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Zika virus (ZIKV) may cause febrile illness and neurological damage, such as microcephaly in fetuses. ZIKV is transmitted to humans by Aedes aegypti, a nearly cosmopolitan mosquito. Understanding the virus-vector molecular interactions has been promising to enhance the knowledge towards disease mitigation. Since ZIKV infection alters gene physiology of mosquitoes, we examined the expression profile of ZIKV-infected Ae. aegypti by several approaches to identify genes altered by viral infection. Transcriptomics were performed by comparing between ZIKV-infected and uninfected Ae. aegypti females, which revealed some differentially expressed genes. Most of these genes appear to be involved with immune response as evidenced by an interactome analysis, and a prominent finding was a calreticulin-like (CRT) gene, which was upregulated during the infection. Expression of CRT was also experimentally quantified by qPCR, however, it revealed no significant differences between infected and uninfected females. Instead, expression levels were highly variable among individuals and negatively correlated to viral load. We also tested the possibility of this gene to be silenced, but the double-stranded RNA did not reduce CRT expression, and actually increased the inter-individuals' expressional variability. Present results differed from our original hypothesis of upregulation by infection. They also diverged between them (comparing qPCR to Transcriptomics) and from the literature which reported augmented CRT levels in Aedes species during viral infection. Present case probably underlies a more complex virus-host interaction system than we expected. Regulation of this gene seems not to be a linear correlation between expression and viremy. As infection takes place, a complex homeostatic mechanism may act to prevent expression and other cellular tasks from drifting. It is also possible that CRT expression is simply randomly disturbed by viral infection. Taken together, results show that CRT expression profile during ZIKV infection is complex and requires different investigative approaches to be understood. Studies focused on the biochemical function of CRT protein and on its role in the native mosquito metabolic network could unravel how it is actually influenced by ZIKV. Current work contributes more by getting incidental findings and by posing new hypotheses than by answering the original questions.

BackgroundThe invasion of the mosquito salivary glands byPlasmodiumsporozoites 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 reducePlasmodium gallinaceumsporozoite invasion ofAedes aegyptisalivary glands, but the specific role of this protein in sporozoite invasion and in other stages of thePlasmodiumlife cycle remains unknown.MethodsRNA interference and CRISPR/Cas9 were used to evaluate the role ofA. aegyptiSGS1 in theP. gallinaceumlife cycle.ResultsKnockdown 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.ConclusionSGS1 facilitates, but is not essential for, invasion ofA. aegyptisalivary glands byP. gallinaceumand 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.

During probing and blood feeding, haematophagous mosquitoes inoculate a mixture of salivary molecules into their vertebrate hosts’ skin. In addition to the anti‐haemostatic and immunomodulatory activities, mosquito saliva also triggers acute inflammatory reactions, especially in sensitized hosts. Here, we characterize the oedema and the cellular infiltrate following Aedes aegypti mosquito bites in the skin of sensitized and non‐sensitized BALB/c mice by flow cytometry. Ae. aegypti bites induced an increased oedema in the ears of both non‐sensitized and salivary gland extract‐ (SGE‐)sensitized mice, peaking at 6 hr and 24 hr after exposure, respectively. The quantification of the total cell number in the ears revealed that the cellular recruitment was more robust in SGE‐sensitized mice than in non‐sensitized mice, and the histological evaluation confirmed these findings. The immunophenotyping performed by flow cytometry revealed that mosquito bites were able to produce complex changes in cell populations present in the ears of non‐sensitized and SGE‐sensitized mice. When compared with steady‐state ears, the leucocyte populations significantly recruited to the skin after mosquito bites in non‐sensitized and sensitized mice were eosinophils, neutrophils, monocytes, inflammatory monocytes, mast cells, B‐cells and CD4(+) T‐cells, each one with its specific kinetics. The changes in the absolute number of cells suggested two cell recruitment profiles: (i) a saliva‐dependent migration; and (ii) a migration dependent on the immune status of the host. These findings suggest that mosquito bites influence the skin microenvironment by inducing differential cell migration, which is dependent on the degree of host sensitization to salivary molecules.

