Allatostatins (ASTs) are neuropeptides that inhibit the biosynthesis and release of juvenile hormone from the corpora allata (CA) of various insects including the cockroach Diploptera punctata. We hypothesized that a similar allatostatin receptor would exist in the cockroach D. punctata that may regulate the numerous physiological effects that this family of peptides exerts on a range of target tissues. Using polymerase chain reaction (PCR) strategies successful in isolating other insect allatostatin receptor sequences utilized primers designed to known mammalian somatostatin receptors and Drosophila allatostatin receptor (AlstR). Once an internal PCR fragment was proven to be consistent with the sequence of an allatostatin receptor (AstR) then the sequence was completed by rapid amplification of cDNA ends (RACE). The putative allatostatin-like receptor sequence encoding 425 amino acid residues was isolated from a cDNA library prepared from corpora allata of D. punctata. We show that dsRNA targeting the allatostatin receptor gene of D. punctata injected into freshly moulted adult cockroaches produced a long-lasting reduction in the mRNA levels in midgut tissues. The effect lasted up to 6 days. Following dsRNA injection, the juvenile hormone (JH) titers in the corpora allata were clearly raised suggest that the putative inhibition of receptor RNA expression may increase JH production. The receptor is expressed in brains, corpora allata, abdominal ganglion, midguts, ovaries, and testes. We have examined these same tissues with regard to changes in expression levels of Dippu-AstR. JH biosynthesis peaks on day 5 post-emergence in mated females. In mated females, Dippu-AstR mRNA is expressed at the highest levels on day 6 post-emergence in brain and corpora allata and day 2 post-emergence in midgut. Dippu-AstR is likely responsible for the decline in JH biosynthesis after day 5 post-emergence. Virgin females midgut and CA Dippu-AstR mRNA expression dramatically elevated on days 6 and 7, respectively. Expression of Dippu-AstR was similar in the abdominal ganglia of mated or virgin females. Ovarian Dippu-AstR expression declines to low levels by day 4. Testes also express peaks of Dippu-AstR expression on days 4 and 7. A role for Dippu-AST in testes is yet unknown. / Thesis (Ph.D, Biology) -- Queen's University, 2008-04-22 15:21:55.107

The objective of this study was to clarify the regulation of production and signal transduction of juvenile hormone (JH) in insects by experimentally examining the function and evolution of a putative receptor (USP/RXR) and a neuropeptide inhibitor (FGLamide allatostatin). To examine the role of USP/RXR, the cDNA sequence of the receptor was obtained from the cockroach Diploptera punctata. Transcript levels during developmentally critical periods for JH sensitivity may suggest USP/RXR is JH responsive. Comparative sequence analysis of evolutionary rates in the Mecopterida support current hypotheses which suggest some gain in function along this lineage, although this acquisition may have occurred more gradually than previously assumed. To examine allatostatin evolution within insects, ancestral peptides inferred using maximum likelihood ancestral reconstruction methods were assayed for in vitro inhibition of JH production in two cockroach species. Shifts in peptide potency in some ancestral peptides reconstructed may be related to peptide copy number evolution.

The objective of this study was to clarify the regulation of production and signal transduction of juvenile hormone (JH) in insects by experimentally examining the function and evolution of a putative receptor (USP/RXR) and a neuropeptide inhibitor (FGLamide allatostatin). To examine the role of USP/RXR, the cDNA sequence of the receptor was obtained from the cockroach Diploptera punctata. Transcript levels during developmentally critical periods for JH sensitivity may suggest USP/RXR is JH responsive. Comparative sequence analysis of evolutionary rates in the Mecopterida support current hypotheses which suggest some gain in function along this lineage, although this acquisition may have occurred more gradually than previously assumed. To examine allatostatin evolution within insects, ancestral peptides inferred using maximum likelihood ancestral reconstruction methods were assayed for in vitro inhibition of JH production in two cockroach species. Shifts in peptide potency in some ancestral peptides reconstructed may be related to peptide copy number evolution.

The FGLa/ASTs are one family of allatostatin peptides and share an amidated C-terminal sequence (FGL-amide). The inhibitory effect of FGLa/ASTs on juvenile hormone (JH) biosynthesis in Diploptera punctata led to their discovery, but there is a lack of allatostatic function across most insect species that suggests this function may not be their primary role. Rather, the FGLa/ASTs are implicated as brain/gut peptides, modulating gut physiology. This thesis demonstrates the pleiotropic nature of FGLa/ASTs in Locusta migratoria and emphasizes the role of FGLa/ASTs as brain/gut peptides involved in homeostatic processes. FGLa/AST-like immunoreactivity (FLI) is associated with the corpus cardiacum (CC) and corpus allatum (CA). FGLa/ASTs increase adipokinetic hormone release from the CC and alter JH biosynthesis from the CA, suggesting roles in energy utilization and in growth and metamorphosis. Each region of the gut exhibits FLI. The gut is dually innervated: neurons in the abdominal ganglia of the central nervous system (CNS) innervate the posterior gut and some contain FLI, while neurons within the stomatogastric nervous system (STNS) that innervate the anterior gut do not seem to contain FLI, indicating that source of FLI on the gut are cells within the CNS, which may release FGLa/ASTs at the gut to alter aspects of gut physiology. FGLa/ASTs are involved in peristalsis, neural control of foregut contractions, and ileal K+ transport. In particular, FGLa/ASTs inhibit contractions of each gut region and also modulate the rhythmic motor output of a central pattern generator within the ventricular ganglion of the STNS. FGLa/ASTs also inhibit ileal K+ efflux, suggesting a diuretic action and implicating FGLa/ASTs in fluid and ion homeostasis. This work provides a comprehensive picture of how FGLa/ASTs play an integral role in nutrient processing, energy mobilization, and growth and metamorphosis to contribute to the overall maintenance of homeostasis. This reinforces the role of FGLa/ASTs as brain/gut peptides and emphasizes their involvement in the flexibility of nervous communication and integration of the endocrine system with the CNS to achieve homeostasis.

