International Training Programme on Bluetongue Vector Identification (Meeting Book)

Transcription

1 International Training Programme on Bluetongue Vector Identification (Meeting Book) 1 st to 5 th July, 2013 Organized by The Pirbright Institute, UK India Bluetongue Vector Network && Tamil Nadu Veterinary and Animal Sciences University Vaccine Research Centre Viral Vaccines Centre for Animal Health Studies Chennai India 2

2 Contents Organizing committee... 4 Background to Meeting... 5 Introduction... 6 Vice-Chancellor s Message... 7 Meeting Summary... 8 Presentations Appendix 1. Details of Participants Appendix 2: Photographs of Meeting

3 Organizing committee UK Dr Simon Carpenter, Head of Entomology, The Pirbright Institute, Ash Road, Woking, Surrey GU24 0NF. Dr Lara Harrup, The Pirbright Institute, Ash Road, Woking, Surrey GU24 0NF. India TANUVAS Chairman: Dr. V. Purushothaman, Director, CAHS, MMC, Chennai -51. Organizing Secretary: Dr. Y. Krishnamohan Reddy, Professor and Head, VRC- VV, CAHS, MMC, Chennai -51. Members: Dr. C. Sreekumar, (Associating Scientist Indo-UK project) Professor, PGRIAS, Kattupakkam. Dr. K. Senthil Vel, (Associating Scientist Indo-UK project) Associate Professor, Dept. of Vety. Parasitology, VC & RI, Namakkal. Dr. A. Subramanian, (Associating Scientist Indo-UK project) Professor, Dept. of Animal Genetics and Feeding, MVC, Chennai 07. Dr. N. Sankaran, Professor, CAHS, MMC, Chennai -51. International Experts Dr. Glenn Bellis (Northern Australia Quarantine Strategy, Australia) Dr. David Gopurenko (NSW Department of Primary Industries, Australia) Ms Karien Labuschagne (Onderstepoort Veterinary Laboratory, South Africa) Dr. Claire Garros (CIRAD, France) Dr. Bruno Mathieu (Faculté de Médecine de Strasbourg, France) 4

4 Background to Meeting Culicoides biting midges (Diptera: Ceratopogonidae) are among the least studied of the major Dipteran groups that spread arboviruses. In this meeting we will explore Culicoides diversity and taxonomic classification in India by bringing together scientists from across India with international experts on this genus. This will allow us to identify and address problems in the classification of Culicoides in this region and produce a practical plan that can be implemented to improve standardisation of this important area in future studies. Our targets were as follows: 1. To assess the current level of taxonomic expertise available across India including understanding what difficulties are faced by new workers in this area. 2. To try and place the India fauna in a global context and suggest areas where new technologies could improve the flow of taxonomic information in this region. 3. Place a level of certainty upon identifications made to date using morphology including a retrospective examination of previously published work in comparison to studies worldwide. 4. To begin the process of integrating molecular techniques (and in particular DNA barcoding) into standard Culicoides identification. The common sense of purpose for all delegates should be to enhance the accuracy and consistency of Culicoides identification in India with a view to better understanding the transmission of arboviruses in this region. The workshop had the following structure: Day 1: Lecture-based presentations from participating individuals. Day 2: Discussion in groups followed by an open session on morphological Culicoides identification. Day 3: Training of participants in groups (Culicoides collection; preparation of samples and slide mounting; molecular Identification). Day 4-5: Identification of samples and collation of results. To promote the exchange of ideas we have invited an international panel of experts from across the world all of whom have significant experience in the study of Culicoides. This will assist in forming a global view of identification of Culicoides in India and enable collaboration in the future in this area across borders. 5

5 Introduction It is our pleasure to introduce the International Training Programme on bluetongue vector identification. This meeting, which is funded by the Biotechnology and Biological Sciences Research Council (UK) has evolved from a requirement to create consensus in identification of Culicoides biting midges, the primary vectors of bluetongue virus. It brings together experts on Culicoides taxonomy from both within India and beyond its borders to discuss this area in detail leading to an assessment of current knowledge and gaps in research. There is also an emphasis in understanding and implementing new technologies to understand evolutionary relationships in the genus. A major aspect of the meeting is in publicising the ongoing IBVNet project ( This study, funded by BBSRC, DFID and the Scottish Government is characterising Culicoides populations systematically across Southern India for the first time. The discussions within the meeting will strengthen both this project and also link with the major centres of Culicoides taxonomy in India. The meeting additional allows collaboration beyond those ICAR centres upon which IBVNet is primarily based. This desire for open collaboration to create freely available and useful resources is integral to the modern approach to insect taxonomy and underpins a desire to view diseases such as bluetongue as global rather than regional issues. The attendance of our international colleagues is additional evidence of this aim. The holding of this meeting in TANUVAS underlines the importance of this centre in combating Culicoides-associated arboviruses in the region. As a key centre in the recent roll out of the bluetongue vaccination programme in Southern India, the expertise already in place was a key determinant of the choice of the centre to lead field aspects of IBVNet. I gratefully acknowledge the involvement of VC Dr R. Prabakaran who has been particularly helpful in ensuring collections of Culicoides were conducted efficiently across Tamil Nadu. DR S CARPENTER THE PIRBRIGHT INSTITUTE, UK DR YK REDDY TANUVAS, INDIA 6

6 Vice-Chancellor s Message Bluetongue is an important viral disease of sheep which is causing financial loss to the small and landless sheep owners in many parts of the country and the disease is transmitted by Culicoides. Tamil Nadu Veterinary and Animal Sciences University in partnership with Indian Council of Agriculture Research has successfully developed safe and potent bluetongue vaccine. In order to complement research already being carried out by All India Network Programme on bluetongue on vaccination strategies, India Bluetongue Vector Network which is a three year Indo-UK Collaborative Project jointly funded by the UK Department for International Development (DFID), the Biotechnology and Biological Sciences Research Council (BBSRC) and Scottish Government via the combating Infectious Diseases of Livestock for International Development (CIDLID) initiative. India Bluetongue Vector Network (IBV Net) is coordinated by the Entomology group based at the Pirbright Institute UK and through Indian Council of Agriculture Research funded All India Network Programme on bluetongue. I am happy to know that the Pirbright Institute UK, India Bluetongue Vector Network and Tamil Nadu Veterinary and Animal Sciences University are jointly conducting International Training Programme on Bluetongue Vector Identification at Vaccine Research Centre Viral Vaccines, Centre for Animal Health Studies, Chennai- 51 during 1 st to 5 th July, With the participation of National and International panel of experts from Australia, France, South Africa and UK who have significant experience in the study of Cuilicoides, I wish the programme a success in identifying the bluetongue vector involved in transmission, understanding the environmental conditions increasing the population of Culicoides and ultimately developing low cost vector control strategies. R. PRABAKARAN VICE-CHANCELLOR TANUVAS 7

7 Meeting Summary The meeting was attended by 41 Indian workers with an interest or expertise in Culicoides taxonomy drawn from 7 states. Of these, 1 attendee had published a peer reviewed paper on the genus, while a large proportion (>50%) had been involved in more general bluetongue virus studies that yielded publishable data. Within the group attending, two students were carrying out PhD s based on Culicoides (both registered at the University of Burdwan, West Bengal). Expertise in Culicoides identification was found to arise in India through two disparate routes: 1) Through veterinary services for bluetongue virus where an interest in the entomological aspects of disease transmission is developed. This largely occurs under the auspices of the Indian Council for Agricultural Research (ICAR) and/or the Project Directorate for Animal Disease Monitoring and Surveillance (PDADMAS). 2) Through Universities with a veterinary background or prior history of working with Culicoides often linked to the Zoological Society of India (ZSI). Here the route into this area more commonly involves completing a degree in Zoology. Specific establishments with a history of involvement in this area are the University of Burdwan and ZSI Kolkata and Chennai. Most attendees had access to at least one major route of funding but expertise in Culicoides identification and entomology in general was seen as being very much secondary to producing effective vaccines for BTV control. From a veterinary viewpoint the perceived importance of taxonomic information regarding vector species varied from high to negligible and there was agreement that the value of this work was not well appreciated by veterinarians in the field. This is largely because control efforts (in particular insecticidal spraying) are not currently centred upon determining differences in Culicoides species ecology and are applied in the same way regardless of region. Discussion sessions 1. What is your experience with Culicoides ID? The majority of attendee s felt sufficiently competent to distinguish Culicoides from other biting flies reflecting basic entomology training at veterinary degree level. Most attendee s from wildlife centres were confident also in identifying major vector groups although this was not reflected in general discussions during the meeting where several concerns were raised regarding identifying members of the C. shultzei group. Limitations also arose in practical sessions on identification where the majority of attendees struggled to consistently identify Avaritia species. 2. What are the current limitations on understanding the taxonomy of Culicoides in India? Current limitations mostly arose from fragmentation between groups of workers involved in identification of Culicoides across India leading to an inability to create 8

8 critical mass for grant proposals. This additionally meant that there was no coherent platform for veterinary workers to contact experts for collaboration on understanding BTV transmission. The lack of funding manifested itself in only a few labs possessing high quality microscopy and imaging facilities (primarily Universities and ZSI) and the expertise to use them effectively. Conversely, in the case of PCR identification of Culicoides this issue was not so prevalent in the veterinary laboratories as the majority of these units possess thermocyclers for use of diagnostic assays and routinely send samples for commercial sequencing. What difficulties do new workers in the field face? A key issue was the lack of both specialist training in this area and a visible community to approach with questions. It was noted that permanent positions in this area are non-existent, even in the traditionally strong regions of Culicoides taxonomy and hence there is limited benefit for students in attempting to pursue a career in this area. This reflects an increasing emphasis on short term funding in India in areas of high return and in general more senior group leaders are now expected to span many disciplines. Overseas training is in some cases possible but tends to be a low priority in comparison to learning molecular techniques. What resources are commonly employed for identification? At least some hard copies of keys are used but access is limited to specialist units. No current resources are available online although the US Army Entomology website reference collection is heavily utilised by at least three workers. In general veterinary workers tend to send their samples to specialist centralised workers most commonly ZSI although some capacity was demonstrated in certain areas outside both these groups and the IBVNet project. There was little evidence of networking across Indian states to standardise identification leading to potential for groups to diverge in their opinions. At least one dedicated key is in preparation for the India Culicoides fauna. What proportion of workers have access to specialist equipment? This varied according to both the workers background and the state concerned. In general facilities for slide mounting and imaging specimens were more readily available to University and ZSI workers while molecular diagnostics were more easily access by veterinary workers. Specialist mounting and dissection equipment was viewed as being particularly difficult to obtain. There was also a heavy centralisation of resources with very limited facilities in field stations and a much better range of equipment in major cities. There was also a general perception that facilities in the south of India were less extensive although this was somewhat counteracted by outsourcing of some work to other areas (e.g. sequencing). Are internet-based resources readily available? While it was generally reported that internet access was available to most workers, there was a general lack of awareness of Culicoides resources. There was also variation in access between city and rural locations with the latter experiencing frequent loss of connectivity and limited availability. Available access also tended to relate to seniority in some cases. The vast majority of workers unconnected with the 9

