Activation of immune pathways in common bed bugs, Cimex lectularius, in response to bacterial immune challenges - a transcriptomics analysis.

The common bed bug, Cimex lectularius, is an urban pest of global health significance, severely affecting the physical and mental health of humans. In contrast to most other blood-feeding arthropods, bed bugs are not major vectors of pathogens, but the underlying mechanisms for this phenomenon are largely unexplored. Here, we present the first transcriptomics study of bed bugs in response to immune challenges. To study transcriptional variations in bed bugs following ingestion of bacteria, we extracted and processed mRNA from body tissues of adult male bed bugs after ingestion of sterile blood or blood containing the Gram-positive (Gr+) bacterium Bacillus subtilis or the Gram-negative (Gr-) bacterium Escherichia coli. We analyzed mRNA from the bed bugs' midgut (the primary tissue involved in blood ingestion) and from the rest of their bodies (RoB; body minus head and midgut tissues). We show that the midgut exhibits a stronger immune response to ingestion of bacteria than the RoB, as indicated by the expression of genes encoding antimicrobial peptides (AMPs). Both the Toll and Imd signaling pathways, associated with immune responses, were highly activated by the ingestion of bacteria. Bacterial infection in bed bugs further provides evidence for metabolic reconfiguration and resource allocation in the bed bugs' midgut and RoB to promote production of AMPs. Our data suggest that infection with particular pathogens in bed bugs may be associated with altered metabolic pathways within the midgut and RoB that favors immune responses. We further show that multiple established cellular immune responses are preserved and are activated by the presence of specific pathogens. Our study provides a greater understanding of nuances in the immune responses of bed bugs towards pathogens that ultimately might contribute to novel bed bug control tactics.

Profiling lipidomic changes in dengue-resistant and dengue-susceptible strains of Colombian Aedes aegypti after dengue virus challenge.

The mosquito Aedes aegypti is the primary vector for all four serotypes of dengue viruses (DENV1-4), which infect millions across the globe each year. Traditional insecticide programs have been transiently effective at minimizing cases; however, insecticide resistance and habitat expansion have caused cases of DENV to surge over the last decade. There is an urgent need to develop novel vector control measures, but these are contingent on a detailed understanding of host-parasite interactions. Here, we have utilized lipidomics to survey the profiles of naturally DENV-resistant (Cali-MIB) or susceptible (Cali-S) populations of Ae. aegypti, isolated from Cali, Colombia, when fed on blood meals containing DENV. Control insects were fed on a DENV-free blood meal. Midguts were dissected from Cali-MIB and Cali-S females at three time points post-infectious blood meal, 18, 24 and 36h, to identify changes in the lipidome at key times associated with the entry, replication and exit of DENV from midgut cells. We used principal component analysis to visualize broad patterns in lipidomic profiles between the treatment groups, and significance analysis of microarray to determine lipids that were altered in response to viral challenge. These data can be used to identify molecules or metabolic pathways particular to the susceptible or refractory phenotypes, and possibly lead to the generation of stable, DENV-resistant strains of Ae. aegypti.

A Novel Model of Pathogenesis of Metarhizium anisopliae Propagules through the Midguts of Aedes aegypti Larvae.

We assessed the effect of the entomopathogenic fungus Metarhizium anisopliae against Aedes aegypti. Conidia of M. anisopliae strains CG 489, CG 153, and IBCB 481 were grown in Adamek medium under different conditions to improve blastospore production. Mosquito larvae were exposed to blastospores or conidia of the three fungal strains at 1 × 107 propagules mL-1. M. anisopliae IBCB 481 and CG 153 reduced larval survival by 100%, whereas CG 489 decreased survival by about 50%. Blastospores of M. anisopliae IBCB 481 had better results in lowering larval survival. M. anisopliae CG 489 and CG 153 reduced larval survival similarly. For histopathology (HP) and scanning electron microscopy (SEM), larvae were exposed to M. anisopliae CG 153 for 24 h or 48 h. SEM confirmed the presence of fungi in the digestive tract, while HP confirmed that propagules reached the hemocoel via the midgut, damaged the peritrophic matrix, caused rupture and atrophy of the intestinal mucosa, caused cytoplasmic disorganization of the enterocytes, and degraded the brush border. Furthermore, we report for the first time the potential of M. anisopliae IBCB 481 to kill Ae. aegypti larvae and methods to improve the production of blastospores.

