Emanuela Clementi

Bacteria of the genus Asaia stably associate with Anopheles stephensi, an Asian malarial mosquito vector

Here, we show that an α-proteobacterium of the genus Asaia is stably associated with larvae and adults of Anopheles stephensi, an important mosquito vector of Plasmodium vivax, a main malaria agent in Asia. Asaia bacteria dominate mosquito-associated microbiota, as shown by 16S rRNA gene abundance, quantitative PCR, transmission electron microscopy and in situ-hybridization of 16S rRNA genes. In adult mosquitoes, Asaia sp. is present in high population density in the female gut and in the male reproductive tract. Asaia sp. from An. stephensi has been cultured in cell-free media and then transformed with foreign DNA. A green fluorescent protein-tagged Asaia sp. strain effectively lodged in the female gut and salivary glands, sites that are crucial for Plasmodium sp. development and transmission. The larval gut and the male reproductive system were also colonized by the transformed Asaia sp. strain. As an efficient inducible colonizer of mosquitoes that transmit Plasmodium sp., Asaia sp. may be a candidate for malaria control.

Asaia, a versatile acetic acid bacterial symbiont, capable of cross-colonizing insects of phylogenetically distant genera and orders

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.

A Novel Bacteroidetes Symbiont Is Localized in Scaphoideus titanus, the Insect Vector of Flavescence Dorée in Vitis vinifera

ABSTRACT Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, “Candidatus Phytoplasma vitis,” which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the “Candidatus Cardinium hertigii” group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of “Ca. Cardinium hertigii.” This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of “Ca. Phytoplasma vitis” were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and “Ca. Phytoplasma vitis” have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.

Multiple symbiosis in the leafhopper Scaphoideus titanus (Hemiptera: Cicadellidae): details of transovarial transmission of Cardinium sp. and yeast-like endosymbionts.

Scaphoideus titanus is the insect vector of flavescence dorée (FD), a yellow disease of grapevines. Observations on adult females and nymphs of S. titanus showed that this insect is associated with a complex microbial community. Ultrastructural analysis showed that the fat body, salivary glands and ovary of the insect harbour microorganisms showing the brush-like structure typically observed in the genus Cardinium. In particular, it has been shown that these symbiotic bacteria are present both in the follicular cells and in the eggs. In addition, cells resembling bacteriocytes, harbouring numerous Cardinium symbionts in the cytoplasm, were observed in the apical portion of the ovary in adult females. These cells are likely responsible for bacterial transmission to the ovary. Optical microscopy showed that the fat body harbours an enormous population of yeast-like symbionts (YLSs). Ultrastructural observations showed that these symbionts are enclosed within specialized cells of the fat body and are also present in the ovary, where they are found in both the follicular cells and the eggs. There is thus evidence that both Cardinium and the YLSs are transovarially transmitted to the offspring. To our knowledge, S. titanus is the sole insect known to transmit two different kinds of symbionts to the eggs, a prokaryote and an eukaryote. Gene sequence analysis and in situ hybridization led to the identification of YLSs as members of the class Sordariomycetes (=Pyrenomycetes). Finally, ultrastructural observation of the midgut content revealed the presence, in both adult females and nymphs, of a complex microbial community, which include a phytoplasma-like microorganism, likely the agent of FD.

Bacterial endosymbiont localization in Hyalesthes obsoletus, the insect vector of Bois Noir in Vitis vinifera

ABSTRACT One emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by “Candidatus Phytoplasma solani” and spread in vineyards by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the “Candidatus Sulcia muelleri” cluster in the Bacteroidetes and in the “Candidatus Purcelliella pentastirinorum” group in the Gammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to the Betaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tick Haemaphysalis longicornis, we propose the new name “Candidatus Vidania fulgoroideae.” Other bacterial endosymbionts identified in H. obsoletus were related to the intracellular bacteria Wolbachia pipientis, Rickettsia sp., and “Candidatus Cardinium hertigii.” Fluorescent in situ hybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As “Ca. Sulcia” is usually reported in association with other symbiotic bacteria, we propose that in H. obsoletus, it may occur in a bipartite or even tripartite relationship between “Ca. Sulcia” and “Ca. Purcelliella,” “Ca. Vidania,” or both.

Detection of a morphogenetically novel Sarcocystis hominis-like in the context of a prevalence study in semi-intensively bred cattle in Italy

The aim of this study was to determine the prevalence of sarcosporidiosis in semi-intensively bred cattle in northwestern Italy. A diagnostic protocol was setup in which infected animals were identified by rapid histological examination of the esophagus, diaphragm, and heart and the detected Sarcocystis spp. were subsequently typed using conventional electron microscopy in combination with molecular techniques. Sarcosporidia cysts were detected in 78.1% of the animals and were seen most often in the esophagus. The cattle is intermediate host for Sarcocystis hominis (final host, humans and some primates), Sarcocystis cruzi (final host, domestic and wild canids), and Sarcocystis hirsuta (final host, wild and domestic cats).All these three species of Sarcocystis were identified, variously associated, with the following prevalence: S. cruzi (74.2%), S. hirsuta (1.8%), and S. hominis (42.7%). Furthermore, a new S. hominis-like (prevalence 18.5%), characterized by hook-like structures of villar protrusion and a different sequence of the 18S rRNA gene, was identified. The cattle sheds testing positive for zoonotic Sarcocystis were assessed for risk factors contributing to the maintenance of the parasite’s life cycle. Significant associations emerged between consumption of raw meat by the farm owner, mountain pasturing, and absence of a sewerage system on the farm and cattle breed. Our study demonstrates that sarcosporidiosis may constitute a public health problem in Italy and indicates several issues to be addressed when planning surveillance and prevention actions. The applied diagnostic approach revealed that cattle can harbor a further type of Sarcocystis, of which life cycle and zoonotic potential should be investigated.

