Lipid metabolism dysfunction following symbiont elimination is linked to altered Kennedy pathway homeostasis.

Lipid metabolism is critical for insect reproduction, especially for species that invest heavily in the early developmental stages of their offspring. The role of symbiotic bacteria during this process is understudied but likely essential. We examined the role of lipid metabolism during the interaction between the viviparous tsetse fly (Glossina morsitans morsitans) and its obligate endosymbiotic bacteria (Wigglesworthia glossinidia) during tsetse pregnancy. We observed increased CTP:phosphocholine cytidylyltransferase (cct1) expression during pregnancy, which is critical for phosphatidylcholine biosynthesis in the Kennedy pathway. Experimental removal of Wigglesworthia impaired lipid metabolism via disruption of the Kennedy pathway, yielding obese mothers whose developing progeny starve. Functional validation via experimental cct1 suppression revealed a phenotype similar to females lacking obligate Wigglesworthia symbionts. These results indicate that, in Glossina, symbiont-derived factors, likely B vitamins, are critical for the proper function of both lipid biosynthesis and lipolysis to maintain tsetse fly fecundity.

Warm Blood Meal Increases Digestion Rate and Milk Protein Production to Maximize Reproductive Output for the Tsetse Fly, Glossina morsitans.

The ingestion of blood represents a significant burden that immediately increases water, oxidative, and thermal stress, but provides a significant nutrient source to generate resources necessary for the development of progeny. Thermal stress has been assumed to solely be a negative byproduct that has to be alleviated to prevent stress. Here, we examined if the short thermal bouts incurred during a warm blood meal are beneficial to reproduction. To do so, we examined the duration of pregnancy and milk gland protein expression in the tsetse fly, Glossina morsitans, that consumed a warm or cool blood meal. We noted that an optimal temperature for blood ingestion yielded a reduction in the duration of pregnancy. This decline in the duration of pregnancy is due to increased rate of blood digestion when consuming warm blood. This increased digestion likely provided more energy that leads to increased expression of transcript for milk-associated proteins. The shorter duration of pregnancy is predicted to yield an increase in population growth compared to those that consume cool or above host temperatures. These studies provide evidence that consumption of a warm blood meal is likely beneficial for specific aspects of vector biology.

Coupling Stable Isotope Labeling and Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight-Tandem Mass Spectrometry for De Novo Mosquito Ovarian Lipid Studies.

There is a need to better understand lipid metabolism during mosquito ovarian development. Lipids are the major source of energy supporting ovarian follicles development in mosquitoes. In this paper, we describe the complementary use of stable isotope labeling (SIL) and high-resolution mass spectrometry-based tools for the investigation of de novo triglycerides (TG) and diglycerides (DG) during the ovarian previtellogenic (PVG) stage (4-6 days posteclosion) of female adult Aedes aegypti. Liquid chromatography coupled to high-resolution trapped ion mobility spectrometry-parallel accumulation sequential fragmentation-time-of-flight tandem mass spectrometry (LC-TIMS-PASEF-TOF MS/MS) allowed the separation and quantification of nonlabeled and 2H/13C-labeled TG and DG species. Three SIL strategies were evaluated (H2O/2H2O with 50:50 and 95:5 mixtures, 13C-sucrose, and 13C-glucose). Results showed wide applicability with no signs of lipid ovarian impairment by SIL induced toxicity. The analytical workflow based on LC-TIMS-TOF MS/MS provided high confidence and high reproducibility for lipid DG and TG identification and SIL incorporation based on their separation by retention time (RT), collision cross section (CCS), and accurate m/z. In addition, the SIL fatty acid chain incorporation was evaluated using PASEF MS/MS. The 2H/13C incorporation into the mosquito diet provided information on how TG lipids are consumed, stored, and recycled during the PVG stage of ovarian development.

Epoxidation of juvenile hormone was a key innovation improving insect reproductive fitness.

