Potentialities of iodine-enhanced micro-CT imaging in the morphological study of adult Aedes (Stegomyia) aegypti (Linnaeus, 1762) mosquitoes.

X-ray micro-computed tomography (CT) produces three-dimensional images of samples on a micrometer scale. This technique has several advantages, such as its nondestructive character and low measurement time, compared with other techniques. However, when applied to biological samples of soft tissue, the low attenuation and low effective contrast between structures pose difficulties in creating appropriate images for morphological studies. Diffusible iodine-based contrast-enhanced CT (DICE-CT), which uses iodine solutions to enhance contrast, is a viable alternative for addressing the aforementioned challenges. Given the variety of biological samples, an appropriate methodology must be adapted depending on the dimensions and morphological characteristics of the investigated object. A specimen that has not been morphologically studied by micro-CT and is of high sanitary importance is the adult Aedes aegypti mosquito. This study investigated the stage of iodine staining in the treatment of the A. aegypti mosquito to determine the most suitable staining time for the morphological study of this mosquito in adulthood. After determining the appropriate staining time, we discuss the potential of applying DICE-CT and methodology to mosquito studies. Seven A. aegypti females were treated using fixation steps with Bouin's solution, dehydration in a graded ethanol series, staining with iodine solution (1%), and washing in absolute ethanol. Only the staining step was different between samples. Each mosquito spent a varying amount of time (6-72 h) in the iodine solution (1%). For comparison, one of the mosquitoes was not stained. After treatment, the samples were scanned using the Bruker SkyScan 1172 micro-CT scanner. The reconstructed volumes and histograms were compared to determine the most suitable time. In addition, a quantitative analysis was performed based on a comparison of the attenuation profiles of the mosquito brains. Thereafter, the most suitable treatment process was selected, and two other samples were scanned after applying the selected process. Although fewer than 18 h was insufficient for an effective increase in attenuation and effective contrast, surpassing 24 h proved unnecessary and resulted in saturating the gray tones visualized through the histograms, leading to information loss. Therefore, a time of approximately 24 h was the most suitable staining time for studying adult A. aegypti. It was possible to isolate the organs of the digestive and reproductive systems of the mosquito stained for 24 h. Thus, micro-CT was confirmed to be an excellent technique in studies of individual structures of adult A. aegypti mosquitoes.

Molecular techniques for the taxonomy of Aedes Meigen, 1818 (Culicidae: Aedini): A review of studies from 2010 to 2021.

The original description of Aedes Meigen in 1818, written in Latin, was very brief and included a single species, Aedes cinereus. In the last two decades the genus Aedes (Meigen, 1818) has undergone several revisions and reclassifications, with the current proposal being described by Wilkerson in 2015. However, the available keys for morphological identification are still not sufficient to differentiate cryptic species, damaged species, or those with confusing taxonomy. The current study aims to identify and describe the main taxonomic proposals and molecular methodologies available for the identification of the genus Aedes published between the years 2010 and 2021. The main molecular techniques used to identify the genus in the last 10 years, are: Multiplex PCR, DNA barcoding, nuclear and mitochondrial markers, environmental DNA, and bacterial microbiome analysis. This review highlights that there are catalogued data for only a few species of the genus Aedes, being restricted to medically important taxa such as Aedes albopictus and Aedes aegypti. The integrative taxonomy approach is a possibility to reconcile morphological and molecular data to improve species delimitation, contributing to future revisions of the genus.

Influence of abiotic factors on the oviposition of Aedes (Stegomyia) aegypti (Diptera: Culicidae) in Northern Paraná, Brazil.

Aedes aegypti is the main vector of dengue in the Americas and is also a transmitter of urban yellow fever arboviruses, Zika, and Chikungunya, all of which have substantial economic impacts on the affected countries. Through mathematical models, the influence of climatic factors on the oviposition of Ae. aegypti was determined. The data were collected in the city of Apucarana, Paraná State, using oviposition traps. Daily data were submitted to a negative binomial regression model (p < 0.05). The analyses were performed using the R statistical program to determine the climatic factors that most influenced oviposition. A Poisson regression showed that the variables temperature, atmospheric pressure, humidity, and precipitation significantly increased the number of eggs. However, using the semi-normal probability graph with a simulation envelope, it was determined that the Poisson regression model was not adequate to explain the relationships between the variables. Thus, a negative binomial regression model was used, which overcame the problem of overdispersion, and showed that only temperature affected the increase in the number of eggs, where an increase of 1 °C was expected to result in a 54.03% increase in the number of Ae. aegypti eggs.

