Microbiome-Gut-Brain-Axis Communication In�uences 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.


Introduction
The brain is a privileged organ in shaping an animal's behavior from lower to higher taxa by guiding and managing the diverse nature of external and internal stimuli.While each and every behavior of any organism is nely orchestrated by multiple organs, it is the brain that directs and exchange decisionmaking actions to regulate distinct organs functions.Unlike human brain, which hosts billions of neurons, it is amazing to know how blood-feeding mosquitoes, having less than 1,00,000 neurons in their tiny brain, regulate diverse functions such as nding a suitable source for sugar feeding and bloodfeeding, searching for a mate-partner, and locating a proper oviposition site for egg-laying, etc. Decades of research highlights that the molecular interaction of olfactory-derived odorant-binding proteins (OBPs), their olfactory receptors (Ors), and environmental chemical cues (external cues) is central to shape these behaviors (Potter 2014;De et al. 2018b).Additionally, the innate physiological status of the mosquitoes such as satiated/starved, mated/unmated, nutritional status, gravid, or not, also account to the successful accomplishement of these behavioral activities (Das De and Dixit 2020).Thus, how the miniature brain of mosquitoes harmonizes internal and external cues and affects decision-making abilities, is yet unknown.
Upon locating a suitable vertebrate host, a positive feeding decision stimulates the salivary glands to facilitate rapid blood meal ingestion by the adult female mosquitoes, and temporarily arrest the olfactory actions until 30h of blood-feeding (De et al. 2018b).Though, a fully engorged female mosquito shows a dramatic suppression of host-seeking behavior until the eggs are laid (~ 72 h post blood meal) however, to accompany complex oviposition site nding behaviors, reactivation of the peripheral sensory system (30-40h post blood meal) is deemed necessary (Takken et al. 2001;Duvall et al. 2019).A recent study by Duvall et. al. indicates that activation of Neuropeptide Y (NPY) signaling is essential in the suppression of host-seeking behavior for several days after blood feeding (Duvall et al. 2019).However, we have limited knowledge on how the mosquito's brain regulates the binary behavioral switch responses (sugar to bloodfeeding), and maintain organismal physiology in blood-fed females.
Fast engorgement of the mosquito's gut with blood causes a drastic metabolic shift in the innate physiological status from sugar to a protein-rich diet, resulting in the alteration of cellular fuel sources.This 'metabolic switch' is expected to drive multiple organs' engagement to perform their respective functions, such as osmoregulation by Malpighian tubules, progressive blood meal digestion by the gut, nutrient mobilization, and activation of vitellogenesis in the fat body, and ovary development for egg maturation (Sanders et al. 2003 Duvall 2019).It is the central nervous system that ensures institutes inter-organ communication to manage the rapidly changing inner physiological activities.Several neuromodulators, such as neuropeptides, neurotransmitters, and neurohormones having a role in neuro-synaptic signal transmission and inter-organ communication, have been characterized in fruit y (Droujinine and Perrimon 2016; Liu and Jin 2017).However, a similar correlation between the gut metabolic switch and brain function modulation in mosquitoes is limited to the Aedes aegypti, where brain secreted Insulin-likepeptide 3 is reported to play a signi cant role in the regulation of blood meal digestion and egg development (Gulia-Nuss et al. 2011).Only a few recent genetic studies have suggested the key role of few neuropeptides e.g.Neuropeptide-Y, Short-neuropeptide F, and Allatostatin-A, and their receptors in the suppression of host-seeking and paternity enforcement in Aedes aegypti mosquitoes (Christ et Fadda et al. 2019).Furthermore, it is becoming increasingly evident in vertebrates that an enteric nervous system (vagus nerve), also referred to as 'Second Brain' (Mayer 2011), not only mediates cross-talk between the gut and the brain but also establishes a bi-directional communication via gut-endosymbionts.This nexus of communication among the microbiota-gut-vagus-brain axis is crucial for maintaining metabolic homeostasis, mood, and perception(Forsythe and Kunze 2013; Ridaura and Belkaid 2015; Fülling et al. 2019).Blood meal signi cantly modulates metabolic energy homeostasis in the mosquito gut, but how the gut's nutrient-sensing mechanism in uences brain function remains unknown (Lampe et al. 2019).Although blood-meal-induced gut-ora proliferation has been well demonstrated in mosquitoes (Romoli and Gendrin 2018), their neuromodulatory functions remain elusive.
Using a comprehensive RNA-Seq analysis of mosquito brain, coupled with extensive transcriptional pro ling of neuro-modulators, comparative metagenomics analysis, and LC/MS-based quantitative estimation of neurotransmitters, here we demonstrated that fast blood meal engorgement and gutmetabolic switching (i) boost the brain's energy-metabolism, which may likely in uence organismal homeostasis, and (ii) favor the rapid establishment of a bidirectional microbiome-gut-brain axis communication, where the gut may also serve as a secondary brain in the blood-fed mosquitoes.Our data suggest that this gut-brain-axis communication is crucial to guide and manage blood meal digestion, and egg development in Anopheles culicifacies mosquitoes, the dominant malaria vector in rural India.A strategy of impairing this communication could reveal an out-of-the-box technique to disrupt mosquito host-seeking and blood-feeding behavior.

Material And Methods
A technical overview of the current investigation was represented graphically in Fig. S1 (supporting information).

