Jiannong Xu

Dynamic Gut Microbiome across Life History of the Malaria Mosquito Anopheles gambiae in Kenya

The mosquito gut represents an ecosystem that accommodates a complex, intimately associated microbiome. It is increasingly clear that the gut microbiome influences a wide variety of host traits, such as fitness and immunity. Understanding the microbial community structure and its dynamics across mosquito life is a prerequisite for comprehending the symbiotic relationship between the mosquito and its gut microbial residents. Here we characterized gut bacterial communities across larvae, pupae and adults of Anopheles gambiae reared in semi-natural habitats in Kenya by pyrosequencing bacterial 16S rRNA fragments. Immatures and adults showed distinctive gut community structures. Photosynthetic Cyanobacteria were predominant in the larval and pupal guts while Proteobacteria and Bacteroidetes dominated the adult guts, with core taxa of Enterobacteriaceae and Flavobacteriaceae. At the adult stage, diet regime (sugar meal and blood meal) significantly affects the microbial structure. Intriguingly, blood meals drastically reduced the community diversity and favored enteric bacteria. Comparative genomic analysis revealed that the enriched enteric bacteria possess large genetic redox capacity of coping with oxidative and nitrosative stresses that are associated with the catabolism of blood meal, suggesting a beneficial role in maintaining gut redox homeostasis. Interestingly, gut community structure was similar in the adult stage between the field and laboratory mosquitoes, indicating that mosquito gut is a selective eco-environment for its microbiome. This comprehensive gut metatgenomic profile suggests a concerted symbiotic genetic association between gut inhabitants and host.

Gene silencing in mosquito salivary glands by RNAi.

Salivary glands are the ultimate site of development in the insect of mosquito born pathogens such as Plasmodium. Mosquito salivary glands also secrete components involved in anti‐haemostatic activities and allergic reactions. We investigated the feasibility of RNAi as a tool for functional analysis of genes expressed in Anopheles gambiae salivary glands. We show that specific gene silencing in salivary glands requires the use of large amounts of dsRNA, condition that differs from those for efficient RNAi in other mosquito tissues. Using this protocol, we demonstrated the role of AgApy, which encodes an apyrase, in the probing behaviour of An. gambiae.

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 ≥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.

Anopheles gambiae APL1 Is a Family of Variable LRR Proteins Required for Rel1-Mediated Protection from the Malaria Parasite, Plasmodium berghei

Background We previously identified by genetic mapping an Anopheles gambiae chromosome region with strong influence over the outcome of malaria parasite infection in nature. Candidate gene studies in the genetic interval, including functional tests using the rodent malaria parasite Plasmodium berghei, identified a novel leucine-rich repeat gene, APL1, with functional activity against P. berghei. Principal Findings Manual reannotation now reveals APL1 to be a family of at least 3 independently transcribed genes, APL1A, APL1B, and APL1C. Functional dissection indicates that among the three known APL1 family members, APL1C alone is responsible for host defense against P. berghei. APL1C functions within the Rel1-Cactus immune signaling pathway, which regulates APL1C transcript and protein abundance. Gene silencing of APL1C completely abolishes Rel1-mediated host protection against P. berghei, and thus the presence of APL1C is required for this protection. Further highlighting the influence of this chromosome region, allelic haplotypes at the APL1 locus are genetically associated with and have high explanatory power for the success or failure of P. berghei parasite infection. Conclusions APL1C functions as a required transducer of Rel1-dependent immune signal(s) to efficiently protect mosquitoes from P. berghei infection, and allelic genetic haplotypes of the APL1 locus display distinct levels of susceptibility and resistance to P. berghei.

Ribosomal DNA difference between species A and D of the Anopheles dims complex of mosquitoes from China

Abstract. Species A and D of the Anopheles dints complex were found in China. Ribosomal DNA second internal transcribed spacers (ITS2) of both species A and D were sequenced and found to be 716 and 710 base‐pairs in length, respectively, with 699c GC content. No evidence of intraspecific variation was detected in the ITS2 sequence of species A, whereas the sequence of species D showed variation at one position in the ITS2. A large number of simple repeat motifs were dispersed throughout the ITS2 sequences. The level of interspecific difference was 5.4% of the nucleotide sequences. Some of the interspecific differences were located in regions with subrepeat structure.

The Hyrcanus Group of Anopheles (Anopheles) in China (Diptera: Culicidae): Species Discrimination and Phylogenetic Relationships Inferred by Ribosomal DNA Internal Transcribed Spacer 2 Sequences

