Transinfection of Wolbachia wAlbB into Culex quinquefasciatus mosquitoes does not alter vector competence for Hawaiian avian malaria (Plasmodium relictum GRW4).

Avian malaria is expanding upslope with warmer temperatures and driving multiple species of Hawaiian birds towards extinction. Methods to reduce malaria transmission are urgently needed to prevent further declines. Releasing Wolbachia-infected incompatible male mosquitoes could suppress mosquito populations and releasing Wolbachia-infected female mosquitoes (or both sexes) could reduce pathogen transmission if the Wolbachia strain reduced vector competence. We cleared Culex quinquefasciatus of their natural Wolbachia pipientis wPip infection and transinfected them with Wolbachia wAlbB isolated from Aedes albopictus. We show that wAlbB infection was transmitted transovarially, and demonstrate cytoplasmic incompatibility with wild-type mosquitoes infected with wPip from Oahu and Maui, Hawaii. We measured vector competence for avian malaria, Plasmodium relictum, lineage GRW4, of seven mosquito lines (two with wAlbB; three with natural wPip infection, and two cleared of Wolbachia infection) by allowing them to feed on canaries infected with recently collected field isolates of Hawaiian P. relictum. We tested 73 groups (Ntotal = 1176) of mosquitoes for P. relictum infection in abdomens and thoraxes 6-14 days after feeding on a range of parasitemias from 0.028% to 2.49%, as well as a smaller subset of salivary glands. We found no measurable effect of Wolbachia on any endpoint, but strong effects of parasitemia, days post feeding, and mosquito strain on both abdomen and thorax infection prevalence. These results suggest that releasing male wAlbB-infected C. quinquefasciatus mosquitoes could suppress wPip-infected mosquito populations, but would have little positive or negative impact on mosquito vector competence for P. relictum if wAlbB became established in local mosquito populations. More broadly, the lack of Wolbachia effects on vector competence we observed highlights the variable impacts of both native and transinfected Wolbachia infections in mosquitoes.

Population genetic structure of Culex tritaeniorhynchus in different types of climatic zones in China.

BACKGROUND: Culex tritaeniorhynchus is widely distributed in China, from Hainan Island in the south to Heilongjiang in the north, covering tropical, subtropical, and temperate climate zones. Culex tritaeniorhynchus carries 19 types of arboviruses. It is the main vector of the Japanese encephalitis virus (JEV), posing a serious threat to human health. Understanding the effects of environmental factors on Culex tritaeniorhynchus can provide important insights into its population structure or isolation patterns, which is currently unclear. RESULTS: In total, 138 COI haplotypes were detected in the 552 amplified sequences, and the haplotype diversity (Hd) value increased from temperate (0.534) to tropical (0.979) regions. The haplotype phylogeny analysis revealed that the haplotypes were divided into two high-support evolutionary branches. Temperate populations were predominantly distributed in evolutionary branch II, showing some genetic isolation from tropical/subtropical populations and less gene flow between groups. The neutral test results of HNQH (Qionghai) and HNHK(Haikou) populations were negative (P < 0.05), indicating many low-frequency mutations in the populations and that the populations might be in the process of expansion. Moreover, Wolbachia infection was detected only in SDJN (Jining) (2.24%), and all Wolbachia genotypes belonged to supergroup B. To understand the influence of environmental factors on mosquito-borne viruses, we examined the prevalence of Culex tritaeniorhynchus infection in three ecological environments in Shandong Province. We discovered that the incidence of JEV infection was notably greater in Culex tritaeniorhynchus from lotus ponds compared to those from irrigation canal regions. In this study, the overall JEV infection rate was 15.27 per 1000, suggesting the current risk of Japanese encephalitis outbreaks in Shandong Province. CONCLUSIONS: Tropical and subtropical populations of Culex tritaeniorhynchus showed higher genetic diversity and those climatic conditions provide great advantages for the establishment and expansion of Culex tritaeniorhynchus. There are differences in JEV infection rates in wild populations of Culex tritaeniorhynchus under different ecological conditions. Our results suggest a complex interplay of genetic differentiation, population structure, and environmental factors in shaping the dynamics of Culex tritaeniorhynchus. The low prevalence of Wolbachia in wild populations may reflect the recent presence of Wolbachia invasion in Culex tritaeniorhynchus.

Fish microbiota repel ovipositing mosquitoes.

