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Lipid metabolism is critical for insect reproduction, especially for species that invest heavily into early developmental stages of their offspring. The role of symbiotic bacteria during this process is unknown but likely essential, especially in the case of obligate microbes that fulfill key biological functions in the host. Using a combined lipidomics, functional genomics and biochemical strategy, we examined the role of lipid metabolism in the interaction between the viviparous tsetse fly (Glossina morsitans morsitans) and its obligate endosymbiotic bacteria (Wigglesworthia glossinidia) during tsetse pregnancy. We observed increasedCTP:phosphocholine cytidylyltransferase(cct1) expression during pregnancy. This gene codes for the enzyme that functions as the rate limiting step in phosphatidylcholine biosynthesis in the Kennedy pathway which is critical for stored lipid metabolism and progeny development. Experimental removal ofWigglesworthiaimpaired lipid metabolism via disruption of the Kennedy pathway, yielding obese mothers whose developing progeny ultimately starve. Functional validation via experimentalcct1suppression revealed a phenotype similar to females lacking obligateWigglesworthiasymbionts. These results indicate that, inGlossina, symbiont-derived factors, likely B vitamins, are critical for proper function of both lipid biosynthesis and lipolysis. Loss of the symbiosis has a dramatic impact onGlossinafecundity, and may be broadly applicable to other insect systems, particularly to species that require symbiotic partners to maximize lipolysis and reproductive output.

The insect juvenile hormone receptor is a basic helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) domain protein, a novel type of hormone receptor. In higher flies like Drosophila, the ancestral receptor germ cell-expressed (gce) gene has duplicated to yield the paralog Methoprene-tolerant (Met). These paralogous receptors share redundant function during development but play unique roles in adults. Some aspects of JH function apparently require one receptor or the other. To provide a foundation for studying JH receptor function, we have recapitulated endogenous JH receptor expression with single cell resolution. Using Bacteria Artificial Chromosome (BAC) recombineering and a transgenic knock-in, we have generated a spatiotemporal expressional atlas of Met and gce throughout development. We demonstrate JH receptor expression in known JH target tissues, in which temporal expression corresponds with periods of hormone sensitivity. Larval expression largely supports the notion of functional redundancy. Furthermore, we provide the neuroanatomical distribution of JH receptors in both the larval and adult central nervous system, which will serve as a platform for future studies regarding JH action on insect behavior.

Tsetse flies are viviparous insects that nurture a single intrauterine progeny per gonotrophic cycle. The developing larva is nourished by the lipid-rich, milk-like secretions from a modified female accessory gland (milk gland). An essential feature of the lactation process involves lipid mobilization for incorporation into the milk. In this study, we examined roles for juvenile hormone (JH) and insulin/IGF-like (IIS) signaling pathways during tsetse pregnancy. In particular, we examined the roles for these pathways in regulating lipid homeostasis during transitions between non-lactating (dry) and lactating periods. The dry period occurs over the course of oogenesis and embryogenesis, while the lactation period spans intrauterine larvigenesis. Genes involved in the JH and IIS pathways were upregulated during dry periods, correlating with lipid accumulation between bouts of lactation. RNAi suppression of Forkhead Box Sub Group O (FOXO) expression impaired lipolysis during tsetse lactation and reduced fecundity. Similar reduction of the JH receptor Methoprene tolerant (Met), but not its paralog germ cell expressed (gce), reduced lipid accumulation during dry periods, indicating functional divergence between Met and gce during tsetse reproduction. Reduced lipid levels following Met knockdown led to impaired fecundity due to inadequate fat reserves at the initiation of milk production. Both the application of the JH analog (JHA) methoprene and injection of insulin into lactating females increased stored lipids by suppressing lipolysis and reduced transcripts of lactation-specific genes, leading to elevated rates of larval abortion. To our knowledge, this study is the first to address the molecular physiology of JH and IIS in a viviparous insect, and specifically to provide a role for JH signaling through Met in the regulation of lipid metabolism during insect lactation.

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.

The Methoprene-tolerant (Met) gene in Drosophila melanogaster has been shown to function in juvenile hormone (JH) action. Met homologs were isolated from three mosquito species, Culex pipiens, Aedes aegypti and Anopheles gambiae. Sequence similarity was found to be high in bHLH and PAS conserved domains, and the majority of the 7–9 introns in AaMet and AgMet are located in either identical or similar positions, indicating evolutionary relatedness. Sequence comparison with Met and the similar germ-cell expressed (gce) gene in D. melanogaster showed that the mosquito genes are more similar to gce than to Met. Moreover, the multiple introns in AgMet and AaMet are more similar in number with the 7 introns in Dmgce than to the single intron in DmMet; in fact, six intron positions in AaMet and AgMet are similar to those in Dmgce. Efforts to identify a second homologous gene in mosquitoes were unsuccessful, suggesting a single gene in lower Diptera, consistent with the single gene uncovered in genomic sequencing of Ae. aegypti and An. gambiae. These results suggest that a gene duplication occurred during the evolution of higher Diptera, resulting in Met and gce.

