Studying Culicoides vectors of BTV in the post-genomic era: Resources, bottlenecks to progress and future directions

Highlights

• •Culicoides sonorensis is the only colonized species of bluetongue virus vector.
• •The development of a fully annotated genome for this species is in progress.
• •Transcriptomic analyses are being employed to investigate functional elements of the genome, particularly genes involved in hematophagy, reproduction, development and vector competence.

Abstract

Culicoides biting midges (Diptera: Ceratopogonidae) are a major vector group responsible for the biological transmission of a wide variety of globally significant arboviruses, including bluetongue virus (BTV). In this review we examine current biological resources for the study of this genus, with an emphasis on detailing the history of extant colonies and cell lines derived from C. sonorensis, the major vector of BTV in the USA. We then discuss the rapidly developing area of genomic and transcriptomic analyses of biological processes in vectors and introduce the newly formed Culicoides Genomics and Transcriptomics Alliance. Preliminary results from these fields are detailed and finally likely areas of future research are discussed from an entomological perspective describing limitations in our understanding of Culicoides biology that may impede progress in these areas.

1. Introduction

Culicoides biting midges (Diptera: Ceratopogonidae) are among the smallest hematophagous vectors of arboviruses known, with some species of major economic importance possessing wing lengths of less than 1 mm. Over 1400 extant and extinct species have been described worldwide and the genus is represented on all major land masses with the exception of New Zealand, the Hawaiian Islands, Iceland and the extreme polar regions (Kettle, 1977, Mellor et al., 2000). While a diverse and ecologically important genus, the study of Culicoides has been driven primarily by the ability of certain species to act as competent biological vectors of arboviruses (Borkent, 2004, Mellor, 2000). The vast majority of these arboviruses are not pathogenic to humans (Carpenter et al., 2013), but may inflict disease in livestock species and/or wildlife (Mellor et al., 2000). The most economically-important livestock-associated arbovirus transmitted by Culicoides currently is bluetongue virus (BTV), which causes bluetongue (BT), a disease of ruminants. Since BT was initially identified in the Republic of South Africa during the 19th century (Erasmus and Potgieter, 2009, Spreull, 1905), it has caused vast losses in livestock revenue both through inflicting trade barriers between endemic and BTV-free regions (Tabachnick, 1996) and in direct clinical cases (Carpenter et al., 2009, Purse et al., 2005). Although other vectors and modes of transmission have been proposed, BTV distribution and persistence is thought to be almost entirely dependent upon the presence or absence of vector Culicoides adults.

In this review, we examine the current resources available for studying Culicoides with specific reference to the application of transcriptomics and genomics. Technological advances within these fields are currently revolutionizing our understanding of vector biology at a molecular level, particular in the family Culicidae (mosquitoes) where full genomes have already been sequenced, accurately annotated and released to the public domain for several major pathogen vectors (Severson and Behura, 2012). We report on similar projects that have been initiated to examine these areas in Culicoides and discuss their probable impact in the mid- to long-term. To inform these predictions we outline current biological resources for Culicoides in order to identify potential bottlenecks to progress in studying this genus that are imposed by their biology. We do not attempt to describe the process of biological transmission of BTV by Culicoides, or examine the distribution and identity of primary vector species worldwide, as these areas have already been addressed in other reviews (Mellor, 2000, Mellor et al., 2009, Tabachnick, 1996). There is also no attempt to summarize information regarding emergence of BTV strains in Europe, which has been addressed elsewhere in this volume and in previous reviews (Carpenter et al., 2009, Purse et al., 2005, Wilson et al., 2009b). Finally, several general reviews of the biology and ecology of Culicoides and are recommended to the reader (Borkent, 2004, Kettle, 1977, Mellor et al., 2000).