Background During the feeding process, the mouthparts of hematophagous mosquitoes break the skin barrier and probe the host tissue to find the blood. The saliva inoculated in this microenvironment modulates host hemostasis, inflammation and adaptive immune responses. However, the mechanisms involved in these biological activities remain poorly understood and few studies explored the potential roles of mosquito saliva on the individual cellular components of the immune system. Here, we report the immunomodulatory activities of Aedes aegypti salivary cocktail on murine peritoneal macrophages. Results The salivary gland extract (SGE) of Ae. aegypti inhibited the production of nitric oxide and inflammatory cytokines such as interleukin-6 (IL-6) and IL-12, as well as the expression of inducible nitric oxide synthase and NF-κB by murine macrophages stimulated by lipopolysaccharide (LPS) plus interferon-γ (IFN-γ). The spare respiratory capacity, the phagocytic and microbicidal activities of these macrophages were also reduced by Ae. aegypti SGE. These phenotypic changes are consistent with SGE suppressing the proinflammatory program of M1 macrophages. On the other hand, Ae. aegypti SGE did not influence M2-associated markers (urea production, arginase-1 and mannose receptor-1 expression), either in macrophages alternatively activated by IL-4 or in those classically activated by LPS plus IFN-γ. In addition, Ae. aegypti SGE did not display any cytokine-binding activity, nor did it affect macrophage viability, thus excluding supposed experimental artifacts. Conclusions Given the importance of macrophages in a number of biological processes, our findings help to enlighten how vector saliva modulates vertebrate host immunity.

Acetaminophen (N-acetyl-p-aminophenol, APAP) overdose is the most common cause of drug-induced liver injury (DILI). Although the primary hepatic damage is induced by APAP-derived toxic intermediates resulting from cytochrome P450 metabolism, immune components also play an important role in DILI pathophysiology.Aedes aegyptisaliva is a source of bioactive molecules within vitroanti-inflammatory and immunomodulatory activities. However, evidences on the therapeutic use ofAe.aegyptisalivary preparations in animal models of relevant clinical conditions are still scarce. Thus, the present study was designed to evaluate the protective role ofAe.aegyptisaliva in a murine model of APAP-induced DILI. C57BL/6 mice were exposed toAe.aegyptibites 2 hours after APAP overdose. Biochemical and immunological parameters were evaluated in blood and liver samples at different time points after APAP administration. Exposure toAe.aegyptisaliva attenuated liver damage, as demonstrated by reduced hepatic necrosis and serum levels of alanine aminotransferase in APAP-overdosed mice. The levels of hepatic CYP2E1, the major enzyme responsible for the bioactivation of APAP, were not changed inAe.aegyptiexposed animals, suggesting no effects in the generation of hepatotoxic metabolites. On the other hand, mice treated withAe.aegyptisaliva following APAP overdose presented lower serum concentration of TNF-α, IL-6, IL-1β and IL-10, as well as reduced frequency of inflammatory cell populations in the liver, such as NKT cells, macrophages and dendritic cells. These findings show thatAe.aegyptisaliva has bioactive molecules with therapeutic properties and may represent a prospective source of new compounds in the management of DILI-associated inflammatory disorders and, perhaps, many other inflammatory/autoimmune diseases.

Insect-specific viruses do not replicate in vertebrates. Here, we report the genome sequence of a novel strain of a Phasi Charoen-like virus (PCLV) that was isolated from a wild Aedes aegypti mosquito collected in Aracajú, Sergipe State, Brazil. The coding-complete genome of the PCLV is described in this report.

Dengue viruses (DENV) are the wildest transmitted arbovirus members of the family Flaviviridae, genus Flavivirus. Dengue viruses are composed of four serotypes, DENV1, 2, 3, and 4, and these viruses can cause dengue fever and dengue haemorrhagic fever or dengue shock syndrome, when infecting humans. RNA interference (RNAi) is a self-defence mechanism, which can be used to prevent invasions of RNA viruses to the host. Genetically engineering a host with an RNAi molecule that targets a single virus serotype may develop escape mutants, and can cause unusual dominance over other serotypes. Therefore, the simultaneous targeting of multiple serotypes is necessary to block DENV transmission. Here, we report the development of transgenic Aedes aegypti based on a bioinformatically designed multiple miRshRNA (microRNA-based shRNA) DNA sequence under the control of a blood-meal induced promoter, Carboxypeptidase A, to induce RNAi for DENV in Aedes aegypti, and demonstrate the expression of a synthetic multiple shRNA polycistronic cluster having RNA interference sequences to target DENV genomes. The transgenic mosquitoes have lower rates of infection, dissemination, and transmission for DENV2 and DENV4 compared to wild mosquitoes, with a significant reduction of dengue copy number and antigen levels in the midgut. These levels of DENV were low enough to make transgenic mosquitoes stop the DENV transmission from infected host to a susceptible host and refractory to DENV2 and DENV4 infection. Such multiple resistance in Ae. aegypti has not been documented previously. Laboratory fitness measurement of transgenic Ae. aegypti showed results comparable to other reported transgenic mosquitoes.

