DNA BARCODING OF FRESHWATER EPHEMEROPTERANS AND ODONATES IMPORTANT IN WATER QUALITY ASSESSMENT OF TWO INDIAN RIVERS

Project Proposal Submitted to Central 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

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 Biotechnology

School of Biosciences Mahatma Gandhi University PD Hills (PO), Kottayam E mail: linubinoi@gmail.com

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)

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 of taxons (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 3

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 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. 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

Statement II

INTRODUCTION The technique of deoxyribonucleic acid (DNA) barcoding, whereby species are identified 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

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 timeconsuming 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

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 misidentification) 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.

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.

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.

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 et al.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 macroinvertebrates are dominated by Crustaceans, Molluscs, Annelids and Platyhelminthes. In view of covering the entire length of River Ganga, all the higher forms of benthic macroinvertebrates may be included in the study.

Table 1 Biological Water Quality Criteria (BWQC) Sl.No Taxonomic Groups Range of Range of Water quality Water saprobic diversity characteristic quality score Score class (BMWP) 0.2 - 1 Clean A Indicator Colour

Ephemeroptera, Plecoptera, Trichoptera, 7 and more Hemiptera, Diptera Ephemeroptera, Plecoptera, Trichoptera, 67 Hemiptera, Planaria, Odonata, Diptera Ephemeroptera, Plecoptera, Trichoptera, Hemiptera, Odonata, 36 Crustacea, Mollusca, Polychaeta, Diptera Hirudinea, Oligochaeta

Blue

0.5 - 1

Slight Pollution

Light blue

0.3 - 0.9

Moderate Pollution

Green

11

Mollusca, Hemiptera, Coleoptera, Diptera, 2 5 Oligochaeta Diptera, Oligochaeta No animals 02

0.4 Less 0 - 0.2

& Heavy Pollution

Orange

Severe Pollution

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 al 2009. The Australian nonbiting 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 (<1 km apart) with restricted geographical distributions. The results suggest that due to the systemic drying of the Australian continent during the Plio-Pleistocene, the historical E. martini populations might have fragmented and, hence, promoted divergence in allopatry. A study was conducted on the amplification of Primers for Mitochondrial COI gene by Folmer et al, 1994 in which he describes the "universal" DNA primers for polymerase chain reaction (PCR) amplification of a 710-bp fragment of the mitochondrial Cytochrome c oxidase subunit I gene (COI) from 11 invertebrate phyla: Echinodermata, Mollusca, Annelida, Pogonophora, Arthropoda, Nemertinea, Echiura, Sipuncula, Platyhelminthes, Tardigrada, Coelenterata and the putative phylum Vestimentifera. Preliminary comparisons revealed that these COI primers generate informative sequences for phylogenetic analyses at the species and higher taxonomic levels.

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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 Kerala and 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 (JanuaryFebruary) and Summer (May-June). At Reference station sampling will be restricted to Post16

monsoon and summer season. For River Pamba, sampling will be carried out during Postmonsoon (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)] or

by 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 at 20C 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, and Tardigrada) (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 HCO2198-t1 (59TGTAAAACGACGGCCAGTGGTCAACAAATCATAAAGATATTGG-39) (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 Taq polymerase 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.

19

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 Alexander, L. C., M. Delion, D. J. Hawthorne, W. O. Lamp, And D. H. Funk. 2009. Mitochondrial lineages and DNA barcoding of closely related species in the mayfly genus 2011] Dna Barcoding Of Stream Macroinvertebrates 213 Ephemerella (Ephemeroptera:Ephemerellidae). Journal of the North American Benthological Society 28:584 595. 20