2. Culicoides colony and cell-line resources

The establishment of laboratory colonies of vectors allows researchers to address fundamental questions regarding the biology and ecology of model species by enabling a continual supply of insects of known physiological state and in large numbers (Jones, 1966). Historically, a key driver for colonization of hematophagous Diptera was to enable large-scale pathogen transmission experiments to be conducted under controlled conditions, following preliminary evidence of vector status in the field. Field surveys and transmission experiments with field-collected vectors conducted in the Republic of South Africa were integral in establishing a potential link between Culicoides and BTV transmission (Du Toit, 1944) and additionally discounted mosquitoes as primary vectors of the virus (Nieschulz et al., 1934a, Nieschulz et al., 1934b). The demonstration of BTV transmission under vector-proof conditions in the USA, however, for the first time provided unequivocal evidence of the role of Culicoides in the epidemiology of the BTV (Wilson et al., 2009a). These studies were enabled by laboratory colonization of the major Nearctic BTV vector species, C. sonorensis from 1955 at the Kerrville, Texas laboratory of the U.S. Department of Agriculture (Foster et al., 1963, Jones, 1959).

While of clear utility, only a very small proportion of Culicoides possess lifecycle traits suitable for laboratory colonization (Table 1). In the case of C. sonorensis, initial attempts to produce colony lines were extremely challenging, with many failing due to poor production at 6–12 generations and full adaptation to the laboratory environment was considered to require more than 20 generations (Jones, 1960). In general, key parameters in determining the probability of successful colonization of vector species include the proportion of field collected individuals that can be consistently blood-fed (Campbell, 1974), the ability of the species to mate facultatively without the requirement for swarming behavior (Jones, 1966, Linley, 1968) and successful adaptation to the high constant temperature regimes required to provide sufficient turnover (Carpenter, 2001, Jones, 1966). In Culicoides, certain species also exhibit biased sex ratios of emerging adults in the F₁ generation, which are hypothesized to be a consequence of variation in larval nutrition and can also disrupt colonization attempts (Boorman, 1985, Kitaoka, 1982, Veronesi et al., 2009). While most of these obstacles can in some way be overcome, any adaptation of methodology that requires either a high level of technical skill or substantial time commitment (e.g. artificial insemination or decapitation to produce egg batches) will contribute to colonies being unsustainable in the long term.

Table 1.

Colonization of Culicoides species worldwide.

Species	Autogeny recorded?	Wing length (mm)	Facultative mating	BTV vector status	Status of colonies?	References to colonization techniques
C. sonorensis	No	1.2	Yes	Proven	Extant	Boorman (1974), Hunt and Schmidtmann (2005), Jones, 1957, Jones, 1960, Jones, 1966 and Jones and Schmidtmann (1980)
C. nubeculosus	No	2.4	Yes	No	Extant	Boorman (1974) and Megahed (1956)
C. riethi	Yes	1.8	Yes	No	Discontinued	Boorman (1974)
C. furens	Yes	0.9	No	No	Discontinued	Koch and Axtell (1978) and Linley (1968)
C. guttipennis	No	1.3	Yes	No	Discontinued	Gazeau and Messersmith (1970) and Hair and Turner (1966)
C. arakawae	No	1.1	Yes	No	Discontinued	Sun (1974)
C. melleus	Yes	1.1	Yes	No	Failed	Koch and Axtell (1978)
C. hollensis	Yes	1.4	Yes	No	Discontinued	Koch and Axtell (1978)
C. oxystoma	No	1.0	Yes	Potential	Discontinued	Sun (1974)
C. wisconsinensis	Yes	1.2	Yes	No	Discontinued	Mullens and Schmidtmann (1981)
C. impunctatus	Yes	1.3	Yes	No	Failed	Carpenter (2001) and Hill (1947)
C. imicola	No	0.9	Yes	Proven	Failed	Veronesi et al. (2009)
C. obsoletus	No	1.2	Unknown	Proven	Failed	Boorman (1985)
C. insignis	No	1.1	Unknown	Proven	Not attempted	–
C. brevitarsis	No	0.8	No	Proven	Failed	Campbell (1974)

