Population dynamics of plague vector fleas in an endemic focus: implications for plague surveillance.

Plague is a zoonotic vector-borne disease caused by the bacterium Yersinia pestis. In Madagascar, it persists in identified foci, where it is a threat to public health generally from September to April. A more complete understanding of how the disease persists could guide control strategies. Fleas are the main vector for transmission between small mammal hosts and humans, and fleas likely play a role in the maintenance of plague. This study characterized the dynamics of flea populations in plague foci alongside the occurrence of human cases. From 2018 to 2020, small mammals were trapped at sites in the central Highlands of Madagascar. A total of 2,762 small mammals were captured and 5,295 fleas were collected. The analysis examines 2 plague vector species in Madagascar (Synopsyllus fonquerniei and Xenopsylla cheopis). Generalized linear models were used to relate flea abundance to abiotic factors, with adjustments for trap location and flea species. We observed significant effects of abiotic factors on the abundance, intensity, and infestation rate by the outdoor-associated flea species, S. fonquerniei, but weak seasonality for the indoor-associated flea species, X. cheopis. A difference in the timing of peak abundance was observed between the 2 flea species during and outside the plague season. While the present study did not identify a clear link between flea population dynamics and plague maintenance, as only one collected X. cheopis was infected, the results presented herein can be used by local health authorities to improve monitoring and control strategies of plague vector fleas in Madagascar.

The most polyphagous insect herbivore? Host plant associations of the Meadow spittlebug, Philaenus spumarius (L.).

A comprehensive list of all known host plant species utilised by the Meadow Spittlebug (Philaenus spumarius (L.)) is presented, compiled from published and unpublished sources. P. spumarius feeds on 1311 host plants in 631 genera and 117 families. This appears, by a large margin, to be the greatest number of host species exploited by any herbivorous insect. The Asteraceae (222 species) and Rosaceae (110) together account for 25% of all host species. The Fabaceae (76) and Poaceae (73), are nearly tied for third and fourth place and these four families, combined with the Lamiaceae (62), Apiaceae (50), Brassicaceae (43) and Caprifoliaceae (34), comprise about half of all host species. Hosts are concentrated among herbaceous dicots but range from ferns and grasses to shrubs and trees. Philaenus spumarius is an "extreme polyphage", which appears to have evolved from a monophage ancestor in the past 3.7 to 7.9 million years. It is also the primary European vector of the emerging plant pathogen Xylella fastidiosa. Its vast host range suggests that it has the potential to spread X. fastidiosa among multiple hosts in any environment in which both the spittlebug and bacterium are present. Fully 47.9% of all known hosts were recorded in the Xylella-inspired BRIGIT citizen science P. spumarius host survey, including 358 hosts new to the documentary record, 27.3% of the 1311 total. This is a strong demonstration of the power of organized amateur observers to contribute to scientific knowledge.

Crossing the Gateless Barriers: Factors Involved in the Movement of Circulative Bacteria Within Their Insect Vectors.

Plant bacterial pathogens transmitted by hemipteran vectors pose a large threat to the agricultural industry worldwide. Although virus-vector relationships have been widely investigated, a significant gap exists in our understanding of the molecular interactions between circulative bacteria and their insect vectors, mainly leafhoppers and psyllids. In this review, we will describe how these bacterial pathogens adhere, invade, and proliferate inside their insect vectors. We will also highlight the different transmission routes and molecular factors of phloem-limited bacteria that maintain an effective relationship with the insect host. Understanding the pathogen-vector relationship at the molecular level will help in the management of vector-borne bacterial diseases.

A Widefield Light Microscopy-Based Approach Provides Further Insights into the Colonization of the Flea Proventriculus by Yersinia pestis.

