No abstract

Mosquitoes have evolved an effective innate immune system. The mosquito gut accommodates various microbes, which play a crucial role in shaping the mosquito immune system during evolution. The resident bacteria in the gut microbiota play an essential role in priming basal immunity. In this study, we show that antibacterial immunity in Anopheles gambiae can be enhanced by priming via a sugar meal supplemented with bacteria. Serratia fonticola S1 and Enterobacter sp. Ag1 are gut bacteria in mosquitoes. The intrathoracic injection of the two bacteria can result in an acute hemocoelic infection in the naïve mosquitoes with mortality of ∼40% at 24 h post-infection. However, the Enterobacter orSerratia primed mosquitoes showed a better 24 h survival upon the bacterial challenge. The priming confers the protection with a certain degree of specificity, the Enterobacter primed mosquitoes had a better survival upon the Enterobacter but not Serratia challenge, and the Serratia primed mosquitoes had a better survival upon the Serratia but not Enterobacter challenge. To understand the priming-mediated immune enhancement, the transcriptomes were characterized in the mosquitoes of priming as well as priming plus challenges. The RNA-seq was conducted to profile 10 transcriptomes including three samples of priming conditions (native microbiota, Serratia priming, and Enterobacter priming), six samples of priming plus challenges with the two bacteria, and one sample of injury control. The three priming regimes resulted in distinctive transcriptomic profiles with about 60% of genes affected by both bacteria. Upon challenges, different primed mosquitoes displayed different transcriptomic patterns in response to different bacteria. When a primed cohort was challenged with a heterogenous bacterium, more responsive genes were observed than when challenged with a homogenous bacterium. As expected, many canonical immune genes were responsive to the priming and challenge, but much more non-immune genes with various functions were also responsive in the contexts, which implies that the prior priming triggers a delicately coordinated systemic regulation that results in an enhanced immunity against the subsequent challenge. Besides the participation of typical immune pathways, the transcriptome data suggest the involvement of lysosome and metabolism in the context. Overall, this study demonstrated a trained immunity via priming with bacteria in diet.

21 22 23 24 25 26 2 ABSTRACT 27Advances in infectious disease control strategies through genetic manipulation of insect 28 microbiomes have heightened interest in microbially produced small molecules within 29 mosquitoes. Herein, 33 mosquito-associated bacterial genomes were mined and over 700 30 putative biosynthetic gene clusters (BGCs) were identified, 135 of which belong to known 31 classes of BGCs. After an in-depth analysis of the 135 BGCs, iron-binding siderophores 32 were chosen for further investigation due to their high abundance and well-characterized 33 bioactivities. Through various metabolomic strategies, eight siderophore scaffolds were 34 identified in six strains of mosquito-associated bacteria. Among these, serratiochelin A 35 and pyochelin were found to reduce female Anopheles gambiae overall fecundity likely by 36 lowering their blood feeding rate. Serratiochelin A and pyochelin were further found to 37 inhibit the Plasmodium parasite asexual blood and liver stages in vitro. Our work supplies 38 a bioinformatic resource for future mosquito microbiome studies and highlights an 39 understudied source of bioactive small molecules. 40 41 KEYWORDS 42 Mosquito-Microbiome; Biosynthetic Gene Clusters; Siderophores; Anopheles; 43 Plasmodium 44 45 46 100 transmit avian malaria, P. gallinaceum (Alavi et al., 2003), as well as human infectious 101 agents including dengue virus, Zika virus, and others (2016). As a relevant disease vector,102 Aedes-associated bacteria were also included in our data set. Due to the limited amount 103 of genome sequencing data on mosquito-associated bacteria, especially when compared 104 5 to human microbiomes, our sample size is smaller than analogous studies (Aleti et al., 105 2019; Donia et al., 2014; Helfrich et al., 2018), however this enabled an in-depth 106 bioinformatic analysis. Despite this size, our data set well-represents the unique gut 107 microbiome of mosquitoes. Our previous metagenomic study demonstrated that within the 108 gut microbiota of field-caught A. gambiae mosquitoes four days post blood-meal, 5 109 bacterial genera (Serratia, Elizabethkingia, Acinetobacter, Enterobacter, and 110 Pseudomonas) constitute 84 % of the 16S rRNA reads (Wang et al., 2011), all of which 111 are represented within our sample set. 112 A phylogenetic tree comparing the 33 bacterial strains was generated (Figure 113 1). The best-represented bacterial phylum in our sample set was Proteobacteria (16 γ-114 Proteobacteria, 1 β-Proteobacteria, and 3 α-Proteobacteria), consistent with reports 115 showing Proteobacteria are often the overwhelming microbial phylum in the midgut of 116 field-caught Anopheles mosquitoes (Wang et al., 2011). Species in the Bacteroidetes 117 phylum, which include Elizabethkingia spp., were the second most recurrent group (4 118 Elizabethkingia spp. and 1 Sphingobacterium sp.) in our analysis. Elizabethkingia spp. are 119 well-established mosquito symbionts that are the dominant species within midguts 4-and 120 7-days post-blood meal (PBM) as well as 7-day...

In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracic injection, and Cas13a transcripts were detectable at least 10 days post-delivery. The target specific crRNA was synthesized in vitro using T7 RNA polymerase. The Cas13a plasmid and target crRNA can be delivered by intrathoracic injection together, or the Cas13a construct can be provided first, and then target crRNA can be given later when appropriate. The machinery was tested in two mosquito species. In Anopheles gambiae, vitellogenin gene was silenced by Cas13a/Vg-crRNA, which was accompanied by a significant reduction in egg production. In Aedes aegypti, the αand δ-subunits of COPI genes were silenced by Cas13a/crRNA, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. In addition to dsRNA or siRNA mediated RNA interference, the programmable CRISPR interference method offers an alternative to knock down genes in mosquitoes.

A decentralised, Secure, Peer-to-Peer Multi-Voting System on Ethereum Blockchain is a distributed ledger technology (DLT) that permits virtual votes to be transacted in a peer-to-peer decentralized network. Those transactions are validated and registered through every node of the network, so creating a transparent and immutable series of registered events whose truthfulness is supplied through a consensus protocol. Smart contract automates the execution of agreement that runs routinely as soon as the conditions are satisfied. Smart contract would not need any third parties consequently prevents time loss. By Eliminating the requirement for third parties, consequently, allows numerous processes to be extra efficient and economical. The system is secure, reliable, and anonymous. Smart contract is enforced for the Ethereum network using the Ethereum wallets and also the Solidity language. Users are capable of submit their votes immediately from their Ethereum wallets, and those transaction requests is handled with the consensus of each single Ethereum node. This creates a transparent environment for evoting. A lot of concerning efficiency of the peer-to-peer decentralized electoral system on Ethereum network along with application and the outcomes of implementation are provided in this paper. Keywords: Blockchain, Distributed Ledger Technology (DLT), Consensus Protocol, Smart Contracts, Ethereum, Solidity