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.

Africa's Bane Tsetse are blood-feeding, fast-flying flies that transmit a range of Trypanosoma spp. protozoan pathogens, which cause sleeping sickness in humans and their nagana in their livestock. The International Glossina Genome Initiative (p. 380 ) sequenced the genome of Glossina morsitans and identified the genes for many attributes of the tsetse's remarkable biology, including viviparity and the expression of analogs of mammalian milk proteins. Tsetse are host to several specific symbionts that appear to synthesize essential nutrients for the fly and also to hitherto undiscovered parasitoid-derived viruses. Deeper exploration of this genome will reveal what makes these fly species so host- and trypanosome specific.

Insect development proceeds through a series of discrete developmental stages called instars. During hexapod evolution, the development of complete metamorphosis introduced a novel mechanism for separating feeding and reproductive stages (Truman & Riddiford, 2002), facilitating the tremendous evolutionary success of holometabolous insects. In contrast to hemimetabolous insects, which progress through a series of instars that appear as smaller iterations of the adult form, holometabolous insects proceed from egg to adult through a progression of isomorphic larval instars and a pupal transitory stage. In each case, the physical boundary for growth during an instar is established by a chitinous exoskeleton, which must be periodically shed. This molting process is under the control of two counteracting hormones. Toward the end of an instar, a pulse of the insect molting hormone, 20-hydroxyecdysone (20E) initiates a transcriptional cascade that carries the molt to a subsequent instar. However, it is the interaction of 20E and the sesquiterpenoid juvenile hormone (JH) that governs the developmental outcome of each molt. During larval development, an elevated JH titer and 20E directs the sequential progression through larval development until the final larval instar, when the JH titer substantially declines. The removal of circulating JH facilitates a 20E-directed developmental switch that initiates the metamorphic molt. Thus, it was proposed that JH can modulate 20E activity, maintaining the status quo during preadult development.

The Drosophila Methoprene-tolerant (Met) and Germ cell-expressed (Gce) bHLH-PAS transcription factors are products of two paralogous genes. Both proteins potentially mediate the effect of juvenile hormone (JH) as candidate JH receptors. Here we report that Met and Gce are partially redundant in transducing JH action. Both Met and gce null single mutants are fully viable, but the Met gce double mutant, Met(27) gce(2.5k), dies during the larval-pupal transition. Precocious and enhanced caspase-dependent programmed cell death (PCD) appears in fat body cells of Met(27) gce(2.5k) during the early larval stages. Expression of Kr-h1, a JH response gene that inhibits 20-hydroxyecdysone (20E)-induced broad (br) expression, is abolished in Met(27) gce(2.5k) during larval molts. Consequently, expression of br occurs precociously in Met(27) gce(2.5k), which may cause precocious caspase-dependent PCD during the early larval stages. Defective phenotypes and gene expression changes in Met(27) gce(2.5k) double mutants are similar to those found in JH-deficient animals. Importantly, exogenous application of JH agonists rescued the JH-deficient animals but not the Met(27) gce(2.5k) mutants. Our data suggest a model in which Drosophila Met and Gce redundantly transduce JH action to prevent 20E-induced caspase-dependent PCD during larval molts by induction of Kr-h1 expression and inhibition of br expression.

Juvenile hormone (JH) is critical for multiple aspects of insect development and physiology. Although roles for the hormone have received considerable study, an understanding of the molecules necessary for JH action in insects has been frustratingly slow to evolve. Methoprene-tolerant (Met) in Drosophila melanogaster fulfills many of the requirements for a hormone receptor gene. A paralogous gene, germ-cell expressed (gce), possesses homology and is a candidate as a Met partner in JH action. Expression of gce was found to occur at multiple times and in multiple tissues during development, similar to that previously found for Met. To probe roles of this gene in JH action, we carried out in vivo gce over- and underexpression studies. We show by overexpression studies that gce can substitute in vivo for Met, alleviating preadult but not adult phenotypic characters. We also demonstrate that RNA interference-driven knockdown of gce expression in transgenic flies results in preadult lethality in the absence of MET. These results show that (1) unlike Met, gce is a vital gene and shows functional flexibility and (2) both gene products appear to promote JH action in preadult but not adult development.

