Andrew B. Nuss

Insulin-like peptides in the mosquito Anopheles stephensi: Identification and expression in response to diet and infection with Plasmodium falciparum

Insulin-like peptides (ILPs) regulate a multitude of biological processes, including metabolism and immunity to infection, and share similar structural motifs across widely divergent taxa. Insulin/insulin-like growth factor signaling (IIS) pathway elements are similarly conserved. We have shown that IIS regulates reproduction, innate immunity, and lifespan in female Anopheles stephensi, a major mosquito vector of human malaria. To further explore IIS regulation of these processes, we identified genes encoding five ILPs in this species and characterized their expression in tissues. Antisera to ILP homologs in Anopheles gambiae were used to identify cellular sources in An. stephensi females by immunocytochemistry. We analyzed tissue-specific ILP transcript expression in young and older females, in response to different feeding regimens, and in response to infection with Plasmodiumfalciparum with quantitative reverse transcriptase-PCR assays. While some ILP transcript changes were evident in older females and in response to blood feeding, significant changes were particularly notable in response to hormonal concentrations of ingested human insulin and to P. falciparum infection. These changes suggest that ILP secretion and action may be similarly responsive in Plasmodium-infected females and potentially alter metabolism and innate immunity.

Molecular characterization of neuropeptide F from the eastern subterranean termite Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae)

Neuropeptide F (NPF)-like immunoreactivity was previously found to be abundant in the eastern subterranean termite, Reticulitermes flavipes. Purification of the NPF from a whole body extract of worker termites was accomplished in the current study by HPLC and heterologous radioimmunoassay for an NPF-related peptide, Helicoverpa zea Midgut Peptide-I. A partial amino acid sequence allowed determination of the corresponding cDNA that encoded an open reading frame deduced for authentic R. flavipes NPF (Ref NPF): KPSDPEQLADTLKYLEELDRFYSQVARPRFa. Effects of synthetic NPFs on muscle contractions were investigated for isolated foreguts and hindguts of workers, with Drm NPF inhibiting spontaneous contractions of hindguts. Phylogenetic analysis of invertebrate NPF sequences reveals two separate groupings, with Ref NPF occurring within a clade composed exclusively of arthropods.

Distribution of neuropeptide F-like immunoreactivity in the eastern subterranean termite, Reticulitermes flavipes

Abstract The nervous system and gut of worker, soldier and alate castes of the eastern subterranean termite, Reticulitermes flavipes Kollar (Isoptera: Rhinotermitidae) were examined for immunoreactivity to an antiserum to Helicoverpa zea (Boddie) (Leipidoptera: Noctuidae) MP-I (QAARPRF-NH2), a truncated form of neuropeptide F. More than 145 immunostained axons and cell bodies were seen in the brain and all ganglia of the ventral nerve cord. Immunoreactive axons exiting the brain projected anteriorly to the frontal ganglion and posteriorly to the corpora cardiaca and corpora allata. In the stomatogastric nervous system, immunoreactive axons were observed over the surface of the foregut, salivary glands, midgut and rectum. These axons originated in the brain and from 15–25 neurosecretory cells on the foregut. Staining patterns were consistent between castes, with the exception of immunostaining observed in the optic lobes of alates. At least 600 immunoreactive endocrine cells were evenly distributed in the midguts of all castes with higher numbers present in the worker caste. Immunostaining of cells in the nervous system and midgut was blocked by preabsorption of the antiserum with Hez MP-I but not by a peptide having only the RF-NH2 in common. This distribution suggests NPF-like peptides coordinate feeding and digestion in all castes of this termite species.

Neuropeptide F and the corn earworm, Helicoverpa zea: a midgut peptide revisited.

The neuropeptide Y family of peptides is implicated in the regulation of feeding across a broad range of animals, including insects. Among vertebrates, neuropeptide Y exerts its actions mainly centrally, whereas peptide YY and pancreatic polypeptide arise from digestive tissues. Among invertebrates, neuropeptide F (NPF) is the sole counterpart of the NPY family. Shared features of NPF sequences derived for Lepidoptera indicate that the midgut peptide (Hez-MP-I) of the corn earworm, Helicoverpa zea, characterized more than a decade ago, is a carboxyl fragment of a full-length NPF. An antibody to Hez-MP-I was used to characterize the peptides distribution in tissues of larvae, pupae, and adults. Immunostaining demonstrated NPF-related material both in nervous tissues and in abundant endocrine cells of the midgut. Radioimmunoassay of Hez-MP-I in the head, midgut and hemolymph of fifth instar larvae revealed concentration changes corresponding to development and feeding state. As with the vertebrate homologs, NPF may arise both centrally and peripherally to modulate the physiology of feeding and digestion of Lepidoptera.

