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DNA BARCODING OF FRESHWATER EPHEMEROPTERANS AND

ODONATES IMPORTANT IN WATER QUALITY ASSESSMENT OF

TWO INDIAN RIVERS

Project Proposal Submitted toCentral Pollution Control Board, New Delhi (CPCB)

(Revised as per reviewers comments)

Principal Investigator : Dr. Linu Mathew

Co- Investigators : 1 Dr. J. G. Ray

2 Dr. Pratima Akolkar

.


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CENTRAL POLLUTION CONTROL BOARD, NEW DELHI (CPCB)

APPLICATION FOR GRANT FOR RESEARCH PROJECT

(To be completed by the Principal investigator)

1. Title of the Project

DNA Barcoding of Freshwater Ephemeropterans and Odonates Important In Water Quality Assessment of Two Indian Rivers

2. Name and Designation of the

Principal-Investigator : Dr. Linu Mathew

3. Postal Address of the Principal

Investigator : Assistant Professor in BiotechnologySchool of Biosciences

Mahatma Gandhi University

PD Hills (PO), Kottayam

E mail: [email protected]

4. Name of the institution/organization

in which the project will be carried out : School of Biosciences,Mahatma Gandhi

University, PD Hills (PO), Kottayam

5. Name of other institution(s)/

Organization (s) involved in the project : Central Pollution Control Board, New Delhi.

6. Duration of the project : 2 Years

7. Total amount of assistance required : 20, 00000(Twenty lakhs only)


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DNA BARCODING OF FRESHWATER EPHEMEROPTERANS AND

ODONATES IMPORTANT IN WATER QUALITY ASSESSMENT OF

TWO INDIAN RIVERS

Statement- 1

ABSTRACT

Ephemeroptera (May flies) and Odonates (dragon flies) are sensitive indicators, very

important to the assessment of water quality. Mayflies and dragon flies typically are indicators

of clean water and a healthy environment, because most species are relatively intolerant to

pollution. River Ganges and River Pampa are two holy rivers of India, in the north and south

respectively. Both the holy rivers are subjected to high pollution load during the pilgrimage

seasons and also from urban and agricultural sources throughout the year. Keeping these

under consideration, a comparative study based on the ecology and taxonomy of

Ephemeropterans and Odonates of the two famous rivers of India (The Pampa and the

Ganges) will be carried out to assess the water quality status based on these well-known water

quality indicator organisms. Classical taxonomical approaches as well as DNA barcoding

technique will be used for species identification and ultimately the identification of insects

using the modern method would be applied to easy interpretation of water quality assessment

of all rivers of India subjected to pollution . The technique of deoxyribonucleic acid (DNA)

barcoding, for identification of species by means of sequencing a short region of DNA

normally the Cytochrome C oxidase I gene (COI)was first described and tested by Hebert et

al. (2003). This illustrates the methodical creation of a library of DNA barcodes that will link

newly collected specimens to a reference library of authoritatively identified specimens. This

will eventually provide the exact knowledge of species diversity, species composition of

unknown faunas, accurate (i.e. avoiding mis-identification) and consistent identification oftaxons (level of taxonomic identification e.g. family/genus/species thereby shedding light on

identification and revealing species not currently described in larval taxonomic keys. This

project incorporates two methods linked together: biomonitoring of Ephemeropterans

important in water quality assessment and DNA barcoding, in an effort to gain a greater

understanding of how traditional taxonomic approaches compare to species identification by


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DNA barcoding and how this may affect interpretation of water quality. In this study a small

sequence of DNA containing an approximately 700 base pair regions of the Mitochondrial

Cytochrome C oxidase I gene (COI) should be isolated from the benthic samples since most

eukaryotic cells containmitochondria,and mitochondrial DNA (mtDNA) has a relatively fast

mutation rate, which results in significant variation in mtDNA sequences between species

and, in principle, a comparatively small variance will exist within species. This avoids the

time-consuming and expensive task of sequencing the entire genome of the animal and

consequently reducing the cost. The basic methodology involved are Collection and

Identification of Ephemeropteran and Odonate larvae from two Indian rivers noted for its

religious and sociological importance namely river Pampa of Kerala and river Ganges,

Specimen Preservation, DNA Isolation from the preserved samples, Polymerase Chain

Reaction (PCR) and DNA Sequencing of the COI gene, DNA Sequencing, Sequence Editing

and Alignment of the COI gene and identification of the Species. Parallel to the collection of

these organisms, field analysis of certain important water quality parameters of respective

sample waters such as, temperature, conductivity, salinity, total solids (TS) total dissolved

solids (TDS), dissolved oxygen (DO), dissolved CO2, dissolved chlorides, fluorides, hardness

and transparency using a Secchi disc also would be carried out to assess the ecology of these

specie
http://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Mitochondriahttp://en.wikipedia.org/wiki/Mutationhttp://en.wikipedia.org/wiki/Mutationhttp://en.wikipedia.org/wiki/Mitochondriahttp://en.wikipedia.org/wiki/Eukaryote


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Statement II

INTRODUCTION

The technique of deoxyribonucleic acid (DNA) barcoding, whereby species areidentified by means of sequencing a short region of DNAnormally the Mitochondrial

Cytochrome C oxidase I gene (COI)was first described and tested by Hebert et al. (2003).

In recent years particularly with the introduction of the concept of DNA barcoding in 2003

efforts have been directed towards building a standard sequence library for all eukaryotes by

focusing DNA sequencing efforts on small, species specific portions of the genome called

DNA barcodes (Marshall, 2005).

Back ground of the work: Over 1.9 M species have been formally described since

Linnaeus first started the task 250 years ago, yet it is estimated that 10100 M species exist on

Earth (Wilson, 1985; Chapman, 2009). Therefore, not only is our characterization of

biodiversity painstakingly slow, but the fact that there is order-of-magnitude uncertainty in

our best estimate for the totality of Earths biodiversity. Wilson, 1985 suggests that current

tools and techniques are inadequate for the task of accurate assessment. What is the species

composition of a particular ecosystem? How does biodiversity change over time, space,

and in relation to future environmental change? are both fundamental questions trying to

answer through biomonitoring programs, by employing biotic surveys to assess change in

threatened habitats.

Molecular genetic analysis can help resolve some of these issues. This approach has

been used in the past to distinguish morphologically cryptic species of macro invertebrates in

ecological and evolutionary studies, but these methods were expensive and time consuming

(e.g., Sweeney et al. 1987, Funk et al. 1988, Funk and Sweeney 1990, Sweeney and Funk

1991, Jackson and Resh 1992, 1998). Recent advances in direct sequencing of

deoxyribonucleic acid (DNA) make molecular methods more readily available to help resolve

taxonomic challenges presented by fauna in general (Hebert et al. 2003) and freshwater

macroinvertebrates in particular (Sharley et al. 2004, Pfenninger et al. 2007, Zhou et al. 2007,

2009, 2010, Sinclair and Gresens 2008, Stahls and Savolainen 2008, Krosch et al. 2009).