New traps for the capture of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) (Diptera: Culicidae) eggs and adults.

The control of arboviruses carried by Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) can be performed with tools that monitor and reduce the circulation of these vectors. Therefore, the efficiency of four types of traps in capturing A. aegypti and A. albopictus eggs and adults, with the biological product Vectobac WG, was evaluated in the field. For this, 20 traps were installed in two locations, which were in the South (Londrina, Paraná) and North (Manaus, Amazonas) Regions of Brazil, from March to April 2017 and January to February 2018, respectively. The UELtrap-E (standard trap) and UELtrap-EA traps captured A. aegypti and A. albopictus eggs: 1703/1866 eggs in Londrina, and 10268/2149 eggs in Manaus, respectively, and presented high ovitraps positivity index (OPI) values (averages: 100%/100% in Londrina, and 100%/96% in Manaus, respectively); and high egg density index (EDI) values (averages: 68/75 in Londrina, and 411/89 in Manaus, respectively), so they had statistically superior efficiency to that of the CRtrap-E and CRtrap-EA traps in both regions, that captured less eggs and adults: 96/69 eggs in Londrina, and 1091/510 eggs in Manaus, respectively. Also presented lower OPI values (averages: 28%/4% in Londrina, and 88%/60% in Manaus, respectively); and lower EDI values (averages: 10.5/9 in Londrina, and 47/30 in Manaus, respectively). The capture ratios of Aedes adults in the UELtrap-EA and CRtrap-EA traps in Londrina and Manaus were 53.3%/29.5% and 0%/9.8%, respectively. UELtrap-EA can be adopted as efficient tool for Aedes monitoring due to their high sensitivity, low cost and ease of use.

Genetic study in Aedes (Stegomyia) aegypti (Linnaeus, 1762) from Londrina (Paraná State, Brazil): an approach to population structure and pyrethroid resistance

Abstract Londrina is the fourth most populous city in southern Brazil. Its subtropical weather with rain in all seasons, as well as its high population density, make the city perfect for the Aedes aegypti (Linnaeus, 1762) life cycle. Over the last few years, Londrina presented high infestation indexes and was one of the cities with the most reported cases of dengue. Uncontrolled use of synthetic insecticides may influence the mosquito's genetic composition. In this paper, we studied mitochondrial DNA and kdr mutations in Aedes aegypti. The analysis of the ND4 gene in 330 specimens showed the presence of 27 haplotypes. The pyrethroid resistance alleles (kdr) evaluated are present in the collected populations, with a 50% frequency of the Val1016Ile and 48% of the Phe1534Cys mutations. Such analysis of the mutations in the populations collected at the State University of Londrina's campus - a microenvironment that differs from the rest of the city - showed frequencies of 57% and 62%, respectively. The low gene flow observed, Nm = 0.11 and Nm = 0.10, along with the elevated differentiation, Fst = 0.19 and Fst = 0.18, among populations suggest an influence of genetic drift. The strong presence of resistance alleles kdr in the city is evident, which demonstrates that even with the interruption of the use of pyrethroids by the National Dengue Control Program, resistance may be maintained due to domestic use. Thus, the results have shown the need for genetic monitoring, alongside other entomological surveillance monitoring tools, to create strategies of mosquito control.

Comparison Between Larval Survey Index and Positive Ovitrap Index in the Evaluation of Populations of Aedes (Stegomyia) aegypti (Linnaeus, 1762) North of Paraná, Brazil.

