Mosquitocidal and Anti-Inflammatory Properties of The Essential Oils Obtained from Monoecious, Male, and Female Inflorescences of Hemp (Cannabis sativa L.) and Their Encapsulation in Nanoemulsions.

Among the various innovative products obtainable from hemp (Cannabis sativa L.) waste biomass originating from different industrial processes, the essential oil (EO) deserves special attention in order to understand its possible application in different fields, such as cosmetics, pharmaceuticals, and botanical insecticides. For the purpose, in the present work, we studied the chemical composition of EOs obtained from different hemp varieties, namely Felina 32 and Carmagnola Selezionata (CS) using monoecious, male, and female inflorescences, and we evaluated their mosquitocidal activities on larvae and pupae of two main malaria vectors, Anopheles gambiae and An. stephensi. Then, in order to evaluate the safe use of hemp EOs for operators, the potential pro- or anti-inflammatory effect of hemp EOs together with their toxicological profile were determined on dermal fibroblasts and keratinocytes. Given the promising results obtained by insecticidal and anti-inflammatory studies, a preliminary evaluation of EOs encapsulation into nanoemulsions (NEs) has been performed with the aim to develop a formulation able to improve their poor physicochemical stability. Felina 32 and CS inflorescences provided EOs with an interesting chemical profile, with monoterpene and sesquiterpene hydrocarbons as the major components. This study highlighted the potential application of male inflorescences, which are usually discharged during hemp product processing. These EOs could be exploited as potential sustainable and eco-friendly insecticides, given their capability to be toxic against mosquitoes and the possibility to use them to prepare stable and safe formulations. The LC50 values found in this study (<80 ppm) are lower, on average, than those of many plant EOs, with the advantage of using an industrial waste product. From MTT assay and gene and protein expression analysis, EOs showed no cytotoxicity at the appropriate doses and exerted an anti-inflammatory effect on the human cell lines tested. These findings encourage further applied research on hemp EOs in order support their industrial exploitation.

A yeast strain associated to Anopheles mosquitoes produces a toxin able to kill malaria parasites.

BACKGROUND: Malaria control strategies are focusing on new approaches, such as the symbiotic control, which consists in the use of microbial symbionts to prevent parasite development in the mosquito gut and to block the transmission of the infection to humans. Several microbes, bacteria and fungi, have been proposed for malaria or other mosquito-borne diseases control strategies. Among these, the yeast Wickerhamomyces anomalus has been recently isolated from the gut of Anopheles mosquitoes, where it releases a natural antimicrobial toxin. Interestingly, many environmental strains of W. anomalus exert a wide anti-bacterial/fungal activity and some of these 'killer' yeasts are already used in industrial applications as food and feed bio-preservation agents. Since a few studies showed that W. anomalus killer strains have antimicrobial effects also against protozoan parasites, the possible anti-plasmodial activity of the yeast was investigated. METHODS: A yeast killer toxin (KT), purified through combined chromatographic techniques from a W. anomalus strain isolated from the malaria vector Anopheles stephensi, was tested as an effector molecule to target the sporogonic stages of the rodent malaria parasite Plasmodium berghei, in vitro. Giemsa staining was used to detect morphological damages in zygotes/ookinetes after treatment with the KT. Furthermore, the possible mechanism of action of the KT was investigated pre-incubating the protein with castanospermine, an inhibitor of ß-glucanase activity. RESULTS: A strong anti-plasmodial effect was observed when the P. berghei sporogonic stages were treated with KT, obtaining an inhibition percentage up to around 90%. Microscopy analysis revealed several ookinete alterations at morphological and structural level, suggesting the direct implication of the KT-enzymatic activity. Moreover, evidences of the reduction of KT activity upon treatment with castanospermine propose a ß-glucanase-mediated activity. CONCLUSION: The results showed the in vitro killing efficacy of a protein produced by a mosquito strain of W. anomalus against malaria parasites. Further studies are required to test the KT activity against the sporogonic stages in vivo, nevertheless this work opens new perspectives for the possible use of killer strains in innovative strategies to impede the development of the malaria parasite in mosquito vectors by the means of microbial symbionts.

A rapid qPCR method to investigate the circulation of the yeast Wickerhamomyces anomalus in humans.

The yeast Wickerhamomyces anomalus has been proposed for many biotechnological applications in the food industry. However, a number of opportunistic pathogenic strains have been reported as causative agents of nosocomial fungemia. Recognition of potentially pathogenic isolates is an important challenge for the future commercialization of this yeast. The isolation of W. anomalus from different matrices and, recently, from mosquitoes, requires further investigations into its circulation in humans. Here we present a qPCR protocol for the detection of W. anomalus in human blood samples and the results of a screening of 525 donors, including different classes of patients and healthy people.