In this study a small sequence of DNA containing an approximately 700 base pair

regions of the Mitochondrial Cytochrome C oxidase I gene (COI) should be isolated from the


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benthic samples since most eukaryotic cells contain mitochondria, and mitochondrial DNA

(mtDNA) has a relatively fast mutation rate, which results in significant variation in mtDNA

sequences between species and, in principle, a comparatively small variance will exist within

species. Using the CO1 gene enables species level identification while eliminating the time-

consuming and expensive task of sequencing the entire genome of the animal and

consequently this significantly reduces the cost. In the past seven years, over 1.1 M

individuals from about 95,000 species have been added to the DNA barcode library

(Ratnasingam, 2007). Another major objective of the series is to illustrate the importance of

careful and methodical creation of a library of DNA barcodes that will link existing Linnaean

names and ecological information associated with them to barcode sequences. This library

building process needs to occur at local, regional, national, and international scales.

Janzen (2004) passionately argues for this technology as a way to promote his idea of

bioliteracy, which he describes as the ability to understand species diversity on a genetic

level to promote greater appreciation and conservation of the natural world. Barcoding has

already been utilized confirm or modify morphological species determinations for insects (i.e.

Astraptes fulgerator complex, Tachinid parasitiods of Costa Rica, and Phyciodes butterflies)

in the United States and in the tropical regions of 6 Costa Rica (Smith et al. 2007, Hajibabaei

et al. 2006, Wahlberg et al. 2003). DNA barcoding is quickly becoming an important

taxonomic tool by allowing taxonomists, ecologists and conservationists to catalog a variety

of species (insects, birds, etc). Spreading the concept of bioliteracy can promote greater

understanding of the habitats that desperately require conservation and restoration efforts.

Fresh water systems are one of those essential ecosystems because water is an obligatory

resource for human beings.

One very practical application of DNA barcoding is biological monitoring for

assessing water quality. Identification of benthic organisms for biomonitoring purposes

presents a significant cost to those programs, and, as shown by Sweeney at al. 2011, DNA

Barcoding has much to offer. It signaled new possibilities for identification of previously

unidentifiable material in benthic samples. Subsequently, the technique has been gaining

wider adoption by benthic scientists (Holzenthal et al.2010). Geraci et al. 2011 showed how

DNA-barcoding approaches could shed light on an unknown faunain this case the


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Trichoptera of Iraq, and how this method can be used to reveal hidden biodiversity in a poorly

studied region of the world.

A longstanding constraint on use of aquatic macroinvertebrates for environmental

assessments has been the difficulty of identifying them to species, especially in the larval

stages. This task is challenging for even the best taxonomists because species identification

keys for the larval stages of many aquatic macroinvertebrates are incomplete, unreliable, and

nonexistent in some cases (Gresens et al. 2007). This also makes it impossible to identify the

larvae of freshwater species, which are highly threatened taxonomic group and to potentially

shed light on their ecology.

In addition,microscopic indicator species because of their life stage, size, or condition

(i.e., damaged), not only prevents full access to the ecological and evolutionary information

they hold (e.g., physiological mechanisms of pollution tolerance), but also leads to errors and

imprecision in assessments of habitat and water quality (Lenat and Resh, 2001, Stribling et al.

2008, Buchwalter and Luoma, 2005, Buchwalter et al. 2008). To complicate matters, cryptic

species continue to be problematic for aquatic taxonomists (e.g., Funk et al.1988, Sweeney

and Funk, 1991, Sharley et al. 2004, Stahls and Savolainen, 2008, Alexander et al. 2009,

Krosch et al. 2009, Zhou et al. 2010).

The molecular approach can provide a finer resolution than traditional taxonomy for

evaluating environmental change associated with both natural and anthropogenic processes.

Last, Pilgrim et al.2011 illustrated that DNA barcoding is now firmly established as a key

tool for the application of benthic science in environmental protection and regulation and that

national regulatory organizations are taking steps to implement this new technology in their

programs to improve data quantity, quality, and immediacy.

As a consequence of the sensitivity of species to pollution and other disturbances

which alter their habitat, environmental agencies are increasingly choosing biomonitoring

approaches to assess ecosystem status and trends. However, accurate (i.e. avoiding mis-

identification) and consistent (level of taxonomic identification e.g. family/genus/species)

taxon identification has proved difficult to achieve using traditional morphological

approaches. This is particularly true for the large scale application of macroinvertebrates

sampling in river biomonitoring, where larval stages are often difficult or impossible to

identify below the level of taxonomic family.


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In particular, this technology is on its way to being mainstreamed as a fundamental

technique in support of both basic and applied benthic science. Moreover, DNA barcoding has

the potential to take the accuracy and precision of the taxonomy of benthic fauna to an

unprecedented level and to increase the overall level of information associated with key

water-quality metrics (e.g., taxon richness) throughout the world by at least 50%. These

breakthroughs would reverberate at all levels of benthic science, from small, tightly focused

benthic studies on one river or stream reach to large-scale national monitoring programs

involving thousands of streams and rivers.

Comprehensive and systematic analysis of the physico-chemical environmental

complex such as water temperature, Secchi depth, electric conductivity, pH, total dissolved

solids, total solids, total alkalinity, hardness, dissolved oxygen and carbon dioxide, dissolved

mineral ions such as Chlorine and Fluorine are significant to the assessment of the ecology of

a natural water body (Ray et al 2009). Therefore, parallel study of these water quality

parameters from the respective spots of collections of these organisms would enable us to

understand the ecological conditions of these insects as well as to assess their value as specific

ecological indicators to pollution status of natural water bodies.

STATE OF THE ART OF THE SUBJECT

In the past seven years, over 1.1 M individuals from about 95,000 species have been

added to the DNA barcode library. This number is not significant in the context of the 1.9 M

known and 10100 M estimated unknown species. Therefore, not only is our characterization

of biodiversity painstakingly slow, but also there is an order-of-magnitude uncertainty in our

best estimate for the totality of Earths biodiversity.

There were earlier studies in India contributing to assessing and monitoring the quality

of water using benthic macro invertebrate fauna, but very little is known about the possibility

of using the DNA barcoding technique for the assessment of water quality. Barcoding, when

combined with traditional aquatic macro invertebrate sampling, provides the most accurate

and cost effective method to determine the water quality of fresh water ecosystems. This

technique help us to gain a greater understanding of how traditional taxonomic approaches

compare to species identification by DNA barcoding and how this may affect interpretation of

water quality.


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The technique eventually provide the knowledge of species diversity, species

composition of unknown faunas, accurate (i.e. avoiding mis-identification) and consistent

(level of taxonomic identification e.g. family/genus/species) taxon identification thereby

shedding light on identification of larval stages and revealing species not currently described

in larval taxonomic keys.

NATIONAL STATUS

There is little available infrastructure for DNA barcoding with aquatic insects in

India and practically little research is happening any where on benthic macro invertebrates.

There is very little understanding by scientists and government officials of the values of this

technology for monitoring water pollution. The little that is known about the species of Indian

aquatic insects and other freshwater macro invertebrates has been acquired mostly by foreign

scientists so that there are no indigenous experts for most of the groups of freshwater insects

and macro invertebrates, and few young scientists are being trained in this field. In Kerala the

diversity of aquatic insects are very high due to low pollution and other suitable

environmental condition for their growth. Since the human population is in an edge of

explosion and other anthropogenic activities are increasing, there is a high possibility of

destruction to the Rivers and thereby leading to the decrease in the diversity of aquatic insects.

Since there is a technical difficulty in long term storage of the intact tissue as such in

preservative medium it is high time that we should maintain a permanent insect library for the

future. So along with the taxonomic identification of benthic macro invertebrates the

technique of DNA barcoding should also be implemented for the most accurate, cost effective

and future storage method to determine the conservation of the insect community before they

get extinct from the face of the Earth.

