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The Black Fly (Diptera: Simuliidae) Genome and EST Project Black Fly Genome Consortium Organizing Laboratories: Marine & Aquatic Genetics, Biology,Creighton University, Omaha, NE. (Brockhouse) Center for Genomics and Biotechnology(CGB), Indiana University, Bloomington, IN.

(Cobourne) Department of Entomology, Natural History Museum, London, U.K. (Post) Biodiversity of Medically Important Arthropods Laboratory, Clemson University, Clemson, SC.

(Adler) Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Accra, Ghana,

(Wilson, Boakye) Freshwater Ecology Laboratory, University of South Alabama, Mobile, AL. (McCreadie)

Abstract: We propose a full genome sequencing project and an accompanying cDNA sequencing project for the Simuliidae (Black Flies). The full genome sequence will be an invaluable resource for the insect genomics community, that will allow order-wide functional genomic comparative analysis of genomic contents and their organization, as well as functional analyses of critical parameters such as insect attributes linked to their capacity to transmit disease agents. These attributes include blood feeding (haematophagy), parasite/pathogen transmission, symbiosis, and insecticide resistance. The EST project is critical to assembling the full genome in the face of the absence of genetic maps and the presence of inversion polymorphisms, and will enormously enhance efforts to genetically map the black fly genome and explore gene regulation, differences among species, and symbiosis. The cDNA project is a critical component of this proposal, to facilitate the annotation of the genome sequence and to produce reliable microarrays that will be used to explore the conservation of transcriptional regulators under conditions that are shared by species that vector the agents of disease. This project aims to make a significant impact in furthering genomic knowledge of vector biology, by promoting comparative research on a disease vector that has close phylogenetic relationships to both mosquitoes (Anopheles, Aedes) and non-haematophagous insects. Biological material will be supplied by participating laboratories (Noblet, Brockhouse, Adler, Post, McCreadie, Wilson, Boakye), while the CGB will carry out the genomic projects in support of the genome sequencing and effort, including cDNA library construction/screening, sequence assembly validations, EST characterization, and related bioinformatics. The principal investigators will solicit the involvement of a growing insect genomics research community for the overall analysis and annotation. The resulting database will be incorporated into VectorBase and within the proposed InsectBase. 1) Justification for Sequencing the Black Fly Genome. Family Simuliidae: Importance to Humans and the Environment Black flies are generally regarded as the second most pernicious group of insects that afflict the health and economic well-being of humans (Adler et al., 2004). The blood-feeding activity of the adult females transmits a variety of pathogens, notably Onchocerca, Leucocytozoon, Mansonella, Trypanosoma, and Dirofilaria (Adler, 2005) Onchocerca volvulus alone infects approximately 19 million people on two continents, and has been the focus of one of the World Health Organization’s largest programs (Onchocerciasis Control Programme).

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In spite of these associated pathogens, much of the negative impact of black flies is not due to transmission of disease agents, but to the effects of sheer mass biting. In the northern hemisphere, vast areas are rendered nearly uninhabitable due to the biting rates (Adler et al., 2004). This is also true of developing countries in the tropics, where biting nuisance is recognized as a major barrier to economic development (Huggard et al., 1998). Ironically, while the adult females pose an enormous burden to human well being, the larvae are keystone species in rivers and constitute an essential ecological resource. Black fly larvae are aquatic filter feeders and are able to capture material classified as “dissolved organic matter” (DOM, in ecological parlance). This material is available to few other macroinvertebrates. As a result, this material becomes available to other macroinvertebrates and other organisms which feed either directly on the black flies or on their fecal pellets (Malmqvist, 2004). Despite the medical and ecological importance of black flies, progress on simuliid molecular biology has been slow. This is largely due to the tremendous difficulty in creating and maintaining laboratory colonies. Only one colony of a single species (Simulium vittatum) currently exists at the University of Georgia (Athens). As a consequence, the bulk of work must be conducted using wild-collected material from populations that are highly polymorphic, often of mixed species, and often parasitized or carrying poorly characterized symbionts and pathogens (for review of black fly pathogens see Crosskey, 1990). Black Flies (The Simuliidae): A Window on the Mosquito Genome. Although significant pests in their own right, black flies pale in comparison to mosquitoes as vectors of disease agents, especially with respect to the range of pathogens transmitted. (several of the mosquito-borne pathogens have been listed as bio-terror agents). A great deal of the vectorial capacity of mosquitoes is due to the simple mechanics of blood-feeding: mosquito mouthparts actually travel into the capillaries to imbibe blood, whereas black flies are pool-feeders that slash the skin and lap the oozing blood; pathogens are more directly inoculated into the host by the hypodermic mechanism of mosquitoes than through the lapping action of simuliids (cf. Clements, 1999; Crosskey, 1990). Given the potential number of mosquito species (>3500) available as vectors, it is not feasible to completely sequence all potentially important haematophagous mosquito genomes. By using an appropriate “outgroup” to the three existing mosquito genome projects, however, it is possible to construct a “consensus” view of the mosquito genome. The black fly genome provides the perfect “outgroup” with which to better understand the mosquito genome. Black flies are members of the Culicimorpha, allied to mosquitoes, but separated from them by several other families (Wood and Borkent, 1989). They are taxonomically closer to the culicids than are other dipterans with sequenced genomes (e.g. the drosophilids), and significantly the Culicidae and Simuliidae have startling parallels in their biology, many of which are independently derived. For example, both mosquitoes and simuliids have obligatory aquatic larvae, both groups are female-hematophagous (possibly independently derived; Black and Kondratieff, 2005), and both include members that have secondarily abandoned blood-feeding in either an obligatory or facultative manner. The females of both groups exhibit complex host-seeking behavior involving visual and olfactory cues. The list of parallels continues: insecticide resistance is a significant problem in black flies and mosquitoes, both exhibit inter- and intra-specific variation in vectorial capacity, and both have a propensity for sibling species, which pose great taxonomic and genetic challenges. Parallel development of many of these traits presents a sterling opportunity for genome-wide