Yersinia pestis (the agent of flea-borne plague) must obstruct the flea's proventriculus to maintain transmission to a mammalian host. To this end, Y. pestis must consolidate a mass that entrapped Y. pestis within the proventriculus very early after its ingestion. We developed a semiautomated fluorescent image analysis method and used it to monitor and compare colonization of the flea proventriculus by a fully competent flea-blocking Y. pestis strain, a partially competent strain, and a noncompetent strain. Our data suggested that flea blockage results primarily from the replication of Y. pestis trapped in the anterior half of the proventriculus. However, consolidation of the bacteria-entrapping mass and colonization of the entire proventricular lumen increased the likelihood of flea blockage. The data also showed that consolidation of the bacterial mass is not a prerequisite for colonization of the proventriculus but allowed Y. pestis to maintain itself in a large flea population for an extended period of time. Taken as the whole, the data suggest that a strategy targeting bacterial mass consolidation could significantly reduce the likelihood of Y. pestis being transmitted by fleas (due to gut blockage), but also the possibility of using fleas as a long-term reservoir. IMPORTANCE Yersinia pestis (the causative agent of plague) is one of the deadliest bacterial pathogens. It circulates primarily among rodent populations and their fleas. Better knowledge of the mechanisms leading to the flea-borne transmission of Y. pestis is likely to generate strategies for controlling or even eradicating this bacillus. It is known that Y. pestis obstructs the flea's foregut so that the insect starves, frantically bites its mammalian host, and regurgitates Y. pestis at the bite site. Here, we developed a semiautomated fluorescent image analysis method and used it to document and compare foregut colonization and disease progression in fleas infected with a fully competent flea-blocking Y. pestis strain, a partially competent strain, and a noncompetent strain. Overall, our data provided new insights into Y. pestis' obstruction of the proventriculus for transmission but also the ecology of plague.

Bacterial Endosymbionts Identified From Leafhopper (Hemiptera: Cicadellidae) Vectors of Phytoplasmas.

Insects often harbor bacterial endosymbionts that provide them with nutritional benefit or with protection against natural enemies, plant defenses, insecticides, and abiotic stresses. Certain endosymbionts may also alter acquisition and transmission of plant pathogens by insect vectors. We identified bacterial endosymbionts from four leafhopper vectors (Hemiptera: Cicadellidae) of 'Candidatus Phytoplasma' species by direct sequencing 16S rDNA and confirmed endosymbiont presence and identity by species-specific conventional PCR. We examined three vectors of Ca. Phytoplasma pruni, causal agent of cherry X-disease [Colladonus geminatus (Van Duzee), Colladonus montanus reductus (Van Duzee), Euscelidius variegatus (Kirschbaum)] - and a vector of Ca. Phytoplasma trifolii, the causal agent of potato purple top disease [Circulifer tenellus (Baker)]. Direct sequencing of 16S identified the two obligate endosymbionts of leafhoppers, 'Ca. Sulcia' and 'Ca. Nasuia', which are known to produce essential amino acids lacking in the leafhoppers' phloem sap diet. About 57% of C. geminatus also harbored endosymbiotic Rickettsia. We identified 'Ca. Yamatotoia cicadellidicola' in Euscelidius variegatus, providing just the second host record for this endosymbiont. Circulifer tenellus harbored the facultative endosymbiont Wolbachia, although the average infection rate was only 13% and all males were Wolbachia-uninfected. A significantly greater percentage of Wolbachia-infected Ci. tenellus adults than uninfected adults carried Ca. P. trifolii, suggesting that Wolbachia may increase this insect's ability to tolerate or acquire this pathogen. Results of our study provide a foundation for continued work on interactions between leafhoppers, bacterial endosymbionts, and phytoplasma.

Flavescence dorée phytoplasma enters insect cells by a clathrin-mediated endocytosis allowing infection of its insect vector.

To perform its propagative and circulative cycle into its insect vector, the flavescence dorée phytoplasma invades different cell types. Clathrin-mediated endocytosis is used by a wide range of bacteria to infect eukaryote cells. Among the insect proteins interacting with the phytoplasma adhesin VmpA, we identified the adaptor protein complex AP-1 and AP-2 suggesting that phytoplasmas could enter the insect cells via clathrin-mediated endocytosis. By infection assays of insect cells in culture, we showed that phytoplasmas entry into Drosophila S2 cells was more efficient than infection of the Euva cell line developed from the insect vector Euscelidius variegatus. Chlorpromazine, cytochalasin D and knockdown of clathrin heavy chain (chc) gene expression using RNA interference inhibited entry of phytoplasmas into S2 cells. During invasion of S2 cells, phytoplasmas were observed very closed to recombinant GFP-labelled clathrin light chain. To verify the role of clathrin in the insect colonization by phytoplasmas, RNAi was performed via artificial feeding of chc dsRNA by the vector E. variegatus. This decreased the expression of chc gene in the midgut and heads of E. variegatus. The chc lower expression correlated to a decreased of midgut and salivary gland cells colonization after the insects had ingested phytoplasmas from infected plants. In conclusion, results indicate that clathrin is important for the FD phytoplasma to enter insect cells and colonize its insect vector.