9 project had no awareness of the IBVNet website or other primary contact sites (e.g. The Ceratopogonid information exchange). What assumptions do workers have on primary BTV vectors in their region? Where workers expressed an opinion there was general consensus on the primary BTV vectors being C. imicola and C. oxystoma in southern India. For the majority of vets attending there was interest in this area due to the potential for species ecology to lead to variable efficacy of control techniques. There was a strong consensus, however, that this fine-scale differentiation was not understood by the vast majority of field veterinary workers who applied treatments in the same way wherever outbreaks occurred. Practical outcomes of workshop All attending scientists either saw demonstrations or took part in morphological and molecular identification of Culicoides. Preliminary key of southern Indian Culicoides of the Avaritia group produced and trialled with workers. Preliminary sequencing of mitochondrial barcode (CO-I region) completed successfully for primary vector species with paired morphological slides. Recommendations for future capacity building in this area 1. Establish an online network for Culicoides identification in India allowing: - Rapid identification of expertise for veterinary workers - Networking of experts including sharing of both specimens and images - Access to international expertise where useful The location of this site was not decided at the meeting but there will be a requirement for users to update details regularly given previous experience in this area and there was general agreement that the website should be focussed rather than trying to include large quantities of information. 2. Improve the frequency of scientists communicating in this area - A yearly meeting could be attached to the ICAR Network Programme meeting - A yearly meeting could be organised as an additional part of a major veterinary conference - Ring trials could be organised for assessing consistency in identification and validated by an expert group 3. Improve the basic knowledge concerning Culicoides taxonomy - Potential for visit of students to other countries via travel grants/phd studentships - Where possible applications for funding should be made across groups rather than individually to build critical mass and improve collaboration 4. Joint applications under the current biodiversity system. 10

10 - The current biodiversity legislation in India which restricts movement of biological materials from India to other countries impinges on the free flow of materials. - Due to the time consuming nature of these applications, joint ownership should be considered across projects and countries and involving multiple collaborators. 11

11 Presentations Culicoides (Diptera: Ceratopogonidae) Identification in South Africa Introduction Karien Labuschagne The ARC-Onderstepoort Veterinary Institute, situated approximately 12 km north of the Pretoria city centre, was founded by Sir Arnold Theiler in He is considered the father of veterinary science in South Africa. Initially the Institute, then known as the Bacteriological Institute, was located at Daspoort to the west of Pretoria. Theiler s research needs soon outgrew the capacity at Daspoort and the farm De Onderstepoort was purchased for the construction of a new Institute. The new facilities were officially opened in 1908 and soon became known as the Onderstepoort Institute (Gutsche 1979; Vandenbergh 2009). Theiler and colleagues produced vaccines for many local diseases. African horse sickness (AHS) was abundant in the Onderstepoort area. Theiler discovered that it was caused by a virus (AHSV), that there were multiple strains and produced the first crude vaccine against it. His pioneering work would prove to be invaluable in the production of an effective live attenuated vaccine (Gutsche 1979; Vandenbergh 2009). Though Theiler suspected that AHSV was transmitted by an insect vector, he and other researchers, focusing on mosquitoes and ticks, were unsuccessful in discovering the vector. In 1943 Rene du Toit obtained a light trap from Germany and noticed thousands of small blood feeding midges belonging to the genus Culicoides in his collections. Within a year he proved that these insects were the vectors of not only bluetongue virus (BTV) but also of AHSV (du Toit 1944). Today we know that at least two species are involved in the transmission of AHSV in South Africa (Meiswinkel & Paweska 2003). Both these species, C. imicola Kieffer and C. bolitinos Meiswinkel, belong to the Imicola group in the subgenus Avaritia Fox. Worldwide more than Culicoides species have been described (Borkent 2012). Some 64 named and 11 unnamed viruses, 33 protozoa and 20 filarial worms may be transmitted by these insects (Meiswinkel et.al. 2004). Though only 41 and 46 species have been implicated in the transmission of micro-organisms and viruses respectively, more species are discovered, described and implicated in disease transmission on an annual basis (Meiswinkel et.al. 2004). Since 1995, 103 species, of which only 72 have been morphologically described, have been recorded in South Africa. Species descriptions are often based on relatively few specimens of one sex. This may complicate the identification of that species if only specimens of the un-described sex are collected. For the majority of species the sub adult stages as well as their breeding sites are unknown. A further complication with many of the yet un-described species is that they are quite often rare species of which only one or two specimens, usually of the same sex, are collected occasionally. To allow for intra species variation it is 12

12 recommended that at least 20 males and 20 females are slide mounted and compared before the species can be described. The application of molecular techniques would require additional specimens for genomic DNA extraction and sequencing. The recent development of a new non-destructive genomic DNA extraction technique implies that the same specimens can be used for molecular and morphological identification (Bellis 2013; Bellis et.al. 2013). For more than 90% of species, wing pattern is the most obvious and reliable character (Meiswinkel 1996), but for species groups or plain wing species this character may not be adequate (Fig. 1). For these species characteristics such as size and shape of the third palpal segment, coeloconica distribution of the antennal segments and colouration of the specimen are used. In the females the number and shape of the spermathecae can be used. The shape of the male genitalia can also be used to differentiate between species. This unfortunately means that the specimen needs to be slide mounted. Slide mounting is time consuming and it can takes up to 3 5 days to mount a single specimen and the subsequent drying process can take up to 4 months. The specimen needs to be adequately dry before examination to prevent damage. Dexterity and experience are needed to dissect each midge into six parts which all need to be mounted separately onto one microscope slide. Identification keys Current identification keys are a major stumbling block for the accurate identification of Culicoides species in Afrotropical countries. The three dichotomous identification keys most often used are: Khamala and Kettle (1971), Boorman and Dipeolu (1979) and Glick (1990). The problem in the South African context is that these keys were developed for East African, Nigerian and Kenyan species respectively. Although Fiedler (1951) did compile an identification key for South African species; the problem is that the number of species in South Africa as recorded by Fiedler has increased from 22 to presently more than 120 species in the southern African region (Meiswinkel 1996). Species of interest for India and South Africa Although most Afrotropical Culicoides species are restricted to sub-saharan Africa, a few species can be found outside Africa. E.g. C. imicola occurs throughout Africa, across most of southern Europe, the middle-, near- and Far East. It is unknown whether the remaining five African species (C. bolitinos, C. sp. # 107 (kwagga), C. loxodontis Meiswinkel, C. miombo Meiswinkel and C. tuttifrutti Meiswinkel, Cornet & Dyce) in the Imicola group occurs outside Africa. With two of the six species suspected to be the primary vectors of AHSV and BTV in South Africa this group is of great interest in disease epidemiology. The six members of the Imicola group can easily be distinguished on wing pattern by looking at cell R5, vein M2 and the 13

13 proximal base of the anal cell (Fig 1). Figure 1. Typical Culicoides wing showing venation and cells Another species group of interest is the Schultzei group. This group consists of eight species. Six (C. schultzei Enderlein, C. rhizophorensis Khamal & Kettle, C. kingi Austen, C. nevilli Cornet & Brunhes, C. enderleini Cornet & Brunhes and C. subschultzei Cornet & Brunhes) of these are known to occur in the Afrotropical region with four (C. nevilli, C. enderleini, C. rhizophorensis and C. subschultzei) only recorded from Africa. Culicoides schultzei and C. kingi have a wider distribution and can be collected across the near East and C. schultzei is suspected to occur as far east as India. Culicoides oxystoma Kieffer occurs in Israel and further east up to India and is suspected to occur in northern Africa. The only known localities for C. neoschultzei Boorman & Meiswinkel are Rumais, Al Khaburah and Haema in Oman. Bluetongue virus has been isolated from the Schultzei group of species (Mellor et.al. 1984) and it is suspected that C. oxystoma plays an important role in bluetongue (BT) outbreaks in India as was shown by the isolation of BTV serotype 1 from these midges (Dadawala et.al. 2012). It is often difficult to distinguish between the species within the Schultzei group. Culicoides neoschultzei, C. nevilli and C. enderleini are more easily distinguished by the spot in cell M4 that is unique to each species. The remaining species are more difficult to separate on wing pattern alone. The only differences between C. oxystoma and C. subschultzei seem to be the post stigmatic spots, with C. oxystoma having three and C. subschultzei having two spots. The wing patterns of the remaining three species, C. kingi, C. schultzei and C. rhizophorensis are very similar, but these species are more readily distinguished on the shape of the male genitalia (Fig. 5). In C. rhizophorensis the dististyles are much stronger and better developed than in the other members of this group. In C. schultzei and C. kingi the space between the horns of the apicolateral processes differ (Cornet & Brunhes 1994). 14

14 Figure 4. Schematic drawing of the male genitalia (Wirth & Hubert, 1989). Figure 5. Schematic drawings of the apicolateral process of the Schultzei group: C. schultzei (a d), C. kingi (e), C. enderleini (f), C. nevilli (g), C. subschultzei (h), C. rhizophorensi (i) and the dististyle of C. rhizophorensis (j) from Cornet & Brunhes, Recommendations: According to Borkent (2012) all the Culicoides species can be divided into 31 subgenera, 38 species groups and 173 species without subgenus- or group affiliation. On breaking this down further into South African species, the 103 species that have been collected since 1995 can be divided into nine subgenera (43 species), six species groups (21 species), eight species with no subgenus- or group affiliation and 31 un-described species. The current subgeneric classification of Culicoides species which is based on morphological traits need to be re-examined. 15