Human IGF1 extends lifespan and enhances resistance to Plasmodium falciparum infection in the malaria vector Anopheles stephensi

SUMMARY The highly conserved insulin/insulin-like growth factor (IGF) signaling (IIS) pathway regulates metabolism, development, lifespan and immunity across a wide range of organisms. Previous studies have shown that human insulin ingested in the blood meal can activate mosquito IIS, resulting in attenuated lifespan and increased malaria parasite infection. Because human IGF1 is present at higher concentrations in blood than insulin and is functionally linked with lifespan and immune processes, we predicted that human IGF1 ingested in a blood meal would affect lifespan and malaria parasite infection in the mosquito Anopheles stephensi. Here we demonstrate that physiological levels of ingested IGF1, like insulin, can persist intact in the blood-filled midgut for up to 30 h and disseminate into the mosquito body, and that both peptides activate IIS in mosquito cells and midgut. At these same levels, ingested IGF1 alone extended average mosquito lifespan by 23% compared with controls and, more significantly, when ingested in infected blood meals, reduced the prevalence of Plasmodium falciparum-infected mosquitoes by >20% and parasite load by 35–50% compared with controls. Thus, the effects of ingested IGF1 on mosquito lifespan and immunity are opposite to those of ingested insulin. These results offer the first evidence that insect cells can functionally discriminate between mammalian insulin and IGF1. Further, in light of previous success in genetically targeting IIS to alter mosquito lifespan and malaria parasite transmission, this study indicates that a more complete understanding of the IIS-activating ligands in blood can be used to optimize transgenic strategies for malaria control.

Dopamine Receptor Antagonists as New Mode-of-Action Insecticide Leads for Control of Aedes and Culex Mosquito Vectors

Background New mode-of-action insecticides are sought to provide continued control of pesticide resistant arthropod vectors of neglected tropical diseases (NTDs). We previously identified antagonists of the AaDOP2 D1-like dopamine receptor (DAR) from the yellow fever mosquito, Aedes aegypti, with toxicity to Ae. aegypti larvae as leads for novel insecticides. To extend DAR-based insecticide discovery, we evaluated the molecular and pharmacological characteristics of an orthologous DAR target, CqDOP2, from Culex quinquefasciatus, the vector of lymphatic filariasis and West Nile virus. Methods/Results CqDOP2 has 94.7% amino acid identity to AaDOP2 and 28.3% identity to the human D1-like DAR, hD1. CqDOP2 and AaDOP2 exhibited similar pharmacological responses to biogenic amines and DAR antagonists in cell-based assays. The antagonists amitriptyline, amperozide, asenapine, chlorpromazine and doxepin were between 35 to 227-fold more selective at inhibiting the response of CqDOP2 and AaDOP2 in comparison to hD1. Antagonists were toxic to both C. quinquefasciatus and Ae. aegypti larvae, with LC50 values ranging from 41 to 208 μM 72 h post-exposure. Orthologous DOP2 receptors identified from the African malaria mosquito, Anopheles gambiae, the sand fly, Phlebotomus papatasi and the tsetse fly, Glossina morsitans, had high sequence similarity to CqDOP2 and AaDOP2. Conclusions DAR antagonists represent a putative new insecticide class with activity against C. quinquefasciatus and Ae. aegypti, the two most important mosquito vectors of NTDs. There has been limited change in the sequence and pharmacological properties of the DOP2 DARs of these species since divergence of the tribes Culicini and Aedini. We identified antagonists selective for mosquito versus human DARs and observed a correlation between DAR pharmacology and the in vivo larval toxicity of antagonists. These data demonstrate that sequence similarity can be predictive of target potential. On this basis, we propose expanded insecticide discovery around orthologous DOP2 targets from additional dipteran vectors.

