A family of evolutionarily old hormones is the glycoprotein cysteine knot-forming heterodimers consisting of alpha- (GPA) and beta-subunits (GPB), which assemble by noncovalent bonds. In mammals, a common glycoprotein hormone alpha-subunit (GPA1) pairs with unique beta-subunits that establish receptor specificity, forming thyroid stimulating hormone (GPA1/TSHβ) and the gonadotropins luteinizing hormone (GPA1/LHβ), follicle stimulating hormone (GPA1/FSHβ), choriogonadotropin (GPA1/CGβ). A novel glycoprotein heterodimer was identified in vertebrates by genome analysis, called thyrostimulin, composed of two novel subunits, GPA2 and GPB5, and homologs occur in arthropods, nematodes and cnidarians, implying that this neurohormone system existed prior to the emergence of bilateral metazoans. In order to discern possible physiological roles of this hormonal signaling system in mosquitoes, we have isolated the glycoprotein hormone genes producing the alpha- and beta-subunits (AedaeGPA2 and AedaeGPB5) and assessed their temporal expression profiles in the yellow and dengue-fever vector, Aedes aegypti. We have also isolated a putative receptor for this novel mosquito hormone, AedaeLGR1, which contains features conserved with other glycoprotein leucine-rich repeating containing G protein-coupled receptors. AedaeLGR1 is expressed in tissues of the alimentary canal such as the midgut, Malpighian tubules and hindgut, suggesting that this novel mosquito glycoprotein hormone may regulate ionic and osmotic balance. Focusing on the hindgut in adult stage A. aegypti, where AedaeLGR1 was highly enriched, we utilized the Scanning Ion-selective Electrode Technique (SIET) to determine if AedaeGPA2/GPB5 modulated cation transport across this epithelial tissue. Our results suggest that AedaeGPA2/GPB5 does indeed participate in ionic and osmotic balance, since it appears to inhibit natriuresis and promote kaliuresis. Taken together, our findings imply this hormone may play an important role in ionic balance when levels of Na⁺ are limited and levels of K⁺ are in excess – such as during the digestion and assimilation of erythrocytes following vertebrate blood-feeding by females.

After a blood meal, Rhodnius prolixus undergoes a rapid diuresis to eliminate excess water and salts. During the voiding of this primary urine, R. prolixus acts as a vector of Chagas’ disease, with the causative agent, Trypanosoma cruzi, infecting the human host via the urine. Diuresis in R. prolixus is under the neurohormonal control of serotonin and peptidergic diuretic hormones, and thus, diuretic hormones play an important role in the transmission of Chagas’ disease. Although diuretic hormones may be degraded or excreted, resulting in the termination of diuresis, it would also seem appropriate, given the high rates of secretion, that a potent antidiuretic factor could be present and act to prevent excessive loss of water and salts after the postgorging diuresis. Despite the medical importance of R. prolixus, no genes for any neuropeptides have been cloned, including obviously, those that control diuresis. Here, using molecular biology in combination with matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry, we determined the sequence of the CAPA gene and CAPA-related peptides in R. prolixus, which includes a peptide with anti-diuretic activity. We have characterized the expression of mRNA encoding these peptides in various developmental stage and also examined the tissue-specific distribution in fifth-instars. The expression is localized to numerous bilaterally paired cell bodies within the central nervous system. In addition, our results show that RhoprCAPA gene expression is also associated with the testes, suggesting a novel role for this family of peptides in reproduction.

