Boosting immunity to treat parasitic infections: Asaia bacteria expressing a protein from Wolbachia determine M1 macrophage activation and killing of Leishmania protozoans.

Leishmaniases are severe vector-borne diseases affecting humans and animals, caused by Leishmania protozoans. Over one billion people and millions of dogs live in endemic areas for leishmaniases and are at risk of infection. Immune polarization plays a major role in determining the outcome of Leishmania infections: hosts displaying M1-polarized macrophages are protected, while those biased on the M2 side acquire a chronic infection that could develop into a deadly disease. The identification of the factors involved in M1 polarization is essential for the design of therapeutic and prophylactic interventions, including vaccines. Infection by the filarial nematode Dirofilaria immitis could be one of the factors that interfere with leishmaniasis in dogs. Indeed, filarial nematodes induce a partial skew of the immune response towards M1, likely caused by their bacterial endosymbionts, Wolbachia. Here we have examined the potential of AsaiaWSP, a bacterium engineered for the expression of the Wolbachia surface protein (WSP), as an inductor of M1 macrophage activation and Leishmania killing. Macrophages stimulated with AsaiaWSP displayed a strong leishmanicidal activity, comparable to that determined by the choice-drug amphotericin B. Additionally, AsaiaWSP determined the expression of markers of classical macrophage activation, including M1 cytokines, ROS and NO, and an increase in phagocytosis activity. Asaia not expressing WSP also induced macrophage activation, although at a lower extent compared to AsaiaWSP. In summary, the results of the present study confirm the immunostimulating properties of WSP highlighting a potential therapeutic efficacy against Leishmania parasites. Furthermore, Asaia was designed as a delivery system for WSP, thus developing a novel type of immunomodulating agent, worthy of being investigated for immuno-prophylaxis and -therapy of leishmaniases and other diseases that could be subverted by M1 macrophage activation.

Development of microsatellite markers for the invasive mosquito Aedes koreicus.

BACKGROUND: Aedes koreicus is a mosquito species native to East Asia which has recently invaded several countries in Europe. In Italy, this mosquito was first detected in the North-East in 2011 and is now widely distributed in the entire northern part of the country. The development of specific genetic markers, such as microsatellites, is necessary to uncover the dispersal routes of this mosquito from its native areas and, eventually, to plan future control interventions. METHODS: Available raw sequences of genomic DNA of Ae. koreicus were screened in silico using BLASTn to identify possible microsatellite-containing sequences. Specific primer pairs were then designed, and their efficiency was determined through polymerase chain reaction (PCR) on 32 individuals of Ae. koreicus collected in Italy. PCR conditions were optimised in three multiplex reactions. Genotyping of individual mosquitoes was performed on both single and multiplex PCR reactions. Finally, analysis of intra-population variation was performed to assess the level of polymorphism of the markers. RESULTS: Mosquito genotyping provided consistent results in both single and multiplex reactions. Out of the 31 microsatellite markers identified in the Ae. koreicus genome raw sequences, 11 were polymorphic in the examined mosquito samples. CONCLUSIONS: The results show that the 11 microsatellite markers developed here hold potential for investigating the genetic structure of Ae. koreicus populations. These markers could thus represent a novel and useful tool to infer the routes of invasion of this mosquito species into Europe and other non-native areas.

Morphological and molecular insights into the diversity of Leptoconops biting midges from a heavily infested Mediterranean area.

The genus Leptoconops Skuse (Diptera: Ceratopogonidae) are blood-sucking midges known to pester humans and domestic animals. In certain Mediterranean areas, midges occur in large numbers during summer and limit the use of recreational areas, also raising serious health and social concerns. Despite such impact, the diversity and distribution of Leptoconops in Maremma Regional Park (Tuscany Region, Italy), a heavily infested area, is not well known, and neither molecular nor detailed morphological studies exist. We sampled adult midge females in six areas and used high-resolution digital stereomicroscopy and scanning electron microscopy to identify species and investigate the morphology of structures involved in host searching/recognition (antennae and maxillary palps) and host attack (mouthparts). We also performed energy-dispersive X-ray spectroscopy to characterize the elemental composition of mouthparts. Finally, the cytochrome c oxidase subunit 1 (cox1) gene was amplified and sequenced, to confirm species identification of collected specimens. We identified two species: Leptoconops (L.) irritans Noé and Leptoconops (L.) noei Clastrier & Coluzzi, with the former being more frequently sampled than the latter and closer to sea coast and rivers. The antennal segments appeared slightly more globular in L. noei than in L. irritans. Five types of trichoid, basiconic and chaetic sensilla were found on the antennae, with some differences between the two species. Mouthparts had the labellum visibly larger in L. noei compared with L. irritans. The maxillary palps possessed a pit filled with bulb-shaped sensilla, which appeared denser in L. noei than in L. irritans. Mouthpart cuticle included Calcium (Ca) and Aluminum (Al) at small but significant concentrations (0.3-1.0%) in both species. Our results suggest that the limited but appreciable differences in sensory system between the studied species of Leptoconops and other Ceratopogonidae may reflect different host or habitat preferences, a scenario potentially suggested also by preliminarily data on their distribution in the studied area. The presence of Ca and Al in the cuticle of mouthparts may help host skin drilling during bite activity. Finally, the gene sequences obtained in this study provide a first reference for future investigations on the taxonomy and dispersal patterns of Leptoconops spp. in the Mediterranean area.

IgE response to Aed al 13 and Aed al 14 recombinant allergens from Aedes albopictus saliva in humans.