In an attempt to control the mosquito-borne diseases yellow fever, dengue, chikungunya, and Zika fevers, a strain of transgenically modified Aedes aegypti mosquitoes containing a dominant lethal gene has been developed by a commercial company, Oxitec Ltd. If lethality is complete, releasing this strain should only reduce population size and not affect the genetics of the target populations. Approximately 450 thousand males of this strain were released each week for 27 months in Jacobina, Bahia, Brazil. We genotyped the release strain and the target Jacobina population before releases began for21,000 single nucleotide polymorphisms (SNPs). Genetic sampling from the target population six, 12, and 27-30 months after releases commenced provides clear evidence that portions of the transgenic strain genome have been incorporated into the target population. Evidently, rare viable hybrid offspring between the release strain and the Jacobina population are sufficiently robust to be able to reproduce in nature. The release strain was developed using a strain originally from Cuba, then outcrossed to a Mexican population. Thus, Jacobina Ae. aegypti are now a mix of three populations. It is unclear how this may affect disease transmission or affect other efforts to control these dangerous vectors. These results highlight the importance of having in place a genetic monitoring program during such releases to detect un-anticipated outcomes.

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.

Malaria is an infectious disease responsible for approximately one million deaths annually. Oligopeptides such as angiotensin II (AII) and its analogs are known to have antimalarial effects against Plasmodium gallinaceum and Plasmodium falciparum. However, their mechanism of action is still not fully understood at the molecular level. In the work reported here, we investigated this issue by comparing the antimalarial activity of AII with that of (i) its diastereomer formed by only d-amino acids; (ii) its isomer with reversed sequence; and (iii) its analogs restricted by lactam bridges, the so-called VC5 peptides. Data from fluorescence spectroscopy indicated that the antiplasmodial activities of both all-D-AII and all-D-VC5 were as high as those of the related peptides AII and VC5, respectively. In contrast, retro-AII had no significant effect against P. gallinaceum. Conformational analysis by circular dichroism suggested that AII and its active analogs usually adopted a β-turn conformation in different solutions. In the presence of membrane-mimetic micelles, AII had also a β-turn conformation, while retro-AII was random. Molecular dynamics simulations demonstrated that the AII chains were slightly more bent than retro-AII at the surface of a model membrane. At the hydrophobic membrane interior, however, the retro-AII chain was severely coiled and rigid. AII was much more flexible and able to experience both straight and coiled conformations. We took it as an indication of the stronger ability of AII to interact with membrane headgroups and promote pore formation.

The global economic cost of Aedes-borne diseases, such as dengue, is estimated to be in the billions of dollars annually. In this scenario, a sustained vector control strategy is the only alternative to control dengue, as well as other diseases transmitted by Aedes, including Zika and chikungunya. The use of transgenic mosquitoes is a promising weapon in the improvement of approaches currently applied in Aedes aegypti control. Field trials using genetically modified mosquitoes for population control have been conducted and offer an excellent opportunity to evaluate what can be improved. In a mass-rearing mosquito facility, the absence of a transgenic line that produces male-only progeny is undoubtedly a limiting factor; thus, being able to manipulate sex determination in this species is a fundamental step for the success of this strategy. Likewise, the possibility of manipulation of the sex determination pathway opens-up a new opportunity for disease control.

Aedes aegypti is the principal vector of the urban arboviruses and the blood ingestion is important to produce the eggs in this species. To analyze the egg production in Ae. aegypti, researchers frequently use small cages or Drosophila vials to collect eggs from gravid females. Although it is affordable, the setup is time- and space-consuming, mainly when many mosquitoes need to be individually analyzed. This study presents an easy, cheap, and space-saving method to perform individual oviposition assays in Ae. aegypti using cell culture plates. This new method to access fecundity rate was named &ldquo, oviplate&rdquo, The oviplates are setup with 12- or 24-well plates, distilled water and filter paper and they are 78 to 88% cheaper than the traditional Drosophila vial assay, respectively. Furthermore, to allocate 72 vitellogenic females in an insectary using Drosophila vial is necessary 4100 cm3 against 1400 cm3 and 700 cm3 when using 12- and 24-well plates, respectively. No statistical differences were found between the number of eggs laid in Drosophila vials and the oviplates, validating the method. The oviplate method is an affordable, and time- and space-efficient device, and it is simpler to perform individual fecundity analyses in Ae. aegypti.

Versatile molecular tools for creating driving transgenes and other invasive genetic factors present regulatory, ethical, and environmental challenges that should be addressed to ensure their safe use. In this article, we discuss driving transgenes and invasive genetic factors that can potentially spread after their introduction into a small proportion of individuals in a population. The potential of invasive genetic factors to increase their number in natural populations presents challenges that require additional safety measures not provided by previous recommendations regarding accidental release of arthropods. In addition to providing physical containment, invasive genetic factors require greater attention to strain management, including their distribution and identity confirmation. In this study, we focus on insects containing such factors with recommendations for investigators who are creating them, institutional biosafety committees charged with ensuring safety, funding agencies providing support, those managing insectaries handling these materials who are responsible for containment, and other persons who will be receiving insects—transgenic or not—from these facilities. We give specific examples of efforts to modify mosquitoes for mosquito-borne disease control, but similar considerations are relevant to other arthropods that are important to human health, the environment, and agriculture.

