Factors that alter the biochemical biomarkers of environmental contamination in Chironomus sancticaroli (Diptera, Chironomidae)

ABSTRACT Changes in physiology of the nervous system and metabolism can be detected through the activity of acetylcholinesterase (AChE), alpha esterase (EST-a) and beta esterase (EST-ß) in chironomids exposed to pollutants. However, to understand the real effect of xenobiotics on organisms, it is important to investigate how certain factors can interfere with enzyme activity. We investigated the effects of different temperatures, food stress and two steps of the enzymatic protocol on the activity of AChE, EST-a and EST-ß in Chironomus sancticaroli. In experiment of thermal stress individuals from the egg stage to the fourth larval instar were exposed to different temperatures (20, 25 and 30 °C). In food stress experiment, larvae were reared until IV instar in a standard setting (25 °C and 0.9 g weekly ration), but from fourth instar on they were divided into groups and offered different feeding regimes (24, 48 and 72 h with/without food). In sample freezing experiment, a group of samples was processed immediately after homogenization and another after freezing for 30 days. To test the effect of centrifugation on samples, enzyme activity was quantified from centrifuged and non-centrifuged samples. The activity of each enzyme reached an optimum at a different temperature. The absence of food triggered different changes in enzyme activity depending on the period of starvation. Freezing and centrifugation of the samples significantly reduced the activity of three enzymes. Based on these results we conclude that the four factors studied had an influence on AChE, EST-a and EST-ß and this influence should be considered in ecotoxicological approaches.

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests. Organophosphate compounds are used in agro-systems, and in programs to control pathogen vectors. Because they are continuously applied, organophosphates often reach water sources and may have an impact on aquatic life. The effects of acute and chronic exposure to the organophosphate insecticide malathion on the midge Chironomus sancticaroli are evaluated. To that end, three biochemical biomarkers, acetylcholinesterase (AChE), alpha (EST-α) and beta (EST-β) esterase were used. Acute bioassays with five concentrations of malathion, and chronic bioassays with two concentrations of malathion were carried out. In the acute exposure test, AChE, EST-α and EST-β activities declined by 66, 40 and 37%, respectively, at 0.251 µg L-1 and more than 80% at 1.37, 1.96 and 2.51 µg L-1. In chronic exposure tests, AChE and EST-α activities declined by 28 and 15% at 0.251 µg L-1. Results of the present study show that low concentrations of malathion can influence larval metabolism, indicating high toxicity for Chironomus sancticaroli and environmental risk associated with the use of organophosphates.

Genetic variability of a population of Aedes aegypti from Paraná, Brazil, using the mitochondrial ND4 gene / Variabilidade genética de uma população de Aedes aegypti do Paraná, Brasil, usando o gene mitocondrial ND4

Genetic variability of a population of Aedes aegypti from Paraná, Brazil, using the mitochondrial ND4 gene. To analyze the genetic variability of populations of Aedes aegypti, 156 samples were collected from 10 municipalities in the state of Paraná, Brazil. A 311 base pairs (bp) region of the NADH dehydrogenase subunit 4 (ND4) mitochondrial gene was examined. An analysis of this fragment identified eight distinct haplotypes. The mean genetic diversity was high (h = 0.702; p = 0.01556). AMOVA analysis indicated that most of the variation (67%) occurred within populations and the F ST value (0.32996) was highly significant. F ST values were significant in most comparisons among cities. The isolation by distance was not significant (r = -0.1216 and p = 0, 7550), indicating that genetic distance is not related to geographic distance. Neighbor-joining analysis showed two genetically distinct groups within Paraná. The DNA polymorphism and AMOVA data indicate a decreased gene flow in populations from Paraná, which can result in increased vectorial competence.