Evaluation of AaDOP2 receptor antagonists reveals antidepressants and antipsychotics as novel lead molecules for control of the yellow fever mosquito, Aedes aegypti

The yellow fever mosquito, Aedes aegypti, vectors disease-causing agents that adversely affect human health, most notably the viruses causing dengue and yellow fever. The efficacy of current mosquito control programs is challenged by the emergence of insecticide-resistant mosquito populations, suggesting an urgent need for the development of chemical insecticides with new mechanisms of action. One recently identified potential insecticide target is the A. aegypti D1-like dopamine receptor, AaDOP2. The focus of the present study was to evaluate AaDOP2 antagonism both in vitro and in vivo using assay technologies with increased throughput. The in vitro assays revealed AaDOP2 antagonism by four distinct chemical scaffolds from tricyclic antidepressant or antipsychotic chemical classes, and elucidated several structure-activity relationship trends that contributed to enhanced antagonist potency, including lipophilicity, halide substitution on the tricyclic core, and conformational rigidity. Six compounds displayed previously unparalleled potency for in vitro AaDOP2 antagonism, and among these, asenapine, methiothepin, and cis-(Z)-flupenthixol displayed subnanomolar IC50 values and caused rapid toxicity to A. aegypti larvae and/or adults in vivo. Our study revealed a significant correlation between in vitro potency for AaDOP2 antagonism and in vivo toxicity, suggesting viability of AaDOP2 as an insecticidal target. Taken together, this study expanded the repertoire of known AaDOP2 antagonists, enhanced our understanding of AaDOP2 pharmacology, provided further support for rational targeting of AaDOP2, and demonstrated the utility of efficiency-enhancing in vitro and in vivo assay technologies within our genome-to-lead pipeline for the discovery of next-generation insecticides.

Isolation of an insulin-like peptide from the Asian malaria mosquito, Anopheles stephensi, that acts as a steroidogenic gonadotropin across diverse mosquito taxa

Many insulin-like peptides (ILPs) have been identified in insects, yet only a few were isolated in their native form for structural and functional studies. Antiserum produced to ILP3 in Aedes aegypti was used in a radioimmunoassay to monitor the purification of an ILP from heads of adult An. stephensi and recognized the ILP in other immunoassays. The structure of the purified peptide matched that predicted for the ILP3 in this species. The native form stimulated ecdysteroid production by ovaries isolated from non-blood fed females. Synthetic forms of An. stephensi ILP3 and ILP4 similarly activated this process in a dose responsive manner. This function was first established for ILP3 and ILP4 homologs in Aedes aegypti, thus suggesting their structural and functional conservation in mosquitoes. We tested the extent of conservation by treating ovaries of An. gambiae, Ae. aegypti, and Culex quinquefasciatus with the An. stephensi ILPs, and both the native and synthetic ILP3 were stimulatory, as was the ILP4. Taken together, these results offer the first evidence for ILP functional conservation across the Anophelinae and Culicinae subfamilies.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Nutritional Quality during Development Alters Insulin-Like Peptides’ Expression and Physiology of the Adult Yellow Fever Mosquito, Aedes aegypti

Mosquitoes have distinct developmental and adult life history, and the vectorial capacity of females has been shown to be affected by the larval nutritional environment. However, little is known about the effect of developmental nutrition on insulin-signaling and nutrient storage. In this study, we used Aedes aegypti, the yellow fever mosquito, to determine whether larval nutrition affects insulin gene expression. We also determined the traits regulated by insulin signaling, such as stored-nutrient levels and fecundity. We raised mosquito larvae on two different diets, containing either high protein or high carbohydrates. Development on a high-carbohydrate diet resulted in several life-history phenotypes indicative of suboptimal conditions, including increased developmental time and decreased fecundity. Additionally, our data showed that insulin transcript levels are affected by a high-carbohydrate diet during development. Females, not males, reared on high-carbohydrate diets had much higher transcript levels of insulin-like peptide 3 (ILP3), a mosquito equivalent of human insulin, and these females more readily converted sugar meals into lipids. We also found that AaILP4, not AaILP3, transcript levels were much higher in the males after a sugar meal, suggesting sex-specific differences in the insulin-signaling pathway. Our findings suggest a conserved mechanism of carbohydrate-mediated hyperinsulinemia in animals.