The Putative AKH Receptor of the Tobacco Hornworm, Manduca sexta, and Its Expression

Adipokinetic hormones are peptide hormones that mobilize lipids and/or carbohydrates for flight in adult insects and activate glycogen Phosphorylase in larvae during starvation and during molt. We previously examined the functional roles of adipokinetic hormone in Manduca sexta L. (Lepidoptera: Sphingidae). Here we report the cloning of the full-length cDNA encoding the putative adipokinetic hormone receptor from the fat body of M. sexta. The sequence analysis shows that the deduced amino acid sequence shares common motifs of G protein-coupled receptors, by having seven hydrophobic transmembrane segments. We examined the mRNA expression pattern of the adipokinetic hormone receptor by quantitative Real-Time PCR in fat body during development and in different tissues and found the strongest expression in fat body of larvae two days after molt to the fifth instar. We discuss these results in relation to some of our earlier results. We also compare the M. sexta adipokinetic hormone receptor with the known adipokinetic hormone receptors of other insects and with gonadotropin releasing hormone-like receptors of invertebrates

Insulin-Like Peptides and the Target of Rapamycin Pathway Coordinately Regulate Blood Digestion and Egg Maturation in the Mosquito Aedes aegypti

Mosquitoes are insects that vector many serious pathogens to humans and other vertebrates. Most mosquitoes must feed on the blood of a vertebrate host to produce eggs. In turn, multiple cycles of blood feeding promote frequent contacts with hosts and make mosquitoes ideal disease vectors. Both hormonal and nutritional factors are involved in regulating egg development in the mosquito, Aedes aegypti. However, the processes that regulate digestion of the blood meal remain unclear.Here we report that insulin peptide 3 (ILP3) directly stimulated late phase trypsin-like gene expression in blood fed females. In vivo knockdown of the mosquito insulin receptor (MIR) by RNA interference (RNAi) delayed but did not fully inhibit trypsin-like gene expression in the midgut, ecdysteroid (ECD) production by ovaries, and vitellogenin (Vg) expression by the fat body. In contrast, in vivo treatment with double-stranded MIR RNA and rapamycin completely blocked egg production. In vitro experiments showed that amino acids did not simulate late phase trypsin-like gene expression in the midgut or ECD production by the ovaries. However, amino acids did enhance ILP3-mediated stimulation of trypsin-like gene expression and ECD production.Overall, our results indicate that ILPs from the brain synchronize blood meal digestion and amino acid availability with ovarian ECD production to maximize Vg expression by the fat body. The activation of digestion by ILPs may also underlie the growth promoting effects of insulin and TOR signaling in other species

Oviposition Preference and Offspring Performance In Container Breeding Mosquitoes: Evaluating the Effects of Organic Compounds and Laboratory Colonisation

1. The preference–performance hypothesis (PPH) predicts that organisms lacking parental care should oviposit in habitats that optimise offspring performance. Preference–performance relationships were investigated for the Asian tiger mosquito (Aedes albopictus Skuse) and the southern house mosquito (Culex quinquefasciatus Say) (Diptera: Culicidae), two medically important container-breeding species, in response to an organic chemical blend mimicking decaying plant matter. Additionally, the effects of long-term laboratory colonisation of Cx. quinquefasciatus using wild and laboratory strains were evaluated. 2. Oviposition bioassays were conducted by releasing gravid mosquitoes into field enclosures with automobile tires containing low and high concentrations of the chemical blend, and water controls. The offspring were then reared in water collected from the tires in which they were deposited. 3. Aedes albopictus and wild Cx. quinquefasciatus laid more eggs in the chemical blend than water controls but did not differentiate between the low and high concentrations. Conversely, laboratory Cx. quinquefasciatus only preferred the high concentration to the low concentration. No statistical associations between oviposition preference and larval survival were found, as the chemical blend did not affect survivorship of either species. 4. The oviposition preference for the chemical blend over water controls suggests that both species oviposit in the best available resource environment, but further studies are needed before conclusions regarding preference–performance relationships can be drawn. 5. It was found that long-term laboratory colonisation affects the oviposition behaviour in Cx. quinquefasciatus, suggesting that behavioural studies on laboratory strains are not always applicable to wild populations

Association of genetic variants in the promoter region of genes encoding p22phox (CYBA) and glutamate cysteine ligase catalytic subunit (GCLC) and renal disease in patients with type 1 diabetes mellitus

