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.     

Insights from the Genome Annotation of Elizabethkingia anophelis from the Malaria Vector Anopheles gambiae

Elizabethkingia anophelis is a dominant bacterial species in the gut ecosystem of the malaria vector mosquito Anopheles gambiae. We recently sequenced the genomes of two strains of E. anophelis, R26^T and Ag1, isolated from different strains of A. gambiae. The two bacterial strains are identical with a few exceptions. Phylogenetically, Elizabethkingia is closer to Chryseobacterium and Riemerella than to Flavobacterium. In line with other Bacteroidetes known to utilize various polymers in their ecological niches, the E. anophelis genome contains numerous TonB dependent transporters with various substrate specificities. In addition, several genes belonging to the polysaccharide utilization system and the glycoside hydrolase family were identified that could potentially be of benefit for the mosquito carbohydrate metabolism. In agreement with previous reports of broad antibiotic resistance in E. anophelis, a large number of genes encoding efflux pumps and β-lactamases are present in the genome. The component genes of resistance-nodulation-division type efflux pumps were found to be syntenic and conserved in different taxa of Bacteroidetes. The bacterium also displays hemolytic activity and encodes several hemolysins that may participate in the digestion of erythrocytes in the mosquito gut. At the same time, the OxyR regulon and antioxidant genes could provide defense against the oxidative stress that is associated with blood digestion. The genome annotation and comparative genomic analysis revealed functional characteristics associated with the symbiotic relationship with the mosquito host.     

Deep sequencing reveals extensive variation in the gut microbiota of wild mosquitoes from Kenya

The mosquito midgut is a hostile environment that vector‐borne parasites must survive to be transmitted. Commensal bacteria in the midgut can reduce the ability of mosquitoes to transmit disease, either by having direct anti‐parasite effects or by stimulating basal immune responses of the insect host. As different bacteria have different effects on parasite development, the composition of the bacterial community in the mosquito gut is likely to affect the probability of disease transmission. We investigated the diversity of mosquito gut bacteria in the field using 454 pyrosequencing of 16S rRNA to build up a comprehensive picture of the diversity of gut bacteria in eight mosquito species in this population. We found that mosquito gut typically has a very simple gut microbiota that is dominated by a single bacterial taxon. Although different mosquito species share remarkably similar gut bacteria, individuals in a population are extremely variable and can have little overlap in the bacterial taxa present in their guts. This may be an important factor in causing differences in disease transmission rates within mosquito populations.     

Role of the gut proteinases from mosquito larvae in the mechanism of action and the specificity of the Bacillus sphaericus toxin

Gut proteinases from larvae of mosquito species both susceptible and not susceptible to Bacillus sphaericus converted the 43-kDa toxin to a 40-kDa polypeptide exhibiting enhanced cytotoxicity to mosquito cell cultures. The toxin was also activated by gut proteinases from the nonsusceptible Lepidoptera Spodoptera littoralis in vitro and in vivo. Therefore, the specificity of Bacillus sphaericus toxin does not seem to be determined by gut proteinase action. However, susceptibility of mosquito cell cultures did not reflect the specificity of the toxin, which must now be investigated at the cellular level in the larvae.     

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.     

Detection and localization of antibody ingested with a mosquito bloodmeal

Mouse immunoglobulins were found to persist in Aedes aegypti (L.) mosquito bloodmeals for 2-3 days after ingestion. Immunoenzyme labelling revealed mouse antibody specifically bound to the mosquito midgut epithelium after ingestion; immunogold labelling of thin sections revealed mouse antibody within the cytoplasm of the microvilli on the midgut epithelium. Ingested mouse antibody was not conclusively demonstrated bound to tissues outside the gut, though antibody was detected in mosquito haemolymph using a sensitive ELISA test. Possible mechanisms by which antibody may cross mosquito gut barriers and actions that in vivo antibody:antigen reactions may have on these bloodfed insects are discussed.     

