Aedes aegypti SGS1 is critical for Plasmodium gallinaceum infection of both the mosquito midgut and salivary glands.

BACKGROUND: The invasion of the mosquito salivary glands by Plasmodium sporozoites is a critical step that defines the success of malaria transmission and a detailed understanding of the molecules responsible for salivary gland invasion could be leveraged towards control of vector-borne pathogens. Antibodies directed against the mosquito salivary gland protein SGS1 have been shown to reduce Plasmodium gallinaceum sporozoite invasion of Aedes aegypti salivary glands, but the specific role of this protein in sporozoite invasion and in other stages of the Plasmodium life cycle remains unknown. METHODS: RNA interference and CRISPR/Cas9 were used to evaluate the role of A. aegypti SGS1 in the P. gallinaceum life cycle. RESULTS: Knockdown and knockout of SGS1 disrupted sporozoite invasion of the salivary gland. Interestingly, mosquitoes lacking SGS1 also displayed fewer oocysts. Proteomic analyses confirmed the abolishment of SGS1 in the salivary gland of SGS1 knockout mosquitoes and revealed that the C-terminus of the protein is absent in the salivary gland of control mosquitoes. In silico analyses indicated that SGS1 contains two potential internal cleavage sites and thus might generate three proteins. CONCLUSION: SGS1 facilitates, but is not essential for, invasion of A. aegypti salivary glands by P. gallinaceum and has a dual role as a facilitator of parasite development in the mosquito midgut. SGS1 could, therefore, be part of a strategy to decrease malaria transmission by the mosquito vector, for example in a transgenic mosquito that blocks its interaction with the parasite.

Morphological aspects of immature stages of Migonemyia migonei (Diptera: Psychodidae, Phlebotominae), an important vector of Leishmaniosis in South America, described by scanning electron microscopy.

We describe the immature stages of Migonemyia migonei, which is the vector of Leishmania (Viannia) braziliensis, the etiological agent of cutaneous leishmaniasis in South America, and a putative vector of Leishmania infantum chagasi. Scanning Electron Microscopy (SEM) was used to refine the description of the structures of the egg, all instar larvae, and the pupa. The eggs have polygonal cells on the egg exochorion, and differences between larval and pupal chaetotaxy have been highlighted. Different sensillary subtypes-trichoidea, basiconica, coelonica and campanoformia-were observed in the larval stages. The results presented herein contribute to the taxonomy of Mg. migonei and may contribute to future studies on the phylogeny of this important vector species.

The first characterization of a cystatin and a cathepsin L-like peptidase from Aedes aegypti and their possible role in DENV infection by the modulation of apoptosis.

Recently, a salivary gland transcriptome study demonstrated that the transcripts of a putative cystatin gene (SeqID AAEL013287; Aacystatins) from Aedes aegypti were increased in DENV2-infected mosquitoes and that silencing of the Aacystatin gene resulted in an increase in DENV titres. In this work, Aacystatin was biochemically characterized; the purified recombinant inhibitor was able to inhibit typical cysteine proteases with a Ki in the nM range. Pulldown assays using Aag2 cell extracts identified a cathepsin L-like peptidase (AaCatL) as a possible target of Aacystatin. Purified recombinant AaCatL had an optimal pH of 5.0 and displayed a preference for Leu, Val and Phe residues at P2, which is common for other cathepsin L-like peptidases. Transcription analysis of Aacystatin and AaCatL in the salivary glands and midgut of DENV2-infected mosquitoes revealed a negative correlation between DENV2 titres and levels of the inhibitor and peptidase, suggesting their involvement in DENV2-mosquito interactions. Considering that apoptosis may play an important role during viral infections, the possible involvement of Aacystatin in staurosporine-induced apoptosis in Aag2 cells was investigated; the results showed higher expression of the inhibitor in treated cells; moreover, pre incubation with rAacystatin was able to increase Aag2 cell viability.

Correction: Mosquito pornoscopy: Observation and interruption of Aedes aegypti copulation to determine female polyandric event and mixed progeny.

[This corrects the article DOI: 10.1371/journal.pone.0193164.].

An Anopheles aquasalis GATA factor Serpent is required for immunity against Plasmodium and bacteria.

Innate immunity is an ancient and conserved defense system that provides an early effective response against invaders. Many immune genes of Anopheles mosquitoes have been implicated in defense against a variety of pathogens, including plasmodia. Nevertheless, only recent work identified some immune genes of Anopheles aquasalis mosquitoes upon P. vivax infection. Among these was a GATA transcription factor gene, which is described here. This is an ortholog of GATA factor Serpent genes described in Drosophila melanogaster and Anopheles gambiae. Gene expression analyses showed an increase of GATA-Serpent mRNA in P. vivax-infected A. aquasalis and functional RNAi experiments identified this transcription factor as an important immune gene of A. aquasalis against both bacteria and P. vivax. Besides, we were able to identify an effect of GATA-Serpent knockdown on A. aquasalis hemocyte proliferation and differentiation. These findings expand our understanding of the poorly studied A. aquasalis-P. vivax interactions and uncover GATA-Serpent as a key player of the mosquito innate immune response.

