Genetic Control of Mosquitoes: population suppression strategies

Ao longo das duas últimas décadas, morbidade e mortalidade da malária e dengue e outros patógenos tem se tornado cada vez mais um problema de Saúde Pública. O aumento na distribuição geográfica de seus respectivos vetores é acompanhada pela emergência de doenças em novas áreas. Não estão disponíveis drogas específicas suficientes e não há vacinas específicas para imunizar as populações alvo. As medidas de controle de mosquitos atuais falharam em atingir os objetivos propostos, principalmente devido à grande capacidade reprodutiva dos mosquitos e alta flexibilidade genômica. O controle químico se torna cada vez mais restrito devido a sua potencial toxicidade aos seres humanos, mortalidade de organismos não alvos, resistência a inseticida além de outros impactos ambientais. Novas estratégias de controle são necessárias. A técnica do inseto estéril (SIT) é um método de supressão populacional espécie específico e ambientalmente amigável, baseia-se na criação em massa, esterilização mediante irradiação e liberação de um grande número de insetos machos. Liberar insetos carregando um gene letal dominante (RIDL) oferece uma solução a muitas limitações impostas pela técnica do inseto estéril (SIT) que limitaram sua aplicação em mosquitos e ainda assim mantém suas características de ambientalmente amigável e espécie específica. A natureza auto-limitante de mosquitos estéreis tende a deixar alguns empecilhos para uso no campo, de certa forma, menos desafiadores quando comparados a sistemas auto-propagação, característicos de estratégias de substituição de população. Sistemas auto-limitantes estão mais próximos para uso no campo, portanto pode ser apropriado considerá-lo primeiro. A perspectiva de métodos de controle genéticos contra mosquitos vetores de doenças que acometem humanos está rapidamente se tornando uma realidade, muitas decisões terão de ser tomadas em âmbito nacional, regional e internacional com relação a aspectos étnicos, sociais, culturais e de biossegurança para o uso e liberação destes métodos de controle de vetores.Over the last two decades, morbidity and mortality from malaria and dengue fever among other pathogens are an increasing Public Health problem. The increase in the geographic distribution of vectors is accompanied by the emergence of viruses and diseases in new areas. There are insufficient specific therapeutic drugs available and there are no reliable vaccines for malaria or dengue, although some progress has been achieved, there is still a long way between its development and actual field use. Most mosquito control measures have failed to achieve their goals, mostly because of the mosquito's great reproductive capacity and genomic flexibility. Chemical control is increasingly restricted due to potential human toxicity, mortality in no target organisms, insecticide resistance, and other environmental impacts. Other strategies for mosquito control are desperately needed. The Sterile Insect Technique (SIT) is a species-specific and environmentally benign method for insect population suppression, it is based on mass rearing, radiation mediated sterilization, and release of a large number of male insects. Releasing of Insects carrying a dominant lethal gene (RIDL) offers a solution to many of the drawbacks of traditional SIT that have limited its application in mosquitoes while maintaining its environmentally friendly and species-specific utility. The self-limiting nature of sterile mosquitoes tends to make the issues related to field use of these somewhat less challenging than for self-spreading systems characteristic of population replacement strategies. They also are closer to field use, so might be appropriate to consider first. The prospect of genetic control methods against mosquito vectored human diseases is rapidly becoming a reality, many decisions will need to be made on a national, regional and international level regarding the biosafety, social, cultural and ethical aspects of the use and deployment of these vector control methods

Baixa diversidade genética em Culex quinquefasciatus (Diptera: Culicidae) infectado por Wolbachia do Brasil e Argentina