The only baseline data of Benthic macro invertebrates in India is confined to

classical taxonomical approaches and the phylogenetic characterization (DNA Barcoding) of

benthic macro invertebrates in India is in an initial stage. Since we hardly have any base line

data for Benthic macro invertebrates in India, the project aims to create a new baseline data

(Phylogenetic characterization) of the benthic macro invertebrates of the two rivers of India

based on already available DNA sequence base data information (Barcode of Life databases,

NCBI etc) in support of International studies.


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INTERNATIONAL STATUS

Protocols for using aquatic insects and macro invertebrates to monitor water quality

have been published and implemented in many developed countries, including Australia

(Australian River Assessment System, 2005),Canada(Rosenberg et al. 2005), the European

Union (European Union Water Framework Directive, 2000),New Zealand (Stark et al. 2001),

United Kingdom (RIVPACS, 2005) and the United States (Barbour etal.1999). Progress has

also been toward developing and implementing regionally appropriate protocols in several

Asian countries including China, Japan, Korea, Malaysia, Mongolia, Thailand and Russia

(Morse et al. 2007). The developed countries have an advantage for using this technology

because the world experts for identifying freshwater organisms have lived in these countries

and have studied their respective freshwater biota for many decades.

DNA barcoding technique has become so successful in the area of biodiversity

discovery that it has generated what is currently one of the largest global biodiversity projects

to date (International Barcode of Life project (iBOL, http://www.ibolproject.org/).

In May 2009, a special session titled Environmental Barcoding: Genomic Solutions

for Biomonitoring was held at the North American Benthological Societys (NABS) 57th

annual meeting in Grand Rapids, Michigan. The purpose of this session was to: 1) facilitate an

exchange of ideas between benthic scientists working in the application of DNA barcoding,

and 2) promote wider understanding among other conference attendees of the latest barcoding

technology, especially the improved robustness and accessibility of the technique.

The success of the special session in 2009 was so great that a follow-up interactive

session, titled Barcoding the Benthos: New Vistas in Laboratory, Museum and Field

Science, was held at the NABS meeting in Santa Fe, New Mexico, in June 2010 and was

met with excellent participation from the NABS member community.

The following Universities are involved in the DNA Barcoding techniques for Benthic

macro invertebrates, Environment Canada at Canadian Rivers Institute, University of New

Brunswick, Canada; Stroud Water Research Center, Pennsylvania, USA; University of

Pennsylvania, USA; Biodiversity Institute of Ontario, Canada; Department of Biology, USA;

Illinois Natural History Survey, USA and Center for Theoretical and Applied Genetics, and

Institute of Marine and Coastal Science, Rutgers University, New Brunswick, New Jersey.


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The present study does not take into consideration of life cycle analysis. Only the

aquatic stages are considered for water quality assessment using Biological Water Quality

Criteria developed by CPCB (Table 1). Unlike fish, benthic macro-invertebrates cannot move

around much so they are less able to escape from the impacts of sediment and other pollutants

that diminish the water quality. Therefore, benthic macro-invertebrates can give us reliable

information on river water quality compared to other aquatic fauna (Table 1).

Among all the fresh water invertebrates, only Arthropods, annelids, Molluscs and

Platihelminthes are considered for Biological Water Quality assessment wherein taxonomic

identification is required up to family/genus/species level. For example, Nymphal stage of

Ephemeroptera, Plecoptera and Odonata, Larval stage of Trichoptera, Diptera and adult stage

of Hemiptera, Coleoptera, Molluscs, Annelida and Platihelminthes are living in water bodies.

However, insects are confined in River Ganga mostly to the shallow reach upto Allahabado

whereas, the deeper reaches from Banaras downstream, population of benthic macro-

invertebrates are dominated by Crustaceans, Molluscs, Annelids and Platyhelminthes. In

view of covering the entire length of River Ganga, all the higher forms of benthic macro-

invertebrates may be included in the study.

Table 1Biological Water Quality Criteria (BWQC)

Sl.No Taxonomic Groups Range ofsaprobicscore

(BMWP)

Range ofdiversityScore

Water qualitycharacteristic Waterqualityclass

IndicatorColour

1 Ephemeroptera,

Plecoptera, Trichoptera,Hemiptera, Diptera

7 and more 0.2 - 1 Clean A Blue

2 Ephemeroptera,

Plecoptera, Trichoptera,Hemiptera, Planaria,

Odonata, Diptera

67 0.5 - 1 Slight Pollution BLightblue

3 Ephemeroptera,Plecoptera, Trichoptera,

Hemiptera, Odonata,

Crustacea, Mollusca,Polychaeta, Diptera

Hirudinea, Oligochaeta

36 0.3 - 0.9Moderate

PollutionC Green


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4 Mollusca, Hemiptera,

Coleoptera, Diptera,Oligochaeta

25 0.4 &Less

Heavy Pollution D Orange

5 Diptera, OligochaetaNo animals 02 0 - 0.2 Severe Pollution E Red

OBJECTIVES

1. Collection of Ephemeropteran and Odonate larvae from river Pampa of Kerala and

Ganges and identification by conventional taxonomy.

2. Account the important field water quality parameters to assess the ecology of these

species and also to understand the value of each of them as ecological indicators of

pollution status of natural waters

3. Finding out the species composition of a particular riverine ecosystem and how does

the biodiversity change over time, space, and in relation to future environmental

changes.

4. To adopt DNA-barcoding approaches to shed light on unknown faunas and how this

method can be used to reveal hidden biodiversity in a poorly studied region.

REVIEW OF LITERATURE

The combined morphological and molecular approach provides a finer resolution for

evaluating environmental change associated with both natural and anthropogenic processes

was studied by Bernard et al. 2011. Macro invertebrates were used to assess community

structure and water quality in White Clay Creek, Pennsylvania, USA. A success rate of 98%

were obtained, of the 1617 specimens used for analysis, including small, larvae , and damaged

specimens, were successfully barcoded (sequenced) for the Mitochondrial Cytochrome c

oxidase subunit I gene. A criterion of 2 to 4% genetic divergence provided good separation of

presumptive species. Barcoding revealed species not currently described in larval taxonomic

keys, including multiple (211) coexisting congeneric species. That 150 species were revealed

by barcoding samples collected on the same date and in the same habitat were unprecedented.

The results revealed a pollution-tolerance gap because barcoding pushed larval taxonomy


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beyond the available pollution-tolerance data. The ability to distinguish larvae at the species

level through barcoding finally puts biodiversity assessments for aquatic communities in

terms comparable to those used for terrestrial ecosystems where estimates of biodiversity for

plants and animals are never quantified at the level of genus or family. The study conclude

that DNA barcodes of stream macro invertebrates will improve descriptions of community

structure and water quality for both ecological and bioassessment purposes.