Beauveria bassiana interacts with gut and hemocytes to manipulate Aedes aegypti immunity.

BACKGROUND: Mosquito-borne diseases affect millions of people. Chemical insecticides are currently employed against mosquitoes. However, many cases of insecticide resistance have been reported. Entomopathogenic fungi (EPF) have demonstrated potential as a bioinsecticide. Here, we assessed the invasion of the EPF Beauveria bassiana into Aedes aegypti larvae and changes in the activity of phenoloxidase (PO) as a proxy for the general activation of the insect innate immune system. In addition, other cellular and humoral responses were evaluated. METHODS: Larvae were exposed to blastospores or conidia of B. bassiana CG 206. After 24 and 48 h, scanning electron microscopy (SEM) was conducted on the larvae. The hemolymph was collected to determine changes in total hemocyte concentration (THC), the dynamics of hemocytes, and to observe hemocyte-fungus interactions. In addition, the larvae were macerated to assess the activity of PO using L-DOPA conversion, and the expression of antimicrobial peptides (AMPs) was measured using quantitative Real-Time PCR. RESULTS: Propagules invaded mosquitoes through the midgut, and blastopores were detected inside the hemocoel. Both propagules decreased the THC regardless of the time. By 24 h after exposure to conidia the percentage of granulocytes and oenocytoids increased while the prohemocytes decreased. By 48 h, the oenocytoid percentage increased significantly (P < 0.05) in larvae exposed to blastospores; however, the other hemocyte types did not change significantly. Regardless of the time, SEM revealed hemocytes adhering to, and nodulating, blastospores. For the larvae exposed to conidia, these interactions were observed only at 48 h. Irrespective of the propagule, the PO activity increased only at 48 h. At 24 h, cathepsin B was upregulated by infection with conidia, whereas both propagules resulted in a downregulation of cecropin and defensin A. At 48 h, blastospores and conidia increased the expression of defensin A suggesting this may be an essential AMP against EPF. CONCLUSION: By 24 h, B. bassiana CG 206 occluded the midgut, reduced THC, did not stimulate PO activity, and downregulated AMP expression in larvae, all of which allowed the fungus to impair the larvae to facilitate infection. Our data reports a complex interplay between Ae. aegypti larvae and B. bassiana CG 206 demonstrating how this fungus can infect, affect, and kill Ae. aegypti larvae.

Characterization of New Defensin Antimicrobial Peptides and Their Expression in Bed Bugs in Response to Bacterial Ingestion and Injection.

Common bed bugs, Cimex lectularius, can carry, but do not transmit, pathogens to the vertebrate hosts on which they feed. Some components of the innate immune system of bed bugs, such as antimicrobial peptides (AMPs), eliminate the pathogens. Here, we determined the molecular characteristics, structural properties, and phylogenetic relatedness of two new defensins (CL-defensin1 (XP_024085718.1), CL-defensin2 (XP_014240919.1)), and two new defensin isoforms (CL-defensin3a (XP_014240918.1), CL-defensin3b (XP_024083729.1)). The complete amino acid sequences of CL-defensin1, CL-defensin2, CL-defensin3a, and CL-defensin3b are strongly conserved, with only minor differences in their signal and pro-peptide regions. We used a combination of comparative transcriptomics and real-time quantitative PCR to evaluate the expression of these defensins in the midguts and the rest of the body of insects that had been injected with bacteria or had ingested blood containing the Gram-positive (Gr+) bacterium Bacillus subtilis and the Gram-negative (Gr-) bacterium Escherichia coli. We demonstrate, for the first time, sex-specific and immunization mode-specific upregulation of bed bug defensins in response to injection or ingestion of Gr+ or Gr- bacteria. Understanding the components, such as these defensins, of the bed bugs' innate immune systems in response to pathogens may help unravel why bed bugs do not transmit pathogens to vertebrates.