Variabilidade genética de uma população de Aedes aegypti do Paraná, Brasil, usando o gene mitocondrial ND4. Para analisar a variabilidade genética de populações de Aedes aegypti, 156 amostras foram coletadas em dez cidades do estado do Paraná, Brasil. Foi examinada a distribuição dos 311 pares de base do gene que codifica a subunidade 4 da enzima NADH desidrogenase. Pela amplificação e o sequenciamento deste fragmento foram identificados 8 haplótipos distintos. Os valores de diversidade genética foram altos (h = 0.702; p = 0.01556). A análise de AMOVA indicou que a maior variação ocorreu dentro das populações com um valor de FST de 0.32996 altamente significativo. Valores de FST foram significativos na maior das comparações entre as cidades. O isolamento por distância não foi significativo (r = -0.1216 e p = 0.755), indicando que a distância genética não está relacionada a distância geográfica. A análise de Neighbor-joining mostrou dois grupos genéticos distintos dentro do estado do Paraná, grupo I e grupo II. O polimorfismo de DNA e os dados de AMOVA indicam decréscimo de fluxo gênico no estado do Paraná, o que pode resultar em aumento da competência vetorial da população.

Crepuscular activity of culicids (Diptera, Culicidae) in the peridomicile and in the remaining riparian forest in Tibagi river, State of Paraná, Brazil

Crepuscular activity of culicids (Diptera, Culicidae) in the peridomicile and in the remaining riparian forest in Tibagi river, State of Paraná, Brazil. Human-attracted mosquitoes were collected for one hour, around sunset time (half hour before and half after), from April to December 2006, in two environments (riparian forest and near houses), in Tibagi river basin, Palmeira municipality, State of Paraná. Seven-hundred forty-nine mosquitoes, belonging to 13 species, were collected. Psorophora champerico Dyar & Knab, 1906 (42.86%) and Psorophora discrucians (Walker, 1856) (40.59%) were the most frequent species. No significant differences between quantities of Ps. champerico (t = -0.792; d.f. = 16; p = 0.43) and Ps. discrucians (t = 0.689; d.f. = 16; p = 0.49) obtained in riparian forest and near houses were observed, indicating similar conditions for crepuscular activity of these species in both environments. Psorophora champerico and Ps. discrucians responded (haematophagic activity) to environmental stimuli associated with the twilight hours differently in distinct habitats studied. The former species is registered for the first time in the Atlantic forest biome.

Atividade crepuscular de culicídeos (Diptera, Culicidae) no peridomicílio e remanescentes de matas ciliares do Rio Tibagi. Estado do Paraná, Brasil. Mosquitos atraídos por humanos foram coletados por uma hora em torno do crepúsculo vespertino (meia hora antes e meia hora depois), de abril a dezembro de 2006, em dois locais (mata ciliar e peridomicílio) na bacia do Rio Tibagi, município de Palmeira, Estado do Paraná. Foram capturados 749 mosquitos distribuídos em 13 espécies. Psorophora champerico Dyar & Knab, 1906 (42,86%) e Ps. discrucians (Walker, 1856) (40,59%) foram as espécies mais freqüentes. Não foram registradas diferenças significativas entre as médias de indivíduos capturados entre os pontos de mata ciliar e peridomicílio para Ps. champerico (t = -0,792; g.l. = 16; p = 0,43) e para Ps. discrucians (t = 0,689; g.l. = 16; p = 0,49). Isto demonstra que os dois ambientes estudados fornecem condições semelhantes para a atividade crepuscular destas espécies. Psorophora champerico e Ps. discrucians responderam (atividade hematofágica) aos estímulos ambientais associados ao horário crepuscular de forma distinta nos diferentes habitats analisados. Foi registrada pela primeira vez a presença de Psorophora champerico no bioma Mata Atlântica.

