Microbiome-Gut-Brain-Axis Communication Influences Metabolic Switch in the Mosquito Anopheles Culicifacies

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 ‘metabolic switch. Although the down-regulation of olfactory factors is key to restrain host-attraction, how the gut ‘metabolic switch’ modulates brain functions, and resilience physiological homeostasis remains unexplored. Here we demonstrate that the protein-rich diet induces the expression of brain transcripts related to mitochondrial function and energy metabolism, possibly to cause a shift of the brain’s engagement to manage organismal homeostasis. A dynamic expression pattern of neuro-signalling and neuro-modulatory genes in both gut and brain, establishes an active brain-distant organ communication. Disruption of this comunication 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 the 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’. Finally, a comparative metagenomics evaluation of gut microbiome population dynamics, highlighted that blood-feeding not only suppresses Enterobacteriaceae family member by 50%, but favors rapid proliferation of Pseudomonadales to 46% of the total community. Notable obesrvation of a rapid proliferation of Pseudomonas bacterial sp. in the gut correlates a possible cause for the suppression of appetite after blood-feeding. Additionally, an altered NTs dynamics of naïve and aseptic mosquitoes provide the initial evidence that gut-endosymbionts are key modulators for the synthesis of major neuroactive molecules. Conclusion: Our data establish a new conceptual understanding of microbiome-gut-brain-axis communication in mosquitoes.Data deposition: Mosquito Brain RNAseq data are accessible under Accession IDs: SRR9853884 (Ac-Br-SF); SRR9853885 (Ac-Br-BF-30Min), SRR9853883 (Ac-Br-BF-30hrs) at NCBI repository. Mosquito Gut metagenomics data are accessible under accession IDs: SRR12579422 (Ac-MG-SF); SRR12622557 (Ac-MG-BF) at NCBI repository.

The oviposition cue indole inhibits animal-host attraction in Aedes aegypti mosquitoes

Mosquitoes represent a major source of disease transmission and possess the uncanny ability to locate suitable animal-hosts, a feature mediated by their exquisite olfactory system. Insect repellents such as N,N-Diethyl-meta-toluamide, also called DEET, have been shown to activate and inhibit mosquito odorant receptors resulting in behavioral modulation. This and other repellents available for personal protection against mosquitoes are topically applied on the skin and operate at short range. In our search for potential long-range odorant repellents, we have hypothesized that the shared chemical similarities between indole and DEET, may confer the former the ability to block odorant receptor function and inhibit human-host attraction. Using the two-electrode voltage clamp of Xenopus laevis oocytes as a pharmacological platform, we provide evidence that indole inhibits the Aedes aegypti (R)-1-octen-3-ol receptor OR8, a receptor involved in the decision of female mosquitoes to identify human hosts. Coincidentally, we also show that indole inhibits the animal-host seeking behavior of female Aedes aegypti. Together, our findings suggests that indole may be a candidate spatial repellent for the long-range protection of humans against mosquito bites.

Microbiome-Gut-Brain-Axis communication influences metabolic switch in the mosquito Anopheles culicifacies

