Winter S, Assogba of the biological and toxicological properties of
-Komlan F, Reeder R Miller SA. 2011. First Aloe barbadensis (Miller). J. Environ. Sci. Health
report of anthracnose caused by Colletotrichum 24:103-154.
gloeosporioides on onion (Allium cepa) in Benin.
New Dis. Reports 23:7. Capasso F, Borrelli F, Capasso R, Di Carlo G,
Izzo AA, Pinto L, Mascolo N, Castaldo S, Longo,
Sergeeva V, Spooner-Hart R, Nair NG. 2008. R. 1998. Aloe and its therapeutic use. Phytother.
Colletotrichum acutatum and C. gloeosporioides Res., 12:S124.
causing leaf spots of olives (Olea europaea) in
Australia. Aus. Pl. Dis. Notes 3:143-144. Dubey R, Pandey AK. 2009. A new report on
collar and root rot disease of Aloe vera caused by
Jadhav SK, Diwakar MP, Sawant UK, Kadam Sclerotium rolfsii. J. Mycol. Pl. Pathol., 39:300-
JJ. 2008. Management of leaf spot disease of 301.
Kokum (Garcinia indica) incited by Colletotrichum
gloeosporioides Penz. J. Pl. Dis. Sc., 3. Rajendran A, Narayanan V, Gnanavel I. 2007.
Sepration and Characterization of the Phenolic
Jadhav SN, Gadre UA, Kadam JJ, Joshi MS, Anthraquinones from Aloe vera. J. App. Sci. Res.,
Sawant UK. 2009. In vitro evaluation of bioagents 3:1407-1415.
against Colletotrichum gloeosporioides Penz.
causing leaf spot of clove. J. Pl. Dis. Sc. 4:104-106.
Baracroft A, Myskja A. 2003. Aloe vera natures

silent healer. BAAM Pub. Ltd. 28-45.

Publication group
Ameliorative effect by calcium on NaCl salinity stress related to reactive

oxygen species metabolism in Amaranthus tricolor L.
Authors: ABSTRACT:
Sunukumar SS, Harish
SR, Manoj GS, Soil salinity affects plant growth and development by way of osmotic
Sreelekshmi SG, Remya imbalance which induces oxidative damage in plant tissues. Oxidative stress is caused
Krishnan, Lubaina AS and
by increased production of Active Oxygen Species (AOS), such as O 2, OH, H2O2 and
Murugan K. 1
O2 these lead to lipid peroxidation, enzyme inactivation and oxidative damage to
DNA. Plants have antioxidant defense systems to protect against the production and
action of the AOSs. Plants with high level of antioxidants, either constitutive or
induced, have been reported to produce greater resistance to this oxidative damage in
plant cells. AOS synthesis and its scavenging were investigated in the control, different
concentrations of NaCl and NaCl + CaCl2 stressed Amaranthus tricolor L. and Phaseolus
Institution: vulgaris L.. AOS such as superoxide anion and H2O2 content showed a steady increase
Plant Biochemistry and in the plants of all NaCl treated media compared to control. When the salinized media
Molecular biology Lab, were supplemented with CaCl2 the AOS level drastically decreased compared to the
Department of Botany, corresponding plants grown on salt alone. Similarly, the activity of antioxidant
University College, enzymes such as superoxide dismutase, ascorbate peroxidase, catalase and
Thiruvananthapuram-695 glutathione reductase under salt stress were higher in NaCl + CaCl 2 supplemented
034, Kerala, India. media than the plants on the salinized media alone. This suggested that the alleviation
effect of calcium under saline condition was through modulation of the enzyme
complexes that accelerate the rate of antioxidant enzymes biosynthesis under salt
stress. Similarly, the level of lipid peroxidation was found to be lower in plants of all
NaCl + CaCl2 media than control.
Murugan K.
Keywords:
Amaranthus tricolor L., Phaseolus vulgaris L., superoxide anion, H2O2; NaCl,
CaCl2, superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase,
lipid peroxidation.
Email: Article Citation:

harimurukan@gmail.com Sunukumar SS, Harish SR, Manoj GS, Sreelekshmi SG, Remya Krishnan, Lubaina AS
and Murugan K.
Ameliorative effect by calcium on NaCl salinity stress related to reactive oxygen
species metabolism in Amaranthus tricolor L.
Web Address: Dates:

http://jresearchbiology.com/ Received: 17 Sep 2011 /Accepted: 08 Oct 2011 /Published: 12 Oct 2011
Ficus Publishers.
cited.
411-418 | JRB | 2011 | Vol 1 | No 6

Sunukumar et al.,2011
INTRODUCTION -nitro-5-sulfophenyl)-2H-tetrazolium-5-
Soil salinity is an inevitable problem for carboxanilideinner salt (XTT sodium salt). The O2.-
agriculture in most parts of the world. Better production rate was calculated using molar
understanding of the mechanisms that enable plants extinction coefficient 2.16 104 M-1 cm-1.
to adapt to salt stress is necessary to make the best Quantification of hydrogen peroxide (H2O2)
use of these saline soils. Cellular mechanisms are Horse radish POX-catalysed oxidation of
especially important to glycophytes, in which TMB was used for the quantification of H2O2 and
physiological and biochemical processes contribute was measured colorimetrically at 450 nm (Gallate
to the adaptation to salt stress. In recent years, and Pracht, 1985),
biochemical responses of plants to salt stress have Lipid Peroxidation
been studied intensively. One of the biochemical The level of lipid peroxidation in the cells
changes possibly induced by salt stress is the was measured in terms of malondialdehyde content
increased production of Active Oxygen Species determined by the thiobarbituric acid reaction as
(AOS) (Yildirim etal., 2006; Vaidyanathan et al. described by Heath and Packer, (1968). The
2003; Michaela et al., 2002 ). ROS are highly concentration of malondialdehyde was calculated
reactive and when the capacity of plant for using the extinction coefficient 155 mm/L.
scavenging is less than ROS production they can Antioxidant enzyme extraction and assay
seriously disrupt normal metabolism through Old and young leaves (0.5 g) were
oxidative damages of lipids, proteins and nucleic homogenized in 25 mm K-phosphate buffer (pH
acids. Plants posses a number of antioxidant 7.8) containing 0.4 mm EDTA, 1 mM ascorbate and
systems that protect them from these potential 2% (w/v) polyvinylpolypyrrolidone (PVPP). The
cytotoxic effects. Antioxidant enzymes are the most homogenate was then centrifuged at 15,000 x g for
important components in the scavenging system of 20 min at 4C. The filtered supernatant was used as
ROS. Superoxide dismutase (SOD) is a major an enzyme extract for APX, CAT and GR
scavenger of O2. and its enzymatic action results in (Yanagida et al. 1999).. For SOD assay, the extract
the formation of H2O2. Catalase (CAT), ascorbate was dialyzed overnight with 10 mm K-phosphate
peroxidase (APX) and a variety of general buffer (pH 7.8) at 4C. The dialyzed extract was
peroxidases catalyze the breakdown of H2O2. centrifuged at 15,000 x g for 20 min at 4 C.
Therefore, these enzymatic systems eliminate the Superoxide dismutase (SOD) assay was
damaging effects of toxic oxygen species. Paraquat calculated following the formula below; (Vb/Vs)-1,
is a redox-active compound wildly used to control where Vb is the reaction rate of the blank and Vs is
existing vegetation (Suntres, 2002; Skorzyn ska- the reaction rate of the sample.
Polit et al., 1998). The mechanisms of paraquat Catalase (CAT) reaction mixture consisted
toxicity involve: 1) the generation of the O2. in the of 50 mm K-phosphate buffer (pH 7.0) containing
light, which can lead to the formation of more toxic 10 mm H2O2 (0.95 ml) and enzyme extract (0.05
AOS, such as OH 2) lipid peroxidation which ml) was prepared. Immediately after adding the
results in the oxidative degeneration of cellular enzyme to the buffer, the initial rate of absorbance
polyunsaturated fatty acids. Therefore, the aim of at 240 nm was determined. The molar absorption
this work was to evaluate the effects of NaCl stress coefficient of H2O2 (0.04/ mm /cm) was used to
and its alleviation by calcium on active oxygen calculate the enzyme activity.
species formation, the lipid peroxidation and the Glutathione reductase (GR) assay mixture
activity of antioxidative enzymes in Amaranthus consisted of 100 mm K-phosphate buffer (pH 7.8)
tricolor L. and Phaseolus vulgaris L. in order to (0.25 ml), 10 mm oxidized glutathione (GSSG)
better understand their differences on salt stress (0.05 ml), distilled water (0.48 ml), 1 mm NADPH
tolerance. (0.12 ml) and enzyme extract (0.1 ml) was
Quantification of superoxide anion ( O2.-) prepared. The assay was started by addition of
The O2.- production rate was measured as GSSG. GR activity was determined from the rate of
described previously (Jiang and Zhang, 2002) with NADPH oxidation measuring the decrease of
some modifications. 0.2 g leaf tissue was absorbance at 340 nm. The molar absorption
homogenized in 1 ml of 50 mM TrisHCl (pH 7.5) coefficient of NADPH (6.1/ mm /cm) was used to
and centrifuged at 5000 g for 10 min at 4C. The calculate the enzyme activity. In ascorbate
reaction mixture (1 ml) contained 200 ml peroxidase (APX) assay, the reaction mixture
supernatant and 800 ml 0.5 mm 3- bis (2-methoxy-4 consisted of 100 mm K-phosphate buffer (pH 7.0)
(0.25 ml), 1 mM ascorbate (0.25 ml), 0.4 mm both the species (data not shown). But NaCl +
EDTA (0.25 ml), distilled water (0.19 ml), 10 mm CaCl2 treated plants shows a significant reduction
H2O2 (0.01 ml) and enzyme extract (0.05 ml) was in O2- and H2O2 content upto 200 mm NaCl in
used. The reaction was started by adding H2O2 and A.tricolor and thereafter the free radical content
the oxidation rate of ascorbate was measured by the slightly increased. In P.vulgaris, external Ca2+ did
initial rate of decrease of absorbance at 290 nm. not significantly reduced O2- and H2O2 content after
The molar absorption coefficient of ascorbate (2.8/ 100 mm NaCl concentration.
mm /cm) was used to calculate the enzyme activity. Ca2+ treatment on MDA Content
Paraquat sensitivity assay Application of external Ca2+ + NaCl could
Ten discs with 4 mm diameter were excised reduce MDA content considerably, compared with
from the NaCl and NaCl + CaCl2 treated leaves NaCl stress media in A.tricolor, where as
and soaked into 3 ml of 0.3 m of paraquat (1,1- marginally in P.vulgaris, compared with those of
dimethyl-4,4-bipiridinium dichloride salt) solution non-external calcium medium contain only NaCl
with their adaxial side up. Paraquat soaked treated (150 mm) (Table 4). However, in control,
leaves were preincubated in the dark for 1 h and application of external calcium made no significant
then irradiated with fluorescent light (260-280 E/ difference to MDA content compared with non-
m2/s) at 25C for 48 h. Chlorophyll contents in the external Ca2+ media.
leaf discs were determined with the modified Paraquat assay
method described by Chappelle et al. (1992). Effect of paraquat on leaf discs preparing
Statistical analysis from the seedlings treated with NaCl + Ca2+ were
The data obtained were analyzed statistically determined by measuring chlorophyll contents (Fig.
and the results are presented as Mean Standard 1 A & B). Chlorophyll contents in the young and
deviation. old leaf discs of A.tricolor both of nontreated and
treated with 0.3 M paraquat slightly increased with
RESULTS AND DISCUSSION increasing NaCl + Ca2+concentration. In contrast,
Application of external NaCl + Calcium (5 chlorophyll contents in the young and old leaf discs
mM CaCl2) elevated greatly FW and DW compared of P. vulgaris were greatly reduced with increasing
to NaCl alone in both species, but differently i.e., NaCl + Ca2+concentration.
FW of root and shoot in A.tricolor ranged between Chlorophyll contents in the disks were
0.4 to 1.88; 0.58 2 fold respectively whereas, in measured to determine the decomposition of the
P.vulgaris it was 0.68 - 0.83; 0.43 0.58 fold. pigment caused by generated AOS. With increasing
Similarly the DW of root and shoot in A.tricolor NaCl concentrations + 5 mm Ca2+, chlorophyll
was 0.74-1.47 and 0.63 1.6 fold. However, in contents in the discs from young and old leaves of
P.vulgaris external calcium treatment had less Table 1 b. Effect of NaCl + CaCl2 (5 mM) on fresh
significant difference on FW and DW of shoot in and dry weights (g/plant) of 14 days old P.vulgaris
comparison with the same parameters in the control seedlings (D= dead).
(Table 1 a & b). NaCl Conc.
External Ca2+ on O2- and H2O2 Content 0 50 100 150
(mM)
The trend of O2- and H2O2 content was Fresh
8.2 0.3 6.8 0.2 5.6 0.4 D
remarkably different between treatments containing weight
external calcium (Table 2 & 3). O2- and H2O2 Root Dry weight 0.6 0.89 0.45 0.3 0.4 0.2 D
content reduced marginally in stress media Shoot
Fresh
7.2 0.6 4.2 0.2 3 0.2 D
containing only external calcium (5 mm CaCl2), in weight
Dry weight 0.86 3 0.58 0.1 0.4 1 D
Table 1 a. Effect of NaCl + CaCl2 (5 mM) on fresh and dry weights (g/plant) of 14 days old
A.tricolor seedlings.
NaCl Conc.
0 50 100 150 200 250
(mM)
Fresh weight 4.1 0.6 6.9 0.3 4.2 0.2 3.4 0.2 2.4 0.3 1.6 0.2
Root Dry weight 0.38 0.04 0.56 0.7 0.42 0.1 0.42 0.1 0.35 2 0.28 0.1
Fresh weight 3.80.4 7.6 0.2 5.04 0.2 5.4 0.1 3.1 0.6 2.2 0.3
Shoot Dry weight 0.760.1 1.2 0.4 0.64 0.3 0.61 0.1 0.53 1 0.48 0.2

Fig. 1 A & B. Effects of paraquat on chlorophyll contents in leaf discs from young leaves (A) and old leaves (B)
of A.tricolor and P. vulgaris treated by 0, 50, 100, 150, 2000 and 250 mM NaCl + 5 mm CaCl 2 for 12 days.
Vertical bars indicate standard errors (n=3). 0 m paraquat; 0.3 m paraquat.
A.tricolor did not decrease. On the contrary, adverse environmental conditions (Pie et al., 2000).
tolerance to paraquat was slightly higher in NaCl + In vitro studies on the cell wall formation in spruce
Ca2+ treated plants. This finding indicates that this hypocotyl cuttings indicated that Ca2+ was essential
species has greater tolerance to the toxicity of AOS. and its absence made the wall more susceptible to
Antioxidative enzyme system in A.tricolor seemed injury (Price et al., 1994). Shabala et al., (1997)
to be effective for elimination of AOS which arises observed that higher Ca2+ concentration was
from salt stress. In case of P. vulgaris, it was important in maintaining cell membrane integrity
difficult to determine whether the treated paraquat under water stressed conditions and that was a
cause reduction of chlorophyll content with the 50 function specific to Ca2+. In this study, application
and 100 mM NaCl treatment, because the leaves of external calcium resulted in higher FW and DW
showed chlorosis and necrosis. content and lower MDA content in cells compared
Ca2+ treatment on APX, SOD, CAT and GR with the contents in media with only NaCl. The
The antioxidant enzyme activity trends in data suggested that more concentrated CaCl2
the species were similar to control plants containing treatment could significantly mitigate the damage
only external calcium (CaCl2). However, a of NaCl stress, and the effect was specific to Ca2+
paramount increase in activities of antioxidant modulated proteins.
machinery was observed in the species when treated Our data showed that the activities of SOD,
with external calcium + NaCl i.e., SOD, CAT, and CAT and APX in cells remained relatively steady
APX activities were higher than in media contain under control conditions compared with those under
only NaCl (Table 5 a & b). GR showed steady NaCl stress conditions. This suggests that SOD,
activity in all the experimental conditions CAT and APX participate in the regulating
irrespective of treatments. mechanism of cells against salt stress and was
In recent years, it has been widely noted that consistent with the result of Shabala et al., (1997).
Ca2+ plays an important role in the adaptation of The result indicated that external calcium could
plants to adverse environments (Bowler and Fluhr, elevate the activities of SOD, CAT, and APX in
2000). It was indicated that Ca2+ had a pertaining cells under NaCl stress conditions. This increase is
role in preventing cell membrane injury and leakage regarded as an adaptive signal to trigger gene
as well as stabilizing cell membrane structure under expression and activate some unknown biochemical
Table 2. Effect of NaCl + CaCl2 (5 mM) on Super Table 3. Effect of NaCl + CaCl2 (5 mM) on hydrogen
oxide anion (O2.-) (molmin-1g-1) of 14 days old peroxide (molmin-1g-1) of 14 days old seedlings.
seedlings. (D= dead). NaCl
Conc. 0 50 100 150 200 250
NaCl (mM)
Conc. 0 50 100 150 200 250
(mM) A.tricolor 0.70.01 1 0.22 1.70.6 2.40.6 2.90.7 3.90.2
A.tricolor 0.290.08 0.450.3 1.30.3 1.90.2 2.30.3 3.20.11
P.vulgaris 0.40.13 2.20.12 5.90.23 12.40.2 D D
P.vulgaris 0.150.4 10.05 2.40.15 40.52 D D
(D=dead)
events to enable plants to adapt to environmental concentrations and was significant at 200 mM NaCl
stress (Rengel, 1992). In addition, the calcium + CaCl2. The results are comparable with that of
receptor protein calmodulin and other calcium- Phyllanthus amarus and Catharanthus roseus under
binding proteins have been found to be involved in water and salt stress (Jaleel et al., 2008; 2007 b).
the regulation of plant responses to environmental The increased ROS accumulation in Arabidopsis
stress (Ishitani et al. 2000). root and leaf cells has been related to the
Ca2+ is also a primary second messenger in maintenance of a more favourable K+/ Na+ ratio in
signal transduction and regulates physiological and the NaCl stressed roots supplied with calcium and it
biochemical processes in the responses of plants to was suggested that the failure to maintain a
extracellular adverse abiotic environments (Bowler favourable K+/ Na+ ratio can inhibit enzyme
and Fluhr, 2000). The important role of calcium functions (Shabala et al., 2006). Studies on salt
signals in the transduction of environmental change tolerance in peanut reveals that the K+/ Na+ ratio
into plant response has been documented over a increased at all levels of external salinity (Girija et
wide range of stimuli (Shabala et al., 1997; Romano al., 2002). The mechanism leading to the increase
et al., 1998). Much of the evidence has shown that in antioxidant enzyme levels in salinized plants by
external calcium treatment can increase tolerance calcium is not clear still. However, it is evident
capacity to adverse environments involving drought from the present study that the AOX enzymes
(Bowler and Fluhr, 2000), cold (Shabala and synthesis is accelerated by some Ca++ dependent
Newman, 2000), heat, and salt stress (White and mechanisms, may be by the formation of Ca++
Broadley, 2003). modulated protein complexes in the cytosol, which
Ca2+ signal patterns can occur as single might have activated enzymes; leading to
transients or repetitive oscillations (Demidchick et scavenging of ROSs (Jaleel et al., 2008). Ca++ is
al., 2002 a; Elphick et al., 2001), and also different reported to reduce the toxic effect of NaCl salinity.
signaling patterns, such as oscillations and waves, Ca++ supplementations apparently alleviate NaCl
may arise from the selective activation of stress, at least in part, by modulating the enzyme
transcriptional regulators. Special calcium signaling protein and a greater reduction in lipid peroxidation
coming from different stimuli/conditions should be to maintain low ROS levels which can be expected
a mechanism that perceives and transudes different to contribute for an ideal level osmoregulation.
signals stimuli and causes different physiological Osmotic adjustment is an important mechanism of a
responses in plants. In this investigation, external plants tolerance to a stress environment (Halfter et
calcium treatment increased total calcium content in al., 2000). Osmotic adjustment and osmotic
cells. For determining the signal role of Ca2+, potential increased slowly in cells under prolonged
intracellular free calcium ion content and stress, but were not affected significantly by
subcellular locations of Ca2+ will be measured in external Ca2+ treatment under salt stress conditions.
our next investigation. The results indicate that the effect of external Ca2+
AOSs such as O2.- and H2O2 content in NaCl on cells was not due to the regulation of osmotic
stressed A.tricolor was comparatively more than potential or osmotic adjustment. Thus, the present
control but its increase was more obvious in study suggests that A.tricolor has higher levels of
P.vulgaris. Thus an unambiguous correlation was antioxidative enzymes both constitutive and
noticed between salt concentrations and ROSs induced resulting in greater resistance to oxidative
content (Table 3 & 4). Interestingly, the magnitude damage caused by NaCl stress and is mitigated
of ROS content in A.tricolor was decreased by efficiently by calcium salts.
supplementing the salinized media with CaCl2
compared to the plants with corresponding NaCl
Table 4. Effect of NaCl + CaCl2 (5 mM) on Lipid peroxidation (nmolg-1) in NaCl + CaCl2 (5 mM) treated
A.tricolor and P. vulgaris 14 days after starting NaCl treatments (D= dead).
NaCl Conc. 0 mM 50 mM 100 mM 150 mM 200mM 250mM
A.tricolor 10.01 1.30.22 20.6 2.80.04 3.20.9 4.20.25
P.vulgaris 0.50.1 2.70.3 60.65 9.50.4 D D