Physiological age and longevity of Anopheles (Kerteszia) cruzii Dyar Knab (Diptera: Culicidae) in the Atlantic Forest of Southern Brazil

We analyzed the reproductive status, ovarian development, daily survival rate, and length of the gonotrophic cycle in females of Anopheles ( Kerteszia ) cruzii Dyar & Knab, to determine how these factors influence the risk of malaria transmission in the coastal region of the state of Paraná, southern Brazil. In the Palmito State Forest, Paranaguá, females were captured at dawn and dusk by aspiration, bimonthly from December 2006 through March 2007. A total of 2,268 females were captured, of which 454 were dissected. Of these, 48 percent were parous, 50 percent not reproductive, 73 percent in Christopher and Mer stages I and II, 23 percent in stages III to V, 55 percent nulliparous, 14 percent uniparous, and 11 percent had blood in their midgut. Daily survival was 0.24 ± 0.03 overall, 0.51 ± 0.04 for females captured at dusk, and 0.25 ± 0.03 for those captured at dawn. The Davidson equation for calculation of the gonotrophic cycle was inadequate for An. cruzii populations. Females captured at dusk had a higher survival rate than those from dawn, which means that more females of the dusk population enter the parasite extrinsic cycle. The continuous activity and abundance of A. cruzii in the Palmito State Forest suggests that the conditions are very favorable for its development, with a potential for participation in the protozoan's transmission cycle.

Review of semiochemicals that mediate the oviposition of mosquitoes: a possible sustainable tool for the control and monitoring of Culicidae / Revisão dos semioquímicos que mediam a oviposição em mosquitos: uma possível ferramenta sustentável para o monitoramento e controle de Culicidae

The choice for suitable places for female mosquitoes to lay eggs is a key-factor for the survival of immature stages (eggs and larvae). This knowledge stands out in importance concerning the control of disease vectors. The selection of a place for oviposition requires a set of chemical, visual, olfactory and tactile cues that interact with the female before laying eggs, helping the localization of adequate sites for oviposition. The present paper presents a bibliographic revision on the main aspects of semiochemicals in regard to mosquitoes' oviposition, aiding the comprehension of their mechanisms and estimation of their potential as a tool for the monitoring and control of the Culicidae.

A seleção de locais adequados pelas fêmeas de mosquitos para depositarem seus ovos é um fator chave para a sobrevivência de seus imaturos (ovos e larvas). O conhecimento das relações ecológicas implicadas neste processo é de grande importância quando se refere a vetores de agentes patogênicos. A determinação do local de oviposição pelas fêmeas grávidas envolve uma rede de mensagens químicas, visuais, olfativas e táteis que facilitam a localização de lugares adequados para depositarem seus ovos. Neste trabalho é apresentada uma revisão bibliográfica dos principais aspectos relacionados com semioquímicos presentes na oviposição dos mosquitos auxiliando no entendimento dos mecanismos de atuação dos mesmos e potencializando a aplicação destes semioquímicos como uma possível ferramenta de monitoramento e controle de Culicidae.

Abundância e agregação de ovos de Aedes aegypti L. e Aedes albopictus (Skuse) (Diptera: Culicidae) no norte e noroeste do Paraná / Abundance and aggregation egg of Aedes aegypti L. and Aedes albopictus (Skuse) (Diptera: Culicidae) in the north and northwest of the State of Paraná, Brazil

Avaliou-se a abundância e agregação dos ovos de Aedes aegypti L. e Aedes albopictus Skuse em Cambé, Ibiporã, Jacarezinho, Maringá e Paranavaí, PR, utilizando-se armadilhas de oviposição. De 225 armadilhas instaladas, 100 registraram-se positivas para ovos. Coletaram-se 4140 ovos, demonstrando distribuição altamente agregada. Ambas as espécies foram encontradas em Cambé, Jacarezinho, Maringá e Paranavaí. Ae. albopictus não foi detectado em Ibiporã, porém onde esteve presente foi menos abundante. A relação entre sexos aproximou-se de 1:1, para Ae. aegypti. Na comparação dos números de adultos coletados entre as duas espécies, obteve-se correlação negativa nas amostras de Maringá e Cambé, o que foi atribuído à sazonalidade dessas populações. A coexistência dessas espécies indica que elas estão sobre pressão dos programas de controle, sendo, portanto necessárias avaliações específicas.