Another study conducted by Tanya Dapkey, 2008 combines DNA Barcoding and

Macro invertebrate Sampling to Assess Water Quality. The importance of Barcoding, when

combined with traditional aquatic macro invertebrate sampling, provides the most accurate

and cost effective method to determine the water quality of fresh water ecosystems. DNA

barcoding (using a standardized sequence of the mitochondrial CO1 gene) was used to

determine the aquatic insect species richness of two sites along White Clay Creek in

Pennsylvania. Water quality assessment at the sites using the barcoding increased the species

richness and provided a much more detailed analysis by detecting cryptic species. Here

Aquatic insect identifications by an amateur biologist and by expert taxonomists using

traditional methods based on morphology were compared to DNA barcoding. The Amateur

biologists identifications were limited to order and family while expert taxonomists were able

to identify 44 different species and DNA barcoding indicated 128 different species. There was

a success rate of 84% of the 1786 specimens that were submitted for barcoding which

generated a successful DNA sequence. DNA barcoding revealed the presence of more species

than expert taxonomists identified as shown in the following listing of insect orders with

comparison of numbers of species identified by expert taxonomists and DNA barcoding:

Diptera (23 expert spp. and 128 barcoding spp.), Ephemeroptera (6 expert spp. and 16

barcoding spp.), Plecoptera (0 expert spp. and 6 barcoding spp), Trichoptera (9 expert spp.

and 14 barcoding spp), and Coleoptera (6 expert spp. and 6 barcoding spp).

Environmental Barcoding: A Next-Generation Sequencing Approach for

Biomonitoring Applications Using River Benthos was carried out by Hajibabaei et al. 2011.

The results showed the ability of 454 pyrosequencing of mini-barcodes to accurately identify

all species with more than 1% abundance in the pooled mixture. The approach failed to

identify 6 rare species in the mixture, but the presence of sequences from 9 species that were


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not represented by individuals in the mixture provides evidence that DNA based analysis may

yet provide a valuable approach in finding rare species in bulk environmental samples.

Ephemeroptera, Plecoptera, and Trichoptera fauna of Churchill (Manitoba, Canada)

was studied by Xin Zhou et al. 2010. The study showed a >53X increase in the EPT fauna,

including 68 caddis fly, 37 mayfly, and 7 stonefly species, recorded from Churchill. DNA

barcoding also allowed identification of unidentifiable life stages, revealing several potentially

new species of caddisflies and mayflies which suggest the presence of cryptic species. There

study also explore the phenology and habitat preferences of Churchills Trichopteran and

demonstrate that comprehensive sampling is important for documenting biodiversity through

DNA barcoding

Cycles of rainforest contraction and expansion of north-eastern Australians dry

sclerophyll forest associated with climatic fluctuations leads to geographical endemism in

terrestrial rainforest taxa. The study was conducted by Matt et al2009. The Australian non-

biting midge species Echinocladius martini Cranston (Diptera: Chironomidae), restricted to

cool, well-forested freshwater streams, was found to disperse among populations located in

isolated rainforest pockets during periods of sclerophyllous forest expansion. In this study,

mitochondrial COI were analyzed for E. martini collected from eight sites spanning the Wet

Tropics bioregion to assess the scale and extent of phylogeographic structure. Analyses of

genetic structure showed several highly divergent cryptic lineages (


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METHODOLOGY

The basic methodologies involved are

1. Collection and Identification and preservation of Ephemeropteran and Odonate larval

samples from the river Pampa of Kerala and river Ganges

2. Assessment of field water quality parameters using a water analysis kit (APHA, 1995)

3. DNA Isolation from the preserved samples.

4. Polymerase Chain Reaction and DNA Sequencing of the COI gene.

5. DNA Sequencing, Sequence Editing and Alignment of the COI gene.

1. Collection, Identification and preservation of Ephemeropteran and Odonate larval

samples from the river Pampa of Kerala and river Ganges.

Benthic (bottom dwelling) larvae will be collected and identified from river Pampa of Keralaand river Ganga from the point of river origin to the discharge. Collection methods include the

use of benthic nets (D net) and kick screens. Using the stream current, to wash specimens

physically from substrates, and examination of stone, wood and other substrates to find the

attached larvae and pupae. Identification of specimens to the most refined taxonomic level

possible will commence immediately after the collection with the help of keys for

identification. The sampling locations on River Pampa will be Pulachimalai Hills,

Sannidhanam, Madamon, Kozhencherry, Aranmula, Mannar and Viapuram. The

Pulachimalai hills is situated in forest area devoid of any anthropogenic activities. Reference

location will be at Pulachimalai.The Ganga basin accounts for a little more than one fourth

(26.3 %) of the countrys total geographical area and is the biggest river basin in India,

covering the entire states of Uttarakhand , Uttar Pradesh , Bihar , Delhi, parts of Punjab,

Haryana, Himachal Pradesh, Rajasthan, Madhya Pradesh and West Bengal. The main river

stream originates in the northern most part of Uttarakhand flows through Uttar Pradesh, Bihar

and West Bengal and finally drains into the Bay of Bengal. Rising from the icy caves of

Gangotri glacier at the height of 4255 above mean sea level, River Ganga starts its long

journey to join River Alaknanda and becomes River Ganga near Devprayag. River Ganga is

the longest river (2,525 km) and has largest river basin (861,404 km2) in India. The main

stretch of River Ganga runs from Haridwar to Allahabad through over Nagal, Bijnor,

Garhmukteshwar, Hasanpur, Anupshahar, Narora, Sahaswan, Kasgang, Ptiali, Kampil,Kaimgang, Fatehgarh, Kannauj, Bithaur, Brahmavart, Kanpur and finally Allahabad. At

Allahabad, it joins with a major tributary River Yamuna and thereafter passing through

Banaras, Patna. At Ganga Sagar in West Bengal, it joins Bay of Bengal. Following sampling

locations have been selected on River Ganga. Reference location will be at Gangotri.

1 Gangotri


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1. Haridwar

2. Nagal

3. Bijnor

4. Garhmukteshwar

5. Hasanpur

6. Anupshahar

7. Narora

8. Sahaswan

9. Kasgang

10. Ptiali

11.Kampil

12.Kaimgang

13.Fatehgarh

14.Kannauj

15.Bithaur

16.Brahmavart

17.Kanpur

18.Allahabad

19.Banaras

20.Patna

21.Ganga Sagar

In the first phase, Main River Pamba and River Ganga will be considered from origin to

discharge. The frequency of sampling will be in three different season. For River Ganga,

sampling will be carried out during Post-monsoon (Octobr-November), Winter (January-

February) and Summer (May-June). At Reference station sampling will be restricted to Post-


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monsoon and summer season. For River Pamba, sampling will be carried out during Post-

monsoon (October-November), Winter (December-February) and Summer (April-June).

2. Specimen Preservation.

To allow DNA isolation, 95% ethanol should be used. The ethanol should generally be

poured off and replaced with new 95% ethanol within a few days of collection to optimize

DNA preservation. DNA has been successfully extracted from formalin-preserved tissue,

including relatively ancient samples and these techniques may be important in examining

previously archived specimens (Fang et al. 2002; France and Kocher, 1996)

3. DNA Isolation

Whole cell DNA was extracted from either fresh tissue immediately after collection of a

specimen or from tissue frozen at - 20C or other preserved samples (Ethanol). DNA from the

CO1 gene is extracted, amplified and sequenced through a set of six protocols viz

Lysis

DNA Extraction

PCR

Sequence Editing and Alignment

For isolation of DNA from the benthic insects we should first standardize the protocol

either manually (Standard barcoding protocols for DNA extraction (Ivanova et al.2006)] orby Kit procedures available. For the manual isolation of DNA from the specimens the

following steps are followed

General Manual Steps for DNA isolation

Lysis and DNA Extraction

1.Mix 5 ml of insect Lysis Buffer and 0.5 ml of Proteinase K, 20 mg/ml in a sterile container.