Uptake and persistence of ingested antibody in the mosquito Anopheles stephensi

A sensitive ELISA was developed to monitor the persistence of a specific antibody, rabbit anti-BSA, in the bloodmeal, haemolymph and tissues of the mosquito Anopheles stephensi Liston. Different concentrations of anti-BSA were fed to female mosquitoes in sheep blood, via a membrane-feeder, and it was found that antibody persisted in the gut as the bloodmeal was digested: concentrations present at 24 h were directly related to those fed. Homogenates of mosquito bodies, from which the intact guts had been removed, were always antibody-positive up to 9 days post-feeding, indicating that undigested antibody had passed through the gut wall into the haemocoele. Haemolymph was extracted from mosquitoes at different times post-feeding, using a microcapillary and manipulator, and antibody was detected in several of the assays. The level of antibodies in the haemolymph 24 h post-feeding was less than half of the level in mosquito heads, indicating removal of antibodies from the haemolymph, perhaps by binding onto haemocoelic tissues. The relevance of these results to the ingestion, survival and fate of antibody against malaria sporozoites is discussed.     

The Search for Novel Malaria Transmission-blocking Targets in the Mosquito Midgut

The need for new malaria control strategies has led to increased efforts to understand more clearly the mosquito stages of Plasmodium. The absolute requirement of gamete maturation and fertilization, transformation of sedentary zygote to motile ookinete, ookinete interaction and invasion of gut epithelium, and the survival of the mosquito against immune attack suggest that numerous unidentified targets exist, which could be modified to achieve transmission-blocking of malaria. In the search for new transmission-blocking targets in the mosquito gut, Mohammed Shahabuddin, Stéphane Cociancich and Helge Zieler here summarize recent studies to identify the cellular and biochemical factors that affect the malaria parasite's development; in particular, factors influencing the early development of Plasmodium, receptor-mediated interactions between the parasite and the mosquito midgut, and the gut-associated immune responses directed against Plasmodium.     

Biochemical and cytoimmunological evidence for the control of Aedes aegypti larval trypsin with Aea-TMOF

Trypsin and chymotrypsin-like enzymes were detected in the gut of Aedes aegypti in the four larval instar and pupal developmental stages. Although overall the amount of trypsin synthesized in the larval gut was 2-fold higher than chymotrypsin, both enzymes are important in food digestion. Feeding Aea-Trypsin Modulating Oostatic Factor (TMOF) to Ae. aegypti and Culex quinquefasciatus larvae inhibited trypsin biosynthesis in the larval gut, stunted larval growth and development, and caused mortality. Aea-TMOF induced mortality in Ae. aegypti, Cx. quinquefasciatus, Culex nigripalpus, Anopheles quadrimaculatus, and Aedes taeniorhynchus larvae, indicating that many mosquito species have a TMOF-like hormone. The differences in potency of TMOF on different mosquito species suggest that analogues in other species are similar but may differ in amino acid sequence or are transported differently through the gut. Feeding of 29 different Aea-TMOF analogues to mosquito larvae indicated that full biological activity of the hormone is achieved with the tetrapeptide YDPA. Using cytoimmunochemical analysis, intrinsic TMOF was localized to ganglia of the central nervous system in larvae and male and female Ae. aegypti adults. The subesophageal, thoracic, and abdominal ganglia of both larval and adult mosquitoes contained immunoreactive cells. Immunoreactive cells were absent in the corpus cardiacum of newly molted 4th instar larvae but were found in late 4th instar larvae. In both males and females, the intrinsic neurosecretory cells of the corpus cardiacum were filled with densely stained immunoreactive material. These results indicate that TMOF-immunoreactive material is synthesized in sugar-fed male and female adults and larvae by the central nervous system cells.     

Morphological and molecular characterization of a Cypovirus (Reoviridae) from the mosquito Uranotaenia sapphirina (Diptera: Culicidae)

A novel cypovirus has been isolated from the mosquito Uranotaenia sapphirina (UsCPV) and shown to cause a chronic infection confined to the cytoplasm of epithelial cells of the gastric ceca and posterior stomach. The production of large numbers of virions and inclusion bodies and their arrangement into paracrystalline arrays gives the gut of infected insects a distinctive blue iridescence. The virions, which were examined by electron microscopy, are icosahedral (55 to 65 nm in diameter) with a central core that is surrounded by a single capsid layer. They are usually packaged individually within cubic inclusion bodies (polyhedra, approximately 100 nm across), although two to eight virus particles were sometimes occluded together. The virus was experimentally transmitted per os to several mosquito species. The transmission rate was enhanced by the presence of magnesium ions but was inhibited by calcium ions. Most of the infected larvae survived to adulthood, and the adults retained the infection. Electrophoretic analysis of the UsCPV genome segments (using 1% agarose gels) generated a migration pattern (electropherotype) that is different from those of the 16 Cypovirus species already recognized. UsCPV genome segment 10 (Seg-10) showed no significant nucleotide sequence similarity to the corresponding segment of the other cypoviruses that have previously been analyzed, and it has different "conserved" termini. A BLAST search of the UsCPV deduced amino acid sequence also showed little similarity to Antheraea mylitta CPV-4 (67 of 290 [23%]) or Choristoneura fumiferana CPV-16 (33 of 111 [29%]). We conclude that UsCPV should be recognized as a member of a new Cypovirus species (Cypovirus 17, strain UsCPV-17).     