The mere presence of predators causes prey organisms to display predation-avoidance strategies. Predator presence is often communicated through predator-released chemical signals. Ovipositing female mosquitoes of several species are repelled by unknown signals released from larvivorous fish. It was previously suggested that in many cases, a predator's microbiota plays an important role in the release of these signals; however, this mechanism is still poorly understood. In this study, we looked into the effects of the microbiota originating from the larvivorous Gambusia affinis (Baird and Girard) on the oviposition behaviour of gravid female mosquitoes. We used fish with altered microbiota and bacterial isolates in a set of outdoor mesocosm experiments to address this aim. We show that interference with the fish microbiota significantly reduces fish's repellent effect. We further show that the bacterium Pantoea pleuroti, isolated from the skin of the fish, repels oviposition of Culex laticinctus Edwards and Culiseta longiareolata Macquart mosquitoes similarly to the way in which live fish repel them. Our results highlight the importance of bacteria in the interspecies interactions of their hosts. Furthermore, this finding may lead to the development of an ecologically friendly mosquito repellent, that may reduce the use of larvivorous fish for mosquito control.

Molecular identification and GC-MS analysis of a newly isolated novel bacterium (Lysinibacillus sp. VCRC B655) for mosquito control.

BACKGROUND: Mosquitoes are widespread globally and have contributed to transmitting pathogens to humans and the burden of vector-borne diseases. They are effectively controlled at their larval stages by biocontrol agents. Unravelling natural sources for microbial agents can lead us to novel potential candidates for managing mosquito-borne diseases. In the present study, an attempt was made to isolate a novel bacterium from the field-collected agricultural soil for larvicidal activity and promising bacterial metabolites for human healthcare. METHODS AND RESULTS: Field-collected soil samples from the Union territory of Puducherry, India, have been used as the source of bacteria. Isolate VCRC B655 belonging to the genus Lysinibacillus was identified by 16S rRNA gene sequencing and exhibited promising larvicidal activity against different mosquito species, including Culex (Cx.) quinquefasciatus, Anopheles (An.) stephensi, and Aedes (Ae.) aegypti. The lethal concentration (LC) of Lysinibacillus sp. VCRCB655 was observed to be high for Cx. quiquefasciatus: LC50 at 0.047 mg/l, LC90 at 0.086 mg/l, followed by An. stephensi and Ae. aegypti (LC50: 0.6952 mg/l and 0.795 mg/l) respectively. Additionally, metabolic profiling of the culture supernatant was carried out through Gas chromatography and Mass spectrophotometry (GC/MS) and identified 15 major secondary metabolites of different metabolic classes. Diketopiperazine (DKPs), notably pyro lo [1, 2-a] pyrazine1, 4-dione, are the abundant compounds reported for antioxidant activity, and an insecticide compound benzeneacetic acid was also identified. CONCLUSIONS: A new bacterial isolate, Lysinibacillus sp. VCRC B655 has been identified with significant larvicidal activity against mosquito larvae with no observed in non-target organisms. GC-MS analysis revealed diverse bioactive compounds with substantial biological applications. In conclusion, Lysinibacillus sp. VCRC B655 showed promise as an alternative biocontrol agent for mosquito vector control, with additional biological applications further enhancing its significance.

Wolbachia infection in natural mosquito populations from Argentina.

The increasing spread of mosquito vectors has made mosquito-borne arboviral diseases a global threat to public health, leading to the urgent need for effective population control methods. Strategies based in the intracellular bacterium Wolbachia Hertig, 1936 are considered environmentally friendly, safe for humans, and potentially cost-effective for controlling arboviral diseases. To minimize undesirable side effects, it is relevant to assess whether Wolbachia is present in the area and understand the diversity associated with native infections before implementing these strategies. With this purpose, we investigated Wolbachia infection status, diversity, and prevalence in populations of Aedes albifasciatus (Macquart, 1838), Aedes fluviatilis (Lutz, 1904), and hybrids of the Culex pipiens (Linnaeus, 1758) complex from Argentina. Aedes albifasciatus and C. pipiens complex samples were collected in the province of Buenos Aires, and A. fluviatilis in the province of Misiones. Aedes albifasciatus was found to be uninfected, while infections with strains wFlu and wPip were detected in A. fluviatilis and hybrids of the C. pipiens complex, respectively. All strains were fixed or close to fixation and clustered within supergroup B. These finding provides valuable information on Wolbachia strains found in natural mosquito populations in Argentina that might be used in heterologous infections in the future or be considered when designing control strategies based on Wolbachia infection.