15 A combination of morphological, DNA extraction and sequencing techniques should be employed to re-examine each species. The morphological descriptions and DNA sequence analysis of all the as yet un-described species in South Africa need to be done. Acknowledgements: The Biological Sciences Research Council, UK (BBSRC) provided the funding to attend the India Bluetongue Vector Network (IBVNet) workshop on Culicoides identification which was hosted by the Tamil Nadu Veterinary and Animal Sciences University in Chennai, India. Drs Gert Venter, Kerstin Junker and Mr Arthur Spickett are thanked for their constructive comments on the manuscript. References BELLIS, G.A Studies on the taxonomy of Australasian species of Culicoides Latreille (Diptera: Ceratopogonidae). PhD thesis, The University of Queensland, Australia. BELLIS, G.A., DYCE, A.L., GOPURENKO, D. & MITCHELL, A Revision of the Immaculatus Group of Culicoides Latreille (Diptera: Ceratopogonidae) from the Australasian Region with description of two new species. Zootaxa, 3680 (1): BOORMAN, J. & DIPEOLU, O.O A taxonomic study of adult Nigerian Culicoides Latreille (Diptera: Ceratopogonidae) species. Occasional Publications of the Entomological Society of Nigeria, 22: BORKENT, A The Subgeneric Classification of Species of Culicoides - thoughts and a warning Last revised: February 20, 2012 Accessed in November CORNET, M. & BRUNHES, J Révision des espèces de Culicoides apparentées à C. schultzei (Enderlein, 1908) dans la region afrotropicale (Diptera, Ceratopogonidae). Bulletin de la Société entomologique de France, 99(2): DADAWALA, A.I., BISWAS, S.K., REHMAN, W., CHAND, K., DE, A., MATHAPATI, B.S., KUMAR, P., CHAUHAN, H.C., CHANDEL, B.S. & MONDAL, B Isolation of Bluetongue Virus Serotype 1 from Culicoides vector Captured in Livestock Farms and Sequence Analysis of the Viral Genome Segment-2. Transboundary and Emerging Diseases, 59: DU TOIT, R.M The transmission of Bluetongue and Horse-Sickness by Culicoides. Onderstepoort Journal of Veterinary Science and Animal Industry, 19: FIEDLER, O.G.H The South African Biting Midges of the Genus Culicoides (Ceratopogonid., Dipt.). Onderstepoort Journal of Veterinary Research, 25: GLICK, J.J Culicoides biting midges (Diptera: Ceratopogonidae) of Kenya. Journal of Medical Entomology, 27 (5): GUTSCHE, T There was a man. The life and times of Sir Arnold Theiler K.C.M.G. of Onderstepoort. Cape Town: Howard Timmins KHAMALA, C.P.M. & KETTLE, D.S The Culicoides Latreille (Diptera: Ceratopogonidae) of East Africa. Transactions of the Royal Entomological Society of London, 123: MEISWINKEL, R. & PAWESKA, J.T Evidence for a new field Culicoides vector of African horse sickness in South Africa. Preventive Veterinary Medicine, 60: MEISWINKEL, R., VENTER, G.J., NEVILL, E.M., Vectors: Culicoides spp. In: Coetzer, J.A.W., Tustin, R.C. (Eds.). Infectious Diseases of Livestock with special reference to southern Africa. Oxford University Press, Cape Town,

16 MEISWINKEL, R Afrotropical Culicoides: biosystematics of the Imicola group, subgenus Avaritia (Diptera: Ceratopogonidae). With special reference to the epidemiology of African horse sickness. M.Sc. thesis, University of Pretoria. MEISWINKEL, R Wing picture atlas. Unpublished data MELLOR, P.S., OSBORNE, R. & JENNINGS, D.M Isolation of bluetongue and related viruses from Culicoides spp. in the Sudan. Journal of Hygiene Cambridge, 93: VANDENBERGH, S.J.E The story of a disease: a social history of African horse sickness c MA. (History) thesis, University of Stellenbosch WIRTH, W.W. & HUBERT, A.A The Culicoides of Southeast Asia (Diptera; Ceratopogonidae). Memoirs of the American Entomological Institute. 44:

17 DNA barcoding for species identifications: applications to research of Culicoides and other arthropods. Dr. David Gopurenko Introduction The Culicoides Latrielle 1809 comprises a species rich genus within Ceratopogonidae (Diptera) found in most terrestrial regions of the globe. Culicoides are generally haematophagous as adults and many species are of biosecurity concern as they carry and spread disease-causing viral pathogens among livestock off which they feed. Species level identifications using traditional taxonomic approaches are mainly reliant on examination of subtle morphological features present at antennae, palps, reproductive organs, and wings. Species identifications are frequently complicated by the subtlety of the morphological characters available for analysis, and in many cases species diagnostic features may be present at one sex but absent in the other. Species identifications of specimens which are degraded or at larval stages are largely impossible. As a consequence, traditional species delimitation and identification of Culicoides is likely to be restrictive under circumstances where rapid bio-inventories are required, or in cases where closely related species are morphologically indistinct and cannot be distinguished. Given the prevalence of pathogen vector association among many of the Culicoides, and the importance of this genus to global biosecurity, it is essential that the provision of accurate species-level diagnostics for this genus is available. DNA barcoding is a genetic methodology which is increasingly used to complement traditional taxonomic practises, particularly for the Culicoides, as it can provide rapid and accurate species diagnostics, and also be used as a means to discover novel or cryptic species diversity. Here I provide some discussion on the theory behind DNA barcoding, its use and applications to insect research, criticisms aimed at the methodology, and finally the exciting prospects of an integrative taxonomic approach incorporating DNA barcoding to improve taxonomic research on important groups such as Culicoides. 18

18 DNA barcoding: description and theory behind the approach The promise of DNA barcoding as a standardised method to provide rapid and accurate species level identification has been widely touted since its first report early this century (Hebert et al. 2003). DNA barcoding is now a global scientific enterprise aiding taxonomy and species discovery. The premise of DNA barcoding is simple: the majority of eukaryotic species on the planet may be genetically identified by their unique nucleotide sequence at a standardised genomic region(s). In the case of animals, the standard diagnostic target advocated for analysis is a > 500 base pair portion of the 5 -portion of the cytochrome c oxidase I (COI) gene present in the mitochondrial genome (Hebert et al. 2003). There are several reasons why this gene region is advocated for DNA barcode analysis, and why nucleotide sequence at this gene can be used as a diagnostic barcode for species identity, given an acceptable range of intra-specific variation. Before addressing that, it is essential to define some terms used here and to elaborate on some of the theoretical underpinnings of DNA barcoding. When we refer to species, we are generally referring to the widely used and accepted biological species concept, which, according to the definition of Mayr (1995), species are defined as groups of interbreeding natural populations that are reproductively isolated from other such groups. The process of speciation describes the splitting of an ancestral species as two or more derived daughter species; the causes of speciation are myriad and are not discussed here. Stochastic and demographic processes operating during and after speciation generally result in a series of temporal changes in the diversity of genetic lineages present within the populations of derived daughter species (refer Avise 2000). For example, immediately after speciation, two daughter species (or sister species) generally share much of their genetic diversity owing to their shared ancestry. If this genetic diversity is measured as a branching tree representative of genetic distances among genetic lineages present within the species, then the recently separated sister species will share many branches (Fig. 1), a condition referred to a polyphyletic sharing. After some time has passed and assuming an absence of gene flow between sister species, the older lineages shared among the sister species go extinct by genetic 19

19 drift and new lineages unique to each species emerge as a result of novel mutations. In some circumstances it is not uncommon for one of the recently diverged species to be paraphyletic or partially share some closer related lineages with respect to the other species. Given sufficient time since speciation, all shared genetic lineages between the species go extinct and the two species attain a state of reciprocal genetic monophyly. From that point in time, genetic distances within each of the species becomes less as older lineages are lost, and genetic distance between the species increase as novel mutations within the species accumulate. DNA barcoding as a method of species identification and discovery is most powerful as a method of species identification and species delimitation when sister species are reciprocally monophyletic. Hebert et al. (2003 and subsequent papers) in their seminal paper referred to the use of threshold genetic distance based approaches to species identification. The idea is simple: when sister species are reciprocally monophyletic at a target gene, they can often be readily distinguished based on genetic distances at that target gene, because under reciprocal monophyly, genetic distances within each species is generally less than the distances between the species (Fig. 2) i.e., there is a residual gap in the genetic distance between species after intraspecific variation is accounted for, and this is referred to as the DNA Barcode gap (Meyer and Paulay, 2005). Empirical evidence collected over the last decade indicates there is no universal measure of the minimal genetic distance threshold or DNA barcode gap between species pairs (such as the adoption of a > 3 % sequence difference criteria, or 10- times gap rule; see later) that can be reliably used as a standard distance metric to distinguish among all species pairs. Rather, a condition of reciprocal monophyly must generally be met in order for the DNA barcoding to accurately distinguish all members of two species compared, and that can only gauged by adequately sampling the diversity of the examined species over their geographic distributions. It is important however, that a gene region used for DNA barcoding can rapidly track the history of a speciation process. Such a gene region would need to be relatively free of recombination which causes reticulations among gene lineages, evolve at a rapid rate to allow emergence of new lineages unique to species, and be sensitive to demographic processes that cause random fixation of lineages within 20

20 species and also loss of shared lineages between sister species. The 5 -portion of the COI gene present in the mitochondrial genome was recognised by Hebert et al. 2003, as having all these attributes. The gene has a rapid rate of mutation and, as it is part of the mitochondrial genome, it is maternally inherited without recombination. As a consequence new mutations at this gene can rapidly accumulate within a species, and pass down from mother to offspring as distinct genetic lineages representative of the genetic diversity within the species. The rate of mutation at COI is sufficiently high to allow analysis of population genetics, and because of its strict maternal inheritance, mitochondrial COI lineages in a species population can be greatly affected by demographic processes such as genetic drift and population bottlenecks. These processes cause some genetic lineages to go to extinct and others to increase in frequency in the population. Subsequently the diversity of mitochondrial COI lineages within a species generally goes to fixation within a species much faster than at nuclear encoded genes (Avise 2000). Genes encoded within the mitochondrial genome such as COI, will as a result of population processes, approach species monophyly much faster than can occur at nuclear genes and will therefore have greater facility for distinguishing recently diverged species. COI has an added attribute as a DNA barcode region, in that primers required for PCR amplification are readily available and generally universal (or near so) among a broad variety of taxa. Finally, COI is a protein coding gene, free of non-coding or indel regions, and this can be used to good advantage for sequence alignment, primer design, and for testing for presence of confounding pseudo-genes and other non-target gene regions. One of the setbacks of using COI for analyses is that it rarely provides reliable resolution of phylogenetic relationships above the sister species level. The rate of mutation is high enough at this gene that nucleotide homoplasy (base saturation) is generally a prominent feature obscuring phylogenetic relationships among more distantly related species. However the goal of DNA barcoding is resolution of alpha-taxonomy (species units); analyses of higher level phylogenetic relationships among species are generally more adequately delivered by assaying genes with slower rates of evolution than COI. 21

