Massimo Pajoro

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.

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.

Seasonal progression of sex ratio and phytoplasma infection in Scaphoideus titanus Ball (Hemiptera: Cicadellidae).

The differences between the seasonal occurrence and likelihood of being infected by FD phytoplasmas, of male and female Scaphoideus titanus Ball, were investigated. Sex ratio (male: female) was calculated by counting males and females sampled by means of yellow sticky traps and sweep-nets and from adults derived from hatched eggs in field-collected grapevine wood. PCR essays were performed to test differences in infection between genders. Overall, the sex ratio on sticky traps was significantly more male biased (1.99:1) if compared to net sweeping (0.62:1) and laboratory rearing (0.60:1). The peak of male presence was recorded in the middle of July in laboratory rearing and sweep net, and in the middle of August on sticky traps; the maximum presence of females was detected at the end of July in laboratory rearing, and at the end of August in sweep net samplings and on sticky traps. The seasonal sex ratio was more male biased at the beginning in laboratory rearing (1.50:1) and sticky traps (9:1), and then decreased in favor of females at the end of the sampling period, both in laboratory rearing (0.17:1) and in sticky traps (0.07:1). This trend was significantly less skewed, although similar, in sweep net samplings that recorded a sex ratio of 1:1 and 0.16:1 at the beginning and at the end of the sampling period, respectively. Concerning phytoplasma detection, an interaction between gender and sampling period was observed, the males showing a peak of infected individuals later in the season (35%). Some possible behavioral explanations of the data obtained are given.

Ethiological agents of rickettsiosis and anaplasmosis in ticks collected in Emilia-Romagna region (Italy) during 2008 and 2009

Ticks are the main vectors of rickettsiae of the spotted fever group, as well as of a variety of other Rickettsiales, including bacteria of the genus Anaplasma, that might cause diseases in humans and animals. Here we present the result of a survey for ticks and for tick-associated Rickettsiales in the Emilia Romagna region (Northern Italy). The study was focused on ticks collected from wild-hunted animals. Out of 392 ticks collected from these animals, 282 (72%) were identified as Ixodes ricinus, 110 (28%) as Dermacentor marginatus. The former was found on four vertebrate species, whereas the latter appeared more specific for wild boar. The presence of rickettsiae was demonstrated in 22.5% of I. ricinus (57/253) and in 29% of D. marginatus (32/110). Five ticks of the species I. ricinus were also positive for Anaplasma phagocytophilum (2%). In addition, we collected ticks by dragging in a natural park of the same region. All of the ticks captured by dragging were identified as I. ricinus. Thirty-six out of 200 analyzed ticks proved positive for Rickettsia monacensis and R. helvetica (16.5 and 1.5%, respectively). Our results highlight that that ticks present in wild areas, widely exploited for recreation and hunting in Emilia-Romagna, represent a risk for the transmission of spotted fevers and anaplasmosis to humans.

Lyme Borreliosis, Po River Valley, Italy

We aimed to determine the presence of Ixodes ricinus ticks in heavily populated areas of the Po River Valley after report of a Lyme disease case. Eighteen percent of ticks examined from 3 locations were positive for Lyme disease borreliae. Lyme disease was diagnosed for 3 workers at risk for tick bite.

Mycobacterium avium paratuberculosis in Italy: Commensal or emerging human pathogen?

BACKGROUND Specific bacterial infections or alterations of the gut microbiota likely trigger immuno-pathological phenomena associated with Crohns disease and ulcerative colitis. Mycobacterium avium subspecies paratuberculosis is a candidate etiological agent of Crohns disease. Definitive causal connection between Mycobacterium avium subspecies paratuberculosis infection and Crohns disease has not been demonstrated. AIMS To determine the circulation of Mycobacterium avium subspecies paratuberculosis in Crohns disease patients and water supplies in an Italian region where this bacterium is endemic in cattle farms. METHODS Mycobacterium avium subspecies paratuberculosis screening was performed on biopsies from human patients, and from water samples, using two different PCR procedures. RESULTS In hospitals where multiple specimens were obtained from different sites in the intestine, the prevalence of Mycobacterium avium subspecies paratuberculosis infection was 82.1% and 40% respectively in Crohns disease and ulcerative colitis patients; in another hospital, where single specimens were obtained from patients, the bacterium was not detected. Control subjects also harboured Mycobacterium avium subspecies paratuberculosis, but at a lower prevalence. Tap water samples collected in the study area contained Mycobacterium avium subspecies paratuberculosis DNA. DISCUSSION The results of screenings for Mycobacterium avium subspecies paratuberculosis in humans are deeply influenced by both the number and location of the collected biopsies. There is a wide circulation of the organism in the study area, considering the prevalence in humans and its presence in drinking water.

Molecular screening for bacterial pathogens in ticks (Ixodes ricinus) collected on migratory birds captured in northern Italy

Migratory birds have an important role in transporting ticks and associated tick-borne pathogens over long distances. In this study, 2,793 migratory birds were captured by nets in a ringing station, located in northern Italy, and checked for the presence of ticks. Two-hundred and fifty-one ticks were identified as nymphs and larvae of Ixodes ricinus (Linnaeus, 1758) and they were PCR-screened for the presence of bacteria belonging to Borrelia burgdorferi sensu lato, Rickettsia spp., Francisella tularensis and Coxiella burnetii. Four species of Borrelia (B. garinii, B. afzelii, B. valaisiana and B. lusitaniae) and three species of Rickettsia (R. monacensis, R. helvetica and Candidatus Rickettsia mendelii) were detected in 74 (30%) and 25 (10%) respectively out of 251 ticks examined. Co-infection with Borrelia spp. and Rickettsia spp. in the same tick sample was encountered in 7 (7%) out of the 99 infected ticks. We report for the first time the presence of Candidatus Rickettsia mendelii in I. ricinus collected on birds in Italy. This study, besides confirming the role of birds in dispersal of I. ricinus, highlights an important route by which tick-borne pathogens might spread across different countries and from natural environments towards urbanised areas.