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.

JH and JHAs: Insecticidal use of hormone agonistsSince the first chemical analysis resolved the sesquiterpenoid structure of endogenous JH (Röller et al., 1967), several homologs have been identified, each bearing opposing, terminal epoxide and methyl ester functions.Variation in the degree and identity of alkyl group substitution at C3, C7, and C11 along the carbon skeleton defines the homologs.The evolutionary importance of multiple JH homologs is unclear.JH 0, I, II, and III have all been isolated from lepidopteran insects, whereas JH III, the presumed evolutionary precursor to the higher homologs, is found in all insects.JH bisepoxide (JHB3) has been identified as a product of the corpus allatum (CA) in higher Diptera including Drosophila melanogaster and Sarcophaga bullata (Richard et al., 1989;Bylemans et al., 1998).Nearly identical in structure to JH III, JHB3 is distinguished by an additional epoxide group spanning C6-C7. www.intechopen.com Gene Duplication 334The major JHs and some juvenile hormone analogs (JHAs) are presented in Figure 1.

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.

Juvenile hormone (JH) signaling underpins both regulatory and developmental pathways in insects. However, the JH receptor is poorly understood. Methoprene tolerant (Met) and germ cell expressed (gce) have been implicated in JH signaling in Drosophila. We investigated the evolution of Met and gce across 12 Drosophila species and found that these paralogs are conserved across at least 63 million years of dipteran evolution. Distinct patterns of selection found using estimates of dN/dS ratios across Drosophila Met and gce coding sequences, along with their incongruent temporal expression profiles in embryonic Drosophila melanogaster, illustrate avenues through which these genes have diverged within the Diptera. Additionally, we demonstrate that the annotated gene CG15032 is the 5' terminus of gce. In mosquitoes and beetles, a single Met-like homolog displays structural similarity to both Met and gce, and the intron locations are conserved with those of gce. We found that Tribolium and mosquito Met orthologs are assembled from Met- and gce-specific domains in a modular fashion. Our results suggest that Drosophila Met and gce experienced divergent evolutionary pressures following the duplication of an ancestral gce-like gene found in less derived holometabolous insects.

Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Here we report that genetic ablation of the corpus allatum cells of the Drosophilaring gland (the JH source) resulted in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice,two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), were significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Droncand Drice. In the Met- and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, we demonstrate that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila.

The Methoprene-tolerant (Met) gene of Drosophila melanogaster is involved in both juvenile hormone (JH) action and resistance to JH insecticides, such as methoprene. Although the consequences of Met mutations on development and methoprene resistance are known, no studies have examined Met+ overexpression. Met+ was overexpressed in transgenic lines with various promoters that drive overexpression to different levels. Flies expressing either genomic or cDNA Met+ transgenes showed higher susceptibility to both the morphogenetic and toxic effects of methoprene, consistent with the hormone-binding property of MET. Both the sensitive period and lethal period were the same as seen for non-overexpressing Met+ flies. However, continual exposure of high-overexpressing Met+ larvae to borderline-toxic or higher methoprene doses advanced the sensitive period from prepupae to first instar and the lethal period from pharate adults to larvae and early pupae. When expression of transgenic UAS-Met+ was driven to high levels by either an actin-GAL4 or tubulin-GAL4 promoter, larvae showed high mortality in the absence of methoprene, indicating that high MET titer is lethal, perhaps resulting from expression in an inappropriate tissue. Adults overexpressing Met+ did not show enhanced oogenesis, ruling out MET as a limiting factor for this hormone-driven physiology.

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.

Juvenile hormone (JH) regulates insect development by a poorly understood mechanism. Application of JH agonist insecticides to Drosophila melanogaster during the ecdysone-driven onset of metamorphosis results in lethality and specific morphogenetic defects, some of which resemble those in mutants of the ecdysone-regulated Broad-Complex (BR-C). The Methoprene-tolerant (Met) bHLH–PAS gene mediates JH action, and Met mutations protect against the lethality and defects. To explore relationships among these two genes and JH, double mutants were constructed between Met alleles and alleles of each of the BR-C complementation groups: broad (br), reduced bristles on palpus (rbp), and 2Bc. Defects in viability and oogenesis were consistently more severe in rbp Met or br Met double mutants than would be expected if these genes act independently. Additionally, complementation between BR-C mutant alleles often failed when MET was absent. Patterns of BRC protein accumulation during metamorphosis revealed essentially no difference between wild-type and Met-null individuals. JH agonist treatment did not block accumulation of BRC proteins. We propose that MET and BRC interact to control transcription of one or more downstream effector genes, which can be disrupted either by mutations in Met or BR-C or by application of JH/JH agonist, which alters MET interaction with BRC.

