Novel Discoveries in the Male Accessory Secretions of the Tsetse fly (A transcriptomic/proteomic analysis)

Tsetse flies are the sole vectors of the human and animal forms of African Trypanosomiasis, neglected diseases that affect the health and development of marginalized populations in sub-Saharan Africa. Vector population control is one of the primary methods to prevent trypanosomiasis transmission. However, little is known about the reproductive biology of tsetse flies. A particularly important aspect of tsetse reproductive biology, male seminal secretions, remains unstudied. Based upon the knowledge derived from other insects such as Drosophila and various mosquito species, the proteins contained within these secretions play an important roles in regulating sperm storage, sperm motility, sperm competition, female sexual receptivity, egg production, ovulation, reproductive tract morphology, feeding behavior and other aspects of fly biology. To establish a foothold on this aspect of tsetse reproductive biology, we undertook a project to sequence the transcriptome of the male accessory tissue in tsetse and to sequence the proteome of the male seminal secretions found within the female after mating. Material for transcriptomic analysis was derived from dissected adult male reproductive tracts at different time points. Samples were collected from teneral, 3 day old (reproductively mature), and 6-8 hours post mating male flies. Male reproductive tracts were dissected into two fractions, testes and accessory glands. RNA isolated from these samples was used to construct 6 illumina libraries which were sequenced using paired end sequencing technology. To complement the transcriptomes, samples of male produced spermatophores were collected for proteomic analysis from the uterus of newly mated female flies. Protein samples were analyzed by LC-MS/MS. Male accessory and testes illumina sequencing data was analyzed to identify the most abundant transcripts within each library, compared between libraries to identify temporal and tissue specific differential expression patterns and compared with transcriptome data from adult female flies to identify male specific transcripts. The results of these analyses were then compared with the proteomic data to confirm transcriptomic predictions of secreted accessory proteins and identify additional unpredicted proteins. Analysis of these data sets resulted in the identification of a novel set of male accessory genes/proteins. Our initial analysis has identified a total of 25 putative accessory gland proteins via cross referencing our tissue specific transcriptome and the spermatophore proteome. Of these proteins, only one of the predicted genes (a serine protease inhibitor from the BPTI/Kunitz family) is orthologus to an accessory protein identified within Drosophila. Many proteins identified are tsetse specific and novel. Three of these novel proteins are the most abundant proteins in the spermatophore. These three proteins form a novel tsetse specific gene family that appears to have arisen through tandem gene duplication events. Further functional analysis will be required to identify the role that these novel proteins play in tsetse reproductive physiology. However, while many of the proteins we have identified are not true orthologs to other characterized accessory proteins, their function appears to have remained orthologus to those of accessory proteins from other Dipteran species. These functions include serine protease inhibitors, odorant binding proteins, antioxidants, immune proteins, glycoproteins, endocuticle proteins and sperm binding proteins.