Table 5 a & b. Antioxidant enzymes such as superoxide dismutase (SOD, U/g), Ascorbate peroxidase (APX,
mol AsA decomposed/gFW/min), Guaiacol peroxidase (POX), Catalase (CAT, mol H2O2 decomposed/gFW/
min) and Glutathione reductase (GR, mol NADPH oxidized/gFW/min) in NaCl + CaCl2 (5 mM) treated
A.tricolor and P. vulgaris 14 days after starting treatments.
Table Young leaves A.tricolor
5a
NaCl conc.
(mm) SOD APX CAT GR
0 672 0.2 4.90.7 4420.2 2.40. 09
50 12890.6 9 0.8 6980.8 3.90.6
100 14060.4 160.5 7440.6 4.50. 9
150 14980.2 18 0.4 9240.9 4.60.8
200 15220.3 19.71 9470.3 4.70.2
NaCl conc.
Young leaves P.vulgaris
(mm)
0 207 0.1 30.03 1580.27 1.40. 09
50 3880.07 8.6 0.07 2310.11 1.90.15
100 2230.04 11.50.06 2010.12 1.60. 43
150 1560.01 6 0.09 1960.12 10.24
200 D D D D
Table 5b. Old leaves A.tricolor

NaCl conc.
(mm) SOD APX CAT GR
0 332 0.9 4.30.17 3690.22 30. 9
50 5120.06 5.9 02.8 4810.28 40.05
100 6990.34 80.35 51402.6 4.80. 06
150 8030.22 9.8 0.44 5670.29 3.90.07
200 8200.23 10.40.81 5770.33 40.02
NaCl conc.
Old leaves P.vulgaris
(mm)
0 197 42 2.40.17 1190.12 1.80. 9
50 2490.26 5 0.81 1410.28 2.30.26
100 1980.64 5.4.90.25 1140.16 1.80.1 9
150 1840.82 4.7 0.24 96.40.39 1.40.38
200 D D D D
The data are the means of four replicates S.E.
ACKNOWLEDGEMENTS Chappelle EW, Kim MS and McMurtrey JE.

The author sincerely acknowledges the 1992. Ratio analysis of reflectance spectra (RARS):
University Grant Commission, New Delhi for the an algorithm for the remote estimation of the
financial assistance connected with the minor concentrations of chlorophyll a, chlorophyll b and
project. carotenoids in soybean leaves. Remote Sens.
Environ., 39:239-247.
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Publication group
Microbial fuel cell an alternative for COD removal of distillery wastewater

Authors: ABSTRACT:
Anupama1, Pradeep NV2,
Hampannavar US3.
Microbial fuel cell (MFC) is a type of renewable technology for electricity
generation since it recovers energy from renewable materials that are difficult to
dispose of, such as organic wastes and wastewaters. In the present study, mixed
Institution:
1, 2
M.Tech Scholar, Dept of consortia from domestic sewage were used in Double Chambered Microbial Fuel Cell
Civil Engg, K.L.Es C.E.T, for the treatment of distillery wastewater. Distillery wastewater was diluted to get
Belgaum, India. different concentrations from 1100 mg COD/L to 10100 mg COD/L and this was given
3 as feed to microbes present in MFC. The COD removal efficiency increased with the
Professor, Dept of Civil
Engg, K.L.Es C.E.T, increase in feed concentrations until 6100 mg COD/L and further increase in feed
Belgaum, India. concentration led to deterioration in the COD removal efficiency and current
generation. The maximum COD removal of 64% was achieved at the feed
concentration of 6100 mg COD/L. MFC produced a maximum current of 0.36 mA and
power density of 18.35 mW/m2.
Anupama
Email:
Keywords:
akkushekhar@gmail.com Distillery wastewater, Microbial Fuel Cell, Agar salt bridge, COD, Catholyte.

http://jresearchbiology.com/ Anupama, Pradeep NV, Hampannavar US.
Documents/RA0112.pdf. Microbial fuel cell an alternative for COD removal of distillery wastewater
Dates:
Ficus Publishers.
cited.
419-423 | JRB | 2011 | Vol 1 | No 6

Anupama et al.,2011
INTRODUCTION treatment, biosensors for on-line monitoring of

Distilleries generate large volumes of high- organic matter and secondary fuel production such
strength wastewater that is of serious environmental as hydrogen gas (Das and Mangwani, 2010). Very
concern. Spentwash handling, treatment and less study has been done on COD removal of
disposal are of great importance and needs special distillery wastewater using double chambered
attention. Various physico-chemical and biological MFC.
treatment options are available for the treatment of In the present study double chambered MFC
distillery wastewaters. was constructed for the generation of electricity and
Microbial fuel cells (MFC) are unique COD removal of varied feed concentrations of
devices that can utilize microorganisms as catalysts distillery wastewater using the mixed consortia
for converting chemical energy directly into present in domestic sewage.
electricity, representing a promising technology for
simultaneous energy production and wastewater MATERIALS AND METHODS
treatment (Liu et al., 2004; Logan and Regan, MFC Reactor Setup
2006). In MFC electrons generated in anode cell Double chambered MFC was constructed
reach the cathode and combine with protons that using non-reactive plastic containers with
diffuse from anode through the membrane or agar dimensions of 8 x 8 x 12 inches. One plastic
salt bridge (Logan et al., 2006; Min and Logan, container was used as anode chamber (to be fed
2004). MFCs are being constructed using a variety with wastewater) and the other as cathode chamber
of materials, and in an ever increasing diversity of as shown in Fig 1. The wastewater was fed to the
configurations. These systems are operated under a anode chamber and Potassium permanganate
range of conditions that include differences in (catholyte) was fed to the cathode chamber
temperature, pH, electron acceptor, electrode (Lefebvre et al., 2008; Behera et al., 2010). The
surface areas, reactor size, and operation time agar salt bridge was used as the proton exchange
(Logan et al., 2006). medium (Momoh and Naeyor., 2010). Hence the
MFCs operated using mixed cultures cathode and anode chamber was connected using
currently achieve substantially greater power agar salt bridge. The length and diameter of agar
densities than those with pure cultures. Community salt bridge was 5 inches and 1.5 inches respectively.
analysis of the microorganisms that exist in MFCs The electrodes were placed in the chambers, then
has so far revealed a great diversity in composition were sealed and made air tight. The electrodes were
(Logan et al., 2006). connected by using copper wire as reported by
Microbial fuel cells (MFCs) have gained a Logan (2005). Graphite rods from pencils were
lot of attention in recent years as a mode of used as both anode and cathode (Logan and Regan,
converting organic waste including low-strength 2006; Logan et al., 2007). The arrangement of the
wastewaters and lignocellulosic biomass into graphite rods was made in such a way, so as to
electricity (Pant et al., 2010). There has been great provide the maximum surface area for the
interest in using MFCs for wastewater treatment development of biofilm on anode. The length and
and power generation has been shown using a
variety of wastewaters including both domestic and
industrial wastewaters (Ahn and Logan, 2010).
MFCs have been developed to generate electricity
directly from complex organic wastewater such as
food processing wastewater, brewery wastewater,
domestic wastewater, chemical wastewater, starch
wastewater, swine manure slurry, manure waste,
landfill leachate, meatpacking wastewater, palm oil
mill effluent, paper mill effluent and for
denitrification of domestic wastewater (Behera et
al., 2010).
MFCs were first used to produce power
from the electrical current generated by bacteria,
but there has been an evolution in these systems
resulting in applications such as wastewater Fig 1: Double chambered MFC.
Anupama et al.,2011
diameter of the graphite rods were 90 mm and 2mm reduced the COD removal efficiency from 64% to
respectively. Pre-treatment was not provided for the 38.6%.
electrode materials. The microorganisms started to become
Distillery Wastewater and Microbial Inoculum inactive or rather they went into decline phase
The distillery wastewater was used as a because of higher feed concentration of 10100 mg
substrate as well as source of inoculum. No other COD/L. The findings of this study are nearer to
nutrients were given for micro-organisms except Feng et al., (2008) who conducted the studies on
the nutrients present in the distillery wastewater. brewery wastewater treatment using air-cathode
MFC. Liu et al., (2004) conducted the studies on
Table 1: Characteristics of distillery wastewater domestic wastewater treatment using single
Characteristics Average Value chamber MFC and observed 50% to 70% COD
removal efficiency. Mathuriya and Sharma (2010)
pH 3.99 obtained 85.8% COD removal efficiency at pH 7
Colour Dark Brown when they utilized plain diluted beer brewery
BOD3 (mg/L) 36666 wastewater. They added 10% glucose and sucrose
COD (mg/L) 89833 solution to the diluted beer brewery wastewater,
Total Solids(mg/L) 74033 and obtained the highest COD removal efficiencies
Dissolved Solids (mg/L) 59733 of 93.8 and 91.92% respectively. Mohanakrishna et
Chlorides (mg/L) 6933
al., (2009) have reported 72.84% COD removal
Conductivity (mS/cm) 20.2
efficiency while treating distillery wastewater using
continuous flow single chamber air-cathode MFC.
Experimental Conditions
The anode chamber was filled with distillery
wastewater so that micro-organisms in the
wastewater could colonize the electrodes and
produce electricity. The samples were drawn from
the anode chamber periodically and analysed. The
ambient room temperature during most of the
period of study varied between 27 oC and 32 oC.
When the reactor reached steady state conditions,
the reactor was loaded with distillery wastewater of
higher concentration.
Analyses
The voltage (V) and Current (I) in the MFC
circuit was monitored at 24hour intervals using a
multimeter (UNI-T , Model Number- DT830D)
(Kim et al., 2005). Analysis of COD was performed
as prescribed in Standard Methods (1995). Current Generation and Power Density
The average values of current and power
RESULTS AND DISCUSSIONS density for each feed concentration in MFC is as
COD Removal Efficiency given in the Fig 3 and 4. The current and power
Distillery wastewater showed its potential density showed a gradual increase with respect to
for COD removal indicating the function of the increase in feed concentration until 6100 mg
microbes, present in wastewaters in metabolizing COD/L and later showed a gradual decrease in both
the carbon source as electron donors. It is evident current and power density. The highest average
from experimental data that current generation and value of current obtained was 0.27 mA at 6100 mg
COD removal showed relative compatibility. COD COD/L feed concentration. Similarly the highest
removal efficiency increased from 45.4% to 64% as power density value obtained was 18.35 mW/m2 at
the feed concentration increased from 1100 mg 6100 mg COD/L feed concentration.
COD/L to 6100 mg COD/L respectively as shown Mohanakrishna et al., (2009) reported the
in Fig 2. The increase in feed concentration from maximum current generation of 2.14 mA at 3600
6100 mg COD/L to 9100 mg COD/L slowly mg COD/L in single chamber air cathode MFC

Anupama et al.,2011
4. The performance of MFC deteriorated at the

feed concentration of 10100 mg/L COD.
Acclimatization of microbes to higher feed
concentration of distillery wastewater might lead to
good efficiency of COD removal even at higher
feed concentrations.
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using the acidophile Acidiphilium cryptum.
Biotechnology Letters 30:1367-1372.
Das S and Mangwani N. 2010. Recent

developments in microbial fuel cells: a review.
Journal of Scientific and Indusrtrail Research.
69:727-731.
Feng Y, Wang X, Logan BE and Lee H. 2008.

Brewery wastewater treatment using air-cathode
microbial fuel cells. Applied Microbiology and
Biotechnology 78:873-880.
Kim JR, Min B and Logan BE. 2005. Evaluation

of procedures to acclimate a microbial fuel cell for
COD reduction for various feed electricity production. Applied Microbiology and
concentrations of distillery wastewater was Biotechnology 68:23-30.
observed along the power generation
(Mohanakrishna et al., 2009). Lefebvre O, Al-Mamun A and Ng HY. 2008. A
microbial fuel cell equipped with a biocathode for
CONCLUSIONS organic reduction and denitrification. Water Science
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MFC was able to treat spent wash successfully. The
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1. MFC produced maximum current of 0.34 mA Production of electricity during wastewater
and voltage of 824 mV at the feed treatment using a single chamber microbial fuel
concentration of 6100 mg COD/L. cell. Environmental Science and Technology
2. Average power density of 18.35 mW/m2 was 38:2281-2285.
observed in MFC at the feed concentration of
6100 mg COD/L. Logan BE and Regan JM. 2006. Microbial fuel
3. The optimal feed concentration for MFC was cells - challenges and applications. Environmental
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W and Rabaey K. 2006. Microbial fuel cells: distillery wastewater in microbial fuel cell
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Publication group
Shoot multiplication and direct organogenesis of an important medicinal

plant Plumbago zeylanica L. (Plumbaginaceae).
Authors: ABSTRACT:
Lubaina AS1, Nair GM2
and Murugan K1. Efficient micropropagation protocol was developed for Plumbago zeylanica
L., a species threatened due to over exploitation for medicinal purposes and habitat
destruction in Southern Peninsular India. Multiple shoot induction was more
successful using nodes as explants on Murashige and Skoogs (MS) medium
Institution:
1. Plant Biochemistry and supplemented with 1mg/L benzyl amino purine (BAP) . Shoots, when transferred to
Molecular biology Lab, MS medium containing 0.2 0.5 mg/L gibberellic acid (GA3) showed variable
Department of Botany, elongation. Further, MS medium fortified with 1.5 mg/L BAP induced highest
University College, frequency of shoots through adventitious de novo organogenesis. Shoots developed
Thiruvananthapuram, were rooted on full strength MS medium with either -naphthalene acetic acid (NAA),
Kerala-695034, India. indole-3-butyric acid (IBA) or indole-3-acetic acid (IAA). Optimum shoots and root
2. Professor of Botany, multiplication were obtained within 8 weeks. In vitro derived plantlets were
University of Kerala, successfully weaned and transferred to soil and showed 90 % survival rate.
Kariavattom.
Corresponding author: Keywords:

Murugan K
Plumbago zeylanica L., medicinal plant, caulogenesis, rhizogenesis, In vitro
rooting, de novo organogenesis.
Abbreviations:
Email:
harimurukan@gmail.com Murashige and Skoogs (MS), Benzyl Amino Purine (BAP), Gibberellic Acid
(GA3), -Naphthalene Acetic Acid (NAA), Indole-3-Butyric Acid (IBA), Indole-3-Acetic
Acid (IAA), Mercuric Chloride (HgCl2).