<p>Abstract</p> <p>Background</p> <p>Oxidative stress is recognized as a major pathogenic factor of cellular damage caused by hyperglycemia. NOX/NADPH oxidases generate reactive oxygen species and NOX1, NOX2 and NOX4 isoforms are expressed in kidney and require association with subunit p22phox (encoded by the <it>CYBA </it>gene). Increased expression of p22phox was described in animal models of diabetic nephropathy. In the opposite direction, glutathione is one of the main endogenous antioxidants whose plasmatic concentrations were reported to be reduced in diabetes patients. The aim of the present investigation was to test whether functional single nucleotide polymorphisms (SNPs) in genes involved in the generation of NADPH-dependent O₂^•-(-675 T → A in <it>CYBA</it>, unregistered) and in glutathione metabolism (-129 C → T in <it>GCLC </it>[rs17883901] and -65 T → C in <it>GPX3 </it>[rs8177412]) confer susceptibility to renal disease in type 1 diabetes patients.</p> <p>Methods</p> <p>401 patients were sorted into two groups according to the presence (n = 104) or absence (n = 196) of overt diabetic nephropathy or according to glomerular filtration rate (GFR) estimated by Modification of Diet in Renal Disease (MDRD) equation: ≥ 60 mL (n = 265) or < 60 mL/min/1.73 m²(n = 136) and were genotyped.</p> <p>Results</p> <p>No differences were found in the frequency of genotypes between diabetic and non-diabetic subjects. The frequency of GFR < 60 mL/min was significantly lower in the group of patients carrying <it>CYBA </it>genotypes T/A+A/A (18.7%) than in the group carrying the T/T genotype (35.3%) (P = 0.0143) and the frequency of GFR < 60 mL/min was significantly higher in the group of patients carrying <it>GCLC </it>genotypes C/T+T/T (47.1%) than in the group carrying the C/C genotype (31.1%) (<it>p </it>= 0.0082). Logistic regression analysis identified the presence of at least one A allele of the <it>CYBA </it>SNP as an independent protection factor against decreased GFR (OR = 0.38, CI95% 0.14-0.88, <it>p </it>= 0.0354) and the presence of at least one T allele of the <it>GCLC </it>rs17883901 SNP as an independent risk factor for decreased GFR (OR = 2.40, CI95% 1.27-4.56, <it>p </it>= 0.0068).</p> <p>Conclusions</p> <p>The functional SNPs <it>CYBA </it>-675 T → A and <it>GCLC </it>rs17883901, probably associated with cellular redox imbalances, modulate the risk for renal disease in the studied population of type 1 diabetes patients and require validation in additional cohorts.</p

Alterations in the Aedes aegypti Transcriptome during Infection with West Nile, Dengue and Yellow Fever Viruses

West Nile (WNV), dengue (DENV) and yellow fever (YFV) viruses are (re)emerging, mosquito-borne flaviviruses that cause human disease and mortality worldwide. Alterations in mosquito gene expression common and unique to individual flaviviral infections are poorly understood. Here, we present a microarray analysis of the Aedes aegypti transcriptome over time during infection with DENV, WNV or YFV. We identified 203 mosquito genes that were ≥5-fold differentially up-regulated (DUR) and 202 genes that were ≥10-fold differentially down-regulated (DDR) during infection with one of the three flaviviruses. Comparative analysis revealed that the expression profile of 20 DUR genes and 15 DDR genes was quite similar between the three flaviviruses on D1 of infection, indicating a potentially conserved transcriptomic signature of flaviviral infection. Bioinformatics analysis revealed changes in expression of genes from diverse cellular processes, including ion binding, transport, metabolic processes and peptidase activity. We also demonstrate that virally-regulated gene expression is tissue-specific. The overexpression of several virally down-regulated genes decreased WNV infection in mosquito cells and Aedes aegypti mosquitoes. Among these, a pupal cuticle protein was shown to bind WNV envelope protein, leading to inhibition of infection in vitro and the prevention of lethal WNV encephalitis in mice. This work provides an extensive list of targets for controlling flaviviral infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses

Blood Feeding and Insulin-like Peptide 3 Stimulate Proliferation of Hemocytes in the Mosquito Aedes aegypti