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searches for selection. Selection leaves imprints on the genome (e.g., differential mutation rates between silent and non-silent sites) that allows the detection of genes involved in the processes being examined (e.g., Savard et al., 2006; Steinke et al., 2006). Having the full gene inventories is also useful to detect lineage-specific gene family expansions (or reductions) as a function of the organisms’ biology. By obtaining the fully characterized simuliid genome, rather than just partial sequences or ESTs alone, the wider dipteran and arthropod genomics communities will be able to engage in functional genomic studies, focusing on haematophagy and ecological traits at the family level, for example. 2) No Member of the Simuliidae is Currently the Focus of a Genome Project. In spite of the very early beginnings of black fly molecular biology (Teshima, 1972; Sohn et al., 1975), no black fly genome has been sequenced nor has another proposal been made to sequence a black fly genome to our knowledge. However, a BAC library is currently being prepared from S. squamosum (a member of the S. damnosum complex and a vector of onchocerciasis) by the Post laboratory. This library will be available for the genome sequencing project. 3) Interest to the Scientific Community The community of black fly workers includes representation on all the populated continents, and is represented by three major professional organizations (North American Black Fly Association, British Simuliid Group, and the European Simuliid Symposium Group.) The North American and British groups meet annually, and the European group every two years, with international meetings held periodically (the September 2006 Novi Sad meeting was the 5th such meeting). Approximately 250 laboratories worldwide either focus on simuliids or include black flies as a major part of their research program. Additionally, black flies have been used for comparative purposes by workers concentrating on other taxa, most notably mosquitoes (e.g., Edwards et al., 1997; Pennington et al., 2002) Specific participants from the black fly community include Brockhouse (Creighton University), Adler (Clemson University), McCreadie (University of South Alabama), Post (Natural History Museum, London) and Lustigman (New York Blood Center), Wilson, and Boakye (both Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Accra) . Non-black fly workers with interest in participating in the project for comparative purposes include Grigliatti (University of British Columbia, Vancouver, Canada) and Tanguay (Université Laval, Québec, Canada). The Center for Genomics and Biotechnology of Indiana University (Bloomington) is willing to perform the technical and bioinformatics aspects of the project, and coordinate annotation of the data. We anticipate great interest from the mosquito community, as well as the wider insect genomics community. The availability of an annotated simuliid genome will greatly enhance efforts toward the functional genomics of insects and a wider effort focusing on arthropods, as envisioned by the organizers of the proposed InsectBase genome data bank (which would include FlyBase and VectorBase). 4) Suitability of the Organism for Experimentation. Although molecular and biochemical studies focused on Simuliidae lag behind those of some other insect families (most notably culicids and drosophilids), a huge amount of information