Lubaina AS, Nair GM and Murugan K.
Shoot multiplication and direct organogenesis of an important medicinal plant
Plumbago zeylanica L. (Plumbaginaceae).
Dates:
Ficus Publishers.
cited.
424-428 | JRB | 2011 | Vol 1 | No 6

Lubaina et al.,2011
INTRODUCTION Medium and culture conditions

Plumbago, reported to comprise 10-20 The explants were cultured in Murashige
species (family Plumbaginaceae), is native to warm and Skoogs (1962) medium (MS) containing
temperate to tropical regions of the World. It can various growth regulators (BAP, Kinetin, NAA,
be propagated by seeds or cuttings. Plumbago IAA, IBA) at different concentrations either alone
zeylanica L. is a perennial rambling subscandent or in combinations. The dispensed media, after
under shrub distributed throughout India. Flowers adjustment of the pH to 5.7 1, were autoclaved at
are white with conspicuous glandular persistent 121C and 15 lbs pressure for 20 min. All the
calyx. The root is a rich source of alkaloid cultures were maintained at 24 2C under 12h
plumbagin, a naturally occurring naphthoquinone, photoperiod with 3000 lux light intensity using
which is reported to have wide pharmaceutical fluorescent lights at 60- 70% relative humidity. Ten
applications such as antibacterial, antifungal, anti- cultures were raised for each treatment and all
fertility and anti-tumor properties (Kini et al, 1996). experiments were repeated thrice.
The roots are used in many Ayurvedic Caulogenesis
preparations for the treatment of diseases like The nodes of 0.5-1.0 cm were inoculated on
diarrhoea, dyspepsia, rheumatism, anasarca and MS medium containing BAP and KIN alone at
piles. It is also efficiently used as a diuretic, causticconcentrations (0.5-3 mg/L) were used for
and expectorant. The root extract is made into a multiplication of shoots whereas BAP or KIN alone
paste and applied externally in leprosy and other (1-4 mg/L) or in combination with IAA (0.1-0.2
skin diseases. The increasing demand of herbal mg/L) were used for direct regeneration from
medicine is due to their fewer side effects in nodes and leaves. Within 10 days, rapid growth has
comparison to synthetic drugs and antibiotics been observed and the explants were sub cultured
(Krishnaswamy and Purushothaman, 1980). The after every 28-30 days.
gradual decline in the population of this species In vitro rooting
demands the launching of conservation efforts so as Two to three centimeters of well-developed
to ensure continuous and ample supply by shoots were excised and inoculated on MS
establishing a balanced cycle of harvest and medium with 3% sucrose containing individual
renewal. Such conservation strategies would ensure concentration of (0.5-2 mg/L) IAA, IBA, and
immense availability of this valuable medicinal NAA. The combination of two different auxins,
herb which is in great demand by the NAA+IBA (data not presented) were also studied.
pharmaceutical industry. Only a small percentage of Hardening and Acclimatization
medicinal plants, used in the industry are cultivated. Micro shoots with well-developed root
Most of them are collected from the wild, very systems were transferred directly to small pots
often in a destructive and unsustainable manner. containing sterile vermiculite and coco peat in (1:1)
Keeping the above facts in mind, the present study ratio and nourished with half strength MS basal
was undertaken to develop a suitable protocol in the liquid medium. Survival rate of the plantlets and the
species for the rapid propagation through shoot plantlets established in the field were recorded.
multiplication and de novo organogenesis. Experimental design and statistical analysis
All experiments were carried out in a
MATERIALS AND METHODS randomized design, ten replicates were raised for
Plant material each treatment and experiments were repeated
Nodal segments and leaf segments of thrice. The data were analyzed statistically using
Plumbago zeylanica L. (Plumbaginaceae), collected one way analysis of variance (ANOVA), and the
from the green house, Department of Botany, data, mean SD of at least three different
University of Kerala, Kariavattom, were washed in experiments were represented and compared using
running tap water followed by soaking and washing Tukey-Kramer multiple comparisons test with the
with 10% labolene solution (a commercial neutral level of significant P < 0.05.
detergent, Qualigens, India) for 15 min. The explants
after washing with sterile distilled water, were surface RESULTS AND DISCUSSION
disinfected with HgCl2 (0.1% w/v for 8 min) . After The morphogenetic responses of nodal
repeated washes with sterile double distilled water to
explants to various cytokinins (BAP, Kinetin) are
remove traces of HgCl2, the explants were trimmed
represented in the table (Table 1). Nodal explants
to appropriate sizes and inoculated.
cultured on growth regulator free MS medium
Lubaina et al.,2011
Table 1. Effect of different concentrations of results were found significant at 5% level. The
cytokinins on MS medium for shoot multiplication stimulatory effect of GA3 in the elongation of shoot
in Plumbago zeylanica bud is because it promotes cell division and
Cytokinin Cytokinin Mean number of elongation in the sub apical zone of the shoots
concentration concentration shoots after 30 (John et al., 1997). The role of GA3 on shoot
BAP BAP days * (mg/L) differentiation and elongation has been reported in
0.5 0 9.0 0.25 Withania somnifera (Sivanesan et al., 2007).
1.0 0 20.2 0.32 Adventitious de novo organogenesis was
1.5 0 15.7 0.12 achieved from leaf explants after 12 days of
2.0 0 8.0 0.84
incubation. Among the various combinations of
plant growth regulators used, cytokinins alone were
2.5 0 5.7 0.12
very effective in inducing direct
3.0 0 4.5 0.12 morphogenesis. The results are in corroboration
0 0.5 6.4 0.54 with the organogenesis obtained in other plants
0 1.0 12.8 0.32 (Faisal and Anis, 2006; Shinde et al.,
0 1.5 8.5 0.18 2009). Presence of BAP (1.5 mg/L) alone in the
0 2.0 6.2 0.74 medium induced the initiation of about fifteen
0 2.5 4.8 0.25 shoots directly from the explants after four weeks
of culture (Fig. 3). Further increase in
0 3.0 3.7 0.25
concentration of BAP or KIN resulted in the
*Values represent mean SD of ten replicates and the development of lesser number of shoots (Table
experiments were repeated thrice. The level of 2). Higher levels of cytokinin may be supra optimal
significance at 5% probability level.
Table 2. Effect of different concentrations of growth
showed no sign of proliferation even after two regulators on MS medium for Direct organogenesis in
weeks. Addition of a cytokinin was essential to Plumbago zeylanica
induce multiple shoot formation from the explants.
Of the two cytokinins tested, BAP was more Hormones mg/L Mean number of shoots after
effective than KIN. The nodal segments responded BA KIN IAA 30 days * (mg/L)
by an initial enlargement of the dormant axillary
buds followed by bud break within a week, and 1 0 0 8.6 0.12
1.5 0 0 15.2 0.25
multiple shoot induction and proliferation within
2 0 0 9.3 0.74
four weeks of culture. The frequency of axillary
3 0 0 5.6 0.29
shoot proliferation and the number of shoots per
4 0 0 3.8 0.35
explant increased with increasing concentration of 0 1 0 6.8 0.18
BAP up to some extent (Table 1). BAP 1mg/L 0 1.5 0 10.3 0.25
showed the highest shoot multiplication (20.2 0 2 0 7.0 0.31
0.32) ability (P < 0.05) (Fig. 1). The results were 0 3 0 4.6 0.54
found significant at 5% level. Thus, the results 0 4 0 3.2 0.32
strongly suggest that the BAP is the most effective 1 0 0.1 6.3 0.23
plant growth regulator for inducing multiple shoots 1.5 0 0.1 10.3 0.20
in P. zeylanica. These results were in consonant 1.5 0 0.2 7.0 0.31
with the multiple shoot induction reported in 2 0 0.1 5.8 0.51
Ceropegia intermedia (Karuppusamy et al., 2009), 3 0 0.1 4.1 0.12
C. bulbosa (John Britto et al., 2003), C. 4 0 0.2 3.0 0.35
candelabrum (Beena et al., 2003) and Centella 0 1 0.1 5.6 0.31
asiatica (Karthikeyan et al., 2009). 0 1.5 0.1 8.8 0.28
Multiple shoots obtained on MS medium 0 1.5 0.2 7.6 0.12
containing 1mg/L BAP after two weeks of culture 0 2 0.1 4.9 0.43
when transferred to medium supplemented with 0.2 0 3 0.1 3.8 0.18
-0.5mg/L GA3 showed variable rate of elongation. 0 4 0.2 2.7 0.25
Maximum elongation was observed when the *Values represent mean SD of ten replicates and the
experiments were repeated thrice. The level of signifi-
medium fortified with 0.5mg/L GA3 (Fig. 2). The
cance at 5% probability level.

Lubaina et al.,2011
for shoot regeneration. Of the two cytokinins used,

BAP was found to be most effective for producing
maximum number of shoots. Superiority of BAP
over KIN on de novo organogenesis was reported
earlier in Eryngium foetidum (Gayathri et al., 2006)
and Phyllanthus niruri (Karthikeyan et al.,
2007). The synergistic effect of cytokinins and
auxins also favored direct regeneration of
shoots. BAP (1.5mg/L) with 0.1mg/L IAA induced
maximum shoot regeneration. Combination of BAP
and IAA favored shoot initiation was noted in the
case of Capsicum annuum (Sobhanakumari and
Lalithakumari,, 2003). The results were found
significant at 5% level.
Individual concentration of IAA, IBA, NAA
and combination of two different auxins were used
for in vitro rooting. IBA 1.5mg/L induced
maximum number of roots. The present result
coincides with the observation obtained by others
(Abri and Staden, 2001; Vadawale et al .,
2004). The roots induced in NAA containing
medium were long but less in number. However,
the combination of two different auxins IBA +
NAA had the maximum impact on the elongation of
roots.
The well developed plantlets were
transferred to plastic cups containing autoclaved
vermiculite and coco peat in 1:1 ratio. The plantlets
were acclimatized in a mist chamber. The humidity Fig. (1-4). In vitro shoot multiplication and direct shoot
regeneration of Plumbago zeylanica L:
was maintained at 95 % in the initial days. Later on,
1. Multiple shoot induction on MS meium + 1mg/L
the percent of humidity was decreased by pricking BAP.
the plastic cover with a needle. On the 30th day, the 2. Elongation of multiple shoots on MS medium + 0.5
plants were transferred to the pots. The survival rate mg/L GA3.
of plantlets was 90% (Fig. 4). The results were 3. Adventitious shoots on MS medium + 1.5 mg/L
found significant at 5% level. The plantlets were BAP.
successfully adapted to the natural environment and 4. Acclimatized plantlet.
exhibited their similarity with that of mother plants.
The present study therefore projects the successful
micropropagation protocol established that can be Beena MR, Martin KP, Kirti B, Hariharan M.
employed in the propagation of Plumbago 2003. Rapid in vitro propagation of medicinally
zeylanica L. for its conservation and domestication. important Ceropegia candelabrum, Plant Cell
Tissue Organ Cult., 72:285-289.
ACKNOWLEDGEMENT
Authors are thankful to the Head, Faisal M, Anis M. 2006. Thidiazuron induced high
Department of Botany, University of Kerala, frequency axillary shoot multiplication in Psoralea
Kariavattom, Thiruvananthapuram for providing the corylifolia. Biol Plant.., 50:437-440.
necessary facilities.
Gayathri MC, Madhu M, Kavyashree R,
REFERENCES Dhananjayan, SP. 2006. A protocol for in vitro
Abri AL, Van Staden J. 2001. Micropropagation regeneration of Eryngium foetidum L., Indian J
of the endangered Aloe polyphylla, Plant Growth Biotechnol., 5:249-251.
Regulat., 33:19-23.

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2003. In vitro flowering and shoot multiplication Plumbagin a study of its anticancer, antibacterial
from nodal explants of Ceropegia bulbosa Roxb. and antifungal properties Indian J Exp Biol., 18:876
Var. bulbosa. Taiwania. 48:106-111. -877.
John S, Soniya EV, Valsala K, Nair GM. 1997. Murashige T, Skoog F. 1962. A revised
In vitro adventitious shoot formation from mature medium for rapid growth and bioassays with
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& A. Indian J Exp Biol., 35:1249-1251.
Shinde AN, Malpathak N, Fulzele DP. 2009.
Karthikeyan K, Chandran C, Kulothungan S. Induced high frequency shoot regeneration and
2009. Rapid clonal multiplication through in vitro enhanced isoflavones production in Psoralea
axillary shoot proliferation of Centella asiatica L., corylifolia, Rec Nat Prod., 3:38-45.
Indian J Biotechnol., 8:232-235.
Sivanesan I. 2007. Direct regeneration from apical
Karthikeyan K, Chandran C, Kulothungan S. bud explants of Withania somnifera Dunal. Indian J
2007. Rapid regeneration of Phyllanthus niruri L. Biotechnol., 6:125-127.
from shoot tip and nodal explants, Indian J Appl
Pure Biol., 22:337-342. Sobhakumari VP, Lalithakumari D. 2003. Direct
plant regeneration from shoot tip cultures of
Karuppusamy S, Kiranmai C, Aruna V, Capsicum annuum L. cv PLR-1, Phytomorphology
Pullaiah T. 2009 In vitro conservation of 53:235-242.
Ceropegia intermedia an endemic plant of south
India, African Journal of Biotechnology., 8 Vadawale AV, Mehta-Bhatt P, Dave AM. 2004.
(17):4052-4057. Rapid in vitro propagation of Aswagandha
(Withania somnifera) through axillary bud
Kini DP, Pandey S, Shenoy BD, Singh UV, multiplication and indirect organogenesis.
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activities of plumbagin scontrolled release
formulations, Indian J Exp Biol., 35:374-379.

Original Research Paper Publication group
Histomorphology of the gastrointestinal tract of domesticated Grasscutter

(Thyronomys swinderianus) in Northern Nigeria
Authors: ABSTRACT:
Byanet Obadiah*,
Abdu PA1 and Shekaro A2. The gastrointestinal tract was dissected from twelve matured domesticated
grasscutters of both sexes. The stomach was J-shaped, simple monogastric relatively
Institution:
* small in relation to the size of the animal. Its histological studies revealed three
Department of Veterinary
regions (cardiac, fundus and pylorus) with their gastric glands. Simple structure of the
Anatomy, College of
Veterinary Medicine, intestine; duodenum, jejunum and ileum and well developed large intestines; cecum,
University of Agriculture, colon and rectum were also observed. Some common histological features of the
Makurdi, Benue State, intestine observed include: intestinal glands, the villi and goblet cells. The cecum with
Nigeria. coma-shaped blind ended sac had three regions (base, body and apex) and Teniae
with intervening haustra (sacculations) was the largest organ in the abdominal cavity
1
Department of Veterinary and the largest segment of the intestine. The structure of the cecum suggests that it is
Surgery and Medicine, the bacterial fermentation (digestion) vat, similar to that of the horse rumen in
Faculty of Veterinary ruminant. The colon was the widest portion of the intestine and had fecal balls which
Medicine, Ahmadu Bello were the indigestible portion of the fed that will pass through the rectum.
University, Zaria, Kaduna
State, Nigeria.
2
National Veterinary
Research Institute, Vom, Jos, Keywords:
Plateau State, Nigeria. Histomorphology, gastrointestinal tract, grasscutter.
Corresponding author: Article Citation:

Byanet Obadiah Byanet Obadiah, Abdu PA and Shekaro A.
Histomorphology of the gastrointestinal tract of domesticated Grasscutter
(Thyronomys swinderianus) in Northern Nigeria.
Email: Journal of research in Biology (2011) 6: 429-434
byaneto@yahoo.com
Dates:
Phone No: Received: 28 May 2011 /Accepted: 21 Jun 2011 /Published: 12 Oct 2011
+2347095291713
Ficus Publishers.
Web Address: This Open Access article is governed by the Creative Commons Attribution License (http://
http://jresearchbiology.com/ creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
Documents/RA0036.pdf. commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.
429-434 | JRB | 2011 | Vol 1 | No 6

Byanet et al.,2011
INTRODUCTION MATERIALS AND METHODS

The grasscutter (Thyronomys swinderianus) Animal source
is a wild herbivorous rodent and a close relative of A total of twelve mature domesticated
porcupines and guinea pigs. It is the second largest grasscutters of all sexes (6 males and 6 females)
African rodent after the Porcupine and is found were used for this study. They were purchased from
only in Africa, South of the Sahara (Baptist and local breeders in Makurdi and Otukpo towns of
Mensah, 1986; Adoun, 1993). Although many Benue State, Nigeria. The animals were transported
varieties have been described (Thomas, 1894), they using laboratory rat cages (2 males for a cage and 2
belong to two different species; the smaller females for a cage) to the Department of Veterinary
(Thryonomys gregorianus) and larger (Thyronomys Anatomy Laboratory, Ahmadu Bello University,
swinderianus). Zaria.
Grasscutters are heavily built rodents with The animals were acclimatized for three
average adult weight of 3 kg for females and 4.5 kg days prior to the research and had free access to
for males (Eben, 2004). They are nocturnal and live elephant grass, commercial feed supplement and
in marshy areas, along the river banks, feeding on water. The animals were observed to be in good
aquatic grasses in the wild. The grasscutter is a nutritional status on physical examination before
monogastric herbivore and very fond of sweet and euthanasia. They were all sedated using gaseous
salty foods. It adapts readily to diet like leguminous chloroform in a confined container and later
fodder, tubers (cassava, sweet potatoes), fruits sacrificed.
(pawpaw, pineapple and mango) and food crops Gross anatomy
(rice, maize), making them a significant pests For each animal, an incision was made on
(Eben, 2004. the ventral midline immediately after sacrifice and
The grasscutter meat is the most expensive the abdominal cavity was exposed. The stomach
and preferred meat in West Africa (Asibey and and intestines were dissected from the mesenteric
Addo, 2000). Apart from its excellent taste like and spread in a straight line. The gross anatomical
most bush meat, it is nutritionally superior to most structures of the stomach and the entire intestine
domestic meat from domestic animals because of its were observed.
higher protein and mineral content and low fat. As a Photomacrographs were taken using a digital
consequence, grasscutters are being raised in cages camera. Tissues samples were collected from the
for sale and so are sometimes refer to as micro segments of the stomach, small intestine
livestock (National Research Council, 1991). (duodenum, jejunum) and large intestine (cecum,
Grasscutters can be used as laboratory colon) and fixed 10 % in buffered formalin for
animals, but the effectiveness of using them in the histology.
laboratory needs the information on their dietary Histology
requirements and feeding habit which depend on The fixed tissues (stomach, duodenum,
the knowledge of their digestive tract. Its jejunum, cecum and colon) were cut into blocks and
domestication is largely in the hands of peasant identified. They were then dehydrated through a
farmers in villages who keep these animals in series of graded alcohols (70%, 80%, 90%, 95%
boxes, empty drums and small pens. The constraint and 100%). The blocks were cleared in xylene and
to large scale production of grasscutter is the then infiltrated with molten paraffin wax. Sections
unavailability of breeding stocking information: on (5 m) microns thick were cut from embedded
housing, feeding, anatomy, physiology, and other tissue using Jung Rotary Microtome (model 42339).
useful knowledge to the few breeders (Adu et al., The tissues were then mounted on grease
1999). free clean glass slides. The slides were prepared at
The morphology of the digestive tract of a room temperature stained alternatively with
given animal species is related to the nature of food, Hematoxylin and Eosin (H & E). The prepared
feeding habits, body size and shape (Smith, 1989). slides were studied using light microscope
The study of healthy grasscutters in this study was (Olympus binocular micr os cope).
aimed at presenting information on the normal Photomicrographs of the prepared slides mounted
morphological and histological pattern of the on the binocular microscope were taken using a
intestine which would contribute to the knowledge digital microscopic objective. These pictures were
of pathological conditions and/or physiological then transferred to a computer and detailed studies
alterations. were carried out.
Byanet et al.,2011
RESULTS Histology of the Stomach

Gross Structure of the Stomach The histological studies of the stomach in
The stomach of the grasscutter was observed this study revealed three regions (cardiac, fundus
to be simple relatively small in relation to the size and pylorus). The stomach wall was observed to
of the animal, thin-walled and J-shaped with small have the same structural layers (the mucosa, the sub
distended bag-like when full. It had two surfaces; mucosa, muscularis mucosae and muscular externa)
the parietal and the visceral, two curvatures; lesser within the three regions (Plate 2 and 3).
and greater; two orifices, cardiac and pyloric and Plate 2 Plate 3
three regions; cardiac, fundus and pylorus (Plate 1).
Gross Structure of the Intestine
The intestine was observed to be divided Gp
into small intestine (duodenum, jejunum and ileum)
and large intestines (cecum, colon and rectum). The
duodenum was long and started at the pylorus Pg
region close to the stomach. The jejunum appeared Fg
to be convoluted or coiled, very long and occupied
the abdominal floor between the stomach urinary
Gp
bladder. The ileum was short and followed the
jejunum and marked the end of the small intestine.
The distal end of the ileum had a thick walled
enlargement, Sacculus rotundus which mark the
junction between the ileum, cecum and colon (Plate Plate 2 and 3: Photomicrographs of the fundic and
1). pyloric region of the stomach respectively showing
The cecum was observed to be the largest the fundic glands (FG), gastric pits (GP) and sub-
mucosa (SM). The lamina propria contain the
segment of the entire gastrointestinal tract and also
pyloric glands (PG). H & E Stain, x 100.
the largest organ in the abdominal cavity. The
cecum was seen to have a coma-shaped blind ended The cardiac region was found to be
sac with three regions (base, body, and apex) and surrounding the point of entry of the esophagus. Its
Teniae with intervening haustra (sacculations) on it lamina propria contained simple/branched tubular
entire length. The colon was the widest portion of cardiac glands or mucosa which extended deep into
the intestine and had fecal balls, particularly at it the mucosa. The gastric pits of this region were
transverse and descending parts. The rectum was deep and lined with a simple columnar epithelium.
short, straight and marked the terminal portion of The pyloric glands were observed to be similar to
the gastrointestinal tract. those of the cardiac glands and were characterized
by deep, large or open gastric pits and short coils
pyloric glands (Plate 3).
The fundic region had the most numerous
types of gastric glands. The region had shallow
gastric pits, long branched tubular glands that
contain several cell types like parietal cells and
chief cells (Plate 2).
Histology of the Intestine
The three segments of the small intestine
(duodenum, jejunum and ileum) were observed to
have some common histological features (the villi
and goblet cells) and some minor structural
differences. The duodenal segment was observed to
have the intestinal villi as an outgrowth of the
mucosa projecting into the lumen. The goblet cells
and duodenal glands (Brunners) in the sub-mucosa
Plate 1: Grasscutter opened up (in situ) showing: were also noted as the major distinguishing features
1, jejunum; 2, ascending colon; 3, transverse colon; observed in the duodenum (Plate 4 and 5).
4, descending colon; 5, urinary bladder.