21 Identification of ambiguous specimens One of the major advantages of using DNA barcoding as an investigative tool for biodiversity analyses, is its unique facility to provide species identifications for specimens that are either physically degraded (dewaard et al. 2010), or of life stages with taxonomically intractable morphology (Hebert et al. 2004; Gopurenko et al. 2013). Typically, these diagnostic efforts construct and or rely on pre-existing DNA barcode libraries of taxonomically described and voucher species which can be used as references in distance based comparisons against query specimens. For example, a recently released DNA barcode analysis of leafhoppers and kin (Hemiptera; Auchenorrhyncha) reported DNA barcode matches of morphologically indistinct nymphs to taxonomically described adult morpho-species (Gopurenko et al. 2013). The same study however also reported a portion of the sampled nymphs had novel DNA barcodes not matched to any existing adult specimens, indicating some of the total adult leafhopper diversity present at the sample location was un-sampled. This novel diversity would have remained undetected if specimen identifications were based solely on the initial sampling effort targeted at the adult specimens. Taxonomic keys available for many insects describe and identify species based primarily on specimens of one sex or life history stage; keys for identification of other forms are either unavailable, or at best, limited to the higher levels of classification (e.g. Dmitriev 2009). As a result, morphological surveys of most insect groups may underestimate species diversity due to presence of morphologically indistinguishable adult species, and excessive presence of physically degraded or immature specimens present at a location are not conspecific with any sampled adults. The extent of this hidden diversity will vary among sampling efforts according to both the sampling methods employed for specimen collection, the seasonality of adult / immature specimen occurrence at sample locations, and most importantly to the levels of morphological convergence present among the taxa examined. To this end, inclusion of DNA barcoding in future insect sampling efforts will provide a means to not only identify presence of hidden diversity, but also provide ecological information concerning the seasonality of occurrence among life stages of particular species (Ahrens et al. 2007). For these reasons, DNA barcoding could effectively inform the design and duration of ongoing Culicoides survey efforts, particularly if a 22

22 predetermined threshold of hidden or seasonal diversity was exceeded in pilot analyses. Criticisms of DNA barcoding: One major criticism of DNA barcoding, as a method for species identification and discovery, concerns the lack of universal operational criteria for assigning a specimen barcode to a given species. Methods of species identification based on sequence distance statistics, such as the DNA barcode gap (Hebert et al. 2003; Hebert et al. 2004b) and other similar threshold distance approaches (Ferri et al. 2009), are frequently used to derive operational standards for species delimitation. Distance based approaches are also frequently used in DNA barcoding studies to signify potential presence of cryptic diversity in morphologically cohesive species when limits are exceeded (Léfebure et al. 2006). These distance based approaches to species identification and delimitation are computationally simple, are based directly on the empirical DNA barcode sequence data and are seemingly applicable across a broad variety of fauna (Hebert et al. 2004b), albeit usually tailored for the focal study taxa. For example, sequence differences greater than 3 % among faunal conspecifics at COI are infrequently observed in empirical population genetic surveys (Ferguson 2002; Hebert et al. 2003; Hebert et al. 2004a& 2004b) and consequently this ~3 % value is used as a general threshold limit for demarcating species in DNA barcode efforts. Such empirically determined threshold distance criteria for species delimitation have been largely successful for species identifications at many groups of focal taxa. However, sequence differences exceeding 3 % (or other similar empirically derived thresholds) in some reproductively cohesive insect species can occur simply as a result of historical allopatric separations in the distribution of a species, and are likely to be more evident as the scale of both geographic sampling and sample replication is increased in DNA barcode surveys (Trewick 2008; Bergstein et al. 2012; but see Lukhtanov et al. 2009). So, adoption of an arbitrary threshold limit used for species delimitation, can result in erroneous over estimation of species diversity when there are deep population divisions present in species exceeding the threshold, and under estimation of species diversity when sister species have very recently diverged and show level of divergence less than the threshold. 23

23 Furthermore, the distance threshold approach has been criticised as computationally naïve regarding macro-evolutionary processes (Will & Rubinoff 2004) and vulnerable to error depending on metrics used (Meier et al. 2008) and the extent of congeneric sampling employed (Jansen et al. 2009). Alternative approaches of species delimitation using DNA barcode data include character based analyses to detect parsimonious sharing of species diagnostic nucleotides (Davis and Nixon 1992; DeSalle et al. 2005) and more recent theory-based statistical approaches which employ population coalescent modelling to predict species boundaries from single or multi-locus data (Pons et al. 2006; Rosenberg 2007; Cummings et al. 2008; Yang & Rannala 2010). These new approaches are likely to increase analytical rigour in future DNA barcoding surveys (Fujita et al. 2012). Regardless, DNA barcoding, like all single gene approaches to species identification ignore issues of genetic paraphyly (Trewick 2008) when single locus gene trees are not in accord with species trees due to a variety of biological, demographic, geographic or temporal processes (Doyle 1997; Avise 2000). Deep genetic divisions observed at a single locus within a morphospecies may equally signal presence of ancestral lineage retention or interspecies introgression as plausible alternatives to sympatry or parapatry of morphologically cryptic species (Funk and Omland 2003). Resolution of these issues can only be arrived at by examining multiple independent forms of data in an integrative context. DNA barcoding and Integrative Taxonomy Increasingly there are calls to the DNA barcode community to treat DNA barcode species identifications as hypotheses to be tested in an integrative taxonomic framework, incorporating multiple independent data (behavioural, ecological, molecular, morphological) to examine the cohesiveness of species boundaries defining examined taxa (Dayrat 2005; DeSalle et al. 2005; Padial et al. 2010; Schlick-Steiner et al. 2010; Goldstein and DeSalle 2011). This integrative taxonomic approach has been successfully used in DNA barcode efforts recently applied to Culicoides. In these studies, independent data from taxonomic, morphological, geographical and ecological analyses, were incorporated with multi gene sequence data including DNA barcodes to address a range of questions, including identification of reproductive boundaries between sympatric sister species (Bellis et al. 2013a), 24

24 identification and description of novel species (Bellis et al. 2013b; Bellis et al. 2014a), and identification of putative exotic pest midge incursions (Bellis et al 2014b in review). These papers serve as models of what can be achieved when data from DNA barcodes and others genes are co-analysed along side independent data describing the taxonomy, ecology and geography of voucher specimens. The power of this approach is readily apparent in the analyses of Culicoides immaculatus morph variants conducted by Bellis et al. (2013a). Those authors examined DNA barcode and independent nuclear gene sequences from sampled C. immaculatus morphs to test the hypothesis that a 2 nd potentially cryptic species (now described as C. shivasi) co-existed with C. immaculatus in northern Australia. The initial cryptic species hypothesis was raised owing solely to evidence of a subtle difference in tergite morphology observed among males specimens present at the area. Subsequent DNA barcoding delineated two genetic species in agreement with the male morph differences observed (Fig. 3). This species schism was similarly confirmed at sequences of an independent nuclear gene. The real power of the test however lay in the fact that the two morpho-species did not share mitochondrial or nuclear lineages at locations where they co-occurred, indicating there was an absence of gene flow between them. This evidence of sympatric genetic segregation was strongly supportive of the independent status of these two species and provided confirmation of the initial subtle evidence suggested by analysis of male morphological features. More information and descriptions of the diagnostic features separating C. immaculatus from C. shivasi can be obtained at Bellis et al. (2013a). Conclusions DNA barcoding using mitochondrial COI is in most cases, a highly effective tool to assist in rapid species identification of insects, particularly when specimens are ambiguously identified using traditional taxonomic analyses. It is also an efficient means of generating initial hypotheses concerning species limits, cryptic species presence, and for providing estimates of species diversity in empirical surveys. Furthermore novel means of DNA barcode sequence analysis are emerging which provide a means for researchers to avoid relying on naïve distance metrics for automated species delimitation and identification. But species hypotheses based on DNA barcoding must always be treated with the caveat that such analyses are based on a single gene that is occasionally prone to historical processes which may distort 25

25 its ability to accurately track a species history. It is therefore not a standalone methodology and should be used in preparation for, or and in concert with independent analyses of taxonomic, ecological, genetic and geographic data in an integrative taxonomic framework to provide greater accuracy of species resolutions. REFERENCES AHRENS, D., MONAGHAN, M.T. AND VOGLER, A.P. (2007). DNA-based taxonomy for associating adults and larvae in multi-species assemblages of chafers (Coleoptera: Scarabaeidae). Molecular Phylogenetics and Evolution 44: AVISE, J.C. (2000). Phylogeography: the History and Formation of Species. Harvard University Press, Cambridge, MA. BELLIS GA, DYCE AL, GOPURENKO D & MITCHELL A. (2013a). Revision of the Immaculatus group of Culicoides Latreille (Diptera: Ceratopogonidae) from the Australasian biogeographic region with description of two new species. Zootaxa 3680(1): BELLIS G, KIM HC, KIM MS, KLEIN TA, LEE DK & GOPURENKO D. (2013b) Three species of Culicoides Latreille (Diptera: Ceratopogonidae) newly recorded from the Republic of Korea. Zootaxa 3718(2): BELLIS GA, DYCE AL, GOPURENKO D, YANASE T, YANASE T, GARROS C, LABUSCHAGNE K & MITCHELL A. (2014a). Revision of the Culicoides (Avaritia) Imicola complex Khamala & Kettle (Diptera: Ceratopogonidae) from the Australasian region. Zootaxa 3768(4): BELLIS G, GOPURENKO D, COOKSON B, POSTLE A, HALLING L, HARRIS N & MITCHELL A (2014b) Identification of incursions of Culicoides Latreille species (Diptera: Ceratopogonidae) in Australasia using morphological techniques and DNA barcoding. Austral Entomology (in review). BERGSTEN, J., BILTON, D.T., FUJISAWA, T., ELLIOT, M., MONAGHAN, M.T., BALKE, M., HENDRICH, L., GEIJER, J., HERRMANN, J., FOSTER, G.N., RIBERA, I., NILSSON, A.N., BARRACLOUGH, T.G. AND VOGLER A.P. (2012). The effect of geographical scale of sampling on DNA barcoding. Systematic Biology 61: CUMMINGS, M.P., NEEL, M.C. AND SHAW, K.L. (2008). A genealogical approach to quantifying lineage divergence. Evolution 62: DAVIS, J.I. AND NIXON, K.C. (1992). Populations, genetic variation, and the delimitation of phylogenetic species. Systematic Biology 41: DAYRAT, B. (2005). Towards integrative taxonomy. Biological Journal of the Linnean Society 85:

26 DESALLE, R., EGAN, M.G. AND SIDDAL, M. (2005). The unholy trinity: taxonomy, species delimitation and DNA barcoding. Philosophical Transactions of the Royal Society B 360: DEWAARD, J.R., MITCHELL, A., KEENA, M.A., GOPURENKO, D., BOYKIN, L.M., ARMSTRONG, K.F., POGUE, M.G., LIMA, J., FLOYD, R., HANNER, R.H., HUMBLE, L.M. (2010). Towards a global barcode library for Lymantria (Lepidoptera: Lymantriinae) tussock moths of biosecurity concern. Public Library of Science One 5: e DMITRIEV, D.A. (2009). Nymphs of some Nearctic leafhoppers (Homoptera, Cicadellidae) with description of a new tribe. ZooKeys 29: DOYLE, J.J. (1997). Trees within trees. Genes and species, molecules and morphology. Systematic Biology 46: FERRI, E., BARBUTO, M., BAIN, O., GALIMBERTI, A., UNI, S., GUERRERO, R., FERTÉ, H. BANDI, C., MARTIN, C. AND CASIRAGHI M. (2009). Integrated taxonomy: traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda). Frontiers in Zoology 6: FERGUSON, J.W.H. (2002). On the use of genetic divergence for identifying species. Biological Journal of the Linnean Society 75: FUJITA, M.K., LEACHÉ, A.D., BURBRINK, F.T., MCGUIRE, J.A. AND MORITZ, C. (2012). Coalescent-based species delimitation in an integrative taxonomy. Trends in Ecology and Evolution 27: FUNK, D.J. AND OMLAND, K.E. (2003). Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annual Review of Ecology, Evolution and Systematics 34: GOLDSTEIN, P.Z. AND DESALLE, R. (2011). Integrating DNA barcode data and taxonomic practice: Determination, discovery, and description. Bioessays 33: GOPURENKO D, FLETCHER MJ, LÖCKER H & MITCHELL A. (2013) Morphological and DNA barcode species identifications of leafhoppers, planthoppers and treehoppers (Hemiptera: Auchenorrhyncha) at Barrow Island. In: Records of the Western Australian Museum, Supplement 83: The Terrestrial Invertebrate Fauna of Barrow Island (eds, Nihara R. Gunawardene, Jonathan D. Majer, Christopher K. Taylor and Mark S. Harvey), pp Western Australian Museum Publications, Perth. ISBN: ISSN X 27

27 JANSEN, G., SAVOLAINEN, R., AND VEPSALAINEN, K. (2009). DNA barcoding as a heuristic tool for classifying undescribed Nearctic Myrmica ants (Hymenoptera: Formicidae). Zoologica Scripta 38: HEBERT, P.D.N., CYWINSKA, A., BALL, S.L. AND DEWAARD, J.R. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B. Biological Sciences 270: HEBERT, P.D.N., PENTON, E.H., BURNS, J.M., JANZEN, D.H. AND HALLWACHS, W. (2004a). Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences of the United States of America 101: HEBERT, P.D.N., STOECKLE, M.Y., ZEMLAK, T.S. AND FRANCIS, C.M. (2004b). Identification of birds through DNA barcodes. Public Library of Science Biology 2: e312. LEFÉBURE, T., DOUADY, C.J., GOUY, M. AND GIBERT, J. (2006). Relationship between morphological taxonomy and molecular divergence within Crustacea: proposal of a molecular threshold to help species delimitation. Molecular Phylogenetics and Evolution 40: LUKHTANOV, V.A., SOURAKOV, A., ZAKHAROV, E.V. and HEBERT P.D. (2009). DNA barcoding Central Asian butterflies: increasing geographical dimension does not significantly reduce the success of species identification. Molecular Ecology Resources 9: MAYR, E. (1995) Species, classification, and evolution. Pp in R. Arai, M. Kato, and Y. Doi (eds.) Biodiversity and Evolution. National Sciences Museum Foundation, Tokyo. MEIER, R., ZHANG, G.Y. AND ALI, F. (2008). The use of mean instead of smallest interspecific distances exaggerates the size of the barcoding gap and leads to misidentification. Systematic Biology 57: MEYER C. P., PAULAY G. (2005) DNA barcoding: Error rates based on comprehensive sampling. PLoS Biol. 3: MONAGHAN, M.T., WILD, R., ELLIOT, M., FUJISAWA, T., BALKE, M., INWARD, D.J.G., LEES, D.C., RANAIVOSOLO, R., EGGLETON, P., BARRACLOUGH, T.G. AND VOGLER, A.P. (2009). Accelerated species inventory on Madagascar using coalescent-based models of species delineation. Systematic Biology 58: PADIAL, J.M., MIRALLES, A., DE LA RIVA I. AND VENCES M. (2010). The integrative future of taxonomy. Frontiers in Zoology 7:16. 28

28 PONS, J., BARRACLOUGH, T.G., GOMEZ-ZURITA, J., CARDOSO, A., DURAN, D.P., HAZELL, S., KAMOUN, S., SUMLIN, W.D. AND VOGLER, A.P. (2006). Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Systematic Biology 55: ROSENBERG, N.A. (2007). Statistical tests for taxonomic distinctiveness from observations of monophyly. Evolution 61: SCHLICK-STEINER, B.C., STEINER, F.M., SEIFERT, B., STAUFFER, C., CHRISTIAN, E. AND CROZIER, R.H. (2010). Integrative taxonomy: A multisource approach to exploring biodiversity. Annual Review of Entomology 55: TREWICK, S.A. (2008). DNA Barcoding is not enough: mismatch of taxonomy and genealogy in New Zealand grasshoppers (Orthoptera: Acrididae). Cladistics 24: WILL, K.W. AND RUBINOFF, D. (2004). Myth on the molecule: DNA barcodes for species cannot replace morphology for identification and classification. Cladistics 20: YANG, Z. AND RANNALA, B. (2010). Bayesian species delimitation using multilocus sequence data. Proceedings of the National Academy of Sciences of the United States of America 107:

29 Figure 1 Evolution of gene genealogies (lineages) over time with splitting of an ancestral species and subsequent speciation as two descendent daughter species A & B. At time in the past equivalent to N generations, daughter species are polyphyletic and equally share the genetic variation present in the ancestral species. At time 2N, some of the older lineages have gone extinct at both daughter species and novel lineages have emerged; species A retains some lineages which are more closely related to species B and is therefore paraphyletic with respect to species A. At time 4N, daughter species A and B share no old lineages, each has an assemblage of unique novel lineages, and the two species can be described as reciprocally monophyletic. 30

30 Figure 2 Reciprocal monophyly present at the gene genealogies of two sister species A & B descendent from a shared ancestor. The genetic distance (d) within each species is less than the genetic distance between the two species. The DNA barcode gap demarcates the residual genetic distance between the species after intraspecific variation is accounted for. The species may or may not have evolved unique diagnostic morphological features, regardless they can be distinguished by DNA barcoding. 31

31 Figure 3 Integrative taxonomic assessment of variant Culicoides immaculatus morphs present in northern Australia. Neighbour joining (NJ) tree of DNA barcodes from C. immaculatus and newly described C. shivasi sampled from Northern Territory (NT) in Australia (NJ tree bootstrap supports > 70% (10,000 reps) as indicated; outgroup = C. molestus; scale bar = 2 % COI sequence divergence). Symbols at NJ tree tips indicate sample sites where the two species co-occur. The red dashed boxes in the tree indicate genetic monophyly of specimens at an independent nuclear gene, supportive of the monophyly detected at the DNA barcodes. Male wing of each species indicated to the right of the tree (C. immaculatus above; C. shivasi below). The Caption box to the left indicates the levels of agreement among the various independent analyses in regards to the species delimitation of C. immaculatus and C. shivasi (refer Bellis et al. 2013a). Here it can be seen that independence of sympatric species C. immaculatus and C. shivasi is supported by COI and nuclear gene DNA barcodes, in agreement with analyses of male tergite morphology. Interestingly wing pattern, which is frequently used to distinguish Culicoides species, is not diagnostic for these two species. 32

32 THE C. IMICOLA AND C. SCHULTZEI GROUPS IN EUROPE AND AFRICA DR. CLAIRE GARROS Dr Garros s paper is located here in published format: IDENTIFICATION OF CULICOIDES IN AUSTRALIA AND SE ASIA DR. GLENN BELLIS Dr Bellis s paper is located here in published format: In addition, Dr Bellis provided the following summary tables. Table 1 BTV Vector status of Austro-Oriental Culicoides species Species Association with hosts Laboratory infection Field isolation/ detection Laboratory transmission C. (Avaritia) actoni# C. (Avaritia) brevitarsis# C. (Avaritia) fulvus# C. (Avaritia) imicola C. (Avaritia) wadai# NT C. (Remmia) oxystoma# NT C. (Hoffmania) peregrinus# NT C. (Avaritia) dumdumi# + + NT NT C. (Avaritia) brevipalpis# + + NT NT C. (Avaritia) nudipalpis + NT + NT C. (Avaritia) asiatica + NT + NT NT = not tested # = present in both India and Australasia 33

33 Table 2. Representation and proposed origins of Culicoides subgeneric groupings in Australasia, SE Asia and India Number of species present in region Subgeneric group Australasia SE Asia SE Asia + India Australasia + SE Asia India Australasia + India Biogeographic Origin (Dyce 2001) Drymodesmyia Neotropical incursion Bancrofti gp Australasian endemic Tokunagahelea Australasian endemic Melanesiae gp Australasian endemic Marksomyia Australasian endemic Antennalis gp Australasian endemic Victoriae gp Australasian endemic Coronalis gp Australasian Kusaiensis gp Australasian Immaculatus gp Australasian Purus gp Australasian Costalis gp Australasian Williwilli gp Australasian Ornatus gp Australasian/Indian Molestus gp Australasian/Indian/African) Clavipalpis gp Gondwanan Trithecoides Indian/African Shermani gp Indian/African Meijerehelea Indian/African Avaritia Indian/African Hoffmania Neotropics, Africa, India Haemophoructus Oriental Shortti gp Oriental Remmia African Monoculicoides/ Wirthomyia/ Pontoculicoides/ Beltranmyia/ Oecacta Palaearctic 34