International audience; Major efforts are currently underway to develop novel, complementary methods to combat mosquito-borne diseases. Mosquito genetic control strategies (GCSs) have become an increasingly important area of research on account of their species-specificity, track record in targeting agricultural insect pests, and their environmentally non-polluting nature. A number of programs targeting Aedes and Anopheles mosquitoes, vectors of human arboviruses and malaria respectively, are currently being developed or deployed in many parts of the world. Operationally implementing these technologies on a large scale however, beyond proof-of-concept pilot programs, is hampered by the absence of adequate sex separation methods. Sex separation eliminates females in the laboratory from male mosquitoes prior to release. Despite the need for sex separation for the control of mosquitoes, there have been limited efforts in recent years in developing systems that are fit-for-purpose. In this special issue of Parasites and Vectors we report on the progress of the global Coordinated Research Program on Exploring genetic, molecular, mechanical and behavioural methods for sex separation in mosquitoes that is led by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture with the specific aim of building efficient sex separation systems for mosquito species. In an effort to overcome current barriers we briefly highlight what we believe are the three main reasons why progress has been so slow in developing appropriate sex separation systems: the availability of methods that are not scalable, the difficulty of building the ideal genetic systems and, finally, the lack of research efforts in this area.

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.

Background The worldwide expansion of new emergent arboviruses such as Chikungunya and Zika reinforces the importance in understanding the role of mosquito species in spreading these pathogens in affected regions. This knowledge is essential for developing effective programs based on species specificity to avoid the establishment of endemic transmission cycles sustained by the identified local vectors. Although the first autochthonous transmission of Chikungunya virus was described in 2014 in the north of Brazil, the main outbreaks were reported in 2015 and 2016 in the northeast of Brazil. Methodology/Principal findings During 5 days of February 2016, we collected mosquitoes in homes of 6 neighborhoods of Aracaju city, the capital of Sergipe state. Four mosquito species were identified but Culex quinquefasciatus and Aedes aegypti were the most abundant. Field-caught mosquitoes were tested for Chikungunya (CHIKV), Zika (ZIKV) and Dengue viruses (DENV) by qRT-PCR and one CHIKV-infected Ae. aegypti female was detected. The complete sequence of CHIKV genome was obtained from this sample and phylogenetic analysis revealed that this isolate belongs to the East-Central-South-African (ECSA) genotype. Conclusions Our study describes the first identification of a naturally CHIKV-infected Ae. aegypti in Brazil and the first report of a CHIKV from ECSA genotype identified in this species in the Americas. These findings support the notion of Ae. aegypti being a vector involved in CHIKV outbreaks in northeast of Brazil., Author summary The chikungunya outbreaks have become one of the main public health problems in the Northeast Region of Brazil. Since the chikungunya virus (CHIKV) was recently introduced in this country, finding vector associations involved in its transmission to the vertebrate host is primordial to better design efficient intervention against mosquitoes. In the present study, field-caught mosquitoes in Aracaju city, Sergipe State, Brazil were evaluated for the presence of CHIKV and other two endemic arboviruses (Zika and Dengue viruses) in this region. We described here the first identification of the East-Central-South-African (ECSA) CHIKV genotype naturally infecting Aedes aegypti mosquitoes in Brazil. This work suggests the involvement of this species in CHIKV outbreaks in Northeast Region of this country and reveals a possible interaction between this vector and the recently introduced CHIKV ECSA genotype.

The Zika virus outbreaks are unprecedented human threat in relation to congenital malformations and neurological/autoimmune complications. Since this virus has high potential to spread in regions presenting the vectors, improvement in mosquito control is a top priority. Thus, Aedes aegypti laboratory strains will be fundamental to support studies in different research fields implicated on Zika-mosquito interactions which are the basis for the development of innovative control methods. In this sense, our aim was to determine the main infection aspects of a Brazilian Zika strain in reference Aedes aegypti laboratory mosquitoes. We orally exposed Rockefeller, Higgs and Rexville mosquitoes to the Brazilian ZIKV (ZIKVBR) and qRT-PCR was applied to determine the infection, dissemination and detection rates of ZIKV in the collected saliva as well as viral levels in mosquito tissues. The three strains sustain the virus development but Higgs showed significantly lower viral loads in bodies at 14 days post-infection (dpi) and the lowest prevalences in bodies and heads. The Rockefeller strain was the most susceptible at 7 dpi but similar dissemination rates were observed at 14 dpi. Although variations exist, the ZIKVBR RNA shows detectable levels in saliva of the three strains at 14 dpi but is only detected in Rockefeller at 7 dpi. Moreover, saliva samples from the three strains were confirmed to be infectious when intrathoracically injected into mosquitoes. The ZIKVBR kinetics was monitored in Rockefeller mosquitoes and virus could be identified in the heads at 4 dpi but was more consistently detected late in infection. Our study presents the first evaluation on how Brazilian Zika virus behaves in reference Aedes aegypti strains and shed light on how the infection evolves over time. Vector competence and hallmarks of the ZIKVBR development were revealed in laboratory mosquitoes, providing additional information to accelerate studies focused on ZIKV-mosquito interactions.