Pseudomonas protegens Affects Mosquito Survival and Development.

This study investigated the pathogenic potential of Pseudomonas protegens on mosquito larvae of the two species Culex pipiens and Aedes albopictus, representing major threats for disease transmission in the Mediterranean area and worldwide. The bacterium achieved to kill over 90% of the mosquito larvae within 72 h after exposition to a bacterial concentration of 100 million CFU/ml. These lethal effects were concentration dependent and a significantly higher susceptibility was associated with younger larvae of both mosquito species. Significant slowdown of immature (larval and pupal) development and decrease in adult emergence rate after treatment with sub-lethal doses of the bacterium were also detected. This study reports for the first time the insecticidal activity of a root-associated biocontrol bacterium against aquatic mosquito larvae.

Cys183 and Cys258 in Cry49Aa toxin from Lysinibacillus sphaericus are essential for toxicity to Culex quinquefasciatus larvae.

The two-component Cry48Aa/Cry49Aa toxin produced by Lysinibacillus sphaericus shows specifically toxic to Culex quinquefasciatus mosquito larvae. Cry49Aa C-terminal domain is responsible for specific binding to the larval gut cell membrane, while its N-terminal domain is required for interaction with Cry48Aa. To investigate functional role of cysteine in Cry49Aa, four cysteine residues at positions 70, 91, 183, and 258 were substituted by alanine. All mutants showed similar crystalline morphology and comparable yield to that of the wild type except that the yield of the C91A mutant was low. Four cysteine residues did not involve in disulfide bond formation within or between Cry49Aa molecules. Cys91, Cys183, and Cys258 are essential for larvicidal activity against C. quinquefasciatus larvae, while Cys70 is not. Substitution at C91, C183, and C258 caused weaker Cry48Aa- Cry49Aa interaction, while mutations at C183 and C258 reduced the binding capacities to the larval gut cell membrane. Thus, Cysteine residues at position 91, 183, and 258 in Cry49Aa are required for full toxicity of Cry48Aa/Cry49Aa toxin.

Pathogenic fungi infection attributes of malarial vectors Anopheles maculipennis and Anopheles superpictus in central Iran.

BACKGROUND: Due to the effect of synthetic and commercial insecticides on non-target organisms and the resistance of mosquitoes, non-chemical and environmentally friendly methods have become prevalent in recent years. The present study was to isolate entomopathogenic fungi with toxic effects on mosquitoes in natural larval habitats. METHODS: Larvae of mosquitoes were collected from Central, Qamsar, Niasar, and Barzok Districts in Kashan County, Central Iran by standard dipping method, from April to late December 2019. Dead larvae, live larvae showing signs of infection, and larvae and pupae with a white coating of fungal mycelium on the outer surface of their bodies were isolated from the rest of the larvae and sterilized with 10% sodium hypochlorite for 2 min, then washed twice with distilled water and transferred to potato-dextrose-agar (PDA) and water-agar (WA) media and incubated at 25 ± 2 °C for 3-4 days. Larvae and fungi were identified morphologically based on identification keys. RESULTS: A total of 9789 larvae were collected from urban and rural areas in Kashan County. Thirteen species were identified which were recognized to belong to three genera, including Anopheles (7.89%), Culiseta (17.42%) and Culex (74.69%). A total of 105 larvae, including Anopheles superpictus sensu lato (s.l), Anopheles maculipennis s.l., Culex deserticola, Culex perexiguus, and Culiseta longiareolata were found to be infected by Nattrassia mangiferae, Aspergillus niger, Aspergillus fumigatus, Trichoderma spp., and Penicillium spp. Of these, Penicillium spp. was the most abundant fungus isolated and identified from the larval habitats, while An. superpictus s.l. was the most infected mosquito species. CONCLUSIONS: Based on the observations and results obtained of the study, isolated fungi had the potential efficacy for pathogenicity on mosquito larvae. It is suggested that their effects on mosquito larvae should be investigated in the laboratory. The most important point, however, is the proper way of exploiting these biocontrol agents to maximize their effect on reducing the population of vector mosquito larvae without any negative effect on non-target organisms.

Preliminary analysis of whether mosquitoes can carry and transmit African swine fever.