Glycoprotein hormone receptors mediate a diverse range of physiological functions in vertebrate and invertebrate organisms. The heterodimeric glycoprotein hormone GPA2/GPB5 and its receptor LGR1, constitute a recently discovered invertebrate neuroendocrine signaling system that remains to be functionally characterized. We previously reported that LGR1 is expressed in the testes of adult Aedes aegypti mosquitoes, where its immunoreactivity is particularly regionalized. Here, we show that LGR1 immunoreactivity is associated with the centriole adjunct of spermatids and is observed transiently during spermatogenesis in mosquitoes, where it may act to mediate the regulation of flagellar development. RNA interference to downregulate LGR1 expression was accomplished by feeding mosquito larvae with bacteria that produced LGR1-specific dsRNA, which led to defects in spermatozoa, characterized with shortened flagella. LGR1 knockdown mosquitoes also retained ∼60% less spermatozoa in reproductive organs and demonstrated reduced fertility compared to controls. To date, the endocrine regulation of spermatogenesis in mosquitoes remains an understudied research area. The distribution of LGR1 and detrimental effects of its knockdown on spermatogenesis in A. aegypti indicates that this heterodimeric glycoprotein hormone signaling system contributes significantly to the regulation of male reproductive biology in this important disease-vector.

Diuresis following blood-gorging in Rhodnius prolixus is the major process leading to the transmission of Chagas’ disease. We have cloned the cDNA of the first receptor known to be involved in an antidiuretic strategy in insects, a strategy that prevents diuresis. This receptor belongs to the insect CAPA receptor family known in other insects to be activated by peptides encoded within the capability gene. We characterize the expression profile in fifth-instars and find expression is localized to the alimentary canal. Highest transcript levels are found in Malpighian tubules and the anterior midgut, which are known targets of the antidiuretic hormone, RhoprCAPA-α2. Two transcripts were identified, capa-r1 and capa-r2 ; however, the latter encodes an atypical G protein-coupled receptor lacking a region ranging between the first and second transmembrane domain. Our heterologous expression assay revealed the expressed capa-r1 receptor is activated by RhoprCAPA-α2 (EC ₅₀ = 385nM) but not by RhoprCAPA-α1. Structural analogs of the inactive RhoprCAPA-α1 were capable of activating the expressed capa-r1 receptor, confirming the importance of the C-terminal consensus sequence common to CAPA-related peptides. In addition, this receptor has some sensitivity to the pyrokinin-related peptide, RhoprCAPA-αPK1, but with an efficacy ≈40-fold less than RhoprCAPA-α2. Other peptides belonging to the PRXamide superfamily were inactive on the capa-r1 receptor. Taken together, the neuroendocrinological relevance of this receptor in facilitating the antidiuretic strategy in R. prolixus may make this receptor a useful target for development of agonists or antagonists that could help influence the transmission of Chagas’ disease that occurs during diuresis in this medically important insect-disease vector.

Environmental factors challenge the physiological homeostasis in animals, thereby evoking stress responses. Various mechanisms have evolved to counter stress at the organism level, including regulation by neuropeptides. In recent years, much progress has been made on the mechanisms and neuropeptides that regulate responses to metabolic/nutritional stress, as well as those involved in countering osmotic and ionic stresses. Here, we identified a peptidergic pathway that links these types of regulatory functions. We uncover the neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown ofCrzand acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs ofCapa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. We show that Crz acts to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown ofCrzRin Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. Here we additionally propose that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body inDrosophila.

SUMMARY: In the haematophagous insect Rhodnius prolixus, diuresis is accomplished through the combined actions of peptidergic diuretic hormones and 5-HT released from neurohaemal sites on the abdominal nerves. Preliminary work on anti-diuresis in this blood-feeder, previously believed to occur through a decrease in the levels of the diuretic factors, indicates that an anti-diuretic hormone, with properties similar to CAP2b (pELYAFPRVamide;recently renamed Mas-CAPA-1), might also be present in R. prolixus. Here, we present evidence from immunohistochemical analysis that suggests a PRXamide-like neuropeptide may be released from the abdominal neurohaemal sites beginning 3–4 h following feeding; a time that coincides with the cessation of diuresis. We also show evidence for an endogenous factor,isolated from the central nervous system using reversed-phase high performance liquid chromatography, which mimics the effects of Mas-CAPA-1. Specifically,this endogenous anti-diuretic factor inhibits rates of 5-HT-stimulated secretion in a dose-dependent manner and elevates intracellular cGMP levels of Malpighian tubules stimulated with 5-HT.