Metagenomic profiling of antibiotic resistance and virulence removal: Activated sludge vs. algal wastewater treatment system.

Conventional activated sludge-based (CAS) wastewater treatment plants are known to be a source of antibiotic resistance genes (ARGs) and virulence genes (VGs). As an alternative, a single-step mixotrophic algal wastewater treatment (A-WWT) system is proposed here to effectively reduce ARGs and VGs in the final effluent while meeting all the discharge standards. In this study, we applied the metagenomic profiling approach to compare the A-WWT system against the CAS system in terms of removal efficacy of ARG and VGs. A total of 111 ARG and 93 VG subtypes belonging to 10 antibiotic resistant classes and 19 virulence classes were detected in this study. Although the CAS system reduced the relative abundance of most classes of ARGs (7 of 10) and VGs (11 of 19), 3 ARG classes and 7 VG classes had increased abundances. On the other hand, the A-WWT system reduced the relative abundance of all classes of ARGs and VGs, and effectively eliminated most subtypes of ARGs and VGs. In the CAS system, the bacterial genera carrying ARGs and VGs was expanded, and the diversity index was increased greatly, suggesting the occurrence of horizontal gene transfer (HGT). In contrast, the A-WWT system narrowed down the potential host range and decreased their diversity substantially. Results of this study highlight the potential risk of ARGs and VGs in CAS system and demonstrate the feasibility of the algal-based system in removing ARGs and VGs.

Historical survey of the kdr mutations in the populations of Anopheles sinensis in China in 1996-2014.

BACKGROUND: Anopheles sinensis has become an important malaria vector in China. The long-term extensive utilization of pyrethroids for ITNs and IRS for mosquito control in the last three decades has resulted in the occurrence of resistant An. sinensis populations in many regions. Knockdown resistance (kdr), caused by point mutations in the VGSC gene, is one of the mechanisms that confer resistance to DDT and pyrethroids. Recently, several investigations revealed the kdr occurrence in some An. sinensis populations, however, no kdr data were available earlier than 2009. A survey tracking the dynamics of the kdr mutations in past decades would provide invaluable information to understand how the kdr alleles spread in mosquito populations temporally and spatially. METHODS: A survey was conducted on the kdr alleles at condon 1014 of the VGSC gene and their distributions in 733 specimens of An. sinensis and 232 specimens of the other eight member species of the Anopheles hyrcanus group that were collected from 17 provinces in China in 1996-2014. RESULTS: A total of three kdr alleles, TTT (F), TTG (F) and TGT (C) were detected, and TGT (C) and TTT (F) were already present in the specimens from Jiangsu and Shandong as early as 1997. The TTT (F) was the most frequent mutant allele, and largely distributed in central China, namely Shandong, Jiangsu, Anhui, Henan, Shanghai, Jiangxi and Hubei. When data were analysed in three time intervals, 1996-2001, 2005-2009, 2010-2014, the prevalence of kdr alleles increased progressively over time in the populations in central China. In contrast, the kdr alleles were less frequent in the samples from other regions, especially in Yunnan and Hainan, despite the documented presence of pyrethroid resistant populations in those regions. Interestingly, no mutant alleles were detected in all 232 specimens of eight other species in the An. hyrcanus group. CONCLUSION: The survey revealed that the kdr occurrence and accumulation in the An. sinensis populations were more frequent in central China than in the other regions, suggesting that the kdr mutations may contribute significantly to the pyrethroid resistance in the mosquitoes in central China.

[Genetic Polymophism and Evolution of SRPN14 Gene in Anopheles sinensis (Diptera : Culicidae)].