A Compartmental Model for Xylella fastidiosa Diseases with Explicit Vector Seasonal Dynamics.

The bacterium Xylella fastidiosa is mainly transmitted by the meadow spittlebug Philaenus spumarius in Europe, where it has caused significant economic damage to olive and almond trees. Understanding the factors that determine disease dynamics in pathosystems that share similarities can help to design control strategies focused on minimizing transmission chains. Here, we introduce a compartmental model for X. fastidiosa-caused diseases in Europe that accounts for the main relevant epidemiological processes, including the seasonal dynamics of P. spumarius. The model was confronted with epidemiological data from the two major outbreaks of X. fastidiosa in Europe, the olive quick disease syndrome in Apulia, Italy, caused by the subspecies pauca, and the almond leaf scorch disease in Mallorca, Spain, caused by subspecies multiplex and fastidiosa. Using a Bayesian inference framework, we show how the model successfully reproduces the general field data in both diseases. In a global sensitivity analysis, the vector-to-plant and plant-to-vector transmission rates, together with the vector removal rate, were the most influential parameters in determining the time of the infectious host population peak, the incidence peak, and the final number of dead hosts. We also used our model to check different vector-based control strategies, showing that a joint strategy focused on increasing the rate of vector removal while lowering the number of annual newborn vectors is optimal for disease control. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Potential of fungi of the genus Trichoderma for biocontrol of Philaenus spumarius, the insect vector for the quarantine bacterium Xylella fastidosa.

BACKGROUND: The meadow spittlebug Philaenus spumarius L. is the vector for the bacterium Xylella fastidiosa subspecies pauca, involved in olive quick decline syndrome (OQDS) in Salento (Italy). Control of P. spumarius is key to limiting transmission of the bacterium, and an innovative approach can be based on effective natural compounds and biocontrol agents. Entomopathogenic fungi are an important source of bioactive natural molecules that play a role in the relationship between microorganisms and insects. RESULTS: Pathogenicity bioassays, performed by dipping adults of P. spumarius in either fungal culture suspension (120 mg mL-1 ) or cell-free culture supernatant of Trichoderma chlorosporum GJS 91-150, showed, respectively, 97% and 87% death within 24 h. The effect was dose-dependent. In laboratory bioassays, the powdered fungal culture of T. chlorosporum GJS 91-150 did not exhibit pathogenic activity when injected into nymph spittle. CONCLUSIONS: T. chlorosporum GJS 91-150 affected the survival of P. spumarius adults. The lethal effect was not associated with the development of mycelium on the cuticle, but seems due, at least partly, to fungal metabolites released in the culture medium. The fungus tested here has good potential for the development of effective low-environmental impact control strategies for P. spumarius and suppression of X. fastidiosa. © 2022 Society of Chemical Industry.

Climate change and the potential distribution of the glassy-winged sharpshooter (Homalodisca vitripennis), an insect vector of Xylella fastidiosa.

Biological invasions represent a major threat for biodiversity and agriculture. Despite efforts to restrict the spread of alien species, preventing their introduction remains the best strategy for an efficient control. In that context preparedness of phytosanitary authorities is very important and estimating the geographical range of alien species becomes a key information. The present study investigates the potential geographical range of the glassy-winged sharpshooter (Homalodisca vitripennis), a very efficient insect vector of Xylella fastidiosa, one of the most dangerous plant-pathogenic bacteria worldwide. We use species distribution modeling (SDM) to analyse the climate factors driving the insect distribution and we evaluate its potential distribution in its native range (USA) and in Europe according to current climate and different scenarios of climate change: 6 General Circulation Models (GCM), 4 shared socioeconomic pathways of gas emission and 4 time periods (2030, 2050, 2070, 2090). The first result is that the climate conditions of the European continent are suitable to the glassy-winged sharpshooter, in particular around the Mediterranean basin where X. fastidiosa is present. Projections according to future climate conditions indicate displacement of climatically suitable areas towards the north in both North America and Europe. Globally, suitable areas will decrease in North America and increase in Europe in the coming decades. SDM outputs vary according to the GCM considered and this variability indicated areas of uncertainty in the species potential range. Both potential distribution and its uncertainty associated to future climate projections are important information for improved preparedness of phytosanitary authorities.

