A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes

Role in Diuresis of a Calcitonin Receptor (GPRCAL1) Expressed in a Distal-Proximal Gradient in Renal Organs of the Mosquito Aedes aegypti (L.)

Evolution of anthropophilic hematophagy in insects resulted in the coordination of various physiological processes for survival. In female mosquitoes, a large blood meal provides proteins for egg production and as a trade-off, rapid elimination of the excess water and solutes (Na(+), Cl(-)) is critical for maintaining homeostasis and removing excess weight to resume flight and avoid predation. This post-prandial excretion is achieved by the concerted action of multiple hormones. Diuresis and natriuresis elicited by the calcitonin-like diuretic hormone 31 (DH(31)) are believed to be mediated by a yet uncharacterized calcitonin receptor (GPRCAL) in the mosquito Malpighian tubules (MTs), the renal organs. To contribute knowledge on endocrinology of mosquito diuresis we cloned GPRCAL1 from MT cDNA. This receptor is the ortholog of the DH(31) receptor from Drosophila melanogaster that is expressed in principal cells of the fruit fly MT. Immunofluorescence similarly showed AaegGPRCAL1 is present in MT principal cells in A. aegypti, however, exhibiting an overall gradient-like pattern along the tubule novel for a GPCR in insects. Variegated, cell-specific receptor expression revealed a subpopulation of otherwise phenotypically similar principal cells. To investigate the receptor contribution to fluid elimination, RNAi was followed by urine measurement assays. In vitro, MTs from females that underwent AaegGPRcal1 knock-down exhibited up to 57% decrease in the rate of fluid secretion in response to DH(31). Live females treated with AaegGPRcal1 dsRNA exhibited 30% reduction in fluid excreted after a blood meal. The RNAi-induced phenotype demonstrates the critical contribution of this single secretin-like family B GPCR to fluid excretion in invertebrates and highlights its relevance for the blood feeding adaptation. Our results with the mosquito AaegGPRCAL1 imply that the regulatory function of calcitonin-like receptors for ion and fluid transport in renal organs arose early in evolution

Immunolocalization of the short neuropeptide F receptor in queen brains and ovaries of the red imported fire ant (Solenopsis invicta Buren)

<p>Abstract</p> <p>Background</p> <p>Insect neuropeptides are involved in diverse physiological functions and can be released as neurotransmitters or neuromodulators acting within the central nervous system, and as circulating neurohormones in insect hemolymph. The insect short neuropeptide F (sNPF) peptides, related to the vertebrate neuropeptide Y (NPY) peptides, have been implicated in the regulation of food intake and body size, and play a gonadotropic role in the ovaries of some insect species. Recently the sNPF peptides were localized in the brain of larval and adult <it>Drosophila</it>. However, the location of the sNPF receptor, a G protein-coupled receptor (GPCR), has not yet been investigated in brains of any adult insect. To elucidate the sites of action of the sNPF peptide(s), the sNPF receptor tissue expression and cellular localization were analyzed in queens of the red imported fire ant, <it>Solenopsis invicta </it>Buren (Hymenoptera), an invasive social insect.</p> <p>Results</p> <p>In the queen brains and subesophageal ganglion about 164 cells distributed in distinctive cell clusters (C1-C9 and C12) or as individual cells (C10, C11) were immuno-positive for the sNPF receptor. Most of these neurons are located in or near important sensory neuropils including the mushroom bodies, the antennal lobes, the central complex, and in different parts of the protocerebrum, as well as in the subesophageal ganglion. The localization of the sNPF receptor broadly links the receptor signaling pathway with circuits regulating learning and feeding behaviors. In ovaries from mated queens, the detection of sNPF receptor signal at the posterior end of oocytes in mid-oogenesis stage suggests that the sNPF signaling pathway may regulate processes at the oocyte pole.</p> <p>Conclusions</p> <p>The analysis of sNPF receptor immunolocalization shows that the sNPF signaling cascade may be involved in diverse functions, and the sNPF peptide(s) may act in the brain as neurotransmitter(s) or neuromodulator(s), and in the ovaries as neurohormone(s). To our knowledge, this is the first report of the cellular localization of a sNPF receptor on the brain and ovaries of adult insects.</p

Genomic Insights Into The Ixodes scapularis Tick Vector Of Lyme Disease

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retrotransposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing B57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing’, prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent

Genomic Insights Into The Ixodes scapularis Tick Vector Of Lyme Disease

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retrotransposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing B57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing’, prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent

Differences in sNPF receptor-expressing neurons in brains of fire ant (Solenopsis invicta Buren) worker subcastes: indicators for division of labor and nutritional status?

In the red imported fire ant, Solenopsis invicta Buren, the neuronal and molecular mechanisms related to worker division of labor are poorly understood. Workers from different subcastes (major, medium and minors) perform different tasks, which are loosely associated with their size. We hypothesized that the short neuropeptide F (sNPF) signaling system (NPY-like) could be involved in mechanisms of worker division of labor and sensing or responding to colony nutritional requirements. Thus, we investigated the expression of the short neuropeptide F receptor (sNPFR) in the brain and subesophageal ganglion (SEG) of workers from colonies with and without brood. Across worker subcastes a total of 9 clusters of immunoreactive sNPFR cells were localized in the brain and the subesophageal ganglion (SEG); some of these cells were similar to those observed previously in the queen. Worker brain sNPFR cell clusters were found in the protocerebrum near mushroom bodies, in the central complex and in the lateral horn. Other sNPFR immunoreactive cells were found at the edge of the antennal lobes. Across subcastes, we observed both a constant and a differential pattern of sNPFR clusters, with a higher number of sNPFR cells found in minor than in major workers. Those sNPFR cells detected in all worker subcastes appear to be involved in olfaction or SEG functions. The differential expression of clusters in subcastes suggests that sNPFR signaling is involved in regulating behaviors associated with specific subcastes and thus, division of labor. Some sNPFR cells appear to be involved in nutrient sensing and/or brood care, feeding behavior and locomotion. In colonies without brood, workers showed a lower cluster number, and an overall reduced sNPFR signal. Our results suggest the sNPF signaling system is a candidate for the neurobiological control of worker division of labor and sensing brood presence, perhaps correlating with protein requirements and availability

Brain immunolocalization of the sNPFR in worker subcastes from colonies without brood and comparisons with those with brood.

<p>(A–H) Schematic of the location of cell clusters expressing the sNPFR in all subcastes, comparing colonies with and without brood. Areas in orange boxes enclose specific brain areas for comparison. Dashed white circles correspond to the expected location of immunoreactive cells in colonies with brood. A–D, G and H show the receptor signal in the posterior brain; E and F show the anterior brain view. In colonies without brood cluster c7 became undetectable both in minor (H, I) and medium workers (D, K); compare with C, G. Additionally, cluster c2 became undetectable in minors from these colonies (F, J). In the posterior view, cluster c9 also became undetectable in minors (H, and insets in M, N) and mediums (D, L, inset). Cluster c14 remained present in minor (O, P) and medium workers (Q, R), but cluster c15 became undetectable in these workers, and only observed in majors, however, composed by lower number of immunoreactive cells (S, T) in comparison to majors in colonies with brood. Cluster c12 also is undetectable in majors (B, and in S, lower right corner) when brood is absent.</p