Culex quinquefasciatus é vetor de patógenos humanos, incluindo nematódeos filarídeos e vários vírus. Embora a sua relevância epidemiológica varie entre as diferentes regiões geográficas, o conhecimento da estrutura genética da população é ainda incipiente. Em vista disso, foram avaliados os níveis de diversidade genética de Cx. quinquefasciatus e de híbridos Cx. quinquefasciatus x Cx. pipiens de nove cidades do Brasil e em La Plata, na Argentina. Para os testes foram utilizados fragmentos dos genes mitocondriais cox1 e nd4, juntamente com coxA e wsp do endossimbionte Wolbachia, herdado maternalmente. O fragmento nd4 não apresentou variação entre as amostras, e o cox1 exibiu quatro haplótipos que separaram dois tipos de Cx. quinquefasciatus, com um deles agrupado no sul do Brasil. Os dados de sequência mostraram baixa diversidade, sendo esta discutida. Ambas as amostras de mosquitos brasileiros e argentinos estão infectados com uma única cepa de Wolbachia. A diversidade apresentada por nd4 e cox1 não é congruente com a estrutura da população revelada por marcadores nucleares e morfologia alar de estudos anteriores com estas mesmas populações. Pesquisas com Cx. quinquefasciatus devem, se possível, avaliar a diversidade por DNA mitocondrial na luz de outros marcadores.Culex quinquefasciatus is a vector of human pathogens, including filarial nematodes and several viruses. Although its epidemiological relevance is known to vary across geographical regions, an understanding of its population genetic structure is still incipient. In light of this, we evaluated the genetic diversity of Cx. quinquefasciatus and Cx. pipiens x Cx. quinquefasciatus hybrids collected from nine localities in Brazil and one site in Argentina. We used mitochondrial genes cox1 and nd4, along with the coxA and wsp genes of the maternally-inherited Wolbachia endosymbiont. The nd4 fragment was invariant between samples, whilst cox1 exhibited four haplotypes that separated two types of Cx. quinquefasciatus, one clustered in southern Brazil. Low sequence diversity was generally observed, being discussed. Both Brazilian and Argentinian mosquitoes were infected with a single Wolbachia strain. As reported in previous studies with these populations, cox1 and nd4 diversity is not congruent with the population structure revealed by nuclear markers or alar morphology. Future Cx. quinquefasciatus research should, if possible, evaluate mtDNA diversity in light of other markers

Detrimental effects of malaria, toxoplasmosis, leishmaniosis and Chagas disease on cardiac and skeletal muscles

The pathogenic mechanisms of several diseases triggered by protozoan parasites, such as the causative agents of Chagas disease, toxoplasmosis, leishmaniosis, and malaria, have demonstrated to cause direct detrimental effect on cardiac and skeletal muscle. These are amongst the most prevalent and epidemiologically relevant protozoan infections worldwide and infecting millions of people per year. As such, this review focuses on the current knowledge on the pathogenic mechanisms of these diseases on muscles. Case studies and original research addressing the mechanisms of action for direct and indirect damage to cardiac and skeletal muscle were analyzed and the main findings summarized. Importantly, all diseases reviewed here produce an intense inflammatory response, with the associated oxidative stress and pro-inflammatory cytokine production leading to or furthering these detrimental effects. Critically, the disruption of cardiac muscle function can lead to minor arrhythmias and even death, and skeletal muscle damage can result in homeostatic imbalances serving to further morbidity and mortality. Strategies for preventing complications and determining the effectiveness of interventions designed with antioxidant and anti-inflammatory molecules to minimize muscle injury and help the millions of people with these diseases are an urgent need

Control of vector populations using genetically modified mosquitoes

Formas químicas de controle de mosquitos vetores são ineficazes, levando ao desenvolvimento de novas estratégias. Assim, foi realizada revisão das estratégias de controle genético de populações de mosquitos vetores baseada na técnica do inseto estéril. Uma delas consiste na liberação de machos esterilizados por radiação, a outra, na integração de um gene letal dominante associado a um promotor específico de fêmeas imaturas. Entre as vantagens sobre outras técnicas biológicas e químicas de controle de vetores estão: alta especificidade, não prejudicial ao meio ambiente, baixo custo de produção e alta eficácia. O uso desta técnica de modificação genética pode vir a ser uma importante ferramenta do manejo integrado de vetoresThe ineffectiveness of current strategies for chemical control of mosquito vectors raises the need for developing novel approaches. Thus, we carried out a literature review of strategies for genetic control of mosquito populations based on the sterile insect technique. One of these strategies consists of releasing radiation-sterilized males into the population; another, of integrating a dominant lethal gene under the control of a specific promoter into immature females. Advantages of these approaches over other biological and chemical control strategies include: highly species-specific, environmentally safety, low production cost, and high efficacy. The use of this genetic modification technique will constitute an important tool for integrated vector managemen