A Role for Early-Phase Transmission in the Enzootic Maintenance of Plague.

Yersinia pestis, the bacterial agent of plague, is enzootic in many parts of the world within wild rodent populations and is transmitted by different flea vectors. The ecology of plague is complex, with rodent hosts exhibiting varying susceptibilities to overt disease and their fleas exhibiting varying levels of vector competence. A long-standing question in plague ecology concerns the conditions that lead to occasional epizootics among susceptible rodents. Many factors are involved, but a major one is the transmission efficiency of the flea vector. In this study, using Oropsylla montana (a ground squirrel flea that is a major plague vector in the western United States), we comparatively quantified the efficiency of the two basic modes of flea-borne transmission. Transmission efficiency by the early-phase mechanism was strongly affected by the host blood source. Subsequent biofilm-dependent transmission by blocked fleas was less influenced by host blood and was more efficient. Mathematical modeling predicted that early-phase transmission could drive an epizootic only among highly susceptible rodents with certain blood characteristics, but that transmission by blocked O. montana could do so in more resistant hosts irrespective of their blood characteristics. The models further suggested that for most wild rodents, exposure to sublethal doses of Y. pestis transmitted during the early phase may restrain rapid epizootic spread by increasing the number of immune, resistant individuals in the population.

Immune defense mechanisms against a systemic bacterial infection in the cat flea (Ctenocephalides felis).

A significant amount of work has been devoted towards understanding the cellular and humoral immune responses in arthropod vectors. Although fleas (Siphonaptera) are vectors of numerous bacterial pathogens, few studies have examined how these insects defend themselves from infection. In this study, we investigated the immune defense mechanisms in the hemocoel of cat fleas (Ctenocephalides felis), currently the most important flea pest of humans and many domestic animals. Using model species of bacteria (Micrococcus luteus, Serratia marcescens, and Escherichia coli), we delivered a systemic infection and measured the following: antimicrobial activity of hemolymph, levels of free radicals resulting from the induction of oxidase-based pathways, number of circulating hemocytes, phagocytosis activity of circulating hemocytes, and in vivo bacteria killing efficiency when phagocytosis activity is limited. Our results show that the antimicrobial activity of flea hemolymph increases in response to certain species of bacteria; yet, a systemic infection with the same bacterial species did not influence levels of hydrogen peroxide (H2O2), a reactive intermediate of oxygen, at the same time. Additionally, the number of circulating hemocytes increases in response to E. coli infection, and these cells display strong phagocytic activity against this bacterium. Moreover, limiting phagocytosis by injecting polystyrene beads subsequently increases flea susceptibility to E. coli infection when compared to injury controls; however, impairing the cellular immune response itself did not increase flea susceptibility to infection when compared to untreated fleas. Overall, this work yields significant insight into how fleas interact with bacterial pathogens in their hemocoel, and suggests that cellular and humoral immune responses cooperate to combat systemic bacterial infections.

Transmission of Xylella fastidiosa subsp. pauca ST53 by the Sharpshooter Cicadella viridis From Different Source Plants and Artificial Diets.

The sharpshooter Cicadella viridis L. (Hemiptera: Cicadellidae) is the most common sharpshooter in Europe and, given its xylem feeding behavior, is considered a potential vector of the plant pathogenic bacterium Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae). We tested X. fastidiosa subsp. pauca ST53 (Xfp) transmission capabilities of C. viridis adults, namely 1) acquisition efficiency from four host plant species-periwinkle, milkwort, lavender, alfalfa-and from two artificial diets (PD3 and Xfm), 2) inoculation efficiency to periwinkle at different times post acquisition from different plant and artificial diet sources. The main European vector species-Philaenus spumarius L. (Hemiptera: Aphrophoridae)-was used as a control. C. viridis was able to acquire Xfp from periwinkle, milkwort, and lavender, although with low efficiency (3-16%) and from artificial diets (23-25%). Successful inoculation on periwinkle was extremely rare, being observed only three times, following feeding on milkwort plant and PD3 artificial diet sources. Our study shows that C. viridis is not a relevant vector of Xfp, given the very low transmission rate in controlled conditions, and the inability to feed on olive. The low efficiency reported here correlates with ecological constraints of the vector (mainly monocots host plants, humid environments) that make it difficult to forecast a relevant role in dispersing X. fastidiosa, at least within the present distribution of the exotic bacterium in Europe. However, a possible role of this species in spreading Xf in other agroecosystems, e.g., vineyard and stone fruits grown in humid areas, cannot be excluded.