OBJECTIVE: To identify and locate the serine protease inhibitor 14 (SRPN14) gene of Anopheles sinensis, and analyze its genetic polymorphism among populations as well as the selective pressure during evolution. METHODS: Primers were designed based on the genomic sequencing data of An. sinensis, and PCR amplification system for the SRPN14 gene was established. The chromosomal location of SRPN14 gene was determined by fluorescence in situ hybridization. The SRPN14 gene of An. sinensis populations collected from 18 sampling sites in 12 provinces (municipality) was sequenced, its genetic variations within and among populations calculated, and the selective pressure during adaptive evolution evaluated. RESULTS: The amplified part of the SRPN14 gene of An. sinensis was 429 bp in length, and had 77%(nt) and 88% (aa)similarities with An. gambiae. The SRPN14 gene located on 2L: 23C of salivary gland chromosomes of An. sinensis. The sequences of 411 individuals from 13 An. sinensis populations were analyzed. In the 411 individuals, the total number of alleles of the SRPN14 gene was 204, among which 51 (25.00% ) showed inter-population consistency. The ranges of SRPN14 allel number and genetic polymorphism were from 11 (Liaoning) to 33 (Chongqing), and from 0.008 (Liaoning) to 0.024 (Hainan), respectively. AMOVA result showed that genetic divergence within populations was significantly higher than that among populations; variation within populations was 95.79% of the total variation. The genetic divergence among populations was small, with FST value of 0.042. The number of synonymous substitutions in SRPN14 was significantly higher than that of non-synonymous substitutions sites, and ω was less than 1 in all populations. CONCLUSION: Genetic polymorphism occurs in SRPN14 gene of An. sinensis populations, and its evolution is under the negative selective pressure.

Transcriptome analysis of cytoplasmic male sterility and restoration in CMS-D8 cotton.

KEY MESSAGE: A global view of differential expression of genes in CMS-D8 of cotton was presented in this study which will facilitate the understanding of cytoplasmic male sterility in cotton. Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants which is incapable of producing functional pollen. However, the male fertility can be restored by one or more nuclear-encoded restorer genes. A genome-wide transcriptome analysis of CMS and restoration in cotton is currently lacking. In this study, Affymetrix GeneChips© Cotton Genome Array containing 24,132 transcripts was used to compare differentially expressed (DE) genes of flower buds at the meiosis stage between CMS and its restorer cotton plants conditioned by the D8 cytoplasm. A total of 458 (1.9 %) of DE genes including 127 up-regulated and 331 down-regulated ones were identified in the CMS-D8 line. Quantitative RT-PCR was used to validate 10 DE genes selected from seven functional categories. The most frequent DE gene group was found to encode putative proteins involved in cell wall expansion, such as pectinesterase, pectate lyase, pectin methylesterase, glyoxal oxidase, polygalacturonase, indole-3-acetic acid-amino synthetase, and xyloglucan endo-transglycosylase. Genes in cytoskeleton category including actin, which plays a key role in cell wall expansion, cell elongation and cell division, were also highly differentially expressed between the fertile and CMS plants. This work represents the first study in utilizing microarray to identify CMS-related genes by comparing overall DE genes between fertile and CMS plants in cotton. The results provide evidence that many CMS-associated genes are mainly involved in cell wall expansion. Further analysis will be required to elucidate the molecular mechanisms of male sterility which will facilitate the development of new hybrid cultivars in cotton.

[Taxonomic composition of metagenomic community in the larval gut of mosquito Anopheles sinensis (Diptera: Culicidae)].

OBJECTIVE: To investigate the bacteria diversity in larval gut of field-collected Anopheles sinensis. METHODS: The 16S rDNA V4 region of An. sinensis larvae collected from paddy on Jiading District of Shanghai (L1/L2) and small seeping water on Wenchang City of Hainan (AS) was sequenced by high-throughput pyrosequencing. Using Qiime and Mothur softwares, the number of sequences and operational taxonomic units (OTUs) for each sample was sorted and calculated, the species abundance and distribution, Alpha diversity index and difference times of species abundance among samples were analyzed. RESULTS: The number of sequences and OTUs for each sample were 253 724/3 930 (L1), 225 203/4 312 (L2) and 73 990/2 380 (AS). The rarefaction curves showed that adequate sampling was achieved. The number of OTUs was close to actual situation. The value of richness index was 5 942.61/6 534.88 (L1), 6 328.17/7 235.89 (L2) and 4228.66/5 651.20 (AS); diversity index was 4.63/0.03 (L1), 5.10/0.02 (L2) and 0.14/3.94 (AS). The dominant species of An. sinensis larvae gut microbiota all belonged to the phylum Proteobacteria, with a percentage of 87% (AS) and 90% (L). In addition, the dominant phyla among them were Firmicutes, Bacteroidetes and Actinobacteria. The comparison of bacterial abundance between L and AS showed that there were 18 phyla with significant difference, except the Proteobacteria and Deinococcus-Thermus; only 9 phyla were different significantly between L1 and L2. CONCLUSION: Evenness and richness of bacteria flora in the An. sinensis larvae gut collected from paddy and small seeping waters were obtained.