Microsatellite loci cross-species transferability in Aedes fluviatilis (Diptera:Culicidae): a cost-effective approach for population genetics studies

Abstract\ud \ud Background\ud \ud Aedes fluviatilis is a neotropical mosquito species thought to be a potential vector of Yellow Fever viruses and can be infected with Plasmodium gallinaceum in laboratory. A better understanding of its genetic structure is very important to understand its epidemiologic potential and how it is responding to urbanization. The objective of this study was to survey the transferability of microsatellites loci developed for other Aedes to Ae. fluviatilis.\ud \ud \ud \ud Findings\ud We tested in Ae. fluviatilis 40 pairs of primers known to flank microsatellite regions in Aedes aegypti, Aedes albopictus and Aedes caspius, and found eight loci that amplified consistently. The number of alleles per locus ranged from 2 to 15, and the expected heterozygosity ranged from 0.09 to 0.85.\ud \ud \ud Conclusions\ud We found that several microsatellite primers successfully transferred to Ae. fluviatilis. This finding opens avenues for cost-effective optimization of high-resolution population genetic tools.The authors would like to thank the Fundação de Amparo à Pesquisa do\ud Estado de São Paulo (FAPESP) for providing financial support (Grant 2013/\ud 15313-4). LCM is a fellow of FAPESP (2013/22347-2). ABBW is the recipient of\ud a postdoctoral fellowship from FAPESP (2012/19117-2)

Taxonomic and systematic analysis among triatomine bug species (Hemiptera, Reduviidae) from colonies of the Special Health Service of Araraquara (SESA), inferred from 16S rDNA mitocondrial sequences

Foram analisadas seqüências de nucleotídeos do gene 16S do rDNA mitocondrial em 14 populações de triatomíneos mantidos em colônias no insetário SESA de Araraquara- SP, comparando-as com seqüências do mesmo gene disponíveis no GenBank. Os fragmentos variaram de 311 a 317 pb com baixa variação intra-específica entre as distâncias genéticas (0% a 0,6%), exceto para os relacionamentos entre espécimes de Triatoma sordida (1%) e espécimes de T. brasiliensis (1,3%) atribuídos a populações geográficas diferentes. A parafilia de Rhodniini e do gênero Panstrongylus foi evidenciada pelas analises, confirmando resultados anteriores entre estes e os estreitos relacionamentos de R. prolixus com R. robustus e de T. infestans e T. platensis. O relacionamento entre T. maculata e T. pseudomaculata não foi solucionado, uma vez que, esses táxons apareceram tanto em monofilia quanto em parafilia: T. pseudomaculata (SESA) está agrupado com T. maculata (seqüência do GenBank) e associados a T . brasiliensis (SESA), enquanto T. maculata (SESA) aparece agrupado com T. pseudomaculata do SESA e do GenBank. Os resultados evidenciam a utilidade do gene 16S como marcador de espécies de triatomíneos e sua importância em questões de sistemática e taxonomia. Há necessidade de novos estudos envolvendo outros marcadores associados a caracteres sistemáticos clássicos de morfologia, ecologia e comportamento para decisões sistemáticas adequadas uma vez, que teriam impacto não apenas sistemático mas, para as estratégias de controle.Nucleotide sequences of mitocondrial 16S rDNA gene were analyzed in 14 populations of triatomine bugs from colonies maintained by SESA, Araraquara, São Paulo, Brazil, comparing them with sequences of same gene found in the GenBank. The fragments varied from 311 to 317 bp, with low intra-specific genetic distance (0% to 0.6%), with exception of the relationship among Triatoma sordida specimens (1%) and T. brasiliensis specimens (1.3%) that were attributed to different geographical populations. The paraphily of Rhodniini and Panstrongylus genus was evidenced by this analyses, confirming previous results between these and the narrow relationships of R. prolixus with R. robustus and of T. infestans with T. platensis. The relationship between T. maculata and T. pseudomaculata has not been resolved, since these taxa appeared both in monophyly and as in paraphyly: T. pseudomaculata (SESA) is grouped with T. maculata (sequence of GenBank) and the associated T. brasiliensis (SESA), while T. maculata (SESA) appears grouped with T. pseudomaculata of SESA and GenBank. The results evidence the utility of the gene 16S as molecular marker in the species of triatomine bugs and its importance in systematic and taxonomy questions. There is a necessity of new studies including new molecular markers associated with classic systematic characters of morphology, ecology and behavior to adjust systematic decisions since those will be not of only systematic impact but, for new strategies of control.Fundação de Amparo à Pesquisa em São Paulo - FAPESP 05/50225-