The Methoprene-tolerant (Met) bHLH-PAS gene is involved in juvenile hormone (JH) action in Drosophila melanogaster as a likely component of a JH receptor. We expressed Met in Drosophila S2 cells and explored for MET partners using pull-down assays. MET-MET interaction was found to occur. The germ-cell expressed (gce) gene is another D. melanogaster bHLH-PAS gene with high homology to Met, and GCE formed heterodimers with MET. In the presence of JH or either of two JH agonists, MET-MET and MET-GCE formation was drastically reduced. Interaction between GCE and MET having N- or C-terminus truncations, bHLH or PAS-A domain deletions, or a point mutation in the PAS-B domain failed to occur. However, JH-dependent interaction occurred between GCE and MET having point mutations in bHLH or PAS-A. During development, changes in JH titer may alter partner binding by MET and result in different gene expression patterns.

Using subtractive hybridization, we have identified 17 genes that are either up- or down-regulated in the hepatopancreas (Hp) of the lobster, Homarus americanus, by acute exposure to the juvenile hormone analog methoprene. The expression of some of the genes obtained from the subtraction libraries was confirmed by real time Q-PCR experiments. These genes encode several different classes of proteins including: structural, enzymatic and regulatory polypeptides. Enzymes represent the predominant genes up-regulated by methoprene. Included in this group are betaine-homocysteine S-methyltransferase (BHMT) and two other enzymes of the methionine cycle. Increased expression of a translation factor (eIF2), as well as of cytosolic (aldose reductase), structural (beta-tubulin, L5A) and plasma membrane (CD42d) proteins was observed. In addition, a major feature of altered gene expression in methoprene treated Hp was increased levels of enzymes associated with protein turnover, including trypsin, ubiquitin conjugating enzyme and ubiquitin carboxyl terminal hydrolase. Down-regulation of the members of the hemocyanin family was observed. Assays confirmed elevated levels of trypsin in the Hp of lobsters after 24 h exposure to methoprene. Our findings suggest a wide variety of cellular targets are altered by methoprene.

The Methoprene-tolerant (Met) bHLH-PAS gene in Drosophila melanogaster is involved in the molecular action of juvenile hormone (JH), and mutants result in resistance to the toxic and morphogenetic effects of JH and JH agonist insecticides such as methoprene. A detailed study of Met mutants can shed light on the poorly understood action of JH as well as the molecular basis of Met resistance to JH insecticides. Nine mutant alleles bearing point mutations in Met were examined for penetrance and expressivity of three phenotypic characters: resistance, defective oogenesis, and a novel eye defect. The collection ranged from two weak alleles having less severe phenotypes to strong alleles with severe phenotypes similar to that of a null allele. The point mutations were located in both conserved and nonconserved domains. Both the eye defect, seen as severely malformed ommatidial facets in the posterior margin of the compound eye, and the oogenesis phenotype are nonconditional, whereas expression of the resistance phenotype requires treatment with JH or JH analogs (JHAs) during early metamorphosis. A proposed basis for all the phenotypic characters centers on MET action as a transcriptional regulator of ecdysone secondary-response target genes during metamorphosis. Disruption of MET function either by mutation or by JHA presence during early metamorphosis results in transcriptional misregulation of different target genes, resulting in the pathology seen in either instance. The variety of amino acid changes in MET that resulted in resistance may portend a rapid rise in resistance in response to increased use of JH insecticides in field insect populations.

Methoprene was a constituent of the pesticide cocktail applied to the Western Long Island Sound (WLIS) watershed area during the summer of 1999. Subsequently, the seasonal lobster catches from the WLIS have decreased dramatically. We have been engaged in ongoing studies of the effects of methoprene on larval, juvenile and adult lobsters. Most recently, we found that Stage IV larvae exposed to 50 ppb methoprene experience > 90% mortality rate after 3 days. Bioaccumulation studies on adult lobsters showed that methoprene concentrated against the gradient of the surrounding seawater (50 ppb) in hepatopancreas (1.55 ppm), gonad (5.18 ppm). epithelial tissue (6.17 ppm) and, most significantly, the eyestalks (28.83 ppm). Exposure to methoprene altered the expression of the stress proteins and the pattern of ubiquitinylation of cytosolic proteins by Day 1 Stage I larvae and by epithelial tissue of postmolt juvenile lobsters. Postmolt juvenile animals also demonstrated an altered pattern of protein phosphorylation in their epithelial tissues following methoprene exposure, indicating that it may interfere with cell signaling pathways. Increasing concentrations of methoprene were associated with increasing chitoproteins in the microsomal fractions of Day 1 Stage I larvae, suggesting that methoprene may compromise the exocytosis of shell matrix precursors from the epithelial cells. Methoprene did not, however, alter the activity of chitin synthase in these larvae. Although it is likely that a combination of harmful events and exposures led to the reduced lobster population in WLIS. methoprene may have contributed to the decline both by direct toxic effects and by disrupting homeostatic processes.

