Symbiosis in insects is accumulating significant amount of studies: the description of a wide array of mutualistic associations across the evolutionary history of insects suggests that resident microbiota acts as a driving force by affecting several aspects of hosts biology. Among arthropods, mosquito midgut microbiota has been largely investigated, providing crucial insights on the role and implications of host-symbiont relationships. However, limited amount of studies addressed their efforts on the investigation of microbiota colonizing salivary glands and reproductive tracts, crucial organs for pathogen invasion and vertical transmission of symbiotic microorganisms. Using 16S rRNA gene sequencing-based approach, we analysed the microbiota of gut, salivary glands and reproductive tracts of several mosquito species, representing some of the main vectors of diseases, aiming at describing the dynamics of bacterial communities within the individual. We identified a shared core microbiota between different mosquito species, although interesting inter- and intra-species differences were detected. Additionally, our results showed deep divergences between genera, underlining microbiota specificity and adaptation to their host. The comprehensive landscape of the bacterial microbiota components may ultimately provide crucial insights and novel targets for possible application of symbionts in innovative strategies for the control of vector borne diseases, globally named Symbiotic Control (SC), and suggesting that the holobiont of different mosquito species may significantly vary. Moreover, mosquito species are characterized by distinctive microbiota in different organs, likely reflecting different functions and/or adaptation processes.

The relative contributions of local T helper cell type 1 (Th1)- and Th2-like responses to the course of primary and secondary gastrointestinal (GI) candidiasis were examined in adult immunocompetent BALB/c mice. Both Th1 cytokines, such as interferon-γ (IFN-γ), and the Th2 cytokines, interleukin (IL)-4 and IL-5, were produced by CD4⁺ cells from Peyer's patches (PP) and mesenteric lymph nodes at a time when the fungus was cleared from the stomach and intestine. Augmentation of antigen-specific Th2-like responses by treatment with cholera toxin did not modify the course of disease. In contrast, treatment with soluble IL-4 receptor, which increased Th1 cells, was associated with enhanced yeast clearance. In addition, IFN-γ but not IL-4 mRNA was present in PP and spleen CD4⁺ cells in mice resistant to subsequent GI inoculation. Activation of Th1- but not Th2-like responses may be responsible locally for controlling GI candidiasis and generating protective immunity.

Malaria still remains a serious health burden in developing countries, causing more than 1 million deaths annually. Given the lack of an effective vaccine against its major etiological agent, Plasmodium falciparum, and the growing resistance of this parasite to the currently available drugs repertoire and of Anopheles mosquitoes to insecticides, the development of innovative control measures is an imperative to reduce malaria transmission. Paratransgenesis, the modification of symbiotic organisms to deliver anti-pathogen effector molecules, represents a novel strategy against Plasmodium development in mosquito vectors, showing the potential to reduce parasite development. However, the field application of laboratory-based evidence of paratransgenesis imposes the use of more realistic confined semi-field environments. Large cages were used to evaluate the ability of bacteria of the genus Asaia expressing green fluorescent protein (Asaia gfp), to diffuse in Anopheles stephensi and Anopheles gambiae target mosquito populations. Asaia gfp was introduced in large cages through the release of paratransgenic males or by sugar feeding stations. Recombinant bacteria transmission was directly detected by fluorescent microscopy, and further assessed by molecular analysis. Here we show the first known trial in semi-field condition on paratransgenic anophelines. Modified bacteria were able to spread at high rate in different populations of An. stephensi and An. gambiae, dominant malaria vectors, exploring horizontal ways and successfully colonising mosquito midguts. Moreover, in An. gambiae, vertical and trans-stadial diffusion mechanisms were demonstrated. Our results demonstrate the considerable ability of modified Asaia to colonise different populations of malaria vectors, including pecies where its association is not primary, in large environments. The data support the potential to employ transgenic Asaia as a tool for malaria control, disclosing promising perspective for its field application with suitable effector molecules.

Sulforaphane is a multi-action drug and its anticancer activity is the reason for the continuous growth of attention being paid to this drug. Sulforaphane shows an in vitro antiproliferative activity against melanoma and other skin cancer diseases. Unfortunately, this natural compound cannot be applied in free form on the skin due to its poor percutaneous permeation determined by its physico-chemical characteristics. The aim of this investigation was to evaluate ethosomes^® and transfersomes^® as ultradeformable vesicular carriers for the percutaneous delivery of sulforaphane to be used for the treatment of skin cancer diseases. The physico-chemical features of the ultradeformable vesicles were evaluated. Namely, ethosomes^® and transfersomes^® had mean sizes <400 nm and a polydispersity index close to 0. The stability studies demonstrated that the most suitable ultradeformable vesicles to be used as topical carriers of sulforaphane were ethosomes^® made up of ethanol 40% (w/v) and phospholipon 90G 2% (w/v). In particular, in vitro studies of percutaneous permeation through human stratum corneum and epidermis membranes showed an increase of the percutaneous permeation of sulforaphane. The antiproliferative activity of sulforaphane-loaded ethosomes^® was tested on SK-MEL 28 and improved anticancer activity was observed in comparison with the free drug.

The circumsporozoite protein (CSP) plays a key role in malaria sporozoite infection of both mosquito salivary glands and the vertebrate host. The conserved Regions I and II have been well studied but little is known about the immunogenic central repeat region and the N-terminal region of the protein. Rodent malaria Plasmodium berghei parasites, in which the endogenous CS gene has been replaced with the avian Plasmodium gallinaceum CS (PgCS) sequence, develop normally in the A. stephensi mosquito midgut but the sporozoites are not infectious. We therefore generated P. berghei transgenic parasites carrying the PgCS gene, in which the repeat region was replaced with the homologous region of P. berghei CS (PbCS). A further line, in which both the N-terminal region and repeat region were replaced with the homologous regions of PbCS, was also generated. Introduction of the PbCS repeat region alone, into the PgCS gene, did not rescue sporozoite species-specific infectivity. However, the introduction of both the PbCS repeat region and the N-terminal region into the PgCS gene completely rescued infectivity, in both the mosquito vector and the mammalian host. Immunofluorescence experiments and western blot analysis revealed correct localization and proteolytic processing of CSP in the chimeric parasites. The results demonstrate, in vivo, that the repeat region of P. berghei CSP, alone, is unable to mediate sporozoite infectivity in either the mosquito or the mammalian host, but suggest an important role for the N-terminal region in sporozoite host cell invasion.