The abundance and aggregation of eggs of Aedes aegypti L. and Aedes albopictus Skuse was evaluated in the municipalities of Cambé, Ibiporã, Jacarezinho, Maringá and Paranavaí, in the State of Paraná, Brazil by means of oviposition traps. Of the 225 installed traps, 100 were registered as positive for eggs; 4140 eggs were collected, thus demonstrating an highly aggregate distribution. Both species were registered in Cambé, Jacarezinho, Maringá and Paranavaí. Ae. albopictus was generally less abundant and was not present in Ibiporã nor in the oviposition traps of a second collection of Maringá. The relation between sexes for Ae. aegypti was approximately 1:1. In the comparison of the number of adults collected between the two species, a negative correlation was obtained in the samples of Maringá and Cambé, what was attributed the seasonality of these populations. The coexistence of these species indicates that both are under pressure by the control programs, therefore specific evaluations are necessary.

Paridade de Anopheles cruzii em Floresta Ombrófila Densa no Sul do Brasil / Anopheles cruzii parity in dense rain forest in Southern Brazil

OBJETIVO: Conhecer a paridade e desenvolvimento ovariano da espécie Anopheles cruzii, durante os períodos estacionais. MÉTODOS: As capturas foram realizadas quinzenalmente, no período matutino, de abril/2004 a abril/2005, no Parque Estadual do Palmito, município de Paranaguá litoral do Estado do Paraná. Mosquitos adultos foram capturados usando a técnica pouso homem. As dissecções foram feitas utilizando-se a técnica de Detinova e de Polovodova e a avaliação do desenvolvimento folicular, segundo os critérios de Christophers e Mer. RESULTADOS: Foram dissecadas 208 fêmeas de Anopheles cruzii. A maioria das fêmeas dissecadas nas estações eram nulíparas. Sendo que 14,4 por cento eram nulíparas com folículo além do estádio II de Christophers & Mer, o que evidencia o exercício da hematofagia previamente à primeira oviposição. Observou-se que as populações de Anopheles cruzii são constituídas de indivíduos jovens, talvez em razão da alta mortalidade de fêmeas paridas. CONCLUSÕES: A provável discordância gonotrófica das fêmeas dissecadas é importante do ponto de vista epidemiológico, considerando que a fêmea pode procurar mais de um hospedeiro para completar a maturação dos seus ovos.

OBJECTIVE: To determine the parity and ovarian development of Anopheles cruzii species during the seasons. METHODS: Collections were carried out fortnightly in the morning in the Palmito State Park in the municipality of Paranaguá, Southern Brazil, between April 2004 and April 2005. Adult mosquitoes were captured using human landing rate. Dissections were performed using Detinova's and Polovodova's methods and follicular development was assessed following Christophers and Mer's criteria. RESULTS: A total of 208 specimens of Anopheles cruzii were dissected. Most females dissected were nulliparous in the seasons; 14.4 percent of them were found to be nulliparous above Christophers and Mer's stage II, which shows previous blood meal prior to the first oviposition. It was observed that Anopheles cruzii populations comprised young mosquitoes, probably due to high mortality among parous females. CONCLUSIONS: The likely gonotrophic discordance is epidemiologically relevant because female mosquitoes can search for more than one host to complete the maturation of their eggs.