LOW GENETIC DIVERSITY IN Wolbachia-INFECTED Culex quinquefasciatus (DIPTERA: CULICIDAE) FROM BRAZIL AND ARGENTINA

Culex quinquefasciatus is a vector of human pathogens, including filarial nematodes and several viruses. Although its epidemiological relevance is known to vary across geographical regions, an understanding of its population genetic structure is still incipient. In light of this, we evaluated the genetic diversity of Cx. quinquefasciatus and Cx. pipiens x Cx. quinquefasciatus hybrids collected from nine localities in Brazil and one site in Argentina. We used mitochondrial genes cox1 and nd4, along with the coxA and wsp genes of the maternally-inherited Wolbachia endosymbiont. The nd4 fragment was invariant between samples, whilst cox1 exhibited four haplotypes that separated two types of Cx. quinquefasciatus, one clustered in southern Brazil. Low sequence diversity was generally observed, being discussed. Both Brazilian and Argentinian mosquitoes were infected with a single Wolbachia strain. As reported in previous studies with these populations, cox1 and nd4 diversity is not congruent with the population structure revealed by nuclear markers or alar morphology. Future Cx. quinquefasciatus research should, if possible, evaluate mtDNA diversity in light of other markers.FAPESP [05/50225-2, 06/02622-5]FAPES

Mitochondrial PCR-RFLP Assay to Distinguish Triatoma brasiliensis macromelasoma

Triatoma brasiliensis sensu lato (s.l.), the main vector of Chagas disease in northeastern Brazil, is a species complex comprising four species, one with two subspecies (T. brasiliensis brasiliensis, T. brasiliensis macromelasoma, T. juazeirensis, T. sherlocki, and T. melanica), and each taxon displaying distinct ecological requirements. In order to evaluate the genetic relationships among nine T. brasiliensis s.l. populations from northeastern Brazil, we analyzed their mitochondrial cytochrome c oxidase subunit 1 sequences and suggested a PCR-RFLP assay to distinguish between T. b. macromelasoma and T. b. brasiliensis subspecies. All the specimens were morphologically identified as T. b. brasiliensis. The resulting phylogenies identified two major clades that are congruent with the geographical populations studied. Based on collection sites and in accordance with type-location, one clade was identified as the subspecies T. b. macromelasoma. The second clade grouped T. b. brasiliensis populations. Restriction endonuclease sites were observed in the sequences and used in PCR-RFLP assays, producing distinct fingerprints for T. b. macromelasoma and T. b. brasiliensis populations. The results suggest that these are different species and that gene flow occurs only among T. b. brasiliensis populations, possibly associated with human activity in the area

Wing geometry of Culex coronator (Diptera: Culicidae) from South and Southeast Brazil