34 CULICOIDES IDENTIFICATION IN EUROPE DR BRUNO MATHIEU Dr Mathieu s paper is located here in published format: STUDY ON CULICOIDES MIDGES IN RELATION TO BLUETONGUE DR.K.GANESH UDUPA, DR. RAVINDRA BHOYAR AND DR. SATHEESHA S.P. KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, KARNATAKA A study on Culicoides species associated with livestock and their relevance to bluetongue infection in Tamil Nadu was undertaken by Dr.K.Ganesh Udupa, as a part of Ph.D programme under the guidance of Dr. J.Ramkrishna, Professor, in TANUVAS, Chennai. In the present study, the Culicoides midges were collected at 13 livestock farms located at different agroclimatic zones of Tamil Nadu during July to September 2000 (monsoon season), December to February 2001 (post monsoon season) and May and June 2001 (summer season) using down-draught light traps. A total of 26 species of Culicoides were collected during the study. Culicoides imicola and C.oxystoma were found in all the 9 farms during the monsoon period, followed by C.peregrinus, C.orientalis and C.similis. In most of the farms, abundance of C.oxystoma was found to be high. Abundance of Culicoides midges was high in post monsoon season than during monsoon in majority of the farms. Culicoides imicola and C.oxystoma were prevalent in all the 12 farms followed by C.peregrinus. The upsurge of C.imicola was noticed during post monsoon season in all the farms while the abundance of C.oxystoma was low during post monsoon season. During summer season (May and June 2001), when Culicoides midges were collected from four livestock farms, the abundance of C.imicola was found to be reduced substantially. Bluetongue virus has been isolated from Culicoides midges (mixed species) in embryonated chicken eggs and BHK21 cells. The presence of BTV was confirmed by AGID and indirect FAT. Bluetongue virus was also isolated from C.imicola, C.oxystoma and C.peregrinus species using embryonated chicken eggs. The presence of bluetongue virus in crushed smears of C.imicola, C.oxystoma and C.peregrinus was identified by using indirect FAT and indirect IPT and 20 (66.67%) out of 30 pools of Culicoides midges were found to be positive to BTV. Antigen capture ELISA was performed on extracts of these midges and presence of BTV was demonstrated. Analysis of association with climatological elements on the abundance of Culicoides midges revealed a positive correlation between relative humidity and vector abundance during post monsoon period. Epidemiological aspects of bluetongue infection in Bidar district of Karnatka was studied by Dr. Ravindra Bhoyar for the M.V.Sc programme under the guidance of Dr.K.Ganesh Udupa and midges were collected in 3 different e cattle sheep and goat 35

35 farms at fortnightly intervals from October 2002 to August 2003 and midges were found to be abundant throughout the year and their abundance was found to be highest during South West monsoon aqnd was lowest during premonsoon season. A total of 14 species were identified among which C.imicola and C.oxystoma were found to be dominant species. There was a positive significant correlation between midges abundance and relative humidity. The presence of BTV antigen was detected in one of the 11 pools of Culicoides midges using AGID test. An attempt was made to find the survival period of Culicoides midges under starvation and feeding of sugar and blood under confinement in jars. The monthly monitoring of seronegative goats and cattle to detect the possible seroconversion to BTV revealed that seroconversions occurred during the periods of higher abundance of midges indicating the active circulation of virus between host and vectors. Study on certain aspects of vector capacity of Culicoides species in relation to bluetongue was studied by Dr. Satheesha, S.P for the M.V.Sc programme under the guidance of Dr.K.Ganesh Udupa, in Veterinary College, Bidar, Karnataka. The study was conducted from June 2004 to July The traits for the vector capacity studied are prevalence, abundance, population dynamics, parity, voltinism, host preference, feeding rate and survival period. The midges were collected using down draught light trap in the sheds of cattle, buffaloes, sheep and goats and their abundances were higher in the month of September 2004 in all the sheds. A totalk of 15 species of Culicoides midges were identified iut of which C.imicola and C. oxystoma were found to be dominant in all these sheds. Based on the abundances of dominant Culicoides species in light trap collections and aspirations from the body of the animals, it was evident that C. imicola prefers goats followed by sheep, cattle and buffaloes. The order of preferences of C. oxystoma is buffaloes, cattle, sheep and goats. The extent of host preferences by these Culicoides species may govern the degree of disease risk in host species. Both C. imicola and C. oxystoma were found to be multivoltine and substantial numbers of nulliparaous females of these species were found during all the seasons indicating their continued breeding activity. Periods of higher abundances of parous females were found in both species indicating the periods of disease risk. Emergence of C. imicola and C. oxystoma were studied using a collapsible semi automatic tent type emergence trap and the suitable breeding habitats were found in the study area. Swarms of both the dominant species were collected using sweep net and found to be male dominant. Swarms may be the markers of midges activity in a locality. Analysis for finding the association between the abundance of dominant species and the climatic factors indicated significantly negative correlation of abundance of C.imicola with maximum temperature and its positive correlation with relative humidity. There was no significant influence of climatic factors on the abundance of C. oxystoma. Both the species were fed with the blood in the laboratory using microcapillary tubes and survival period of 36-60m hours upon blood feeding was found. Based on the results of above traits of vector capacity, it was concluded that C.imicola and C. oxystoma rank first and second respectively as candidate vector species of BTV and related species. 36

36 A project was undertaken by Dr. K.Ganesh Udupa and co-workers in Bidar, Karnataka, to identify the potential Culicoides species as vectors of bluetongue disease of sheep,under the financial assistance of Karnataka Sheep and Wool Development Corporation Limited, Hebbal,Bangalore The study comprised of mapping for the prevalence of Culicoides midges associated with sheep in different agroclimatic zones of Karnataka. Mapping for the prevalence of Culicoides midges associated with sheep in different agroclimatic zones was conducted probably for the first time in Karnataka. Culicoides midges were collected near sheep flocks at 12 districts covering 8 Agroclimatic zones of Karnataka during monsoon and post monsoon seasons. Substantial Culicoides abundances were noticed both in rainy and post rainy seasons ranging from 365 to and 69 to respectively. Thus, Culicoides midges were found to be widely being prevalent in the state. However, there was no significant difference (P 0.050) in their abundances in rainy and post rainy seasons.a total of 19 species of Culicoides midges found to prevalent near the sheep flocks at different agroclimatic zones of Karnataka. Culicoides imicola, C. oxystoma and C. perigrinus were commonly been found in various agroclimatic zones.potentiality of Culicoides imicola and C. oxystoma as vectors of bluetongue with the ranking of first and second respectively was identified on the basis of their wider distribution, consistent presence with higher abundances. Seasonality of C. imicola was identified based on its abundances increasing in post monsoon season coinciding the months of occurrences bluetongue outbreaks in Karnataka. Swarms of Culicoides midges near the sheds of domestic animals have been studied. Practical utility of appreciation of swarms near the flock has been explored. Since swarms indicate the activity of Culicoides midges near the flock, visual appreciation of the swarms with their identification of the species composition of may help to immediate suitable measures to repel the midges and thus to control their bites. Association between occurrence of bluetongue and prevalence and abundance of species of Culicoides midges was analyzed to find the potential vector species of Culicoides for bluetongue virus. It has been noted that in 9 out of 12 districts where Culicoides imicola and C.oxystoma were found, bluetongue antibodies and occurrences bluetongue disease in sheep were also recorded. It suggests the presence of bluetongue disease, evidence of exposure to the bluetongue virus and the presence of potential vector species of Culicoides midges for bluetongue virus in a geographical area. In addition, when Culicoides midges were collected from the seven sheep flocks at the time of bluetongue outbreak; substantially higher abundances of Culicoide imicola were found than other species in all these flocks. Thus, it can be considered that bluetongue virus is actively circulating between host population and vectors (Culicoides species), bringing a clinical outbreaks in sheep and goats. Since common species are more likely to be important vectors than uncommon species, C. imicola can be considered as primarily a potential vector of bluetongue. Presence of bluetongue virus antigen in Culicoides imicola midges has been demonstrated through indirect immunofluorescent test. Culicoides imicola has identified as prime vector of bluetongue based on its wider distribution, higher abundances in post monsoon season coinciding with the 37

37 occurrences of bluetongue outbreaks, its higher abundances in the flocks having bluetongue, detection of BTV antigen in them and as this species is a proven vector of bluetongue virus. It has been demonstrated through the presence of bluetongue antibodies in sheep, clinical disease in sheep, preference of C. imicola to sheep and the presence of BTV in C. imicola that the bluetongue virus may be actively circulating between the sheep population and vector, C. imicola bringing a clinical disease in susceptible sheep and goats when there are suitable environmental factors favour the upsurge in C. imicola. TAXONOMIC STUDIES ON CULICOIDES SPP. IN INDIA, VECTOR FOR BLUETONGUE VIRUS: GAPS, CHALLENGES AND OPPORTUNITIES Background DR. VIKAS KUMAR, DR. DHRITI BANERJEE CENTRE FOR DNA TAXONOMY& FLY RESEARCH LAB MOLECULAR SYSTEMATICS DIVISION ZOOLOGICAL SURVEY OF INDIA Culicoides are small flies in the family Ceratopogonidae, commonly referred to as biting midges, no-see-ums, or punkies. Culicoides is the most species rich genus of Ceratopogonidae encompassing about 20 % of total species in the family Ceratopogonidae (Borkent 2012). Members of Culicoides have a worldwide distribution, Antarctica being the only exception (Borkent 2004). Culicoides is the main pest genus of the family because females of many species feed on vertebrate blood for maturation of eggs. Because of the medical and veterinary concerns associated with Culicoides, the study of the genus has focused largely on the medical and veterinary aspects of these flies, resulting in gaps in our knowledge of their taxonomy at species and sub-generic level. A little is known about the evolutionary relationships of members of Culicoides. Culicoides encompasses more than 1400 species worldwide out of which about 60 are from India. Taxonomic studies on Culicoides have been largely focused on females as they have medical and veterinary importance. However, in some cases male must be used for identification as they possess more distinguishable taxonomic features than females. As of now we are lacking in identification keys for most biogeographic regions of India and structured taxonomic and ecological studies on Culicoides in India have never been attempted. Gaps and Challenges To date around 60 species of Culicoides have been reported from India in various scattered publications. Taxonomic studies on Culicoides/Ceratopogonids in India spin around the untiring efforts of Dr. Das Gupta and co-workers spanning over a period of more than 35 years. In last 40 years a number of outbreaks of bluetongue virus have been reported from India and Culcioides spp. have been claimed to be vector, however, vector status has never been confirmed by substantial scientific evidence. Since BTV is vectored by Culicoides spp., the identification of vector is important in order to understand the vector-virus relationship and in planning control/management strategies. A number of significant studies have revealed that 38