APHA, 1995. Standard methods for the examination of water and wastewater. American Public Health Association, American Water Works Association/ Water Pollution Control Federation, Washington DC Bernard W. Sweeney and Donald J. Baird, 2011, Applying DNA barcoding in benthology: the state of the science. J. N. Am. Benthol. Soc., 2011, 30(1):122124 Buchwalter, D. B., And S. N. Luoma. 2005. Differences in dissolved cadmium and zinc uptake among stream insects: mechanistic explanations. Environmental Science and Technology 39:498504. Buchwalter, D. B., D. J. Cain, C. A. Martin, L. Xie, S. N. Luoma, And T. Garland. 2008. Aquatic insect ecophysiological traits reveal phylogenetically based differences in dissolved cadmium susceptibility. Proceedings of the National Academy of Sciences of the United States of America 105:83218326. Chapman AD. 2009. Numbers of Living Species in Australia and the World: A Report for the Australian Biological Resources Study. Toowoomba. Fang SG, Wan QH, Fijihara N. 2002. Formalin removal from archival tissue by critical point drying. BioTechniques 33:604-611. France SC, Kocher TD. 1996. DNA sequencing of formalin-fixed crustaceans from archival research collections. Mol Mar Biol Biotechnol 5:304-313. Folmer, M. Black, W. Hoeh,* R. Lutz, and R. Vrijenhoek+ 1994 DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology (1994) 3(5), 294-299 Funk, D. H., And B. W. Sweeney. 1990. Electrophoretic analysis of species boundaries and phytogenetic relationships of taeniopterygid stoneflies (Plecoptera). Transactions of the American Entomological Society 116:727751 Funk, D. H., B. W. Sweeney, And R. L. Vannote. 1988. Electrophoretic study of Eastern North American Eurylophella (Ephemeroptera: Ephemerellidae) with the discovery of morphologically cryptic species. Annals of the Entomological Society of America 81:174186. Geraci, C. J., M. A. Al-Saffar, And X. Zhou. 2011. DNA barcoding facilitates description of unknown faunas: a case study on Trichoptera in the headwaters of the Tigris River, Iraq. Journal of the North American Benthological Society 30:163173. Gresens, S. E., K. T. Belt, J. A. Tang, D. C. Gwinn, And P. A. Banks. 2007. Temporal and spatial responses of Chironomidae (Diptera) and other benthic invertebrates to urban stormwater runoff. Hydrobiologia 575:173190. 21

Hall T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symp. Ser. 41, 958. Hajibabaei, Mehrdad, Daniel H. Janzen, John M. Burns, Winnie Hallwachs and Paul D. N. Hebert. 2006. DNA barcodes distinguish species of tropical Lepidoptera. Proceedings of the Natural Academy of Sciences of the United States of America. 103(4): 968-971. Hebert, P. D. N., A. Cywinska, S. L. Ball, And J. R. Dewaard. 2003. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London Series B: Biological Sciences 270:313321. Holzenthal, R. W., D. R. Robertson, S. U. Pauls, And P. K. Mendez. 2010. Taxonomy and systematics: contributions to benthology and J-NABS. Journal of the North American Benthological Society 29:147169. IBOL (INTERNATIONAL BARCODE OF LIFE). 2010. Official launch of iBOL activates worlds largest biodiversity genomics project. Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada. (Available from: http://ibol.org/official-launch/) Ivanova, N. V., J. R. Dewaard, And P. D. N. Hebert. 2006. An inexpensive, automation-friendly protocol for recovering high-quality DNA. Molecular Ecology Notes 6: 9981002. Jackson, J. K., And V. H. Resh. 1992. Variation in genetic structure among populations of the caddisfly Helicopsyche borealis from three streams in northern California, U.S.A. Freshwater Biology 27:2942. Jackson, J. K., And V. H. Resh. 1992. Variation In Genetic Structure Among populations of the caddisfly Helicopsyche borealis from three streams in northern California, U.S.A. Freshwater Biology 27:2942. Janzen, D. H. 2004. Now is the Time. Philosophical Transactions of the Royal Society London, B DOI 10.1098/rstb.2003.1444. Krosch, M. N., A. M. Baker, B. G. Mckie, P. B. Mather, And P. S. Cranston. 2009. Deeply divergent mitochondrial lineages reveal patterns of local endemism in Chironomids of the Australian Wet Tropics. Austral Ecology 34: 317328. Lenat, D. R., And V. H. Resh. 2001. Taxonomy and stream ecology: the benefits of genus- and species-level identifications. Journal of the North American Benthological Society 20:287298.