Draft genome sequence of Pseudomonas sp. strain Ag1, isolated from the midgut of the malaria mosquito Anopheles gambiae.

A Pseudomonas sp. bacterium was isolated from the midguts of Anopheles gambiae mosquitoes. Here we present the annotated Pseudomonas sp. draft genome sequence as a contribution to the efforts of characterization of the mosquito gut microbiome.

Draft genome sequences of Enterobacter sp. isolate Ag1 from the midgut of the malaria mosquito Anopheles gambiae.

An isolate of Enterobacter sp. was obtained from the microbial community within the gut of the Anopheles gambiae mosquito, a major malaria vector in Africa. This genome was sequenced and annotated. The genome sequences will facilitate subsequent efforts to characterize the mosquito gut microbiome.

Aryl hydrocarbon receptor and Krüppel like factor 10 mediate a transcriptional axis modulating immune homeostasis in mosquitoes.

Immune responses require delicate controls to maintain homeostasis while executing effective defense. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. The Krüppel-like factor 10 (KLF10) is a C2H2 zinc-finger containing transcription factor. The functions of mosquito AhR and KLF10 have not been characterized. Here we show that AhR and KLF10 constitute a transcriptional axis to modulate immune responses in mosquito Anopheles gambiae. The manipulation of AhR activities via agonists or antagonists repressed or enhanced the mosquito antibacterial immunity, respectively. KLF10 was recognized as one of the AhR target genes in the context. Phenotypically, silencing KLF10 reversed the immune suppression caused by the AhR agonist. The transcriptome comparison revealed that silencing AhR and KLF10 plus challenge altered the expression of 2245 genes in the same way. The results suggest that KLF10 is downstream of AhR in a transcriptional network responsible for immunomodulation. This AhR-KLF10 axis regulates a set of genes involved in metabolism and circadian rhythms in the context. The axis was required to suppress the adverse effect caused by the overactivation of the immune pathway IMD via the inhibitor gene Caspar silencing without a bacterial challenge. These results demonstrate that the AhR-KLF10 axis mediates an immunoregulatory transcriptional network as a negative loop to maintain immune homeostasis.

Fine pathogen discrimination within the APL1 gene family protects Anopheles gambiae against human and rodent malaria species.

Genetically controlled resistance of Anopheles gambiae mosquitoes to Plasmodium falciparum is a common trait in the natural population, and a cluster of natural resistance loci were mapped to the Plasmodium-Resistance Island (PRI) of the A. gambiae genome. The APL1 family of leucine-rich repeat (LRR) proteins was highlighted by candidate gene studies in the PRI, and is comprised of paralogs APL1A, APL1B and APL1C that share > or =50% amino acid identity. Here, we present a functional analysis of the joint response of APL1 family members during mosquito infection with human and rodent Plasmodium species. Only paralog APL1A protected A. gambiae against infection with the human malaria parasite P. falciparum from both the field population and in vitro culture. In contrast, only paralog APL1C protected against the rodent malaria parasites P. berghei and P. yoelii. We show that anti-P. falciparum protection is mediated by the Imd/Rel2 pathway, while protection against P. berghei infection was shown to require Toll/Rel1 signaling. Further, only the short Rel2-S isoform and not the long Rel2-F isoform of Rel2 confers protection against P. falciparum. Protection correlates with the transcriptional regulation of APL1A by Rel2-S but not Rel2-F, suggesting that the Rel2-S anti-parasite phenotype results at least in part from its transcriptional control over APL1A. These results indicate that distinct members of the APL1 gene family display a mutually exclusive protective effect against different classes of Plasmodium parasites. It appears that a gene-for-pathogen-class system orients the appropriate host defenses against distinct categories of similar pathogens. It is known that insect innate immune pathways can distinguish between grossly different microbes such as Gram-positive bacteria, Gram-negative bacteria, or fungi, but the function of the APL1 paralogs reveals that mosquito innate immunity possesses a more fine-grained capacity to distinguish between classes of closely related eukaryotic pathogens than has been previously recognized.

Metagenomic insights into dissemination of antibiotic resistance across bacterial genera in wastewater treatment.