Since the Zika outbreaks are unprecedented human threat in relation to congenital malformations and neurological/autoimmune complications as well as its high potential to spread in regions presenting the vectors, improvements in mosquito control is a top priority. Thus, Aedes aegypti laboratory strains will be fundamental to support studies in different research fields implicated on Zika-mosquito interactions which are the basis for the development of innovative control methods. In this sense, we determined the main infection aspects of the Brazilian Zika strain in reference Aedes aegypti laboratory mosquitoes.We orally exposed Rockefeller, Higgs and Rexville mosquitoes to a Brazilian ZIKV (ZIKVBR) and qRT-PCR was applied to determine the infection and dissemination rates, and viral levels in mosquito tissues as well as in the saliva. The ZIKVBR kinetics was monitored during the infection in Rockefeller mosquitoes. Rockefeller strain was the most susceptible at 7 days post-infection but all strains presented similar infection levels at 14 days post-infection. Although variations in the saliva detection rates were observed, we confirmed that ZIKVBR was present in saliva from Rockefeller, Higgs and Rexville females at detectable levels at 14 days post-infection. The ZIKVBR kinetics in Rockefeller mosquitoes showed that the virus could be detected in the heads at 4 days post-infection but was more consistently detected late in infection. The viral levels peaked at 11 days post-infection in the mosquito bodies, remaining stable until 14 days post-infection, in contrast to the heads, where the mean viral levels only peaked at 14 days post-infection.Our study presents the first evaluation on how Brazilian Zika virus behaves in reference Aedes aegypti strains and shed light on how the infection evolves over time. Vector competence and basic hallmarks of the ZIKVBR development were revealed in laboratory mosquitoes. This study provides additional information to accelerate studies focusing on ZIKV-mosquito interactions.

The number of mosquito populations resistant to insecticides is increasing along with the reemerging of vector-borne diseases. New technologies are under evaluation to complement the strategies used against these mosquitoes. Transgenic mosquitoes are one approach that some countries are considering and they are being evaluated to control the wild population. Although they have achieved success in population suppression of Aedes aegypti (L.) (Diptera: Culicidae), these studies have not demonstrated what the outcomes are when releases are interrupted (ceased). In this study, after demonstrating suppression of Ae. aegypti using transgenic technology, changes in the spatial distribution of the infestation and the abundance of the vector Ae. aegypti were assessed in the post-release period, along with fluctuation of transgenic mosquitoes in two areas of Brazil. In both pilot trials, there was an average suppression of ca. 70% of the wild population due to the release of transgenic males compared to the pre-release period. In Juazeiro (Mandacaru), in the post-release phase, the number of eggs per trap ranged between 0.06 and 14.41 (mean ± SE = 4.44 ± 0.44), and the ovitrap index (OI = number of ovitraps with eggs/total number of ovitraps recovered) ranged from 0.01 to 0.43 (0.13 ± 0.01). In Jacobina (Pedra Branca), during the post-release phase, the number of eggs per trap ranged between 1 and 7.2 (1.72 ± 0.72), and the OI ranged from 1 to 0.83 (0.095 ± 0.032). The mosquito population in Juazeiro (Mandacaru) remained suppressed for 17 weeks after the release interruption, whereas in Jacobina (Pedra Branca) suppression lasted 32 weeks. In Juazeiro, transgenic larvae were detected up to 5 months after the interruption of the over-flooding releases of transgenic males. In Jacobina, they were found up to 2 months after the release interruption. The number of eggs collected increased 4–5 months after the release interruption, which indicated that the Ae. aegypti population had been re-established after the interruption of releases. The results demonstrate that the technique requires a continuous release in the treated areas, and after suppression, the release rate can be decreased and used as a barrier against external migration.