Background: 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 ‘metabolic switch. Although the down-regulation of olfactory factors is key to restrain host-attraction, how the gut ‘metabolic switch’ modulates brain functions, and resilience physiological homeostasis remains unexplored. Methods: To uncover a possible correlation of gut metabolic switching and brain function, we carried out a comparative RNAseq analysis of naïve and blood-fed mosquito’s brain. Spatio-temporal expression of neuro-signaling and neuro-modulatory genes was monitored through Real-Time PCR. To establish a proof-of-concept, we followed LC/MS-based absolute quantification of different neurotransmitters (NT) and compared their levels in the brain as well as in the gut of the mosquitoes. To correlate how microbiome influences gut-brain-axis communication, we performed a comparative gut metagenomic analysis. Results: Our findings demonstrate that the protein-rich diet induces the expression of brain transcripts related to mitochondrial function and energy metabolism, possibly to cause a shift of the brain’s engagement to manage organismal homeostasis. A dynamic expression pattern of neuro-signaling and neuro-modulatory genes in both gut and brain, presumably a key to establish an active brain-distant organ communication. Disruption of this comunication 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 the 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’. Finally, a comparative metagenomics evaluation of gut microbiome population dynamics, highlighted that blood-feeding not only suppresses Enterobacteriaceae family member by 50%, but favors rapid proliferation of Pseudomonadales to 46% of the total community. Notable obesrvation of a rapid proliferation of Pseudomonas bacterial sp. in the gut correlates a possible cause for the suppression of appetite after blood-feeding. Additionally, an altered NTs dynamics of naïve and aseptic mosquitoes provide the initial evidence that gut-endosymbionts are key modulators for the synthesis of major neuroactive molecules. Conclusion: Our data establish a new conceptual understanding of microbiome-gut-brain-axis communication in mosquitoes.

Volatile allosteric antagonists of mosquito odorant receptors inhibit human-host attraction

Odorant-dependent behaviors in insects are triggered by the binding of odorant ligands to the variable subunits of heteromeric olfactory receptors. Previous studies have shown, however, that specific odor binding to ORco, the common subunit of odorant receptor heteromers, may allosterically alter olfactory receptor function and profoundly affect subsequent behavioral responses. Using an insect cell-based screening platform, we identify and characterize several antagonists of the odorant receptor co-receptor of the African malaria vector Anopheles gambiae (AgamORco) in a small collection of natural volatile organic compounds (VOCs). Because some of the identified antagonists were previously shown to strongly repel Anopheles and Culex mosquitoes, we examined the bioactivities of the identified antagonists against Aedes, the third major genus of the Culicidae family. The tested antagonists inhibited the function of Ae. aegypti ORco ex vivo and repelled adult Asian tiger mosquitoes (Ae. albopictus). Binary mixtures of specific antagonists elicited higher repellency than single antagonists, and binding competition assays suggested that this enhanced repellence is due to antagonist interaction with distinct ORco sites. Our results also suggest that the enhanced mosquito repellency by antagonist mixtures is due to additive rather than synergistic effects of the specific antagonist combinations on ORco function. Taken together, these findings provide novel insights concerning the molecular aspects of odorant receptor function. Moreover, our results demonstrate that a simple screening assay may be used for the identification of allosteric modifiers of olfactory-driven behaviors capable of providing enhanced personal protection against multiple mosquito-borne infectious diseases.

The Role of Antennae in Heat Detection and Feeding Behavior in the Bed Bug (Hemiptera: Cimicidae)

The common bed bug (Cimex lectularius L.) is an obligate hematophagous ectoparasite that has significant impacts on human health and well-being. All life stages of bed bugs (except eggs) feed solely on blood, which is required to molt and reproduce. Bed bugs use multiple cues to locate their hosts, including heat, CO2, and body odors. Of these cues, detection of heat appears limited to a short distance of <3 cm. However, it remains unclear if bed bugs can detect radiant heat, what structure(s) are responsible for heat detection, and if heat detection via the antennae is required for feeding. In this study, bed bug response to radiant heat was evaluated using the two-choice T-maze assay with the heat source either in contact with the surface (i.e., conduction) or not in contact (i.e., radiation) in nonantennectomized bed bugs. Further, we systematically ablated the bed bug’s antennal segments (distal tip, first segment, and all four segments) and assessed their responses to heat and feeding in a unique two-choice T-maze assay and individual feeding assays, respectively. Our two-choice assays with contact to or no contact with the surface indicated that bed bugs cannot detect radiant heat. Later, we found that the distal tip of the terminal antennal segment is responsible for orientation toward a heat source. However, >50% of the bed bugs fed even when the entire antenna was removed, suggesting redundancy in sensory cues that drive feeding. These results will be used to better understand the role heat plays in bed bug host attraction and design of traps.

