The immune signaling pathways of Manduca sexta.

Signal transduction pathways and their coordination are critically important for proper functioning of animal immune systems. Our knowledge of the constituents of the intracellular signaling network in insects mainly comes from genetic analyses in Drosophila melanogaster. To facilitate future studies of similar systems in the tobacco hornworm and other lepidopteran insects, we have identified and examined the homologous genes in the genome of Manduca sexta. Based on 1:1 orthologous relationships in most cases, we hypothesize that the Toll, Imd, MAPK-JNK-p38 and JAK-STAT pathways are intact and operative in this species, as are most of the regulatory mechanisms. Similarly, cellular processes such as autophagy, apoptosis and RNA interference probably function in similar ways, because their mediators and modulators are mostly conserved in this lepidopteran species. We have annotated a total of 186 genes encoding 199 proteins, studied their domain structures and evolution, and examined their mRNA levels in tissues at different life stages. Such information provides a genomic perspective of the intricate signaling system in a non-drosophiline insect.

Programmed autophagy in the fat body of Aedes aegypti is required to maintain egg maturation cycles.

Autophagy plays a pivotal role by allowing cells to recycle cellular components under conditions of stress, starvation, development and cancer. In this work, we have demonstrated that programmed autophagy in the mosquito fat body plays a critical role in maintaining of developmental switches required for normal progression of gonadotrophic cycles. Mosquitoes must feed on vertebrate blood for their egg development, with each gonadotrophic cycle being tightly coupled to a separate blood meal. As a consequence, some mosquito species are vectors of pathogens that cause devastating diseases in humans and domestic animals, most importantly malaria and Dengue fever. Hence, deciphering mechanisms to control egg developmental cycles is of paramount importance for devising novel approaches for mosquito control. Central to egg development is vitellogenesis, the production of yolk protein precursors in the fat body, the tissue analogous to a vertebrate liver, and their subsequent specific accumulation in developing oocytes. During each egg developmental cycle, the fat body undergoes a developmental program that includes previtellogenic build-up of biosynthetic machinery, intense production of yolk protein precursors, and termination of vitellogenesis. The importance of autophagy for termination of vitellogenesis was confirmed by RNA interference (RNAi) depletions of several autophagic genes (ATGs), which inhibited autophagy and resulted in untimely hyper activation of TOR and prolonged production of the major yolk protein precursor, vitellogenin (Vg). RNAi depletion of the ecdysone receptor (EcR) demonstrated its activating role of autophagy. Depletion of the autophagic genes and of EcR led to inhibition of the competence factor, betaFTZ-F1, which is required for ecdysone-mediated developmental transitions. Moreover, autophagy-incompetent female mosquitoes were unable to complete the second reproductive cycle and exhibited retardation and abnormalities in egg maturation. Thus, our study has revealed a novel function of programmed autophagy in maintaining egg maturation cycles in mosquitoes.

microRNA miR-275 is indispensable for blood digestion and egg development in the mosquito Aedes aegypti.

The mosquito Aedes aegypti is the major vector of arboviral diseases, particularly of Dengue fever, of which there are more than 100 million cases annually. Mosquitoes, such as A. aegypti, serve as vectors for disease pathogens because they require vertebrate blood for their egg production. Pathogen transmission is tightly linked to repeated cycles of obligatory blood feeding and egg maturation. Thus, the understanding of mechanisms governing egg production is necessary to develop approaches that limit the spread of mosquito-borne diseases. Previous studies have identified critical roles of hormonal- and nutrition-based target of rapamycin (TOR) pathways in controlling blood-meal-mediated egg maturation in mosquitoes. In this work, we uncovered another essential regulator of blood-meal-activated processes, the microRNA miR-275. The depletion of this microRNA in A. aegypti females after injection of its specific antagomir resulted in severe defects in blood digestion, fluid excretion, and egg development, clearly demonstrating that miR-275 is indispensable for these physiological processes. miR-275 exhibits an expression profile that suggests its regulation by a steroid hormone, 20-hydroxyecdysone (20E). In vitro organ culture experiments demonstrated that miR-275 is induced by this hormone in the presence of amino acids, indicative of a dual regulation by 20E and TOR. This report has uncovered the critical importance of microRNAs in controlling blood-meal-activated physiological events required for completion of egg development in mosquito disease vectors.

Pathogenomics of Culex quinquefasciatus and meta-analysis of infection responses to diverse pathogens.