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Marshall E. 2005. Will DNA bar codes breathe life into classification? Science 307: 1037. Matt N. Krosch, Andrew M. Baker, Brendan G. Mckie, Peter B. Mather And Peter S. Cranston. 2009. Deeply divergent mitochondrial lineages reveal patterns of local endemism in chironomids of the Australian Wet Tropics. Austral Ecology 34, 317328 Mehrdad Hajibabaei1, Shadi Shokralla, Xin Zhou, Gregory A. C. Singer, Donald J. Baird. 2011. Environmental Barcoding: A Next-Generation Sequencing Approach for Biomonitoring Applications Using River Benthos. PLoS ONE 6(4): e17497. doi:10.1371/journal.pone.0017497 Pfenninger, M., C. Nowak, K. C. Steinke, And B. Streit. 2007. Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species. Molecular Ecology 16:1957 1968. Pilgrim, E. M., S. A. Jackson, S. Swenson, I. Turcsanyi, E. Friedman, L. Weigt, And M. J. Bagley. 2011. Incorporation of DNA barcoding into a large-scale biomonitoring program: opportunities and pitfalls. Journal of the North American Benthological Society 30: 217231. Ratnasingham S, Hebert PDN.2007. BOLD: The Barcode of Life Data System (www.barcodinglife.org). Mol Ecol Notes 7: 355364 Ray J G, Krishnan J., Unni K. S. and Shobha V., (2009) Physico-chemical environmental complex of a commercially exploited tropical freshwater system within a wildlife sanctuary, Kerala, India. Ecology and Noospherology, 20: (3-4): 124-144. http://uenj.cv.ua/Ecology_and_noospherology_2009_20_3-4/Rey2.pdf Sharley, D. J., V. Pettigrove, And Y. M. Parsons. 2004. Molecular identification of Chironomus spp. (Diptera) for biomonitoring of aquatic ecosystems. Australian Journal of Entomology 43:359365. Smith, M. Alex, D. Monty Wood, Daniel H. Janzen, Winnie Hallwachs, and Paul D. N. Hebert. 2007. DNA barcodes affirm that 16 species of apparently generalist tropical parasitoid flies (Diptera, Tachinidae) are not all generalists. Proceedings of the Natural Academy of Sciences of the United States of America. 104 (12): 4967-4972. Stahls, G., And E. Savolainen. 2008. MtDNA COI barcodes reveal crytic diversity in the Baetis vernus group (Ephemeroptera, Baetidae). Molecular Phylogenetics and Evolution 46:8287. Stribling, J. B., K. L. Pavlik, S. M. Holdsworth, And E. W. Leppo. 2008. Data quality, performance, and uncertainty in taxonomic identification for biological assessments. Journal of the North American Benthological Society 27: 906919. 23

Sweeney, B. W., And D. H. Funk. 1991. Population genetics of the burrowing mayfly Dolania americana: Geographic variation and the presence of a cryptic species. Aquatic Insects 13:1727. Sweeney, B. W., J. M. Battle, J. K. Jackson, And T. Dapkey. 2011. Can DNA barcodes of stream macroinvertebrates improve descriptions of community structure and water quality? Journal of the North American Benthological Society 30:195216. Tanya Dapkey, 2008. Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Master of Environmental Studies. Wahlberg, Niklas, Rita Oliveira, and James A. Scott. 2003. Phylogenetic relationships of Phyciodes butterfly species (Lepidoptera: Nymphalidae): complex mtDNA variation and species delimitation. Systematic Entomology. 28: 1-17. Wilson EO.1985. Time to Revive Systematics. Science 230: 1227. Xin Zhou, Luke M. Jacobus, R. Edward DeWalt, Sarah J. Adamowicz, and Paul D. N. Hebert. 2010. Ephemeroptera, Plecoptera, and Trichoptera fauna of Churchill (Manitoba, Canada): insights into biodiversity patterns from DNA barcoding Journal of the North American Benthological Society, 29(3):814-837. 2010. Zhou, X., K. M. Kjer, And J. C. Morse. 2007. Associating larvae and adults of Chinese Hydropsychidae caddisflies (Insecta:Trichoptera) using DNA sequences. Journal of the North American Benthological Society 26: 719742. Zhou, X., L. M. Jacobus, R. E. Dewalt, S. J. Adamowicz, And P. D. N. Hebert. 2010. Ephemeroptera, Plecoptera, and Trichoptera fauna of Churchill (Manitoba, Canada): insights into biodiversity patterns from DNA barcoding. Journal of the North American Benthological Society 29: 814837. Zhou, X., S. J. Adamowicz, L. M. Jacobus, R. E. Dewalt, And P. D. N. Hebert. 2009. Towards a comprehensive barcode library for artic life Ephemeroptera, Plecoptera, and Trichoptera of Churchill, Manitoba, Canada. Frontiers in Zoology 6:30.