The Screening of Resistance against Meloidogyne graminicola in Oats

Including pest resistance elements against the major local concern is naturally important in the breeding process. Oat (Avena sativa L.) has been recently reintroduced into Taiwan as a winter alternative forage crop, and its agronomic performance has been evaluated at different locations in the country. This study examined the resistance to root-knot nematode, Meloidogyne graminicola, in four oat (Avena sativa L.) breeding lines of mass planting potential for winter in Taiwan. The host attraction level to the nematode, and the penetration and reproduction ability of nematode towards host roots were evaluated by chemotaxis assay, root staining assay, root galling, and nematode extraction. Based on the gall index (GI) and multiplication factor (R), the resistance of each oat line was evaluated. At 24 h postinoculation, second-stage juvenile (J2) nematodes appeared most attracted by oat breeding lines UFRGS136104-3 and UFRGS136119-2. The number of J2s successfully penetrated into the two breeding lines were also high. However, at 40 days postinoculation, observation of the oats in the newly developed culture bag nematode-inoculation system revealed that the amount of root galls and 2nd generation nematodes were significantly higher in line LA08085BS-T2 than in other lines. In sum, oat breeding line UFRGS136104-3 was highly resistant to M. graminicola by inhibiting the gall formation and nematode reproduction, while UFRGS136106-3 and UFRGS136119-2 showed relatively weak resistance and oat line LA08085BS-T2 would be a moderately susceptible host to M. graminicola, with high numbers of root gall formation. The outcome of this study provides ground information for nematode-resistant oat cultivar breeding.

Salivary AsHPX12 influence pre-blood meal associated behavioral properties in the mosquito Anopheles stephensi

In the adult female mosquito, successful blood meal acquisition is accomplished by salivary glands, which releases a cocktail of proteins to counteract vertebrate host’s immune-homeostasis. However, the biological relevance of many salivary proteins remains unknown. Here, we characterize a salivary specific Heme peroxidase family member HPX12, originally identified from Plasmodium vivax infected salivary RNAseq data of the mosquito Anopheles stephensi. We demonstrate that dsRNA silencing mediated mRNA depletion of salivary AsHPX12 (80-90%), causes enhanced host attraction but reduced blood-meal acquisition abilities, by increasing probing propensity (31%), as well as probing time (100–200s, P<0.0001) as compared to control (35-90s) mosquitoes group. Altered expression of the salivary secretory and antennal proteins may account for an unusual fast release of salivary cocktail proteins, but the slowing acquisition of blood meal, possibly due to salivary homeostasis disruption of AsHPX12 silenced mosquitoes. A parallel transcriptional modulation in response to blood feeding and P. vivax infection, further establish a possible functional correlation of AsHPX12 role in salivary immune-physiology and Plasmodium sporozoites survival/transmission. We propose that salivary HPX12 may have a vital role in the management of ‘pre- and post’-blood meal associated physiological-homeostasis and parasite transmission.Graphical abstractFigure 1:Schematic representation of mosquito’s blood meal acquisition and upshot on blood-feeding after silencing of salivary gland HPX-12. (A) After landing over host skin, mosquito mouthparts (proboscis) actively engaged to search, probe, and pierce the skin followed by a rapid release of the pre-synthesized salivary cocktail, which counteracts the host homeostasis, inflammation, and immune responses, during blood meal uptake. (B) Silencing of HPX-12 disrupts salivary gland homeostasis, enhancing mosquito attraction, possibly by up-regulating odorant-binding proteins genes-OBP-7,10 and OBP-20 expression in the Olfactory System. However, HPX-12 disruption may also cause significant effects on pre-blood meal associated probing abilities, which may be due to fast down-regulation of salivary cocktail proteins such as Anopheline, Apyrase, D7L proteins.

