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.

Human semen is heterogeneous in quality, not only between males but also within a single ejaculate. Differences in quality are evident, both when examining the classical parameters of sperm number, motility and morphology and in the integrity of the sperm nucleus. The aim of this study was to determine the efficiency of the PureSperm^®, Percoll^® and swim-up preparation techniques to eliminate spermatozoa with nuclear anomalies. Semen samples were collected, washed and one part of the semen spread on a slide, the remainder was prepared using the swim-up, PureSperm^® or Percoll^® techniques. Spermatozoa from different fractions were fixed on slides and assessed. Sperm samples (n) from different men were stained using the chromomycin A₃ (CMA₃) fluorochrome, which indirectly demonstrates a decreased presence of protamine (n = 31 for swim-up; n = 45 for PureSperm^®; n = 39 for Percoll^®). Spermatozoa prepared using PureSperm^® (n = 35) and Percoll^® (n = 37) were also examined for the presence of endogenous DNA nicks. Good quality spermatozoa should not possess DNA nicks and not stain (i.e. fluoresce) with CMA₃. When prepared using the swim-up technique the spermatozoa recovered showed no significant improvement with the CMA₃ staining. When spermatozoa were prepared using the PureSperm^® and Percoll^® techniques, a significant (P < 0.001) decrease in both CMA₃ positivity and DNA strand breakage was observed. These results indicate that both the PureSperm^® and Percoll^® techniques can enrich the sperm population by separating out those with nicked DNA and with poorly condensed chromatin.

Summary: Bacterial symbionts of insects have been proposed for blocking transmission of vector‐borne pathogens. However, in many vector models the ecology of symbionts and their capability of cross‐colonizing different hosts, an important feature in the symbiotic control approach, is poorly known. Here we show that the acetic acid bacterium Asaia , previously found in the malaria mosquito vector Anopheles stephensi , is also present in, and capable of cross‐colonizing other sugar‐feeding insects of phylogenetically distant genera and orders. PCR, real‐time PCR and in situ hybridization experiments showed Asaia in the body of the mosquito Aedes aegypti and the leafhopper Scaphoideus titanus , vectors of human viruses and a grapevine phytoplasma respectively. Cross‐colonization patterns of the body of Ae. aegypti , An. stephensi and S. titanus have been documented with Asaia strains isolated from An. stephensi or Ae. aegypti , and labelled with plasmid‐ or chromosome‐encoded fluorescent proteins (Gfp and DsRed respectively). Fluorescence and confocal microscopy showed that Asaia , administered with the sugar meal, efficiently colonized guts, male and female reproductive systems and the salivary glands. The ability in cross‐colonizing insects of phylogenetically distant orders indicated that Asaia adopts body invasion mechanisms independent from host‐specific biological characteristics. This versatility is an important property for the development of symbiont‐based control of different vector‐borne diseases.

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.

Wolbachia 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. Different 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. Here 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. A 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.

Cytosine DNA methylation has been demonstrated in numerous eukaryotic organisms and has been shown to play an important role in human disease. The function of DNA methylation has been studied extensively in vertebrates, but establishing its primary role has proved difficult and controversial. Analysing methylation in insects has indicated an apparent functional diversity that seems to argue against a strict functional conservation. To investigate this hypothesis, we here assess the data reported in four different insect species in which DNA methylation has been analysed more thoroughly: the fruit fly Drosophila melanogaster , the cabbage moth Mamestra brassicae , the peach‐potato aphid Myzus persicae and the mealybug Planococcus citri .