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on natural populations exist that will be enormously useful in combination with functional genomic studies. Laboratory colonization of black fly species. It proved to be extraordinarily difficult to establish laboratory colonies of black flies. Only one colony of one species is extant, Simulium vittatum, maintained by the Noblet lab at the University of Georgia, Athens. Brockhouse and Adler (2003) give a summary of the history of this colony, and a cytogenomic analysis of its genetic variability with reference to wild populations, and its now extinct sister colony at the University of Arizona. This colony is free from parasites, including the ubiquitous trichomycete fungi (Beard and Adler, unpublished) and the endobacterium Wolbachia (Dobson and Brockhouse, unpublished). While black flies are difficult to breed in the lab, most species are easily maintained for a limited time (months), and so it is possible for molecular labs to obtain material for nucleic acid and protein extractions. Black Flies as Study Organisms Cytogenomics The genomes of no other arthropod family have been so well characterized at the natural population level, ironically because simuliids are so difficult to breed in the laboratory. The laboratory of Klaus Rothfels and its many offshoots have focused on cytogenetic studies of wild populations since 1949. Phenomena as diverse as intra- and interspecific genome rearrangements (Rothfels, 1979), chiasma localization (Rothfels and Nambiar, 1975), polytene chromosome DNA replication (Bedo, 1975), sex-determination (Rothfels and Nambiar, 1981; Post, 1982; Feraday, 1984; Brockhouse, 1985), and supernumerary chromosomes (Procunier, 1982b; Brockhouse et al., 1989) have all been studied intensively. Critically, this body of population cytogenomics work has enabled simuliidologists to understand speciation and species delineations throughout the family at a level without parallel in animal systems (cf. Adler et al., 2004). Systematics The systematics of black flies can truthfully be said to be among the most advanced of any eukaryote taxa. Gamma level taxonomy has been applied (largely, though not exclusively through application of population cytogenomics) for approximately 50 years (cf. Adler et al., 2004). Higher level taxonomy (above species level) has also been intensively studied, both within the family and in comparison with allied dipterans, using both classical and DNA comparison methods (Moulton, 2000, 2003). Systematics studies have been integrated with biogeographical problems, for example the colonization of Polynesia and other Pacific island groups (Craig et al., 2001). Molecular systematics studies have included sibling species resolution (e.g., Post and Flook, 1992; Brockhouse et al., 1993; Duncan et al., 2004; Krüger et al., 2000, 2006; Rodriguez-Perez et al., 2006) and higher level relationships within the family (Moulton, 2000, 2003). To date, no molecular approach has matched the success of polytene cytogenomic analysis, although some of the studies approach this resolution. The African S. damnosum complex, which includes the most significant vectors of onchocerciasis, has 55 sibling species and cytotypes, which is more than any other insect, and probably any other organism of any sort. This emphasizes how well characterized black flies are at the natural population level.

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Physiology Relatively little has been done concerning simuliid physiology, largely because of the difficulty in obtaining species-pure material and developmentally staged specimens in sufficient quantity. Cupp’s group has conducted a program relating to the blood-feeding of the adult female focusing on anticoagulant pathways (e.g., Abebe et al., 1994, 1996; Cupp and Cupp, 1997). Literature on larval hemolymph physiology exists (Gordon and Bailey, 1976), but little recent information is available. The behavioral physiology of host location by adult females has been examined by Sutcliffe’s group (Sutcliffe, 1986; Sutcliffe et al., 1994, 1995). Ecology The Simuliidae have been the focus of extensive ecological investigations (cf. Kim and Merritt, 1988; Crosskey, 1990; Adler et al., 2004). The ecology of the larvae is particularly well advanced, in great part because larvae can be identified to the species level using cytogenomic analysis. Macro- and microhabitat studies, species interactions, community structure, and symbioses are all the foci of active study. Studies of adult ecology have focused primarily on oviposition (Imhof and Smith, 1979; McCall et al., 1997), flight activity (Grace & Shipp, 1988), dispersal (Baldwin et al., 1975), and relationships with the vertebrate hosts (e.g., Hunter et al., 1993; Rohner et al., 2000). Genome structure Typically, black flies have n = 3 submetacentric chromosomes, which are greatly amplified in

the larval silk glands (Figure 1; the polytene chromosomes of S. vittatum). The chromosome complement has been reduced to n=2 independently in several simuliid taxa, including the subgenus Eusimulium and certain members of the genus Cnephia (Leonhardt 1985, Procunier 1982a). The only black fly genome size to be measured was 1.87 x 108bp (Prosimulium multidentatum; Sohn et al. 1975). Rothfels and Nambiar (1975) indicated that this species has unusually large meiotic/mitotic chromosomes, so this size may

well be on the large end for the family. All species examined by Sohn et al. (1975) had large amounts of highly repetitive DNA (~30-40%) and high A-T genomes (~70% A-T).

