Insulin-like peptides and ovary ecdysteroidogenic hormone differentially stimulate physiological processes regulating egg formation in the mosquito Aedes aegypti.

Mosquitoes including Aedes aegypti are human disease vectors because females must blood feed to produce and lay eggs. Blood feeding triggers insulin-insulin growth factor signaling (IIS) which regulates several physiological processes required for egg development. A. aegypti encodes 8 insulin-like peptides (ILPs) and one insulin-like receptor (IR) plus ovary ecdysteroidogenic hormone (OEH) that also activates IIS through the OEH receptor (OEHR). In this study, we assessed the expression of A. aegypti ILPs and OEH during a gonadotrophic cycle and produced each that were functionally characterized to further understand their roles in regulating egg formation. All A. aegypti ILPs and OEH were expressed during a gonadotrophic cycle. Five ILPs (1, 3, 4, 7, 8) and OEH were specifically expressed in the head, while antibodies to ILP3 and OEH indicated each was released after blood feeding from ventricular axons that terminate on the anterior midgut. A subset of ILP family members and OEH stimulated nutrient storage in previtellogenic females before blood feeding, whereas most IIS-dependent processes after blood feeding were activated by one or more of the brain-specific ILPs and/or OEH. ILPs and OEH with different biological activities also exhibited differences in IIS as measured by phosphorylation of the IR, phosphoinositide 3-kinase/Akt kinase (AKT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK). Altogether, our results provide the first results that compare the functional activities of all ILP family members and OEH produced by an insect.

The mosquito Aedes aegypti requires a gut microbiota for normal fecundity, longevity and vector competence.

Mosquitoes shift from detritus-feeding larvae to blood-feeding adults that can vector pathogens to humans and other vertebrates. The sugar and blood meals adults consume are rich in carbohydrates and protein but are deficient in other nutrients including B vitamins. Facultatively hematophagous insects like mosquitoes have been hypothesized to avoid B vitamin deficiencies by carryover of resources from the larval stage. However, prior experimental studies have also used adults with a gut microbiota that could provision B vitamins. Here, we used Aedes aegypti, which is the primary vector of dengue virus (DENV), to ask if carryover effects enable normal function in adults with no microbiota. We show that adults with no gut microbiota produce fewer eggs, live longer with lower metabolic rates, and exhibit reduced DENV vector competence but are rescued by provisioning B vitamins or recolonizing the gut with B vitamin autotrophs. We conclude carryover effects do not enable normal function.

The selective regulation of immune responses by matrix metalloproteinase MMP14 in Ostrinia furnacalis.

Matrix metalloproteinases (MMPs) are crucial for tissue remodeling and immune responses in insects, yet it remains unclear how MMPs affect the various immune processes against pathogenic infections and whether the responses vary among insects. In this study, we used the lepidopteran pest Ostrinia furnacalis larvae to address these questions by examining the changes of immune-related gene expression and antimicrobial activity after the knockdown of MMP14 and bacterial infections. We identified MMP14 in O. furnacalis using the rapid amplification of complementary DNA ends (RACE), and found that it was conserved and belonged to the MMP1 subfamily. Our functional investigations revealed that MMP14 is an infection-responsive gene, and its knockdown reduces phenoloxidase (PO) activity and Cecropin expression, while the expressions of Lysozyme, Attacin, Gloverin, and Moricin are enhanced after MMP14 knockdown. Further PO and lysozyme activity determinations showed consistent results with gene expression of these immune-related genes. Finally, the knockdown of MMP14 decreased larvae survival to bacterial infections. Taken together, our data indicate that MMP14 selectively regulates the immune responses, and is required to defend against bacterial infections in O. furnacalis larvae. Conserved MMPs may serve as a potential target for pest control using a combination of double-stranded RNA and bacterial infection.

Multiple endocrine factors regulate nutrient mobilization and storage in Aedes aegypti during a gonadotrophic cycle.

Anautogenous mosquitoes must blood feed on a vertebrate host to produce eggs. Each gonadotrophic cycle is subdivided into a sugar-feeding previtellogenic phase that produces primary follicles and a blood meal-activated vitellogenic phase in which large numbers of eggs synchronously mature and are laid. Multiple endocrine factors including juvenile hormone (JH), insulin-like peptides (ILPs), ovary ecdysteroidogenic hormone (OEH), and 20-hydroxyecdysone (20E) coordinate each gonadotrophic cycle. Egg formation also requires nutrients from feeding that are stored in the fat body. Regulation of egg formation is best understood in Aedes aegypti but the role different endocrine factors play in regulating nutrient mobilization and storage remains unclear. In this study, we report that adult female Ae. aegypti maintained triacylglycerol (TAG) stores during the previtellogenic phase of the first gonadotrophic cycle while glycogen stores declined. In contrast, TAG and glycogen stores were rapidly mobilized during the vitellogenic phase and then replenishment. Several genes encoding enzymes with functions in TAG and glycogen metabolism were differentially expressed in the fat body, which suggested regulation was mediated in part at the transcriptional level. Gain of function assays indicated that stored nutrients were primarily mobilized by adipokinetic hormone (AKH) while juvenoids and OEH regulated replenishment. ILP3 further showed evidence of negatively regulating certain lipolytic enzymes. Loss of function assays indicated AKH depends on the AKH receptor (AKHR) for function. Altogether, our results indicate that the opposing activities of different hormones regulate nutrient stores during a gonadotrophic cycle in Ae. aegypti.

