Insulin-Like Peptide Signaling in Mosquitoes: The Road Behind and the Road Ahead

Insulin signaling is a conserved pathway in all metazoans. This pathway contributed toward primordial metazoans responding to a greater diversity of environmental signals by modulating nutritional storage, reproduction, and longevity. Most of our knowledge of insulin signaling in insects comes from the vinegar fly, Drosophila melanogaster, where it has been extensively studied and shown to control several physiological processes. Mosquitoes are the most important vectors of human disease in the world and their control constitutes a significant area of research. Recent studies have shown the importance of insulin signaling in multiple physiological processes such as reproduction, innate immunity, lifespan, and vectorial capacity in mosquitoes. Although insulin-like peptides have been identified and functionally characterized from many mosquito species, a comprehensive review of this pathway in mosquitoes is needed. To fill this gap, our review provides up-to-date knowledge of this subfield

RNAi reveals proteins for metabolism and protein processing associated with Langat virus infection in Ixodes scapularis (black-legged tick) ISE6 cells

Tick-borne flaviviruses (TBFs) cause thousands of human cases of encephalitis worldwide each year, with some TBF infections progressing to hemorrhagic fever. TBFs are of medical and veterinary importance and strategies to reduce flavivirus transmission by the tick vector may have significant application. Analyses of the proteome of ISE6 cells derived from the black legged tick, Ixodes scapularis infected with the TBF, Langat virus (LGTV), have provided insights into proteins and cellular processes involved with LGTV infection. Methods: RNA interference (RNAi)-induced knockdown of transcripts was used to investigate the role of ten tick proteins in the LGTV infection cycle in ISE6 cells. LGTV-infected cells were separately transfected with dsRNA corresponding to each gene of interest and the effect on LGTV genome replication and release of infectious virus was assessed by RT-qPCR and plaque assays, respectively. Results: RNAi-induced knockdown of transcripts for two enzymes that likely function in amino acid, carbohydrate, lipid, terpenoid/polykeytide and vitamin metabolism, and a transcript for one protein of unknown function were associated with decreased replication of the LGTV genome and release of infectious virus from cells. The knockdown of transcripts for five enzymes predicted to function in metabolism, a protein likely associated with folding, sorting and degradation, and a protein of unknown function was associated with a decrease only in the amount of infectious LGTV released from cells. Conclusions: These data suggest tick proteins potentially associated with metabolism and protein processing may be involved in LGTV infection of ISE6 cells. Our study provides information to begin to elucidate the function of these proteins and identify targets for the development of new interventions aimed at controlling the transmission of TBFs

Dynamics of Insulin Signaling in the Black-Legged Tick, Ixodes scapularis

Insulin-like peptides (ILPs) have been identified in several invertebrates, particularly insects, and work on these ILPs has revealed many roles including regulation of energy homeostasis, growth, development, and lifespan to name a few. However, information on arthropod ILPs outside of insects is sparse. Studies of Ixodid tick ILPs are particularly scarce, despite their importance as vectors of infectious agents, most notably Lyme disease. The recent publication of the genome of the black-legged tick, Ixodes scapularis, has advanced opportunities to study this organism from a molecular standpoint, a resource sorely needed for an organism with challenging life history requirements for study in the laboratory, such as a long life cycle and obligate, prolonged, blood-feeding at each life stage. Through bioinformatics searches of the tick genome and other available I. scapularis databases, we identified four putative ILP sequences. Full-length sequences of these ILP transcripts were confirmed, and quantitative RT-PCR was used to examine expression levels of these ILPs in different life stages, feeding states, and adult tissues. This work serves as an initial characterization of ILP expression in ticks and provides the foundation for further exploration of the roles of ILPs in these important arthropod vectors

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 retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing approximately 57% 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 \u27questing\u27, 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 retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ~57% 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 \u27questing\u27, 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