Polytene chromosome replication studies by Bedo (1982) indicate that the entire genome is amplified during polytenization and that centromeric regions may even be amplified to a greater degree than the “euchromatic” areas. The “unique” fraction of the genome, determined by CoT plotting, was reported at 49% (Sohn et al., 1975). Detailed polytene chromosome maps and/or annotated idiograms of approximately 500 species have been published, with considerably more available from various laboratories (cf. Adler et al., 2004). To date, there have been very few genes mapped to the polytenes by in situ hybridization, largely because so few cloned genes are available that no significant coverage can be achieved. Those that have include antihemostatic proteins in Simulium vittatum (Procunier et al., 2005) and insecticide resistance loci in Simulium sanctipauli

Fig.1

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(Boakye et al., 2000). Otherwise, genetic maps consist solely of cytologically recognizable loci such as nucleolar organizers, centromeres, and uniquely sex-differential regions.

Sex-determination is based on a positively-male determining Y chromosome (i.e. like that of mammals, rather than the balance system of Drosophila). There are, however, no permanent X & Y chromosomes. Rather, areas of the genome become sex-differential by linkage of chromosome rearrangements (inversions, most typically) with the sex-determiner. Males, then, are heterozygous for a particular inversion while females are homozygous. Figure 2 shows the X/Y chromosome pair of Simulium vernum IIS-1 (Brockhouse, 1984). Any area of the genome can be sex-determining and is often the major or sole cytological difference among sibling species. The nature of the primary sex-determiner, whether a transposon or a multigene system, is still unknown.

5) Rationale for the Sequencing Strategy.

A) Full Genome Sequence. The full simuliid genome sequence would greatly facilitate functional genomic studies, well beyond simply presence/absence of given genes. Character evolution would be traceable with sequence evolution on a genome wide level, greatly enhancing our understanding of a wide variety of taxa. The full genome would also greatly enhance the quality of the EST banks (see below), and permit studies such as intron boundary determination. The genome sequence, coupled with cDNA sequencing, would also greatly enhance the development of technologies to investigate gene functions and regulation. To minimize problems with inversion polymorphisms, which are extraordinarily common in the Simuliidae, a small number of larvae will be individually cytotyped (by the removal of one silk gland). The remainder of the typed-larvae will be used for DNA extraction, followed by whole genome amplification. The CGB will create BAC libraries for transfer to the appropriate sequencing center (the CGB is prepared to sequence in-house if other facilities are not available). The CGB will handle the bioinformatics, coordinating annotation, and database management. The black fly genome will be incorporated into FlyBase, thereby making the data available to the wider community of genome workers.

B) Accompanying EST Project A black fly EST project was listed as a high priority project by the Special Program for Research and Training in Tropical Diseases (TDR) group of the World Health Organization (WHO, 2003). Specific applications of the DNA arrays include producing a genetic map of Simulium vittatum. This will be a critical aid in the assembly of the full genome, as there are currently no genetic maps for this family. At the moment we are conducting pilot experiments with Drosophila melanogaster to use microarrays as genome mapping tools. Polytene chromosome regions will be microdissected and amplified (“Genomify”), and used as probes on Drosophila EST arrays to test the feasibility of assigning genes to chromosome regions.

Fig. 2

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Other applications include gene induction during symbiosis, gene expression differences among sibling species, and detection of environmental stress responses. In addition, we believe that many unanticipated projects will come about once the new tools are available for investigators. An EST project would also encourage the recruitment of new students and investigators into the simuliidology community by creating the tools necessary for modern experiments. Investigations, for example, on sex-determination, symbiosis, environmental stress, sibling speciation, and modes of insecticide resistance would be possible. More generally, the tropical health community will benefit with the addition of a hematophagous dipteran model system. The Simuliidae is placed among the Nematocera (“primitive” dipterans), taxonomically far removed from the drosophilids and more closely allied with the Culicidae. While Drosophila is a powerful model system, there are many things that Drosophila simply does not do, such as blood-feeding. Mosquito workers would benefit greatly by the availability of ESTs of another hematophagous fly vector for use in comparative genomics and “in silico physiology”. EST strategy: (1) The Brockhouse lab and his colleagues will ship on dry-ice minimally 5-10 micrograms of total RNA from tissues/conditions deemed important for the genome annotation project. (2) The CGB will measure the quality of the samples before proceeding to making cDNA libraries. If the samples fail quality assurance tests (primarily based on running the Agilent Bioanalyser), we will obtain more material from our collaborators. (3) The CGB will produce 12 normalized full-length enriched cDNA libraries, representing tissues and conditions deemed important for black fly biology (male larval, female larval, male adult, female adult, heat shocked, parasitized, blood fed adult female, and sublethal insecticide treated), using methods described at https://daphnia.cgb.indiana.edu/456.html and at https://projects.cgb.indiana.edu/display/genomics/Sea+Urchin+cDNA+Library+Project. (4) The CGB will deliver the following quality control data for each library and will ship glycerol/LB stocks on dry-ice to the sequencing center. >- Titer (cfu/mL) >- Size distribution of inserts as measured by agarose gel electorphoresis >- Sequences quality pass rates >- Number of Vector Sequences >- Number of mtDNA Sequences >- Number of Sequences Failing Adaptor Screen >- Number of Sequences to Cluster >- Number of Assembled Contigs + Singlets >- Percent Redundancy (gene discovery rate) (5) The sequencing center will array the libraries in 384-well plates and