The aim of this study was to evaluate the impacts of conventional wastewater treatment processes including secondary treatment and chlorination on the removal of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB), and to assess the association of ARGs with their potential hosts in each treatment process. The results showed chlorination with subinhibitory concentration (<8 mg/L) resulted in an increased ARB number in the disinfection effluent. qPCR analysis indicated secondary treatment increased relative abundance of ARGs in remaining bacteria whereas disinfection reduced the relative abundance of those genes effectively. Metagenomic analysis revealed a significant shift of dominating bacterial genera harboring ARGs. Along the treatment train, 48, 95 and 80 genera were identified to be the ARG carriers in primary effluent, secondary effluent, and disinfection effluent, respectively. It was also found that secondary treatment increased the diversity of potential ARG hosts while both secondary treatment and chlorination broadened the host range of some ARGs at the genus level, which may be attributed to the spread of antibiotic resistance across bacterial genera through horizontal transfer. This study highlights the growing concerns that wastewater treatment plants (WWTPs) may disseminate ARGs by associating this effect to specific treatment stages and by correlating ARGs with their bacterial hosts.

Trained Immunity in Anopheles gambiae: Antibacterial Immunity Is Enhanced by Priming via Sugar Meal Supplemented With a Single Gut Symbiotic Bacterial Strain.

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.

Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes.

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 α- and 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 Systematic Analysis of Mosquito-Microbiome Biosynthetic Gene Clusters Reveals Antimalarial Siderophores that Reduce Mosquito Reproduction Capacity.

Advances in infectious disease control strategies through genetic manipulation of insect microbiomes have heightened interest in microbially produced small molecules within mosquitoes. Herein, 33 mosquito-associated bacterial genomes were mined and over 700 putative biosynthetic gene clusters (BGCs) were identified, 135 of which belong to known classes of BGCs. After an in-depth analysis of the 135 BGCs, iron-binding siderophores were chosen for further investigation due to their high abundance and well-characterized bioactivities. Through various metabolomic strategies, eight siderophore scaffolds were identified in six strains of mosquito-associated bacteria. Among these, serratiochelin A and pyochelin were found to reduce female Anopheles gambiae overall fecundity likely by lowering their blood-feeding rate. Serratiochelin A and pyochelin were further found to inhibit the Plasmodium parasite asexual blood and liver stages in vitro. Our work supplies a bioinformatic resource for future mosquito-microbiome studies and highlights an understudied source of bioactive small molecules.

In Silico Identification of Three Types of Integrative and Conjugative Elements in Elizabethkingia anophelis Strains Isolated from around the World.

Elizabethkingia anophelis is an emerging global multidrug-resistant opportunistic pathogen. We assessed the diversity among 13 complete genomes and 23 draft genomes of E. anophelis strains derived from various environmental settings and human infections from different geographic regions around the world from 1950s to the present. Putative integrative and conjugative elements (ICEs) were identified in 31/36 (86.1%) strains in the study. A total of 52 putative ICEs (including eight degenerated elements lacking integrases) were identified and categorized into three types based on the architecture of the conjugation module and the phylogeny of the relaxase, coupling protein, TraG, and TraJ protein sequences. The type II and III ICEs were found to integrate adjacent to tRNA genes, while type I ICEs integrate into intergenic regions or into a gene. The ICEs carry various cargo genes, including transcription regulator genes and genes conferring antibiotic resistance. The adaptive immune CRISPR-Cas system was found in nine strains, including five strains in which CRISPR-Cas machinery and ICEs coexist at different locations on the same chromosome. One ICE-derived spacer was present in the CRISPR locus in one strain. ICE distribution in the strains showed no geographic or temporal patterns. The ICEs in E. anophelis differ in architecture and sequence from CTnDOT, a well-studied ICE prevalent in Bacteroides spp. The categorization of ICEs will facilitate further investigations of the impact of ICE on virulence, genome epidemiology, and adaptive genomics of E. anophelisIMPORTANCEElizabethkingia anophelis is an opportunistic human pathogen, and the genetic diversity between strains from around the world becomes apparent as more genomes are sequenced. Genome comparison identified three types of putative ICEs in 31 of 36 strains. The diversity of ICEs suggests that they had different origins. One of the ICEs was discovered previously from a large E. anophelis outbreak in Wisconsin in the United States; this ICE has integrated into the mutY gene of the outbreak strain, creating a mutator phenotype. Similar to ICEs found in many bacterial species, ICEs in E. anophelis carry various cargo genes that enable recipients to resist antibiotics and adapt to various ecological niches. The adaptive immune CRISPR-Cas system is present in nine of 36 strains. An ICE-derived spacer was found in the CRISPR locus in a strain that has no ICE, suggesting a past encounter and effective defense against ICE.