Different mosquito species host Wickerhamomyces anomalus (Pichia anomala): perspectives on vector-borne diseases symbiotic control.

The genetic manipulation of the microbial community associated with hematophagus insects is particularly relevant for public health applications. Within mosquito populations, this relationship has been overlooked until recently. New advances in molecular biotechnology propose the genetic manipulation of mosquito symbionts to prevent the transmission of pathogens to humans by interfering with the obligatory life cycle stages within the insect through the use of effector molecules. This approach, defined as 'paratransgenesis', has opened the way for the investigation and characterization of microbes residing in the mosquito body, particularly those localised within the gut. Some interesting bacteria have been identified as candidates for genetic modification, however, endosymbiotic yeasts remain largely unexplored with little information on the symbiotic relationships to date. Here we review the recent report of symbiotic relationship between Wickerhamomyces anomalus (Pichia anomala) and several mosquito vector species as promising methods to implement control of mosquito-borne diseases.

Killer yeasts exert anti-plasmodial activities against the malaria parasite Plasmodium berghei in the vector mosquito Anopheles stephensi and in mice.

BACKGROUND: Wickerhamomyces anomalus is a yeast associated with different insects including mosquitoes, where it is proposed to be involved in symbiotic relationships with hosts. Different symbiotic strains of W. anomalus display a killer phenotype mediated by protein toxins with broad-spectrum antimicrobial activities. In particular, a killer toxin purified from a W. anomalus strain (WaF17.12), previously isolated from the malaria vector mosquito Anopheles stephensi, has shown strong in vitro anti-plasmodial activity against early sporogonic stages of the murine malaria parasite Plasmodium berghei. RESULTS: Here, we provide evidence that WaF17.12 cultures, properly stimulated to induce the expression of the killer toxin, can directly affect in vitro P. berghei early sporogonic stages, causing membrane damage and parasite death. Moreover, we demonstrated by in vivo studies that mosquito dietary supplementation with activated WaF17.12 cells interfere with ookinete development in the midgut of An. stephensi. Besides the anti-sporogonic action of WaF17.12, an inhibitory effect of purified WaF17.12-killer toxin was observed on erythrocytic stages of P. berghei, with a consequent reduction of parasitaemia in mice. The preliminary safety tests on murine cell lines showed no side effects. CONCLUSIONS: Our findings demonstrate the anti-plasmodial activity of WaF17.12 against different developmental stages of P. berghei. New studies on P. falciparum are needed to evaluate the use of killer yeasts as innovative tools in the symbiotic control of malaria.

Asaia Activates Immune Genes in Mosquito Eliciting an Anti-Plasmodium Response: Implications in Malaria Control.

In mosquitoes, the discovery of the numerous interactions between components of the microbiota and the host immune response opens up the attractive possibility of the development of novel control strategies against mosquito borne diseases. We have focused our attention to Asaia, a symbiont of several mosquito vectors who has been proposed as one of the most potential tool for paratransgenic applications; although being extensively characterized, its interactions with the mosquito immune system has never been investigated. Here we report a study aimed at describing the interactions between Asaia and the immune system of two vectors of malaria, Anophelesstephensi and An. gambiae. The introduction of Asaia isolates induced the activation of the basal level of mosquito immunity and lower the development of malaria parasite in An. stephensi. These findings confirm and expand the potential of Asaia in mosquito borne diseases control, not only through paratransgenesis, but also as a natural effector for mosquito immune priming.

Denaturing Gradient Gel Electrophoresis Analysis of Bacteria in Italian Ticks and First Detection of Streptococcus equi in Rhipicephalus bursa from the Lazio Region.

Tick-borne diseases are an increasing problem for the community. Ticks harbor a complex microbial population acquired while feeding on a variety of animals. Profiling the bacterial population by 16S rDNA amplification and denaturing gradient gel electrophoresis enables detection of the broad spectrum of bacteria that settles in the ticks. This study identified known and unknown tick-infecting bacteria in samples from Italy. Seven adult ticks from different hosts and origins were analyzed: two Rhipicephalus sanguineus ticks from dogs (Lombardia), two Rhipicephalus bursa ticks from bovines (Lazio), and three Ixodes ricinus ticks from humans (Marche). The major result was the first report of the zoonotic agent Streptococcus equi in ticks. S. equi is a species complex of highly contagious pathogens. Subsequent to S. equi detection in a R. bursa tick removed from a bovine of Lazio in 2012, we studied 95 R. bursa samples collected from 3 bovines, 3 ponies, and 1 sheep grazing in the same area in 2012 and from 6 ponies grazing there in 2017. The results of a specific PCR assay indicated a not sporadic occurrence of S. equi in ticks. This finding provides a basis for assessing the potential of ticks to harbor and disperse S. equi.