Mosquito rearing and maintenance
A cyclic colony of An. culicifacies mosquito, sibling species A was reared and maintained at 28 ± 2°C temperature and relative humidity of 80% in the central insectary facility of the ICMR-National Institute of Malaria Research.For routine rearing, adult female mosquitoes were fed on the rabbit.All protocols for rearing and maintenance of the mosquito culture were approved by the ethical committee of the institute.
RNA isolation and transcriptome sequencing analysis: For RNA-Seq analysis, the brain tissues were dissected from 0-1-day old, 30 min post-blood-fed, and 30 h post-blood-fed cold anesthetized An. culicifacies mosquitoes by decapitation of the heads followed by application of gentle pressure over the head to pull out the brain tissue from the head cuticle and were collected in Trizol reagent.Then, total RNA was extracted from the collected brain tissues (approximately 30 mosquitoes were pooled to form one single sample), and a double-stranded cDNA library for each set of naïve, 30min, and 30h post-bloodfed was prepared by a prior well-established PCR-based protocol (Sharma et al. 2015).For transcriptome sequencing, the Illumina MiSeq 2 X 150 paired-end library preparation protocol was followed.
The bioinformatics data analysis pipeline is shown in Fig S1 .Brie y, raw reads from each set were processed to remove the adaptors and low-quality bases (< 20).A de-novo clustering was used to build the nal contigs/transcripts dataset using CLC Genomics Workbench (V6.2) (31) with default parameters (contig length ≥ 200, Automatic word size: Yes, Perform Scaffolding: Yes, Mismatch cost: 2, Insertion cost: 3, Deletion cost: 3, length fraction: 0.5, Similarity fraction: 0.8).Finally, the assembled transcriptome was used for CDS prediction and annotation using transdecoder software and BLASTX at e-value 1e − 6 respectively.For a comprehensive differential gene expression analysis, we used the same protocol as mentioned previously (Sharma et al. 2015;De et al. 2018b).Additionally, to identify the differentially expressed genes associated with certain biological and molecular processes, we performed gene-list enrichment analysis using the Kobas 3.0 web server.The unique appearance of certain pathways in different brain samples was screened depending on the p-value (< 0.5).

PCR-based gene expression analysis
To establish the concept of the metabolic switch and inter-organ communication in mosquitoes, we targeted An. culicifacies brain, midgut, Malpighian tubule, and ovary tissues.The respective tissues were dissected and collected from both naïve sugar-fed and blood-fed mosquitoes originated from the same cohort at different time points.At rst, the tissues were collected from 5-6-day old 25-30 naïve sugar-fed adult female mosquitoes.Next, adult female mosquitoes from the same cohort were offered blood-meal by offering a live animal (rabbit), and the desired tissues were collected as per the technical design of the experiments.In general, the fully engorged females were separated and kept in a proper insectary condition and the tissues were collected at the selected time points of post-blood-meal (PBM) such as 5min PBM, 2h PBM, 8-10h PBM, 24-30h PBM, 48h PBM, and 72h PBM from 25-30 mosquitoes for tissuespeci c detailed expression analysis of the respective genes.The different tissues were pooled accordingly in Trizol and total RNA was extracted, followed by cDNA preparation.Differential gene expression analysis was performed using the normal RT-PCR and agarose gel electrophoresis protocols.The nal steps of PCR at 95 o C for 15 secs followed by 55 o C for 15 secs, and again 95 o C for 15 secs were completed before deriving a melting curve.Each experiment was performed in three independent biological replicates for a better evaluation of the relative expression.The actin or Rps7 gene was used as an internal control in all the experiments, and the relative quanti cation was analyzed by the 2 −ΔΔCt method(Livak and Schmittgen 2001), which was further statistically analyzed by applying the student 't' test and two-way ANOVA.The detailed list of primer sequences used in the study is mentioned in Table S1.

ROS determination assay of blood-fed mosquitoes brain
To unravel the origin of the oxidative stress response in the blood-fed brain, we performed reactive oxygen species (ROS) determination assay by incubating the brain tissue dissected from naïve and blood-fed mosquitoes with a 2 mM solution of the oxidant-sensitive uorophores, CM-H2DCFDA [5-(and-6)-chloromethyl-29,79-dichloro-dichloro uorescein diacetate, acetyl ester] (Sigma).After a 20-min incubation at room temperature in the dark, the brain tissues were washed thrice with PBS, and then transferred to a glass slide in a drop of PBS and checked the uorescence intensity at wavelength 490 nm under a uorescent microscope.

Antibiotic treatment of mosquitoes
To establish the concept of microbiome-gut-brain-axis communication, we disrupted the gut-commensal bacteria through antibiotic treatment.For the removal of gut bacteria, the pupae emerged in a washed and aseptic mosquito cage made up of muslin cloth.The antibiotic diet was provided to the newly emerged mosquitoes for 4-5days by mixing 10% sucrose solution with 10 µg of penicillinstreptomycin/ml and 15 µg gentamicin sulfate in it.To avoid any contamination, the antibiotic regimen was changed daily.After 4-5days of antibiotic treatment, blood-meal was provided to mosquitoes through the rabbit by maintaining proper sterile conditions such as (i) removed the extra hairs of rabbit pinnate/ears for easy access to blood meal, (ii) wipe the body of the rabbit with 70% ethanol, (iii) wipe the rabbit cage with 70% alcohol.

Decapitation Experiment
To test mosquito's gut ability to function as a second brain, we offered a blood meal to 5-6 days old naïve sugar-fed mosquitoes, and decapitated ~ 100 mosquitoes after one hour of blood-feeding.Next, the decapitated mosquitoes were securely kept back in the insectary for recovery.The head tissues were submerged in 1X PBS to avoid desiccation.As per the technical design, post decapitation, the percentage of mosquito's survival was recorded at different time points until we observed 100% mortality (mosquitoes that vibrate/move their legs or other body parts are considered as live and non-movable mosquitoes with visible shrinkage of the body parts at the respective time points are considered as dead).The brain and the gut tissues of surviving mosquitoes were dissected and collected at different time points for further gene expression analysis.