BACKGROUND: Mosquitoes are important insect vectors, but whether they can carry and transmit African swine fever virus (ASFV) in large-scale pig farms in China is unknown. RESULTS: In this study, probe-based qPCR analysis was performed on mosquitoes from five pig farms with ASF virus (ASFV). Analysis of ASFV in 463 mosquitoes yielded negative cycle threshold (CT) value), and detection remained negative after mixing samples from all five pig farms. CONCLUSIONS: Therefore, mosquitoes appear unlikely to transmit ASFV, and pose little threat to large-scale pig farms. Thus, farms should continue to follow normal mosquito control procedures when formulating strategies for the prevention and control of ASF.

The cellular phenotype of cytoplasmic incompatibility in Culex pipiens in the light of cidB diversity.

Wolbachia are maternally inherited endosymbiotic bacteria, widespread among arthropods thanks to host reproductive manipulations that increase their prevalence into host populations. The most commonly observed manipulation is cytoplasmic incompatibility (CI). CI leads to embryonic death in crosses between i) infected males and uninfected females and ii) individuals infected with incompatible Wolbachia strains. CI can be conceptualized as a toxin-antidote system where a toxin deposited by Wolbachia in the sperm would induce embryonic death unless countered by an antidote produced by Wolbachia present in the eggs. In Drosophila melanogaster, transgenic expression of Wolbachia effector cidB revealed its function of CI-inducing toxin. Moreover in Culex pipiens, the diversity of cidB variants present in wPip strains accounts for the diversity in crossing-types. We conducted cytological analyses to determine the CI mechanisms that lead to embryonic death in C. pipiens, and assess whether diversity in crossing-types could be based on variations in these mechanisms. We revealed that paternal chromatin condensation and segregation defects during the first embryonic division are always responsible for embryonic death. The strongest observed defects lead to an exclusion of the paternal chromatin from the first zygotic division, resulting in haploid embryos unable to hatch. The proportion of unhatched haploid embryos, developing with only maternal chromatin, which reflects the frequency of strong defects can be considered as a proxy of CI intensity at the cellular level. We thus studied the putative effect of variations in crossing types and cidB diversification on CI defects intensity. Incompatible crosses involving distinct wPip strains revealed that CI defects intensity depends on the Wolbachia strains hosted by the males and is linked to the diversity of cidB genes harbored in their genomes. These results support that, additionally to its implication in C. pipiens crossing type variability, cidB diversification also influences the strength of CI embryonic defects.

Wolbachia transinfections in Culex quinquefasciatus generate cytoplasmic incompatibility.

Culex quinquefasciatus is an important mosquito vector of a number of viral and protozoan pathogens of humans and animals, and naturally carries the endosymbiont Wolbachia pipientis, strain wPip. Wolbachia are used in two distinct vector control strategies: firstly, population suppression caused by mating incompatibilities between mass-released transinfected males and wild females; and secondly, the spread of pathogen transmission-blocking strains through populations. Using embryonic microinjection, two novel Wolbachia transinfections were generated in C. quinquefasciatus using strains native to the mosquito Aedes albopictus: a wAlbB single infection, and a wPip plus wAlbA superinfection. The wAlbB infection showed full bidirectional cytoplasmic incompatibility (CI) with wild-type C. quinquefasciatus in reciprocal crosses. The wPipwAlbA superinfection showed complete unidirectional CI, and therefore population invasion potential. Whereas the wAlbB strain showed comparatively low overall densities, similar to the native wPip, the wPipwAlbA superinfection reached over 400-fold higher densities in the salivary glands compared to the native wPip, suggesting it may be a candidate for pathogen transmission blocking.

Novel Wolbachia-transinfected Aedes aegypti mosquitoes possess diverse fitness and vector competence phenotypes.