Identification of a Killer Toxin from Wickerhamomyces anomalus with ß-Glucanase Activity.

The yeast Wickerhamomyces anomalus has several applications in the food industry due to its antimicrobial potential and wide range of biotechnological properties. In particular, a specific strain of Wickerhamomyces anomalus isolated from the malaria mosquito Anopheles stephensi, namely WaF17.12, was reported to secrete a killer toxin with strong anti-plasmodial effect on different developmental stages of Plasmodium berghei; therefore, we propose its use in the symbiotic control of malaria. In this study, we focused on the identification/characterization of the protein toxin responsible for the observed antimicrobial activity of the yeast. For this purpose, the culture medium of the killer yeast strain WaF17.12 was processed by means of lateral flow filtration, anion exchange and gel filtration chromatography, immunometric methods, and eventually analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on this concerted approach, we identified a protein with a molecular weight of approximately 140 kDa and limited electrophoretic mobility, corresponding to a high molecular weight ß-glucosidase, as confirmed by activity tests in the presence of specific inhibitors.

Mosquitoes can harbour yeasts of clinical significance and contribute to their environmental dissemination.

There is still a lack of studies on fungal microbiota in mosquitoes, compared with the number available on bacterial microbiota. This study reports the identification of yeasts of clinical significance in laboratory mosquito species: Anopheles gambiae, Anopheles stephensi, Culex quinquefasciatus, Aedes albopictus and Aedes aegypti. Among the yeasts isolated, they focused on the opportunistic pathogen Candida parapsilosis, since there is a need to better understand breakthrough candidaemia with resistance to the usual antifungals, which requires careful consideration in the broad-spectrum therapy, as documented in many clinical reports. C. parapsilosis occurs widely and has been isolated from diverse sources, including insects, which may contribute to its dissemination. In this study, it was isolated from the gut of An. gambiae and its presence in developmental stages and organs of different mosquito species was studied. Our results indicated that there was a stable association between C. parapsilosis and reared mosquitoes during the entire life cycle, and in adult male and female gut and gonads. A wide occurrence of C. parapsilosis was also documented in several populations of wild mosquitoes. Based on these findings, it can be said that mosquitoes might participate in the spreading of this opportunistic pathogen, not only as a carrier.

Paratransgenesis to control malaria vectors: a semi-field pilot study.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Mutual exclusion of Asaia and Wolbachia in the reproductive organs of mosquito vectors.

BACKGROUND: Wolbachia is a group of intracellular maternally inherited bacteria infecting a high number of arthropod species. Their presence in different mosquito species has been largely described, but Aedes aegypti, the main vector of Dengue virus, has never been found naturally infected by Wolbachia. Similarly, malaria vectors and other anophelines are normally negative to Wolbachia, with the exception of an African population where these bacteria have recently been detected. Asaia is an acetic acid bacterium stably associated with several mosquito species, found as a dominant microorganism of the mosquito microbiota. Asaia has been described in gut, salivary glands and in reproductive organs of adult mosquitoes in Ae. aegypti and in anophelines. It has recently been shown that Asaia may impede vertical transmission of Wolbachia in Anopheles mosquitoes. Here we present an experimental study, aimed at determining whether there is a negative interference between Asaia and Wolbachia, for the gonad niche in mosquitoes. METHODS: Different methods (PCR and qPCR, monoclonal antibody staining and FISH) have been used to address the question of the co-localization and the relative presence/abundance of the two symbionts. PCR and qPCR were performed to qualitatively and quantitatively verify the distribution of Asaia and Wolbachia in different mosquito species/organs. Monoclonal antibody staining and FISH were performed to localize the symbionts in different mosquito species. RESULTS: Here we provide evidence that, in Anopheles and in other mosquitoes, there is a reciprocal negative interference between Asaia and Wolbachia symbionts, in terms of the colonization of the gonads. In particular, we have shown that in some mosquito species the presence of one of the symbionts prevented the establishment of the second, while in other systems the symbionts were co-localized, although at reduced densities. CONCLUSIONS: A mutual exclusion or a competition between Asaia and Wolbachia may contribute to explain the inability of Wolbachia to colonize the female reproductive organs of anophelines, inhibiting its vertical transmission and explaining the absence of Wolbachia infection in Ae. aegypti and in the majority of natural populations of Anopheles mosquitoes.