Modelo de simulación para el control del mosquito Aedes aegypti, transmisor del dengue y la fiebre amarilla, por el crustáceo Mesocyclops spp / A simulation model for the control of the Aedes aegypti, the mosquito vector of dengue and yellow fever, by the crustacean Mesocyclops spp

OBJETIVOS: Se presenta un modelo de simulación que muestra la dinámica de depredación de Mesocyclops spp., sobre Aedes aegypti MÉTODOS: Representado por cuatro ecuaciones diferenciales: H'(t), cantidad de huevos; L'(t), cantidad de larvas; A'(t), cantidad de adultos y C'(t), cantidad de copépodos. Inicialmente las ecuaciones son del tipo clásico presa-depredador, según Lotka y Volterra. Posteriormente se modifica en un sistema con respuesta funcional para invertebrados, según Holling. RESULTADOS: El primer sistema controla y estabiliza la población de mosquitos, el segundo muestra que los copepodos son inefectivos como controladores. CONCLUSIONES: Se reconoce la necesidad de estudiar sistemas presa depredador (mosquitos - copepodos) con trabajos que integren pruebas de laboratorio y de campo. Solo así será posible establecer la validez en el uso de estos últimos como controladores biológicos efectivos de mosquitos.

Development, longevity, gonotrophic cycle and oviposition of Aedes albopictus Skuse (Diptera: Culicidae) under cyclic temperatures / Desenvolvimento, longevidade, ciclo gonadotrófico e oviposição de Aedes albopictus Skuse (Diptera: Culicidae) sob temperaturas cíclicas

Os efeitos de temperaturas cíclicas no desenvolvimento, longevidade, ciclo gonadotrófico e número de ovos postos de Aedes albopictus Skuse foram analisados por meio de experimentoslaboratoriais. Os experimentos foram conduzidos com mosquitos oriundos de Registro, SP e mantidos em insetário por dois anos. O desenvolvimento do inseto foi acompanhado desde o ovo até a emergência do adulto, a 25/18ºC e 27/20ºC e o adulto a 27/20ºC, ambos associados com fotoperíodo de 12h. Os ovos receberam dois tratamentos: (A) manutenção do volume da água; (B) troca periódica e completa da água. A alimentação sangüínea era oferecida diariamente aos mosquitos com interrupção quando o repasto era realizado, e retomada após a primeira oviposição. O desenvolvimento dos imaturos foi significativamente diferente sob os diferentes regimes de temperatura (P < 0,05), e a velocidade de desenvolvimento foi afetada positivamente pelo aumento da temperatura. A combinação do regime detemperaturas mais altas com a troca periódica e completa da água permitiu maior viabilidade dos ovos e encurtou o período de incubação. A longevidade dos adultos não foi diferente entre machos e fêmeas e a mortalidade foi regular ao passar do tempo. A comparação da longevidade do mosquito sob temperaturas constantes e cíclicas sugere que a menor temperatura do regime cíclico seja um fator limitante para a sobrevivência do mosquito. Esse fato pode ser um importante limitador da distribuição de A. albopictus por sugerir que o mosquito está restrito a ocupar áreas com temperaturas mínimas não muito inferiores a 20ºC.

The effects of cyclic temperatures on Aedes albopictus Skuse development, longevity, gonotrophic cycle and the number of oviposited eggs were assessed by means of laboratory experiments. The experiments were carried out with mosquitoes from Registro, São Paulo, Brazil, kept in insectary for two years. The development of the insect was followed from egg to adult emergence under 25/18ºC and 27/20ºC and adult stage under 27/20ºC, both associated with LD 12:12h. The eggs received two treatments: (A) maintenance of water volume; (B) periodical and complete change of water. Blood meal was offereddaily and it was interrupted after haematophagy and restarted after first oviposition. The immature development was significantly different under the temperature regimes (P < 0.05) and the increased temperatures positively affected the development speed. The combination of higher temperaturesregime and periodical and complete change of water increased the eggs viability and shortened the incubation time. Adult longevity was not different between males and females and the mortality was regular through the time. Comparing the mosquito longevity under constant and cyclic temperatures, itis suggested that the lowest temperature of the cyclic regime is a limiting factor for mosquito survival.This fact may limit the A. albopictus distribution range to areas where the minimal temperatures are not much bellow 20ºC.