The colonization of C. sonorensis (the founding line of which was designated ‘AA’ or ‘Sonora 000’), was a landmark advance, providing what remains the sole system for detailed studies of BTV infection, although recent studies of alternative insect models have been conducted (Shaw et al., 2012). Advances were greatly enhanced by a collaborative relationship between the USDA and what was then the Animal Virus Research Institute (now The Pirbright Institute), which culminated in a parallel daughter line of the AA colony being established in the United Kingdom during 1969 (Boorman, 1974). Studies carried out both in the USA and UK using C. sonorensis as a model vector included direct visualization of BTV dissemination (Chandler et al., 1985, Fu et al., 1999, Nunamaker et al., 1997, Sieburth et al., 1991), which almost entirely underpins our understanding of this process in the genus (Mellor et al., 2009). In addition, investigations of the genetic basis underlying competence for BTV have been based entirely on selective breeding of this species (Mellor et al., 2009, Tabachnick, 1991, Tabachnick, 1996), alongside preliminary physical mapping of the C. sonorensis genome (Nunamaker et al., 1999, Nunamaker et al., 1998).

Following development of the ‘AA’ colony in the USA, additional lines of C. sonorensis were developed, originating from Idaho (‘AK’: colonized in 1973), Nebraska (‘AX’: colonized in 1981), Colorado (‘Ausman’: colonized in 1986) and California (‘Van Ryn’: colonized in 1995) (Jones and Foster, 1978, Mullens et al., 1995, Tabachnick, 1990). At present, the AK, Van Ryn and Ausman lines are maintained by USDA and the daughter AA line remains extant at The Pirbright Institute (having been continuously produced for over 55 years and approximately 700 generations). The parental AA line was discontinued in the USA along with the AX lines due to low productivity in 2000, but the AK, Ausman and Van Ryn lines and the daughter AA line remain publically available from the USDA and The Pirbright Institute under material transfer agreement.

A second key resource generated following the successful colonization of C. sonorensis was the development of embryonic cell lines (Table 2). Initially, these cell lines (designated CuVa) were produced from two day old AA colony C. sonorensis eggs according to a protocol originally designed to initiate cell lines from Drosophila melanogaster (Wechsler et al., 1989). While initially containing diverse cell types, later passages became more uniform and fibroblast-like and increasingly lacked a visible cytopathic response to BTV infection. Following production, a picorna-like virus (termed CUV) was discovered in the CuVa line and its use was limited in subsequent studies, despite the demonstration that this infection did not have a pathogenic effect on either the CuVa cells or inoculated suckling mice (Wechsler et al., 1991). As a response to this, a secondary line was initiated using eggs from the AK colony and these cells did not contain detectable CUV infection when examined using electron microscopy (Wechsler et al., 1991). This latter line, designated CuVaK (and more commonly referred to as ‘KC’) has since been used by a large number of laboratories worldwide as a major research and diagnostic tool (Mertens et al., 1996, Nunamaker et al., 1999, Schnettler et al., 2013, Xu et al., 1997). A major advantage of the CuVaK line has been the fact that it allows replication of BTV to high titers in a comparatively natural environment. The degree of selection of cells that has occurred during passage, however, remains undetermined and has led to the production of further lines to better represent the diversity of C. sonorensis cell types (Table 2).

Table 2.

Culicoides sonorensis cell lines including colony of origin and maintenance conditions.