Add 50 l of Lysis Mix to each Eppendorf tubes.

2.DNA is isolated from a piece of each specimen (a leg for most insects, posterior parapods

for chironomid Diptera, or a piece of body tissue for other dipteran insects, oligochaetes,

crustaceans, and snails).

3. Add a small amount of tissue (e.g. 2-4 mm of insect leg or 2-3 mm3 of ethanol preserved

tissue) to each Eppendorf tubes.


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4. Incubate at 56C for a minimum of 6 hours or overnight to allow digestion.

5. Centrifuge at 1500 g for 15 sec for proper mixing of the samples.

6. Add 100 l of Binding Mix to each sample using micro pipette and centrifuge at 1000 g

for 20 sec.

7. Transfer the lysate (about 150 l) from the Eppendorf tubes into a tube containing GF

membrane.

8. Centrifuge at 5000 g for 5 min to bind DNA to the GF membrane.

9. First wash step: Add 180 l of Protein Wash Buffer (PWB) to each tube containing GF

membrane and centrifuge at 5000 g for 2 min.

10.Second wash step: Add 750 l of Wash Buffer (WB) to each tube containing GF

membrane and centrifuge at 5000 g for 5 min.

11.Remove the GF membrane and incubate at 56C for 30 min to evaporate residual ethanol.

12.Collect the DNA eluted from the GF membrane and is resuspended using 30 60 l of

ddH20 (prewarmed to 56C) into a vial and incubate at room temperature for 1 min.

13.DNA can be temporarily stored at 4C or at20C for long-term storage and further use.

Kit Procedures Available

Total genomic DNA was extracted from larval tissue using the Qiagen DNeasy extraction

kit, following the manufacturers guidelines.

In addition to standard methods, there are commercial kits (e.g. Sigma-Aldrich product

number GDI-3) that are inexpensive and have high success in recovering DNA.

The Genomic DNA isolated by this procedure contains an approximately 700 base pair

(bp) barcoding region of the Mitochondrial Cytochrome C oxidase subunit 1 (COI) gene. The

next step is to amplify and sequence the gene using standard barcoding protocols (Ivanova et

al. 2006). Broad range primers are available that will amplify an approximately 700-bp

segment from diverse invertebrates (including Annelida, Arthropoda, Coelenterata,

Echinodermata, Echiura, Mollusca, Nemertina, Platyhelminthes, Pogonophora, Sipuncula, andTardigrada) (Folmer et al.1994)

Polymerase Chain Reaction and DNA Sequencing

Most eukaryote cells contain mitochondria,and mitochondrial DNA (mtDNA) has a

relatively fast mutation rate, which results in significant variation in mtDNA sequences


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between species and, in principle, a comparatively small variance within species. A 648-bp

region of the mitochondrial Cytochrome c oxidase subunit I (COI) gene was proposed as a

potential 'barcode'

The COI gene was used for this analysis as it is fast evolving and thus is considered to

be an optimal marker for intraspecific population analysis (Avise 1986; Moriyama & Powell

1997). A 639-base pair (bp) fragment of the Cytochrome c oxidase subunit I (COI) gene was

amplified using universal invertebrate COI primers LCO1490 and HCO2198 (Folmer et al.

1994).

Full-length COI barcodes were amplified using the M13-tailed versions of the Folmer

primers:

LCO1490-t1 (59TGTAAAACGACGGCCAGTGGTCAACAAATCATAAAGATATTGG-39)

HCO2198-t1 (59-CAGGAAACAGCTATGACTAAACTTCAGGGTGACCAAAAAATCA-39)

Poly LCO and Poly HCO primers were used when full-length polymerase chain

reaction (PCR) amplification was not successful (Carr 2010).

Primers play a very important role in the PCR reaction steps. The primers are some

times common or may be specific depending upon the specimens selected. We have to

standardize the annealing temperature for each reaction. The amount of DNA used will

depend on the concentration of the sample. The COI amplification reaction mix contains 41.5

l of template DNA from extractions, 0.5 l of 10 M Primer (5 pmol), 4.5 l of 10X PCR

Buffer, 2.5 l of 50 mM MgCl2 (2.5 mM), 0.25 l of 10 mM dNTP, 0.2 l Taqpolymerase

and were adjusted to a final volume of 50 l with dH2O.

Steps for COI amplification (Polymerase Chain Reaction)

Step 1 is an initial 94C soak to completely denature the original template, particularly

if it is genomic DNA.

Step 2 is the denaturing step.

Step 3 is the annealing step whose temperature will depend on the sequence of the

primers. For COI, it is ideal to begin annealing at a low temperature (45C) for a few

initial cycles to allow the primers to bind to the template and then raise the

temperature (51C) to avoid excessive non-specific binding of primers.

Step 4 is the extension step whose time depends on the length of the product.

Generally, extension steps should be at least 1 min/1000 bp.
http://en.wikipedia.org/wiki/Base_pairhttp://en.wikipedia.org/wiki/Mitochondrialhttp://en.wikipedia.org/wiki/Cytochrome_c_oxidasehttp://en.wikipedia.org/wiki/Cytochrome_c_oxidasehttp://en.wikipedia.org/wiki/Mitochondrialhttp://en.wikipedia.org/wiki/Base_pair


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Step 5 repeats steps 2, 3, and 4 five more times.

Steps 6, 7, 8, and 9 denature, anneal at 51C, and extend for 36 cycles.

Step 10 is a soak at 72C that will allow the Taq polymerase to complete any

unfinished products.

Step 11 is a 4C soak for 10 min.

Cloning of COI gene into a suitable host bacteria for further studies

The amplified COI gene sequence has to be cloned into a suitable vector and introduction

into host bacteria for further studies.

DNA Sequencing, Sequence Editing and Alignment

The amplified PCR products were examined using 2% Agarose Gel Electrophoresis

(AGE) and the amplified COI gene present in the gel is imaged using an Alpha Imager .

Successful PCR products were sent for cycle sequencing. The cytochrome c oxidase subunit I

sequences generated after the DNA sequencing were aligned and edited by using software

called bioedit (Hall, 1999). Edited sequences were aligned using software known as BLAST

and uploaded onto the Barcode of Life Datasystems (BOLD) website

(http://www.barcodinglife.com/) or NCBI (http://www.ncbi.com). Sequences and detailed

information about all specimens are stored on Gen-Bank and are publicly accessible to all. A

phylogenetic Analysis Tree (Dendrogram) drawn after the process will create a library of

DNA barcodes that will link existing Linnaean names and ecological information associated

with them to barcode sequences we submit. This signals new possibilities for identification of

previously unidentifiable material in benthic samples and also about the species diversity,

composition and richness.

Reference

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Gresens, S. E., K. T. Belt, J. A. Tang, D. C. Gwinn, And P. A. Banks. 2007. Temporaland spatial responses of Chironomidae (Diptera) and other benthic invertebrates tourban stormwater runoff. Hydrobiologia 575:173190.


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Ivanova, N. V., J. R. Dewaard, And P. D. N. Hebert. 2006. An inexpensive,automation-friendly protocol for recovering high-quality DNA. Molecular EcologyNotes 6: 9981002.