Midgut Microbiota of the Malaria Mosquito Vector Anopheles gambiae and Interactions with Plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.     

First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.     

Organization and evolution of heterochromatin in malaria mosquitoes

The African malaria mosquito Anopheles gambiae was the first disease vector chosen for genome sequencing. Although its genome assembly has been facilitated by physical mapping, large gaps still pose a serious problem for accurate annotation and genome analysis. The majority of the gaps are located in regions of pericentromeric and intercalary heterochromatin. Genomic analysis has identified protein-coding genes and various classes of repetitive elements in the Anopheles heterochromatin. Molecular and cytogenetic studies have demonstrated that heterochromatin is a structurally heterogeneous and rapidly evolving part of the malaria mosquito genome.     

Effects of insect hormones on hemagglutination activity in two members of the Culex pipiens complex

Effects of methoprene and 20-hydroxyecdysone on the development and hemagglutination activity (HA) were studied in both sexes of two members of the Culex pipiens complex-anautogenous C. p. quinquefasciatus and autogenous C. p. molestus. Juvenile hormone analogue (methoprene) and 20-hydroxyecdysone caused developmental changes in both mosquito strains. High larval mortality, prolongation of intermolt period in each larval instar and in the pupal stage, and morphological changes in the larval-pupal and pupal-adult transformations were also observed. Developmental changes were accompanied with some differences in the HA. HA was found in both sexes of both experimental mosquito strains. The juvenile hormone analogue used in the larval stage caused significant decrease of HA in the gut of adults of both sexes. On the other hand, 20-hydroxyecdysone decreased HA only in the female gut. Results obtained indicate that HA depends on the sex, the studied organ, and the level of hormones.     

Expression of mosquito-larvicidal toxin genes under the control of a native promoter in Enterobacter amnigenus An11

Enterobacter amnigenus An11, that can colonize the gut of mosquito larva, is an alternative toxin-producing host to be used as a mosquito control since it is able to float in the feeding zone of mosquito larvae. To produce mosquito-larvicidal toxins in this bacterium, a native promoter has been identified from its genomic DNA. The promoter exhibited consensus sequences for ?35 and ?10 regions of bacterial promoters and constitutively drove the expression of gfp. This promoter was inserted into recombinant plasmids upstream of promoter-free cyt2Aa2 from Bacillus thuringiensis and mtx2 from Bacillus sphaericus. Results demonstrated that Cyt2Aa2 and Mtx2 are constitutively produced without induction. The recombinant E. amnigenus showed toxicity against mosquito larvae, demonstrating a potential to be applied in a mosquito control program.     

In vivo imaging of malaria parasites--recent advances and future directions

A new view into the life of malaria parasites is now possible owing to recent advances in imaging techniques and to the generation of tagged parasites. Insights into how parasites interact with their insect vectors and mammalian hosts have been gained by the study of various parasitic forms in their natural environment. Quantitative analysis of Plasmodium ookinete motility has revealed different modes of motility in parasite invasion of the mosquito gut and the extrusion of invaded gut cells from the epithelium. Similar analysis with Plasmodium sporozoites has revealed the importance of parasite motility in transmission from the mosquito vector to the mammalian host.     

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo

When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.     

Mosquito transposable elements

The completion of the genome assembly for the African malaria mosquito, Anopheles gambiae, and continuing genomic efforts for the yellow fever mosquito, Aedes aegypti, have allowed the use of bioinformatics tools to identify and characterize a diverse array of transposable elements (TEs) in these and other mosquito genomes. An overview of the types and number of both RNA-mediated and DNA-mediated TEs that are found in mosquito genomes is presented. A number of novel and interesting TEs from these species are discussed in more detail. These findings have significant implications for our understanding of mosquito genome evolution and for future modifications of natural mosquito populations through the use of TE-mediated genetic transformation.