Sample processing and MS analysis for neurotransmitter quanti cation
For absolute quanti cation of neurotransmitters, mosquitoes were decapitated and brains pulled out from the head cuticle and quickly collected in an Eppendorf containing 50µl of 1% ascorbic acid and immediately freeze it.For each set, ~ 60-65 mosquito brains or guts were pooled in a single tube.All samples were stored at -80 o C until further use.Each sample was extracted with 3X volume of extraction solvent.Samples were vortexed and refrigerated for 10-15 minutes at 4°C.Samples were then subjected to sonication in a bath-type ultra-sonicator in pulses (twice, for 1 min each).Samples were then centrifuged at 14500 rpm for 5 mins at 4°C.The supernatants were separated and dried under a vacuum.
Dried samples were spiked with internal standards (ISTDs) and derivatized, cleaned up, and prepared for LC-MS injections as per the protocol described earlier (Natarajan et al. 2015).Brie y, Standards (STDs) were spiked in 200µl of extraction solvent (acidic acetone (0.1% FA) containing 0.5mM ascorbic acid) and dried under vacuum.ISTDs were spiked to dried STDs, followed by the addition of 80µL borate buffer (200mM, pH 8.8) containing 1mM ascorbic acid.To the above mixture 10µl of 0.1 N NaOH was added, followed by the addition of AQC (from 1 mg mL − 1 stock).Samples were incubated at 55°C for 10 min.The reaction was stopped by the addition of 500µL of acidic water (0.1% FA).The derivatized standards were cleaned-up using the RP-SPE cartridges using the previously optimized protocol (Ramesh and Brockmann 2019) (Natarajan et al. 2015): activation with methanol, equilibration with water (0.1% FA), loading of samples, washing (twice) with water (0.1% FA), and elution with acetonitrile: methanol (80:20) containing 2% FA.The eluate was dried under vacuum and reconstituted in 50µL of 0.5% acetonitrile.10µL of reconstituted standards were injected for UHPLC-MS/SRM analysis.
Data were acquired on a TSQ Vantage (triple stage quadrupole) mass spectrometer (Thermo Fisher Scienti c, San Jose, CA, USA) coupled with an Agilent 1290 In nity series UHPLC system (Agilent Technologies India Pvt.Ltd.).The UHPLC system was equipped with a column oven (set at 40°C) and a thermo-controller for maintaining the auto-sampler at 10°C.A C-18 column (2.1 × 100 mm, 1.8µm, Agilent, Inc.) was used to perform the separation.The mobile phase solvent A was 10mM ammonium acetate in water containing 0.1% formic acid, and solvent B was acetonitrile containing 0.1% formic acid.The gradient was optimized to get maximum separation (2% B at 0 min, 2% B at 3 min, 20% B at 20 min, 35% B at 25 min, 80% B at 25-27 min, 2% B at 27-35 min) at a ow rate of 200µL min − 1.The operating conditions were as follows: ionization mode: positive; spray voltage: 3700 V; capillary temperature: 270°C; source temperature: 80°C; sheath gas: 30 (arbitrary units); auxiliary gas: 10 (arbitrary units); collision gas: argon.Parent and product masses, S-lens voltages, and collision energies were used as per the previously optimized method (Natarajan et al. 2015; Ramesh and Brockmann 2019).
Metagenomics analysis & microbiome pro ling: For the metagenomics study, we collected gut from 3-4 days old sugar-fed adult female mosquitoes (n = 50).While for blood-fed mosquito gut samples, 3-4 days old adult female mosquitoes from the same cohort were provided blood-meal by offering a live animal (rabbit), and midguts were collected after 24-30 hrs of blood-feeding.Before dissection, the mosquitoes were surface sterilized with 70% ethanol for 1 min in the highly sterilized condition of the Laminar air ow, and dissected tissues were collected in 1X Saline Tris-EDTA (100 mM NaCl/10 mM Tris-HCl, pH 8.0/1 mM EDTA, pH 8.0) buffer.At least 50 whole guts either from naïve sugar-fed or blood-fed mosquitoes, originating from the same cohort were collected into the minimal volume (20µl) of sterile icecold 1X STE and whole DNA was extracted as described earlier (Sharma et al. 2020).The quality of extracted genomic DNA (gDNA) was checked by loading the 5µl aliquot on 1 % agarose gel under the condition of 110 V for 30 min. 1 µl of each sample was loaded in NanoDrop 8000 for determining the A 260/280 ratio.The DNA was quanti ed using the Qubit dsDNA BR Assay kit (Thermo Fisher Scienti c Inc.). 1 µl of each sample was used for determining concentration using Qubit® 2.0 Fluorometer.For the preparation of amplicon libraries, V3-V4 hyper-variable region primers were used according to the library preparation protocol for the 16S Metagenomic Sequencing.The library for the sequenced fragments was obtained as per the standard Illumina protocol.Corresponding gut metagenomics data has been deposited to public repository NCBI, and freely accessible under accession IDs: SRR12579422 (Ac-MG-SF); SRR12622557 (Ac-MG-BF).QIIME software was used for maximum-likelihood phylogeny inference (Kuczynski et al. 2011), generating OTUs for taxonomic identi cation and diversity estimation was performed using Megan software (Huson et al. 2007) (Table S2, Fig. S2).To validate the metagenomics data, the abundancy of the selected bacterial species was pro led through Real-Time PCR as described earlier (Sharma et al. 2020).