Methyl farnesoate (MF) plays hormonal regulatory roles in crustaceans. An epoxidated form of MF, known as juvenile hormone (JH), controls metamorphosis and stimulates reproduction in insects. To address the evolutionary significance of MF epoxidation, we generated mosquitoes completely lacking either of the two enzymes that catalyze the last steps of MF/JH biosynthesis and epoxidation, respectively: the JH acid methyltransferase (JHAMT) and the P450 epoxidase CYP15 (EPOX). jhamt-/- larvae lacking both MF and JH died at the onset of metamorphosis. Strikingly, epox-/- mutants, which synthesized MF but no JH, completed the entire life cycle. While epox-/- adults were fertile, the reproductive performance of both sexes was dramatically reduced. Our results suggest that although MF can substitute for the absence of JH in mosquitoes, it is with a significant fitness cost. We propose that MF can fulfill most roles of JH, but its epoxidation to JH was a key innovation providing insects with a reproductive advantage.

Following de novo triglyceride dynamics in ovaries of Aedes aegypti during the previtellogenic stage.

Understanding the molecular and biochemical basis of egg development is a central topic in mosquito reproductive biology. Lipids are a major source of energy and building blocks for the developing ovarian follicles. Ultra-High Resolution Mass Spectrometry (UHRMS) combined with in vivo metabolic labeling of follicle lipids with deuterated water (2H2O) can provide unequivocal identification of de novo lipid species during ovarian development. In the present study, we followed de novo triglyceride (TG) dynamics during the ovarian previtellogenic (PVG) stage (2-7 days post-eclosion) of female adult Aedes aegypti. The incorporation of stable isotopes from the diet was evaluated using liquid chromatography (LC) in tandem with the high accuracy (< 0.3 ppm) and high mass resolution (over 1 M) of a 14.5 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (14.5 T FT-ICR MS) equipped with hexapolar detection. LC-UHRMS provides effective lipid class separation and chemical formula identification based on the isotopic fine structure. The monitoring of stable isotope incorporation into de novo incorporated TGs suggests that ovarian lipids are consumed or recycled during the PVG stage, with variable time dynamics. These results provide further evidence of the complexity of the molecular mechanism of follicular lipid dynamics during oogenesis in mosquitoes.

Common structural features facilitate the simultaneous identification and quantification of the five most common juvenile hormones by liquid chromatography-tandem mass spectrometry.

This study reports the development and application of a liquid chromatography method coupled to electrospray tandem mass spectrometry (LC-MS/MS) for the identification and quantification of the five most common juvenile hormone (JH) homologs and methyl farnesoate (MF). The protocol allows the simultaneous analysis in a single LC run of JH I, JH II, JH III, JH III bisepoxide (JHB3) and JH III skipped bisepoxide (JHSB3). The identification of JHs is based on multiple reaction monitoring (MRM), using two of the most abundant fragmentation transitions for each hormone. Addition of deuterated JH III as an internal standard permits the absolute quantification of the different JHs. The JH homologs common structural features led to similar chromatographic behavior, as well as related fragmentation patterns, which facilitated the simultaneous detection of all the homologs in a single LC-MS/MS run. The protocol detects JHs in the low femtomole range, allowing often the analysis of JH in individual insects. Fragmentation of each of the JH homologs generates unique diagnostic ions that permitted the identification and quantification of JHs from samples of different species of Diptera, Lepidoptera, Heteroptera and Hymenoptera. Having a simple protocol, which can undisputedly determine the identity of the homologs present in a particular species, provides us with the opportunity to identify and quantify JHs existing in insects that are pests, vector of diseases or important research models.

Three Dimensional Secondary Ion Mass Spectrometry Imaging (3D-SIMS) of Aedes aegypti ovarian follicles.