Global warming and plant–pollinator mismatches

The mutualism between plants and their pollinators provides globally important ecosystem services, but it is likely to be disrupted by global warming that can cause mismatches between both halves of this interaction. In this review, we summarise the available evidence on (i) spatial or (ii) phenological shifts of one or both of the actors of this mutualism. While the occurrence of future spatial mismatches is predominantly theoretical and based on predictive models, there is growing empirical evidence of phenological mismatches occurring at the present day. Mismatches may also occur when pollinators and their host plants are still found together. These mismatches can arise due to (iii) morphological modifications and (iv) disruptions to host attraction and foraging behaviours, and it is expected that these mismatches will lead to novel community assemblages. Overall plant–pollinator interactions seem to be resilient biological networks, particularly because generalist species can buffer these changes due to their plastic behaviour. However, we currently lack information on where and why spatial mismatches do occur and how they impact the fitness of plants and pollinators, in order to fully assess if adaptive evolutionary changes can keep pace with global warming predictions.

Microbiome-Gut-Brain-Axis communication influences metabolic switch in the mosquito Anopheles culicifacies

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 ‘metabolic switch. Although the down-regulation of olfactory factors is key to restrain host-attraction, how the gut ‘metabolic switch’ modulates brain functions, and resilience physiological homeostasis remains unexplored. Here, we demonstrate that the protein-rich diet induces mitochondrial function and energy metabolism, possibly shifting the brain’s engagement to manage organismal homeostasis. A dynamic expression pattern of neuro-signaling and neuro-modulatory genes in both the brain and gut indicates an optimal brain-distant organ communication. Even after decapitation, significant modulation of the neuro-modulator receptor genes as well as quantitative estimation of neurotransmitters (NTs), together confer the gut’s ability to serve as a ‘second brain’. Finally, data on comparative metagenomic analysis and altered NTs dynamics of naïve and aseptic mosquitoes provide the initial evidence that gut-endosymbionts are key modulators for the synthesis of major neuroactive molecules. Conclusively, our data establish a new conceptual understanding of microbiome-gut-brain-axis communication in mosquitoes.Abstract FigureGraphical abstractHighlightsHighly proteinaceous blood meal uptake causes gut ‘metabolic switch’ activity in mosquitoes.Gut’s calling shifts the brain’s administrative function from external communication to inter-organ management.‘Gut’, as a ‘Second brain’ plays a crucial role in the maintenance of physiological homeostasis.Metabolic switch and proliferation of symbiotic bacteria establish microbiome-gut-brain axis communication in mosquitoes.

Bed Bug (Hemiptera: Cimicidae) Attraction to Human Odors: Validation of a Two-Choice Olfactometer

Bed bugs (Cimex lectularius L.) (Hemiptera: Cimicidae) are obligate hematophagous ectoparasites, and, therefore, must locate suitable hosts to ensure survival and reproduction. Their largely nocturnal activity suggests that chemosensory and thermosensory cues would play critical roles in host location. Yet, the importance of olfaction in host attraction of bed bugs remains unclear. We developed and validated a Y-tube, two-choice olfactometer and tested its suitability for investigating attraction to human odors (from skin swabs). Olfactometer orientation significantly affected the percentage of bed bugs that were activated by human odors, with significantly more bed bugs responding when the olfactometer was oriented vertically (bug introduced at bottom of the olfactometer) compared with all other orientations. Starved (7–10 d) adult males, mated females, and nymphs responded (47–77% moved up the olfactometer and made a choice) when human odors were present in the olfactometer, while starved, unmated females did not respond. Skin swabs from all five human participants elicited high response rates (65–82%), and bed bugs from four different populations responded to skin swabs (40–82% response rate). However, in all assays including those resulting in relatively low response rates, bed bugs exhibited &gt;90% preference for human odors over blank controls. These results provide strong evidence that bed bugs can respond and orient towards human odors, independently of all other host cues. Furthermore, the validated olfactometer should enable rapid and efficient evaluations of bed bug behavioral responses to semiochemicals.