Bessem Chouaia

Plant-mediated interspecific horizontal transmission of an intracellular symbiont in insects

Intracellular reproductive manipulators, such as Candidatus Cardinium and Wolbachia are vertically transmitted to progeny but rarely show co-speciation with the host. In sap-feeding insects, plant tissues have been proposed as alternative horizontal routes of interspecific transmission, but experimental evidence is limited. Here we report results from experiments that show that Cardinium is horizontally transmitted between different phloem sap-feeding insect species through plants. Quantitative PCR and in situ hybridization experiments indicated that the leafhopper Scaphoideus titanus releases Cardinium from its salivary glands during feeding on both artificial media and grapevine leaves. Successional time-course feeding experiments with S. titanus initially fed sugar solutions or small areas of grapevine leaves followed by feeding by the phytoplasma vector Macrosteles quadripunctulatus or the grapevine feeder Empoasca vitis revealed that the symbionts were transmitted to both species. Explaining interspecific horizontal transmission through plants improves our understanding of how symbionts spread, their lifestyle and the symbiont-host intermixed evolutionary pattern.

Acetic Acid Bacteria, Newly Emerging Symbionts of Insects

ABSTRACT Recent research in microbe-insect symbiosis has shown that acetic acid bacteria (AAB) establish symbiotic relationships with several insects of the orders Diptera, Hymenoptera, Hemiptera, and Homoptera, all relying on sugar-based diets, such as nectars, fruit sugars, or phloem sap. To date, the fruit flies Drosophila melanogaster and Bactrocera oleae, mosquitoes of the genera Anopheles and Aedes, the honey bee Apis mellifera, the leafhopper Scaphoideus titanus, and the mealybug Saccharicoccus sacchari have been found to be associated with the bacterial genera Acetobacter, Gluconacetobacter, Gluconobacter, Asaia, and Saccharibacter and the novel genus Commensalibacter. AAB establish symbiotic associations with the insect midgut, a niche characterized by the availability of diet-derived carbohydrates and oxygen and by an acidic pH, selective factors that support AAB growth. AAB have been shown to actively colonize different insect tissues and organs, such as the epithelia of male and female reproductive organs, the Malpighian tubules, and the salivary glands. This complex topology of the symbiosis indicates that AAB possess the keys for passing through body barriers, allowing them to migrate to different organs of the host. Recently, AAB involvement in the regulation of innate immune system homeostasis of Drosophila has been shown, indicating a functional role in host survival. All of these lines of evidence indicate that AAB can play different roles in insect biology, not being restricted to the feeding habit of the host. The close association of AAB and their insect hosts has been confirmed by the demonstration of multiple modes of transmission between individuals and to their progeny that include vertical and horizontal transmission routes, comprising a venereal one. Taken together, the data indicate that AAB represent novel secondary symbionts of insects.

Delayed larval development in Anopheles mosquitoes deprived of Asaia bacterial symbionts

BackgroundIn recent years, acetic acid bacteria have been shown to be frequently associated with insects, but knowledge on their biological role in the arthropod host is limited. The discovery that acetic acid bacteria of the genus Asaia are a main component of the microbiota of Anopheles stephensi makes this mosquito a useful model for studies on this novel group of symbionts. Here we present experimental results that provide a first evidence for a beneficial role of Asaia in An. stephensi.ResultsLarvae of An. stephensi at different stages were treated with rifampicin, an antibiotic effective on wild-type Asaia spp., and the effects on the larval development were evaluated. Larvae treated with the antibiotic showed a delay in the development and an asynchrony in the appearance of later instars. In larvae treated with rifampicin, but supplemented with a rifampicin-resistant mutant strain of Asaia, larval development was comparable to that of control larvae not exposed to the antibiotic. Analysis of the bacterial diversity of the three mosquito populations confirmed that the level of Asaia was strongly decreased in the antibiotic-treated larvae, since the symbiont was not detectable by PCR-DGGE (denaturing gradient gel electrophoresis), while Asaia was consistently found in insects supplemented with rifampicin plus the antibiotic-resistant mutant in the diet, and in those not exposed to the antibiotic.ConclusionsThe results here reported indicate that Asaia symbionts play a beneficial role in the normal development of An. stephensi larvae.