The mobilization of nutrient reserves into the ovaries of Aedes aegypti mosquitoes after sugar-feeding plays a vital role in the female's reproductive maturation. In the present work, three-dimensional secondary ion mass spectrometry imaging (3D-SIMS) was used to generate ultrahigh spatial resolution (~1 µm) chemical maps and study the composition and spatial distribution of lipids at the single ovarian follicle level (~100 µm in size). 3D-Mass Spectrometry Imaging (3D-MSI) allowed the identification of cellular types in the follicle (oocyte, nurse and follicular cells) using endogenous markers, and revealed that most of the triacyglycerides (TGs) were compartmentalized in the oocyte region. By comparing follicles from water-fed and sugar-fed females (n=2), 3D-MSI-Time of Flight-SIMS showed that TGs were more abundant in ovarian follicles of sugar-fed females; despite relative sample reproducibility per feeding condition, more biological replicates will better support the trends observed. While the current 3D-MSI-TOF-SIMS does not permit MS/MS analysis of the lipid species, complementary LC-MS/MS analysis of the ovarian follicles aided tentative lipid assignments of the SIMS data. The combination of these MS approaches is giving us a first glimpse of the distribution of functionally relevant ovarian lipid molecules at the cellular level. These new tools can be used to investigate the roles of different lipids on follicle fitness and overall mosquito reproductive output.

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

BACKGROUND: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. RESULTS: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. CONCLUSIONS: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

Inhibition of juvenile hormone synthesis in mosquitoes by the methylation inhibitor 3-deazaneplanocin A (DZNep).

Juvenile hormone (JH), synthesized by the corpora allata (CA), controls development and reproduction in mosquitoes through its action on thousands of JH-responsive genes. These JH-dependent processes can be studied using tools that increase or decrease JH titers in vitro and in vivo. Juvenile hormone acid methyl transferase (JHAMT) is a critical JH biosynthetic enzyme. JHAMT utilizes the methyl donor S-adenosyl-methionine (SAM) to methylate farnesoic acid (FA) into methyl farnesoate (MF), releasing the product S-adenosyl-L-homocysteine (AdoHcy), which inhibits JHAMT. S-adenosyl-homocysteine hydrolase (SAHH) catalyzes AdoHcy hydrolysis to adenosine and homocysteine, alleviating AdoHcy inhibition of JHAMT. 3-deazaneplanocin A (DZNep), an analog of adenosine, is an inhibitor of SAHH, and an epigenetic drug for cancer therapy. We tested the effect of DZNep on in vitro JH synthesis by CA of mosquitoes. DZNep inhibited JH synthesis in a dose-response fashion. Addition of MF, but not of FA relieved the inhibition, demonstrating a direct effect on JHAMT. In vivo experiments, with addition of DZNep to the sugar ingested by mosquitoes, resulted in a dose-response decrease in JH synthesis and JH hemolymphatic titers, as well as expression of early trypsin, a JH-dependent gene. Our studies suggest that DZNep can be employed to lower JH synthesis and titer in experiments evaluating JH-controlled processes in mosquitoes.

Juvenile hormone controls ovarian development in female Anopheles albimanus mosquitoes.

Anophelinae mosquitoes are vectors of human malaria, a disease that infects hundreds of millions of people and causes almost 600,000 fatalities annually. Despite their medical importance, laboratory studies on key aspects of Anophelinae reproductive biology have been limited, and in particular, relatively little is known about the role of juvenile hormone (JH) in the control of female reproduction. The study presented here attempts to fill a gap of knowledge in our understanding of the JH control of ovarian development in female Anophelinae mosquitoes, using Anopheles albimanus as a model. Our studies revealed that JH controls the tempo of maturation of primary follicles in An. albimanus in a similar manner to that previously described in Aedes aegypti. At adult eclosion JH hemolymph titer was low, increased in 1-day old sugar-fed insects, and decreased in blood fed individuals. JH titers decreased if An. albimanus females were starved, and were reduced if insects emerged with low teneral reserves, precluding previtellogenic ovarian development. However, absolute hemolymph titers were lower than Ae. aegypti. Decapitation experiments suggested that if teneral reserves are sufficient, factors from the head activate JH synthesis by the corpora allata (CA) during the first 9-12 h after adult emergence. In conclusion, our studies support the hypothesis that JH controls previtellogenic ovarian development in female An. albimanus mosquitoes, in a similar manner that have been described in Culicinae.

Effect of antibiotic treatment and gamma-irradiation on cuticular hydrocarbon profiles and mate choice in tsetse flies (Glossina m. morsitans).