Abstract\ud \ud Background\ud The Coronator Group encompasses Culex coronator Dyar & Knab, Culex camposi Dyar, Culex covagarciai Forattini, Culex ousqua Dyar, Culex usquatissimus Dyar, Culex usquatus Dyar and Culex yojoae Strickman. Culex coronator has the largest geographic distribution, occurring in North, Central and South America. Moreover, it is a potential vector-borne mosquito species because females have been found naturally infected with several arboviruses, i.e., Saint Louis Encephalitis Virus, Venezuelan Equine Encephalitis Virus and West Nile Virus. Considering the epidemiological importance of Cx. coronator, we investigated the wing shape diversity of Cx. coronator from South and Southeast Brazil, a method to preliminarily estimate population diversity.\ud \ud \ud Methods\ud Field-collected immature stages of seven populations from a large geographical area in Brazil were maintained in the laboratory to obtain both females and males linked with pupal and/or larval exuviae. For each individual female, 18 landmarks of left wings were marked and digitalized. After Procrustes superimposition, discriminant analysis of shape was employed to quantify wing shape variation among populations. The isometric estimator centroid size was calculated to assess the overall wing size and allometry.\ud \ud \ud Results\ud Wing shape was polymorphic among populations of Cx. coronator. However, dissimilarities among populations were higher than those observed within each population, suggesting populational differentiation in Cx. coronator. Morphological distances between populations were not correlated to geographical distances, indicating that other factors may act on wing shape and thus, determining microevolutionary patterns in Cx. coronator. Despite the population differentiation, intrapopulational wing shape variability was equivalent among all seven populations.\ud \ud \ud Conclusion\ud The wing variability found in Cx. coronator populations brings to light a new biological problem to be investigated: the population genetics of Cx. coronator. Because of differences in the male genitalia, we also transferred Cx. yojoae to the Apicinus Subgroup.Fundação de Amparo à Pesquisa do Estado de São\ud Paulo (FAPESP No. 2010/02658-5 to BDS; FAPESP No. 2011/20397-7 to MAMS;\ud FAPESP No. 2006/02622-6 to LS), Conselho Nacional de Desenvolvimento\ud Científico e Tecnológico (CNPq, No. 301666/2011-3 to MAMS), CAPES\ud Research Grants# 23038.005274/2011-24 and 1275/201

Controle de vetores utilizando mosquitos geneticamente modificados

Formas químicas de controle de mosquitos vetores são ineficazes, levando ao desenvolvimento de novas estratégias. Assim, foi realizada revisão das estratégias de controle genético de populações de mosquitos vetores baseada na técnica do inseto estéril. Uma delas consiste na liberação de machos esterilizados por radiação, a outra, na integração de um gene letal dominante associado a um promotor específico de fêmeas imaturas. Entre as vantagens sobre outras técnicas biológicas e químicas de controle de vetores estão: alta especificidade, não prejudicial ao meio ambiente, baixo custo de produção e alta eficácia. O uso desta técnica de modificação genética pode vir a ser uma importante ferramenta do manejo integrado de vetores.The ineffectiveness of current strategies for chemical control of mosquito vectors raises the need for developing novel approaches. Thus, we carried out a literature review of strategies for genetic control of mosquito populations based on the sterile insect technique. One of these strategies consists of releasing radiation-sterilized males into the population; another, of integrating a dominant lethal gene under the control of a specific promoter into immature females. Advantages of these approaches over other biological and chemical control strategies include: highly species-specific, environmentally safety, low production cost, and high efficacy. The use of this genetic modification technique will constitute an important tool for integrated vector management.Formas químicas de control de mosquitos vectores son ineficaces, llevando al desarrollo de nuevas estrategias. Así, fue realizada revisión de las estrategias de control genético de poblaciones de mosquitos vectores basada en la técnica del insecto estéril. Una de ellas consiste en la liberación de machos esterilizados por radiación, la otra, en la integración de un gen letal dominante asociado a un promotor específico de hembras inmaduras. Entre las ventajas sobre otras técnicas biológicas y químicas de control de vectores están: la alta especificidad, no prejudicial al ambiente, bajo costo de producción y alta eficiencia. El uso de esta técnica de modificación genética puede ser una importante herramienta del manejo integrado de vectores