The U.S. Department of Energy (DOE) Office of River Protection’s Waste Treatment Plant (WTP) is being designed and built to pre-treat and then vitrify a large portion of the wastes in Hanford’s 177 underground waste storage tanks. The WTP consists of three primary facilities—pretreatment, low-activity waste (LAW) vitrification, and high-level waste (HLW) vitrification. The pretreatment facility will receive waste piped from the Hanford tank farms and separate it into a high-volume, low-activity liquid stream stripped of most solids and radionuclides and a much smaller volume of HLW slurry containing most of the solids and most of the radioactivity. Many of the vessels in the pretreatment facility will contain pulse jet mixers (PJM) that will provide some or all of the mixing in the vessels. Pulse jet mixer technology was selected for use in black cell regions of the WTP, where maintenance cannot be performed once hot testing and operations commence. The PJMs have no moving mechanical parts that require maintenance. The vessels with the most concentrated slurries will also be mixed with air spargers and/or steady jets in addition to the mixing provided by the PJMs. Pulse jet mixers are susceptible to overblows that can generate large hydrodynamic forces, forces more » that can damage mixing vessels or their internal parts. The probability of an overblow increases if a PJM does not fill completely. The purpose of the testing performed for this report was to determine how reliable and repeatable the primary and safety (or backup) PJM control systems are at detecting drive overblows (DOB) and charge vessel full (CVF) conditions. Testing was performed on the ABB 800xA and Triconex control systems. The controllers operated an array of four PJMs installed in an approximately 13 ft diameter × 15 ft tall tank located in the high bay of the Pacific Northwest National Laboratory (PNNL) 336 Building test facility. The PJMs were fitted with 4 inch diameter discharge nozzles representative of the nozzles to be used in the WTP. This work supplemented earlier controller tests done on PJMs with 2 inch nozzles (Bontha et al. 2007). Those earlier tests enabled the selection of appropriate pressure transmitters with associated piping and resulted in an alternate overblow detection algorithm that uses data from pressure transmitters mounted in a water flush line on the PJM airlines. Much of that earlier work was only qualitative, however, due to a data logger equipment failure that occurred during the 2007 testing. The objectives of the current work focused on providing quantitative determinations of the ability of the BNI controllers to detect DOB and CVF conditions. On both control systems, a DOB or CVF is indicated when the values of particular internal functions, called confidence values, cross predetermined thresholds. There are two types of confidence values; one based on a transformation of jet pump pair (JPP) drive and suction pressures, the other based on the pressure in the flush line. In the present testing, we collected confidence levels output from the ABB and Triconex controllers. These data were analyzed in terms of the true and noise confidence peaks generated during multiple cycles of DOB and CVF events. The distributions of peak and noise amplitudes were compared to see if thresholds could be set that would enable the detection of DOB and CVF events at high probabilities, while keeping false detections to low probabilities. Supporting data were also collected on PJM operation, including data on PJM pressures and levels, to provide direct experimental evidence of when PJMs were filling, full, driving, or overblowing. « less

The U.S. Department of Energy (DOE) Office of River Protection’s Waste Treatment Plant (WTP) is being designed and built to pretreat and then vitrify a large portion of the wastes in Hanford’s 177 underground waste storage tanks. The WTP consists of three primary facilities: pretreatment, low-activity waste (LAW) vitrification, and high-level waste (HLW) vitrification. The pretreatment facility will receive waste feed from the Hanford tank farms and separate it into 1) a high-volume, low-activity liquid stream stripped of most solids and radionuclides and 2) a much smaller volume of HLW slurry containing most of the solids and most of the radioactivity. Many of the vessels in the pretreatment facility will contain pulse jet mixers (PJMs) that will provide some or all of the mixing in the vessels. This technology was selected for use in so-called “black cell” regions of the WTP, where maintenance capability will not be available for the operating life of the WTP. PJM technology was selected for use in these regions because it has no moving mechanical parts that require maintenance. The vessels with the most concentrated slurries will also be mixed with air spargers and/or steady jets in addition to the mixing provided by the PJMs. This report contains the results of single and multiple PJM overblow tests conducted in a large, ~13 ft-diameter × 15-ft-tall tank located in the high bay of the Pacific Northwest National Laboratory (PNNL) 336 Building test facility. These single and multiple PJM overblow tests were conducted using water and a clay simulant to bound the lower and upper rheological properties of the waste streams anticipated to be processed in the WTP. Hydrodynamic pressures were measured at a number of locations in the test vessel using an array of nine pressure sensors and four hydrophones. These measurements were made under normal and limiting vessel operating conditions (i.e., maximum PJM fluid emptying velocity, maximum and minimum vessel contents for PJM operation, and maximum and minimum rheological properties). Test data collected from the PJM overblow tests were provided to Bechtel National, Inc. (BNI) for assessing hydrostatic, dynamic, and acoustic pressure loadings on in-tank structures during 1) single overblows; 2) multiple overlapping overblows of two to four PJMs; 3) simultaneous overblows of pairs of PJMs.

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.