Byanet et al.,2011
Plate 4 Plate 5 DISCUSSION

The stomach in this study was observed to
V
V be the simply monogastric type. This result
corresponded well with those of the previous study
V documented for man and rabbit (Harold, 1992:
Cathy, 2006). The stomach of dog though simple
monogastric is in the form of a C-shape and rotated
at 900 in a clock wise direction, with the divisions
being similar to those of the monogastric animals
M (Millar, 1964).
Bg
Sm Contrary to the stomach of grasscutter and
mm
dog, those of the ruminants (ox, sheep and goat),
Plate 4: Photomicrograph of the duodenum
have compound stomach (Frandson, 1981; Ojo et
showing the duodenal villi (V), the intestinal glands
(Brunners glands (Bg) and the wall of the intestine: al., 1987), the first three compartments (rumen,
mucosa (M), sub-mucosa (Sm), and muscularis reticulum and omasum) are non-glandular, whereas,
mucosa (Mm). H & E Stain, x 50. the fourth compartment (abomasum) is glandular
and contains typical cardiac, fundus and pylorus
Plate 5: Photomicrograph of the jejunum showing regions similar to that observed in monogastric
the wall of the intestine: mucosa (M), sub-mucosa stomach (Banks, 1993).
(Sm), muscularis mucosa (Mm) and the muscularis Histological results of this study indicate
externa (Me), the villi (V) and the intestinal glands that the wall of the stomach was lined with
(crypts of Lieberkuhn) (Cl). H & E Stain, x 100. glandular mucosa and having all the layers of a
typical tubular organ (George, 1973; Dellmann and
The jejunum had numerous goblet cell and Brown, 1987; Kansas, 2007; Thomas, 2007). It is
long leaf-like villi. Between these villi were the generally accepted that based on the histological
openings of the simple tubular glands called, the characteristics, the stomach of different species can
intestinal glands (crypts or glands of Liberkuhn). be classified into different types. For example,
Each villus of the jejunum was observed to line by simple unisaccular stomach (found in man and
simple columnar epithelium cells. The striated carnivores) lined with glandular mucosa only
border formed by microvilli present on the surface (Dellmann, 1971), the compound unisaccular
of the cell was clearly visible (Plate 4). stomach (example horse and pig) has a small part
The large intestinal segments (the cecum, colon and lined with cutaneous mucosa and the rest with
rectum) had four intestinal wall tunics or layers glandular mucosa (Rudolf and Stromberg, 1976),
similar to that observed in the small intestine. The
intestinal glands were seen to be longer than that of Plate 6: Plate 7:
the small intestine and the goblet cells were more
numerous than were observed in the intestine (Plate
6 and 7).
The cecum had numerous goblet cell
intestinal glands (crypts). Other prominent feature
observed in the cecum was the external longitudinal
muscles coat which has three bands, the taeniae
coli. These longitudinal muscles push the excess
mucosa to form pouches called the hastra (Plate 6).
The colon was observed to have a wider lumen than Plate 6: Photomicrography of the cecum of the
any segment of the intestine and the mucosal grasscutter showing intestinal glands (crypts of
surface lined by simple columnar epithelia cells. Lieberkuhn) (CL) and the thick band of longitudinal
Crypts of Lieberkuhn, with abundant goblet cells muscle, the taeniacoli (T).H & E Stain, x 100.
were also observed in the colon. The muscularis
external layer as in cecum was observed to have Plate 7: Photomicrograph of the colon showing the
thick longitudinal bands called taeniae coli (Plate thick longitudinal muscle band, the taenia coli (T),
7). the lumen (L) and the intestinal villi (V). H & E
Stain, x 50.

Byanet et al.,2011
while the compound multisaccular stomach vat, similar to the rumen in cow. The microbes in
(ruminants) consists of a non glandular part (rumen, the cecum break down feed that was not digested in
reticulum, and omasum) and glandular part the small intestine, particularly fibrous feeds like
(abomasum) (Eurell and Dellman, 1998). hay or pasture.
The grasscutter stomach observed in this Monogastric herbivores like the rabbits and
study may be more of chemical digestion and not rodents have shorter but prominent cecum (George
fermentative digestion as experienced in animals 1973) documented that the cecum in rabbit is the
with compound stomach like cattle and sheep. largest internal organ in the abdomen and
Chemical digestion relies mainly on the fundic fermentation of the intestinal contents occurs there.
glands which has chief cells that secrete gastric And that periodically the cecum contracts and the
enzymes (pepsin, rennin and gastric lipase) and the fermented ingesta are propelled into the colon and
parietal cells (oxyntic cells) secret hydrochloric then out of the anus.
acid, potassium and intrinsic factor essential for In addition to the vitamins and fatty acids
intestinal absorption of vitamin B12 (Banks, 1993; absorption in the colon, water is also said to be
Paul, 2007). This has also been reported in the cat absorbed, resulting in the fecal ball formations.
(Dellman, 1971). These fecal balls (as observed in our study), is said
In the present study, the tunica submucosa of to be the indigestible portion of the fed which will
the duodenum was observed to contain the then pass through the rectum as in the horse (Ohio,
Brunners glands. The lamina propria also had 2007). In rabbit, the fecal pellets are directly
abundant intestinal glands (crypts of Lieberkuhn) in ingested by the animal in a process called
addition to the goblet cells within the epithelial coprophagy or cecotrophy, meaning the ingestion of
cells. Charlotte (2004) had determined the location feces. The cecotropes or night feces as they are
of the Brunners glands to be in the initial and called are coated with mucus which acts as a barrier
middle part of the duodenum in the dog, cat, man to the acidic pH of the stomach, ensuring that the
and small ruminants. But in the pig, horse and large content will be absorbed from the small intestine.
ruminants, they were beyond the duodenum. The
research on the glands, the villi and goblet cells has REFERENCES
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1971; Harold, 1992). Small holder farming of the Cane rat in southern
The absorption capacity of the small Ghana: A baseline survey of management practices.
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villus surface was larger than the duodenal villus Aduon C. 1993. Place De L aulacode (Thryonomy
surface; suggesting that the absorption capacity swinderianus) Dans Le Regne Animal ETSa
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intestine of weaned piglets. promising animal for meat production. In: Turnham
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cellulose by this animal cannot take place in the Zimbabwe). 120.
stomach because of the lack of fermentation like
ruminants. The cecum described in this study Banks WJ. 1993. Applied Veterinary Histology,
resembled the ruminant rumen (abundant papillae) Baltimore, MD William and Wilkins.
and that of the horse. Many authors (Dellmann,
1971; Ohio, 2007) have reported that in Baptist R and Mensah GA. 1986. Benin and West
monogastric herbivores (especially in the horse) the Africa: The cane rat- farm animal of the future?
cecum act as the bacterial fermentation (digestion) World Animal Review 60:2-6.

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Cathy AJ. 2006. Anatomy and Physiology of the Miller EM. 1964. The Digestive System. In: The
Rabbit and Rodent Gastrointestinal System. Anatomy of the Dog. W.B. Saunders Company,
Eastside and Exotic Medical Centre, PLLC 100th Philadelphia.
Ave NE, WA 98034, USA. 9-14.
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histologyReferenc. 1-17. Ojo SA, Adogwa AO and J.O. Hambolu, 1987
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Introduction to Functional Anatomy 171-185.
National Research Council (NRC). 1991.
Delamnn H, 1971: The histology of digestive Grasscutter. In: Microlivestock little- Known Small
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Wilkins. 155 195. Academic Press 331-421.
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Comparative Anatomy of the Vertebrates 7th ed. Wise F, Simon O and Weyrauch KD. 2003.
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Harold E. 1992. The gastrointestinal tract, In: Anatomia. Histologia. Embryologia 32:102-109.
Clinical Anatomy A revision and applied Anatomy
for Clinical Students 8th Ed., 73-97.

Publication group
Conservation threats to the water birds in Deepor Beel, Assam

Authors: ABSTRACT:
Jyotismita Das1 and
Saikia PK2. Water birds are becoming increasingly exposed to human activity as human
population expands. We initiated this study to document the current conservation
threats of water birds in Deepor Beel, the lone Ramsar site in Assam. The study
Institution:
revealed the presence of 39 species of water birds from 16 different families. Out of
1. Research Scholar, Animal
Ecology and Wildlife these species nine species were winter visitors to the wetland and the rest 29 species
Biology Laboratory, were breeding residents. Currently, Deepor Beel is facing dangers from various angles.
Department of Zoology, Most of these are anthropogenic in nature. The prominent conservation threats were
Gauhati University. soil digging, encroachment, agricultural practices, hunting and trapping of water birds,
excessive fishing, fragmentation and degradation of wetland habitat due to the
2. Associate Professor, establishment of railway line were most severe in nature. All these anthropogenic
Animal Ecology and factors were bringing danger to the proper survival of water birds. Thus, the present
Wildlife Biology Laboratory, study will greatly helped in perusing conservation strategies for proper management
Department of Zoology, of the water birds as well as their habitat.
Gauhati University.
Keywords:
Water bird, Ramsar, Anthropogenic, Anatidae, Encroachment, Soil digging,
Jyotismita Das
Deepor Beel.
Email:
deeporbeel@gmail.com Article Citation:
Jyotismita Das and Saikia PK.
Conservation threats to the water birds in Deepor Beel, Assam
Dates:
Web Address: Received: 24 Sep 2011 /Accepted: 28 Sep 2011 /Published: 12 Oct 2011
Ficus Publishers.
cited.
435-439 | JRB | 2011 | Vol 1 | No 6

Jyotismita et al.,2011
INTRODUCTION biodiversity and sociocultural importance. Again,

Human conflicts with animals go back at considering the varieties of bird species found in
least as far as humans have been a species the Beel, Birdlife International has also declared
(Cansdale 1952, Fisher and Lockley, 1954). Birds Deepor Beel as an Important Bird Area (IBA). At
are widely recognized as good bioindicators of the maximum flooding the Beel becomes above four
quality of ecosystems (Gill, 1994) and health of the meters in deep and during the dry season the depth
environment. Kumar et al. (2006) gives a clear drops to about 1-1.5 meter. Deepor Beel
status of wetland birds in his work. Wetland birds (Coordination: 260326260926N and 9036
play a significant cultural and social role in local 39904125E) is situated on the Southern bank
communities as well as being an important of the river Brahmaputra and Village Maj Jalukbari,
component of wetland ecosystem (Kumar et al. Pachim Jalukbari, Dharapur and National Highway
2006). Due to continuous degradation of wetland No.37 lie on the North; Dakhin Jalukbari, Tetelia
ecosystems, it becomes a great matter of concern and Pachim Baragoan to the East; Gorbhanga
regarding the status of wetland dependent birds. Reserve Forest, Chakardew Hill and Chilla Hill to
Increasing attention of conservation of this water the South West and the Village Azara and
bodies brings an extensive research to this field. Kahikuchi to the west. Deepor Beel has a meso-
Deepor Beel which is the lone Ramsar site of thermal climate. The temperature ranges between
Assam is facing danger from various angles in this 10.6C to 32C. Deepor Beel appears to be
regard. In this study, attempt had been made to relatively high with respect to the biodiversity of
identify the current conservation threats to the water free floating, emergent and submerged aquatic
birds in and around the Beel periphery. Thus the macrophyte (Saikia and Bhattacharjee, 1987).
study provides immense scope in conservation of Avian data as well as data on threat factors
the water birds and its habitat. The importance of were collected from March 2007 to March 2010.
wintering areas in the dynamics of water bird For watching, counting and identifying birds
populations is suggested by the potentially crucial Binocular (10X50), telescope (25-40X), camera
role these areas play in courtship or pairing and (Cannon 110 PS), note book, guide book, pen,
deposition of fat stores used later as energy for pencil etc were used. Birds were identified by
reproduction (Heitmeyer and Fredrickson, 1981; seeing their characteristics feature in accordance
James, 1989; Baldassarrea and Bolen, 1994). with the identification keys involved in Ali and
Winter habitat quality has been linked to annual Ripley (1983), Grimmett et al. (1999). Data on
survival recruitment, and reproductive success of threat factors were collected by direct observation,
waterfowl (Baldassarre and Bolen, 1994).The area personal interviews.
has not received much attention especially from the
biological point of view Bera et al.2008). The RESULTS
present correspondence is an attempt regarding The present survey reveals the presence of
conservation and preservation of the lone Ramsar 38 species of water birds from 16 different families
site of Assam by identifying different threat factors (Table 1).Of all these nine species were species are
prevailing in and within the Beel periphery. winter visitors to the wetland and the rest 29 species
were breeding residents. The study also
MATERIAL AND METHODS documented the presence of endangered, vulnerable
Study area and Schedule I species under the Wildlife
Deepor Beel is a large natural wetland Protection Act, 1972. These are greater adjutant
having great biological and environmental stork, lesser adjutant stork and large whistling teal
importance (Deka and Goswami, 1992). This large etc.
water body is a great food source and breeding The study documented different threat
ground for a variety of migratory birds, amphibians, factors which were continuously prevailing within
reptiles, insects, micro and macrophytes, terrestrial the Beel periphery. Most of these factors were
weeds and important taxa of ecological and anthropogenic in nature. Soil digging,
economic importance (Bera et al. 2008). The encroachment, agricultural practices around the
Deepor Beel Ramsar site has a total area of 40 Km2 Beel, hunting and trapping of water birds, excessive
of which 4.14 Km2 had declared as a Bird Sanctuary fishing, habitat fragmentation for the construction
(Das et al. 2011). In November 2002, it was listed of railway line, brick making factories within the
as a Ramsar site owing to its rich wetland Beel ecosystem was the prominent threat factors
which are continuously prevailing in and within the Beel. Different people were engaged in this work
Beel periphery. Encroachment in various forms like apart from using heavy vehicles for the
house construction and other development activities transportation of soil to different localities. Thus, it
were degrading the Beel ecosystem in utmost level. was seen that the Beel bed was digged out in
For construction and developmental works people extreme level and water birds were forced to leave
were engaged to clear the aquatic vegetation which the Beel ecosystem. In winter season many water
is very important for the survival of water birds birds were illegally netted by using various nets and
inhabiting there. Apart from this, soil digging traps. Again Deepor Beel acts as a staging ground
processes were carried out day and night within the for winter migratory birds. Many migratory and
Beel for fulfilling the need of people. The intensity local breeding ducks were netted down illegally
of soil digging was documented high within the which brings major threat to their survival.
TABLE 1. List of the name of the water birds of Deepor Beel along with their scientific names
Common Name Scientific Name Family Comments
Large whistling teal Dendrocygna bicolor Anatidae Br(*)
Lesser whistling teal Dendrocygna javanica Anatidae Br
Gaganey Anas querquedula Anatidae Wv(**)
Northern Pintail Anas acuta Anatidae Wv
Red crested poachard Rhodonessa rufina Anatidae Wv
Ruddy shellduck Tadorna ferruginea Anatidae Wv
Mallard Anas platyrhynchos Anatidae Wv
Little egret Egretta garzetta Ardeidae Br
Great egret Casmerodius albus Ardeidae Br
Cattle egret Bulbulcus ibis Ardeidae Br
Intermediate egret Mesophoyx intermedia Ardeidae Br
Purple heron Ardea purpurea Ardeidae Br
Indian pond heron Ardeola grayii Ardeidae Br
Yellow bittern Ixobrychus sinensis Ardeidae Br
White throated kingfisher Halycyon smyrensis Alcidinidae Br
Pied kingfisher Ceryle rudis Alcidinidae Br
Ruddy kingfisher Halcyon coromanda Alcidinidae Wv
Asian open billed stork Anastomus oscitans Ciconiidae Br
Lesser adjutant stork Leptoptilos javanicus Ciconiidae Br
Greater adjutant stork Leptoptilos dubius Ciconiidae Br
Brahminy kite Haliastur Indus Accipitridae Br
Black Kite Milvus migrans Accipitridae Br
Little cormorant Phalacrocorax niger Phalacrocoracidae Br
Great cormorant Phalacrocorax carbo Phalacrocoracidae Br
Black Dongo Dicrurus macrocercus Corvidae Br
Bronze Winged Jacana Metopidius indicus Jacanidae Br
Pheasant tailed jacana Hydrophasianus chirurgus Jacanidae Br
White breasted waterhen Amaurornis phoenicurus Rallidae Br
Common coot Fulica atra Rallidae Br
Common moorhen Gallinula chloropus Rallidae Br
White wagtail Motacilla alba Passeridae Wv
Yellow wagtail Motacilla flava Passeridae Wv
Barn Swallow Hirundo rustica Hirundinidae Br
Common hoopoe Upupa epops Upopidae Br
Red wattled lapwing Vanellus indicus Charadridae Br
Green bee eater Merops orientalis Meropidae Br
Asian palm swift Cyprsiurus balasiensis Apodidae Br
Black headed gull Larus ridibundus Laridae Wv
* Br means Breeding migrant; ** Wv means Winter visitor

generally forcing the biodiversity of the Beel to

become extinct. Bildstein et al. (1991) had reported
that as a result of human impacts, many coastal
wetlands in the Western Hemisphere have already
been lost. Saikia and Kakati (2010) had reported the
same case of anthropogenic dangers within the Beel
periphery. Barman (1997) had also reported that the
highest value of threats in Deepor Beel. Habitat loss
(a) (b) is the most important threat factors for bird species.
This is also correlated with the work of Collar et al.
(1994) where they found that habitat loss not only
affects the anatids but also other bird species. Kafle
et al. (2008) had reported that anthropogenic factors
are the root causes for lake degradation and habitat
destruction of waterbirds. For these threat factors
the birds might be shifting their place to other for
proper survival or they become forced to restrict
(c) (d) themselves in their distribution. This work
correlates with the work of Barman (1997) where
Plate 1 (a.b.c.d). Showing different conservation he found that the wintering bird populations are
threats prevailing within Deepor Beel more affected in the valley due to the anthropogenic
(a) & (b) Soil digging within the Beel, pressures on their wintering sites and thus are
(c) Transportation of soil using vehicle forced either to shift their wintering location or to
(d) Construction processes restrict their distribution to protected areas only
subject to changes of their finding suitable
Pesticides and fertilizers were used in large wintering sites invade the protected area. Berthold
scale in agricultural practices around the Beel (1993) had also reported in his work that four
which enter the Beel as runoff and thus accelerated factors have impact on migratory birds population
eutrophication. Excessive fishing practices were at their stop-over sites and winter quarters:
done within Beel by using different jals, traps etc. restriction of habitats, hunting and trapping,
for the purpose of consumption and selling. disturbance, and effects of biocides. In Yunnan
Sometimes it was seen that peoples were using province habitat destruction and over-hunting were
water pumps for fishing purposes. Thus, fishes the major threats to the wetlands species (Wen et al.
including smaller and larger ones were caught up in 1995). In Similarity, the major threats to the water
heavy rates and this acted as a potential threat to the birds at Deepor Beel Ramsar site were habitat loss,
survival of water birds as some water birds disturbance, and bird trapping.
exclusively depended on fishes for their survival. Deepor Beel, the lone Ramsar site of Assam
Again the construction of railway line along the is facing dangers from various angles. In spite of
Southern and Eastern boundary of Deepor Beel heavy destruction processes, the water birds are still
divided the Beel into two parts, also acted as a visiting the wetland. So, proper management
factor of wetland encroachment. practices should be taken for preventing the heavy
destruction processes so that the biodiversity of this
DISCUSSION Ramsar site can be protected.
Saikia (2005) had reported 232 species of
aquatic avian fauna belonging to 42 different ACKNOWLEDGMENTS
families in his study in Deepor Beel. Among these The authors offer their thanks to the Head,
species 137 species were residential and 97 species Department of Zoology, Gauhati University,
were migratory. But the present study explored only Guwahati: 781014 for providing help to carry out
38 species of birds from 18 different families. Of all the research work. The authors are again thankful to
these species 9 species are winter visitors to the the University grant commission (UGC). We are
wetland and the rest 29 species are breeding highly grateful to the forest personals as well as
residents. Thus it can be seen that the species different boatmen, without their help, this work
numbers are quite declining. These threats are were not possible to be completed.
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H David, Erwin Michael, Nol Erica, Payne X wetlands conditions in the Mississippi Delta
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York.