Background: Mosquito bite is normally associated with mild allergic responses, but severe localized or systemic reactions are also possible. Reliable tools for the diagnosis of mosquito allergy are still unavailable. Here, we investigated the IgE response to 3 potential salivary allergens identified in the saliva of the tiger mosquito Aedes albopictus. Methods: Serum from 55 adult individuals (28 controls and 27 allergic people), were analysed using an in-house Enzyme Linked ImmunoSorbent Assay (ELISA) against the Salivary Gland Extract (SGE) and the recombinant proteins albD7l2 (Aed al 2), albAntigen5-3 (Aed al 13) and albLIPS-2 (Aed al 14). Results: Fifteen of the 27 (56%) individuals having hypersensitive reactions to mosquito bites had IgE serum levels recognizing SGE. Negative sera did not show detectable levels of IgE targeting the SGE from the most common sympatric mosquito Culex pipiens. Among the positive individuals, 2 subjects displayed IgE targeting Aed al 2 (13%), while IgE recognizing Aed al 13 and Aed al 14 were detected in ten (67%) and seven (47%) individuals, respectively. Two sera from non-hypersensitive subjects had detectable levels of IgE targeting Aed al 13, suggesting possible cross-reaction with the homologue salivary proteins of multiple mosquito species or, more generally, of hematophagous insects. Conclusions: Our results indicate that Aed al 13 and Aed al 14 hold the potential to be developed as tools for the diagnosis of allergy to Ae. albopictus bites. Such tools would facilitate epidemiological studies on tiger mosquito allergy in humans and might foster the development of further protein-based assays to investigate cross-species allergies.

A salivary factor binds a cuticular protein and modulates biting by inducing morphological changes in the mosquito labrum.

The mosquito proboscis is an efficient microelectromechanical system, which allows the insect to feed on vertebrate blood quickly and painlessly. Its efficiency is further enhanced by the insect saliva, although through unclear mechanisms. Here, we describe the initial trigger of an unprecedented feedback signaling pathway in Aedes mosquitoes affecting feeding behavior. We identified LIPS proteins in the saliva of Aedes mosquitoes that promote feeding in the vertebrate skin. LIPS show a new all-helical protein fold constituted by two domains. The N-terminal domain interacts with a cuticular protein (Cp19) located at the tip of the mosquito labrum. Upon interaction, the morphology of the labral cuticle changes, and this modification is most likely sensed by proprioceptive neurons. Our study identifies an additional role of mosquito saliva and underlines that the external cuticle is a possible site of key molecular interactions affecting the insect biology and its vector competence.

Assessing the distribution of invasive Asian mosquitoes in Northern Italy and modelling the potential spread of Aedes koreicus in Europe.

In the last decade, Aedes koreicus and Aedes japonicus japonicus mosquitoes, which are competent vectors for various arboviruses of public health relevance, colonised Italy and other European countries. Nevertheless, information about their current and potential distribution is partial. Accordingly, in this study four regions of Northern Italy (Lombardy, Liguria, Piedmont and Aosta Valley) were surveyed during 2021 for the presence of eggs, larvae and pupae of these two invasive species. We found evidence for a widespread presence of Ae. koreicus in pre-Alpine territories of Lombardy and Piedmont. Larvae from the invasive subspecies of Ae. j. japonicus were also collected in the same geographic areas, though they were less frequent. Occurrence data from this study and results from previous monitoring campaigns were used to generate a Maxent model for the prediction of habitat suitability for Ae. koreicus mosquitoes in Northern Italy and the rest of Europe. Peri-urban areas located in proximity to forests, pastures and vineyards were revealed as highly suitable environments for colonisation by this invasive species. Maps of the potential distribution also suggest the presence of further suitable areas in currently uncolonized countries. We conclude that this invasive mosquito species has the potential for a broad expansion at the European level in the coming decades.

Evidence for the spread of the alien species Aedes koreicus in the Lombardy region, Italy.

BACKGROUND: Aedes koreicus is a mosquito species characterized by marked anthropophilic behavior, and a potential vector of nematodes and viruses. It is native to East Asia, but its presence has recently been reported in many regions of Europe. In Italy, these mosquitoes had been detected in the northeast since 2011 and are now spreading towards the southwest of the country. METHODS: In 2020, during a surveillance program for invasive mosquito species in the district of Bergamo (Lombardy Region, Italy), about 6000 mosquito larvae were collected. Emerged adults were assigned to mosquito species according to morphological analyses, followed by amplification and sequencing of genetic markers (COI, ND4, ITS2 and D2). RESULTS: According to the morphological and genetic data, about 50 individuals belonged to the species Ae. koreicus. CONCLUSION: We report the presence of Ae. koreicus in the district of Bergamo, which confirms the spread of this species in the north of Italy and raises concerns about its possible role as a vector of diseases in the Alpine area.

Mosquito Trilogy: Microbiota, Immunity and Pathogens, and Their Implications for the Control of Disease Transmission.

In mosquitoes, the interaction between the gut microbiota, the immune system, and the pathogens that these insects transmit to humans and animals is regarded as a key component toward the development of control strategies, aimed at reducing the burden of severe diseases, such as malaria and dengue fever. Indeed, different microorganisms from the mosquito microbiota have been investigated for their ability to affect important traits of the biology of the host insect, related with its survival, development and reproduction. Furthermore, some microorganisms have been shown to modulate the immune response of mosquito females, significantly shaping their vector competence. Here, we will review current knowledge in this field, focusing on i) the complex interaction between the intestinal microbiota and mosquito females defenses, both in the gut and at humoral level; ii) how knowledge on these issues contributes to the development of novel and targeted strategies for the control of mosquito-borne diseases such as the use of paratransgenesis or taking advantage of the relationship between Wolbachia and mosquito hosts. We conclude by providing a brief overview of available knowledge on microbiota-immune system interplay in major insect vectors.