Wolbachia pipientis from Drosophila melanogaster (wMel) is an endosymbiotic bacterium that restricts transmission of human pathogenic flaviviruses and alphaviruses, including dengue, Zika, and chikungunya viruses, when introduced into the mosquito vector Aedes aegypti. To date, wMel-infected Ae. aegypti have been released in field trials in 5 countries to evaluate the effectiveness of this strategy for disease control. Despite the success in establishing wMel-infected mosquitoes in wild populations, and the well-characterized antiviral capabilities of wMel, transinfecting different or additional Wolbachia strains into Ae. aegypti may improve disease impact, and perhaps more importantly, could provide a strategy to account for the possible evolution of resistant arboviruses. Here, we report the successful transinfection of Ae. aegypti with the Wolbachia strains wMelCS (D. melanogaster), wRi (D. simulans) and wPip (Culex quinquefasciatus) and assess the effects on Ae. aegypti fitness, cytoplasmic incompatibility, tissue tropism and pathogen blocking in a laboratory setting. The results demonstrate that wMelCS provides a similar degree of protection against dengue virus as wMel following an infectious blood meal, and significantly reduces viral RNA levels beyond that of wMel following a direct challenge with infectious virus in mosquitoes, with no additional fitness cost to the host. The protection provided by wRi is markedly weaker than that of wMelCS, consistent with previous characterisations of these lines in Drosophila, while wPip was found to substantially reduce the fitness of Ae. aegypti. Thus, we determine wMelCS as a key candidate for further testing in field-relevant fitness tests and viremic blood feeding challenges in a clinical setting to determine if it may represent an alternative Wolbachia strain with more desirable attributes than wMel for future field testing.

Variation in Wolbachia cidB gene, but not cidA, is associated with cytoplasmic incompatibility mod phenotype diversity in Culex pipiens.

Endosymbiotic Wolbachia bacteria are, to date, considered the most widespread symbionts in arthropods and are the cornerstone of major biological control strategies. Such a high prevalence is based on the ability of Wolbachia to manipulate their hosts' reproduction. One manipulation called cytoplasmic incompatibility (CI) is based on the death of the embryos generated by crosses between infected males and uninfected females or between individuals infected with incompatible Wolbachia strains. CI can be seen as a modification-rescue system (or mod-resc) in which paternal Wolbachia produce mod factors, inducing embryonic defects, unless the maternal Wolbachia produce compatible resc factors. Transgenic experiments in Drosophila melanogaster and Saccharomyces cerevisiae converged towards a model where the cidB Wolbachia gene is involved in the mod function while cidA is involved in the resc function. However, as cidA expression in Drosophila males was required to observe CI, it has been proposed that cidA could be involved in both resc and mod functions. A recent correlative study in natural Culex pipiens mosquito populations has revealed an association between specific cidA and cidB variations and changes in mod phenotype, also suggesting a role for both these genes in mod diversity. Here, by studying cidA and cidB genomic repertoires of individuals from newly sampled natural C. pipiens populations harbouring wPipIV strains from North Italy, we reinforce the link between cidB variation and mod phenotype variation fostering the involvement of cidB in the mod phenotype diversity. However, no association between any cidA variants or combination of cidA variants and mod phenotype variation was observed. Taken together our results in natural C. pipiens populations do not support the involvement of cidA in mod phenotype variation.

Enhancing Nanos expression via the bacterial TomO protein is a conserved strategy used by the symbiont Wolbachia to fuel germ stem cell maintenance in infected Drosophila females.

The toxic manipulator of oogenesis (TomO) protein has been identified in the wMel strain of Wolbachia that symbioses with the vinegar fly Drosophila melanogaster, as a protein that affects host reproduction. TomO protects germ stem cells (GSCs) from degeneration, which otherwise occurs in ovaries of host females that are mutant for the gene Sex-lethal (Sxl). We isolated the TomO homologs from wPip, a Wolbachia strain from the mosquito Culex quinquefasciatus. One of the homologs, TomOwPip 1, exerted the GSC rescue activity in fly Sxl mutants when lacking its hydrophobic stretches. The GSC-rescuing action of the TomOwPip 1 variant was ascribable to its abilities to associate with Nanos (nos) mRNA and to enhance Nos protein expression. The analysis of structure-activity relationships with TomO homologs and TomO deletion variants revealed distinct modules in the protein that are each dedicated to different functions, i.e., subcellular localization, nos mRNA binding or Nos expression enhancement. We propose that modular reshuffling is the basis for structural and functional diversification of TomO protein members.

Culex torrentium mosquitoes from Germany are negative for Wolbachia.