A fat body transcriptome analysis of the immune responses of Rhodnius prolixus to artificial infections with bacteria.

BACKGROUND: Rhodnius prolixus is an important vector of Trypanosoma cruzi, the causal agent of Chagas disease in humans. Despite the medical importance of this and other triatomine vectors, the study of their immune responses has been limited to a few molecular pathways and processes. Insect immunity studies were first described for holometabolous insects such as Drosophila melanogaster, and it was assumed that their immune responses were conserved in all insects. However, study of the immune responses of triatomines and other hemimetabolous insects has revealed discrepancies between these and the Drosophila model. METHODS: To expand our understanding of innate immune responses of triatomines to pathogens, we injected fifth instar nymphs of R. prolixus with the Gram-negative (Gr-) bacterium Enterobacter cloacae, the Gram-positive (Gr+) bacterium Staphylococcus aureus, or phosphate-buffered saline (PBS), and evaluated transcript expression in the fat body 8 and 24 h post-injection (hpi). We analyzed the differential expression of transcripts at each time point, and across time, for each treatment. RESULTS: At 8 hpi, the Gr- bacteria-injected group had a large number of differentially expressed (DE) transcripts, and most of the changes in transcript expression were maintained at 24 hpi. In the Gr+ bacteria treatment, few DE transcripts were detected at 8 hpi, but a large number of transcripts were DE at 24 hpi. Unexpectedly, the PBS control also had a large number of DE transcripts at 24 hpi. Very few DE transcripts were common to the different treatments and time points, indicating a high specificity of the immune responses of R. prolixus to different pathogens. Antimicrobial peptides known to be induced by the immune deficiency pathway were induced upon Gr- bacterial infection. Many transcripts of genes from the Toll pathway that are thought to participate in responses to Gr+ bacteria and fungi were induced by both bacteria and PBS treatment. Pathogen recognition receptors and serine protease cascade transcripts were also overexpressed after Gr- bacteria and PBS injections. Gr- injection also upregulated transcripts involved in the metabolism of tyrosine, a major substrate involved in the melanotic encapsulation response to pathogens. CONCLUSIONS: These results reveal time-dependent pathogen-specific regulation of immune responses in triatomines, and hint at strong interactions between the immune deficiency and Toll pathways.

Evolutionary history of orangutan plasmodia revealed by phylogenetic analysis of complete mtDNA genomes and new biogeographical divergence dating calibration models.

During the past 15 years, researchers have shown a renewed interest in the study of the Plasmodium parasites that infect orangutans. Most recently, studies examined the phylogenetic relationships and divergence dates of these parasites in orangutans using complete mitochondrial DNA genomes. Questions regarding the dating of these parasites, however, remain. In the present study, we provide a new calibration model for dating the origins of Plasmodium parasites in orangutans using a modified date range for the origin of macaques in Asia. Our Bayesian phylogenetic analyses of complete Plasmodium sp. mitochondrial DNA genomes inferred two clades of plasmodia in orangutans (Pongo 1 and Pongo 2), and that these clades likely represent the previously identified species Plasmodium pitheci and Plasmodium silvaticum. However, we cannot identify which Pongo clade is representative of the morphologically described species. The most recent common ancestor of both Pongo sp. plasmodia, Plasmodium. hylobati, and Plasmodium. inui dates to 3-3.16 million years ago (mya) (95% highest posterior density [HPD]: 2.09-4.08 mya). The Pongo 1 parasite diversified 0.33-0.36 mya (95% HPD: 0.12-0.63), while the Pongo 2 parasite diversified 1.15-1.22 mya (95% HPD: 0.63-1.82 mya). It now seems likely that the monkey Plasmodium (P. inui) is the result of a host switch event from the Pongo 2 parasite to sympatric monkeys, or P. hylobati. Our new estimates for the divergence of orangutan malaria parasites, and subsequent diversification, are all several hundred thousand years later than previous Bayesian estimates.

Time- and tissue-specific antimicrobial activity of the common bed bug in response to blood feeding and immune activation by bacterial injection.