Pheromone biosynthesis activating neuropeptide family in insects: a review.

Neuropeptides are involved in almost all physiological activities of insects. Their classification is based on physiological function and the primary amino acid sequence. The pyrokinin (PK)/pheromone biosynthesis activating neuropeptides (PBAN) are one of the largest neuropeptide families in insects, with a conserved C-terminal domain of FXPRLamide. The peptide family is divided into two groups, PK1/diapause hormone (DH) with a WFGPRLa C-terminal ending and PK2/PBAN with FXPRLamide C-terminal ending. Since the development of cutting-edge technology, an increasing number of peptides have been sequenced primarily through genomic, transcriptomics, and proteomics, and their functions discovered using gene editing tools. In this review, we discussed newly discovered functions, and analyzed the distribution of genes encoding these peptides throughout different insect orders. In addition, the location of the peptides that were confirmed by PCR or immunocytochemistry is also described. A phylogenetic tree was constructed according to the sequences of the receptors of most insect orders. This review offers an understanding of the significance of this conserved peptide family in insects.

Transcriptomic analysis provides insights into the immune responses and nutrition in Ostrinia furnacalis larvae parasitized by Macrocentrus cingulum.

Macrocentrus cingulum is a principal endoparasite of Ostrinia furnacalis larvae. M. cingulum larvae repress host immune responses for survival and ingest host nutrients for development until emerging. However, most investigations focused on the mechanisms of how wasps repress the host immunity, the triggered immune responses and nutrient status altered by wasps in host are neglected. In this study, we found that parasitized O. furnacalis larvae activated fast recognition responses and produced some effectors such as lysozyme and antimicrobial peptides, along with more consumption of trehalose, glucose, and even lipid to defend against the invading M. cingulum. However, the expression of peroxidase 6 and superoxide dismutase 2 (SOD 2) was upregulated, and the messenger RNA (mRNA) levels of cellular immunity-related genes such as thioester-containing protein 2 (TEP 2) and hemocytin were also reduced, suggesting that some immune responses were selectively shut down by wasp parasitization. Taken together, all the results indicated that parasitized O. furnacalis larvae selectively activate the immune recognition response, and upregulate effector genes, but suppress ROS reaction and cellular immunity, and invest more energy to fuel certain immune responses to defend against the wasp invading. This study provides useful information for further identifying key components of the nutrition and innate immune repertoire which may shape host-parasitoid coevolutionary dynamics.

Novel RNA Viruses from the Transcriptome of Pheromone Glands in the Pink Bollworm Moth, Pectinophora gossypiella.

In this study, we analyzed the transcriptome obtained from the pheromone gland isolated from two Israeli populations of the pink bollworm Pectinophora gossypiella to identify viral sequences. The lab population and the field samples carried the same viral sequences. We discovered four novel viruses: two positive-sense single-stranded RNA viruses, Pectinophora gossypiella virus 1 (PecgV1, a virus of Iflaviridae) and Pectinophora gossypiella virus 4 (PecgV4, unclassified), and two negative-sense single-stranded RNA viruses, Pectinophora gossypiella virus 2 (PecgV2, a virus of Phasmaviridae) and Pectinophora gossypiella virus 3 (PecgV3, a virus of Phenuiviridae). In addition, sequences derived from two negative-sense single-stranded RNA viruses that belong to Mononegavirales were found in the data. Analysis of previous transcriptome sequencing data derived from the midgut of pink bollworm larvae of a USA population only identified PecgV1, but no other viruses. High viral sequence coverages of PecgV1 and PecgV4 were observed in both field and lab populations. This is the first report of viral sequences discovered from the pink bollworm. Results from this investigation suggest that the pink bollworm harbors multiple viruses. Further investigation of the viral pathogens may help to develop novel pest management strategies for control of the pink bollworm.

Functional identification of fatty acyl reductases in female pheromone gland and tarsi of the corn earworm, Helicoverpa zea.

Most moths utilize sex pheromones released by the female to attract a mate. Females produce the sex pheromone in the pheromone gland in a biosynthetic pathway which consists of several key enzymes. Fatty acyl-CoA reductase is one of the key enzymes, which catalyzes the conversion of fatty acyl-CoA to the corresponding alcohol, playing an important role in producing the final proportion of each pheromone component. In Helicoverpa zea, (Z)-11-hexadecenal is the major sex pheromone component in female pheromone glands and previously a large amount of hexadecanal was also found in female and male tarsi. In our previous study, we compared the transcriptome between pheromone glands and tarsi and found 20 fatty acyl-CoA reductases in both tissues. In this study, we functionally characterized four FARs which were expressed at high levels according to the transcriptome of pheromone glands and tarsi. Fatty acyl-CoA reductase 1 was homologous to other moth pheromone gland specific fatty acyl-CoA reductases, and it was also present in male tarsi. Functional expression in yeast cells indicates that only fatty acyl-CoA reductase 1 was able to produce fatty alcohols. In addition, a decreased mRNA level of fatty acyl-CoA reductase 1 in female pheromone glands and male tarsi by RNAi knockdown caused a significant decrease in the production of (Z)-11-hexadecenal in pheromone glands and hexadecanal in male tarsi. This study is the first to demonstrate the direct function of a fatty acyl-CoA reductase in male tarsi and also confirms its role in sex pheromone biosynthesis in H. zea.

