Bidirectional Microbiome-Gut-Brain-Axis Communication Influences Metabolic Switch-Associated Responses in the Mosquito Anopheles culicifacies.

The periodic ingestion of a protein-rich blood meal by adult female mosquitoes causes a drastic metabolic change in their innate physiological status, which is referred to as a 'metabolic switch'. While understanding the neural circuits for host-seeking is modestly attended, how the gut 'metabolic switch' modulates brain functions, and resilience to physiological homeostasis, remains unexplored. Here, through a comparative brain RNA-Seq study, we demonstrate that the protein-rich diet induces the expression of brain transcripts related to mitochondrial function and energy metabolism, possibly causing a shift in the brain's engagement to manage organismal homeostasis. A dynamic mRNA expression pattern of neuro-signaling and neuro-modulatory genes in both the gut and brain likely establishes an active gut-brain communication. The disruption of this communication through decapitation does not affect the modulation of the neuro-modulator receptor genes in the gut. In parallel, an unusual and paramount shift in the level of neurotransmitters (NTs), from the brain to the gut after blood feeding, further supports the idea of the gut's ability to serve as a 'second brain'. After blood-feeding, a moderate enrichment of the gut microbial population, and altered immunity in the gut of histamine receptor-silenced mosquitoes, provide initial evidence that the gut-microbiome plays a crucial role in gut-brain-axis communication. Finally, a comparative metagenomics evaluation of the gut microbiome highlighted that blood-feeding enriches the family members of the Morganellaceae and Pseudomonadaceae bacterial communities. The notable observation of a rapid proliferation of Pseudomonas bacterial sp. and tryptophan enrichment in the gut correlates with the suppression of appetite after blood-feeding. Additionally, altered NTs dynamics of naïve and aseptic mosquitoes provide further evidence that gut-endosymbionts are key modulators for the synthesis of major neuroactive molecules. Our data establish a new conceptual understanding of microbiome-gut-brain-axis communication in mosquitoes.

Symbiotic Bacteria as Potential Agents for Mosquito Control.

Xenorhabdus and Photorhabdus species are symbiotic bacteria of the insect-pathogenic soil nematodes that produce insecticidal compounds lethal to prey insects. Recently, there has been much interest in adapting these insecticidals for mosquito control. Here, I advocate the potential of Xenorhabdus/Photorhabdus as natural sources of mosquitocides (larvicides, adulticides) and feeding-deterrents.

Bacteria: A novel source for potent mosquito feeding-deterrents.

Antibiotic and insecticidal bioactivities of the extracellular secondary metabolites produced by entomopathogenic bacteria belonging to genus Xenorhabdus have been identified; however, their novel applications such as mosquito feeding-deterrence have not been reported. Here, we show that a mixture of compounds isolated from Xenorhabdus budapestensis in vitro cultures exhibits potent feeding-deterrent activity against three deadly mosquito vectors: Aedes aegypti, Anopheles gambiae, and Culex pipiens. We demonstrate that the deterrent active fraction isolated from replicate bacterial cultures is highly enriched in two compounds consistent with the previously described fabclavines, strongly suggesting that these are the molecular species responsible for feeding-deterrence. The mosquito feeding-deterrent activity in the putative fabclavine-rich fraction is comparable to or better than that of N,N-diethyl-3-methylbenzamide (also known as DEET) or picaridin in side-by-side assays. These findings lay the groundwork for research into biologically derived, peptide-based, low-molecular weight compounds isolated from bacteria for exploitation as mosquito repellents and feeding-deterrents.

Host plant induced variation in gut bacteria of Helicoverpa armigera.

Helicoverpa are important polyphagous agricultural insect pests and they have a worldwide distribution. In this study, we report the bacterial community structure in the midgut of fifth instar larvae of Helicoverpa armigera, a species prevalent in the India, China, South Asia, South East Asia, Southern & Eastern Africa and Australia. Using culturable techniques, we isolated and identified members of Bacillus firmus, Bacillus niabense, Paenibacillus jamilae, Cellulomonas variformis, Acinetobacter schindleri, Micrococcus yunnanesis, Enterobacter sp., and Enterococcus cassiliflavus in insect samples collected from host plants grown in different parts of India. Besides these the presence of Sphingomonas, Ralstonia, Delftia, Paracoccus and Bacteriodetes was determined by culture independent molecular analysis. We found that Enterobacter and Enterococcus were universally present in all our Helicoverpa samples collected from different crops and in different parts of India. The bacterial diversity varied greatly among insects that were from different host plants than those from the same host plant of different locations. This result suggested that the type of host plant greatly influences the midgut bacterial diversity of H. armigera, more than the location of the host plant. On further analyzing the leaf from which the larva was collected, it was found that the H. armigera midgut bacterial community was similar to that of the leaf phyllosphere. This finding indicates that the bacterial flora of the larval midgut is influenced by the leaf surface bacterial community of the crop on which it feeds. Additionally, we found that laboratory made media or the artificial diet is a poor bacterial source for these insects compared to a natural diet of crop plant.

Investigations on the role of a lysozyme from the malaria vector Anopheles dirus during malaria parasite development.