Wolbachia (Rickettsiales: Anaplasmataceae) infects a wide range of arthropods, including several mosquito species. The bacterium is known to induce a plethora of phenotypes in its host, examples being the reproductive phenotype cytoplasmic incompatibility or resistance against infection with arboviruses. The latter is especially relevant when assessing the vector competence of mosquito species for emerging arboviruses. Thus, knowledge of Wolbachia infection status is important for the assessment of vector competence. To facilitate Wolbachia screening in mosquito populations, a quantitative polymerase chain reaction (qPCR) assay was developed to enable high-throughput analysis of mosquito samples. Using this assay, the Wolbachia infection status of the two most common Culex mosquito species in Germany, Culex pipiens biotype pipiens Linnaeus (Diptera: Culicidae) and Culex torrentium Martini (Diptera: Culicidae), was assessed. About 93% of all tested C. pipiens biotype pipiens individuals were positive for Wolbachia, whereas none of the C. torrentium samples was found to be infected. Furthermore, other applications of the qPCR assay were explored by assessing a potential link between the levels of Wolbachia and West Nile virus (WNV) infections in German C. pipiens biotype pipiens mosquitoes. No relationship was found between the two variables, indicating that a Wolbachia-induced antiviral phenotype in this mosquito population is not exclusively attributable to the general level of bacterial infection.

Detection and distribution of Wolbachia endobacteria in Culex quinquefasciatus populations (Diptera : Culicidae) from Metropolitan Manila, Philippines.

BACKGROUND & OBJECTIVES: Culex quinquefasciatus is a peridomestic mosquito known for its ability to transmit pathogenic diseases such as filariasis and Japanese encephalitis. The development and use of novel and innovative vector control measures such as the utilization of Wolbachia, along with the existing ones, are necessary to prevent the transmission of these diseases. Studies exploring the diversity of Wolbachia, particularly in Cx. quinquefasciatus are very limited in the Philippines. Thus, the aim of the study was to detect the presence, distribution, and phylogenetic relationship of Wolbachia infections in Cx. quinquefasciatus in Metropolitan Manila, Philippines. METHODS: Adult Cx. quinquefasciatus mosquitoes were collected using a commercially available light-trap from May 2014-January 2015. Based on their sampling grids (n = 51), the adult mosquito abdomens were pooled and subjected to Wolbachia surface protein (wsp) gene amplification assay. Five selected wsp-positive samples were then sequenced and further analyzed to infer their phylogenetic relationship with known Wolbachia strains. RESULTS: A total of 1090 adult Cx. quinquefasciatus mosquitoes were collected. Pooled abdomens (n = 53) were then sorted based on their sampling grids for subsequent screening of wsp gene. Wolbachia infection rate was 59% (31/53). These infections were located at 29 (57%) sampling grids, and were observed to be widely distributed in the study area. Phylogenetic analysis indicated that the sample sequences were Wolbachia pipientis isolated from known hosts, Cx. pipiens and Cx. quinquefasciatus belonging to supergroup B clade. INTERPRETATION & CONCLUSION: The study was able to demonstrate the prevalence and distribution of Wolbachia in Cx. quinquefasciatus in Metropolitan Manila, Philippines. The findings of this study are geared towards proposing a vector control program that utilizes the potential of Wolbachia as a biological control agent in preventing the transmission of Culex-borne diseases.

Temporal Variations of Microbiota Associated with the Immature Stages of Two Florida Culex Mosquito Vectors.

Microbiota associated with mosquito vector populations impact several traits of mosquitoes, including survival, reproduction, control, and immunity against pathogens. The influence of seasonal variations and mosquito species on mosquito gut microbiota is poorly understood. We sought to determine whether the mosquito microbiota associated with immature stages of two congeners (Culex coronator and Culex nigripalpus) differ temporally and between the two species. Using high throughput 16S rRNA gene sequence analysis, we characterized bacterial and archaeal communities found in the immature stages of the two Culex mosquito species sampled over three seasons to compare the diversity of bacteria between the two species. Beta diversity analyses of the larval microbiota sequences revealed that the two Culex species differed significantly, both temporally within each species and between the two species. Bacteria in Cx. coronator larvae were dominated by Alphaproteobacteria, mainly associated with Roseoccocus and unidentified species of Rhizobiales, and two unidentified species of Cyanobacteria. In contrast, Cx. nigripalpus was dominated by Thorsellia anophelis (Gammaproteobacteria), Clostridium, an unidentified species of Ruminococcacae (Clostridiales), and additional unidentified species associated with Erysipelotrichaceae (Erysipelotrichales), Bacteroidales, and Mollicutes. Results of our study revealed both seasonal and interspecies differences in bacterial community composition associated with the immature stages of Cx. coronator and Cx. nigripalpus vector populations in Florida. These results have important implications for our understanding of the underlying factors of variations in disease transmission among seasons, susceptibility to various pesticides, and other biotic factors, including the role of the microbiota on the spread of invasive species. In addition, our results suggest close associations of certain bacteria species with each of the two Culex species that will be further targeted for their potential in the development of microbial-based control strategies.