Angiotensin II (Ang II) is a natural mammalian hormone that has been described to exhibit antiplasmodial activity therefore constituting a promising alternative for the treatment of malaria. Despite its promise, the development of Ang II as an antimalarial is limited by its potent induction of vasoconstriction and its rapid degradation within minutes. Here, we used peptide design to perform targeted chemical modifications to Ang II to generate conformationally restricted (disulfide-crosslinked) peptide derivatives with suppressed vasoconstrictor activity and increased stability. Designed constrained peptides were synthesized chemically and then tested for antiplasmodial activity. Two lead constrained peptides were identified (i.e., peptides 1 and 2), each composed of 10 amino acid residues. These peptides exhibited very promising activity in both our Plasmodium gallinaceum (>80%) and Plasmodium falciparum (>40%) models, an activity that was equivalent to that of Ang II, and led to complete suppression of vasoconstriction. In addition, peptide 5 exhibited selective activity towards the pre-erythrocytic stage (98% of activity against P. gallinaceum), thus suggesting that it may be possible to design peptides that target specific stages of the malaria life cycle. The Ang II derived stable scaffolds presented here may provide the basis for development of a new generation of peptide-based drugs for the treatment of malaria.

Anopheles (Nyssorhynchus) aquasalis Curry, 1932, is an anopheline that plays an important role in Plasmodium transmission in the New World. Few studies have explored the molecular reproductive biology of this vector species. In the current study, we isolated and sequenced a partial, 886 bp, cDNA fragment of a vitellogenin (Vtg) gene from An. aquasalis. The transcription profile of this Vtg gene in adult females was characterized, and it is similar to other members of the Culicidae. We also detected a smaller subunit of An. aquasalis Vtg proteins in ovary tissue and characterized its translational profile in previtellogenic and vitellogenic females. Our results represent the first molecular characterization of An. aquasalis vitellogenin expression products.

Angiotensin II (AII) as well as analog peptides shows antimalarial activity against Plasmodium gallinaceum and Plasmodium falciparum, but the exact mechanism of action is still unknown. This work presents the solid-phase synthesis and characterization of eight peptides corresponding to the alanine scanning series of AII plus the amide-capped derivative and the evaluation of the antiplasmodial activity of these peptides against mature P. gallinaceum sporozoites. The Ala screening data indicates that the replacement of either the Ile5 or the His6 residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala6]-AII (79%), and [Ala5]-AII (75%). Analogs [Ala3]-AII, [Ala1]-AII, and AII-NH2 showed antiplasmodial activity around 65%, whereas the activity of the [Ala8]-AII, [Ala7]-AII, [Ala4]-AII, and [Ala2]-AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a β-fold conformation in different solutions. All AII analogs, except [Ala4]-AII and [Ala8]-AII, show contractile responses and interact with the AT1 receptor, [Ala5]-AII and [Ala6]-AII. In conclusion, this approach is helpful to understand the contribution of each amino acid residue to the bioactivity of AII, opening new perspectives toward the design of new sporozoiticidal compounds. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

When considering a mosquito release programme, one of the first issues to be addressed is how to eliminate/separate the females. The greatest number of options might eventually be available for those who can use transgenic mosquitoes, but the inherent characteristics of the target species may also provide possibilities for interim measures until more efficient methods can be developed. Differences in intrinsic size, in behaviour and in development rate between females and males are often available and useful for sexing. Efficient species-specific systems for eliminating females at the embryo stage have been developed, but most have since been discarded due to lack of use. Ideal systems specifically kill female embryos using some treatment that can be manipulated during production. Such killing systems are far more efficient than using intrinsic sexual differences, but they systems require selectable genetic markers and sex-linkage created by rare random chromosomal rearrangements. While intrinsic sexual differences should not be considered as long-term candidates for the development of robust and efficient sexing approaches, in the absence of these, the accessibility and integration of less efficient systems can provide a stop-gap measure that allows rapid start up with a minimum of investment. The International Atomic Energy Agency is funding over a 5 year period (2013-2018) a new Coordinated Research Project on "Exploring Genetic, Molecular, Mechanical and Behavioural Methods of Sex Separation in Mosquitoes" to network researchers and to address the critical need of genetic sexing strains for the implementation of the sterile insect technique (using radiation-sterilised or transgenic male mosquitoes) and for insect incompatibility technique programmes against disease-transmitting mosquitoes.

Mosquitoes are responsible for the transmission of pathogens that cause devastating human diseases such as malaria and dengue. The current increase in mean global temperature and changing sea level interfere with precipitation frequency and some other climatic conditions which, in general, influence the rate of development of insects and etiologic agents causing acceleration as the temperature rises. The most common strategy employed to combat target mosquito species is the Integrated Vector Management (IVM), which comprises the use of multiple activities and various approaches to preventing the spread of a vector in infested areas. IVM programmes are becoming ineffective; and the global scenario is threatening, requiring new interventions for vector control and surveillance. Not surprisingly, there is a growing need to find alternative methods to combat the mosquito vectors. The possibility of using transgenic mosquitoes to fight against those diseases has been discussed over the last two decades and this use of transgenic lines to suppress populations or to replace them is still under investigation through field and laboratory trials. As an alternative, the available transgenic strategies could be improved by coupling suppression and substitution strategies. The idea is to first release a suppression line to significantly reduce the wild population, and once the first objective is reached a second release using a substitution line could be then performed. Examples of targeting this approach against vectors of malaria and dengue are discussed.