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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 of two rivers (Pampa and Ganga).

Second Year

First half (6 months)

25

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 Lab Setting, Site fixing, Collection of Aquatic insects from the two rivers (Pampa and Ganga) of India. Taxonomical identification and

Duration

Duration=6 months

Preservation of Insects. assessment of water quality

Duration Duration=6 months

SECOND YEAR

26

Work Plan DNA isolation, Amplification of the isolated DNA with the help of universal Primers for COI

Duration

Duration=6 months

Duration . 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=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

27

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 A Salaries & Wages* Junior Research Fellow(JRF)
[*The monthly salary of JRF@Rs.14,000/-] No I Year (in Rupees) II Year (in Rupees) total

2 Total

3,36,000

3,36,000 672,000

B C

Major and minor equipments Multi-parameter Portable Water Analysis Kit: Expendables 1.Chemicals 2.Glassware (including Plastic wares) 3.Stationaries Total Travel 1. Travel and Field work

300,000.00

1,00,000 50,000 10,000

200,000 50,000 10,000

3,00,000 100,000 20,000 4,20,000 5 250,000

2,00,000

50,00,0

28

0 , 0 0 0 E Other Project costs 1.Books F Contingencies 1. Analytical charges. 2.Contingent expenses Total Total Institutional Overhead 8,000 100,000 100,000 8,000 1,00,000 150,000 19,00000 100,000

50,000

Grand total Statement VI

Rs 20,00000 ( Rupees Twenty lakhs only)

Facilities available with the host institution

Optical Stereo Microscope Thermo 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. Equipments For analyzing water quality in the field itself C. Expendables/Consumables DNA 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

29

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 Rivers is 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

30

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 the Department/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 Biotechnology School of Biosciences Mahatma Gandhi University PD Hills (PO), Kottayam Ph. 09447505690 E mail: linubinoi@gmail.com

Co- Investigators 1 Dr. J. G. Ray M Sc, M E & E, Ph D Professor, School of Biosciences Mahatma Gandhi University PD Hills (PO), Kottayam Ph. 09446119626 E mail: jgray@rediffmail.com , josephray@rediffmail.com

2 Dr. Prathima Akolkar Scientist D Head, In- charge, Biolab, Central Pollution Control Board, Parivesh Bhawan, East Arjun Nagar, Delhi-110 032

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Ph. 09818146122 E mail: pratimacpcb@yahoo.co.in

Curriculam Vitae of principal Investigator

Dr. Linu Mathew

Assistant professor (Sr.grade) school of Biosciences Mahatma Gandhi University Priyadarshini Hills P O Keralam-686 560 linubinoi@gmail.com 9447505690 w/o Binoi Antony Pichakapallil Amalagiri P O Kottayam-36 Keralam 0481 2591790

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Educational Qualifications
Degree S. S. L. C. Board State Board of Public Examinations Mahatma Gandhi University Kerala Agricultural University Tamilnadu Agricultural University Mahatma Gandhi University Subjects Basic Sciences, Languages Physics, Chemistry, Biology, English, Hindi Agronomy, Horticulture, Plant Pathology, Entomology, Genetics, Plant Breeding Genetic Engineering, Molecular Biology, Enzymology, Biochemistry Year Mark (%) Class/Rank Distinction

1990 94

Pre- Degree

1992 82

First Class

BSc. (Agriculture)

1997 93

Distinction

MSc. Biotechnology

1999 96

First Rank with distinction

Ph. D. in Biotechnology

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 19901992 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

33

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 years experience)

Publications
a) Papers Published 1 Linu 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 Kerala Preethy MR, Jyothis Mathew, Linu Mathew 2010 3Genetic variation in populations of Clarias dussumieri dussumieri in different geographical locations of Kerala Aneesha devassi, Smitha Thomas, Linu Mathew World congress on biotechnology,2011 4 Genetic variation in populations of Monochoria vaginalis in different geographical areas of coastal Kerala, Smitha thomas. K., Aneesha devassy and Linu Mathew, World congress on biotechnology,2011 5 Endophytic fungal diversity and colonization in Achyranthes aspera Linn. Reshma John, Siji Raju, Jyothis Mathew, Liinu Mathew 24th Kerala Science Congress, KSCSTE, Thiruvanthapuram 2012

34

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., 568 bp DNA

Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 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 VRT 28-FEB-2012 DEFINITION Clarias gariepinus isolate CLGP1 16S ribosomal RNA gene, partial sequence; 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 VRT 28-FEB-2012 DEFINITION 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 VRT 28-FEB-2012 DEFINITION 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 DNA linear VRT 28-FEB-2012 DEFINITION Clarias batrachus isolate CLBT3 16S ribosomal RNA gene, partial sequence; mitochondrial.