Abstract The Hyrcanus group comprises many closely related species with wide distributions in the Oriental and Palaearctic regions. The sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA were determined for 12 species in China—An. crawfordi, An. hyrcanus, An. junlianensis, An. kunmingensis, An. kweiyangensis, An. lesteri, An. liangshanensis, An. peditaeniatus, An. pullus, An. sinensis, and two unknown species within the group. The length of the ITS2 ranged from 436 bp in An. hyrcanus to 469 bp in An. crawfordi, with GC contents of 44.9–46.8%. Intraspecific variation was found in three species (An. junlianensis, An. liangshanensis, and An. pullus) at the level of 0.0–0.4%, whereas interspecific differences ranged from 1.6% between An. liangshanensis and An. kunmingensis to 50.8% between An. peditaeniatus and sp. 1. The ITS2 comparisons revealed two unknown species, verified the valid species status for An. kunmingensis, and found An. pullus in China. We agree that An. anthropophagus is a junior synonym of An. lesteri. The validation of An. junlianensis awaits recognition of the molecular identity of the entity identified as An. yatsushiroensis. The ITS2 divergences were used for inferring phylogenetic relationships among 12 species in China. The estimation revealed close relationships among An. liangshanesis, An. kunmingensis, An. kweiyangensis, An. lesteri, and An. sinensis. Our study emphasizes the need for the molecular identity of the species members in integrated studies in systematics, bionomics, and population genetics for the Hyrcanus group.

Molecular Genetics of Mosquito Resistance to Malaria Parasites

Malaria parasites are transmitted by the bite of an infected mosquito, but even efficient vector species possess multiple mechanisms that together destroy most of the parasites present in an infection. Variation between individual mosquitoes has allowed genetic analysis and mapping of loci controlling several resistance traits, and the underlying mechanisms of mosquito response to infection are being described using genomic tools such as transcriptional and proteomic analysis. Malaria infection imposes fitness costs on the vector, but various forms of resistance inflict their own costs, likely leading to an evolutionary tradeoff between infection and resistance. Plasmodium development can be successfully completed onlyin compatible mosquito-parasite species combinations, and resistance also appears to have parasite specificity. Studies of Drosophila, where genetic variation in immunocompetence is pervasive in wild populations, offer a comparative context for understanding coevolution of the mosquito-malaria relationship. More broadly, plants also possess systems of pathogen resistance with features that are structurally conserved in animal innate immunity, including insects, and genomic datasets now permit useful comparisons of resistance models even between such diverse organisms.

Anopheles gambiae genome reannotation through synthesis of ab initio and comparative gene prediction algorithms

BackgroundComplete genome annotation is a necessary tool as Anopheles gambiae researchers probe the biology of this potent malaria vector.ResultsWe reannotate the A. gambiae genome by synthesizing comparative and ab initio sets of predicted coding sequences (CDSs) into a single set using an exon-gene-union algorithm followed by an open-reading-frame-selection algorithm. The reannotation predicts 20,970 CDSs supported by at least two lines of evidence, and it lowers the proportion of CDSs lacking start and/or stop codons to only approximately 4%. The reannotated CDS set includes a set of 4,681 novel CDSs not represented in the Ensembl annotation but with EST support, and another set of 4,031 Ensembl-supported genes that undergo major structural and, therefore, probably functional changes in the reannotated set. The quality and accuracy of the reannotation was assessed by comparison with end sequences from 20,249 full-length cDNA clones, and evaluation of mass spectrometry peptide hit rates from an A. gambiae shotgun proteomic dataset confirms that the reannotated CDSs offer a high quality protein database for proteomics. We provide a functional proteomics annotation, ReAnoXcel, obtained by analysis of the new CDSs through the AnoXcel pipeline, which allows functional comparisons of the CDS sets within the same bioinformatic platform. CDS data are available for download.ConclusionComprehensive A. gambiae genome reannotation is achieved through a combination of comparative and ab initio gene prediction algorithms.

Population Structure of the Malaria Vector Anopheles sinensis (Diptera: Culicidae) in China: Two Gene Pools Inferred by Microsatellites

Background Anopheles sinensis is a competent malaria vector in China. An understanding of vector population structure is important to the vector-based malaria control programs. However, there is no adequate data of A. sinensis population genetics available yet. Methodology/Principal Findings This study used 5 microsatellite loci to estimate population genetic diversity, genetic differentiation and demographic history of A. sinensis from 14 representative localities in China. All 5 microsatellite loci were highly polymorphic across populations, with high allelic richness and heterozygosity. Hardy–Weinberg disequilibrium was found in 12 populations associated with heterozygote deficits, which was likely caused by the presence of null allele and the Wahlund effect. Bayesian clustering analysis revealed two gene pools, grouping samples into two population clusters; one includes six and the other includes eight populations. Out of 14 samples, six samples were mixed with individuals from both gene pools, indicating the coexistence of two genetic units in the areas sampled. The overall differentiation between two genetic pools was moderate (F ST = 0.156). Pairwise differentiation between populations were lower within clusters (F ST = 0.008–0.028 in cluster I and F ST = 0.004–0.048 in cluster II) than between clusters (F ST = 0.120–0.201). A reduced gene flow (Nm = 1–1.7) was detected between clusters. No evidence of isolation by distance was detected among populations neither within nor between the two clusters. There are differences in effective population size (Ne = 14.3-infinite) across sampled populations. Conclusions/Significance Two genetic pools with moderate genetic differentiation were identified in the A. sinensis populations in China. The population divergence was not correlated with geographic distance or barrier in the range. Variable effective population size and other demographic effects of historical population perturbations could be the factors affecting the population differentiation. The structured populations may limit the migration of genes under pressures/selections, such as insecticides and immune genes against malaria.

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.