Controlling the dissemination of malaria requires the development of new drugs against its etiological agent, a protozoan ofthe Plasmodium genus. Angiotensin II and its analog peptides exhibit activity against the development of immature andmature sporozoites of Plasmodium gallinaceum. In this study, we report the synthesis and characterization of angiotensin IIlinear and cyclic analogs with anti-plasmodium activity. The peptides were synthesized by a conventional solid-phase methodon Merrifield's resin using the t-Boc strategy, purified by RP-HPLC and characterized by liquid chromatography/ESI (+) MS(LC-ESI(+)/MS), amino acid analysis, and capillary electrophoresis. Anti-plasmodium activity was measured in vitro byfluorescence microscopy using propidium iodine uptake as an indicator of cellular damage. The activities of the linear andcyclic peptides are not significantly different (p

Classical studies have shown that Aedes aegypti salivary secretion is responsible for the sensitization to mosquito bites and many of the components present in saliva are immunogenic and capable of inducing an intense immune response. Therefore, we have characterized a murine model of adjuvant-free systemic allergy induced by natural exposure to mosquito bites. BALB/c mice were sensitized by exposure to A. aegypti mosquito bites and intranasally challenged with phosphate-buffered saline only or the mosquito's salivary gland extract (SGE). Blood, bronchoalveolar lavage (BAL) and lung were collected and evaluated for cellularity, histopathological analyses, cytokines and antibody determination. Respiratory pattern was analyzed by Penh measurements and tracheal segments were obtained to study in vitro reactivity to methacholine. BAL recovered from sensitized mice following challenge with SGE showed an increased number of eosinophils and Th2 cytokines such as IL-4, IL-5 and IL-13. Peribronchoalveolar eosinophil infiltration, mucus and collagen were also observed in lung parenchyma of sensitized mice, suggesting the development of a typical Th2 response. However, the antibody profile in serum of these mice evidenced a mixed-type response with presence of both, IgG1/IgE (Th2-related) and IgG2a (Th1-related) isotypes. In addition, changes in breathing pattern and tracheal reactivity to methacholine were not found. Taken together, our results show that A. aegypti bites trigger an atypical allergic reaction, with some classical cellular and soluble Th2 components in the lung, but also systemic Th1 and Th2 antibody isotypes and no change in either the respiratory pattern or the trachea responsiveness to agonist.

New studies and strategies are under development for mosquito control. The resulting new information might be evaluated and shared with the local population. They must face the possibility that those strategies will result in increased numbers of mosquitoes in local houses, gardens, and streets. Here is presented a brief collection of studies using this kind of technology and the outcomes from the public engagement used by them, from failures as in India to the one demonstrating a lot of success in Brazil. We describe the set of activities and discuss about the need to adapt any action plan to every target population. Based on the Brazilian experience, contrasted against Cayman Island, the most important characteristic that shifts from a good community engagement is the lack of transparency and distance from local population.

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types.

Vector-borne diseases are currently the worst challenge of public health in Brazil and worldwide, being the current control measures inefficient. Innovations concerning vector control point to a new perspective to handling with gene engineering, as they may interfere with disease transmission, preventing the pathogen to complete its cycle in the vector, ie, reducing mosquito population. We present some of the mean genetic tools available, SIT and RIDL for example. We discuss how these tools work, some risks and benefits

Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites.

To analyze the genetic relatedness and phylogeographic structure of Aedes aegypti, we collected samples from 36 localities throughout the Americas (Brazil, Peru, Venezuela, Guatemala, US), three from Africa (Guinea, Senegal, Uganda), and three from Asia (Singapore, Cambodia, Tahiti). Amplification and sequencing of a fragment of the mitochondrial NADH dehydrogenase subunit 4 gene identified 20 distinct haplotypes, of which 14 are exclusive to the Americas, four to African/Asian countries, one is common to the Americas and Africa, and one to the Americas and Asia. Nested clade analysis (NCA), pairwise distribution, statistical parsimony, and maximum parsimony analyses were used to infer evolutionary and historic processes, and to estimate phylogenetic relationships among haplotypes. Two clusters were found in all the analyses. Haplotypes clustered in the two clades were separated by eight mutational steps. Phylogeographic structure detected by the NCA was consistent with distant colonization within one clade and fragmentation followed by range expansion via long distance dispersal in the other. Three percent of nucleotide divergence between these two clades is suggestive of a gene pool division that may support the hypothesis of occurrence of two subspecies of Ae. aegypti in the Americas.

