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.

Multiple dengue virus serotypes resistant transgenic Aedes aegypti fitness evaluated under laboratory conditions.

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.

Evaluation of inflammatory skin infiltrate following Aedes aegypti bites in sensitized and non-sensitized mice reveals saliva-dependent and immune-dependent phenotypes.

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.

Endogenously-expressed NH2-terminus of circumsporozoite protein interferes with sporozoite invasion of mosquito salivary glands.

BACKGROUND: The circumsporozoite protein is the most abundant polypeptide expressed by sporozoites, the malaria parasite stage capable of infecting humans. Sporozoite invasion of mosquito salivary glands prior to transmission is likely mediated by a receptor/ligand-like interaction of the parasites with the target tissues, and the amino (NH2)-terminal portion of CSP is involved in this interaction but not the TSR region on the carboxyl (C)-terminus. Peptides based on the NH2-terminal domain could compete with the parasites for the salivary gland receptors and thus inhibit penetration. METHODS: Peptides based on the NH2-terminus and TSR domains of the CSP from avian or human malaria parasites, Plasmodium gallinaceum and Plasmodium falciparum, respectively, were expressed endogenously in mosquito haemolymph using a transient (Sindbis virus-mediated) or stable (piggyBac-mediated transgenesis) system. RESULTS: Transient endogenous expression of partial NH2-terminus peptide from P. falciparum CSP in P. gallinaceum-infected Aedes aegypti resulted in a reduced number of sporozoites in the salivary glands. When a transgenic approach was used to express a partial CSP NH2-terminal domain from P. gallinaceum the number of sporozoites in the salivary glands did not show a difference when compared to controls. However, a significant difference could be observed when mosquitoes with a lower infection were analysed. The same result could not be observed with mosquitoes endogenously expressing peptides based on the TSR domain from either P. gallinaceum or P. falciparum. CONCLUSION: These results support the conclusion that CSP partial NH2-terminal domain can be endogenously expressed to promote a competition for the receptor used by sporozoites to invade salivary glands, and they could be used to block this interaction and reduce parasite transmission. The same effect cannot be obtained with peptides based on the TSR domain.

Evidences for the action mechanism of angiotensin II and its analogs on Plasmodium sporozoite membranes.

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 ß-turn conformation in different solutions. In the presence of membrane-mimetic micelles, AII had also a ß-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.

New linear antiplasmodial peptides related to angiotensin II.

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 ß-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.

Antiplasmodial activity study of angiotensin II via Ala scan analogs.

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 Ile(5) or the His(6) residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala(6) ]-AII (79%), and [Ala(5) ]-AII (75%). Analogs [Ala(3) ]-AII, [Ala(1) ]-AII, and AII-NH2 showed antiplasmodial activity around 65%, whereas the activity of the [Ala(8) ]-AII, [Ala(7) ]-AII, [Ala(4) ]-AII, and [Ala(2) ]-AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a ß-fold conformation in different solutions. All AII analogs, except [Ala(4) ]-AII and [Ala(8) ]-AII, show contractile responses and interact with the AT1 receptor, [Ala(5) ]-AII and [Ala(6) ]-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.

The First Molecular Characterization of Anopheles (Nyssorhynchus) aquasalis (Diptera: Culicidae) Vitellogenin Expression Products and a Partial cDNA Sequence Isolation.

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.

A study of the anti-plasmodium activity of angiotensin II analogs.

Controlling the dissemination of malaria requires the development of new drugs against its etiological agent, a protozoan of the Plasmodium genus. Angiotensin II and its analog peptides exhibit activity against the development of immature and mature sporozoites of Plasmodium gallinaceum. In this study, we report the synthesis and characterization of angiotensin II linear and cyclic analogs with anti-plasmodium activity. The peptides were synthesized by a conventional solid-phase method on 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 by fluorescence microscopy using propidium iodine uptake as an indicator of cellular damage. The activities of the linear and cyclic peptides are not significantly different (p < 0.05). Kinetics studies indicate that the effects of these peptides on plasmodium viability overtime exhibit a sigmoidal profile and that the system stabilizes after a period of 1 h for all peptides examined. The results were rationalized by partial least-square analysis, assessing the position-wise contribution of each amino acid. The highest contribution of polar amino acids and a Lys residue proximal to the C-terminus, as well as that of hydrophobic amino acids in the N-terminus, suggests that the mechanism underlying the anti-malarial activity of these peptides is attributed to its amphiphilic character.