The FGLa/ASTs are one family of allatostatin peptides and share an amidated C-terminal sequence (FGL-amide). The inhibitory effect of FGLa/ASTs on juvenile hormone (JH) biosynthesis in Diploptera punctata led to their discovery, but there is a lack of allatostatic function across most insect species that suggests this function may not be their primary role. Rather, the FGLa/ASTs are implicated as brain/gut peptides, modulating gut physiology. This thesis demonstrates the pleiotropic nature of FGLa/ASTs in Locusta migratoria and emphasizes the role of FGLa/ASTs as brain/gut peptides involved in homeostatic processes. FGLa/AST-like immunoreactivity (FLI) is associated with the corpus cardiacum (CC) and corpus allatum (CA). FGLa/ASTs increase adipokinetic hormone release from the CC and alter JH biosynthesis from the CA, suggesting roles in energy utilization and in growth and metamorphosis. Each region of the gut exhibits FLI. The gut is dually innervated: neurons in the abdominal ganglia of the central nervous system (CNS) innervate the posterior gut and some contain FLI, while neurons within the stomatogastric nervous system (STNS) that innervate the anterior gut do not seem to contain FLI, indicating that source of FLI on the gut are cells within the CNS, which may release FGLa/ASTs at the gut to alter aspects of gut physiology. FGLa/ASTs are involved in peristalsis, neural control of foregut contractions, and ileal K+ transport. In particular, FGLa/ASTs inhibit contractions of each gut region and also modulate the rhythmic motor output of a central pattern generator within the ventricular ganglion of the STNS. FGLa/ASTs also inhibit ileal K+ efflux, suggesting a diuretic action and implicating FGLa/ASTs in fluid and ion homeostasis. This work provides a comprehensive picture of how FGLa/ASTs play an integral role in nutrient processing, energy mobilization, and growth and metamorphosis to contribute to the overall maintenance of homeostasis. This reinforces the role of FGLa/ASTs as brain/gut peptides and emphasizes their involvement in the flexibility of nervous communication and integration of the endocrine system with the CNS to achieve homeostasis.

<p>DH31 and drosokinin are known promoters of fluid secretion in <em>Drosophila</em> Malpighian tubules, while the effect of allatostatin A on Malpighian tubule fluid secretion is unknown. The expression of these peptides and their receptors is widespread in the larval gut and central nervous system. The scanning ion-selective electrode technique (SIET) was used to measure changes in epithelial K⁺ flux in the gut as a proxy for the flow of osmotically-obliged water between the gut lumen and the haemolymph. The primary goal of this study was to investigate the effects of DH31 and drosokinin on the gut as an indicator of coordination of diuretic activity between this tissue and the Malpighian tubules. Such coordination, whereby Malpighian tubule fluid secretion is stimulated concomitantly with fluid uptake by the gut, would be physiologically essential for the maintenance of haemolymph volume and osmolarity. Secondarily, this study sought to characterize the function of allatostatin A with respect to its effect on gut K⁺ transport. DH31 stimulated K⁺ absorption across the anterior midgut epithelium and reduced K⁺ absorption in the middle midgut copper cell zone. Drosokinin increased K⁺ absorption across the anterior midgut epithelium and was also stimulatory in the posterior midgut neutral zone. Allatostatin A stimulated K⁺ absorption across the epithelia of the anterior midgut and middle midgut copper cell zone, but was inhibitory in the large flat cell zone. The larger surface area of the anterior midgut relative to the middle midgut means that all three peptides were likely net stimulators of K⁺ uptake. The reduction in K⁺ absorption in the middle midgut by DH31 and allatostatin A may relate to a redistribution of fluid uptake among the regions of the midgut to preserve lumenal pH. DH31 and drosokinin also independently increased the contraction frequency of the anterior midgut, while the contraction frequency of the pyloric sphincter was increased by combined application of both peptides. Stimulation of gut contractions has previously been attributed to these and other diuretic factors in insects. The findings of this investigation implicate DH31, drosokinin, and allatostatin A in the stimulation of midgut K⁺ absorption, which may suggest a role for these peptides in altering fluid transport across this epithelium in larval <em>Drosophila</em>.</p> / Master of Science (MSc)

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