Mosquito-Bacteria Symbiosis: The Case of Anopheles gambiae and Asaia

The symbiotic relationship between Asaia, an α-proteobacterium belonging to the family Acetobacteriaceae, and mosquitoes has been studied mainly in the Asian malaria vector Anopheles stephensi. Thus, we have investigated the nature of the association between Asaia and the major Afro-tropical malaria vector Anopheles gambiae. We have isolated Asaia from different wild and laboratory reared colonies of A. gambiae, and it was detected by PCR in all the developmental stages of the mosquito and in all the specimens analyzed. Additionally, we have shown that it localizes in the midgut, salivary glands and reproductive organs. Using recombinant strains of Asaia expressing fluorescent proteins, we have demonstrated the ability of the bacterium to colonize A. gambiae mosquitoes with a pattern similar to that described for A. stephensi. Finally, fluorescent in situ hybridization on the reproductive tract of females of A. gambiae showed a concentration of Asaia at the very periphery of the eggs, suggesting that transmission of Asaia from mother to offspring is likely mediated by a mechanism of egg-smearing. We suggest that Asaia has potential for use in the paratransgenic control of malaria transmitted by A. gambiae.

Acetobacter tropicalis Is a Major Symbiont of the Olive Fruit Fly (Bactrocera oleae)

ABSTRACT Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with Bactrocera oleae, one of the major agricultural pests in olive-producing countries. Bacterial 16S rRNA gene libraries and ultrastructural analyses revealed the presence of several bacterial taxa associated with this insect, among which Acetobacter tropicalis was predominant. The recent increased detection of acetic acid bacteria as symbionts of other insect model organisms, such as Anopheles stephensi (G. Favia et al., Proc. Natl. Acad. Sci. USA 104:9047-9051, 2007) or Drosophila melanogaster (C. R. Cox and M. S. Gilmore, Infect. Immun. 75:1565-1576, 2007), prompted us to investigate the association established between A. tropicalis and B. oleae. Using an A. tropicalis-specific PCR assay, the symbiont was detected in all insects tested originating from laboratory stocks or field-collected from different locations in Greece. This acetic acid bacterium was successfully established in cell-free medium, and typing analyses, carried out on a collection of isolates, revealed that different A. tropicalis strains are present in fly populations. The capability to colonize and lodge in the digestive system of both larvae and adults and in Malpighian tubules of adults was demonstrated by using a strain labeled with a green fluorescent protein.

Molecular Evidence for Multiple Infections as Revealed by Typing of Asaia Bacterial Symbionts of Four Mosquito Species

ABSTRACT The recent increased detection of acetic acid bacteria (AAB) of the genus Asaia as symbionts of mosquitoes, such as Anopheles spp. and Aedes spp., prompted us to investigate the diversity of these symbionts and their relationships in different mosquito species and populations. Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with four mosquito species, Anopheles stephensi, Anopheles gambiae, Aedes aegypti, and Aedes albopictus, which are important vectors of human and/or animal pathogens. Denaturing gradient gel electrophoresis (DGGE) analysis based on the 16S rRNA gene revealed the presence of several bacterial taxa, among which Asaia sequences were among the dominant in most of the samples. A collection of 281 Asaia isolates in cell-free media was established from individuals belonging to the four species. The isolates were typed by internal transcribed spacer (ITS)-PCR, tRNA-PCR, BOX-PCR, and randomly amplified polymorphic DNA (RAPD)-PCR, revealing that different Asaia strains are present in different mosquito populations, and even in single individuals.

Modulation of Fecal Clostridiales Bacteria and Butyrate by Probiotic Intervention with Lactobacillus paracasei DG Varies among Healthy Adults

BACKGROUND The modulation of gut microbiota is considered to be the first target to establish probiotic efficacy in a healthy population. OBJECTIVE This study was conducted to determine the impact of a probiotic on the intestinal microbial ecology of healthy volunteers. METHODS High-throughput 16S ribosomal RNA gene sequencing was used to characterize the fecal microbiota in healthy adults (23-55 y old) of both sexes, before and after 4 wk of daily consumption of a capsule containing at least 24 billion viable Lactobacillus paracasei DG cells, according to a randomized, double-blind, crossover placebo-controlled design. RESULTS Probiotic intake induced an increase in Proteobacteria (P = 0.006) and in the Clostridiales genus Coprococcus (P = 0.009), whereas the Clostridiales genus Blautia (P = 0.036) was decreased; a trend of reduction was also observed for Anaerostipes (P = 0.05) and Clostridium (P = 0.06). We also found that the probiotic effect depended on the initial butyrate concentration. In fact, participants with butyrate >100 mmol/kg of wet feces had a mean butyrate reduction of 49 ± 21% and a concomitant decrease in the sum of 6 Clostridiales genera, namely Faecalibacterium, Blautia, Anaerostipes, Pseudobutyrivibrio, Clostridium, and Butyrivibrio (P = 0.021), after the probiotic intervention. In contrast, in participants with initial butyrate concentrations <25 mmol/kg of wet feces, the probiotic contributed to a 329 ± 255% (mean ± SD) increment in butyrate concomitantly with an ∼55% decrease in Ruminococcus (P = 0.016) and a 150% increase in an abundantly represented unclassified Bacteroidales genus (P = 0.05). CONCLUSIONS The intake of L. paracasei DG increased the Blautia:Coprococcus ratio, which, according to the literature, can potentially confer a health benefit on the host. The probiotic impact on the microbiota and on short-chain fatty acids, however, seems to strictly depend on the initial characteristics of the intestinal microbial ecosystem. In particular, fecal butyrate concentrations could represent an important biomarker for identifying subjects who may benefit from probiotic treatment. This trial was registered at www.controlled-trials.com/isrctn as ISRCTN56945491.