Widespread insecticide resistance in Aedes aegypti L. from New Mexico, U.S.A.

BACKGROUND: Aedes aegypti mosquitoes are vectors of a variety of emerging viral pathogens, including yellow fever, dengue, chikungunya, and Zika virus. This species has established endemic populations in all cities across southern New Mexico sampled to date. Presently, control of Aedes-borne viruses relies on deployment of insecticides to suppress mosquito populations, but the evolution of insecticide resistance threatens the success of vector control programs. While insecticide resistance is quite common in Ae. aegypti field populations across much of the U.S., the resistance status of this species in populations from New Mexico has not previously been assessed. RESULTS: First, we collected information on pesticide use in cities in southern New Mexico and found that the most commonly used active ingredients were pyrethroids. The use of insecticides with the same mode-of-action over multiple years is likely to promote the evolution of resistance. To determine if there was evidence of resistance in some cities in southern New Mexico, we collected Ae. aegypti from the same cities and established laboratory strains to assess resistance to pyrethroid insecticides and, for a subset of populations, to organophosphate insecticides. F2 or F4 generation mosquitoes were assessed for insecticide resistance using bottle test bioassays. The majority of the populations from New Mexico that we analyzed were resistant to the pyrethroids permethrin and deltamethrin. A notable exception to this trend were mosquitoes from Alamogordo, a city that did not report using pyrethroid insecticides for vector control. We screened individuals from each population for known knock down resistance (kdr) mutations via PCR and found a strong association between the presences of the F1534C kdr mutation in the para gene of Ae. aegypti (homologue to F1534C in Musca domestica L.) and pyrethroid resistance. CONCLUSION: High-level pyrethroid resistance is common in Ae. aegypti from New Mexico and geographic variation in such resistance is likely associated with variation in usage of pyrethroids for vector control. Resistance monitoring and management is recommended in light of the potential for arbovirus outbreaks in this state. Also, alternative approaches to mosquito control that do not involve insecticides should be explored.

Wolbachia pipientis occurs in Aedes aegypti populations in New Mexico and Florida, USA.

The mosquitoes Aedes aegypti (L.) and Ae. albopictus Skuse are the major vectors of dengue, Zika, yellow fever, and chikungunya viruses worldwide. Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found that Wolbachia is commonly detected in Ae. albopictus but rarely detected in Ae. aegypti. In this study, we used a two-step PCR assay to detect Wolbachia in wild-collected samples of Ae. aegypti. The PCR products were sequenced to validate amplicons and identify Wolbachia strains. A loop-mediated isothermal amplification (LAMP) assay was developed and used for detecting Wolbachia in selected mosquito specimens as well. We found Wolbachia in 85/148 (57.4%) wild Ae. aegypti specimens from various cities in New Mexico, and in 2/46 (4.3%) from St. Augustine, Florida. Wolbachia was not detected in 94 samples of Ae. aegypti from Deer Park, Harris County, Texas. Wolbachia detected in Ae. aegypti from both New Mexico and Florida was the wAlbB strain of Wolbachia pipientis. A Wolbachia-positive colony of Ae. aegypti was established from pupae collected in Las Cruces, New Mexico, in 2018. The infected females of this strain transmitted Wolbachia to their progeny when crossed with males of Rockefeller strain of Ae. aegypti, which does not carry Wolbachia. In contrast, none of the progeny of Las Cruces males mated to Rockefeller females were infected with Wolbachia.

Redox state affects fecundity and insecticide susceptibility in Anopheles gambiae.

Redox reactions play a central role in the metabolism of an organism. It is vital to maintain redox homeostasis in response to the fluctuation of redox shift in various biological contexts. NADPH-dependent reducing capacity is one of the key factors contributing to the redox homeostasis. To understand the redox capacity and its impact on mosquito fecundity and susceptibility to insecticides in Anopheles gambiae, we examined the dynamics of elevated oxidative state via induction by paraquat (PQ) and the inhibition of NADPH regeneration by 6-aminonicotinamide (6AN). In naïve conditions, inherent oxidative capacity varies between individuals, as measured by GSSG/GSH ratio. The high GSSG/GSH ratio was negatively correlated with fecundity. Both PQ and 6AN feeding increased GSSG/GSH ratio and elevated protein carbonylation, a marker of oxidative damage. Both pro-oxidants lowered egg production. Co-feeding the pro-oxidants with antioxidant lycopene attenuated the adverse effects on fecundity, implying that oxidative stress was the cause of this phenotype. Pre-feeding with 6AN increased insecticide susceptibility in DDT resistant mosquitoes. These results indicate that oxidative state is delicate in mosquitoes, manipulation of NADPH pool may adversely affect fecundity and insecticide detoxification capacity. This knowledge can be exploited to develop novel vector control strategies targeting fecundity and insecticide resistance.