Name	Colony of origin	Tissue source	Cell growth requirements (growth media, temperature)	Virus susceptibility	Comments	Reference
CuVa	AA colony (Original Jones colony)	Embryo	S; 20 °C; lightly adhesive	BTV-11	Picorna-like virus contaminant	Wechsler et al. (1989)
CuVaK (KC)	AK colony	Embryo	S or MEM and L-15; 20 °C or 30–36 °C; lightly adhesive	BTV-2, 10, 11, 13, 17; EHDV-1, 2	Bluetongue virus core particle contaminant	Wechsler et al. (1991)
KC-E3	Clone of KC (AK colony)	n/a	MEM; 20 °C and 30 °C	BTV-10, 17	No contamination detectable by EM	Unpublished
KC-E3.1	Clone of KC-E3 (AK colony)	n/a	MEM; 20 °C	No data on susceptibility	No contamination detectable by EM.
KC-E3.2	Clone of KC-E3 (AK colony)	n/a	MEM; 20 °C	No data on susceptibility	No data on possible contamination.
CuVaX	AX colony	Embryo	S; 20 °C; lightly adhesive	No data on susceptibility	Picorna-like viral contaminant
CuVaW3#	Ausman farm	Embryo	S; 20 °C and 30 °C; mod. adhesive	No data on susceptibility	Epithelial-like	McHolland and Mecham (2003)
CuVaW8A (W8A) and CuVaW8B (W8B)	Ausman farm	Embryo	S and MEM; 20 °C and 30 °C; lightly adhesive	BTV-2, 10, 11,13, 17; EHDV-1, 2	No contamination detected W8A by EM or PCR for BTV No contamination by EM for W8B
CuVaW16 (W16)	Ausman farm	Embryo	S; 20 °C; mod. adhesive	No data on susceptibility	Myoblast-like. No contamination detected by EM.

Growth media abbreviations: S = Schneiders, MEM = Minimal essential media.

3. Culicoides sonorensis transcriptome and genome projects

The advantages of C. sonorensis colonies and associated cell lines as models for transcriptomic and genomic analysis over field collections of vector species are substantial. Unlike field populations of other Culicoides BTV vectors, all physiological stages of development can be conveniently produced in substantial numbers, enabling production of consistent biological replicates or pooled samples. In addition, the levels of microbial and fungal contamination in individuals can be substantially minimized in production of adults for analyses and specific treatments to elicit physiological responses demarcated with certain knowledge of prior exposure (e.g. blood feeding). Massively parallel second generation DNA sequencing technologies are transforming de novo ‘omic sequencing by allowing the generation of billions of reads at relatively low costs with options of combining multiple samples and hybrid approaches with new analysis algorithms (Severson and Behura, 2012). Unlike many other important hematophagous insect vectors, such as mosquitoes, the genome and transcriptome(s) of C. sonorensis have not been an available as a resource for vector biologists, preventing the exploration of genetics and functional genomics of parameters such as BTV vector competence. However, recent combined efforts of several institutions including the Agricultural Research Service of the United States Department of Agriculture (ARS-USDA), Clemson University Genomics Institute (CUGI), The Pirbright Institute, and the European Bioinformatics Institute (part of the European Molecular Biology Laboratory; EBI-EMBL) have led to the first transcriptome and genome projects as part of the newly formed Culicoides Genome and Transcriptome Alliance (CGTA). The following sections describe progress in these two areas and provide suggestions of future areas of research.

3.1. The adult female Culicoides sonorensis transcriptome

The transcriptome of a cell is dynamic and in constant flux, as opposed to a static genome content, continuously changing in response to various stimuli to meet cellular needs. RNA-sequencing (RNAseq) is a technology that captures and measures the transcriptome in a spatial context at depths that reach even the rarest transcripts in a population and has transformed the way in which we view the extent and complexity of eukaryotic transcriptomes (Wang et al., 2009). This technique is now commonly used to catalog and explore the functional elements of the genome with wide applications across vector biology (Severson and Behura, 2012). In the first application of this technology to Culicoides, the adult Culicoides sonorensis transcriptome was investigated in response to feeding on blood.

Previously, Culicoides sonorensis tissue-specific transcriptomes were sequenced and annotated in the USA (Campbell et al., 2005, Campbell and Wilson, 2002). These studies generated the first mid-gut and salivary-gland expressed-sequence tag (EST) libraries for C. sonorensis, and several genes (i.e. cDNA sequences) involved in hematophagy, reproduction and defense were identified. Additional studies identified transcripts that were differentially expressed in C. sonorensis during EHDV-1 infection using both differential display and suppression subtractive hybridization (Campbell and Wilson, 2002). As a follow up to these studies, our aim was to use deep sequencing technologies to capture a comprehensive view of the transcriptome of adult female C. sonorensis and to examine differentially expressed genes across several conditions from a transcriptome-wide perspective. Our conditions included several feeding states (teneral/unfed, blood fed, and sucrose fed) as well as multiple time points after feeding (2, 6, 12 and 36 h), and we utilized female midges from our AK colony at USDA-ARS-ABADRU. RNAseq data for these transcriptomes were collected on an Illumina HiSeq2000 using paired-end reads.