Unlike almost all hematophagous insects, common bed bugs, Cimex lectularius, are not known to transmit pathogens to humans. To help unravel the reasons for their lack of vector competence, we studied the time- and tissue-dependent expression of innate immune factors after blood feeding or immune activation through the intrathoracic injection of bacteria. We used minimum inhibitory concentration (MIC1) bioassays and the Kirby-Bauer protocol to evaluate antimicrobial peptide (AMP2) activity in tissue extracts from the midguts or 'rest of body' (RoB3) tissues (containing hemolymph and fat body AMPs) against Gram-positive and Gram-negative bacteria. We compared AMP activity between blood-fed female bed bugs and yellow fever mosquitoes, Aedes aegypti and determined how female and male bed bugs respond to immune challenges, and how long AMP gene expression remains elevated in bed bugs following a blood meal. Blood meal-induced AMP activity is 4-fold stronger in female bed bugs than in female mosquitoes. Male bed bugs have elevated AMP activity within 8 h of a blood meal or an intrathoracic injection with bacteria, with the strongest activity expressed in RoB tissue 24 h after the immune challenge. Female bed bugs have a stronger immune response than males within 24 h of a blood meal. The effects of blood meal-induced elevated AMP activity lasts longer against the Gram-positive bacterium, Bacillus subtilis, than against the Gram-negative bacterium Escherichia coli. Unravelling the specific immune pathways that are activated in the bed bugs' immune responses and identifying the bed bug-unique AMPs might help determine why these insects are not vectors of human parasites.

Transcriptome comparison of dengue-susceptible and -resistant field derived strains of Colombian Aedes aegypti using RNA-sequencing.

BACKGROUND: Forty percent of the world's population live in areas where they are at risk from dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Dengue viruses are transmitted primarily by the mosquito Aedes aegypti. In Cali, Colombia, approximately 30% of field collected Ae. aegypti are naturally refractory to all four dengue serotypes. OBJECTIVES: Use RNA-sequencing to identify those genes that determine refractoriness in feral mosquitoes to dengue. This information can be used in gene editing strategies to reduce dengue transmission. METHODS: We employed a full factorial design, analyzing differential gene expression across time (24, 36 and 48 h post bloodmeal), feeding treatment (blood or blood + dengue-2) and strain (susceptible or refractory). Sequences were aligned to the reference Ae. aegypti genome for identification, assembled to visualize transcript structure, and analyzed for dynamic gene expression changes. A variety of clustering techniques was used to identify the differentially expressed genes. FINDINGS: We identified a subset of genes that likely assist dengue entry and replication in susceptible mosquitoes and contribute to vector competence. MAIN CONCLUSIONS: The differential expression of specific genes by refractory and susceptible mosquitoes could determine the phenotype, and may be used to in gene editing strategies to reduce dengue transmission.

Rhodnius prolixus uses the peptidoglycan recognition receptor rpPGRP-LC/LA to detect Gram-negative bacteria and activate the IMD pathway.

Insects rely on an innate immune system to recognize and eliminate pathogens. Key components of this system are highly conserved across all invertebrates. To detect pathogens, insects use Pattern recognition receptors (PRRs) that bind to signature motifs on the surface of pathogens called Pathogen Associated Molecular Patterns (PAMPs). In general, insects use peptidoglycan recognition proteins (PGRPs) in the Immune Deficiency (IMD) pathway to detect Gram-negative bacteria, and other PGRPs and Gram-negative binding proteins (GNBPs) in the Toll pathway to detect Gram-positive bacteria and fungi, although there is crosstalk and cooperation between these and other pathways. Once pathogens are recognized, these pathways activate the production of potent antimicrobial peptides (AMPs). Most PRRs in insects have been reported from genome sequencing initiatives but few have been characterized functionally. The initial studies on insect PRRs were done using established dipteran model organisms such as Drosophila melanogaster, but there are differences in the numbers and functional role of PRRs in different insects. Here we describe the genomic repertoire of PGRPs in Rhodnius prolixus, a hemimetabolous hemipteran vector of the parasite Trypanosoma cruzi that causes Chagas disease in humans. Using a de novo transcriptome from the fat body of immune activated insects, we found 5 genes encoding PGRPs. Phylogenetic analysis groups R. prolixus PGRPs with D. melanogaster PGRP-LA, which is involved in the IMD pathway in the respiratory tract. A single R. prolixus PGRP gene encodes isoforms that contain an intracellular region or motif (cryptic RIP Homotypic Interaction Motif-cRHIM) that is involved in the IMD signaling pathway in D. melanogaster. We characterized and silenced this gene using RNAi and show that the PGRPs that contain cRHIMs are involved in the recognition of Gram-negative bacteria, and activation of the IMD pathway in the fat body of R. prolixus, similar to the PGRP-LC of D. melanogaster. This is the first functional characterization of a PGRP containing a cRHIM motif that serves to activate the IMD pathway in a hemimetabolous insect.