Methoprene is a pesticide that acts as a juvenile hormone agonist. Although developed initially against insects, it has since been shown to have toxic effects on larval and adult crustaceans. Methoprene was one of several pesticides applied to the Western Long Island Sound (WLIS) watershed area during the summer of 1999; the other pesticides were malathion, resmethrin, and sumethrin. These pesticides were applied as part of a county-by-county effort to control the mosquito vector of West Nile Virus. Subsequently, the seasonal lobster catches from the WLIS have decreased dramatically. The lethality of the pesticides to lobsters had been unknown. We studied the effects of methoprene while other investigators studied effects of the other pesticides. We questioned whether methoprene, through its effects on larvae, adults or both, could have contributed to this decline. We found that low levels of methoprene had adverse effects on lobster larvae. It was toxic to stage II larvae at 1 ppb. Stage IV larvae were more resistant, but did exhibit significant increases in molt frequency beginning at exposures of 5 ppb. Juvenile lobsters exhibited variations in tissue susceptibility to methoprene: hepatopancreas appeared to be the most vulnerable, reflected by environmental concentrations of methoprene inhibiting almost all protein synthesis in this organ.Our results indicated that methoprene concentrates in the hepatopancreas, nervous tissue and epidermal cells of the adult lobster. Methoprene altered the synthesis and incorporation of chitoproteins (cuticle proteins) into adult postmolt lobster explant shells. SDS PAGE analyses of adult post-molt shell extracts revealed changes in the synthesis of chitoproteins in the methoprene-treated specimens, suggesting that methoprene affects the normal pathway of lobster cuticle synthesis and the quality of the post-molt shell. Although it is likely that a combination of factors led to the reduced lobster population in WLIS, methoprene may have contributed both by direct toxic effects and by disrupting homeostatic events under endocrine control.

Synthetic insecticides have been used intensively for the past 50 years in many parts of the world. Insect populations, both target and nontarget, have responded by evolving resistance. One of the nontarget insects is Drosophila melanogaster, which is well-suited for genetic analysis and has been particularly well-studied in both laboratory and field populations. Resistance to several insecticides, including two for which significant resistance in field populations has not been found, has been generated in susceptible laboratory strains following mutagenesis, allowing comprehensive study of the resistance genes. Field populations of D. melanogaster have evolved resistance to many, but not all, insecticides in use today. Both the genetic and biochemical mechanisms that underlie resistance in this insect are similar to those in other insects. Therefore, D. melanogaster can be a sentinel organism for long-term release of toxicants into the environment. While it remains useful for genetic analysis of resistance, a better understanding of the movement and population structures of this insect will be a prerequisite for its sentinel utilization at specific locales.

Studies in a variety of insects during the past four decades has deepened our understanding of juvenile hormone (JH) physiology, but how this hormone works at the molecular level remains elusive. Similarly, the mechanism of toxicity of JH analogue insecticides is still in question. There is much evidence from laboratory usage that JHAs act as JH agonists and generally show the highest toxicity when applied at the onset of metamorphosis. A physiological basis for the toxicity and morphogenetic effects has been suggested by recent work linking these effects with interference with the expression or action of certain genes, particularly the Broad-Complex (BR-C) transcription factor gene, that direct metamorphic change. Misexpressed BR-C then leads to improper expression of one or more downstream effector genes controlled by BR-C gene products, resulting in abnormal developmental and physiological changes that disrupt metamorphosis. Therefore, JH is a necessary molecule at certain times in insect development but becomes toxic when present during metamorphosis.

Over 4 years have elapsed since the first National Health and Medical Research Council (NHMRC) guidelines were published for the management of patients after potentially curative resection of colorectal cancer. New information has now been published indicating that more intensive follow up than was originally recommended might provide a survival benefit for patients. This new information should be considered when formulating new NHMRC guidelines. In particular, meta-analyses of published individual trials have suggested a survival advantage that was not evident in the individual studies. There have been significant developments in chemotherapy with new individual agents and use of agents in combination that have proved far more effective than previous protocols. The therapeutic effect of these developments is the downstaging of some patients with metastatic disease, which was previously unresectable, to undergo resection. Furthermore, there is now some evidence that palliation of patients with advanced disease is more effective if commenced before the development of symptoms and this needs to be considered in the assessment of the benefits of follow up. There have been limited studies of cost-effectiveness, but international analyses suggest that the costs associated with more intensive follow-up regimes are within the accepted cost parameters associated with the management of many other conditions.