Towards novel Cry toxins with enhanced toxicity/broader: a new chimeric Cry4Ba / Cry1Ac toxin.

Attempts have been made to express or to merge different Cry proteins in order to enhance toxic effects against various insects. Cry1A proteins of Bacillus thuringiensis form a typical bipyramidal parasporal crystal and their protoxins contain a highly conserved C-terminal region. A chimerical gene, called cry(4Ba-1Ac), formed by a fusion of the N-terminus part of cry4Ba and the C-terminus part of cry1Ac, was constructed. Its transformation to an acrystalliferous B. thuringiensis strain showed that it was expressed as a chimerical protein of 116 kDa, assembled in spherical to amorphous parasporal crystals. The chimerical gene cry(4Ba-1Ac) was introduced in a B. thuringiensis kurstaki strain. In the generated crystals of the recombinant strain, the presence of Cry(4Ba-1Ac) was evidenced by MALDI-TOF. The recombinant strain showed an important increase of the toxicity against Culex pipiens larvae (LC50 = 0.84 mg l-1 ± 0.08) compared to the wild type strain through the synergistic activity of Cry2Aa with Cry(4Ba-1Ac). The enhancement of toxicity of B. thuringiensis kurstaki expressing Cry(4Ba-1Ac) compared to that expressing the native toxin Cry4Ba, might be related to its a typical crystallization properties. The developed fusion protein could serve as a potent toxin against different pests of mosquitoes and major crop plants.

Bacterial diversity of cosmopolitan Culex pipiens and invasive Aedes japonicus from Germany.

Symbiotic bacteria have gained significant attention in recent years. For example, microbiota of some mosquito species seems to influence the development and transmission of pathogens. Furthermore, several attempts using bacteria as a paratransgenetic tool have been made in order to assist the control of mosquito-borne diseases. In this study, we examined the bacterial diversity of wild-caught adult Culex (Cx.) pipiens and laboratory-reared adult Aedes japonicus (Ae. japonicus) in Germany using a culture-independent method. Genomic DNA was extracted from each specimen and submitted to PCR amplification of eubacterial 16S rDNA. After the cloning reaction, 28 bacterial transformants per sample containing the 16S rDNA inserts were selected per each sample for sequencing. The analysed specimens of Cx. pipiens as well as of Ae. japonicus showed a diverse bacterial community including some common bacterial genera. Blast analysis allowed to identify 21 bacterial genera belonging to 2 phyla among the 23 specimens of Cx. pipiens. The 14 analysed Ae. japonicus revealed 11 bacterial genera belonging to 3 phyla. In both mosquito species, identified isolates were mainly Proteobacteria. Only 4 of the bacterial genera were found in both mosquito species, with the most prevalent genera Sphingomonas and Rahnella in Cx. pipiens and in Ae. japonicus respectively. Most of the bacterial genera found in our study have been identified in other mosquito species before. Due to the currently scarce data situation, ongoing examinations on the very abundant bacterial genera or species are strongly required to determine their relevance for the biology and adaptiveness of mosquitoes including pathogen-host relationship.

Resistance in bacteria and indirect beta-lactamase detection in E. coli isolated from Culex pipiens detected by matrix-assisted laser desorption ionization time of flight mass spectrometry.

The aim of this study was detections of antibiotic resistance and resistance mechanism in bacteria isolated from mosquitos (Culex pipiens) living near humans. Therefore, antibiotic resistance in bacteria isolated from Culex pipiens was investigated by disk diffusion test and MIC E-test in this study. MALDI-TOF mass spectrometry was used for detection of resistant mechanism. In this study, hydrolytic breakdown products after a few hours of incubation of the bacteria isolated from Culex pipiens were detected. Results show that enzymatic destruction of ampicillin by beta-lactamases is able to be detected by MALDI-TOF mass spectrometry from wild strains of potential pathogens. The MALDI-TOF mass spectrometry is useful method for routine detection of beta-lactamases resistant mechanism, but overnight incubation of pure culture is necessary. The results are important for proper and fast intervention to limit the spread of beta-lactamase-producing wild bacteria and provide information for appropriate initial therapy of the infections caused by these microbes.