A total of 257 million read pairs amounting to 52 gigabases of transcriptome sequence were produced. To construct a female C. sonorensis unigene for annotation and differential expression analysis, we performed a de novo assembly using all reads for each condition with the Trinity software package (Grabherr et al., 2011). In order to filter a large fraction of likely misassembled, chimeric, or artificial contigs from the unigene assembly, we applied a binning approach by first screening the unigene assembly for homology to the close relatives Aedes aegypti and Culex pipiens, and the non-redundant protein database at NCBI using putative open reading frames (ORFs) with BLASTX alignments and then filtering remaining unique unigenes that have internal stop codons or are unlikely genuine coding sequences using the TransDecoder supplement to the Trinity software package (Grabherr et al., 2011), which resulted in a ∼36% reduction in unigenes. Next, we clustered and filtered for redundancy using high stringency parameters (98%) and the cdHIT software (Li and Godzik, 2006) with a final round of manual inspection to further remove spurious contigs. The final female C. sonorensis unigene set is composed of 19,041 contigs with sizes ranging from 300 bp to ∼8 kb with a mean transcript size of 1.3 kb. The Culicoides sonorensis unigene aligned well with the Aedes aegypti and Culex quinquefasciatus transcriptomes covering ∼76% and ∼84% of the genes, respectively. Additionally, 76% of the unigene matched a protein sequence in the nr database, leaving ∼3000 unigenes unique to Culicoides.

With a robust unigene assembly and alignment of the Culicoides unigene to the Gene Ontology (Ashburner et al., 2000), a significant compliment of genes involved in immune response and defense, reproduction, hematophagy, digestion and transport, and many other important biological processes was identified. These genes were classed into functional sub-categories to determine candidate genes underlying physiological processes; for example, the immune response and defense genes were sub-categorized into components such as the humoral immune response, hemolymph coagulation, and regulation. Changes in these adaptive immune response processes during blood feeding were observed, and through category analysis, we identified a short list of candidate genes annotated with cytokine production and function that may in the future serve as targets for further functional analysis. Some key genes associated with antimicrobial defense response that were revealed in the transcriptome included the antimicrobial peptides defensin, cecropin and attacin which have been correlated with vector competence in other hematophagous arthropods (Carlsson et al., 1987, Carlsson et al., 1998, Chernysh et al., 1996, Hu et al., 2013, Lee and Brey, 1994, Nayduch and Aksoy, 2007, Nygaard et al., 2012, Sugiyama et al., 1995). As for these other vectors, we hypothesize that innate immune and apoptosis genes and pathways are of critical importance in vector competence of Culicoides for pathogens such as arboviruses.

The studies conducted also provide a glimpse into the genetic compliment involved with blood feeding, which includes secreted salivary proteins, catalytic and transport processes associated with digestion and egg production (vitellogenesis). As was found in the transcriptome analyses of the malaria vector Anopheles gambiae (Dana et al., 2005, Marinotti et al., 2006), characteristic genes were identified that are associated with hematophagy, including serine proteases, trypsins, cytochrome oxidases, anti-coagulation factors and vitellogenins which have recently been reported to play a major functional role in acquisition, processing and utilization of the blood meal and its components (Gulia et al., 2010, Kokoza et al., 2001, Prasad, 1987, Ramalho-Ortigao et al., 2003). The identification and expression profiles of genes associated with hematophagy as well as defense can be critical in understanding the biological functioning of Culicoides and may well provide a platform for the development of molecular targets for altering vector competence or controlling vector populations.