All vector mosquito species must feed on the blood of a vertebrate host to produce eggs. Multiple cycles of blood feeding also promote frequent contacts with hosts, which enhance the risk of exposure to infectious agents and disease transmission. Blood feeding triggers the release of insulin-like peptides (ILPs) from the brain of the mosquito Aedes aegypti, which regulate blood meal digestion and egg formation. In turn, hemocytes serve as the most important constitutive defense in mosquitoes against pathogens that enter the hemocoel. Prior studies indicated that blood feeding stimulates hemocytes to increase in abundance, but how this increase in abundance is regulated is unknown. Here, we determined that phagocytic granulocytes and oenocytoids express the A. aegypti insulin receptor (AaMIR). We then showed that: 1) decapitation of mosquitoes after blood feeding inhibited hemocyte proliferation, 2) a single dose of insulin-like peptide 3 (ILP3) sufficient to stimulate egg production rescued proliferation, and 3) knockdown of the AaMIR inhibited ILP3 rescue activity. Infection studies indicated that increased hemocyte abundance enhanced clearance of the bacterium Escherichia coli at lower levels of infection. Surprisingly, however, non-blood fed females better survived intermediate and high levels of E. coli infection than blood fed females. Taken together, our results reveal a previously unrecognized role for the insulin signaling pathway in regulating hemocyte proliferation. Our results also indicate that blood feeding enhances resistance to E. coli at lower levels of infection but reduces tolerance at higher levels of infection

Development of the Bi-Partite Gal4-UAS System in the African Malaria Mosquito, Anopheles gambiae

Functional genetic analysis in Anopheles gambiae would be greatly improved by the development of a binary expression system, which would allow the more rapid and flexible characterisation of genes influencing disease transmission, including those involved in insecticide resistance, parasite interaction, host and mate seeking behaviour. The Gal4-UAS system, widely used in Drosophila melanogaster functional genetics, has been significantly modified to achieve robust application in several different species. Towards this end, previous work generated a series of modified Gal4 constructs that were up to 20 fold more active than the native gene in An. gambiae cells. To examine the Gal4-UAS system in vivo, transgenic An. gambiae driver lines carrying a modified Gal4 gene under the control of the carboxypeptidase promoter, and responder lines carrying UAS regulated luciferase and eYFP reporter genes have been created. Crossing of the Gal4 and UAS lines resulted in progeny that expressed both reporters in the expected midgut specific pattern. Although there was minor variation in reporter gene activity between the different crosses examined, the tissue specific expression pattern was consistent regardless of the genomic location of the transgene cassettes. The results show that the modified Gal4-UAS system can be used to successfully activate expression of transgenes in a robust and tissue specific manner in Anopheles gambiae. The midgut driver and dual reporter responder constructs are the first to be developed and tested successfully in transgenic An. gambiae and provide the basis for further advancement of the system in this and other insect species

Functional genomics of the horn fly, Haematobia irritans (Linnaeus, 1758)

<p>Abstract</p> <p>Background</p> <p>The horn fly, <it>Haematobia irritans </it>(Linnaeus, 1758) (Diptera: Muscidae) is one of the most important ectoparasites of pastured cattle. Horn flies infestations reduce cattle weight gain and milk production. Additionally, horn flies are mechanical vectors of different pathogens that cause disease in cattle. The aim of this study was to conduct a functional genomics study in female horn flies using Expressed Sequence Tags (EST) analysis and RNA interference (RNAi).</p> <p>Results</p> <p>A cDNA library was made from whole abdominal tissues collected from partially fed adult female horn flies. High quality horn fly ESTs (2,160) were sequenced and assembled into 992 unigenes (178 contigs and 814 singlets) representing molecular functions such as serine proteases, cell metabolism, mitochondrial function, transcription and translation, transport, chromatin structure, vitellogenesis, cytoskeleton, DNA replication, cell response to stress and infection, cell proliferation and cell-cell interactions, intracellular trafficking and secretion, and development. Functional analyses were conducted using RNAi for the first time in horn flies. Gene knockdown by RNAi resulted in higher horn fly mortality (protease inhibitor functional group), reduced oviposition (vitellogenin, ferritin and vATPase groups) or both (immune response and 5'-NUC groups) when compared to controls. Silencing of ubiquitination ESTs did not affect horn fly mortality and ovisposition while gene knockdown in the ferritin and vATPse functional groups reduced mortality when compared to controls.</p> <p>Conclusions</p> <p>These results advanced the molecular characterization of this important ectoparasite and suggested candidate protective antigens for the development of vaccines for the control of horn fly infestations.</p

Proteases of haematophagous arthropod vectors are involved in blood-feeding, yolk formation and immunity : a review

Ticks, triatomines, mosquitoes and sand flies comprise a large number of haematophagous arthropods considered vectors of human infectious diseases. While consuming blood to obtain the nutrients necessary to carry on life functions, these insects can transmit pathogenic microorganisms to the vertebrate host. Among the molecules related to the blood-feeding habit, proteases play an essential role. In this review, we provide a panorama of proteases from arthropod vectors involved in haematophagy, in digestion, in egg development and in immunity. As these molecules act in central biological processes, proteases from haematophagous vectors of infectious diseases may influence vector competence to transmit pathogens to their prey, and thus could be valuable targets for vectorial control