Antifungal immune responses in mosquitoes (Diptera: Culicidae): A review.

Mosquitoes transmit many parasites and pathogens to humans that cause significant morbidity and mortality. As such, we are constantly looking for new methods to reduce mosquito populations, including the use of effective biological controls. Entomopathogenic fungi are excellent candidate biocontrol agents to control mosquitoes. Understanding the complex ecological, environmental, and molecular interactions between hosts and pathogens are essential to create novel, effective and safe biocontrol agents. Understanding how mosquitoes recognize and eliminate pathogens such as entomopathogenic fungi may allow us to create insect-order specific biocontrol agents to reduce pest populations. Here we summarize the current knowledge of fungal infection, colonization, development, and replication within mosquitoes and the innate immune responses of the mosquitoes towards the fungal pathogens, emphasizing those features required for an effective mosquito biocontrol agent.

Molecular relationships of introduced Aedes japonicus (Diptera: Culicidae) populations in British Columbia, Canada using mitochondrial DNA.

Aedes japonicus japonicus (Theobald) is a relatively recent immigrant to the Pacific Northwest, having been collected in Washington State in 2001 and in British Columbia (BC) since 2014. We applied a molecular barcoding approach to determine the phylogenetic relationship of Ae. j. japonicus populations in BC with those from around the world. We sequenced a 617 base-pair segment of the cytochrome c oxidase 1 gene and a 330 base-pair region of the NADH dehydrogenase 4 gene to find genetic variation and characterize phylogenetic and haplotypic relationships based on nucleotide divergences. Our results revealed low genetic diversity in the BC samples, suggesting that these populations arose from the same introduction event. However, our approach lacked the granularity to identify the exact country of origin of the Ae. j. japonicus collected in BC. Future efforts should focus on detecting and preventing new Ae. j. japonicus introductions, recognizing that current molecular techniques are unable to pin-point the precise source of an introduction.

The Role of Bacterial Symbionts in Triatomines: An Evolutionary Perspective.

Insects have established mutualistic symbiotic interactions with microorganisms that are beneficial to both host and symbiont. Many insects have exploited these symbioses to diversify and expand their ecological ranges. In the Hemiptera (i.e., aphids, cicadas, and true bugs), symbioses have established and evolved with obligatory essential microorganisms (primary symbionts) and with facultative beneficial symbionts (secondary symbionts). Primary symbionts are usually intracellular microorganisms found in insects with specialized diets such as obligate hematophagy or phytophagy. Most Heteroptera (true bugs), however, have gastrointestinal (GI) tract extracellular symbionts with functions analogous to primary endosymbionts. The triatomines, are vectors of the human parasite, Trypanosoma cruzi. A description of their small GI tract microbiota richness was based on a few culturable microorganisms first described almost a century ago. A growing literature describes more complex interactions between triatomines and bacteria with properties characteristic of both primary and secondary symbionts. In this review, we provide an evolutionary perspective of beneficial symbioses in the Hemiptera, illustrating the context that may drive the evolution of symbioses in triatomines. We highlight the diversity of the triatomine microbiota, bacterial taxa with potential to be beneficial symbionts, the unique characteristics of triatomine-bacteria symbioses, and the interactions among trypanosomes, microbiota, and triatomines.