Publication group
Scanning Electron Microscopic study on few Diatoms from KRP Dam,

Tamil Nadu- India.
Authors: ABSTRACT:
Ramanibai R and
Kanniga S
A study was carried out to examine the morphology of diatom species in
Krishnagiri Reservoir, Tamil Nadu. The sediment samples were collected and
transferred immediately in to zip lock bags to avoid the oxidation. Then the samples
Institution:
Department of Zoology were stored at 5C in an ice box and carefully transferred to the laboratory. The
University of Madras sediment sample was treated with acid wash before detailed observation under the
Guindy Campus, Chennai - light and electron microscopes. All samples were identified up to species level based
25. on Scanning Electron Microscope (SEM) observations. The SEM photographs provided
fine morphological features of the species for easy identification. Qualitative aspects
of the differences between LM (Light Microscope) and SEM approaches are discussed
in the text.
Ramanibai R
Email: Keywords:
rramani8@hotmail.com
Diatom, KRP Dam, SEM, Sediment diatom.

Ramanibai R and Kanniga S.
Scanning Electron Microscopic study on few Diatoms from KRP Dam, Tamil Nadu-
India.
Dates:
Received: 27 Sep 2011 /Accepted: 08 Oct 2011 /Published: 14 Oct 2011
Ficus Publishers.
cited.
440-443 | JRB | 2011 | Vol 1 | No 6

Ramanibai et al.,2011
INTRODUCTION MATERIALS AND METHODS

Diatoms are single celled microscopic algae Study Area
that posses highly ornamented cell wall composed History and Morphometric of Krishnagiri
of glass silica (SiO2) which provide variety of reservoir
shapes from nano to micro-scale structures. They Krishnagiri dam is located in Krishnagiri
are free floating Planktonic or attached to a Dharmapuri district of Tamil Nadu (fig.1) at the
substrate, benthic forms (Werner, 1977). Diatoms latitude of 1228 north on the longitude of 7811
are key components present nearly in all types of east. The Krishnagiri dam was constructed across
fresh and saline environments. All most all species the Ponnaiyar River (also called as Ponnaiyar) near
are good indicators for a range of water quality Periyamuttur village about 10 km from Krishnagiri
variables because they have narrow optima and town. The reservoir has two main canals, one on
tolerances for many environmental variables (Van the left side called Left Main Canal (LMC) and
Dam et al., 1994). In the last 20 years, numerous other on the right side Right Main Canal (RMC)
ecological studies were conducted regarding the use running almost parallel to the Ponnaiyar River.
of diatoms as indicators of water ecosystem (Agbeti The Ponnaiyar takes its source near Nandi durg in
and Dickman :1989; Underwood et al ., 1998). Karnataka of Chennakesava hills. It is known as
Diatoms are mostly identified based on the Dhakshina Pinakini in Karnataka. It enters Tamil
structure of their cell wall features. The diatom Nadu at places near Bagalur village in Hosur taluk.
frustules have two valves namely epitheca (larger The river is called Ponnaiyar from this point of
upper valve) and hypotheca (smaller lower valve) Tamil Nadu. It is located at the latitude of 12 28N
structures. Although their ecological importance is and longitude of 78 11E. Irrigation projects are
evident, the taxonomy of the genus is confused and constructed across Ponnaiyar by Kelavarapalli
has created much controversy (Round, 1981). reservoir project, Krishnagiri reservoir project and
Earlier many researchers worked on the diatom and Sathanur reservoir project. The reservoir is being
their relationship with physico chemical parameters used for multipurpose utility such as irrigation,
and nutrients absorption. With an increasing fishing and washing.
amount of information on details of the siliceous Sediment samples were collected by PVC
diatom cell wall, especially observed under electron corer to a depth of 20 cm. Samples were transferred
microscope will be helpful in its easy identification. immediately in to zip lock bags to avoid the
Additionally, SEM observations have a remarkable oxidation of sediment samples and stored at 5C in
impact on diatom taxonomy rendering traditional an ice box and carefully transferred to the
identification methods insufficient for recognition laboratory. All extractions were done in duplicate.
of newly created taxa. The consequent lack of Preparation of diatoms:
taxonomic resolution at the LM level may be Detailed diatom studies were done following
leading to over estimation of geographical hot HCl and KMnO4 method (recommended
distributions, ranges of tolerance to environmental technique of acid digestion) by Taylor et al. (2005).
parameters and optimal conditions for growth. The benthic diatom samples subjected to Scanning
Qualitative aspects of the differences between LM Electron Microscopic observations. The SEM
and SEM micrographs are discussed in the present Photos were taken at CAS in Botany, University of
paper. Madras, Guindy campus, Chennai -25.
Fig.1 Map showing Krishnagiri Dam

Scanning Electron Microscopy (SEM) Striae were coarse and often visibly punctuate. But
The specimens were cleaned by adopting the under light microscope only cell wall can be seen
same procedure as described earlier. Acid washed visible and other information are lacking in LM
samples were placed onto a clean glass cover slip. view (fig. 5).
Samples were left air dried overnight. The samples
were coated using gold-platinum using a JEOL JFC
-1600 Auto Fine Coater (JEOL, Tokyo, Japan). The
samples were then examined under a JEOL JSM-
6390 LA Analytical SEM (JEOL, Tokyo, Japan)
and digital images were taken using the system.
RESULT
The topography of diatoms cells were
observed in light microscope and as well as fig. 4 SEM view of fig.4 LM view of
scanning electron microscope. The identified Gomphonema parvulum Gomphonema parvulum
diatoms are Cyclotella menghiniana, Gomphonema
parvulum and Aulacoseira granulatae. The SEM image of Aulacoseira granulate
Under the light microscope (fig.2) the cell (fig. 6) showed valves which were 4-17 m in
wall of diatom looked drum-shaped one. Valves are diameter, with a mantle height of 4-20 m. The
circular with a tangential undulation in the central mantle had straight sides and the valve face was
zone. With this information, identification of the flat. The mantle areolae are square. Linking spines
genus is easy but up to species level, it is difficult to are located at the end of pervalvar costa but it got
be concluded. Under the Scanning Electron damaged during the sediment process. Under LM
Microscopic view (fig.3) the cell wall was drum (fig. 7) the valves and mantle are not clearly
shaped and the margin of the cell well defined and focused and at one end of pervalvar costa the spine
clearly differentiated from the central part of the is present.
valve face. The marginal zone had 6-10 radial striae
measured at 10 m, each composed of a single
alveolus which was open on the inside of the valve.
This gave the marginal zone a chambered
appearance. A single marginal rimoportula was
present. The central zone was structure less which
are radially streaked. Hence with SEM image the
Cyclotella is identified as Cyclotella meneghiniana.
fig.6 SEM of Aulacoseira fig. 7 LM of

granulate Aulacoseira granulate
DISCUSSION:
Despite the high resolution offered by TEM,
the introduction of scanning electron microscopical
analysis had a more profound impact on diatom
taxonomy (Masse et al., 2001). Phycologists
fig.2 LM view of fig.3 SEM view of appreciate the three dimensional structure of diatom
Cyclotella meneghiniana Cyclotella meneghiniana valves and the highly characteristic architecture of
the silica shell through SEM analysis ( Gerloff and
SEM image of Gomphonema parvulum Helmcke ;1974) and Round and Crawfold (1990).
(fig.4) showed that, they were slightly asymmetrical Fine morphological structures of three genus
to transapical axis, symmetrical to apical axis. of diatom namely Cyclotella meneghiniana,
Apices rounded raphe are slightly sinuous. A single Gomphonema parvulum and Aulacoseira granulate
stigma was present on one side of the central area. had been documented in this study. Fine tune of

morphological structure studies of other diatom Masse G, Poulin ST, Belt JM, Robert A,
species from various locations are currently Barreau, Rincea Y and Rowland SJ. 2001. A
underway so as to provide a comprehensive simple methods for SEM examination of Secitoned
documentation of diatom species in KRP DAM. diatom frustules. J. Micros., 204(1):87-92.
The epical valve and also the spines were very clear
in the SEM images when compared to light Round FE. 1981. The diatom genus
microscopic images. At higher magnification the Stephanodiscus: An Electron microscopic view of
valve features were observed in even greater detail. the classical species. Arch. Prostistenk., 124:447-
Above all the SEM images were helpful to explore 465.
the unexplored fine structure of diatoms collected
especially from sub and bottom sediment of Round FE, Crawford RM, Mann DG. 1990. The
aquatic ecosystems. diatoms: Biology & Morphology of the Genera.
Cambridge University Press, United Kingdom. 27-
ACKNOWLEDGEMENTS 28.
Special thanks are given to the DST-ES New
Delhi for their financial support for the pilot study. Taylor JC, Le la Rey PA and Van rensburg L.
Project ref no SP/S4/ES-458/2009. 2005. Recommendations for the collection,
preparation and Enumeration of Diatom from
REFERENCES Riverine Habitats for water Quality Monitoring in
Agbeti M and Dickman M. 1989. Use of lake South Africa, African journal of Aquatic Sciences 3
fossil diatom assemblages to determine historical (1):65-75.
changes in trophic status. Canadian Journal of
Fisheries and Aquatic Sciences 46:1013-1021. Underwood G, Phillips J and Saunders K. 1998.
Distribution of estuarine benthic diatom species
Dam H, Mertens A and Sinkeldam J. 1994. A along salinity and nutrient gradients. European
coded checklist and ecological indicator values of Journal of Phycology 33:173-183.
freshwater diatoms from the Netherlands.
Netherlands Journal of Aquatic Ecology 28:117- Werner D. 1997. The Biology of Diatoms.
133. University of California Press. 498.
Gerloff J and Helmcke JG. 1974.

Diatomeenschalen im electron enmikroskopischen
Bild (ed by J.G. Helmcke, W. Krigeger and J.
Gerloff), 8: J. Cramer Verlag, Ehre., 1

Publication group
Laboratory toxicity evaluation of Diflubenzuron, a chitin-synthesis

inhibitor, against Anopheles darlingi (Diptera, Culicidae).
Authors: ABSTRACT:
Costa FM, Tadei WP.
We evaluated Diflubenzuron toxicity against larvae and pupae of Anopheles
darlingi. A series of bioassays were developed to assess the lethal concentrations LC50
and LC90 to larvae after two exposure periods (24 and 48 hours), and to evaluate its
toxicity to 20-minute- and 24-hour-old pupae. The LC50 and LC90 obtained were 0.006
and 0.013 ppm, respectively. For concentrations of 0.01 to 0.1 ppm, a mortality of
100% was observed, mainly during the larval stage. With concentrations between
0.009 and 0.001 ppm, mortality varied from 1.7% to 25.8% in the larval stage; from
21.7% to 44.2% in the pupal stage; and from 15.0% to 4.2% in adults. No
differentiation in emergence inhibition was observed between the exposure periods
of 24 (82.0%) and 48 hours (86.7%). These results revealed that the minimum
exposure period of 24 hours significantly inhibits emergence. The 20-minute-old
pupae were more susceptible than the 24-hour-old ones (61.1% emergence inhibition,
against 93.0% of the former). Diflubenzuron toxicity to pupae indicates that this
Corresponding author: insecticide may also act by contact. These results showed that A. darlingi larvae and
Costa FM pupae are susceptible to the inhibitory action of Diflubenzuron.
Keywords:
IGR, larvicide, mosquito, pesticide, vector control.
Article Citation:
Web Address: Costa FM, Tadei WP.
Laboratory toxicity evaluation of Diflubenzuron, a chitin-synthesis inhibitor, against
Anopheles darlingi (Diptera, Culicidae).
Dates:
Received: 15 Sep 2011 /Accepted: 09 Oct 2011 /Published: 14 Oct 2011
Ficus Publishers.
cited.
444-450 | JRB | 2011 | Vol 1 | No 6

Costa et al.,2011
INTRODUCTION undergoing severe environmental disturbance from

Insect growth regulators (IGR) are a group the exploitation of natural resources (such as wood,
of insecticides with a heterogeneous chemical ores, and oil extraction), rural settlement projects,
nature that interfere with physiological processes and fish farming (Tadei et al., 2007).
essential for insect development. They generally The aim of this study was to evaluate the
cause physiological and morphological alterations insecticidal activity of Diflubenzuron against larvae
to insects, mainly during the immature stages, and pupae of A. darlingi, under laboratory
hindering development to the adult. These conditions.
compounds include juvenile hormone mimics,
antijuvenile hormone analogs, and chitin-synthesis MATERIALS AND METHODS
inhibitors (Eisler, 1992). Diflubenzuron was the Mosquito field collection and laboratory
first IGR synthesized, and currently is the breeding. Females of A. darlingi were collected in
insecticide that is most studied and used against two localities near Manaus, Amazonas, Brazil:
insect pests. Puraquequara (338.63S; 595337.52W) and
Diflubenzuron is a benzoylphenylurea- Brasileirinho (3210.47S; 595217.22W). The
derived insecticide that acts by interfering with females were placed into 350-mL paraffined cups,
chitin synthesis and deposition in the insect cuticle sealed with a tulle mesh. They were then taken to
(Cohen, 1987). Immature stages are more the laboratory and fed with domestic duck (Cairina
susceptible to this insecticide because of their moschata Linnaeus, 1758) blood meal and 10%
successive molts. A large proportion of larvae glucose solution. After feeding, mosquitoes were
exposed to Diflubenzuron die during molting, individually transferred to 50-ml disposable cups.
because they cannot cast their exuvia. Some pupae Moistened cotton and filter paper were placed on
may survive but eventually die in subsequent stages the bottoms of the cups to function as substrates for
(Mulla, 1995). egg laying. After eclosion, larvae were raised
The World Health Organization has according to the methodology described by
recommended the use of Diflubenzuron for the Scarpassa & Tadei (1990), until they reached the
control of mosquito vectors since 1982. appropriate ages for the bioassays.
Diflubenzuron in very low concentrations, from Experimental design.
0.001 to 0.002 ppm, is highly toxic to mosquito Obtaining Diflubenzuron LC50 and LC90 lethal
larvae (Mulla, 1995). The use of Diflubenzuron is concentrations
recommended for special situations, mostly in Diflubenzuron (1-(4-chlorophenyl)-3-(2,6-
disturbed environments, because this compound difluorobenzoyl)urea) serial dilutions were made in
also adversely affects other organisms that are DMSO (dimethyl sulfoxide), to obtain the
phylogenetically close to mosquitoes, or even those concentrations required for the bioassays. The
coexisting in the same habitat. Diflubenzuron methodology followed that described by Mulla et
toxicity to vertebrates and humans is considered al. (1974), with some adjustments.
low (Mulla, 1995; Floore, 2006). Groups of 40 early 4th-instar larvae were
Many mosquito species have been targeted placed in 100-ml plastic jars. Each jar contained
by research on the potential insecticide effect of 200 ml of distilled water, fish meal, and the
Diflubenzuron; members of Anopheles, Culex and corresponding dose of Diflubenzuron for each
Aedes are the most frequently studied. Laboratory- bioassay.
esti mated Diflubenzur on CL90 lethal Each jar had its sidewalls polished and its
concentrations for Anopheles albimanus Wiedman, entrance covered with tulle, in order to prevent the
1820, Culex quinquefasciatus Say, 1823, and Aedes emerged mosquitoes from perching and flying. For
aegypti Linnaeus, 1762 are 0.001, 0.002 and 0.0035 each concentration evaluated, three replicates of 40
ppm, respectively (Mulla et al., 1974; Mulla, 1995; individuals were made, totaling 120 exposed
Fournet et al., 1993). individuals per bioassay. The same conditions were
We studied Anopheles darlingi Root, 1926, a adopted for the control group, which received only
wild mosquito with anthropophilic habits, which is 0.5 ppm DMSO.
the main vector of the three Plasmodium species Dead and emerged individuals were
that causes human malaria in Brazil (Tadei et al., removed and counted daily, and the stage of each
1998). Anopheles darlingi is the main vector of was recorded. The mosquitoes considered alive
malaria in the Amazon, especially in areas were only those that completely emerged. This
Costa et al.,2011
process was repeated until the total number of The mean percentages of larval, pupal and
exposed individuals was reached. adult mortality with the different Diflubenzuron
Larvae exposure periods to Diflubenzuron LC50 concentrations evaluated were submitted to a
Two exposure periods of early 4th-instar Kruskal-Wallis test or H-test (KW-H), using a
larvae to Diflubenzuron LC50 were evaluated: in 24 probability level of 0.05 as the critical level of
and 48 hours. For each exposure period, the significance in all tests. Mean pupal mortality in the
experiment was carried out in plastic cups susceptibility and larvae-exposure-period tests were
containing 50 mL distilled water, meal, 10 A. converted to arcsine , and then submitted to one-
darlingi larvae, and the corresponding insecticide way ANOVA and a post-hoc Tukey-HSD test to
dose (LC50). Each experiment had five replicates, determine the significant differences between group
and all replicates had a control group that received means.
only 0.5 ppm DMSO. The experiments were
repeated three different times. RESULTS AND DISCUSSION
Larvae were kept in the cups only for the The data for Diflubenzuron lethal
respective exposure period. Afterward, they were concentrations to A. darlingi are shown in Table I.
individually transferred to depression slides, The LC50 and LC90 lethal concentrations obtained
washed three times in distilled water, and finally in the bioassays were 0.006 and 0.013 ppm,
placed in 50-ml cups with distilled water and meal. respectively.
Each cup was covered with tulle. Dead individuals A total of 11 tested concentrations caused
and emerged adults were removed and counted mortality in the larval, pupal and adult stages
daily, until the total number of exposed individuals (Figure 1). The mortality of larvae, pupae and
was reached. adults differed significantly from the control group
Pupae susceptibility to Diflubenzuron at all concentrations evaluated (F3.11 = 4.99; P =
Pupae susceptibility tests were carried out 0.001).
only with 0.1 ppm Diflubenzuron. This At concentrations between 0.01 to 0.1 ppm
concentration was selected based on the results of (the highest used), mosquito mortality reached
preliminary bioassays with lower concentrations. 100%. The highest percentages occurred during the
Pupae were divided into two groups: 1) larval stages were 64.2 to 97.5% (Fig. 1A).
pupae aged up to 20 minutes after molting; and 2) Mortality during the pupal stage ranged from 35.0
pupae aged up to 24 hours after molting. The to 2.5% (Fig. 1B). The highest pupal mortality was
experiment was carried out in cups containing 10 recorded with the concentration of 0.01 ppm (35.0
pupae of the same age, 50 ml distilled water, and %), the only concentration that caused mortality
the corresponding dose of insecticide necessary to during the adult phase 0.8% (Fig. 1C).
reach the desired concentration. The experiment At concentrations between 0.009 and 0.01
had three replicates, and the control group received ppm, mortality was observed in all three stages.
only DMSO. Each cup was covered with tulle to Mortality ranged from 1.7 to 25.8% during the
prevent the adults from dispersing after their larval stage (Fig. 1A), from 21.7% to 44.2% during
emergence. The experiment was repeated three the pupal stage (Fig. 1B), and from 15.0% to 4.2%
different times. during the adult stage (fig. 1C). The highest
The contents of the cups were inspected mortality for pupae and adults was recorded with
daily, dead pupae, dead and living adults were the concentration of 0.009 ppm (44.2% and 15.0%,
removed. The mosquitoes counted as alive were respectively).
only those that were completely cast from their
exuvia. The experiment continued until the last
Table I. Diflubenzuron lethal concentrations LC50 and
pupa or adult died, or until the last adult completely LC90 for 4th instar larvae of Anopheles darlingi, under
emerged. laboratory conditions. Confidence Interval (CI) = 95%.
Statistical analysis
To obtain LC50 and LC90 lethal Lethal concentrations ppm (CI 95%)
concentrations, the mortality data were corrected by
means of Abbotts formula (Abbott, 1925). The LC50 0.006 (0.005 0.007)
data were then submitted to a probit analysis, using LC90 0.013 (0.010 0.019)
the program Polo PC (Le Ora Software, 1987).
Regression equation y = 8.46 + 5 + 3.79 * log x