Background Aedes aegypti, the principal vector of dengue fever, have been genetically engineered for use in a sterile insect control programme. To improve our understanding of the dispersal ecology of mosquitoes and to inform appropriate release strategies of ‘genetically sterile’ male Aedes aegypti detailed knowledge of the dispersal ability of the released insects is needed. Methodology/Principal Findings The dispersal ability of released ‘genetically sterile’ male Aedes aegypti at a field site in Brazil has been estimated. Dispersal kernels embedded within a generalized linear model framework were used to analyse data collected from three large scale mark release recapture studies. The methodology has been applied to previously published dispersal data to compare the dispersal ability of ‘genetically sterile’ male Aedes aegypti in contrasting environments. We parameterised dispersal kernels and estimated the mean distance travelled for insects in Brazil: 52.8m (95% CI: 49.9m, 56.8m) and Malaysia: 58.0m (95% CI: 51.1m, 71.0m). Conclusions/Significance Our results provide specific, detailed estimates of the dispersal characteristics of released ‘genetically sterile’ male Aedes aegypti in the field. The comparative analysis indicates that despite differing environments and recapture rates, key features of the insects’ dispersal kernels are conserved across the two studies. The results can be used to inform both risk assessments and release programmes using ‘genetically sterile’ male Aedes aegypti., Author Summary Vector control using releases of sterile insects is a well-known approach. ‘Genetically sterile’ male Aedes aegypti have been developed and released in a modern realisation of the sterile insect technique. Released engineered males seek out and mate with wild females, with the resultant offspring dying before they reach maturity. Control of a wild vector population can therefore be achieved by maintaining sustained releases of sterile males whilst ensuring sufficient distribution and coverage of released males across the target area. In order to efficiently plan releases of these, individuals’ detailed knowledge of how they disperse in the field is required. We present an analysis of the dispersal of these engineered male Aedes aegypti using data from field experiments in Brazil. Our results provide detailed information on how the mosquitoes disperse over their potential flight range.

Background Antiplasmodial activities of angiotensin II and its analogues have been extensively investigated in Plasmodium gallinaceum and Plasmodium falciparum parasite species. Due to its vasoconstrictor property angiotensin II cannot be used as an anti-malarial drug. Methods This work presents the solid-phase syntheses and liquid chromatography and mass spectrometry characterization of ten linear peptides related to angiotensin II against mature P. gallinaceum sporozoites and erythrocyte invasion by P. falciparum. Conformational analyses were performed by circular dichroism. IC50 assays were performed to identify the ideal concentration used on the biological tests and haemolytical erythrocytic assays were made to verify the viability of the biological experiments. The contractile responses of the analogues were made to evaluate if they are promising candidates to be applied as antiplasmodial drugs. Results The results indicate two short-peptides constituted by hydrophobic residues (5 and 6) with antiplasmodial activity in these models, 89 and 94 % of biological activity against P. gallinaceum sporozoite, respectively, and around 50 % of activity against P. falciparum. Circular dichroism spectra suggested that all the peptides adopted β-turn conformation in different solutions, except peptide 3. Besides the biological assays IC50, the haemolysis assays and contractile response activities were applied for peptides 5 and 6, which did not present expressive results. Conclusions The hydrophobic portion and the arginine, tyrosine, proline, and phenylalanine, when present on peptide primary sequence, tend to increase the antiplasmodial activity. This class of peptides can be explored, as anti-malarial drugs, after in vivo model tests.Graphical abstract:The most active peptide presented 94 % activity on P. gallinaceum sporozoites and 53 % inhibited P. falciparum ring forms invasion Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-0974-y) contains supplementary material, which is available to authorized users.

Malaria is an infectious disease responsible for approximately one million deaths annually. Oligopeptides such as angiotensin II (AII) and its analogs are known to have antimalarial effects against Plasmodium gallinaceum and Plasmodium falciparum. However, their mechanism of action is still not fully understood at the molecular level. In the work reported here, we investigated this issue by comparing the antimalarial activity of AII with that of (i) its diastereomer formed by only d-amino acids; (ii) its isomer with reversed sequence; and (iii) its analogs restricted by lactam bridges, the so-called VC5 peptides. Data from fluorescence spectroscopy indicated that the antiplasmodial activities of both all-D-AII and all-D-VC5 were as high as those of the related peptides AII and VC5, respectively. In contrast, retro-AII had no significant effect against P. gallinaceum. Conformational analysis by circular dichroism suggested that AII and its active analogs usually adopted a β-turn conformation in different solutions. In the presence of membrane-mimetic micelles, AII had also a β-turn conformation, while retro-AII was random. Molecular dynamics simulations demonstrated that the AII chains were slightly more bent than retro-AII at the surface of a model membrane. At the hydrophobic membrane interior, however, the retro-AII chain was severely coiled and rigid. AII was much more flexible and able to experience both straight and coiled conformations. We took it as an indication of the stronger ability of AII to interact with membrane headgroups and promote pore formation.