Mosquito pornoscopy: Observation and interruption of Aedes aegypti copulation to determine female polyandric event and mixed progeny.

Ades aegypti is the most important arbovirus vector in the world, and new strategies are under evaluation. Biological studies mentioning the occurrence of a second mate in Aedes aegypti can interfere with vector control program planning, which involves male mosquito release technique. This study presents different experiments to show the occurrence of mixed progeny. Mixed male crosses (using a combination of different type of males in confinement with virgin females) showed no polyandric female. Individual crosses with male substitution in every gonotrophic cycle also did not show any polyandric female. Individual crosses with a 20 minutes interval, with subsequent male change, showed that only a few females presented mixed offspring. The copulation breach in three different moments, group A with full coitus length, group B the coitus was interrupted in 5-7 seconds after the start; and group C, which the copulation was interrupted 3 seconds after started. In summary, group A showed a majority of unique progeny from the first male; group B showed the higher frequency of mixed offspring and group C with the majority of the crosses belonging to the second male. To conclude, the occurrence of a viable second mate and mixed offspring is only possible when the copulation is interrupted; otherwise, the first mate is responsible for mixed progeny.

Aedes aegypti Control Strategies in Brazil: Incorporation of New Technologies to Overcome the Persistence of Dengue Epidemics.

Dengue is considered to be the most important mosquito-borne viral disease in the world. The Aedes aegypti mosquito, its vector, is highly anthropophilic and is very well adapted to urban environments. Although several vaccine candidates are in advanced stages of development no licensed dengue vaccine is yet available. As a result, controlling the spread of dengue still requires that mosquitoes be targeted directly. We review the current methods of dengue vector control focusing on recent technical advances. We first examine the history of Brazil's National Dengue Control Plan in effect since 2002, and we describe its establishment and operation. With the persistent recurrence of dengue epidemics, current strategies should be reassessed to bring to the forefront a discussion of the possible implementation of new technologies in Brazil's mosquito control program.

The role of reactive oxygen species in Anopheles aquasalis response to Plasmodium vivax infection.

Malaria affects millions of people worldwide and hundreds of thousands of people each year in Brazil. The mosquito Anopheles aquasalis is an important vector of Plasmodium vivax, the main human malaria parasite in the Americas. Reactive oxygen species (ROS) have been shown to have a role in insect innate immune responses as a potent pathogen-killing agent. We investigated the mechanisms of free radicals modulation after A. aquasalis infection with P. vivax. ROS metabolism was evaluated in the vector by studying expression and activity of three key detoxification enzymes, one catalase and two superoxide dismutases (SOD3A and SOD3B). Also, the involvement of free radicals in the mosquito immunity was measured by silencing the catalase gene followed by infection of A. aquasalis with P. vivax. Catalase, SOD3A and SOD3B expression in whole A. aquasalis were at the same levels of controls at 24 h and upregulated 36 h after ingestion of blood containing P. vivax. However, in the insect isolated midgut, the mRNA for these enzymes was not regulated by P. vivax infection, while catalase activity was reduced 24 h after the infectious meal. RNAi-mediated silencing of catalase reduced enzyme activity in the midgut, resulted in increased P. vivax infection and prevalence, and decreased bacterial load in the mosquito midgut. Our findings suggest that the interactions between A. aquasalis and P. vivax do not follow the model of ROS-induced parasite killing. It appears that P. vivax manipulates the mosquito detoxification system in order to allow its own development. This can be an indirect effect of fewer competitive bacteria present in the mosquito midgut caused by the increase of ROS after catalase silencing. These findings provide novel information on unique aspects of the main malaria parasite in the Americas interaction with one of its natural vectors.

Inhibition of tissue inflammation and bacterial translocation as one of the protective mechanisms of Saccharomyces boulardii against Salmonella infection in mice.