38 morphological identification alone cannot be reliable for accurate species identification as Culicoides have a number of species complexes, for example schultzei complex involve more than 8 species. Further, inaccessible morphology of Culicoides is a stumbling block in accurate species or even genus identification. Opportunities and ZSI Initiatives Keeping in view the economic importance of Culicoides as vector of important viral and other disease across the globe including India and insufficient taxonomic data, ZSI has initiated to work on an integrated (morphological and molecular) approach to foster a clear understanding of Taxonomy of Culicoides which may play a pivotal role in planning a control/management strategies for the Culicoides vectors. ZSI in its National Zoological Collection (NZC) have almost all the type specimens of Culicoides species described by Dr. Das Gupta. Serious taxonomic studies (in light of new taxonomic characters evolved in last two decades that were not available at the time of original description of the species) along with molecular studies on topotypes (specimens collected from type locality) and other specimens from BTV affected area are being attempted in combination with phylogenetic tools. SYSTEMATICS OF INDIAN CULICOIDES (DIPTERA: CERATOPOGONIDAE): AN UNFINISHED WORK GIRISH MAHESHWARI School of Entomology, St. John s College, Agra , India Culicoides, a family of hematophagous diptera, feed on blood of the most chordate and some invertebrates and are distributed throughout the India except high elevation of the Himalaya above 4000 msl. Culicoides have attracted the attention of Indian Dipterologists in the beginning of 20 th century and yielded about 50 spp. from the various zones of the country. They play an important role in the epidemiology of some veterinary diseases like bluetongue as these are the vector of arboviruses and effect ruminants and other mammals. Since most existing type material is not available in the museum of India comparison of the new material is very costly and difficult. Bluetongue disease has created havoc in many states of the country and therefore it is essential to commence the formulation a monograph to aid in the field identification of Culicoides spp.this task can be achieved through the Integrated Nation Project and a collaborative network embracing all biting midges specialists currently active in the country. CULICOIDES IN TAMIL NADU DR K LLANGO, ZSI SOUTHERN REGIONAL STATION The paper forming Dr Llango s presentation is available here: 39

39 Systematics and Bionomics of Culicoides spp. prevalent in West Bengal Entomology Research Unit, Department of Zoology, The University of Burdwan. Team members: 1. Dr. A. Mazumdar, Associate Professor & Principal Investigator, (M); (R) 2. Ms. M. Nandi, Research Fellow 3. Ms. R. Harsha, Senior Research Fellow (UGC) 4. Mr. S. Sarkar, Project Assistant (UGC) Work summary 1. Review of systematic study of Indian species of Culicoides Latreille. The genus Culicoides Latreille (1809) is distributed almost worldwide, excluding Antarctica and New Zealand, ranging from the tropics to the tundra and from sea level to 4000 m (Mathieu et al., 2010; Talavera et al., 2011). The first taxonomic study on the Culicoides fauna of India started with Kieffer (1910) who later enriched the knowledge by a few more contributions (1911a, b; 1913; 1914a) followed by Patton (1913), Dover (1921), Edwards (1922), Smith (1929), Mukerji (1931), Smith & Swaminath (1932) and Macfie (1932). After a long interval, Sen & Das Gupta (1959) attempted a comprehensive study on the Indian Culicoides mainly on the basis of materials collected in and around Kolkata. Borkent & Wirth (1997) published the first World catalog of Ceratopogonidae following updating by updating in subsequent years by Borkent (2006, 09, 11, 12) with inclusion of newly described species. There are 1354 worldwide species including 46 fossil species of Culicoides Latreille placed in 31 subgenera and 38 species groups (Borkent, 2012) but it is likely that more species may be included in the list. There are 15 species whose systematic status is still obscure. Till now, there about 71 species are recorded from India under 12 subgenera and 5 species groups including 17 new. According to Borkent (2012), the present classification of species of Culicoides is in terrible condition for many years. The arrangement of species of the genus Culicoides is almost entirely phenetic (based on overall similarity). At present it is uncertain whether most of the subgenera or species groups are monophyletic, although it is believed that some of them to be so, based on unpublished synapomorphies. Meiswinkel & Dyce (1989) provided a rare and welcome overview of some character states of phylogenetic importance. Aside from the species placed to subgenus, there are numbers placed in separate species groups of uncertain affiliation and a list of miscellaneous species which no one knows what to do with. There is a great need to study Culicoides on a worldwide basis, using exemplars of the various groups from each region. 40

40 Checklists of Indian species of Culicoides Latreille with special reference to the species occurring in West Bengal No of subgenera 11 No. of species recorded 55 No. of new species (proposed) 15 SUBGENERA No. of species No. of new species (proposed) 1. Avaritia Beltranmyia Diphaomyia Haemophoructus Hoffmania Meigerehelea Monoculicoides Pontoculicoides Remmia Oecacta Trithecoides GROUPS UNPLACED TO GENERA 12. Chaetophthalmus group Clavipalpis group Shermani Group Shortti Group Miscelleneous Group 02 41

41 1.Subgenus AVARITA C. actoni Smith C. annandalei Majumdar & Das Gupta C. autumnalis Sen & Das Gupta C. boophagus Macfie C. brevitarsis Kieffer C. certus Das Gupta C. definitus Sen & Das Gupta C. dumdumi Sen & Das Gupta C. fulvus Sen & Das Gupta C. himalayae Kieffer C. imicola Kieffer C. inexploratus Sen & Das Gupta C. molestior Kieffer C. odiosus Kieffer C. sikkimensis Das Gupta 2.Subgenus BELTRANMYIA C. circumscriptus Kieffer 3.Subgenus DIPHAOMYIA C. mukerjii Majumdar & Das Gupta 4.Subgenus HAEMOPHORUCTUS C. rariradialis Das Gupta 5. Subgenus HOFFMANIA C. indianus Macfie C. innoxius Sen & Das Gupta C. isoregalis Majumdar & Das Gupta C. neoregalis Majumdar & Das Gupta C. paraliui Das Gupta C. pararegalis Majumdar & Das Gupta C. peregrinus Kieffer C. pseudoregalis Majumdar & Das Gupta C. quasiregalis Majumdar & Das Gupta C. regalis Majumdar & Das Gupta C. subregalis Majumdar & Das Gupta 6. Subgenus MEIJEREHELEA C. arakawai (Arakawa) C. hegneri Causey C. magnithecalis Majumdar & Das Gupta 7.Subgenus MONOCULICOIDES C. homotomus Kieffer C. rarus Das Gupta 8.Subgenus OECACTA C. causeyi Majumdar & Das Gupta C. distinctipalpis Majumdar & Das Gupta C. gutsevichi Sen & Das Gupta C. swaminathi Majumdar & Das Gupta C. yadongensis Chu 9.Subgenus PONTOCULICOIDES C. kamrupi Sen & Das Gupta 10.Subgenus REMMIA C. oxystoma Kieffer C. schultzei (Enderlein) 11. Subgenus SYNHELEA C. similis Carter, Ingram & Macfie 12.Subgenus TRITHICOIDES C. anophelis Edwards C. inornatithorax Das Gupta C. insolens Choudhuri & Das Gupta C. macfie Causeyi C. palpifer Das Gupta & Ghosh C. parararipalpis Das Gupta C. raripalpis Smith UNPLACED Chaetophthalmus group C. majorinus Chu Clavipalpis group C. clavipalpis Mukerji C. distinctus Sen & Das Gupta C. huffi Causey Shermani Group C. dryadeus Shortii Group C. shortii Smith & Swaminath C. magnificus Sen & Das Gupta C. turgidus Sen & Das Gupta 42

42 3. Study of seasonality and incidence of the biting midges and probable vectors by using light traps (UV & White light) Adults were obtained by battery operated down draft light traps (UV & White light) which was designed and fabricated by the USIC, University of Burdwan. The traps were fabricated with locally available material.. The traps were made durable as well as portable and were installed within the sheds. The relative abundance has been depicted in the pie diagram. The site specific habitats (Table 1) were screened for immature stages by floatation methods and hand picking. Monthly sampling of adults was made from two locations: Adisaptagram and Paharhati to record the seasonal abundance. Light traps and other collecting devices were also operated in other sampling sites of the following districts of West Bengal chiefly in areas where livestock density (indigenous cattle, goat, and sheep density occurred in moderate numbers (XVII All India Livestock Census, 2003). The specimens were preserved in 70% ethyl alcohol sorted and age graded species wise. Molecular identification of the Culicoides species are being verified with the help of Barcodes. Table 1. The collection sites and the habitats. Collection sites Longitude Latitude Habitats Arambagh 87 50' E 22 53' N Cattle shed, pond margin, marshland Bolpur E N Cattle shed, marsh land Baharampur E 24 6 N Cattle shed, marsh land Burdwan 87 54' E 23 16' N Cattle shed, river bank, marsh land, swamps Kandi 88 1 E N Cattle shed, pond margin, marsh land Kotulpur 87 38' E 23 1' N Cattle shed, agricultural field, pond side, marsh land Kakdwip 88 11'E 21 53'N Cattle shed, marsh land Sagardwip 88 1' E 21 4' N Marsh land, swamps Namkhana 88 14' E 21 46'N Marsh land, swamps Bakkhali 'E 'N Sea beach, pond margin, marsh land, swamps Pandabeshwar 87 10' E 23 88' N Cattle shed, marsh land Raniganj ' E 25 52' N Cattle shed, marsh land Durgapur 87 20' E 23 30' N Cattle shed, marsh land Panagarh 87 26' E 23 27' N Swamps, marsh land 43

43 Table 2. Catch percentage of Ceratopogonidae from the collection sites Month Mean Temperature (C) Mean Humidity (%) Nilobezzia Clinohelea Stilobezzia Dasyhelea Forcipomyia Atrichopogon Allaudomyia Culicoides M F M F M F M F M F M F M F M F 06/ _ / / / / _ 02/ _ / / / / / / _ 09/ _ Bionomics of probable vector species A. Culicoides innoxius Sen & Das Gupta, 1959 a) Rearing of immature stages on natural/artificial rearing media to record the suitability for colonisation b) Longevity related studies of adults fed on sucrose solution c) Recording of associated life stages in the laboratory d) Taxonomic study of the immatures B. Culicoides peregrinus and Culicoides oxystoma a) Studies on seasonality, parity and fecundity b) Laboratory rearing of immature stages on artificial media c) Biting activity and biting cycles of adults using molecular tools d) Taxonomic study and description of the immature has been done 44