Potential of citrus endophyte Bacillus subtilis L1-21 in the control of Candidatus Liberibacter asiaticus in Asian citrus psyllid, Diaphorina citri.

BACKGROUND: Asian citrus psyllid (ACP), also known as Diaphorina citri, is the natural vector of Candidatus Liberibacter asiaticus (CLas), which is responsible for Huanglongbing (HLB), a devastating citrus disease. Previously, the pathogen was successfully excluded from diseased citrus plants by using the indigenous endophyte Bacillus subtilis L1-21. However, the pathogen elimination and colonization potential of B. subtilis L1-21 in the carrier vector ACP, as well as the recruitment of native microbial communities of psyllid in the presence of endophytes, are still unknown. RESULTS: Initially, we suggested that endophyte L1-21 reduced the CLas copies in ACP from 6.58 × 106 to 5.04 × 104 per insect after 48 h, however, the pathogen copies remained stable in the negative control. The endophyte was stable for 48 h after application. Among the bacterial genera those highlighted in ACP were Candidatus Liberibacter, Pseudomonas, Candidatus Profftella, Methylobacterium-Methylorubrum, Pantoea, Curtobacterium, Wolbachia, Actinomycetospora, and Bacillus. Interestingly, B. subtilis L1-21 easily colonizes the midgut of ACP but cannot be detected in eggs. When ACP with endophyte L1-21 was allowed to feed on new citrus leaves, the highest colonization was observed. We also found that psyllids carrying endophyte L1-21 after feeding on citrus leaves reduced the CLas copies in leaves on the 0, 3rd and 5th day from 8.18 × 10,4 2.6 × 10,3 and 0 pathogen copies/g fresh midvein, respectively. CONCLUSIONS: We propose that B. subtilis L1-21 is a native endophyte in citrus and psyllid, which efficiently reduces the CLas pathogen in both citrus and psyllids, provides a more protective effect by increasing the number of cultivable endophytes, and successfully colonizes the midgut of ACP. © 2022 Society of Chemical Industry.

Wolbachia infection and genetic diversity of Italian populations of Philaenus spumarius, the main vector of Xylella fastidiosa in Europe.

Philaenus spumarius is a cosmopolitan species that has become a major threat to European agriculture being recognized as the main vector of the introduced plant pathogen Xylella fastidiosa, the agent of the "olive quick decline syndrome", a disease which is devastating olive orchards in southern Italy. Wolbachia are bacterial symbionts of many insects, frequently as reproductive parasites, sometime by establishing mutualistic relationships, able to spread within host populations. Philaenus spumarius harbors Wolbachia, but the role played by this symbiont is unknown and data on the infection prevalence within host populations are limited. Here, the Wolbachia infection rate was analyzed in relation to the geographic distribution and the genetic diversity of the Italian populations of P. spumarius. Analysis of the COI gene sequences revealed a geographically structured distribution of the three main mitochondrial lineages of P. spumarius. Wolbachia was detected in half of the populations sampled in northern Italy where most individuals belonged to the western-Mediterranean lineage. All populations sampled in southern and central Italy, where the individuals of the eastern-Mediterranean lineage were largely prevalent, were uninfected. Individuals of the north-eastern lineage were found only in populations from the Alps in the northernmost part of Italy, at high altitudes. In this area, Wolbachia infection reached the highest prevalence, with no difference between north-eastern and western-Mediterranean lineage. Analysis of molecular diversity of COI sequences suggested no significant effect of Wolbachia on population genetics of P. spumarius. Using the MLST approach, six new Wolbachia sequence types were identified. Using FISH, Wolbachia were observed within the host's reproductive tissues and salivary glands. Results obtained led us to discuss the role of Wolbachia in P. spumarius, the factors influencing the geographic distribution of the infection, and the exploitation of Wolbachia for the control of the vector insect to reduce the spread of X. fastidiosa.

Rapid Molecular Diagnostics in the Field and Laboratory to Detect Plant Pathogen DNA in Potential Insect Vectors.