Growing evidences suggest that Saccharomyces boulardii (SB) is efficacious against bacterial infections and inflammatory bowel diseases. This study investigated the effects of treatment with SB provided in a murine model of typhoid fever. Mice were divided into two groups: (1) control animals challenged with Salmonella Typhimurium (ST), and (2) animals receiving SB, and then challenged with ST. At days 0, 1, 5, 10 and 15 post-challenge, animals were euthanized and tissues collected to analyze bacterial translocation, cytokines, signaling pathways and histological analysis. Survival rate and animal weight were also evaluated. Treatment with SB increased survival rate and inhibited translocation of bacteria after ST challenge. Histological data showed that SB also protected mice against liver damage induced by ST. SB decreased levels of inflammatory cytokines and activation of mitogen-activated protein kinases (p38, JNK and ERK1/2), phospho-IκB, p65-RelA, phospho-jun and c-fos in the colon, signal pathways involved in the activation of inflammation induced by ST. Further experiments revealed that probiotic effects were due, at least in part, to the binding of ST to the yeast. Such binding diminishes ST translocation, resulting in decreased activation of signaling pathways which lead to intestinal inflammation in a murine model of typhoid fever.

A tetravalent dengue nanoparticle stimulates antibody production in mice.

BACKGROUND: Dengue is a major public health problem worldwide, especially in the tropical and subtropical regions of the world. Infection with a single Dengue virus (DENV) serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients experiencing secondary infection with a different serotype progresses to the severe form of the disease, dengue hemorrhagic fever/dengue shock syndrome. Currently, there are no licensed vaccines or antiviral drugs to prevent or treat dengue infections. Biodegradable nanoparticles coated with proteins represent a promising method for in vivo delivery of vaccines. FINDINGS: Here, we used a murine model to evaluate the IgG production after administration of inactivated DENV corresponding to all four serotypes adsorbed to bovine serum albumin nanoparticles. This formulation induced a production of anti-DENV IgG antibodies (p < 0.001). However, plaque reduction neutralization assays with the four DENV serotypes revealed that these antibodies have no neutralizing activity in the dilutions tested. CONCLUSIONS: Our results show that while the nanoparticle system induces humoral responses against DENV, further investigation with different DENV antigens will be required to improve immunogenicity, epitope specicity, and functional activity to make this platform a viable option for DENV vaccines.

The JAK-STAT pathway controls Plasmodium vivax load in early stages of Anopheles aquasalis infection.

Malaria affects 300 million people worldwide every year and 450,000 in Brazil. In coastal areas of Brazil, the main malaria vector is Anopheles aquasalis, and Plasmodium vivax is responsible for the majority of malaria cases in the Americas. Insects possess a powerful immune system to combat infections. Three pathways control the insect immune response: Toll, IMD, and JAK-STAT. Here we analyze the immune role of the A. aquasalis JAK-STAT pathway after P. vivax infection. Three genes, the transcription factor Signal Transducers and Activators of Transcription (STAT), the regulatory Protein Inhibitors of Activated STAT (PIAS) and the Nitric Oxide Synthase enzyme (NOS) were characterized. Expression of STAT and PIAS was higher in males than females and in eggs and first instar larvae when compared to larvae and pupae. RNA levels for STAT and PIAS increased 24 and 36 hours (h) after P. vivax challenge. NOS transcription increased 36 h post infection (hpi) while this protein was already detected in some midgut epithelial cells 24 hpi. Imunocytochemistry experiments using specific antibodies showed that in non-infected insects STAT and PIAS were found mostly in the fat body, while in infected mosquitoes the proteins were found in other body tissues. The knockdown of STAT by RNAi increased the number of oocysts in the midgut of A. aquasalis. This is the first clear evidence for the involvement of a specific immune pathway in the interaction of the Brazilian malaria vector A. aquasalis with P. vivax, delineating a potential target for the future development of disease controlling strategies.

Oral treatment with Saccharomyces cerevisiae strain UFMG 905 modulates immune responses and interferes with signal pathways involved in the activation of inflammation in a murine model of typhoid fever.

Salmonella spp. are Gram-negative, facultative, intracellular pathogens that cause several diarrheal diseases ranging from self-limiting gastroenteritis to typhoid fever. Previous results from our laboratory showed that Saccharomyces cerevisiae strain UFMG 905 isolated from 'cachaça' production presented probiotic properties due to its ability to protect against experimental infection with Salmonella enterica serovar Typhimurium. In this study, the effects of oral treatment with S. cerevisiae 905 were evaluated at the immunological level in a murine model of typhoid fever. Treatment with S. cerevisiae 905 inhibited weight loss and increased survival rate after Salmonella challenge. Immunological data demonstrated that S. cerevisiae 905 decreased levels of proinflammatory cytokines and modulated the activation of mitogen-activated protein kinases (p38 and JNK, but not ERK1/2), NF-κB and AP-1, signaling pathways which are involved in the transcriptional activation of proinflammatory mediators. Experiments in germ-free mice revealed that probiotic effects were due, at least in part, to the binding of Salmonella to the yeast. In conclusion, S. cerevisiae 905 acts as a potential new biotherapy against S. Typhimurium infection due to its ability to bind bacteria and modulate signaling pathways involved in the activation of inflammation in a murine model of typhoid fever.