Aaron A. Baumann

Drosophila Met and Gce are partially redundant in transducing juvenile hormone action.

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.

Paralogous Genes Involved in Juvenile Hormone Action in Drosophila melanogaster

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.

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.

Juvenile hormone and insulin suppress lipolysis between periods of lactation during tsetse fly pregnancy

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.

A Novel Highly Divergent Protein Family Identified from a Viviparous Insect by RNA-seq Analysis: A Potential Target for Tsetse Fly-Specific Abortifacients

In tsetse flies, nutrients for intrauterine larval development are synthesized by the modified accessory gland (milk gland) and provided in mothers 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.

Molecular Evolution of Juvenile Hormone Signaling

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.

Genetic tools to study juvenile hormone action in Drosophila

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.

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.