The Midgut Microbiota of Colombian Aedes aegypti Populations with Different Levels of Resistance to the Insecticide Lambda-cyhalothrin.

Insecticide resistance in Aedes aegypti populations is a problem that hinders vector control and dengue prevention programs. In this study, we determined the susceptibility of Ae. aegypti populations from six Colombian regions to the pyrethroid lambda-cyhalothrin and evaluated the presence of the V1016I mutation in the sodium channel gene, which has been broadly involved in the resistance to this insecticide. The diversity of the gut microbiota of these mosquito populations was also analyzed. Only mosquitoes from Bello were susceptible to lambda-cyhalothrin and presented a lower allelic frequency of the V1016I mutation. Remarkably, there was not an important change in allelic frequencies among populations with different resistance ratios, indicating that other factors or mechanisms contributed to the resistant phenotype. Treatment of mosquitoes with antibiotics led us to hypothesize that the intestinal microbiota could contribute to the resistance to lambda-cyhalothrin. Beta diversity analysis showed significant differences in the species of bacteria present between susceptible and resistant populations. We identified 14 OTUs of bacteria that were unique in resistant mosquitoes. We propose that kdr mutations are important in the development of resistance to lambda-cyhalothrin at low insecticide concentrations but insect symbionts could play an essential role in the metabolization of pyrethroid insecticides at higher concentrations, contributing to the resistant phenotype in Ae. aegypti.

The impact of baculovirus challenge on immunity: The effect of dose and time after infection.

Understanding how hosts respond to pathogen attack is crucial to disease management. The response of a host can be particularly important if hosts have to defend against multiple pathogens which could either benefit from or be suppressed by prior pathogen exposure. Insect defence against viruses is less well understood than responses to other entomopathogens and much of the information available relates to in vitro studies and model systems. Baculoviruses are natural pathogens of insects, particularly Lepidoptera, and have been well-studied in terms of their ecology, pest control potential and molecular biology. In order to examine how an insect reacts to baculovirus challenge, we measured components of the cellular and humoral immune response of the cabbage looper Trichoplusia ni to Trichoplusia ni SNPV, a narrow-host range nucleopolyhedrovirus (NPV), over four doses and three times after pathogen challenge (18, 42 and 90 h). We found that total haemocyte numbers peaked at 42 h post-exposure at all doses, and declined linearly with increasing dose after the 18 h time point. Two immune-related enzymes, phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD), showed very different responses. PO levels were lowest at the 42 h time point and were not influenced by virus dose when each time point was examined separately. GLD levels declined over time but they interacted with virus dose in a non-linear manner, such that there was an increase in levels at intermediate virus doses after 18 h, no effect at 42 h, and then declined as infection progressed at 90 h post-infection. These data suggest that baculoviruses can rapidly infect haemocytes (or cause a reduction in their numbers) in a dose-dependent manner once the infection is systemic, likely reducing the ability of the host to counter subsequent infections. However, the data do not support a direct role for PO in defence against baculoviruses. Whether GLD plays a role in virus defence is still unclear.

Rhodnius prolixus: Identification of missing components of the IMD immune signaling pathway and functional characterization of its role in eliminating bacteria.

The innate immune system in insects is regulated by specific signalling pathways. Most immune related pathways were identified and characterized in holometabolous insects such as Drosophila melanogaster, and it was assumed they would be highly conserved in all insects. The hemimetabolous insect, Rhodnius prolixus, has served as a model to study basic insect physiology, but also is a major vector of the human parasite, Trypanosoma cruzi, that causes 10,000 deaths annually. The publication of the R. prolixus genome revealed that one of the main immune pathways, the Immune-deficiency pathway (IMD), was incomplete and probably non-functional, an observation shared with other hemimetabolous insects including the pea aphid (Acyrthosiphon pisum) and the bedbug (Cimex lectularius). It was proposed that the IMD pathway is inactive in R. prolixus as an adaptation to prevent eliminating beneficial symbiont gut bacteria. We used bioinformatic analyses based on reciprocal BLAST and HMM-profile searches to find orthologs for most of the "missing" elements of the IMD pathway and provide data that these are regulated in response to infection with Gram-negative bacteria. We used RNAi strategies to demonstrate the role of the IMD pathway in regulating the expression of specific antimicrobial peptides (AMPs) in the fat body of R. prolixus. The data indicate that the IMD pathway is present and active in R. prolixus, which opens up new avenues of research on R. prolixus-T. cruzi interactions.