BACKGROUND: Symbiotic microbes represent a driving force of evolutionary innovation by conferring novel ecological traits to their hosts. Many insects are associated with microbial symbionts that contribute to their host's nutrition, digestion, detoxification, reproduction, immune homeostasis, and defense. In addition, recent studies suggest a microbial involvement in chemical communication and mating behavior, which can ultimately impact reproductive isolation and, hence, speciation. Here we investigated whether a disruption of the microbiota through antibiotic treatment or irradiation affects cuticular hydrocarbon profiles, and possibly mate choice behavior in the tsetse fly, Glossina morsitans morsitans. Four independent experiments that differentially knock down the multiple bacterial symbionts of tsetse flies were conducted by subjecting tsetse flies to ampicillin, tetracycline, or gamma-irradiation and analyzing their cuticular hydrocarbon profiles in comparison to untreated controls by gas chromatography - mass spectrometry. In two of the antibiotic experiments, flies were mass-reared, while individual rearing was done for the third experiment to avoid possible chemical cross-contamination between individual flies. RESULTS: All three antibiotic experiments yielded significant effects of antibiotic treatment (particularly tetracycline) on cuticular hydrocarbon profiles in both female and male G. m. morsitans, while irradiation itself had no effect on the CHC profiles. Importantly, tetracycline treatment reduced relative amounts of 15,19,23-trimethyl-heptatriacontane, a known compound of the female contact sex pheromone, in two of the three experiments, suggesting a possible implication of microbiota disturbance on mate choice decisions. Concordantly, both female and male flies preferred non-treated over tetracycline-treated flies in direct choice assays. CONCLUSIONS: While we cannot exclude the possibility that antibiotic treatment had a directly detrimental effect on fly vigor as we are unable to recolonize antibiotic treated flies with individual symbiont taxa, our results are consistent with an effect of the microbiota, particularly the obligate nutritional endosymbiont Wigglesworthia, on CHC profiles and mate choice behavior. These findings highlight the importance of considering host-microbiota interactions when studying chemical communication and mate choice in insects.

Gene expression in reproductive organs of tsetse females - initial data in an approach to reduce fecundity.

BACKGROUND: Tsetse flies are vectors of African trypanosomes, and their vectorial capacity results in a major public health emergency and vast economic losses in sub-Saharan Africa. Given the limited ability of trypanosome prevention and eradication, tsetse vectors remain major targets of control efforts. Larvae of all three instars are developed in mothers' uteri, nourished through milk, and 'larviposited' shortly before pupation. The past few years have witnessed the emergence of approaches based on knockdown of genes involved in milk production, resulting in a significant reduction of fecundity. RESULTS: In order to identify further genes applicable in the control of tsetse flies, we determined the expression of protein-coding genes in ovaries and uteri from both virgin and heavily pregnant Glossina morsitans morsitans females. Comparison of expression profiles allowed us to identify candidate genes with increased expression in pregnant individuals. Lists with the highest increases include genes involved in oocyte and embryonic development, or nourishment. Maximum ovarian fold change does not exceed 700, while the highest uterine fold change reaches to more than 4000. Relatively high fold changes of two neuropeptide receptors (for corazonin and myosuppressin) propose the corresponding genes alternative targets. CONCLUSIONS: Given the higher fold changes in the uterus, targeting gene expression in this tissue may result in a more evident reduction of fecundity. However, ovaries should not be neglected, as manifested by several genes with top fold changes involved in early developmental stages. Apart from focusing on the highest fold changes, neuropeptide receptors with moderate increases in expression should be also verified as targets, given their roles in mediating the tissue control. However, this data needs to be considered initial, and the potential of these genes in affecting female fecundity needs to be verified experimentally.

JH biosynthesis and hemolymph titers in adult male Aedes aegypti mosquitoes.