A cDNA encoding a lysozyme was obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) from females of the malaria vector Anopheles dirus A (Diptera: Culicidae). The 623 bp lysozyme (AdLys c-1) cDNA encodes the 120 amino acid mature protein with a predicted molecular mass of 13.4 kDa and theoretical pI of 8.45. Six cysteine residues and a potential calcium binding motif that are present in AdLys c-1 are highly conserved relative to those of c-type lysozymes found in other insects. RT-PCR analysis of the AdLys c-1 transcript revealed its presence at high levels in the salivary glands both in larval and adult stages and in the larval caecum. dsRNA mediated gene knockdown experiments were conducted to examine the potential role of this lysozyme during Plasmodium berghei infection. Silencing of AdLys c-1 resulted in a significant reduction in the number of oocysts as compared to control dsGFP injected mosquitoes.

A new role for an old antimicrobial: lysozyme c-1 can function to protect malaria parasites in Anopheles mosquitoes.

BACKGROUND: Plasmodium requires an obligatory life stage in its mosquito host. The parasites encounter a number of insults while journeying through this host and have developed mechanisms to avoid host defenses. Lysozymes are a family of important antimicrobial immune effectors produced by mosquitoes in response to microbial challenge. METHODOLOGY/PRINCIPAL FINDINGS: A mosquito lysozyme was identified as a protective agonist for Plasmodium. Immunohistochemical analyses demonstrated that Anopheles gambiae lysozyme c-1 binds to oocysts of Plasmodium berghei and Plasmodium falciparum at 2 and 5 days after infection. Similar results were observed with Anopheles stephensi and P. falciparum, suggesting wide occurrence of this phenomenon across parasite and vector species. Lysozyme c-1 did not bind to cultured ookinetes nor did recombinant lysozyme c-1 affect ookinete viability. dsRNA-mediated silencing of LYSC-1 in Anopheles gambiae significantly reduced the intensity and the prevalence of Plasmodium berghei infection. We conclude that this host antibacterial protein directly interacts with and facilitates development of Plasmodium oocysts within the mosquito. CONCLUSIONS/SIGNIFICANCE: This work identifies mosquito lysozyme c-1 as a positive mediator of Plasmodium development as its reduction reduces parasite load in the mosquito host. These findings improve our understanding of parasite development and provide a novel target to interrupt parasite transmission to human hosts.

Expression profile of prophenoloxidase-encoding (acppo6) gene of Plasmodium vivax-refractory strain of Anopheles culicifacies.

Anopheles culicifacies is the main vector for transmission of Plasmodium vivax malaria in the Indian subcontinent. A strain of An. culicifacies isolated from its natural niche displayed complete refractoriness to P. vivax by melanotic encapsulation of ookinetes. Prophenoloxidases are key components of the phenoloxidase cascade that leads to recognition and melanization of invading organisms. We isolated and cloned prophenoloxidase-encoding acppo6 gene of An. culicifacies and analyzed its expression profile under various regimens of immune challenge. The acppo6 was differentially expressed during various stages of larval development. The acppo6 transcription was also up-regulated in response to bacteria and Plasmodium vinckei petteri challenge. The transcript levels of the acppo6 gene were higher in naive adult refractory female mosquitoes as compared with female susceptible mosquitoes. Furthermore, the induction of acppo6 in the susceptible strain upon Plasmodium infection was negligible as compared with that of the refractory strain. The observation is suggestive of the role of acppo6 in effectuating a melanotic response in Plasmodium-incompetent naturally occurring refractory An. culicifacies strain.

Characterization of expression, activity and role in antibacterial immunity of Anopheles gambiae lysozyme c-1.

There are eight lysozyme genes in the Anopheles gambiae genome. Transcripts of one of these genes, LYSC-1, increased in Anopheles gambiae cell line 4a3B by 24 h after exposure to heat-killed Micrococcus luteus. Lysozyme activity was also identified in conditioned media from the cell line from which the protein was purified to homogeneity using ion exchange and gel filtration. Mass spectrometric analysis of the purified protein showed 100% identity to lysozyme c-1. Purified lysozyme c-1 was tested against non-mosquito-derived as well as culturable bacteria isolated from mosquito midguts. Lysozyme c-1 had negligible effects on the growth of most mosquito-derived bacteria in vitro but did inhibit the growth of M. luteus. Although Lys c-1 did not directly kill most bacteria, knockdown of LYSC-1 resulted in significant mortality in mosquitoes subjected to hemocoelic infections with Escherichia coli but not M. luteus thus suggesting that this protein plays an important role in antibacterial defense against selected bacteria.

Elucidating the biosynthesis of chitin filaments and their configuration with specific proteins and electron microscopy.

To deepen the knowledge of chitin synthesis, a yeast mutant has been used as a model. Purified chitin synthase I-containing vesicles (chitosomes) with a diameter of 85 to 120 nm are identified by electron microscopy to eject tiny fibers upon addition of UDP-N-acetylglucosamine. The filigree of extruded filaments fused gradually into a large three-dimensional network, which is degradable by a chitinase. The network is targeted and restructured by the Streptomyces chitin-binding protein CHB1, which has a very high affinity only for alpha-chitin. Within the chitosomes, filaments are found to be highly condensed within consecutive oval fibroids, which are specifically targeted by the alpha-chitin-binding protein. The presented data give new insights to the generation of chitin filaments with an antiparallel (alpha) configuration. [image: see text]