Bacteriocyte-like cells harbour Wolbachia in the ovary of Drosophila melanogaster (Insecta, Diptera) and Zyginidia pullula (Insecta, Hemiptera)

Wolbachia is the most widespread bacterial endosymbiont in insects. It is responsible for a variety of reproductive alterations of the hosts. Wolbachia is transmitted through the germline from mother to offspring and, in rare cases, between individuals. This implies that acquired properties (through symbiosis with Wolbachia) can become heritable. We investigated the transovarial inheritance of Wolbachia in two phylogenetically distant insects, Drosophila melanogaster and Zyginidia pullula. We detected in both systems bacteriocyte-like cells, densely packed with Wolbachia endosymbionts, at the tip of the ovarioles. Bacteriocytes are cells specialized to harbour bacteria, typical of mutualistic insect symbiosis. Our observations of bacteriocyte-like cells harbouring Wolbachia in the ovary emphasize the plasticity of the female reproductive system of insects, which maintains its function while some cells are densely colonized by bacteria. In summary, there is evidence from different insects that bacteria which behave as parasites of reproduction are harboured by cells resembling bacteriocytes, which appear to mediate transmission of the bacteria to the progeny. It seems a valid hypothesis that the bacteriocyte-like cells that we observed are not the result of a co-evolution of host and symbiont, considering that Wolbachia is not an obligatory symbiont in Drosophila and Zyginidia.

Francisella tularensis: No Evidence for Transovarial Transmission in the Tularemia Tick Vectors Dermacentor reticulatus and Ixodes ricinus

Background Tularemia is a zoonosis caused by the Francisella tularensis, a highly infectious Gram-negative coccobacillus. Due to easy dissemination, multiple routes of infection, high environmental contamination and morbidity and mortality rates, Francisella is considered a potential bioterrorism threat and classified as a category A select agent by the CDC. Tick bites are among the most prevalent modes of transmission, and ticks have been indicated as a possible reservoir, although their reservoir competence has yet to be defined. Tick-borne transmission of F. tularensis was recognized in 1923, and transstadial transmission has been demonstrated in several tick species. Studies on transovarial transmission, however, have reported conflicting results. Objective The aim of this study was to evaluate the role of ticks as reservoirs for Francisella, assessing the transovarial transmission of F. tularensis subsp. holarctica in ticks, using experimentally-infected females of Dermacentor reticulatus and Ixodes ricinus. Results Transmission electron microscopy and fluorescence in situ hybridization showed F. tularensis within oocytes. However, cultures and bioassays of eggs and larvae were negative; in addition, microscopy techniques revealed bacterial degeneration/death in the oocytes. Conclusions These results suggest that bacterial death might occur in oocytes, preventing the transovarial transmission of Francisella. We can speculate that Francisella does not have a defined reservoir, but that rather various biological niches (e.g. ticks, rodents), that allow the bacterium to persist in the environment. Our results, suggesting that ticks are not competent for the bacterium vertical transmission, are congruent with this view.

NMR as Evaluation Strategy for Cellular Uptake of Nanoparticles

Advanced nanostructured materials, such as gold nanoparticles, magnetic nanoparticles, and multifunctional materials, are nowadays used in many state-of-the-art biomedical application. However, although the engineering in this field is very advanced, there remain some fundamental problems involving the interaction mechanisms between nanostructures and cells or tissues. Here we show the potential of (1)H NMR in the investigation of the uptake of two different kinds of nanostructures, that is, maghemite and gold nanoparticles, and of a chemotherapy drug (Temozolomide) in glioblastoma tumor cells. The proposed experimental protocol provides a new way to investigate the general problem of cellular uptake for a variety of biocompatible nanostructures and drugs.

Isolation of a Trypanosome Related to Trypanosoma theileri (Kinetoplastea: Trypanosomatidae) from Phlebotomus perfiliewi (Diptera: Psychodidae)

The Trypanosoma theileri group includes several trypanosome species hardly distinguishable due to the lack of discriminating morphological characters. Trypanosomes belonging to this group have been isolated from different bovine, ovine, and cervids in Europe, Africa, Asia, and Americas. The principal vectors of the T. theileri group are considered tabanid flies; however, T. melophagium is transmitted exclusively by sheep keds. In 2016, 128 sand flies out of 2,728 trapped in Valsamoggia municipality, Italy, were individually dissected and an unknown trypanosome strain, named TrPhp1, was isolated from a female of the sand fly Phlebotomus perfiliewi. Sequence analysis placed this trypanosome in the T. theileri group with very high homology to other trypanosomes detected in European cervids. This is the first report of the T. theileri group isolation from a sand fly, and the possible role of this insect group in the trypanosome transmission cycle is discussed. Within the T. theileri group, the phylogenetic analysis distinguished several lineages, which, unfortunately, do not correspond with their host specificity and their taxonomic status remains ambiguous.