Costa et al.,2011
Figure 1. Mean percentages of larval (A) (KW-H (11.44) = 38.54; P<0.001), pupal (B) (KW-H (11.44) = 30.34;
P<0.001), and adult mortality (C) (KW-H (11.44) = 32.76; P<0.001) for the concentrations of Diflubenzuron
evaluated in the bioassays against 4th-instar larvae of Anopheles darlingi.
The data from the exposure tests of early 4th-

Table II. Mean percentage of emergence inhibition for
instar larvae to Diflubenzuron for 24 and 48 hours
two 4th instar larvae of Anopheles darlingi exposed for
are shown in Table II. Larval mortality did not 24 and 48 hours to 0.006 ppm-concentrated
differ significantly between the two exposure Diflubenzuron (LC50), under laboratory conditions.
periods. The concentration of 0.006 ppm
Diflubenzuron was very effective within the Exposure period Emergence inhibition
n
(hours) (%) *
exposure period of 24 hours. Emergence inhibition
24 150 82.0 2.0 a
mean percentage was 82% for 24-hours exposure,
and 86.7% for 48-hours exposure. In both Control 24 30 6.7 5.8 b
treatments, the bulk of mortality occurred during
48 150 86.7 6.1 a
the larva-to-pupa molt, although mortality during
the pupa-to-adult molt was also observed. Control 48 30 20.0 0.0 b
The use of insecticides with low toxicity,
Means followed by the same letter are not
high residual effect and high specificity has been statistically different (P< 0.05).
much emphasized recently. A product with these
Costa et al.,2011
features provides effective control of populations of adult phases. Therefore, low concentrations of
insect pests, mainly those of medical importance Diflubenzuron retard individual mortality. The way
(such as culicids), and also provides environmental that the larvae died, especially in the pupa and adult
protection (Resende & Gama, 2006). Consequently, emergence phases (during molting), reveals the
the use of Diflubenzuron and other IGRs for the potential effect of Diflubenzuron to interfere with
control of mosquitoes has been thoroughly physiological processes during cuticle chitin
evaluated in many countries including Brazil. deposition. Borges et al. (2004) described severe
However, because these insecticides adversely injuries to the cuticle and epidermal cells of Aedes
affect arthropod fauna, their use is recommended aegypti, which caused larvae death at the time of
only for certain limited habitats without vulnerable molting.
environments (Floore, 2006). Our results also showed that Diflubenzuron
Diflubenzuron was shown to be highly toxic is effective for both exposure periods evaluated.
to 4th-instar A. darlingi larvae, inhibiting emergence Because no significant difference was observed
at low concentrations. The LC50 and LC90 results when comparing the exposure intervals (24 and 48
obtained in this study are similar to results hours), it is concluded that the stress caused to the
previously obtained for other anophelines under mosquitoes during their removal and washing had
similar conditions. Jakob (1973) reported LC95 little or no influence on individual mortality.
lethal concentrations for Anopheles albimanus and According to these results, it is evident that the
Anopheles stephensi of 0.001 ppm and 0.0025 ppm, minimal exposure period of 24 hours is sufficient to
respectively. Mulla et al. (1974) estimated the inhibit mosquito emergence. The results obtained in
LC90 for A. albimanus of 0.001 ppm. Lowe et al. this study indicate that Diflubenzuron acting for a
(1975) obtained an LC90 of 0.0098 ppm for the minimum of 24 hours against A. darlingi larvae is
same species, and 0.004 ppm for Anopheles highly effective in inhibiting emergence. Sacher
quadrimaculatus Say, 1912. These studies reported (1971) demonstrated a 95% emergence inhibition
no values for the LC50 lethal concentration. Ali & when using IGR MON-0585 CL90 of 0.1 ppm
Nayar (1987) estimated for A. albimanus and A. against 4th-instar Ae. aegypti larvae. In the same
quadrimaculatus, LC50s of 0.00142 ppm and study, Sacher observed a significant decrease in
0.0014 ppm, respectively. Dame et al. (1976), adult emergence when individuals were exposed to
working with a DDT-susceptible and a resistant the insecticide for only three hours.
strain of A. quadrimaculatus, observed The interference with A. darlingi
Diflubenzuron LC50 and LC90 ranging from 0.002 development and consecutive emergence of adults
to 0.004 ppm, respectively. after 24 hours of exposure to Diflubenzuron reveals
The results obtained in this study indicate its ability to penetrate within the larva body and its
that A. darlingi seems to be less susceptible to rapid interference with the biochemical mechanisms
Diflubenzuron than other previously studied of chitin metabolism. Although this study showed
anophelines. This difference may be a result of the the efficiency of Diflubenzuron after 24 hours of
high heterogeneity observed among the individuals exposure to larvae, further studies are necessary to
from the first generation (F1) used in the bioassays. determine the minimal exposure period where this
Anopheles darlingi is a wild species, and all insecticide interferes with the development of A.
attempts to breed it in the laboratory for more than darlingi larvae.
one generation have failed. The previous The results for the pupae susceptibility tests
investigations mentioned of other anopheline are shown in Table III. The Diflubenzuron
species used strains bred for years under laboratory concentration evaluated caused toxicity to pupae.
conditions, resulting in individuals that were even There was a significant difference in the toxicity to
more susceptible, mainly to insecticides such as pupae between the two ages tested, with the 20-
Diflubenzuron. minute-old pupae being more susceptible to the
In relation to larval, pupal and adult insecticide. Pupal emergence inhibition was 93.0%
mortality, our results confirm those of Mulla et al. for 20-minute-old pupae, and 61.1% for 24-hour-
(1974), who observed that high Diflubenzuron old pupae. Among the 20-minute-old pupae, the
concentrations caused great mortality to A. mortality was higher during the pupal stage than
albimanus during the larval phase. Lower among adults. For the 24-hour-old pupae, mortality
concentrations caused mortality during pupal and was higher at molting onset; the deaths of adults

Costa et al.,2011
Table III. Mean percentage of emergence inhibition of ACKNOWLEDGEMENTS.

two ages of pupae of Anopheles darlingi exposed to 0.1 The authors thank the Conselho Nacional de
ppm-concentrated Diflubenzuron, under laboratory Desenvolvimento Cientfico e Tecnolgico (CNPq)
conditions. for grants, the Instituto Nacional de Pesquisas da
Pupa age n
Emergence inhibition Amaznia (INPA) for supporting the research (PPI
(%) * project), the Fundao de Vigilncia em Sade do
20 minutes 90 93.0 6.7 a Estado do Amazonas (FVS) for sampling support,
Control 20 minutes 30 16.7 5.8 bc the company Champion Farmoqumico for
24 hours 90 61.1 19.2 b
providing technical grade Diflubenzuron, and
Professor M.Sc. Flvio A. L. Fonseca for assisting
Control 24 hours 30 3.3 5.8 d with the statistical analysis.
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Publication group
Identification of prokaryotic organisms in goat rumen based on

metagenomic DNA sequences
Authors: ABSTRACT:
Jing Zhou, Brian
Copeland, Cheng Zhang,
Zong Liu, Sarabjit Bhatti,
Roger Sauve, Suping Zhou.
A protocol for isolating pure DNA from goat ruminal fluid was developed.
DNA was partially digested using ApoI enzymes and ligated onto Lambda ZAP II
Institution: Predigested EcoR l/CIAP-treated vector to make a metagenomic DNA library. DNA
Department of Agricultural sequence analyses showed that positive clones carried complete or partial sequences
Sciences, School of for structural genes in different strains of Escherichia coli. Several clones did not
Agriculture and Consumer share significant similarity with nucleotide in the databases. Research results indicate
Sciences, Tennessee State that the population of prokaryotes in the goat rumen contained E. coli, and possibly
University, 3500 John A other organisms that have not been characterized. This is the first report of the use of
Merritt Blvd, Nashville, molecular approaches to characterize microbial genes or organisms from goat
TN 37209. rumens.
Charles Lee, USDA-ARS,
800 Buchanan St., Albany,
CA 94710.
Ryszard Puchala
School of Agriculture and
Applied Sciences, Langston
University, Langston, OK Keywords:
73050. Goat, rumen, microbes, metagenome, Escherichia coli.

Suping Zhou Jing Zhou, Brian Copeland, Cheng Zhang, Zong Liu, Sarabjit Bhatti, Roger Sauve,
Suping Zhou.
Identification of prokaryotic organisms in goat rumen based on metagenomic DNA
Email: sequences.
suping@tnstate.edu
Phone No:
6159632146. Dates:
Received: 06 Aug 2011 /Accepted: 11 Aug 2011 /Published: 18 Oct 2011
Fax:
615-9631557. Ficus Publishers.
Web Address: commercial, distribution, and reproduction in all medium, provided the original work is properly
http://jresearchbiology.com/ cited.
451-455 | JRB | 2011 | Vol 1 | No 6
Zhou et al.,2011
INTRODUCTION from a mixed population communities based on

Goats are known for having an extremely DNA segments of the bulk metagenomic DNA
varied diet (Stoneberg 1989). These animals feed sequences, such as the virus strain responsible for
on the tips of woody shrubs and trees, Honey Bee Colony Collapse disorder (Cox-Foster
lignocellulosic agricultural by-products such as et al., 2007), the virus for several human diseases
cereal straws and stovers. There have been claims (Victoria et al., 2009) and other pathogenic
that these animals can feed on newspapers. organism (Nakamura et al., 2008). DNA sequences
Symbiont microbes in the rumen of these in a metagenomic DNA library can reveal the
herbivores play key roles in providing the hosts genetic diversity under various environments
with various nutrients (Hungate 1966). Enzymes (Biddle et al. 2008; Woyke et al. 2006).
secreted by ruminal microbes are needed for the The metagenome method provides the
conversion of cellulose and hemi-cellulose into investigator with the global microbial gene pool
simple sugars. These sugars serve as a source of without the need to culture these microorganisms
energy for these animals (Miyagi et al.1995). (Yun et al., 2004). This method has been widely
Ruminants also obtain the majority of their essential used to map microbiomes in ruminants such as the
nitrogen needs from the microbial degradation of cow (Morrison et al. 2005). For this study, a
dietary protein within the rumen (Brooker et al. metagenomic DNA library was constructed using
1995). Moreover, the microbial activity is the prokaryotic genomic DNA from goats ruminal
responsible for detoxification and synthesis of fluid. Based on sequence analyses of cloned DNA
essential substances for the host animals fragments, gene identities and the classification of
(Wroblewski et al. 2001; Bas et al. 2003; Kronberg microbes present were predicted. The
et al. 2006). The composition of the microbial determination of microbial populations inhabiting
population differs with goat species as well as with goat rumens is very important for understanding
their food (Shi et al., 2008). nutrient absorption from high-fiber diets and
Analysis of microorganisms in the ruminal consequently enhancing productivity of such
fluid of different herbivores revealed the presence animals (Onodera et al. 1998; Sajiad et al. 2008).
of bacteria (10101011 cells/ml, representing more
than 50 genera), ciliate protozoa (104106/ml, from MATERIALS AND METHODS
25 genera), anaerobic fungi (103105 zoospores/ml, Collection of goat ruminal fluid
representing five genera) and bacteriophages (108 Eight mature goats were ruminally
109/ml) (Hobson, 1989; Sajiad et al. 2008). These cannulated following the procedure developed by
numbers show only a small fraction of the the American Institute for Goat Research. After
microbial species found in rumens of animals on recovery from the surgery, these goats were fed a
fiber based diets since less than 10-20% of diet that consisted of fiber supplemented with
microbial populations these microbes are cultivable minerals and vitamins. Goats were fed at 0900 h
on synthetic media (Amman et al. 1992; and were given free access to water. After being on
McSweeney et al. 1999). this diet for 14 days, collection of ruminal fluid
A recently developed molecular approach to samples were initiated. Samples were collected at
obtain microbial genes in a microenvironment 4, 6, 8 and 10 h after feeding for 4 days. These
allows for the identification of the entire genome samples were pooled before DNA extraction.
present. This method involves cloning genomic Isolation of metagenomic DNA
materials into bacterial chromosome (BAC), or The ruminal fluid composite sample was
plasmid cloning vectors hosted in a cultivable centrifuged at 13,000 rpm for 10 min. After
bacterium such as Escherichia coli (Brady et al., discarding the supernatant, pellet was used to
2004; Rhee et al., 2005). Subsequent functional extract bacterial and prokaryotic microbial DNA
screening of this cultivable bacterium has resulted using the CLS-TC buffer in FastDNA Spin Kit
in the isolation of target genes encoding for the (Cat#6540-600, MP Biomedicals, LLC, Solon,
antibiotic turbomycin (Gillespie et al. 2002), or the Ohio). Extracted DNA was further purified
enzyme xylanase (xyn8) (Lee et al. 2002) and novel utilizing Geneclean Kit (MP Biomedicals).
lipases (Lee et al. 2006) and forage specific Partial digestion of purified genomic DNA
glycoside hydrolases in the bovine rumen was carried out at 50oC for 50 min with FastDigest
microbiome (Brulca et al., 2009) and many others. ApoI (Fermentas, Maryland). Digestion products
Moreover, individual organisms can be identified were separated in 0.8% agarose gels. Bands
Zhou et al.,2011
between 4-10 kb and those > 10kB, <4kb were BW2952; GQ343316 for tRNA(Ile)-lysidine
separately collected from the gel. DNA was synthetase in E. coli BW2952; GQ343317 for a
recovered from gel slices using Qiagen Gel predicted lactam utilization protein in E. coli
Extraction Kit (Qiagen Sciences, Maryland). BW2952; GQ343318 for fused DNA-binding
Construction of metagenomic library transcriptional regulator/proline dehydrogenase in
The recovered DNA was ligated onto E. coli BW2952; GQ343319 for Cpn60 chaperonin
Lambda ZAP II Predigested EcoR l/CIAP-treated GroEL, large subunit of GroESL in E. coli
Vector and packaged into the Gigapack III Gold BW2952; GQ343320 for glyceraldehyde-3-
Packaging Extracts (Stratagene, CA). Titering of phosphate dehydrogenase C (pseudogene) in E. coli
the packaging reaction was on NZY agar plates. str. K12 substrain DH10B; GQ343321 for predicted
The background of the genomic DNA library was methyltransferase in E. coli BL21(DE3);
determined by the Blue and White color selection GQ343324 for Cytochrome b in E. coli; GQ343326
method on NZY plates supplemented with 3.8 mM for fused predicted transporter subunits of ABC
IPTG (isopropyl -D-1-thiogalactopyranoside) and superfamily in E. coli BL21(DE3); GQ343327 for
50 mg/ml final concentration X-gal (bromo-chloro- protein associated with replication fork in E. coli
indolyl-galactopyranoside), both chemicals were BW2952; GQ343328 for ferrochelatase acetyl
obtained from Sigma (Missouri, USA). The color esterase in E. coli BW2952; GQ343329 for the
of background plaques was blue, while recombinant predicted pyruvate formate lyase in E. coli
plaques were white. The white plaques were BW2952; GQ343331 for D-alanyl-D-alanine
randomly selected for singleclone excision to carboxypeptidase in E. coli BW2952; GQ343332
isolate the phagemid (pBluescript SK-). for fused ribonuclease E: endoribonuclease/RNA-
Identification of genes and microbial species binding protein in E. coli BL21(DE3); and
Single stranded DNA were recovered, GQ343333 for periplasmic sensory protein
processed and sequenced using M13 forward (-20) associated with the TorRS two - component
and M13 reverse (20mer) primers from both regulatory system in E. coli O157:H7 strain
directions using the Bigdye Terminator version 3 on TW14359.
a Avant 3100 DNA Analyzer (Applied Biosystems, Dehority and Grubb (1977) isolated 44
CA) following a previously described procedure different bacterial strains from the rumen contents
(Zhou et al. 2006). The identity of cloned gene of goats. These strains were grouped based on their
fragments were determined by BLASTing against morphology, Gram stain reaction, anaerobiosis,
NCBI nucleotide database, (others excluding motility, fermentation end products, and 16S
human and mouse databases. In addition, DNA ribosomal DNA-restriction fragment length
sequences were also translated into protein using polymorphism analysis. Members of E. coli strains
the web-based translation tool (http:// are phenotypically diverse but have similar
www.expasy.ch/tools/dna.html). Longest peptide chromosomal organizations (Welch et al. 2002).
sequences were searched for conserved domains Bacterial clones identified in this study were (99-
and protein identities (Marchler-Bauer et al., 2004, 100%) identical to the DNA sequences reported in
2009). All clones were submitted to the NCBI the database. This suggests that these sequences
databank at http://www.ncbi.nlm.nih.gov. were cloned from E. coli strains present in goats
ruminal fluid.
RESULTS AND DISCUSSIONS In rumens of different herbivores, bacteroids
The following clones contained full-length (obligate anaerobic organisms) are responsible for
sequences that encode for structural genes in E. the degradation of cellulosic polysaccharides from
coli: GQ343311 for L-arabinose isomerase in E. plant sources (Ramirez and Dixon, 2003; Franklund
coli str. K12 substrain; W3110. GQ343312 for Glass, 1987). These organisms could play a similar
tyramine oxidase, copper-requiring in E. coli BL21 role in goats. In this study, several clones had no
(DE3); GQ343313 for dehydrogenase in E. coli; significant similarity when searched in the DNA
BW2952 and other strains; GQ343314 for cysteine database. However one of the six frame translation-
synthase B (O-acetylserine sulfhydrolase B) in E. predicted peptide sequence from one clone matched
coli BW2952; GQ343309 for a conserved protein a fragment of a glucanase homologs from
in E coli BW2952; GQ343315 for citrate lyase, bacteroids, suggesting that cellulosic microbes are
citrate-ACP transferase (alpha) subunit in E. coli presenting in goat rumen. Other clones had no