The innate immune system of kissing bugs, vectors of chagas disease.

Kissing bugs have long served as models to study many aspects of insect physiology. They also serve as vectors for the parasite Trypanosoma cruzi that causes Chagas disease in humans. The overall success of insects is due, in part, to their ability to recognize parasites and pathogens as non-self and to eliminate them using their innate immune system. This immune system comprises physical barriers, cellular responses (phagocytosis, nodulation and encapsulation), and humoral factors (antimicrobial peptides and the prophenoloxidase cascade). Trypanosoma cruzi survives solely in the gastrointestinal (GI) tract of the vector; if it migrates to the hemocoel it is eliminated. Kissing bugs may not mount a vigorous immune response in the GI tract to avoid eliminating obligate symbiotic microbes on which they rely for survival. Here we describe the current knowledge of innate immunity in kissing bugs and new opportunities using genomic and transcriptomic approaches to study the complex triatomine-trypanosome-microbiome interactions.

Glycosylation on proteins of the intestine and perimicrovillar membrane of Triatoma (Meccus) pallidipennis, under different feeding conditions.

Trypanosoma cruzi, the causative agent of Chagas disease, interacts with molecules in the midgut of its insect vector to multiply and reach the infective stage. Many studies suggest that the parasite binds to midgut-specific glycans. We identified several glycoproteins expressed in the intestine and perimicrovillar membrane (PMM) of Triatoma (Meccus) pallidipennis under different feeding conditions. In order to assess changes in protein-linked glycans, we performed lectin and immunoblot analyses on glycoprotein extracts from these intestinal tissues using well-characterized lectins, and an antibody, which collectively recognize a wide range of different glycans epitopes. We observed that the amount and composition of proteins and glycoproteins associated with different glycans structures changed over time in the intestines and PMM under different physiological conditions. PMM extracts contained a wide variety of glycoproteins with different sugar residues, including abundant high-mannose and complex sialylated glycans. We propose that these molecules could be involved in the process of parasite-vector interactions.

Immune response-related genes associated to blocking midgut dengue virus infection in Aedes aegypti strains that differ in susceptibility.

Aedes (Stegomyia) aegypti, the principal global vector of dengue viruses, has differences in its susceptibility to dengue virus infection. We compared the global expression of genes in the midguts of Colombian Ae. aegypti dengue-susceptible (Cali-S) and dengue-refractory (Cali-MIB) field derived strains after ingesting either a sugarmeal, a bloodmeal, or a bloodmeal containing dengue virus serotype 2 (DENV-2). Microarray-based transcriptome analysis among treatments indicated a total of 4725 transcripts with differential expression between the two strains. Eleven genes were selected from different functional groups based on their significant up or down expression levels as well as reports in the literature suggesting they are associated with dengue virus elimination. We measured mRNA abundance of these 11 genes at 0, 8, 24, and 36 h postinfection using quantitative real time PCR (qPCR) to confirm the microarray results and assess any temporal patterns. Four genes were selected (Gram-negative binding protein-GNBP [AAEL009176], Niemann Pick Type-C2-NPC2 [AAEL015136], Keratinocyte lectin [AAEL009842], and Cathepsin-b [AAEL007585]) for knockdown experiments using RNA interference (RNAi) methodology to determine the phenotype (DENV-2 susceptible or refractory). Silencing GNBP, Cathepsin-b and Keratinocyte lectin reduced the percentage of mosquitoes with disseminated virus in the Cali-S strain to 8%, 20%, and 12% respectively compared with 96% in the controls. Silencing of NPC2 increased the percentage of mosquitos with disseminated virus infections in Cali-MIB to 66% compared with 35% in the controls. This study provides insight into genes that may contribute to the Cali-S susceptible and Cali-MIB refractory phenotypes in Ae. aegypti.