Multigenerational expression of antimicrobial peptides in Aedes aegypti exposed to Metarhizium anisopliae: Is trans-generational immune priming involved?

We assessed, for the first time, a multigenerational expression of antimicrobial peptides (AMPs) in Aedes aegypti larvae exposed to the entomopathogenic fungus, Metarhizium anisopliae, and correlated it with a possible involvement in trans-generational immune priming (TGIP). Aedes aegypti larvae were first exposed to blastospores or conidia of M. anisopliae CG 489 for 24 and 48 h, and the relative expression of AMPs were measured using quantitative Real-Time PCR. A suspension of conidia was prepared, and two different survival tests were conducted with different larval generations (F0, F1, and F2). In the first bioassay, the survival curves of the three generations were conducted separately and compared with their respective control groups. In the other bioassay, the survival curves of the F0, F1, and F2 generations were compared simultaneously against a naïve group exposed to Tween 80. In both survival tests, the F0 generation was more susceptible to M. anisopliae than subsequent generations. For molecular analyses related to TGIP, F0, F1, and F2 larvae were exposed to conidia, and their expression of AMPs was compared with their control groups and a naïve group. There was no differential expression of cecropin, defensin A or cathepsin B between generations. Lysozyme C, however, showed an increase in expression across generations, suggesting a role in TGIP. These discoveries may help us develop biological insecticides against mosquito larvae based on entomopathogenic fungi.

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.

Expansion of viral genomes with viral protein genome linked copies.

Virus protein-linked genome (VPg) proteins are required for replication. VPgs are duplicated in a subset of RNA viruses however their roles are not fully understood and the extent of viral genomes containing VPg copies has not been investigated in detail. Here, we generated a novel bioinformatics approach to identify VPg sequences in viral genomes using hidden Markov models (HMM) based on alignments of dicistrovirus VPg sequences. From metagenomic datasets of dicistrovirus genomes, we identified 717 dicistrovirus genomes containing VPgs ranging from a single copy to 8 tandem copies. The VPgs are classified into nine distinct types based on their sequence and length. The VPg types but not VPg numbers per viral genome followed specific virus clades, thus suggesting VPgs co-evolved with viral genomes. We also identified VPg duplications in aquamavirus and mosavirus genomes. This study greatly expands the number of viral genomes that contain VPg copies and indicates that duplicated viral sequences are more widespread than anticipated.

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.

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.

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.

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.