Zhou et al.,2011
matching sequences (at neither peptide nor DNA Brulca JM, Antonopoulosb DA, Millera MEB.
level), these clones could be genomic sequences 2009. Gene-centric metagenomics of the fiber-
from organisms that have not been characterized adherent bovine rumen microbiome reveals forage
previously. These clones will be deposited in the specific glycoside hydrolases. PNAS 106:1948-
database after the full-length gene sequences 1953.
become available.
Cox-Foster DL, Conlan S, Holmes EC. 2007 A
CONCLUSION metagenomic survey of microbes in honey bee
Gene sequences that encode for structural colony collapse disorder. Science 318:283-287.
and functional proteins have been identified in the
goat rumen. This study provides molecular Dehority BA, Grubb JA. 1977. Characterization
evidence for the presence of microbial organisms of the predominant bacteria occurring in the rumen
that have not previously been observed in the goat of goats (Capra hircus). Appl Environ Microbiol.,
rumen and demonstrates that functional genes can 33:1030-1036.
be cloned directly from bulky DNA extracts from
goat ruminal fluid. Franklund CV, Glass TL. 1987. Glucose uptake
by the cellulolytic ruminal anaerobe Bacteroides
ACKNOWLEDGEMENT succinogenes. J Bacteriol 169:500-506.
This research was financially supported by
National Institute of Food and Agriculture, Gillespie DE, Brady SF, Bettermann AD,
Capacity Building Research Grant Award No. 2010 Cianciotto NP, Liles MR, Rondon MR, Clardy J,
-38821-21598, and Evan Allens funds from the Goodman RM, Handelsman J. 2002. Isolation of
United States Department of Agriculture. The antibiotics turbomycin A and B from a
authors wish to thank Dr. Johnson Terrace for metagenomic library of soil microbial DNA. Appl
assistance in conducting the research. Environ Microbiol., 68:4301-4306.
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Publication group
Survey of nature and extent of damage caused by bird pest to pearl millet
crop in Patan District (North Gujarat)
Authors: ABSTRACT:
Patel KB.
Pearl millet is the main crop of Patan district of Gujarat state. It is grown in
two seasons viz. kharif and summer. This research was conducted during 2005 and
2006 at Hansapur village of Patan district. Two varieties viz. Usha-23 and Pioneer of
pearl millet were sown for study purpose. During this research work, bird pests were
identified, and nature of damage and assessment of losses in grain yields were carried
Institution:
out. The bird species observed visiting the pearl millet crop at sowing and
Department of biology
Sheth M. N. Science germinating stages were House Crows (Corvus splendens), Blue Rock Pigeons
College, Patan 384 265. (Columba livia), Cattle Egret (Ardea alba), Grey Francolin (Francolinus pondicerianus)
Dove (Streptopelia decaocto), Red - wattled Lapwing (Vanellus indicus), Common
Myna (Acridotheres tristis), Bank Myna (Acridotheres ginginianus), Common Babbler
(Turdoides caudatus), and Yellow-eyed Babbler (Chrysomma sinense). Blue Rock
Pigeon, House Crow and Grey Francolin were found damaging the germinating seeds
of pearl millet. The bird damage in germinating seeds of pearl millet varied from 7.50
to 9.35(Mean=8.35%) and 4.28 to 8.87(Mean=6.05%), respectively during
Corresponding author: summer,2005-06 and kharif, 2005-06. The birds visited fields sown with pearl millet
Patel KB. during morning hours i.e., from 6.00 to 10.00 a.m. and again in the evening from
4.00 to 6.00 p.m. but some times House Crows, Common Babbler occasionally visit the
crop during noon time. The birds damaged 6.99 and 11.99 per cent of earheads
during summer season and 6.96 and 18.00 per cent during kharif season in the
treatment of manual bird scaring and unprotected (control) plots of pearl millet,
respectively. The avoidable loss in grain yield of pearl millet varied from 8.87 to 15.73
during summer and 24.17 and 36.70 per cent during kharif season, in the treatment of
Email:
kbpatel@gmail.com manual bird scaring and unprotected (control) plots, respectively.

http://jresearchbiology.com/ Patel KB.
Survey of nature and extent of damage caused by bird pest to pearl millet crop in
Patan District (North Gujarat).
Dates:
Ficus Publishers.
cited.
456-460 | JRB | 2011 | Vol 1 | No 6

Patel.,2011
INTRODUCTION seeds sown and no. of seeds damaged or eaten

Patan district is situated in the northern part by birds per row in the net plot area after
of Gujarat state. It is bound on the northwest and germination were ascertained by counting
north by rann of Kutch and Banaskantha district. number of plumules left behind by the
Patan district lies between 23 41 to 23 55 N Frankolin.
latitude and 71 31 to 72 20 E longitude. Pearl 2. Total number of ear heads per row were
millet (Bajara) Pennisetum typhoides is an counted and recorded at the time of tillering
important cereal heavily depredated by birds. stage.
Seed of this crop is exposed and so attract several 3. Number of ear heads sowing bird damage was
bird species during the entire period of seed setting sorted out and recorded.
to harvesting stage and so suffer heavy losses. Birds 4. Ear heads damaged by insects were sorted
may be insectivorous or granivorous, but they out from the total ear heads nipped from a
have a significant impact on the production of plot were counted and recorded separately.
millet crops (Mathew et al., 1998). The pearl 5. After counting the ear heads damaged by birds
millet is highly susceptible to bird damage in and insects were, then sun dried and threshed
northwest India(Beri et al., 1968; Mehrotra and separately. The grains were cleaned and
Bhatnagar, 1979 and Dhindsa et al., 1984). weighed separately and recorded. Similarly,
The extent of bird damage to the pearl millet undamaged ear heads were sun dried and
varies between fields and at different localities threshed; then cleaned grains were weighted
depending upon several factors (Mathew et and recorded separately.
al.,1998). Crop fields attract many birds from 6. Birds present in each plot were observed at
sowing to grain maturity stage (Bhalodia et different stages viz., seedling and milk, dough,
al.,1997). Various species of bird pests affecting maturity stage, harvesting stage and recorded in
different stages of growth to pearl millet was each plot.
studied. Further, the nature and extent of 7. During kharif and summer seasons of 2005-
damage was also studied and analysed in this 2006, following treatments were evaluated.
work. i. Nylon net enclosure
ii. Manual bird scaring
MATERIALS AND METHODS iii. Control (Unprotected).
Survey for the bird pests of pearl millet
crop and assessment of damage in cultivators' RESULTS AND DISCUSSION
field were conducted during 2005 to 2006 at Results of the field survey carried out
Hansapur village in Patan district. Hansapur regarding the bird species in kharif and summer
village lies between 23 50 N latitude and 71 seasons (2005-2006) damaging pearl millet variety
09 E longitude. The fields having crop of Usha-23 and Pioneer in Patan district are given in
pearl millet were randomly selected at each site. Table-1.
Birds visiting pearl millet fields were observed The birds visiting fields sown with pearl
using a binocular (8-16 X 40), and counted twice in millet were classified into three groups based on
a day during the crop season. The observations their food habits namely (i) useful (feeding on
were recorded for one hour duration. Birds were insect, pests, rats, molluscs etc.), (ii) harmful
identified as per the descriptions of birds given by (feeding on grains) and (iii) facultative (feeding on
Ali and Replay (1983, 1987). grains when insect and other animal origin food
In April 2005, fourteen villages which was not available). These bird pests visit the crop
constituted a major belt for this crop in Patan during morning hours i.e., from 6.00 to 10.00 a.m.
district were surveyed. The varieties grown in and again in the evening from 4.00 to 6.00 p.m.
this area were Usha-23, and Pioneer. To estimate but some times House Crows, Common Babbler
the amount of crop loss from each of the above occasionally visit the crop during noon time.
three plots an area of each plot measuring 6 X 4.5m Damage to the crop was initiated after the
was marked in fields sown with above stated first irrigation, immediately after the sowing,
cultivars of pearl millet. when the seeds get soaked and the activity of
Method of recording observation the embryo starts. At this stage, bird pests
1. Number of seeds per row damaged/ eaten by were recorded infesting the crop, picking the
birds during seedling stage. Total number of soaked seeds from the soil and feeding on
Patel.,2011
them. Feeding on the moist seeds by Blue Rock plots. The scaring employed was by human
Pigeon was limited for three days after sowing, till shouting in treatment 1. In treatment 2, limited
the germination of the seeds, but House Crows (6.00m X 4.5m) area of the crop was enclosed with
continue to infest the crop even after the nylon net to prevent bird damage to the earheads. In
emergence of plumule and radicle from the the treatment 3, crop was kept without netting and
seed, and were observed feeding on the young bird scaring. The damage by bird pests to the pearl
seedlings by pulling them out from the soil and millet crop was assessed by taking counts of the
feeding on the remaining portion of the food total number of earheads, no. of healthy earheads
material left in the seed. and earheads at the time of harvest in each plot
Grey Francolin were visited in groups were noted separately which is given in Table 3
and disturbed sown and moist seeds and feeding and 4.
on them. Young seedlings were damaged by Results given in the table 3 revealed that the
removing the arial portion and feeding on the pearl millet earheads were completely protected
remaining food material present in the seed. from bird damage when crop was enclosed in nylon
The results presented in Table 1 and 2 net during both the seasons. On the other hand, bird
revealed that the bird damage in germinating seeds damage was higher in unprotected earheads
of pearl millet varied from 7.50 to 9.35 (135=11.99%) than that in the plot where manual
(Mean=8.35%) and 4.28 to 8.87(Mean=6.05%), bird scarring was employed (80=6.99%).
respectively during summer,2005-06 and kharif, Interestingly, insect damage was higher
2005-06.Since the experimental plot was located in (107=9.99%) in nylon netting than those without
the farm area, where other plant breeding material netting (Treatment 1 =2.01% and Treatment 3
of pearl millet crop were also sown and at =3.02%) during summer season.
germinating stage, bird scaring was employed in More or less similar trend of bird and insect
those plots which were adjacent to this plot. Hence damage was observed during kharif season also.
due to the impact of bird scaring, the bird pests The number of undamaged earheads of
were less and so the damage is not significant, pearl millet differed greatly among various
otherwise the damage could be more. treatments and as such maximum numbers of
Bird pests at earhead stage and assessment of undamaged earheads were harvested from plot
loss covered with nylon nets during both the seasons
Observations for the bird fauna infesting (Table 3 & 4), it was however, higher in summer
pearl millet crop, nature and extent of damage season(964/plot) than kharif season(619/plot).
by the bird pests were recorded in the experimental Whereas, minimum undamaged earheads were
Table 1: Percentage of damage to pearl millet seedling by bird pests in summer 2005-2006
No. of rows Av. No. of Total No. Damage to
Plot size No. of seeds
Plot No. Variety in each seeds in of seeds in germinating
in damaged
plot each row each plot seeds(%)
1. 6.00m X 4.5m Usha-23 10 62 620 58 9.35
2. 6.00m X 4.5m Usha-23 10 61 610 50 8.20
3. 6.00m X 4.5m Usha-23 10 60 600 45 7.50
Mean 10 61 610 51 8.35
Table 2: Percentage of damage to pearl millet seedling by bird pests in kharif 2005-2006
No. of rows Av. No. of Total No. No. of Damage to
Plot No. Plot size in Variety in each seeds in of seeds in seeds dam- germinating
plot each rows each plot aged seeds(%)
1. Pioneer 10 62 620 55 8.87
4.00 X
2. Pioneer 10 63 630 27 4.28
4.5m
3. Pioneer 10 58 580 29 5.00
Mean 10 61 610 37 6.05

Patel.,2011
Table 3: Assessment of damage to pearl millet ear head stage at the harvesting stage during summer 2005-06
Total No. No. of No. of ear heads Ear head damage
Treatment Area of ear healthy ear damaged by (%) by
heads heads Bird Insect Bird Insect
1. Manual bird
6.00m X 4.5m 1144 1041 80 23 6.99 2.01
scaring
2. Enclosed
6.00m X 4.5m 1071 964 00 107 0.00 9.99
in nylon net
3. Control
6.00m X 4.5m 1126 956 135 34 11.99 3.02
(Unprotected)
Mean 1113.67 987 71.67 54.67 6.33 5.01
Table 4: Assessment of insect and bird damage to pearl millet ear heads at the harvesting Kharif 2005-06
Total No. No. of No. of ear heads Ear head damage(%)

Treatment Area of ear healthy damaged by by
heads ear heads
Bird Insect Bird Insect
1. Manual bird
6.00m X 4.5m 618 526 43 49 6.96 7.93
scaring
2. Enclosed
6.00m X 4.5m 688 619 00 69 00.00 11.14
in nylon net
3. Control
6.00m X 4.5m 550 429 99 22 18.00 5.13
(Unprotected)
Mean 618.67 524.67 47.33 46.67 8.32 8.10
found in plots exposed without bird scaring in employed in terms of loud human shouting in
summer season(956/plot) which in turn was higher treatment one. In treatment two, limited area ( 6m
than that found in kharif season (429/plot). X 4.5m) of the crop was enclosed with nylon net to
The percentage of earheads damaged due prevent bird damage to the earheads, in order to
to birds differed greatly and as such the assess the yield under fully protected condition.
earheads were completely free from bird In treatment 3, the crop was totally unprotected i.e.
damage in the plots covered by nylon net. without nylon netting and bird scaring as well.
Maximum bird damage was found in earheads from Assessment of avoidable losses in yield
the plots exposed without bird scaring (11.99 %) in of pearl millet grains was calculated in terms
summer season(Table 3). Similar experiment was of kilogram per hectare as well as percentage
conducted in kharif 2005-2006. The percentage shown table 5 and 6.
of damage to the pearl millet earheads was 18.00 As per the observation recorded Rose
per cent as shown in table 4. During this period, ringed Parakeet and House Crows are the bird
large area surrounding the experimental plot pests which cause damage to the pearl millet
was under cultivation with crops like pearl crop at the milky and dough stage of the crop.
millet, sorghum, pulses and cotton. The bird They damage the earhead by cutting and
pests were observed visiting and damaging the removing the sheath (husk), thus exposing the
above mentioned crops, with the results that the grains. These birds infest the crop during morning
percentage of damage was reduced in the hours i.e. from 6.00 to 10.00 a.m. and again in the
experimental plot. evening from 4.00 to 7.00 p.m. They either visit
In summer and kharif (2005-2006) pearl the crop singly or in groups of 10 to 25.
millet experiment was conducted to study the Perusal of results (Table 5) revealed that the
yield loss. For this purpose, the crop was highest grain yield of pearl millet was registered in
raised on an area of one acre and the trial the crop protected by nylon net (2837.03 kg/ha)
consisted of three treatments, viz. crop exposed with whereas, minimum grain yield was recorded in
bird scaring, crop enclosed with nylon net but unprotected plots which suffered heavy bird
without bird scaring . The manual bird scaring was depredation (2390.73 kg/ha) during summer, 2005-
Patel.,2011
Table 5: Assessment of loss in yield of pearl millet (cv.Usha-23) grains by bird pests during summer 2005-06
Grain yield(kg) Avoidable loss in grain
Treatment Area in
Per Plot Per ha yield (%)
1. Manual bird
6m X 4.5m 6.980 2585.18 8.87
scaring
2. Enclosed in
6m X 4.5m 7.660 2837.03 0.00
nylon net
3. Control
6m X 4.5m 6.455 2390.73 15.73
(Unprotected)
Table 6: Assessment of loss in yield of pearl millet (cv. Pioneer) grains by bird pests during kharif 2005-06
Grain yield(kg) Avoidable loss in grain
Treatment Area in
Per Plot Per Plot yield (%)
1. Open and with 3.45
6m X 4.5m 1277.77 24.17
bird scaring
2. Enclosed in
6m X 4.5m 4.55 1685.18 00.00
nylon net
3. Control
6m X 4.5m 2.88 1066.66 36.70
(Unprotected)
06. Manual bird scaring provided some protection Beri YP, Jotwani MG, Mehra SS and Chander
against bird damage and as such the grain yield was D. 1968. Studies on relative bird damage to
some what higher (2585.18 kg/ha) than that of different experiment hybrids of bajara, Indian J.
unprotected plots. The avoidable loss was minimum ENT. 31(1):69-71.
(8.87%) in plots protected against birds by manual
scaring, whereas, it was maximum in unprotected Bhalodia, Ketan, Shukla, Madhavi and Soni VC.
plots (15.73). 1997. Evaluation of Beneficial And Harmful Role
More or less similar trend of bird damage Of Birds In Agroecosystem Of Morzar Village Of
was observed during kharif 2005-06 in pearl millet Jamnagar District,PAVO 35:1-5
(Table 6). The highest grain yield of pearl millet
was registered in the crop protected by nylon net Dhindsa MS, Toor HS and Sandhu PS. 1984.
(1685.18 kg/ha), whereas, minimum grain yield was Community structure of birds damaging Pearl
recorded in unprotected plots which suffered heavy Millet and Sorghum and estimation of grain loss.
bird depredation (1066.66 kg/ha) during summer, Indian J. Ecol., 11(1):154-159.
2005-06. Manual bird scaring resulted in some
protection against bird damage and as such the Mathew KL, Parasharya BM, Dodia JF and
grain yield was some what higher (1277.77 kg/ha) Yadav DN. 1998. Environmental features
than that of unprotected plots. The avoidable loss associated with bird damage to cereal crops, Society
was minimum (24.17%) in plots protected against for applied ornithology, Hyderabad 21-29.
birds by manual scaring, whereas, it was maximum
in unprotected plots (36.70%). Mehrotra KN and BhatnagarRK. 1979. Status of
economic ornithplogy in India(New Delhi, India:
REFERENCES ICAR).
Ali S and Ripley SD. 1983. A pictorial Guide to
the birds of Indian Subcontinent.BNHS, Oxford
University press, Bombay.
Ali S and Ripley SD. 1987. compact handbook of

the birds of india and Pakistan, BNHS, oxford
University press, Delhi.

Publication group
Enumeration of medicinal plants along kanher and mahadare reservoir

from Satara District, Maharashtra, India.
Authors: ABSTRACT:
Pawar SM and
*Sonawane SR.
Institution:
Department of Zoology,
Padmabhushan Dr. The present study deals with traditional knowledge of medicinal plants
Vasantraodada Patil recorded along the two reservoirs. Local people are using these plants for treatment
Mahavidyalaya, of various diseases to cure it. The study records 56 different medicinal plants.
Tasgaon. Dist.: Sangli.
416 312 (India).
*Department of Zoology,
Dr. Babasaheb Ambedkar
Marathwada University,
Aurangabad. - 431 004,
Maharashtra, India.