35

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 linear VRT 28-FEB-2012 DEFINITION Clarias batrachus isolate CLBT4 16S ribosomal RNA gene, partial sequence; 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 linear VRT 28-FEB-2012 DEFINITION Clarias batrachus isolate CLBT5 16S ribosomal RNA gene, partial sequence; 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 linear VRT 28-FEB-2012 DEFINITION Clarias dussumieri isolate CLDU1 16S ribosomal RNA gene, partial sequence; 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 linear VRT 28-FEB-2012 DEFINITION Clarias dussumieri isolate CLDU2 16S ribosomal RNA gene, partial sequence; 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 DNA linear VRT 28-FEB-2012 DEFINITION Clarias dussumieri isolate CLDU3 16S ribosomal RNA gene, partial sequence; mitochondrial. SOURCE mitochondrion Clarias 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 linear VRT 28-FEB-201 DEFINITION Clarias dussumieri isolate CLDU4 16S ribosomal RNA gene, partial sequence; 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 VRT 28-FEB-2012

36

DEFINITION Clarias dussumieri isolate CLDU5 16S ribosomal RNA gene, partial sequence; 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 linear VRT 28-FEB-2012 DEFINITION 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 linear VRT 28-FEB-2012 DEFINITION 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 linear VRT 28-FEB-2012 DEFINITION Clarias gariepinus isolate CLGP3 cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial. SOURCE mitochondrion Clarias gariepinus (North African catfish) 16 ACCESSION JQ699202 Aneesha,D., Linu,M., Padmakumar,K.G., Gopalakrishnan,A., Raj,K.,Basheer,V.S. and Jena,J.K 671 bp DNA linear VRT 28-FEB-2012 DEFINITION Clarias gariepinus isolate CLGP4 cytochrome 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 VRT 28-FEB-2012 DEFINITION 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-2012 DEFINITION Clarias batrachus isolate CLBT1 cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial.

37

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 VRT 28-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 VRT 28-FEB-2012 DEFINITION 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 linear VRT 28-FEB-2012 DEFINITION 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 VRT 28-FEB-2012 DEFINITION 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 VRT 28-FEB-2012 DEFINITION 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 VRT 28-FEB-2012 DEFINITION 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-2012 DEFINITION 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 linear VRT 28-FEB-2012 DEFINITION 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 VRT 28-FEB-2012 DEFINITION Clarias dussumieri isolate CLDU5 cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial. SOURCE mitochondrion Clarias dussumieri 28 ACCESSION JQ699214 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-2012 DEFINITION Clarias gariepinus isolate CLGP1 cytochrome b (Cytb) gene, partial cds; mitochondrial. SOURCE mitochondrion Clarias gariepinus (North African catfish) 29 ACCESSION JQ699215 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-2012 DEFINITION Clarias gariepinus isolate CLGP2 cytochrome b (Cytb) gene, partial cds; 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 VRT 28-FEB-2012 SOURCE 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 VRT 28-FEB-2012 DEFINITION Clarias gariepinus isolate CLGP4 cytochrome b (Cytb) gene, partial cds; mitochondrial. SOURCE mitochondrion Clarias gariepinus (North African catfish)

39

32 ACCESSION JQ699218 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-2012 DEFINITION Clarias gariepinus isolate CLGP5 cytochrome b (Cytb) gene, partial cds; mitochondrial. SOURCE mitochondrion Clarias gariepinus (North African catfish) 33 ACCESSION JQ699219 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-2012 DEFINITION 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-2012 DEFINITION 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 VRT 28-FEB-2012 DEFINITION 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. (21-122002 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 1003-07

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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. 10.

Kerala Womens Science Congress August 2010 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. 2. Production of phytoecdysones from callus cultures of Achyranthes aspera. Micropropagation and Molecular Characterization of Somaclones of Adathoda vassica. 3. Micropropagation and Molecular characterization of Boerrhavia diffusa from 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

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