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WORK PLAN

First Year

First half (6 months)

The study is selected for three years. A total of 2 Holy Rivers, Pampa from the

Southern Kerala and river Ganga from North India should be taken showing high diversity of

aquatic insects.

Procurement of equipments, chemicals, glass wares etc, setting up of laboratory,

reference collection, visits and selection of sites, design of field and laboratory work.

Second half (6 months)

Taxonomical identification and Preservation of Insects. Insects should be collected

from 20 different sites oftwo rivers(Pampa and Ganga).

Second Year

First half (6 months)


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Isolation of the DNA from the Benthic Samples collected from the two Rivers and

Standardization of the isolation Protocol. We can either isolate the DNA manually or by using

Kit procedures.

The DNA obtained after the isolation procedure should be given for PCR reactions.

Amplification of the isolated DNA fragment is done with the help of Primers and

standardization of the annealing temperature for the PCR reaction should be carried out for

specific amplification of the desired gene of interest.

Second half (6 months)

The amplified gene of interest will be given for sequencing, comparison of the sequence will be

done with the help of Bio-Informatics tool such as BLAST and Bio-edit and the data will be

submitted to National Centre for Biotechnology Information (NCBI) or Barcode of Life

Database (BOLD). The result obtained should be compared and the species diversity,

establishing DNA library and thereby biomonitoring of the water quality will be achieved

Preparation of Project report, Submission and presentation. Publication of Manuscript in

Research Journals.

PERT CHART

FIRST YEAR

Work Plan Duration

Lab Setting, Site fixing, Collection of

Aquatic insects from the two rivers (Pampa

and Ganga) of India.

Duration=6 months

Taxonomical identification and

Preservation of Insects. assessment of

water quality

Duration

Duration=6 months

SECOND YEAR


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Work Plan Duration

DNA isolation,

Amplification of the isolated DNA with the

help of universal Primers for COI

Duration=6

months

. The amplified gene of interest will be

given for sequencing, comparing the

sequence with the help of Bio-Informatics

tool such as BLAST and Bio-edit and

submitting the data to NCBI or BOLD ,

paper publication and project winding up

Duration

Duration=6

months

Practical relevance or utility of the project

1) Data gathered during the proposed project would be useful as a sound data for

researchers, Taxonomist, Scientists, conservationists and development agencies.

2) Data on DNA Barcoding would be useful to compare with the baseline data

gathered by the Taxonomist for the species level classification.

3) Data collected during the proposed project would be useful for implementing

measures for conservation of endangered species and species composition of

unknown faunas and identification of larval forms of benthic aquatic insects,

which are very difficult for identification.

4) Data will give a biological indication about the pollution status of the river.

Agencies, which can utilize the results of the project


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This project is expected to reveal fruitful data which would be directly useful for

researchers, various government departments, administrators and conservationists.

Accordingly, a list of users of the results of the project is drawn as follows:

1. Research students

2. Scientists

3. Taxonomist

4. School and university Teachers

5. Science Popularization movements and agencies

6. Government agencies like Department of Forests, Department of Science,

Technology and Environment.

7. Administrators.

8. National and International, Development Research and Consultancy

Agencies.

9. Forest and wild life extension workers Conservationists

Statement V

Project budget in the prescribed forma1

Particulars NoI Year

(in Rupees)

II Year

(in Rupees)total

A Salaries & Wages*

Junior Research Fellow(JRF) 2 3,36,000 3,36,000

Total 672,000

[*The monthly salary [email protected],000/-]

B

Major and minor equipments

Multi-parameter Portable Water Analysis Kit: 300,000.00

C Expendables

1.Chemicals 1,00,000 200,000 3,00,000

2.Glassware (including Plastic wares) 50,000 50,000 100,000

3.Stationaries 10,000 10,000 20,000

Total 4,20,000

D Travel

1. Travel and Field work 2,00,000 50,00,0 5 250,000
mailto:[email protected],000/-mailto:[email protected],000/-


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0

,00

0

E Other Project costs

1.Books 8,000 - - 8,000F Contingencies

1. Analytical charges. 100,000 1,00,000

2.Contingent expenses 50,000 100,000 150,000

Total

Total 19,00000

Institutional Overhead 100,000

Grand total Rs 20,00000 ( Rupees Twenty lakhs only)

Statement VI

Facilities available with the host institutionOptical Stereo MicroscopeThermo cycler (PCR)

Deep-freezer (-200C)

Refrigerator Centrifuge

Gel Documentation system

Micropipettes

Weigh balance

Water bath

AGE Apparatus

Justification of the budget proposal

A. Salaries and Wages (Man power)

Salary for two project fellows (Two scholar having post graduate in Biotechnology with

experience in the field of Classical taxonomy and molecular taxonomy)

B. EquipmentsFor analyzing water quality in the field itself

C. Expendables/ConsumablesDNA isolation process needed costly chemicals having quality grade particularly

Sigma, Merck etc.

D. Travel

During the period of study, visit of concerned offices/ Departments in and outside the

State for data collection and sample collection along entire the major rivers in Kerala is


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required. For meeting the traveling fare for this purpose and throughout the study area in

different periods, an amount of Rs 250,000 (Two lakhs fifty thousand only) is proposed.

E. Books and Journals

To purchase books and journals related to research works for procuring update works

in this field.

F. Contingency

Department of Biosciences does not have automated sequencing facility for DNA

sequencing. So the samples have to be sent to another well equipped laboratory having DNA

sequencing facilities.

ANNEXURE II (E): CERTIFICATE

To:

Central Pollution Control Board,

Parivesh Bhawan,

East Arjun Nagar,

Delhi-110 032

Sir,

1. A research project entitled,

DNA Barcoding of Freshwater Ephemeropterans and Odonates Important in Water

Quality Assessment of Two Indian Riversis forwarded herewith for consideration of grant funding by the Ministry.

2. It is certified that the same project or another project with similar objectives has not


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been submitted to any other funding agency by the Investigator(s).

3. We have carefully read the terms and conditions of sanctioning the project and agree to

abide by them.4. The organization will provide all necessary infrastructural facilities (both laboratory and

Administrative) if the project is sanctioned.

5. The organization is fully responsible in regard to matters pertaining to the project.6. Certified that the equipment/instruments proposed in the project are available in theDepartment/Institution and are available for dedicated project use.

Yours faithfully,

Place:

Date:

(Registrar/Director/Head of the Organization)

1. Principal Investigator

Dr.Linu Mathew

Assistant Professor in BiotechnologySchool of Biosciences

Mahatma Gandhi UniversityPD Hills (PO), Kottayam

Ph. 09447505690

E mail:[email protected]

Co- Investigators

1 Dr. J. G. Ray M Sc, M E & E, Ph D

Professor,School of BiosciencesMahatma Gandhi UniversityPD Hills (PO), Kottayam

Ph. 09446119626E mail:[email protected] ,[email protected]

2 Dr. Prathima AkolkarScientist D

Head, In- charge, Biolab,

Central Pollution Control Board,Parivesh Bhawan,

East Arjun Nagar,

Delhi-110 032
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]


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Ph. 09818146122

E mail:[email protected]

Curriculam Vitae of principal Investigator

Dr. Linu Mathew

Assistant professor (Sr.grade) w/o Binoi Antony

school of Biosciences Pichakapallil

Mahatma Gandhi University Amalagiri P O

Priyadarshini Hills P O Kottayam-36

Keralam-686 560 Keralam

[email protected] 0481 2591790

9447505690
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]


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Educational Qualifications

Degree Board Subjects YearMark(%)

Class/Rank

S. S. L. C. State Board of

PublicExaminations

Basic Sciences,

Languages

1990 94 Distinction

Pre- Degree MahatmaGandhiUniversity

Physics, Chemistry,Biology, English, Hindi

1992 82 First Class

BSc.(Agriculture)

KeralaAgriculturalUniversity

Agronomy, Horticulture,Plant Pathology,Entomology, Genetics,Plant Breeding

1997 93 Distinction

MSc.Biotechnology

TamilnaduAgriculturalUniversity

Genetic Engineering,Molecular Biology,Enzymology,Biochemistry

1999 96 First Rankwithdistinction

Ph. D. inBiotechnology

MahatmaGandhiUniversity

2008

Title of the thesis

Studies on the anticancer effects of Cassia fistula L.