A variety of sensitive and specific molecular diagnostic assays has been described for detecting nucleic acids in biological samples that may harbor pathogens of interest. These methods include very rapid, isothermal nucleic acid amplification methods that can be deployed outside of the laboratory environment, such as loop-mediated isothermal DNA amplification (LAMP) and recombinase-polymerase amplification (RPA). However, all molecular diagnostic assays must be preceded by nucleic acid extraction from the biological samples of interest, which provides suitable template molecules for the assays. To exploit the features of the amplification assays and be utilized outside of the lab, these methods must be rapid and avoid the need for typical laboratory chemicals and equipment. We describe a protocol for the extraction of DNA from field-collected insects that can be implemented at the point of collection and used to detect the presence of DNA sequences from potential plant pathogens that may be vectored by the insects. This protocol provides template DNA that is suitable for PCR, LAMP, and RPA. The FTA PlantSaver card-based DNA extraction product was also confirmed to amplify the mitochondrial cytochrome oxidase 1 (CO1) universal barcode that could later be sequenced to identify any insect. Lastly, we provide an example using field-collected insects, Neokolla (Graphocephala) heiroglyphica, and demonstrate the detection of the plant pathogen Xylella fastidiosa in carrier insects using PCR, RPA, and LAMP.

Control of arboviruses vectors using biological control by Wolbachia pipientis: a short review.

The number of arbovirus cases has increased in recent years, demonstrating a need for investing in effective control actions. Among these actions, are strategies using biological control vectors, a field where Wolbachia pipientis has shown itself as useful. Wolbachia pipientis, an obligatory intracellular Gram-negative bacteria, which parasites arthropods naturally or through laboratory-induced infections, is capable of manipulating the reproduction of its host. A systematic literature review gathering studies on this bacteria over last 10 years (2007-2021) was performed given its important role in the reduction of insect disease vectors. 111 articles were found, from which 78 were used in this study. Information on the Wolbachia biology, mechanism of action and potential for the biological control of insect disease vectors was gathered. The present study may contribute to the knowledge surrounding the bacterium, as well as stimulate the production of other studies with the same theme.

Epidemiologically-based strategies for the detection of emerging plant pathogens.

Emerging pests and pathogens of plants are a major threat to natural and managed ecosystems worldwide. Whilst it is well accepted that surveillance activities are key to both the early detection of new incursions and the ability to identify pest-free areas, the performance of these activities must be evaluated to ensure they are fit for purpose. This requires consideration of the number of potential hosts inspected or tested as well as the epidemiology of the pathogen and the detection method used. In the case of plant pathogens, one particular concern is whether the visual inspection of plant hosts for signs of disease is able to detect the presence of these pathogens at low prevalences, given that it takes time for these symptoms to develop. One such pathogen is the ST53 strain of the vector-borne bacterial pathogen Xylella fastidiosa in olive hosts, which was first identified in southern Italy in 2013. Additionally, X. fastidiosa ST53 in olive has a rapid rate of spread, which could also have important implications for surveillance. In the current study, we evaluate how well visual surveillance would be expected to perform for this pathogen and investigate whether molecular testing of either tree hosts or insect vectors offer feasible alternatives. Our results identify the main constraints to each of these strategies and can be used to inform and improve both current and future surveillance activities.

Fluid dynamic simulations at the interface of the blue-green sharpshooter functional foregut and grapevine xylem sap with implications for transmission of Xylella fastidiosa.

Xylella fastidiosa is a multi-continental, lethal, plant pathogenic bacterium that is transmitted by sharpshooter leafhoppers (Insecta: Hemiptera: Cicadellidae: Cicadellinae) and adult spittlebugs (Hemiptera: Aphrophoridae). The bacterium forms biofilms in plant xylem and the functional foregut of the insect. These biofilms serve as sources of inoculum for insect acquisition and subsequent inoculation to a healthy plant. In this study, 3D fluid dynamic simulations were performed for bidirectional cibarial propulsion of xylem sap through tube-like grapevine xylem and an anatomically accurate model of the functional foregut of the blue-green sharpshooter, Graphocephala atropunctata. The analysis supports a model of how fluid dynamics influence X. fastidiosa transmission. The model supports the hypothesis that X. fastidiosa inoculation is mostly driven by detachment of bacteria from the foregut due to high-velocity flow during egestion (outward fluid flow from the stylets). Acquisition occurs by fluid dynamics during both egestion and ingestion (fluid uptake through the stylets and swallowing). These simulation results are supported by previously reported X. fastidiosa colonization patterns in the functional foregut and sharpshooter stylet probing behaviors. The model indicates that xylem vessel diameter influences drag forces imposed on xylem wall-adherent bacteria; thus, vessel diameter may be an important component of the complex transmission process. Results from this study are directly applicable to development of novel grapevine resistance traits via electropenetrographic monitoring of vector acquisition and inoculation behaviors.