Pheromone gland transcriptome of the pink bollworm moth, Pectinophora gossypiella: Comparison between a laboratory and field population.

The pink bollworm, Pectinophora gossypiella, is a world-wide pest of cotton and in some parts of the cotton growing region is controlled by the mating disruption technique using synthetic sex pheromone. The sex pheromone consists of two compounds, (Z,Z)- and (Z,E)-7,11-hexadecadienyl acetates, in about a 50:50 ratio. However, recently, a population with sex pheromone compound ratios of about 62:38 were found in cotton fields that use mating disruption in Israel. To investigate how the change developed, we compared the pheromone gland transcriptomes between a reference laboratory population and a population obtained from an Israeli cotton field utilizing mating disruption. We analyzed four biological replicates from each population and found transcripts encoding 17 desaturases, 8 reductases, and 17 candidate acetyltransferases in both populations, which could be involved in sex pheromone biosynthesis. The expression abundance of some genes between the two populations was different. Some desaturases and candidate acetyltransferases were found to have mutated in one of the populations. The differentially expressed genes play potential roles in sex pheromone biosynthesis and could be involved in causing altered female sex pheromone ratios in the field population.

Transcriptional comparison between pheromone gland-ovipositor and tarsi in the corn earworm moth Helicoverpa zea.

The corn earworm, Helicoverpa zea, utilizes (Z)-11-hexadecenal as the major sex pheromone component. The saturated fatty acid derivative hexadecanal is also found in the pheromone gland and recently a large amount (0.5-1.5 µg) was found in male tarsi with lower amounts (0.05-0.5 µg) in female tarsi. In this study, we compared the transcriptome between female pheromone glands (including the ovipositor) and female and male tarsi to identify differences between these tissues, particularly the genes involved in sex pheromone biosynthesis and chemosensation. We found transcripts encoding 9 fatty acyl-CoA desaturases, 20 fatty acyl-CoA reductases, 8 alcohol oxidases, some G protein-coupled receptors and many transcripts involved in signal transduction and pheromone transportation. Also we found gustatory and olfactory receptors associated with the tarsi and ovipositor. Differential expression analysis showed that there were many genes differentially expressed between tissues, including the candidate desaturases, fatty acyl-CoA reductases, and alcohol oxidases. We discuss how some of these genes produce proteins that could be involved in the biosynthesis of hexadecanal in tarsi and (Z)-11-hexadecenal in the pheromone gland and the possible role of proteins in chemosensation of the tarsi and ovipositor.

Refolding Proteins from Inclusion Bodies using Differential Scanning Fluorimetry Guided (DGR) Protein Refolding and MeltTraceur Web.

Differential Scanning Fluorimetry Guided Refolding (DGR) is a simple methodology that can be used to rapidly screen for and identify conditions capable of accurately refolding protein preparations, such as those obtained from Escherichia coli inclusion bodies. It allows for the production in E. coli of functional proteins that would otherwise require far more expensive production methods. This unit describes how to set up a DGR refolding assay, perform DGR refolding trials in microplate format, use MeltTraceur Web software to interactively analyze the resulting data, scale-up protein production via refolding, and lastly, validate that the protein is properly folded. © 2018 by John Wiley & Sons, Inc.

Comprehensive Analysis of Constraint on the Spatial Distribution of Missense Variants in Human Protein Structures.

The spatial distribution of genetic variation within proteins is shaped by evolutionary constraint and provides insight into the functional importance of protein regions and the potential pathogenicity of protein alterations. Here, we comprehensively evaluate the 3D spatial patterns of human germline and somatic variation in 6,604 experimentally derived protein structures and 33,144 computationally derived homology models covering 77% of all human proteins. Using a systematic approach, we quantify differences in the spatial distributions of neutral germline variants, disease-causing germline variants, and recurrent somatic variants. Neutral missense variants exhibit a general trend toward spatial dispersion, which is driven by constraint on core residues. In contrast, germline disease-causing variants are generally clustered in protein structures and form clusters more frequently than recurrent somatic variants identified from tumor sequencing. In total, we identify 215 proteins with significant spatial constraints on the distribution of disease-causing missense variants in experimentally derived protein structures, only 65 (30%) of which have been previously reported. This analysis identifies many clusters not detectable from sequence information alone; only 12% of proteins with significant clustering in 3D were identified from similar analyses of linear protein sequence. Furthermore, spatial analyses of mutations in homology-based structural models are highly correlated with those from experimentally derived structures, supporting the use of computationally derived models. Our approach highlights significant differences in the spatial constraints on different classes of mutations in protein structure and identifies regions of potential function within individual proteins.