Areas of Interests

Plant Tissue Culture, DNA barcoding

Scholarships

1. Mahatma Gandhi University Merit Scholarship based on S. S. L. C. marks from 1990-

1992

2. Kerala Agricultural University Merit scholarship from 1992-1996

3. D. B. T. Scholarship from 1997-1999

4. CSIR. JRF and NET in the year 1998

Merit Awards


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M. S. Swaminathan Award for the best student in Biotechnology for the year 1999 of

Centre for Plant Molecular Biology, TNAU

Teaching Experience

Teaching Biotechnology at PG level since 26 March, 2001 till date. (11 yearsexperience)

Publications

a) Papers Published

1Linu Mathew, R. Chandra Babu, J. Souframanien, P. Chezhian, P. Shanmugasundaram, P.

Nagarajan and S. Sadashivam, (2000), DNA Polymorphism among Rice (Oryza sativa L. )

accessions differing in draught tolerance, J. Plant Biol, Vol 27 (2) pp 145-152

2 Linu Mathew, Sankar Sashidhar, Chemopreventive potential of methanol extract of stem

bark of Cassia fistula in mice,Journal of pharmacognosy and herbal formulations,vol2

,2012,(ISSN2229-6840)

b) Paper presented at conferences

1 Micropropagation and secondary metabolite isolation from Adahthoda vassica

Nees ,Rashmi PA, Reshma John, Linu Mathew ,Kerala women,s science congress

august 2010

2 Endophytic associations in Neem Plants (Azadirachta indica A juss.)in KeralaPreethy MR, Jyothis Mathew, Linu Mathew 2010

3Genetic variation in populations of Clarias dussumieri dussumieri in differentgeographical locations of Kerala Aneesha devassi, Smitha Thomas, Linu MathewWorld congress on biotechnology,2011

4 Genetic variation in populations of Monochoria vaginalis in different geographicalareas of coastal Kerala, Smitha thomas. K., Aneesha devassy and Linu Mathew,World congress on biotechnology,2011

5 Endophytic fungal diversity and colonization in Achyranthes asperaLinn. Reshma

John, Siji Raju, Jyothis Mathew, Liinu Mathew 24th Kerala Science Congress,

KSCSTE, Thiruvanthapuram 2012


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6 Studies on endophytic associations in Achyranthes aspera . Reshma John, Siji

Raju, Jyothis Mathew, Liinu Mathew, International congress and symposium in plant

sciences,at MACFAST ,Thiruvalla, Kerala 2011

c) Gene Bank Accessions

1 ACCESSION JQ699184 Aneesha,D.,Linu,M.,Padmakumar,K.G.,

Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 568 bp DNA

linearVRT28-FEB-2012

DEFINITION Clarias gariepinus isolate CLGP1 16S ribosomal RNA gene,

partialsequence;mitochondrial.

SOURCE mitochondrion Clarias gariepinus (North African catfish)

2 ACCESSION JQ699184 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 568 bp DNA linear VRT28-FEB-2012DEFINITION Clarias gariepinus isolate CLGP1 16S ribosomal RNA gene, partialsequence; mitochondrial.SOURCE mitochondrion Clarias gariepinus (North African catfish)

3 ACCESSION JQ699185 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K., Basheer,V.S. and Jena,J.K 568 bp DNA linear VRT28-FEB-2012DEFINITION Clarias gariepinus isolate CLGP2 16S ribosomal RNA gene, partial

sequence; mitochondrial. .SOURCE mitochondrion Clarias gariepinus (North African catfish).

4 ACCESSION JQ699186 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 568 bp DNA linear VRT28-FEB-2012DEFINITION Clarias gariepinus isolate CLGP3 16S ribosomal RNA gene, partial

sequence; mitochondrial. .SOURCE mitochondrion Clarias gariepinus (North African catfish)ORGANISM Clarias gariepinus

.

5 ACCESSION JQ699191 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 573 bp DNAlinear VRT 28-FEB-2012DEFINITION Clarias batrachus isolate CLBT3 16S ribosomal RNA gene, partialsequence; mitochondrial.


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SOURCE mitochondrion Clarias batrachus (walking catfish)

6 ACCESSION JQ699192 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K. 573 bp DNA linearVRT 28-FEB-2012

DEFINITION Clarias batrachus isolate CLBT4 16S ribosomal RNA gene, partialsequence; mitochondrial.SOURCE mitochondrion Clarias batrachus (walking catfish)

7 ACCESSION JQ699193 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 573 bp DNA linearVRT 28-FEB-2012

DEFINITION Clarias batrachus isolate CLBT5 16S ribosomal RNA gene, partialsequence; mitochondrial.SOURCE mitochondrion Clarias batrachus (walking catfish)

8 ACCESSION JQ699194 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 568 bp DNA linearVRT 28-FEB-2012DEFINITION Clarias dussumieri isolate CLDU1 16S ribosomal RNA gene, partialsequence; mitochondrial.SOURCE mitochondrion Clarias dussumieri

9 ACCESSION JQ699195 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K., Basheer,V.S. and Jena,J.K 568 bp DNA linearVRT 28-FEB-2012DEFINITION Clarias dussumieri isolate CLDU2 16S ribosomal RNA gene, partialsequence; mitochondrial.SOURCE mitochondrion Clarias dussumieri

10 ACCESSION JQ699196 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K., Basheer,V.S. and Jena,J.K 568 bp DNAlinear VRT 28-FEB-2012 DEFINITION Clarias dussumieri isolate CLDU3 16Sribosomal RNA gene, partial sequence; mitochondrial. SOURCE mitochondrionClarias dussumieri

11 ACCESSION JQ699197 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K., Basheer,V.S. and Jena,J.K 568 bp DNA linearVRT 28-FEB-201

DEFINITION Clarias dussumieri isolate CLDU4 16S ribosomal RNA gene, partialsequence; mitochondrial.SOURCE mitochondrion Clarias dussumieri

12 ACCESSION JQ699198 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 568 bp DNA linear VRT28-FEB-2012


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DEFINITION Clarias dussumieri isolate CLDU5 16S ribosomal RNA gene, partialsequence; mitochondrial.SOURCE mitochondrion Clarias dussumieri

13 ACCESSION JQ699199 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K. 671 bp DNA linearVRT 28-FEB-2012DEFINITION Clarias gariepinus isolate CLGP1 cytochrome oxidase subunit I (COI)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias gariepinus (North African catfish)