Citation: Gulia-Nuss, M., Nuss, A. B., Meyer, J. M., Sonenshine, D. E., Roe, R. M., Waterhouse, R. M., . . . Hill, C. A. (2016). Genomic insights into the Ixodes scapularis tick vector of Lyme disease. Nature Communications, 7, 13. doi:10.1038/ncomms10507Additional Authors: Koren, S.;Hostetler, J. B.;Thiagarajan, M.;Joardar, V. S.;Hannick, L. I.;Bidwell, S.;Hammond, M. P.;Young, S.;Zeng, Q. D.;Abrudan, J. L.;Almeida, F. C.;Ayllon, N.;Bhide, K.;Bissinger, B. W.;Bonzon-Kulichenko, E.;Buckingham, S. D.;Caffrey, D. R.;Caimano, M. J.;Croset, V.;Driscoll, T.;Gilbert, D.;Gillespie, J. J.;Giraldo-Calderon, G. I.;Grabowski, J. M.;Jiang, D.;Khalil, S. M. S.;Kim, D.;Kocan, K. M.;Koci, J.;Kuhn, R. J.;Kurtti, T. J.;Lees, K.;Lang, E. G.;Kennedy, R. C.;Kwon, H.;Perera, R.;Qi, Y. M.;Radolf, J. D.;Sakamoto, J. M.;Sanchez-Gracia, A.;Severo, M. S.;Silverman, N.;Simo, L.;Tojo, M.;Tornador, C.;Van Zee, J. P.;Vazquez, J.;Vieira, F. G.;Villar, M.;Wespiser, A. R.;Yang, Y. L.;Zhu, J. W.;Arensburger, P.;Pietrantonio, P. V.;Barker, S. C.;Shao, R. F.;Zdobnov, E. M.;Hauser, F.;Grimmelikhuijzen, C. J. P.;Park, Y.;Rozas, J.;Benton, R.;Pedra, J. H. F.;Nelson, D. R.;Unger, M. F.;Tubio, J. M. C.;Tu, Z. J.;Robertson, H. M.;Shumway, M.;Sutton, G.;Wortman, J. R.;Lawson, D.;Wikel, S. K.;Nene, V. M.;Fraser, C. M.;Collins, F. H.;Birren, B.;Nelson, K. E.;Caler, E.;Hill, C. A.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 retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing similar to 57% 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

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

Anaplasma phagocytophilum infection subverts carbohydrate metabolic pathways in the tick vector, Ixodes scapularis

The obligate intracellular pathogen, Anaplasma phagocytophilum, is the causative agent of human, equine, and canine granulocytic anaplasmosis and tick-borne fever (TBF) in ruminants. A. phagocytophilum has become an emerging tick-borne pathogen in the United States, Europe, Africa, and Asia, with increasing numbers of infected people and animals every year. It has been recognized that intracellular pathogens manipulate host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. However, our current knowledge on how A. phagocytophilum affect these processes in the tick vector, Ixodes scapularis is limited. In this study, a genome-wide search for components of major carbohydrate metabolic pathways was performed in I. scapularis ticks for which the genome was recently published. The enzymes involved in the seven major carbohydrate metabolic pathways glycolysis, gluconeogenesis, pentose phosphate, tricarboxylic acid cycle (TCA), glyceroneogenesis, and mitochondrial oxidative phosphorylation and β-oxidation were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis major carbohydrate metabolic pathway components in response to A. phagocytophilum infection of tick tissues and cultured cells. The results showed that major carbohydrate metabolic pathways are conserved in ticks. A. phagocytophilum infection inhibits gluconeogenesis and mitochondrial metabolism, but increases the expression of glycolytic genes. A model was proposed to explain how A. phagocytophilum could simultaneously control tick cell glucose metabolism and cytoskeleton organization, which may be achieved in part by up-regulating and stabilizing hypoxia inducible factor 1 alpha in a hypoxia-independent manner. The present work provides a more comprehensive view of the major carbohydrate metabolic pathways involved in the response to A. phagocytophilum infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.Peer reviewedVeterinary Pathobiolog

The Neuropeptide Allatostatin A Regulates Metabolism and Feeding Decisions in Drosophila

Coordinating metabolism and feeding is important to avoid obesity and metabolic diseases, yet the underlying mechanisms, balancing nutrient intake and metabolic expenditure, are poorly understood. Several mechanisms controlling these processes are conserved in Drosophila, where homeostasis and energy mobilization are regulated by the glucagon-related adipokinetic hormone (AKH) and the Drosophila insulin-like peptides (DILPs). Here, we provide evidence that the Drosophila neuropeptide Allatostatin A (AstA) regulates AKH and DILP signaling. The AstA receptor gene, Dar-2, is expressed in both the insulin and AKH producing cells. Silencing of Dar-2 in these cells results in changes in gene expression and physiology associated with reduced DILP and AKH signaling and animals lacking AstA accumulate high lipid levels. This suggests that AstA is regulating the balance between DILP and AKH, believed to be important for the maintenance of nutrient homeostasis in response to changing ratios of dietary sugar and protein. Furthermore, AstA and Dar-2 are regulated differentially by dietary carbohydrates and protein and AstA-neuronal activity modulates feeding choices between these types of nutrients. Our results suggest that AstA is involved in assigning value to these nutrients to coordinate metabolic and feeding decisions, responses that are important to balance food intake according to metabolic needs

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