Juvenile hormone (JH) is a major hormonal regulator in insects. In Aedes aegypti females, JH signals the completion of the ecdysis to the adult stage and initiates reproductive processes. Although the regulation of JH synthesis and titer in Ae. aegypti females has been extensively studied, relatively little is known about changes of JH synthesis and titers in male mosquitoes, as well as on the roles of JH controlling male reproductive biology. A better understanding of male mosquito reproductive biology, including an improved knowledge of the hormonal control of reproduction, could increase the likelihood of success of male-targeting vector control programs. Using a high performance liquid chromatography coupled to electrospray tandem mass spectrometry method, we measured JH biosynthesis and hemolymph levels in male mosquitoes during pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers. Synthesis of JH III was very low in late pupae, significantly increased during the first 24 h after adult eclosion, and then remained relatively constant during the first six days after adult eclosion. Feeding high sugar diets resulted in an increase of JH synthesis and titers, and starvation significantly decreased JH synthesis, but this effect could be reversed by changing the males back to a high sugar diet. JH synthesis rates were similar in virgin and mated males, but hemolymph JH levels were different in well-nourished virgin and mated males. Starvation resulted in a significant reduction in insemination rates; with well-nourished males inseminating 2 times more females than water-fed. Giving a 20% sugar meal for 24 h to those mosquitoes that were previously starved for 6 days, caused a significant rise in insemination rates, restoring them to levels similar to those recorded for 20% fed males. These results suggest that nutrition plays a role on male fecundity, and this effect might be mediated by JH.

Rapid autophagic regression of the milk gland during involution is critical for maximizing tsetse viviparous reproductive output.

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.

The Spermatophore in Glossina morsitans morsitans: Insights into Male Contributions to Reproduction.

Male Seminal Fluid Proteins (SFPs) transferred during copulation modulate female reproductive physiology and behavior, impacting sperm storage/use, ovulation, oviposition, and remating receptivity. These capabilities make them ideal targets for developing novel methods of insect disease vector control. Little is known about the nature of SFPs in the viviparous tsetse flies (Diptera: Glossinidae), vectors of Human and Animal African trypanosomiasis. In tsetse, male ejaculate is assembled into a capsule-like spermatophore structure visible post-copulation in the female uterus. We applied high-throughput approaches to uncover the composition of the spermatophore in Glossina morsitans morsitans. We found that both male accessory glands and testes contribute to its formation. The male accessory glands produce a small number of abundant novel proteins with yet unknown functions, in addition to enzyme inhibitors and peptidase regulators. The testes contribute sperm in addition to a diverse array of less abundant proteins associated with binding, oxidoreductase/transferase activities, cytoskeletal and lipid/carbohydrate transporter functions. Proteins encoded by female-biased genes are also found in the spermatophore. About half of the proteins display sequence conservation relative to other Diptera, and low similarity to SFPs from other studied species, possibly reflecting both their fast evolutionary pace and the divergent nature of tsetse's viviparous biology.

Adenotrophic viviparity in tsetse flies: potential for population control and as an insect model for lactation.

Tsetse flies (Glossina spp.), vectors of African trypanosomes, are distinguished by their specialized reproductive biology, defined by adenotrophic viviparity (maternal nourishment of progeny by glandular secretions followed by live birth). This trait has evolved infrequently among insects and requires unique reproductive mechanisms. A key event in Glossina reproduction involves the transition between periods of lactation and nonlactation (dry periods). Increased lipolysis, nutrient transfer to the milk gland, and milk-specific protein production characterize lactation, which terminates at the birth of the progeny and is followed by a period of involution. The dry stage coincides with embryogenesis of the progeny, during which lipid reserves accumulate in preparation for the next round of lactation. The obligate bacterial symbiont Wigglesworthia glossinidia is critical to tsetse reproduction and likely provides B vitamins required for metabolic processes underlying lactation and/or progeny development. Here we describe findings that utilized transcriptomics, physiological assays, and RNA interference-based functional analysis to understand different components of adenotrophic viviparity in tsetse flies.

Vitamin B6 generated by obligate symbionts is critical for maintaining proline homeostasis and fecundity in tsetse flies.

The viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B6) as a cofactor. This vitamin can be synthesized by tsetse's obligate symbiont, Wigglesworthia glossinidia. In this study, we examined the role of Wigglesworthia-produced vitamin B6 for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse's reproductive cycle. We found that expression of agat, as well as genes involved in vitamin B6 metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B6 in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast or Wigglesworthia extracts. RNA interference-mediated knockdown of agat in wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated with agat short interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence of Wigglesworthia in tsetse is critical for the maintenance of proline homeostasis through vitamin B6 production.

Aquaporins are critical for provision of water during lactation and intrauterine progeny hydration to maintain tsetse fly reproductive success.

Tsetse flies undergo drastic fluctuations in their water content throughout their adult life history due to events such as blood feeding, dehydration and lactation, an essential feature of the viviparous reproductive biology of tsetse. Aquaporins (AQPs) are transmembrane proteins that allow water and other solutes to permeate through cellular membranes. Here we identify tsetse aquaporin (AQP) genes, examine their expression patterns under different physiological conditions (blood feeding, lactation and stress response) and perform functional analysis of three specific genes utilizing RNA interference (RNAi) gene silencing. Ten putative aquaporins were identified in the Glossina morsitans morsitans (Gmm) genome, two more than has been previously documented in any other insect. All organs, tissues, and body parts examined had distinct AQP expression patterns. Two AQP genes, gmmdripa and gmmdripb ( = gmmaqp1a and gmmaqp1b) are highly expressed in the milk gland/fat body tissues. The whole-body transcript levels of these two genes vary over the course of pregnancy. A set of three AQPs (gmmaqp5, gmmaqp2a, and gmmaqp4b) are expressed highly in the Malpighian tubules. Knockdown of gmmdripa and gmmdripb reduced the efficiency of water loss following a blood meal, increased dehydration tolerance and reduced heat tolerance of adult females. Knockdown of gmmdripa extended pregnancy length, and gmmdripb knockdown resulted in extended pregnancy duration and reduced progeny production. We found that knockdown of AQPs increased tsetse milk osmolality and reduced the water content in developing larva. Combined knockdown of gmmdripa, gmmdripb and gmmaqp5 extended pregnancy by 4-6 d, reduced pupal production by nearly 50%, increased milk osmolality by 20-25% and led to dehydration of feeding larvae. Based on these results, we conclude that gmmDripA and gmmDripB are critical for diuresis, stress tolerance and intrauterine lactation through the regulation of water and/or other uncharged solutes.

The homeodomain protein ladybird late regulates synthesis of milk proteins during pregnancy in the tsetse fly (Glossina morsitans).

Regulation of tissue and development specific gene expression patterns underlies the functional specialization of organs in multi-cellular organisms. In the viviparous tsetse fly (Glossina), the female accessory gland is specialized to generate nutrients in the form of a milk-like secretion to support growth of intrauterine larva. Multiple milk protein genes are expressed specifically in the female accessory gland and are tightly linked with larval development. Disruption of milk protein synthesis deprives developing larvae of nutrients and results in extended larval development and/or in abortion. The ability to cause such a disruption could be utilized as a tsetse control strategy. Here we identify and delineate the regulatory sequence of a major milk protein gene (milk gland protein 1:mgp1) by utilizing a combination of molecular techniques in tsetse, Drosophila transgenics, transcriptomics and in silico sequence analyses. The function of this promoter is conserved between tsetse and Drosophila. In transgenic Drosophila the mgp1 promoter directs reporter gene expression in a tissue and stage specific manner orthologous to that of Glossina. Analysis of the minimal required regulatory region of mgp1, and the regulatory regions of other Glossina milk proteins identified putative homeodomain protein binding sites as the sole common feature. Annotation and expression analysis of Glossina homeodomain proteins identified ladybird late (lbl) as being accessory gland/fat body specific and differentially expressed between lactating/non-lactating flies. Knockdown of lbl in tsetse resulted in a significant reduction in transcript abundance of multiple milk protein genes and in a significant loss of fecundity. The role of Lbl in adult reproductive physiology is previously unknown. These results suggest that Lbl is part of a conserved reproductive regulatory system that could have implications beyond tsetse to other vector insects such as mosquitoes. This system is critical for tsetse fecundity and provides a potential target for development of a reproductive inhibitor.