The mosquito Culex quinquefasciatus poses a substantial threat to human and veterinary health as a primary vector of West Nile virus (WNV), the filarial worm Wuchereria bancrofti, and an avian malaria parasite. Comparative phylogenomics revealed an expanded canonical C. quinquefasciatus immune gene repertoire compared with those of Aedes aegypti and Anopheles gambiae. Transcriptomic analysis of C. quinquefasciatus genes responsive to WNV, W. bancrofti, and non-native bacteria facilitated an unprecedented meta-analysis of 25 vector-pathogen interactions involving arboviruses, filarial worms, bacteria, and malaria parasites, revealing common and distinct responses to these pathogen types in three mosquito genera. Our findings provide support for the hypothesis that mosquito-borne pathogens have evolved to evade innate immune responses in three vector mosquito species of major medical importance.

A caspase-like decoy molecule enhances the activity of a paralogous caspase in the yellow fever mosquito, Aedes aegypti.

Caspases are cysteine proteases that play critical roles in apoptosis and other key cellular processes. A mechanism of caspase regulation that has been described in mammals and nematodes involves caspase-like decoy molecules, enzymatically inactive caspase homologs that have arisen by gene duplication and acquired the ability to regulate other caspases. Caspase-like decoy molecules are not found in Drosophila melanogaster, raising the question of whether this type of caspase regulation exists in insects. Phylogenomic analysis of caspase genes from twelve Drosophila and three mosquito species revealed several examples of duplicated caspase homologs lacking critical catalytic residues, making them candidate caspase-like decoy molecules. One of these, CASPS18 from the mosquito Aedes aegypti, is a homolog of the D. melanogaster caspase Decay and contains substitutions in two critical amino acid positions, including the catalytic cysteine residue. As expected, CASPS18 lacked caspase activity, but co-expression of CASPS18 with a paralogous caspase, CASPS19, in mosquito cells or co-incubation of CASPS18 and CASPS19 recombinant proteins resulted in greatly enhanced CASPS19 activity. The discovery of potential caspase-like decoy molecules in several insect species opens new avenues for investigating caspase regulation in insects, particularly in disease vectors such as mosquitoes.

Caspase inhibitor P35 is required for the production of robust baculovirus virions in Trichoplusia ni TN-368 cells.

Apoptosis can protect lepidopteran insects against baculovirus infection by limiting viral replication. Baculoviruses counter this response by expressing anti-apoptotic proteins such as the caspase inhibitor P35, which is expressed by several baculoviruses including Autographa californica mutiple nucleopolyhedrovirus (AcMNPV). Mutants of AcMNPV that lack the p35 gene induce apoptosis in Spodoptera frugiperda cells, and replication of these mutants is severely curtailed in S. frugiperda cell lines and larvae. However, cells from another lepidopteran species, Trichoplusia ni, do not undergo apoptosis when infected with AcMNPV mutants lacking p35, and p35 mutant and wild-type viruses replicate to equivalent levels in the T. ni cell line TN-368 and have equivalent infectivity in T. ni larvae by either oral or intrahaemocoelic injection. This has led to the conclusion that p35 is not required for AcMNPV replication in T. ni. However, in this study it was found that stocks of p35 mutant virus produced in TN-368 cells had defects in virion stability and infectivity. TN-368 cells infected with p35 mutant AcMNPV exhibited caspase activity, despite a lack of apoptosis, and propagation of the mutant virus in the presence of a chemical caspase inhibitor restored the normal infection phenotype to the progeny virus. These results suggest that caspases can directly or indirectly damage baculovirus virions, and reveal a novel aspect of the role of apoptosis in antiviral defence.

Annotation and expression profiling of apoptosis-related genes in the yellow fever mosquito, Aedes aegypti.

Apoptosis has been extensively studied in Drosophila by both biochemical and genetic approaches, but there is a lack of knowledge about the mechanisms of apoptosis regulation in other insects. In mosquitoes, apoptosis occurs during Plasmodium and arbovirus infection in the midgut, suggesting that apoptosis plays a role in mosquito innate immunity. We searched the Aedes aegypti genome for apoptosis-related genes using Drosophila and Anopheles gambiae protein sequences as queries. In this study we have identified eleven caspases, three inhibitor of apoptosis (IAP) proteins, a previously unreported IAP antagonist, and orthologs of Drosophila Ark, Dnr1, and BG4 (also called dFadd). While most of these genes have been previously annotated, we have improved the annotation of several of them, and we also report the discovery of four previously unannotated apoptosis-related genes. We examined the developmental expression profile of these genes in Ae. aegypti larvae, pupae and adults, and we also studied the function of a novel IAP antagonist, IMP. Expression of IMP in mosquito cells caused apoptosis, indicating that it is a functional pro-death protein. Further characterization of these genes will help elucidate the molecular mechanisms of apoptosis regulation in Ae. aegypti.