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Tsetse flies are important vectors of human and animal trypanosomiasis. Ability to reduce tsetse populations is an effective means of disease control. Lactation is an essential component of tsetse’s viviparous reproductive physiology and requires a dramatic increase in the expression and synthesis of milk proteins by the milk gland organ in order to nurture larval growth. In between each gonotrophic cycle, tsetse ceases milk production and milk gland tubules undergo a nearly two-fold reduction in width (involution). In this study, we examined the role autophagy plays during tsetse fly milk gland involution and reproductive output. Autophagy genes show elevated expression in tissues associated with lactation, immediately before or within two hours post-parturition, and decline at 24-48h post-parturition. This expression pattern is inversely correlated with that of the milk gland proteins (lactation-specific protein coding genes) and the autophagy inhibitor fk506-bp1. Increased expression of Drosophila inhibitor of apoptosis 1, diap1, was also observed in the milk gland during involution, when it likely prevents apoptosis of milk gland cells. RNAi-mediated knockdown of autophagy related gene 8a (atg8a) prevented rapid milk gland autophagy during involution, prolonging gestation, and reducing fecundity in the subsequent gonotrophic cycle. The resultant inhibition of autophagy reduced the recovery of stored lipids during the dry (non-lactating) periods by 15–20%. Ecdysone application, similar to levels that occur immediately before birth, induced autophagy, and increased milk gland involution even before abortion. This suggests that the ecdysteroid peak immediately preceding parturition likely triggers milk gland autophagy. Population modeling reveals that a delay in involution would yield a negative population growth rate. This study indicates that milk gland autophagy during involution is critical to restore nutrient reserves and allow efficient transition between pregnancy cycles. Targeting post-birth phases of reproduction could be utilized as a novel mechanism to suppress tsetse populations and reduce trypanosomiasis., Author summary Tsetse flies are vectors for trypanosomes that cause both African sleeping sickness in humans and Nagana in animals. The reduction of tsetse populations is the most efficient way to reduce the prevalence of this economically important disease with current control methods including pesticide application, traps, and sterile insect techniques. Tsetse pregnancy and milk production represent a species-specific target for population control and milk gland transition during each larval growth cycle could represent a novel target for tsetse control. Within one day after birth, the milk gland organ, essential for provisioning nutrients to the intrauterine larva, undergoes involution marked by an ecdysone driven increase in autophagy that allows breakdown of this gland. Inhibiting the process of autophagy prevents the timely transition from the lactation phase to the dry phase, triggering a delay in subsequent pregnancy cycle. This misregulation of milk gland involution leads to an overall decrease in the number of offspring that each female can produce per lifetime. This study has determined the molecular components of this process, and reveals new targets of interference for vector control.

In tsetse flies, nutrients for intrauterine larval development are synthesized by the modified accessory gland (milk gland) and provided in mother's milk during lactation. Interference with at least two milk proteins has been shown to extend larval development and reduce fecundity. The goal of this study was to perform a comprehensive characterization of tsetse milk proteins using lactation-specific transcriptome/milk proteome analyses and to define functional role(s) for the milk proteins during lactation. Differential analysis of RNA-seq data from lactating and dry (non-lactating) females revealed enrichment of transcripts coding for protein synthesis machinery, lipid metabolism and secretory proteins during lactation. Among the genes induced during lactation were those encoding the previously identified milk proteins (milk gland proteins 1–3, transferrin and acid sphingomyelinase 1) and seven new genes (mgp4–10). The genes encoding mgp2–10 are organized on a 40 kb syntenic block in the tsetse genome, have similar exon-intron arrangements, and share regions of amino acid sequence similarity. Expression of mgp2–10 is female-specific and high during milk secretion. While knockdown of a single mgp failed to reduce fecundity, simultaneous knockdown of multiple variants reduced milk protein levels and lowered fecundity. The genomic localization, gene structure similarities, and functional redundancy of MGP2–10 suggest that they constitute a novel highly divergent protein family. Our data indicates that MGP2–10 function both as the primary amino acid resource for the developing larva and in the maintenance of milk homeostasis, similar to the function of the mammalian casein family of milk proteins. This study underscores the dynamic nature of the lactation cycle and identifies a novel family of lactation-specific proteins, unique to Glossina sp., that are essential to larval development. The specificity of MGP2–10 to tsetse and their critical role during lactation suggests that these proteins may be an excellent target for tsetse-specific population control approaches., Author Summary Tsetse flies are the sole vector for African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Transcriptome and proteome analyses were utilized to examine the underlying mechanisms of tsetse lactation that occur during each reproductive cycle. These analyses revealed a dramatic shift to the synthesis of milk proteins during lactation and a novel milk-specific protein family. All members of this family were co-localized, shared sequence similarity and were expressed at 40× basal levels during milk secretion. Suppression of gene from this lactation-associated family impaired progeny development by reducing milk protein content and altering milk homeostasis. These novel genes represent an excellent target for tsetse-specific reproductive-based control mechanisms. In addition, the characterization of tsetse milk production revealed multiple factors that are functionally analogous between tsetse and mammalian lactation.