Exposure to Trypanosoma parasites induces changes in the microbiome of the Chagas disease vector Rhodnius prolixus.

BACKGROUND: The causative agent of Chagas disease, Trypanosoma cruzi, and its nonpathogenic relative, Trypanosoma rangeli, are transmitted by haematophagous triatomines and undergo a crucial ontogenetic phase in the insect's intestine. In the process, the parasites interfere with the host immune system as well as the microbiome present in the digestive tract potentially establishing an environment advantageous for development. However, the coherent interactions between host, pathogen and microbiota have not yet been elucidated in detail. We applied a metagenome shotgun sequencing approach to study the alterations in the microbiota of Rhodnius prolixus, a major vector of Chagas disease, after exposure to T. cruzi and T. rangeli focusing also on the functional capacities present in the intestinal microbiome of the insect. RESULTS: The intestinal microbiota of R. prolixus was dominated by the bacterial orders Enterobacterales, Corynebacteriales, Lactobacillales, Clostridiales and Chlamydiales, whereas the latter conceivably originated from the blood used for pathogen exposure. The anterior and posterior midgut samples of the exposed insects showed a reduced overall number of organisms compared to the control group. However, we also found enriched bacterial groups after exposure to T. cruzi as well as T rangeli. While the relative abundance of Enterobacterales and Corynebacteriales decreased considerably, the Lactobacillales, mainly composed of the genus Enterococcus, developed as the most abundant taxonomic group. This applies in particular to vectors challenged with T. rangeli and at early timepoints after exposure to vectors challenged with T. cruzi. Furthermore, we were able to reconstruct four metagenome-assembled genomes from the intestinal samples and elucidate their unique metabolic functionalities within the triatomine microbiome, including the genome of a recently described insect symbiont, Candidatus Symbiopectobacterium, and the secondary metabolites producing bacteria Kocuria spp. CONCLUSIONS: Our results facilitate a deeper understanding of the processes that take place in the intestinal tract of triatomine vectors during colonisation by trypanosomal parasites and highlight the influential aspects of pathogen-microbiota interactions. In particular, the mostly unexplored metabolic capacities of the insect vector's microbiome are clearer, underlining its role in the transmission of Chagas disease. Video Abstract.

Prevalence of Trypanosoma and Sodalis in wild populations of tsetse flies and their impact on sterile insect technique programmes for tsetse eradication.

The sterile insect technique (SIT) is an environment friendly and sustainable method to manage insect pests of economic importance through successive releases of sterile irradiated males of the targeted species to a defined area. A mating of a sterile male with a virgin wild female will result in no offspring, and ultimately lead to the suppression or eradication of the targeted population. Tsetse flies, vectors of African Trypanosoma, have a highly regulated and defined microbial fauna composed of three bacterial symbionts that may have a role to play in the establishment of Trypanosoma infections in the flies and hence, may influence the vectorial competence of the released sterile males. Sodalis bacteria seem to interact with Trypanosoma infection in tsetse flies. Field-caught tsetse flies of ten different taxa and from 15 countries were screened using PCR to detect the presence of Sodalis and Trypanosoma species and analyse their interaction. The results indicate that the prevalence of Sodalis and Trypanosoma varied with country and tsetse species. Trypanosome prevalence was higher in east, central and southern African countries than in west African countries. Tsetse fly infection rates with Trypanosoma vivax and T. brucei sspp were higher in west African countries, whereas tsetse infection with T. congolense and T. simiae, T. simiae (tsavo) and T. godfreyi were higher in east, central and south African countries. Sodalis prevalence was high in Glossina morsitans morsitans and G. pallidipes but absent in G. tachinoides. Double and triple infections with Trypanosoma taxa and coinfection of Sodalis and Trypanosoma were rarely observed but it occurs in some taxa and locations. A significant Chi square value (< 0.05) seems to suggest that Sodalis and Trypanosoma infection correlate in G. palpalis gambiensis, G. pallidipes and G. medicorum. Trypanosoma infection seemed significantly associated with an increased density of Sodalis in wild G. m. morsitans and G. pallidipes flies, however, there was no significant impact of Sodalis infection on trypanosome density.