Hypothesis
Several independent studies highlighted the impact of age, sex, and circadian rhythm on the olfactory derived host-seeking behavioral properties of different mosquito species(De et al. 2018b; Omondi et al. 2019;Tallon et al. 2019).However, a holistic understanding that how a coordinated action of the neuroolfactory system in uences the host-seeking, and blood-feeding behavioral properties of the adult female mosquitoes, remains unresolved.Since the neuro-system is highly sensitive and versatile centre for chemical information exchange, we hypothesize that a minor change in the innate physiological status may have a strong impact on the mosquito's everyday life.Importantly, after blood meal ingestion, a drastic change in the innate physiological status of the gut metabolic machinery i.e. "gut metabolic switch", may trigger brain to drive the engagement of multiple organs.This unusual, and condtional modulation of brain action is crucial to successfully manage the complex process of gonotrophic cycle..Therefore, it is very much plausible to propose that fast engorgement of mosquito gut with blood meal may shift mosquitoes' brain functions from external communication to inter-organ management, such as (a) initiation of diuresis; b) nding a resting site for digestion of blood meal in the midgut; (c) distribution of amino acids, generated through the degradation of protein-rich blood meal; (d) active engagement of the fat body and ovary for egg maturation and life cycle maintenance (Fig. 1).We recently demonstrated that both mating and circadian rhythm have an important role in driving olfactory guided pre-blood-mealassociated behavioral properties in the aging adult female mosquito An. culicifacies (De et al. 2017(De et al. , 2018b;;Das De et al. 2018).Aligning to olfactory responses, here, we aimed to decode and establish a possible molecular correlation between brain and gut-metabolic switch, and desgined a similar RNA-Seq strategy (Fig. 1),, in the mosquito An. culicifacies.

Blood meal ingestion boosts the brain's energy metabolism
A comparative RNA-Seq data analysis of naïve sugar-fed, 30min, and 30h post-blood-fed mosquito's brain showed a gradual suppression of brain-speci c transcript abundance (Fig. 2a).Surprisingly, we also observed an exceptional enrichment of oxidation-reduction process associated transcripts in response to blood-feeding (Fig. 2b) (Table S3a, S3b).Though, we failed to detect any signal of oxidative stress in a 2mM solution of the oxidant-sensitive uorophores, CM-H2DCFDA (data are not shown), but we observed an enrichment of several mitochondrial activity proteins such as 2-oxoglutarate dehydrogenase, NADH dehydrogenase, glutathione peroxidase, etc.A comparative metabolic pathway prediction analysis further con rmed the exclusive induction of several unique pathways linked to (a) energy metabolism, (b) neurotransmitter synthesis, and (c) neurite outgrowth and synaptic transmission (Fig. 2c).Together these data indicated that blood meal-associated gut metabolic switch may trigger a "hyper energy" state, and alter the expression of neuro-modulatory factors in the mosquito brain.
To verify the above presumption, we pro led and compared the expression pattern of the PGC-1 gene (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), an important transcriptional coactivator that regulates genes involved in energy metabolism (Lin et al. 2005; Liang and Ward 2006; Austin and St-Pierre 2012).A persistent elevation of PGC-1 (P ≤ 0.009 at 8h PBM, P ≤ 0.007 at 30h PBM), and a parallel enrichment of glycolysis and TCA cycle gene pyruvate kinase (P ≤ 0.0176) and oxoglutarate dehydrogenase (P ≤ 0.0019) respectively, indicated an enhanced mitochondrial activity in the brain of blood-fed mosquitoes (Fig. 2d, e).Next, we tested whether the amino acids generated through blood meal digestion or trehalose, a non-reducing disaccharide, acts as raw material for the brain's energy metabolism.Although trehalose serves as a primary energy source in the insects' brain (Shukla et al. 2015; Mattila and Hietakangas 2017), we observed a sequential increment in the amino acid transporter (P ≤ 0.0515) as well as trehalose (P ≤ 0.0071) transporter genes in the blood-fed brain (Fig. 2f).Together, these data indicate that both amino acids and trehalose moities may synergistically communicate the nutritional signal to the brain for active management of multi-organ communication(Gulia-Nuss et al.

Spatial and temporal modulations of neuro-signaling in uences metabolic switch-associated physiological activities
In naive sugar-fed mosquitoes, olfactory guided neuro-signaling and the brain's energy consumption is optimal to drive external stimuli associated with routine behavioral events like ight, mating, and hostseeking.Recently, we have demonstrated that prior host-exposure sex, and circadian have a signi cant impact on the olfactory responses in the aging adult-female An. culicifacies mosquito (De et al. 2017, 2018b; Das De et al. 2018).Likewise, to test and evaluate the possible correlation of age with neuroregulation, we monitored the expression pro le of at least 14 neuro-modulatory genes in aging naïve adult female mosquito's brain (Fig. S3).A limited modulation of expression suggested that the rst exposure to the host may have an important role in cognitive learning, and blood-feeding behavioral adaptation in mosquitoes, though further studies are needed to clarify and establish this correlation.However, surprisingly, after blood feeding an increase in the brain's energy consumption, prompted us to test the functional correlation of the brain with gut metabolic switch activities.Here, we hypothesize that blood meal uptake may temporarily pause the external communication, and increased energy state possibly may favor the shifting of the brain's engagement for the maintenance of organismal homeostasis.Thus, we identi ed and shortlisted transcripts encoding proteins, likely involved in the key events of the synaptic signal transmission process, i.e., crucial for the brain's functioning (Fig. 3a).
Thereafter, we evaluated the blood-meal-associated transcriptional response of selected transcripts regulating either receptor-mediated neuronal or cellular signaling processes during synaptic transmission (Fig. 3a).Surprisingly, we observed a limited change in the expression of neurotransmitters and biogenic amine receptor genes such as serotonin receptor, dopamine receptor, octopamine receptor, and GABA receptor, etc. in response to blood-meal (Fig. 3b).While, on the contrary, cellular signal transduction proteins such as cGMP protein kinase, phospholipase C, GABA gated chloride channel, and serine-threonine protein kinase, exhibited a signi cant modulation in response to metabolic switch (Fig. 3c).Together, these ndings support the idea that a rapid blood meal ingestion may drive brain engagement to manage metabolic switch-associated activities and distant organs' function (Fig. 3d).