Sonawane Pawar SM and Sonawane SR.
Enumeration of medicinal plants along kanher and mahadare reservoir from Satara
District, Maharashtra, India.
Web Address: Dates:

http://jresearchbiology.com/ Received: 21 Sep 2011 /Accepted: 28 Sep 2011 /Published: 24 Oct 2011
Ficus Publishers.
cited.
461-466 | JRB | 2011 | Vol 1 | No 6

Pawar et al.,2011
INTRODUCTION office Satara, (Kanher).

India is one of the richest floristic regions of Mahadare reservoir
the world and has been a source of plants and their It is also a natural reservoir, situated in
products. The forest provides major as well as Mahadare village. It is about 8 km away from city.
minor products of commercial importance to the Late Honorable Chh. Pratapsingh Maharaj
local inhabitants. Forest area is of natural type constructed this tank at the foot of Yawateshwar for
consisting trees, shrubs, herbs, aquatic plants. In supplying water to Satara city. Location of reservoir
India almost all parallel systems of medicine like sampling sites S1, S2, S3 is on latitude 1740" 58
ayurveda, unani, allopathy and homeopathy are 43"N and longitude 7358" 2292"E. (Google
used by large section of society mainly in rural and Earth, 2009). Length of this reservoir is 260 feet,
remote areas. The value of medicinal plants to width is 257 feet and depth is 30 feet. This reservoir
human livelihood is essentially infinite. They is constructed by stones. To the south of this tank,
obviously make fundamental contributions to there is a percolation tank called Hatti Talab. The
human is based on knowledge of plants used by water is accumulated from the Yawateshwar hills
human is based on thousands of years experience. into the reservoir. Daily 5000 gallons of water from
Plant used in traditional medicine may constitute an this reservoir is supplied to some part of the city.
important source of new biologically active The main purpose of reservoir is to supply water for
compounds. Utilization of plants for medicinal drinking, domestic purpose and irrigation. These
purposes in India has been documented long back reservoirs store rainwater received from adjoining
in ancient literature. Dradhbala et al (1996). catchments areas through small channels.
Human beings use them in different ways according
to their needs, particularly as food and medicines. MATERIAL AND METHODS
Indigenous medicinal plants as only alternative to The areas were visited regularly at an
antibiotic are said to play a significant role here. interval of one month from June 2008 to June 2010,
Among the entire flora more than 43% of total for collection and field observation of plant which
flowering plants are used for medicinal purpose. has medicinal importance. The survey consisting
Pushpangdanp (1995). The aim of present study is ten different sites of reservoir. Information was
to know the knowledge & distribution of medicinal gathered with the help of some proper knowledge
plants around these reservoirs. of informants, elderly people and local inhabitants.
Study area- A questionnaire was prepared to generate data for
The study area is confined to Sahayadri this purpose. All efforts were made to determine the
ranges of Maharashtra includes Kanher dam and species properly with correct nomenclature by
Mahadare reservoir of Satara district. following standard flora & ethnobotanical method.
Kanher Dam The required material is collected; herbaria of these
It is a medium irrigation project plants were maintained in the laboratory of
constructed by Irrigation Department, Government department. They were identified with the help of
of Maharashtra on Venna river near Kanher. It is renowed floras; Cooke (1901-1903), Jain (1973),
situated in Northwest of Medha Tahsil of Satara Mnimh (1996), Singh et al., (2001) & Bhattacharjee
district. (M. S.). Location of sampling sites S1, S2, (2002) and enumeration were compared with earlier
S3 is on latitude 1744" 1602"N and longitude 73 published literature. Ambasta, (1986) and Jain
53" 4310" E.( Google Earth, 2009). It is about 15 (1991), Heinrich (2000) and Bhagat et al., (2008).
km away from the Satara city. The construction of
Kanher dam began in 1976 and completed in 1988. RESULT AND DISCUSSION
It is 1955 mts. in length and 50.30 mts. in height. Similar type of work has been reported by
The dam has a capacity to store 286 million cubic no. of workers. Yoirentomba Meetei and Singh
mt. of water. The main canal runs 1.68 kms on left, (2007) recorded 160 medicinal plants from Manipur
divided into right, and left the former runs for district. Ranu Lahri (2007) studied 154 species of
58km. covers 10070 hectare land and the latter runs medicinal plant in Sanjay National park of Sidhi
for 21 kms. covering 1605 hectares land. district (M.P.). Sharma et al (2007) listed 25
The main purpose of reservoir is to supply medicinal plants from Balaghat district (M.P.).
water for drinking, domestic purpose and irrigation Thakur and Patil (2010) found 35 medicinally
as well as fishing practices (culture and capture important plants in Nashik district of Maharashtra.
fishery) are carried out under fishery development Sharma et al, (2010) recorded 24 medicinal plants
Pawar et al.,2011
remote areas of Chhindwara district of (M.P.). Trust, India. New Delhi. 127-140.
Uikely et al (2010) listed 35 medicinal plants from
Balaghat district (M.P.). The present investigation Mnimh AC. 1996. The encyclopedia of medicinal
comprises 56 plant species of enthomedicinal plants. Dorling Kindersely Ltd., London.
importance distributed in 42 genera and 19 families.
Some important medicinal plants of the district Pushpangdan P. 1995. Ethno biology of India. A
were enlisted alphabetically, with their botanical Status Report a Govt. of India. New Delhi. India.
name followed by family, local name and plant part
used with medicinal value, in Table .1. The study Ranu Lahiri. 2007. Entho medicinal Survey of
would be very much helpful to the future Sanjay National Park of District Sidhi (M.P.). India
researchers to bridge up the gap between the Journal of Flora and Fauna 16(II):289-299.
traditional knowledge of the people and scientific
research in the field. The tribal communities are Yoirentomba Meetei S and Singh PK. 2007. Survey
Ketkadi, Bhoi, Mahadev Koli, Pradhan, Tambat and for medicinal plants of Thoubal District, Manipur
Bhavsar resides in the hilly region of this area and Journal of Flora and Fauna 13(II):355-358.
local people are highly depend on these plants for
meeting their health care needs. Hence there is a Sairam TV. 1999. Home Remedies Vol- II, Penguin
need of conservation of biodiversity and sustainable Books, India 318-324.
use of plant resources. Several similar studies are
Sharma R, Kantishree DE and Upadhyay. 2007.
carried out in various regions of the country.
Ethnobotanical knowledge of plants used by rural
Sairam (1999), Yadav and Sardesai (2002), Warrier
community of Waraseoni block of Balaghat district.
et al (2010). (M.P.) India. Journal of Flora and Fauna 13(II):285-
288.
REFERENCES
Abasta SP. 1986. The useful plants of India, Sharma V, Diwan RK, Saxena RC and
Publication and Information Directorate, CSIR, Shrivastava PN. 2010. Ethno medicinal studies on
New Delhi, India. edible plant species used by Gond and Bhariatribes of
Chgindwara district of (M.P.) India. Journal of Flora
Bhagat RB, Shimpale VB and Deshmukh RB. and Fauna 16(1):213-216.
2008. Flora of Baramati. Pawar Public Charitable
Trust, Mumbai. Singh NP, Lakshiminarasimhan P, Karthikeyan S
and Prasanna PV. 2001. Flora of Maharashtra State.
Bhattacharjee SK. 2002. Handbook of Medicinal D i c o t y l e d e n e s - V o l . I I ( C o m b r e t a c e a e -
Plants. Ceratophyllaceae) B.S.I., Culcutta.1-1080.
Cooke T. 1901-03. The flora of presidency of Thakur HA and Patil DA. 2010. Wild edible plants
Bombay, Vol. I-III, London (B.S.I.Repr.ed.) of tribal of Nashik district, (M.S.) Journal of Flora and
Calcutta. Fauna 16(I):85-88.
Dradhbala C, Sastri K, Chaturved GN, Sastri R, Uikely SK, Yadav AS, Sharma AK, Rai AK,
Raghuwanshi DK and Badkhane Yogesh. 2010.
Upadhaya Y, Pandeya GS, Gupta B and Mishra
Traditional treatment of various skin diseases in
B. 1996. The Charak Samitha 22nd Revised Edn.
Balaghat district of (M.P.) India. Journal of Flora and
Chaukhamba Bharti Academy Varanasi. Fauna 16(II):217-224.
Heinrich M. 2000. Ethno botany & its role in drug Warrier PK, Nambiar VPK and Ramamabkutty
development. Phototherapy. Res., 14:479-488. C. 2010. (Eds) Indian Medicinal Plants. Vol. I-V,
Universities Press, Hyderabad.
Jain SK. 1991. Dictionary of India Folk Medicine
& Ethnobotany. Deep Publication Paschim Vihar. Yadav SR and Sardesai MM. 2002. Flora of
New Delhi. Kolhapur District. Shivaji University Publication,
Kolhapur.
Jain SK. 1973. Medicinal Plants National Book

Pawar et al.,2011
Table. 1. Medicinal Plants along Kanher and Mahadare reservoirs

Sr. LOCAL
SCIENTIC NAME FAMILY MEDICINAL VALUE
No. NAME
Bark Cleansing property protects skin
Acacia caesia Linn.
1. Wild.
Mimosaceae Babhul against micro-organisms.
Flowers- in menstrual disorders.
Acacia catechu (L.f.) Bark - useful in conjunctivitis;
2. Willd.
Mimosaceae Khair
Heart wood - high medicinal value.
Bark -astringent, anthelmintic, diuretic,
nutritive, used in Pitta, chronic
3. Acacia indica Benth. Mimosaceae ------
dysentery & skin diseases.
Gum- sweet, liver tonic, cough.
Bark - astringent, anathematic, diuretic,
and nutritive used in kapha and pitta,
Acacia nilotica (L.)
4. Willd.ex.Del.
Mimosaceae Babhul chronic dysentery, skin diseases.
Gum sweet, liver tonic, cough,
asthma.
Root, Bark, leaves & fruits - used in
Aegle
5. marmelos Corr.
Rutaceae Bel intestinal disorders,
Leaves -used for treatment of diabetes.
Aeschynomene aspera Shoot- pneumonia, Menstrual disorder,
6. L.
Fabaceae Khujumpere
Leaves- Pains, swellings.
Whole plant- Healing of skin wounds,
sore, rashes, onjunctivitiss, wrinkles,
7. Aloe vera (L.) Burm.f. Liliaceae Korphad
from aging, prevent infection in HIV,
AIDS.
Sahadevi/ Leaves- used as blood coagulant to heel
8. Ageratum conyzoides L. Asteraceae
Osadi the wound, leprosy & skin diseases.
Ammania baccifera Kanjali/ Leaves are appetizer, laxative and
9. subsp. baccifera Cl.
Lythraceae
Dadmari aphrodisiac.
Argemone Mexicana L. Stem latex used in dropsy, jaundice,
10. Papaveraceae Pivala Dhotra
healing of wounds.
Asparagus racemosus
Leaves and tubers- used against skin
11. Willd. Liliaceae Shatavari
disease.
Var. javanica
Tender shoot -cures toothache, gum
Limb/
12. Azadirachta indica Juss. Meliaceae
Kadulimb
diseases,
Leaves- are used as insect repellant.
Whole plant- astringent, bitter used in
Bacopa monnieri (L.) digestive, anti inflammatory, cardio
13. Penn.
Scrophulariaceae Nir Brahmi
tonic, epilepsy, leprosy, elephantiasis,
fever and general debility condition.
Root -decoction used to cure fever.
Butea monosperma
14. (Lam.)Taub
Fabaceae Palas Seed- in constipation & skin diseases.
Gum- in diarrhea & dysentery.
Whole plant used against diarrhorea,
15. Butea superba Roxb. Fabaceae Palas vel dysentery, intestinal worm, diabetes,
leucorrhoea, & retention of urine.
Amaltaas/ Fruits-Pharmaceutical codex,
16. Cassia fistula L. Caesalpiniaceae
Bahava Pulp- medicinal value.
17. Cassia occidentalis L. Caesalpiniaceae Kaswinda In Cough, asthmas, snake bite, scabies.
Cassia pumila L.
Plant is sedative and used in removal of
18. Caesalpiniaceae Rantakkala
intestinal worms.

Pawar et al.,2011
Decoctions of parts- used to improve

vision, analgesic, antifungal and against
19. Cassia tora L. Caesalpiniaceae Takala
skin diseases, obesity, ring worm,
hypertension.
Coccinea grandis (L.)
20. Voight
Cucurbitaceae Tondali Fruit -used in Diabetes.
Roots- astringent and constipating;
useful in diarrhoea and dysentery,
Dalbergia latifolia Shisav/ Leaves- gonorrhea, menorrhagia,
21. Roxb.
Fabaceae
Shisam dyspepsia, colic, vomiting,
haemorrhoids, burning sensation,
diarrhoea and dysentery.
Roots astringent & constipating
L eaves digestive, stimulant
Dalbergia sisso
22. ex.DC.
Fabaceae Sissu and used in diarrhorea & burning
Bark and heartwood skin diseases,
scabies, bronchitis
Whole plant useful in asthama, cough,
23. Datura metel L. Solanaceae Kala Dhotra
fever, ulcer, skin diseases.
White Whole plant is used in hyascyamine,
24. Datura stramonium L. Solanaceae
Dhattura bronchitis, as antispasmodic & narcotic.
Dendrophthoe falcata Bark- astringent, in wounds and
25. L.f.
Loranthaceae Banda
Menstrual cycle
Whole plant - used in pitta, cough,
Desmodium triflorum
26. (L.)DC
Fabaceae Ran Methi bronchitis, wounds, sores & burning
sensation.
Whole plant - used in intestinal worm,
27. Eclipta prostrata (L.) L. Asteraceae Maka
leprosy, fever, hypertension.
Emilia sonchifolia (L.) Whole Plant- used in gastropathy,
28. DC.
Asteraceae Sadmandi
diarrhoea, opthalmia, wounds & asthma.
Whole plant- in gastropathy, bronchitis,
asthma, inflammations, splenomegaly,
Euphorbia cutaneous diseases, dropsy, dyspepsia,
29. ligularia Roxb.
Euphorbiaceae Shend
flatulence, intermitant fever, jaundice,
leprosy rheumatism and ulcers.
Milky juice- in otalgia and opthalmia.
Entire plant- used in relaxation of
bronchioles, asthma, cough, gonorrhea
30. Euphorbia hirta L. Euphorbiaceae Dudhani
and urinogenital complaints and on
warts
Whole plant used in bronchitis, asthma,
Evolvulus alsinoides (L.) Shankhapushp
31. L
Convolvulaceae
i/ Vishnukrant
internal hemorrhage, falling & graying
of hair &general debility.
Roots- in inflammation, ascites, vesicle
calculi, Jaundice,flatulence, dysentery,
and vitiated conditions of vata.Leaves
i n j au nd ice ,d ro p s y,r he u ma t is m,
Hygrophila schulli Kolshinda/ urinogenital disorders, cough,arthritis.
32. (Buch.-Ham.)
Acanthaceae
Talimkhana Seeds- in gonrrhoea,promoting sexual
vigour and strength, arresting
abortion,burning sensation
anaemia,diarrhea,dysentery, gout
rheumatism, and general debility.
Useful in cardiac diseases, coronary
Indigofera
33. linnaei Ali.
Fabaceae Shuiguli artery diseases, coma, dizziness &
kidney inflammation.
Pawar et al.,2011
Ipomoea Root Snake and dog bite,
34. pes-tigridis L.
Convolvulaceae Besharam
Leaves- pimples, sores
Ipomoea carnea Jacq.
35. Subsp.fistulosa (Mart. Ex Convolvulaceae Besharam Used as purgative, in wound and dog bite.
Choisy.)Austin
Dried seeds used as a purgative drug. Fresh
36. Ipomoea nil (L.)Roth. Convolvulaceae Besharam
fruit are eaten as vegetables.
37. Ipomoea aquatica Forssk. Convolvulaceae Besharam Forks as laxative or purgative.
Bark is used in body swelling, cuts, wounds,
Lannea cormandelica Moya,Moi,
38. (Houtt.) Merr.
Anacardiaceae
Shimati
ulcers, & dysentery. Leaves useful in
elephantiasis.
Lantata camara L. Root powder used for stomachache, colic
39. var.aculeata
Verbenaceae Ghaneri
pain.
Leaf decoction is used in sore throat & seeds
40. Lawsonia inermis L. Lythraceae Mehandi
in urinary trouble.
Limnophila Plant -used in galactic, ulcers, constipation &
41. Aromatic (Lam.) Merr.
Schrophulariaceae
fever.
Root and Barks-used in wounds, ulcers,
42. Mangifera indica L. Anacardiaceae Aamba vomiting, diarrhorea and rheumatism. Raw
fruits- are boiled and used as cooling agent.
Leaves -used in wound cuts, diarrhorea,

43. Mentha arvensis Linn. Lamiaceae --------
asthma, dental caries, and general weakness.
Root- vata, pitta, constipation, ulcers and
Mucuna pruriens
44. (Linn) DC.
Fabaceae Khaj kuhiri delirium. Leaves- tonic, anathematic. Seeds-
sterility, gonorrhoea.
Oxalis corniculata L. Leaf extract used in malaria, jaundice.
45. Var. corniculata Edg. Oxalidaceae Ambushi Leaves & root are used in dysentery &
&Hook.f. diarrhorea.
Oxalis deilis H.B. & Cooling effect, Beneficial made in ointment
46. K.Var.corymbosa (DC.)
Oxalidaceae Ambushi
for cuts, scrapes rashes & skin diseases.
Plant - insecticidal & veterinary medicinal

Pongamia pinnata (L.) important.
47. Pierre
Fabaceae Karanj
Leaves- used as antifungal & antibacterial.
Seed oil kills insects.
Tectona
48. grandis L.f.
Verbenaceae Saag/Sagwan Plant oil- ring worm, eczema &itches
Tamarindus Fruits and leaves - used as carminative,
49. indica L.
Caesalpiniaceae Chinch
digestive, laxative and astringent.
Whole plant- antiseptic, anti-inflammatory
Sesbania and antimicrobial properties. Used in
50. seban L. (Merr.)
Fabaceae Shevari
ointment to cure itching, and various skin
eruptions.
Syzygium
51. cumini L. Skeels
Myrtaceae Jambhul The seeds are used in diabetes.
Vitex negundo L.
52. Verbanaceae Nirgudi Leaves- Medicinal importance
Withania Whole plant- rheumatism, weakness,
53. somnifera (L.)Dunal
Solanaceae Ashwagandha
antibacterial activity
Woodfordia fruticosa
54. (L.) Kurz.
Lythraceae Dhaiti / Dhayati Flowers- juice is used to control dysentery.
Bark, leaves and seeds- used as astringent,
Zanthoxylum rhetsa Chirphal/ digestive, antiseptic, and disinfective, in
55. (Roxb.) DC.
Rutaceae
Tirphal kapha, asthma, pyorrhea and traumatic eye
injury.
Whole plant used in fever, tonic,
56. Ziziphus mauritiana Lam. Rhamnaceae Bor purgative, appetizer, astringent, dysentery &
diarrhorea.

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