44 e) Laboratory mating and blood feeding experiments of both species are in progress. Relevant publications Biting midges of the genus Leehelea Debenham (Diptera: Ceratopogonidae) in India. Zootaxa [Magnolia Press, New Zealand], 3399:53-60, 2012 (co-authors: M.Nandi & P.K.Chaudhuri) Two new biting midges of Brachypogon Kieffer (Diptera: Ceratopogonidae) from India. Polish Journal of Entomology, Bydgoszcz, 79: (Co-authors: Das, N., & Chaudhuri, P.K.). - Blood sucking midges of Leptoconops (Holoconops Kieffer) (Diptera: Ceratopogonidae) from India. Zootaxa, [Magnolia Press, New Zealand] 2619: (Co-authors: Saha,N.C. & Chaudhuri,P.K.). ISSN: Biting flies of the genus Homohelea of India. (Diptera: Ceratopogonidae) Folia Heyrovskyana Series A, Czech Republic 16(4): (Co-authors: Ghosh,S., Dasgupta,S.K. & Chaudhuri,P.K.) ISSN: New insectivorous midges of the genus Nilobezzia Kiffer from India (Diptera: Ceratopogonidae: Sphaeromini), Entomologist monthly magazine, UK, 145: (Co-authors: Chaudhuri, P.K. & Dasgupta, S.K.) ISSN - Five new species of the genus Sphaeromias Curtis (Diptera: Ceratopogonidae) from India. Journal of Asia-Pacific Entomology, Korea 12: (Co-authors: Saha,N.C..,& Chaudhuri,P.K.)] - Predaceous biting midges of the genus Bezzia Kieffer (Diptera: Ceratopogonidae) from India. Far Eastern Entomologist, Russia. 203: 1-17 (Co-authors: Saha,N.C. & Chaudhuri,P.K.) ISSN: X - Indian Species of the Genus Dasyhelea Kieffer (Diptera: Ceratopogonidae), International Journal of Dipterological Research, Russia, 20 (4): (Coauthor: Chaudhuri,P.K.) ISSN: Biting midges of the genus Palpomyia Meigen (Diptera: Ceratopogonidae) in India. Bonner Zoologische Beitrage, Germany, 56 (1/2) (Co-authors: Dasgupta,S.K. & Chaudhuri,P.K.) New species of predaceous midges of the Genus Alluaudomyia Kieffer 1913 (Insecta, Diptera, Ceratopogonidae) from the coastal region of West Bengal, India. Zoosystema [Muséum national d'histoire naturelle France] 27(1): (Coauthors: Sinha,S.,& Chaudhuri,P.K.) Biting midges of the genus Atrichopogon Kieffer (Diptera :Ceratopogonidae) from India. Tijdschrift Voor Entomologie, The Netherlands.146: (Co-authors: Bose,M., Dasgupta,S.K. & Chaudhuri,P.K.) 45

45 - Biting midges of the genus Forcipomyia Meigen (Diptera: Ceratopogonidae) from West Bengal,India, The Japanese Journal of Systematic Entomology, Japan 9(1): (Co-authors: Sinha,S & Chaudhuri,P.K.) - Biting midges of the genus Bezzia Kieffer (Diptera: Ceratopogonidae) from the coastal areas of West Bengal, India. Bangladesh Journal of Zoology, Bangladesh 31(1): (Co-authors: Sinha,S. Dasgupta,S.K. & Chaudhuri,P.K.) 46

46 Appendix 1. Details of Participants SI. No. Name and Address of the Participants 1 Dr. Simon Carpenter, UK Collaborator of Indo UK Project and Head of Entomology, The Pirbright Institute, Ash Road, Woking, Surrey GU24 0NF, United Kingdom simon.carpenter@pirbright.ac.uk 3 Dr. Glenn Bellis Northern Australia Quarantine Strategy (NAQS)PO Box Winnellie NT 0821, Australia Telephone: glenn.bellis@daff.gov.au 5 Ms Karien Labuschagne Parasites vectors and vector borne diseases, Entomology Onderstepoort Veterinary Institute PO Box X05 Onderstepoort 0110 South Africa Tel: + 27 (0) Fax: + 27 (0) Cell: + 27 (0) labuschagnek@arc.agric.za 7 Dr.Bruno Mathieu Institut de Parasitologieet de, PathologieTropicale Faculté de Médecine de Strasbourg, 3 rue Koeberlé, Strasbourg, France Telephone: bmathieu@unistra.fr 9 Dr. Minakshi Prasad, Indian Coordinator, (Indo-UK Project), SI. No Name and Address of the Participants. 2 Dr. Lara Harrup The Pirbright Institute, Ash Road, Woking, Surrey GU24 0NF, United Kingdom lara.harrup@pirbright.ac.uk 4 Dr. David Gopurenko Research Scientist Molecular Systematics Science and Research, Biosecurity NSW Department of Primary Industries Wagga Wagga Agricultural Institute Pine Gully Rd Wagga Wagga NSW 2650 PMB Wagga Wagga NSW 2650 Australia P: +61 (0) M: +61(0) david.gopurenko@dpi.nsw.gov.au 6 Dr.Claire Garros, CIRAD UMR 15 Département BIOS Campus International de Baillarguet TA A15/G Montpellier Cedex 5 France Telephone : claire.garros@cirad.fr 8 Ms Alison Bettis London School of Hygiene & Tropical Medicine Room 443, Keppel St London WC1E 7HT United Kingdom T: +44 (0) F: +44 (0) Dr. Y. Krishnamohan Reddy, Field Co-Ordinator, (Indo-UK Project) and Professor and 47

47 & Professor and Head, Department of Animal Biotechnology, College of Veterinary Science, LLRUVAS, Hisar Haryana Dr. P. Kalyani Co Principal Investigator, (Indo-UK Project) and Assistant Professor Department of Veterinary Biotechnology, College of Veterinary Science, Sri Venkateswara Veterinary University, Rajendra Nagar, Hyderabad , Andhra Pradesh 13 Dr. S. Nandi, Principal Investigator, AINP on Bluetongue, CADRAD, Indian Veterinary Research Institute, Izatnagar Uthar Pradesh Dr. S.N. Joardar, Principal Investigator, AINP on Bluetongue, College of Veterinary Science, WBUAFS, Kolkata Dr. Varsha Sharma, Principal Investigator and Professor, AINP on Bluetongue, Deptt. Of Vety. Microbiology, College of Vety. Sc. & A.H. South Civil Lines, NDVSU Jabalpur (M.P) Madhya Pradesh Head, Vaccine Research Centre-Viral Vaccines, Centre for Animal Health Studies, Madhavaram Milk Colony, Chennai Dr. S.M. Byregowda, Principal Investigator, (Indo-UK Project) and Joint Director, Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore Dr. D. Hemadri, Principal Scientist, Project Directorate on ADMAS, Hebbal, Bangalore, Karnataka Dr. B.S. Chandel, Professor and Head & Principal Investigator, AINP on Bluetongue, College of Veterinary Science, SDAU, S.K. Nagar, Distt. Banas Kantha , Gujarat Dr. S.N. Singh, Managing Director, Biovet private limited, Plot No 308, 3 rd phase, KIADB Industrial Area, Malur , Kolar District singhsn@biovet.in 48

48 19 Dr. Dhriti Banerjee, Scientist C, Diptera Section & Molecular Systematics Division, ZSI Kolkata, Dr. H. R. Parsani Associate Professor Department of Parasitology College of Veterinary Science and AH, SDAU, Sardarkrushinagar-Dantiwada , (B.K.), Gujarat Dr. K. Ilango Scientist D Zoological Surrey of India, Southern Regional Centre, 130, Santhome High Road, Chennai 28. kilangozsi@rediffmail.com 25 Dr T. Battacharya, General Manager, Biovet private limited, Plot No 308, 3 rd phase KIADB Industrial Area, Malur , Kolar District tapasb@biovet.in 27 Dr. Ravindra, B.G., Assistant Professor, Veterinary Hospital, Disease Diagnostic and Information Centre, Bida 29 Dr.N.S.Manoharan, Forest Veterinary Officer, O/o The Conservator of Forests, Coimbatore-43, 31 Dr. W. Panner Selvam, MVSc Veterinary Officer, National Zoological Park, New Delhi Dr. Vikas Kumar, Sceintist C Centre for DNA Taxonomy, Molecular Systematics Division, ZSI Kolkata, vikaszsi77@gmail.com Dr. Girish Maheshwari Associate Professor, School of Entomology St. John's College, Agra , India girish_maheswari@yahoo.com Dr. K. Ganesh Udupa, Professor and Head, TVCC, Veterinary College, Vinoba Nagar, Shimoga ganudupa@yahoo.com 26 Mr. B. Ranjith, Scientist, Biovet Pvt., Ltd., Plot No 308, 3 rd phase KIADB Industrial Area Malur , Kolar District 28 Dr. Satheesha S.P., Assistant Professor, Dept of Vety Public Health and Veterinary Epidemiology, Veterinary College, Bidar 30 Dr. D. Kotresha Principal, KSPL Degree College, Karnataka 32 Dr. S. Logeswari, Research Assistant Institute of Veterinary Preventive Medicine, Ranipet 49

49 33 Dr. V. Purushothaman, Director, Centre for Animal Health Studies, Madhavaram Milk Colony, Chennai dcahs@tanuvas.org.in 35 Dr. K. Senthilvel, (Associating Scientist Indo-UK Project) Associate Professor, Dept. of Vety. Parasitology, VC & RI, Namakkal kansen@rediffmail.com 37 Dr. A. Sangaran, Professor, Directorate, Centre for Animal Health Studies, Madhavaram Milk Colony, Chennai Dr. R. Anil Kumar Associate Professor, Sheep Breeding and Research station, Sandyanallah, Ooty, Nilgiris. 41 Dr. R. Ramprabhu Professor and Head, Teaching Veterinary Clinical Complex, VC & RI, Tirunelveli Dr. K. Sivakumar Associate Professor, Veterinary University Training and Research Centre, Kalapatty Pirivu, Coimbatore Ms L.Swathi, M.Sc., Zoology, No.2, Duraisamy Street, Redhills, Chennai Mr Asit Sharma Research Associate AINP on Bluetongue, CADRAD, Indian Veterinary Research Institute, Izatnagar Uthar Pradesh 34 Dr. C. Sreekumar, (Associating Scientist Indo-UK project) Professor, Postgraduate Research Institute of Animal Sciences, Kattupakkam sreekumar@tanuvas.org.in 36 Dr. A. Subramanian, (Associating Scientist Indo-UK Project) Professor, Dept. of Animal Genetics and Feeding, Madras Veterinary College, Chennai Dr. R. Yasothai Assistant Professor and Head, Veterinary University Training and Research Centre, Erode 40 Dr. M. Chellapandian Professor and Head, Department of Animal Nutrition, Veterinary College and Research Institute, Tirunelveli Dr. N. Murali Professor and Head, Mecheri Sheep Research Station, Pottaneri, Salem District 44 Mr N. Naveen Kumar, Senior Research Fellow (Indo-UK Project) Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore Dr. S. Israel Stalin Research Associate (Indo-UK Project), (Indo-UK Project) VRC-VV, CAHS, MMC, Chennai Mr Sunil Raghunath Patil Junior Research Fellow (Indo-UK Project) Department of Veterinary Microbiology, College of Veterinary Science, Sri Venkateswara Veterinary University, 50

50 49 Ms Rupa Harsha Junior Research Fellow Department of Zoology Entomology Research Unit, The University of Burdwan, Burdwan West Bengal Rajendra Nagar, Hyderabad Andhra Pradesh 50 Ms. Mou Nandi Research Scholar, Entomology Research Unit, Department of Zoology, The University of Burdwan, Burdwan, West Bengal Kolkata

51 Appendix 2: Photographs of Meeting 52