Innate physiological status differentially modulates tissue-speci c neuromodulators/receptors transcripts expression
To further validate and correlate brain-inter-organ communication, we monitored the temporal and spatial expression of at least 21 key genes (Table 1) having the blood-meal-associated function in their targeted tissue such as midgut (MG), ovary (Ov), and Malpighian tubules (MT).Notably, we observed a signi cant upregulation of ILP3(p < 0.0002), and also time-dependent modulation of other neuropeptides (Neuropeptide Y, Leukokinin) and neuro-hormones (OEH, DH44, and ARMAA) in the blood-fed mosquitoes brain (Fig. 4a, b, c).We correltes that a gradual induction of ILP3 synthesis and OEH secretion from the brain's neurosecretory cells may activate the ovaries for the synthesis of ecdysteroids to initiate the vitellogenesis process (Brown et

Malpighian tubule
Next, we asked how the dynamic changes of the neuromodulators in the blood-fed brain in uence distant organ responses, such as diuresis regulation by the Malpighian tubule, blood digestion process in the midgut, and oocyte maturation in the ovary.Transcriptional pro ling of selected neuropeptide, neurotransmitter receptor transcripts (Table 1) indicated that blood meal triggers an immediate and prolonged (~ 48h PBM) impact on the expression of the gut-neuro transcript (Fig. 4d).Parallel observation of an early induction (2h PBM) of serine threonine-protein kinase (MAPK activated protein kinase) and late expression of Akt kinase (48h PBM) in the ovary suggested a controlled regulation of the nutritional signaling pathway favors the vitellogenesis process ( Gut, the 'second brain' communicates the nutritional status through neurotransmitter synthesis In vertebrates and also in the fruit ies, it is well evident that effective communication between the gut and brain has a paramount effect in shaping optimal health(Mayer 2011; Fülling et al. 2019), but a very limited knowledge exists in the mosquitoes(Gulia-Nuss et al. 2011).Prolonged modulation of the neuromodulators expression in the blood-fed mosquitoes' gut invigorates us to presume the existence of bi-directional gut-brain axis communication.An enriched expression pattern of neurotransmitter receptor genes, even after decapitation, re ected that the gut may also perform neuro-modulatory actions independently (Fig. S3).To further establish a proof-of-concept, we followed LC/MS-based absolute quanti cation of different neurotransmitters (NT) and compared their levels in the brain as well as in the gut of naïve and blood-fed mosquitoes.
Our data revealed that in naïve sugar-fed mosquitoes, although the brain serves as the primary source of NT synthesis, the midgut also synthesizes a substantial amount of NTs (Fig. 5a).However, blood-feeding causes a drastic shift in the NTs level in the midgut than in the brain (Fig. b, c).Notably, we observed an unpredictable increase in most NTs except glutamic acid, tyrosine, and tyramine in the gut (Fig. 5c).
Whereas, the brain tissue showed a notable decrease in the majority of the NTs synthesis, except for histamine, tyrosine, and tryptophan (Fig. 5b).We also observed that tyrosine amino acid was exclusively induced in the brain after blood-feeding, but remained below the threshold level in the gut (Fig. 5b, c).Although our data support previous studies that in addition to the brain, the gut also serves as a major source of multiple neurotransmitters in vertebrates and fruit y(Mayer 2011; Solari et al. 2017), the mechanism of nutrition-dependent NTs modulation remains unclear.Especially, in mosquitoes our understanding of the complex nature of blood meal digestion and gut-brain axis communication is obscure.Thus, our unusual observation of a thousand-fold increase in the levels of histidine, serine, aspartic acid, and tryptophan in the blood-fed mosquito's gut emanated few key questions: 1) whether increased levels of amino acids in the gut during blood meal digestion may act as an NT? 2) Do bloodmeal-induced proliferation of the gut microbiota has any effect on NT dynamics?3) Do the gut endosymbionts of mosquitoes have any impact on gut-brain axis communication? (Fig. 5d).