3.2. Transcriptome-level differential expression analysis in C. sonorensis

Coupled with the development of a novel unigene set for Culicoides, transcript abundance was determined through deep sequencing of the Culicoides transcriptome in response to teneral (newly-emerged, unfed), blood-fed, and sugar-fed states. The largest physiological response was observed during blood feeding conditions, when compared to teneral (not shown) or sugar feeding midges (Table 3). Since Culicoides sonorensis females are anautogenous, requiring a blood meal in order to produce eggs, it was not surprising that many of these unigenes were classified under reproduction and lipid metabolism, categories which include processes associated with vitellogenesis. Also as expected, a difference was observed in the transcription of genes associated with processing the blood meal including catalytic activity (e.g. digestive enzymes), transport, and interestingly, defense both compared to sugar feeding (within time) and across time in blood fed midges (Table 3). Intriguingly, many of the innate immune genes appear to be induced by blood feeding alone, in the absence of pathogens, which has previously been reported in other hemaophagous arthropods (Jochim et al., 2008, Lai et al., 2004, Munks et al., 2001, Nayduch and Aksoy, 2007).

Table 3.

Unigene sequences shared and differentially expressed in female Culicoides sonorensis transcriptomes. Culicoides sonorensis were fed blood or sugar diets and transcriptomes were generated from whole adult females at early (2, 6, 12 h pooled) and late (36 h) times post-ingestion. The number of genes that were significantly differentially expressed (DE) were determined by the Cufflinks statistical analysis package (Trapnell et al., 2010) (P < 0.01).

	Number unigenes shared	Number unigenes DE (%)
Effect of diet within time interval
Early sugar vs. Early blood	16,306	5712 (35%)
Late sugar vs. Late blood	16,665	3301 (20%)
Effect of time interval within diet
Early sugar vs. Late sugar	16,687	114 (0.7%)
Early blood vs. Late blood	16,297	7334 (45%)

3.3. Genomic studies of C. sonorensis

A high quality reference genome assembly and comprehensively annotated gene complement is critical for deciphering the biology of an organism and performing comparative genomics at the population or species level (Severson and Behura, 2012). Earlier attempts at de novo sequencing of the Culicoides genome were unsuccessful due to a very high level of bacterial and fungal contamination of genomic DNA samples. To mitigate this problem, genomic DNA is being used from the KC cell line (daughter AK line Table 2) maintained at The Pirbright Institute. While still at an early stage, the first full de novo sequencing project to be carried out for Culicoides has already yielded promising preliminary data. Once a draft genome sequence is available from this source, the genome assembly will be verified by resequencing vector competent and refractory individuals of C. sonorensis from the AA line. A stated aim of the project is to generate ∼150-fold coverage of the genome using paired-end reads using Illumina HiSeq2000 sequencing methodologies, supplemented with 50-fold coverage of two large insert libraries (3 kb and 8 kb) for higher level scaffolding.

High repeat content is often the major impediment to genome assembly from short reads. Promisingly, early estimates of genome size in C. sonorensis are in the order of 200 MB, similar to Anopheline species and considerably smaller than Aedes aegypti suggesting that the substantial transposable element content found in this species is not present (Nene et al., 2007). Once a draft reference genome is available, the task of annotation will commence utilizing similar approaches to those previously used to annotate dipteran vector species based on the integrating gene builder MAKER (Holt and Yandell, 2011). This process will be informed by available Culicoides transcriptome resources, proteomes from other arthropod species as well as ab initio approaches. The definition of a high quality, annotated reference sequence will provide researchers with a powerful resource for the elucidation of the mechanisms of the Culicoides vector/pathogen/host relationship, for use in comparative studies amongst Culicoides species and between these and other hematophagous vector groups, and a template that will enable the rapid assembly of individual genomes in population-level studies of species. The data will be hosted by the Ensembl Metazoa portal (http://metazoa.ensembl.org), which will provide access to genome sequence, functional annotation, alignments and evolutionary reconstructions of gene families, and facilitating integrated analysis with a wide range of arthropods through a common set of interactive and programmatic interfaces.