Investigation of the seasonal microbiome of Anopheles coluzzii mosquitoes in Mali.

The poorly understood mechanisms of dry season persistence of Anopheles spp. mosquitoes through the dry season in Africa remain a critical gap in our knowledge of Plasmodium disease transmission. While it is thought that adult mosquitoes remain in a dormant state throughout this seven-month dry season, the nature of this state remains unknown and has largely not been recapitulated in laboratory settings. To elucidate possible connections of this state with microbial composition, the whole body microbiomes of adult mosquitoes in the dry and wet seasons in two locations of Mali with varying water availability were compared by sequencing the 16S ribosomal RNA gene. These locations were a village near the Niger River with year-round water sources (N'Gabakoro, "riparian"), and a typical Sahelian area with highly seasonal breeding sites (Thierola Area, "Sahelian"). The 16S bacterial data consisted of 2057 sequence variants in 426 genera across 184 families. From these data, we found several compositional differences that were seasonally and spatially linked. Counter to our initial hypothesis, there were more pronounced seasonal differences in the bacterial microbiome of riparian, rather than Sahelian areas. These seasonal shifts were primarily in Ralstonia, Sphingorhabdus, and Duganella spp. bacteria that are usually soil and water-associated, indicating these changes may be from bacteria acquired in the larval environment, rather than adulthood. In Sahelian dry season mosquitoes, there was a unique intracellular bacteria, Anaplasma, which likely was acquired through non-human blood feeding. Cytochrome B analysis of blood meals showed greater heterogeneity in host choice of An. coluzzii independent of season in the Thierola area compared to N'Gabakoro (77.5% vs. 94.6% human-origin blood meal, respectively), indicating a relaxation of anthropophily. Overall, this exploratory study provides valuable indications of spatial and seasonal differences in bacterial composition which help refine this difficult to study state.

The impact of metagenomic interplay on the mosquito redox homeostasis.

Mosquitoes are exposed to oxidative challenges throughout their life cycle. The primary challenge comes from a blood meal. The blood digestion turns the midgut into an oxidative environment, which imposes pressure not only on mosquito fecundity and other physiological traits but also on the microbiota in the midgut. During evolution, mosquitoes have developed numerous oxidative defense mechanisms to maintain redox homeostasis in the midgut. In addition to antioxidants, SOD, catalase, and glutathione system, sufficient supply of the reducing agent, NADPH, is vital for a successful defense against oxidative stress. Increasing evidence indicates that in response to oxidative stress, cells reconfigure metabolic pathways to increase the generation of NADPH through NADP-reducing networks including the pentose phosphate pathway and others. The microbial homeostasis is critical for the functional contributions to various host phenotypes. The symbiotic microbiota is regulated largely by the Duox-ROS pathway in Drosophila. In mosquitoes, Duox-ROS pathway, heme-mediated signaling, antimicrobial peptide production and C-type lectins work in concert to maintain the dynamic microbial community in the midgut. Microbial mechanisms against oxidative stress in this context are not well understood. Emerging evidence that microbial metabolites trigger host oxidative response warrants further study on the metagenomic interplay in an oxidative environment like mosquito gut ecosystem. Besides the classical Drosophila model, hematophagous insects like mosquitoes provide an alternative model system to study redox homeostasis in a symbiotic metagenomic context.

Complete Circularized Genome Sequences of Four Strains of Elizabethkingia anophelis, Including Two Novel Strains Isolated from Wild-Caught Anopheles sinensis.

We provide complete circularized genome sequences of two mosquito-derived Elizabethkingia anophelis strains with draft sequences currently in the public domain (R26 and Ag1), and two novel E. anophelis strains derived from a different mosquito species, Anopheles sinensis (AR4-6 and AR6-8). The genetic similarity of all four mosquito-derived strains is remarkable.