Symbiotic gut ora in uences gut-brain axis communication
The mechanism of gut-brain axis communication in vertebrates primarily involves neuronal stimulation through the vagus nerve, where endosymbionts play key role in the regulation of the gut endocrine system, and associated biochemical pathways ( Therefore, to uncover the gut microbiome complexity, and establish their possible relations with neurotransmitter abundancy, we evaluated the nature and diversity of gut microbiome population dynamics alteration in response to blood-feeding.A comparative metagenomic analysis revealed that the naïve sugar-fed mosquito harbors 90% of the Enterobacteriaceae family of gram-negative gammaproteobacteria such as Enterobacter cloacae complex sp., Chonobacter sp., Escherichia coli; 6% Psedomonodales family of gram-negative gamma-proteobacteria such as (a) Acinetobacter sp.members e.g.Acinetobacter guillouiae, Acinetobacter iwo i, (b) Pseudomonas aeruginosa sp. group e.g.Pseudomonas alcaligenes, P. nitroreducens, P. veronii, P. stutzeri and P. viridi ava; and other bacterial family members of Bacteroidetes e.g.Flavobacteriacae -Chryseobacterium sp., Elizabethkingia meningospetica; beta-proteobacteria-Alcaligenaceae-Alcaligenes faecalis (Fig. 6/ Fig. S4a, b, c).Furthermore, we also observed that blood-feeding not only suppresses Enterobacteriaceae family member by 50%, but favors rapid proliferation of Pseudomonadales to 46% of the total community, where we observed dominant association of Pesudomonas sp., Acinetobacter johnsonii; Acinetobacter rhizosphaerae, and other members from Alpha-proteobacteria family such as Sphingobium sp., Gluconacetobacter diazotrophicus, Achromobacter sp., Sphingomonas azoti gens, Methylobacterium sp. as well as Beta-proteobacteria-Burkholderiales family members such as Acidovorex sp., Delftia ramlibacter; Janthinobacterium lividum (Fig. S4a, b, c).Our microbial pro ling data further suggested that blood meal signi cantly alters the abundance of the gram-negative bacteria such as Pseudomonas and Elizabethkingia (Fig. 6), compared to gram-positive e.g.Agromonas and Rubrobacter (Actiobacteria) (Fig. S5).
Although the correlation of microbiome-gut-brain axis communication in the blood-feeding mosquitoes is yet not fully established, however, we opined that amino-acids resulting from rapid digestion of proteinrich blood meal, and its metabolite products may serve as an additional potent source of neuromodulators(1999).Here, our observation of Enterobacteriaceae family member abundancy and low NTs level in the gut than the brain of naïve sugar-fed mosquitoes indicate the basal-level of gut-brain-axis communication is enough to maintain physiological homeostasis.However, a rapid proliferation of Pseudomonadales family members, and a multi-fold enrichment of NTs in the gut, while mild suppression of the majority of NTs in the brain except for Histamine, Tyrosine and Tryptophan of the blood feed mosquitoes suggests that members of Pseudomonas species, may likely have a neuromodulatory role in protein-rich diet-induced gut-brain-axis communication.
To further strengthen our hypothesis, we tested and evaluated the effect of gut ora removal on the neurotransmitters dynamics.We performed an absolute quanti cation of the potent neuroactive molecules, and compared their levels in the gut and brain of the naïve and antibiotic-treated mosquitoes.
A signi cant elevation of tryptophan and consequent downregulation of serotonin levels in both the gut and brain of aseptic non-blood fed mosquitoes (Fig. 7a, b), corroborate with the previous observations that depletion of microbial ora may signi cantly delimit the de-novo-synthesis of serotonin, resulting in increased tryptophan concentration in the gut and brain(O'Mahony et al. 2015).Additionally, we also observed that antibiotic treatment not only caused a notable increase in histidine and histamine levels in both the gut and brain, also favored an exclusive induction of Dopa, and signi cant enrichment of GABA in the gut of the aseptic mosquitoes (Fig. 7a, b).
Together, these data indicated that gut bacteria removal may also in uence the systemic level of amino acid concentration in naïve mosuitoes (Fig. 7a, b).
To understand how blood-feeding in uences gut-brain axis communication, we requanti ed and compared the level of the neurotransmitters of naïve and antibiotic-treated blood-fed mosquitoes.A similar pattern of NTs synthesis was observed in both naïve blood-fed and antibiotic-treated blood-fed mosquitoes, but the level of modulation gets heightened in antibiotic-treated blood-fed gut and brain (Fig. S7, Table S4).To further support the above observation, we also monitored and compared the expression patterns of neurotransmitter receptor genes (Glycine R, glutamate R, serotonin R, dopamine R), insulinlike-peptide, and one of the junction protein gene (lachesin) in the gut and brain of naïve vs. antibiotictreated mosquitoes (Fig. 7c).Consistent with NT quantitative data, the respective receptor genes also showed a signi cant difference in their abundance throughout the gut-brain axis.We also noticed a differential expression pattern of ILP3, ARMAA (Aromatic-L-amino-acid decarboxylase/serotonin synthesizing enzyme), and lachesin transcript between naïve and antibiotic-treated mosquitoes undergoing metabolic switch event (Fig. 7c).
With our current data, we propose that a bi-directional gut-brain axis communication may exist to manage complex gut immune-physiological responses via gut-microbiome association during the blood meal digestion process in gravid females.Although, it is yet to be established as on how this cross-talk directly in uences brain-speci c responses such as mood and cognition.
The mosquito brain maintains basal immunity The immune system plays a crucial role in maintaining brain health by protecting it from both external and internal stress (Aguilera et al. 2018).Since the central nervous system and the immune system are the most energy-consuming organs, we consider that the immune system may play an important role to overcome blood-meal-induced metabolic stress, such as oxidative stress, osmotic stress, and elevated levels of dietary heme molecules.To trace the possible linkage of the brain-immune function, we identi ed and cataloged a total of 913 immune transcripts from brain tissue transcriptome data (Fig. 8a).Among the 18 classi ed immune family proteins, autophagy, CLIP-domain serine proteases, and peroxidases were observed the most predominant, accounting for more than 50% of the total immune transcripts.Furthermore, a comparative transcript abundance analysis showed that blood meal may cause a moderate change in the immune transcript expression (Fig. 8b).Increased percentage of peroxidases and CLIP-domain serine protease transcripts in the blood-fed brain suggested that these immune transcripts may prevent brain tissue from oxidative stress-induced damage and facilitate its recovery (Fig. 8b).Further, functional analysis of the immune transcripts in the central nervous system may unravel the novel regulatory mechanism of the immune genes to maintain the brain in shape.

Discussion
Host-seeking and blood-feeding behavior evolution make it di cult to resolve the complexity of decision- ).Thus, our observation of a limited change in the neuro-modulatory genes expression in the mosquito An. culicifacies, suggests that cognitive learning and memory response towards host-attraction are poorly developed until mosquitoes are exposed to hosts.
Post-blood-feeding an exclusive induction of oxidation-reduction family proteins and a comparative pathway analysis predicts that blood meal may enhance the brain's energy metabolic activities.Shreds of evidence from Drosophila, vertebrates, and limited studies in mosquitoes also suggest that altered metabolic physiology in uences the cross-talk between the brain, and peripheral tissues for the maintenance of systemic energy homeostasis( increase the ROS level which could have a deleterious impact on neuro action.Our observation of the unique appearance of the pentose phosphate pathway and glutathione peroxidase transcript (Oxidationreduction category gene), along with the upregulation of CLIP-domain serine proteases and peroxidases immune transcripts may attribute to the scavenging of ROS generated due to enhanced mitochondrial activity.Moreover, blood-meal-induced expression of amino acid transporters and trehalose transporter indicated that both trehalose and amino acids may serve as a raw material for enhanced energy metabolism.Furthermore, we also observed a signi cant alteration of transcripts involved in intracellular signaling than neurotransmitter receptors in the blood-fed mosquitoes' brains.Taken together, we hypothesize that an internal nutritional stimulus may shift brain engagement from external communication to inter-organ management, which requires a rapid and continuous synaptic transmission, neurotransmitter recycling, and axo-dendritic transport, resulting in enhanced energy metabolism in the brain (Roh et al. 2016;Yellen 2018).