Transcriptome profiling and Calreticulin expression in Zika virus -infected Aedes aegypti.

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.

Transgene-induced cell death following dengue-2 virus infection in Aedes aegypti.

Dengue viruses (DENVs) are mosquito-borne flaviviruses causing millions of human infections each year and pose a challenge for public health systems worldwide. Aedes aegypti is the principal vector species transmitting DENVs to humans. Controlling Ae. aegypti is difficult due to the abundance of breeding sites and increasing insecticide resistance in the vector populations. Developing new vector control strategies is critical for decreasing the disease burden. One potential approach is genetically replacing Ae. aegypti populations with vector populations highly resistant to DENV transmission. Here, we focus on an alternative strategy for generating dengue 2 virus (DENV-2) resistance in genetically-modified Ae. aegypti in which the mosquitoes express an inactive form of Michelob_x (Mx), an antagonist of the Inhibitor of Apoptosis (IAP), to induce apoptosis in those cells in which actively replicating DENV-2 is present. The inactive form of Mx was flanked by the RRRRSAG cleavage motif, which was recognized by the NS2B/NS3 protease of the infecting DENV-2 thereby releasing and activating Mx which then induced apoptosis. Our transgenic strain exhibited a significantly higher mortality rate than the non-transgenic control when infected with DENV-2. We also transfected a DNA construct containing inactive Mx fused to eGFP into C6/36 mosquito cells and indirectly observed Mx activation on days 3 and 6 post-DENV-2 infections. There were clear signs that the viral NS2B/NS3 protease cleaved the transgene, thereby releasing Mx protein into the cytoplasm, as was confirmed by the detection of eGFP expression in infected cells. The present study represents proof of the concept that virus infection can be used to induce apoptosis in infected mosquito cells.

Hemorrhagic fever viruses: Pathogenesis, therapeutics, and emerging and re-emerging potential.

Hemorrhagic fever viruses (HFVs) pose a threat to global public health owing to the emergence and re-emergence of highly fatal diseases. Viral hemorrhagic fevers (VHFs) caused by these viruses are mostly characterized by an acute febrile syndrome with coagulation abnormalities and generalized hemorrhage that may lead to life-threatening organ dysfunction. Currently, the events underlying the viral pathogenicity associated with multiple organ dysfunction syndrome still underexplored. In this minireview, we address the current knowledge of the mechanisms underlying VHFs pathogenesis and discuss the available development of preventive and therapeutic options to treat these infections. Furthermore, we discuss the potential of HFVs to cause worldwide emergencies along with factors that favor their spread beyond their original niches.

Aedes aegypti mosquito saliva ameliorates acetaminophen-induced liver injury in mice.

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 aegypti saliva is a source of bioactive molecules with in vitro anti-inflammatory and immunomodulatory activities. However, evidences on the therapeutic use of Ae. aegypti salivary preparations in animal models of relevant clinical conditions are still scarce. Thus, the present study was designed to evaluate the protective role of Ae. aegypti saliva in a murine model of APAP-induced DILI. C57BL/6 mice were exposed to Ae. aegypti bites 2 hours after APAP overdose. Biochemical and immunological parameters were evaluated in blood and liver samples at different time points after APAP administration. Exposure to Ae. aegypti saliva 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 in Ae. aegypti exposed animals, suggesting no effects in the generation of hepatotoxic metabolites. On the other hand, mice treated with Ae. aegypti saliva following APAP overdose presented lower serum concentration of TNF-α, IL-6, IL-1ß 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 that Ae. aegypti saliva 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.

A Metagenomic Approach Identified a Novel Phasi Charoen-Like Virus Coinfecting a Chikungunya Virus-Infected Aedes aegypti Mosquito in Brazil.

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.

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.

Transgenic Aedes aegypti Mosquitoes Transfer Genes into a Natural Population.

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 for >21,000 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.

Aedes aegypti saliva impairs M1-associated proinflammatory phenotype without promoting or affecting M2 polarization of murine macrophages.

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.

Sex determination and Aedes population control.

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.

Oviplate: A Convenient and Space-Saving Method to Perform Individual Oviposition Assays in Aedes aegypti.

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 "oviplate". 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.

Recommendations for Laboratory Containment and Management of Gene Drive Systems in Arthropods.

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.