Bacterial population and biodegradation potential in chronically crude oil-contaminated marine sediments are strongly linked to temperature

Two of the largest crude oil-polluted areas in the world are the semi-enclosed Mediterranean and Red Seas, but the effect of chronic pollution remains incompletely understood on a large scale. We compared the influence of environmental and geographical constraints and anthropogenic forces (hydrocarbon input) on bacterial communities in eight geographically separated oil-polluted sites along the coastlines of the Mediterranean and Red Seas. The differences in community compositions and their biodegradation potential were primarily associated (P < 0.05) with both temperature and chemical diversity. Furthermore, we observed a link between temperature and chemical and biological diversity that was stronger in chronically polluted sites than in pristine ones where accidental oil spills occurred. We propose that low temperature increases bacterial richness while decreasing catabolic diversity and that chronic pollution promotes catabolic diversification. Our results further suggest that the bacterial populations in chronically polluted sites may respond more promptly in degrading petroleum after accidental oil spills.

The yeast Wickerhamomyces anomalus (Pichia anomala) inhabits the midgut and reproductive system of the Asian malaria vector Anopheles stephensi

While symbiosis between bacteria and insects has been thoroughly investigated in the last two decades, investments on the study of yeasts associated with insects have been limited. Insect-associated yeasts are placed on different branches of the phylogenetic tree of fungi, indicating that these associations evolved independently on several occasions. Isolation of yeasts is frequently reported from insect habitats, and in some cases yeasts have been detected in the insect gut and in other organs/tissues. Here we show that the yeast Wickerhamomyces anomalus, previously known as Pichia anomala, is stably associated with the mosquito Anopheles stephensi, a main vector of malaria in Asia. Wickerhamomyces anomalus colonized pre-adult stages (larvae L(1)-L(4) and pupae) and adults of different sex and age and could be isolated in pure culture. By a combination of transmission electron microscopy and fluorescent in situ hybridization techniques, W. anomalus was shown to localize in the midgut and in both the male and female reproductive systems, suggesting multiple transmission patterns.

Mutual exclusion of Asaia and Wolbachia in the reproductive organs of mosquito vectors

BackgroundWolbachia is a group of intracellular maternally inherited bacteria infecting a high number of arthropod species. Their presence in different mosquito species has been largely described, but Aedes aegypti, the main vector of Dengue virus, has never been found naturally infected by Wolbachia. Similarly, malaria vectors and other anophelines are normally negative to Wolbachia, with the exception of an African population where these bacteria have recently been detected. Asaia is an acetic acid bacterium stably associated with several mosquito species, found as a dominant microorganism of the mosquito microbiota. Asaia has been described in gut, salivary glands and in reproductive organs of adult mosquitoes in Ae. aegypti and in anophelines. It has recently been shown that Asaia may impede vertical transmission of Wolbachia in Anopheles mosquitoes. Here we present an experimental study, aimed at determining whether there is a negative interference between Asaia and Wolbachia, for the gonad niche in mosquitoes.MethodsDifferent methods (PCR and qPCR, monoclonal antibody staining and FISH) have been used to address the question of the co-localization and the relative presence/abundance of the two symbionts. PCR and qPCR were performed to qualitatively and quantitatively verify the distribution of Asaia and Wolbachia in different mosquito species/organs. Monoclonal antibody staining and FISH were performed to localize the symbionts in different mosquito species.ResultsHere we provide evidence that, in Anopheles and in other mosquitoes, there is a reciprocal negative interference between Asaia and Wolbachia symbionts, in terms of the colonization of the gonads. In particular, we have shown that in some mosquito species the presence of one of the symbionts prevented the establishment of the second, while in other systems the symbionts were co-localized, although at reduced densities.ConclusionsA mutual exclusion or a competition between Asaia and Wolbachia may contribute to explain the inability of Wolbachia to colonize the female reproductive organs of anophelines, inhibiting its vertical transmission and explaining the absence of Wolbachia infection in Ae. aegypti and in the majority of natural populations of Anopheles mosquitoes.