Neuromodulatory responses establish brain-distant organ communication
To support our hypothesis, we pro led a selected class of neuromodulators, neuropeptides, and neurohormones gene expression in the brain and correlated their impact on distant organs.
Corresponding to the innate physiological status, we observed a time-dependent change in the expression pattern of the respective transcripts in the brain, and other targeted tissues of mosquito such as midgut, Malpighian tubule, and ovaries.But, in turn, how these neuromodulatory responses reinforce brain action remains unknown.Recent studies in Drosophila suggest that leukokinin neuropeptide regulates protein diet-induced post-prandial sleep and minimized movement (Murphy et al. 2016).We also observed a transient increase in leukokinin, and its receptor gene in the brain, and sustained up-regulation of the leukokinin receptor gene in the gut till 30h of blood-feeding.These data support the idea that until the blood meal gets digested in the gut, the brain may undergo 'food coma' and restrict external communication, but may actively engage in managing inter-organ communications (through ILPs and other neuro-hormones e.g.DH44, OEH, etc.).Compared to the brain, signi cant modulation of neuromodulators, and sustained expression even in the gut of decapitated blood-fed mosquitoes, further suggested a specialized ability of the gut to serve as a "second brain" possibly to share and minimize the function of the primary brain (Mayer 2011).Taken together, we interpret that gut-metabolic-switching may favor the establishment of a bidirectional 'gut-brain-axis' communication in the gravid female mosquitoes, though the detailed molecular mechanism is yet to unravel.
Neurotransmitter signaling and microbiome alteration in uences Gut-brain-axis communications Neurotransmitters, including both biogenic amines and amino acids, are well-known endogenous chemicals, that in uence rapid inter-organ signal transmission and decision-making abilities (Holzer and Farzi 2014;Mittal et al. 2017).To clarify and establish a possible functional correlation between the gut metabolic switch and gut-brain axis communication, we quanti ed the levels of neurotransmitters secreted from both gut and brain tissues.When compared to the naïve sugar-fed status, an unusual and paramount shift from the brain to the gut was observed for almost all the neurotransmitter levels after blood feeding.A signi cant upregulation of aspartic acid, glutamic acid, histidine, and histamine levels in blood-fed mosquito gut and brain may be a consequence of the rapid degradation of protein-rich blood meal in the mosquito gut (Imamura et al. 1972).
Although the effect of tyrosine enrichment in the brain is intriguing, however, an undetectable level of tyrosine in the gut supports previous ndings that the scavenging of toxic tyrosine from the gut is essential for the safeguarding journey of blood-fed mosquitoes (Sterkel et al. 2016).A substantial body of literature also suggests that the biogenic amines such as dopamine and serotonin are the critical regulators of feeding, host-seeking, and cognitive functions (French et  ).Thus, it would be worth testing whether an increase in the precursor molecules of dopamine i.e. tyrosine, in the blood-fed mosquito's brain, improves the cognitive power of the mosquito's host-seeking and blood-feeding behavioral activities.Likewise, an enrichment of tryptophan, a precursor of serotonin, may favor the minimization of the host-seeking behavioral activities of gravid females (Fig. 5b) (Ngai et al. 2019).Additionally, ~ 25-fold upregulation of GABAergic neurotransmission upon blood-feeding in the midgut highlights its possible function in the regulation of innate immune response by activating the autophagy due to gut ora expansion (Fig. 5c) ).However, studies on mosquitoes' gut-symbionts are predominantly limited to their impact on parasite growth and their potentiality for para-transgenic approaches (Blumberg et al. 2015).
Our observation of a rapid proliferation of Pseudomonas bacterial sp. in the gut of blood-fed mosquitoes may likely due to increased consumption of dietary Tryptophan for the synthesis of serotonin, correlating a possible cause for the suppression of appetite (Fig. 6a, c & Fig. 5c) (Jenkins et al. 2016;Kaur et al. 2019).Additionally, a signi cant reduction of the excitatory neurotransmitters Glutamic acid and Aspartic acid in the brain may help to restrict the foraging behavior in gravid females (Taylor and Brown 1943).
However, intriguingly a parallel thousand-fold increase in aspartic acid in the gut is whether a result of gut-microbial metabolism and/or any correlation with gut-neuro-endocrine regulation for egg development remains uncertain.Previous biochemical characterization of Locust's vitellogenin protein showed that it carries high content of aspartic acid, glutamic acid, and leucine (Chen et al. 1978).An independent in-silico amino-acid composition analysis of mosquito An. culicifacies vitellogenin protein (AEO51020.1)also revealed a high content of aspartic acid (6.2%), glutamic acid (6.7%), phenylalanine (7.6%), and serine (8.7%).Furthermore, previous literature indicated that disruption of gut-microbiota by antibiotic treatment not only reduces the anti-Plasmodium immunity but also hinder the egg development in the blood-feeding mosquitoes (Gaio et al. 2011;Romoli and Gendrin 2018).Therefore, we correlate that blood-meal-induced gut-microbial metabolism and activation of the vitellogenesis process may sequester the majority of amino acids to nurture the eggs (Hansen et al. 2014).But, the remaining fraction of amino acids either serves as an energy reservoir in the fat body (Hansen et al. 2014) or functions as a neurotransmitter, possibly to maintain gut-brain-axis communication, though further studies are needed to prove these presumptions.
A noteworthy modulation of gut neurotransmitters reinforces us to test how the blood-meal induced rapid proliferation of gut ora also in uence gut-brain axis communications.We disrupted the gut symbionts by providing an antibiotic diet orally supplement to the newly emerged mosquitoes for 4-5 days, and observed a signi cant difference in the abundance of neurotransmitters in both the gut and the brain.
Surprisingly, we also noticed that aseptic adult female mosquitoes carry assertive feeding behavior towards a vertebrate host (personal observation).In animals, an earlier study showed that germ-free mice exhibited stress-induced altered behavioral response, which was restored after complete microbiota recolonization (Sudo et al. 2004).Studies further signify that the microbial antigens such as lipopolysaccharide (LPS) and lipoteichoic acids generated in response to antibiotic treatment elicit immune responses, and favors early development of the gut-brain axis communication via gut neuronal sensing (Mazzoli and Pessione 2016).In the mosquito An. stephensi, the antibiotic treatment also enhances the transcriptional responses of gut-immune peptides, but the effect on neuro-sensing remains unclari ed(De et al. 2018a).We interpret that a higher abundance of histamine in the brain and GABA in the gut of antibiotic-treated mosquitoes may be accountable for the enhanced host-seeking behavioral activities, either directly through neuro-stimulation or indirectly through the vagal pathway(Bushey et al.