14 ACCESSION JQ699200 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K., Basheer,V.S. and Jena,J.K. 671 bp DNA linearVRT 28-FEB-2012DEFINITION Clarias gariepinus isolate CLGP2 cytochrome oxidase subunit I (COI)

gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias gariepinus (North African catfish )

15 ACCESSSION JQ699201 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 671 bp DNA linearVRT 28-FEB-2012DEFINITION Clarias gariepinus isolate CLGP3 cytochrome oxidase subunit I (COI)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias gariepinus (North African catfish)

16 ACCESSION JQ699202Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 671 bp DNAlinear VRT 28-FEB-2012 DEFINITION Clarias gariepinus isolate CLGP4cytochrome oxidase subunit I (COI)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias gariepinus (North African catfish)

17 ACCESSION JQ699203 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 671 bp DNA linear VRT28-FEB-2012DEFINITION Clarias gariepinus isolate CLGP5 cytochrome oxidase subunit I(COI)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias gariepinus (North African catfish)

18 ACCESSION JQ699204 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 660 bp DNA linear VRT 28-FEB-2012DEFINITION Clarias batrachus isolate CLBT1 cytochrome oxidase subunit I (COI)gene, partial cds; mitochondrial.


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SOURCE mitochondrion Clarias batrachus (walking catfish)

19 ACCESSION JQ699205 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 660 bp DNA linear VRT28-FEB-2012

DEFINITION Clarias batrachus isolate CLBT2 cytochrome oxidase subunit I (COI)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias batrachus (walking catfish)

20 ACCESSION JQ699206 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 660 bp DNA linear VRT28-FEB-2012DEFINITION Clarias batrachus isolate CLBT3 cytochrome oxidase subunit I (COI)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias batrachus (walking catfish)

21 ACCESSION JQ699207 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 660 bp DNA linearVRT 28-FEB-2012DEFINITION Clarias batrachus isolate CLBT4 cytochrome oxidase subunit I (COI)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias batrachus (walking catfish)

22 ACCESSSION JQ699208 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 660 bp DNA linear VRT28-FEB-2012DEFINITION Clarias batrachus isolate CLBT5 cytochrome oxidase subunit I (COI)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias batrachus (walking catfish)

23 ACCESSION JQ699209 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 662 bp DNA linear VRT28-FEB-2012DEFINITION Clarias dussumieri isolate CLDU1 cytochrome oxidase subunit I(COI)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias dussumieri

24 ACCESSION JQ699210 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 662 bp DNA linear VRT28-FEB-2012DEFINITION Clarias dussumieri isolate CLDU2 cytochrome oxidase subunit I (COI)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias dussumieri

25 ACCESSION JQ699211 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 662 bp DNA linear


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VRT 28-FEB-2012DEFINITION Clarias dussumieri isolate CLDU3 cytochrome oxidase subunit I (COI)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias dussumieri

26 ACCESSION JQ699212 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 662 bp DNA linearVRT 28-FEB-2012DEFINITION Clarias dussumieri isolate CLDU4 cytochrome oxidase subunit I (COI)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias dussumieri

27 ACCESSION JQ699213 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 662 bp DNA linear VRT28-FEB-2012DEFINITION Clarias dussumieri isolate CLDU5 cytochrome oxidase subunit I (COI)

gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias dussumieri

28ACCESSION JQ699214 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 578 bp DNA linear VRT28-FEB-2012DEFINITION Clarias gariepinus isolate CLGP1 cytochrome b (Cytb) gene, partialcds; mitochondrial.SOURCE mitochondrion Clarias gariepinus (North African catfish)

29 ACCESSION JQ699215Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 578 bp DNA linear VRT28-FEB-2012DEFINITION Clarias gariepinus isolate CLGP2 cytochrome b (Cytb) gene, partialcds; mitochondrial.SOURCE mitochondrion Clarias gariepinus (North African catfish)

30 ACCESSION JQ699216 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 578 bp DNA linear VRT28-FEB-2012SOURCE mitochondrion Clarias gariepinus (North African catfish)

31 ACCESSION JQ699217 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 578 bp DNA linear VRT28-FEB-2012 DEFINITION Clarias gariepinus isolate CLGP4 cytochrome b (Cytb)gene, partial cds; mitochondrial.SOURCE mitochondrion Clarias gariepinus (North African catfish)


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32 ACCESSION JQ699218Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 578 bp DNA linearVRT 28-FEB-2012DEFINITION Clarias gariepinus isolate CLGP5 cytochrome b (Cytb) gene, partialcds; mitochondrial.

SOURCE mitochondrion Clarias gariepinus (North African catfish)

33 ACCESSION JQ699219Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 578 bp DNA linear VRT28-FEB-2012DEFINITION Clarias batrachus isolate CLBT1 cytochrome b (Cytb) gene, partial cds;mitochondrial.SOURCE mitochondrion Clarias batrachus (walking catfish)

34 ACCESSSION JQ699220 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 578 bp DNA linear VRT

28-FEB-2012DEFINITION Clarias batrachus isolate CLBT2 cytochrome b (Cytb) gene, partial cds;mitochondrial. SOURCE mitochondrion Clarias batrachus (walking catfish)

35 ACCESSION JQ699221 Aneesha,D., Linu,M., Padmakumar,K.G.,Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 578 bp DNA linear VRT28-FEB-2012DEFINITION Clarias batrachus isolate CLBT3 cytochrome b (Cytb) gene, partial

cds; mitochondrial.SOURCE mitochondrion Clarias batrachus (walking catfish)

Workshops and Seminar attended

1. Workshop on recombinant DNA Technology conducted by CFBTR, Madurai. (21-12-

2002 to 31-02-2002)

2. 3 Day Seminar cum Workshop on Animal Cell Culture in School of Medical Education,

M. G. U., Gandhi Nagar, from 03-11-03 to 05-11-03

3. UGC Sponsored orientation Course from 14-10-04 to 10-11-04 at Bharathiar

University

4. Workshop on Statistical Applications in Research Studies By School of Environmental

Sciences, M. G. U. from 20-07-04 to 25-07-04

5. UGC Sponsored Refresher Course on Life Sciences by CUSAT from 19-02-07 to 10-

03-07


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6. National Seminar on Biosciences from 14-02-07to 15-02-07 organized by School of

Biosciences , M. G. U.

7. (convenor ) National seminar series in connection with Birth Bicentenary of Charles

Darwin) at School of Biosciences

8. National seminar on Role of women in climate change at KSCSTE

Thiruvananthapuram Jan 2010

9. Kerala Womens Science Congress August 2010

10. Workshop on Plant DNA barcoding at CMS college, Kottayam, MG University

December 2011

11. National Seminar on Life style and Diseases

Ongoing Research Programmes

1. Production of phytoecdysones from callus cultures ofAchyranthes aspera.

2. Micropropagation and Molecular Characterization of Somaclones ofAdathoda

vassica.

3. Micropropagation and Molecular characterization of Boerrhavia diffusafrom different

Geographical areas.

4. Molecular Characterization of Brackish water fauna of Vembanadu Lake..

Projects

Principal investigator in the KSCSTE funded major research Project Barcoding and

genetic diversity Analysis of Clarias Spp of Kerala (ongoing)

Declaration

The information give above are true to the best of my knowledge and belief.

LINU MATHEW

dna barcoding2 1 revised

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