A Wickerhamomyces anomalus killer strain in the malaria vector Anopheles stephensi.

The yeast Wickerhamomyces anomalus has been investigated for several years for its wide biotechnological potential, especially for applications in the food industry. Specifically, the antimicrobial activity of this yeast, associated with the production of Killer Toxins (KTs), has attracted a great deal of attention. The strains of W. anomalus able to produce KTs, called "killer" yeasts, have been shown to be highly competitive in the environment. Different W. anomalus strains have been isolated from diverse habitats and recently even from insects. In the malaria mosquito vector Anopheles stephensi these yeasts have been detected in the midgut and gonads. Here we show that the strain of W. anomalus isolated from An. stephensi, namely WaF17.12, is a killer yeast able to produce a KT in a cell-free medium (in vitro) as well as in the mosquito body (in vivo). We showed a constant production of WaF17.12-KT over time, after stimulation of toxin secretion in yeast cultures and reintroduction of the activated cells into the mosquito through the diet. Furthermore, the antimicrobial activity of WaF17.12-KT has been demonstrated in vitro against sensitive microbes, showing that strain WaF17.12 releases a functional toxin. The mosquito-associated yeast WaF17.12 thus possesses an antimicrobial activity, which makes this yeast worthy of further investigations, in view of its potential as an agent for the symbiotic control of malaria.

Interactions between Asaia, Plasmodium and Anopheles: new insights into mosquito symbiosis and implications in malaria symbiotic control.

BACKGROUND: Malaria represents one of the most devastating infectious diseases. The lack of an effective vaccine and the emergence of drug resistance make necessary the development of new effective control methods. The recent identification of bacteria of the genus Asaia, associated with larvae and adults of malaria vectors, designates them as suitable candidates for malaria paratransgenic control.To better characterize the interactions between Asaia, Plasmodium and the mosquito immune system we performed an integrated experimental approach. METHODS: Quantitative PCR analysis of the amount of native Asaia was performed on individual Anopheles stephensi specimens. Mosquito infection was carried out with the strain PbGFPCON and the number of parasites in the midgut was counted by fluorescent microscopy.The colonisation of infected mosquitoes was achieved using GFP or DsRed tagged-Asaia strains.Reverse transcriptase-PCR analysis, growth and phagocytosis tests were performed using An. stephensi and Drosophila melanogaster haemocyte cultures and DsRed tagged-Asaia and Escherichia coli strains. RESULTS: Using quantitative PCR we have quantified the relative amount of Asaia in infected and uninfected mosquitoes, showing that the parasite does not interfere with bacterial blooming. The correlation curves have confirmed the active replication of Asaia, while at the same time, the intense decrease of the parasite.The 'in vitro' immunological studies have shown that Asaia induces the expression of antimicrobial peptides, however, the growth curves in conditioned medium as well as a phagocytosis test, indicated that the bacterium is not an immune-target.Using fluorescent strains of Asaia and Plasmodium we defined their co-localisation in the mosquito midgut and salivary glands. CONCLUSIONS: We have provided important information about the relationship of Asaia with both Plasmodium and Anopheles. First, physiological changes in the midgut following an infected or uninfected blood meal do not negatively affect the residing Asaia population that seems to benefit from this condition. Second, Asaia can act as an immune-modulator activating antimicrobial peptide expression and seems to be adapted to the host immune response. Last, the co-localization of Asaia and Plasmodium highlights the possibility of reducing vectorial competence using bacterial recombinant strains capable of releasing anti-parasite molecules.

Symbiotic control of mosquito borne disease.

It is well accepted that the symbiotic relationships insects have established with several microorganisms have had a key role in their evolutionary success. Bacterial symbiosis is also prevalent in insects that are efficient disease vectors, and numerous studies have sought to decrypt the basic mechanisms of the host-symbiont relationships and develop ways to control vector borne diseases. 'Symbiotic control', a new multifaceted approach that uses symbiotic microorganisms to control insect pests or reduce vector competence, seems particularly promising. Three such approaches currently at the cutting edge are: (1) the disruption of microbial symbionts required by insect pests; (2) the manipulation of symbionts that can express anti-pathogen molecules within the host; and (3) the introduction of endogenous microbes that affect life-span and vector capacity of the new hosts in insect populations. This work reviews current knowledge on microbial symbiosis in mosquitoes that holds promise for development of symbiotic control for mosquito borne diseases.