2015; Chen et al. 2017a
).Furthermore, blood-feeding to aseptic mosquitoes resulted in a multi-fold upregulation of serine and glutamic acid suggesting a limited usage of the respective amino acids, in the lack of a microbial population (Fig. S7) (Table S4), which consumes crucial amino acids to synthesize the building blocks of bacterial cell wall components in the healthy blood-fed mosquitoes (Cava et

Conclusion
The current investigation provides a novel insight into how gut-metabolic-switch-induced transcriptional modulation shifts mosquito's brain engagement from external communication (pre-blood meal hostseeking and host selection) to manage inter-organ communication (post-blood meal physiological homeostasis) for the tness of the mosquitoes.Although, evidences are available on the physiological effects of the gut microbiota on whole-body function in health and disease [103], but role in gut-brain-axis communication is very limited in insects.To the best of our knowledge, our data provide initial evidence that correlates the potential role of gut endosymbionts in microbiome-gut-brain-axis communication in the mosquito.We believe our conceptual framework may be valuable to modify mosquitoes' olfactory perception and cognition through the alteration of gut bacteria, and hence for new vector control tool development.A proposed working to establish the correlation between the gut metabolic switch and brain functions in adult female mosquitoes.The behavior of any organism is a very complex event that needs tight coordination between the sensory and neuronal systems.After emergence from pupae, the dynamic changes in the neuro-olfactory system coordinate and regulate different behavioral activities such as mating, sugar feeding, and vertebrate host-seeking, etc.These pre-blood meal-associated behaviors are guided by external stimuli, followed by neuronal decision making.Once the female mosquitoes take a blood meal, their olfactory responses are temporarily arrested to minimize brain and environmental (external) communication.But blood-feeding causes a global change in the physiological homeostasis, and drives multiple tissues (midgut, Malpighian tubule, ovary, and fat body) engagement to manage the systemic equilibrium.Here, we hypothesize that an 'internal stimulus' of gut-metabolic-switch may modulate brain functions to ensure optimal inter-organ communication, at least for the rst 30h until blood meal digestion is completed in the gut.However, after 30-40h of blood-feeding reactivation of the olfactory system, restores olfactory-neuro co-ordination to perform the next level of behavioral activities, such as oviposition and initiation of the second gonotrophic cycle.Blue and red lines indicate the possible functional patterns of the olfactory system (OLF) and the brain, respectively.

Declarations
For relative gene expression analysis, SYBR Green qPCR master mix and Biorad CFX 96 Real-Time PCR machine was used.PCR cycle parameters involved an initial denaturation at 95 o C for 5 min, 40 cycles of 10 s at 95 o C, 15 s at 52 o C, and 22 s at 72 o C. Fluorescence readings were taken at 72 o C after each cycle.

Figures
Figures
(Omondi et al. 2019;Tallon et al. 2019ncides with the age-dependent gradual change in the relative abundance of gene transcripts and increase in sensitivity of the olfactory sensory neurons (OSNs), where older females are likely to be more responsive to salient human odorants, and hence active host-seeker than teneral young female mosquitoes(Omondi et al. 2019;Tallon et al. 2019 (Duvall et al. 201916)n hematophagous insects(Das De and Dixit 2020).Recently, Benjamin J. Matthews et.al., cataloged hundreds of genes that are differentially expressed in the blood-fed brain(Matthews et al. 2016), of which the brain-encoded neuropeptide Y has been suggested to play a crucial role in host-seeking suppression following blood feeding(Duvall et al. 2019).But, in-depth analysis of the gut-metabolic-switching, and modulation of brain function is unexplored.Our study attempts to establish a molecular relationship of gut-brain-axis (GBA) communication, and explore a possible functional correlation of gut-endosymbionts on neuro-transmitters dynamics in uencing GBA communication.Gut-metabolic switch modulates the brain's energy metabolism and functional engagementTo understand that how the engorgement of the gut with blood meal modulate brain functioning, we performed a comparative RNA-Seq analysis of naïve sugar-fed and blood-fed of adult female mosquitoes' brain in An. culicifacies.In contrast to the pre-blood meal olfactory responses, which are signi cantly in uenced by age/sex/circadian even in the absence of host-exposure(De et al. 2017, 2018b; Das De et al. 2018), we did not observe any signi cant alteration of neuro-modulator genes expression in the non-blood-fed (host-unexposed) aging An. culicifacies mosquitoes.