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sequence 100,000 clones (both 5' and 3' EST sequences) distributed across the libraries. Sequences will be clustered/aligned to the Anolis genome sequence assembly for selection of clones for full-length cDNA sequencing by the sequencing center and by the CGB. EST-sequenced clones will be returned to CGB for duplication and community access. Brockhouse will commit 20% of his time towards obtaining biological material and co-ordination of project communications. Colbourne (Genomics Director, CGB) will commit 15% of his time, overseeing the laboratory and bioinformatics efforts of the project. 6) Availability of DNA for Sequencing. The methods for extraction of DNA from black fly larvae are now well established (e.g., Brockhouse et al., 1993). Individual larvae can be cytotyped (to minimize inversion heterozygosity and identify the orientation of any polymorphisms present), and then used for DNA extraction. Choice of Species: As a first choice, we propose focusing on Simulium squamosum, one of the major vector species in the S. damnosum complex. Post is preparing a BAC library from this species, which will be made available for the genome project. In terms of genome synteny, S. squamosum is central in the damnosum complex, and is thus a logical choice for a candidate species. Biological material (eggs, larvae, pupae, sexed adults) for EST projects are available through project participants at the Noguchi Memorial Institute for Medical Research, University of Ghana. Wilson will rear single egg batches from blood-fed females. This means that the species identification of every single specimen could be confirmed (by cytotaxonomy of one of the reared larvae) and they could be fed on a sterilised and DNA-free diet Alternatively, Simulium vittatum, from the laboratory colony, could be used as our model system. It is the only species from which we can readily obtain pathogen-, parasite- and symbiont-free material. Additionally, this species can be sexed throughout its development. Significantly, it has served as the model system for physiological studies, and testing of insecticides, including its use as a surrogate host for Onchocerca volvulus. Closely allied sibling species are readily available from wild sources for comparative studies once DNA arrays are constructed, and old DNA-DNA re-association data suggest that significant amounts (~80%) of the “unique fraction” of the genomes of different Simulium species base-pair (Teshima, 1972; Sohn et al., 1975). The genus Simulium is by far the largest within the Simuliidae and it includes all the major disease vectors, indicating that S. vittatum arrays would prove valuable for most black fly applications. The technique for mRNA extraction is also well established. The method of McDonald et al. (1987) yields mRNA that can be translated in vitro to high MW polypeptides. Tanguay et al. (unpublished) have constructed cDNA expression libraries from Simulium decorum and S. longistylatum. Shibata and Brockhouse (unpublished) are refining the previous work of Stiles et al. (unpublished) to establish black fly embryonic cell culture lines. 7) Availability of Other Funding Sources.

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Black fly genomics will have both practical and theoretical interest in a wide variety of contexts (see above). We would be willing to apply to such organizations as the World Health Organization, NSF, and DOE for support of this genome effort. 8) Literature Cited Abebe, M., M. S. Cupp, F. B. Ramberg and E. W. Cupp. 1994. Anticoagulant activity in salivary gland

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Figure Credits: Figure 1: Rothfels lab archives (Marine & Aquatic Genetics, Creighton University) Figure 2: Brockhouse, C.L. 1984. M.Sc. thesis, University of Toronto. Contact Information: Dr. Charles Brockhouse Biology Department Creighton University Omaha, NE 68178 Email: charlesbrockhouse@creighton.edu Tel: (402) 280-3393, -2811 Fax: (402) 280-5595 Dr. John Kenneth Colbourne Center for Genomics and Bioinformatics Indiana University 915 East Third Street Bloomington, Indiana 47405-7107 Emai:l jcolbour@cgb.indiana.edu Tel: (812) 856-0099 -0418 Fax: (812) 855-6082