4. Impact, applications and ongoing projects

The parallel development of a high-quality transcriptome assembly and genome sequencing project for C. sonorensis has significant complimentary benefits in affording the opportunity to measure gene dynamics under response to particular conditions while providing a template of spliced coding sequences for annotation purposes. Analysis of the transcriptional landscape will decipher gene expression signatures associated with key biological events during feeding over time and allow investigation of vector competence. Our observation of an intense physiological response to feeding has underscored critical pathways and candidate genes for validation and potential functional intervention, using quantitative reverse-transcriptase PCR (qRT-PCR) and RNA interference (RNAi), respectively. While preliminary studies have been conducted in the KC cell line (Schnettler et al., 2013), it is abundantly clear that there is a fundamental gap in understanding the immune/defense response in Culicoides as whole organisms and our future studies stemming from our differential expression analyses will help us test hypothesis that mechanisms of vector competence can be unraveled with the appropriate sampling, depth and quality of sequencing, and proper statistical analysis. The collaborative efforts of the CGTA demonstrate the utility and potential for genome and transcriptome enablement for Culicoides, and our current efforts are being focused on enhancing the unigene set through additional extensive sampling of tissues during a series of development stages.

A potential bottleneck to progress in this area lies in understanding the degree to which the C. sonorensis lines used in these studies are representative of field populations and how the various fundamental processes investigated relate to other members of the genus. As has been stated for mosquitoes (Tabachnick, 2013), it is not currently known whether underlying drivers of vector competence vary at an inter-specific level and this has historically led to suggestions of regional incompatibility between species of Culicoides and circulating BTV strains (Wilson et al., 2009a). In this regard, an additional potential avenue of research would be to examine the extant colonies of C. nubeculosus, which are currently held at The Pirbright Institute and several other collaborating laboratories. While taxonomically closely related to C. sonorensis and placed within the same subgenus (Monoculicoides), rates of arbovirus competence in this line are far lower and the colony provides the same intrinsic advantages for study as the C. sonorensis lines (Boorman, 1974, Mellor, 2000). It is clear, also, that there may be value in re-establishing colonies of Culicoides that exhibit specific phenotypic traits and which can be maintained using existing systems, a possible example being C. riethi, which unlike C. sonorensis and C. nubeculosus exhibits autogeny (Boorman, 1974).

With the advantages of both a framework genome generated from C. sonorensis and concurrent advances in sequencing technologies, however, the targeting of field populations of species according to their importance as vectors of BTV and their behavior in the laboratory is also likely to become increasingly attainable, particularly for transcriptome-based analysis. An obvious candidate for future study is C. imicola, a proven BTV vector whose distribution spans a vast area across sub-Saharan Africa, the middle-east, southern Europe and southern Asia (Mellor et al., 2009). A limited degree of progress has been made in colonizing this species (Veronesi et al., 2009), but the definition of artificial means to allow consistent feeding and infection with arboviruses is a major advantage in study of this species (Venter et al., 1991). In contrast, both the vector status and potential for laboratory colonization of northern European Culicoides at the center of major recent arbovirus outbreaks remains poorly explored and the apparent involvement of several species in transmission complicates this picture further (Carpenter et al., 2009, Hoffmann et al., 2009).

In summary, genomic resources will be of future global significance in creating an entirely new area in Culicoides research. The C. sonorensis genome will provide not only a stand-alone resource for investigating fundamental genetic and immunological processes, but also inform arthropod phylogenetic relationships and allow comparative genomics between these and other available genomes to elucidate the genetic drivers of vector competence. While substantial difficulties remain to be resolved in producing biological material suitable for this process, the availability of C. sonorensis as a framework for future study is likely to alleviate this issue, at least in part. Further studies to correctly define the identity and behavioral malleability of major vector species worldwide, however, are of significant importance in targeting of future research.