Aedes (Stegomyia) aegypti (Linnaeus, 1762) is one of the world's most widely distributed mosquitoes and is the vector of the dengue virus, one of the most important reemerging diseases. Besides dengue, A. aegypti can also transmit urban yellow fever, chikungunya, and Zika virus, making it of great medical importance. Thus, it is of extreme importance to find reliable methods to evaluate the presence of A. aegypti in urban areas. In Brazil, rapid index surveys of Aedes aegypti by means of larval survey (LIRAa) is the official method to estimate the Breteau (BI) and property infestation (PII) indexes, which indicates how many infested containers with larvae of A. aegypti were found by the total number of properties surveyed and the proportion of houses infested, respectively. As the LIRAa requires access to private residences and trained personal to find breeding sites and do not reveal the mosquito's presence when in low density, it has not demonstrated efficacy in determining the presence of A. aegypti. To evaluate an alternative method, the LIRAa method was compared with an oviposition trap, made with hay infusion and a hardboard pallet, to evaluate the BI and the PII. The 2 methods were carried out simultaneously through 4 surveys, sampling 60 homes per survey. To evaluate the best configuration of ovitraps for surveillance of A. aegypti, the ovitraps were installed in intradomicile and peridomicile areas, with 1 to 5 traps per residence and with 1 to 3 pallets per trap, and these different configurations were compared using the positive ovitrap index (POI) and egg density index (EDI). The ovitraps showed greater sensitivity for detecting the presence of A. aegypti, with a BI of 72.5% and PII of 54.2%, whereas the LIRAa revealed only 2.1% for the BI and 1.3% for the PII. Therefore, the use of sentinel traps can provide information in a more rapid and precise manner. As there were no differences in the ovitraps distributions patterns, the ovitraps can be installed in the peridomicile area, with 2 traps per surveillance point and 1 pallet per trap, making their installation easier and more cost-efficient, facilitating the work of health agents in future surveillances complementing LIRAa's actions for efficient monitoring.

Morphological study of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) eggs by X-ray computed microtomography.

Aedes aegypti and Aedes albopictus are vectors of several arboviruses responsible for causing dengue, chikungunya, Zika, and yellow fever viruses and microcephaly, resulting in a public health problem in several countries worldwide. Even in this scenario, studies on the morphology of mosquito eggs are still lacking. In this paper, X-ray microtomography was used to study mosquito egg morphology. Several parameters such as length, surface area, volume, area-by-volume ratio, eccentricity and center of mass of the eggs were determined. The results obtained showed that micro-CT is an efficient technique for the morphological study of insect eggs and provides information that cannot be obtained with other techniques.

Aquatic insect communities in small stream in the south of Brazil.

Pollution of rivers and streams, by anthropic action, is characterized as an environmental, social, and sanitary problem. Factors such as the association between the marginal vegetation, the distribution of the substrates in the riverbed, and the availability of allochthonous organic matter influence the distribution and composition of the aquatic entomofauna. The objective of this study was to analyze the structure of aquatic insect communities in a pasture stream in northern Paraná, southern Brazil, with emphasis on the groups of indicators of good water quality, thus inferring the conditions of its preservation. Samples were collected from July to October in three parts of the stream (P1, P2, and P3), where the insect faune was collected with the aid of a sieve in the foliage substrate and washing of rocks and decomposing pieces of wood. A total of 1323 individuals were collected, being Chironomidae (Diptera) the most abundant taxon. The analysis of the biotic indices (EPT/Chironomidae, IBF, BMWP, and BMWP/ASPT) and diversity indicated better preservation conditions at points P1 and P3 where the riparian forest was well preserved, with less exposure to the stream bed. In P2, the entomofauna presented less diversity and the biotic indexes indicated loss of water quality, showing the impacts of changes in the marginal vegetation of this section. In a generally preserved aquatic environment, small changes in its vegetation are sufficient to cause an imbalance in the aquatic insect community, showing the efficiency of these organisms as bio-indicators and the sensitivity of biotic indexes.

Oviposition of Aedes aegypti Linnaeus, 1762 and Aedes albopictus Skuse, 1894 (Diptera: Culicidae) under laboratory and field conditions using ovitraps associated to different control agents, Manaus, Amazonas, Brazil

ABSTRACT The aim of this study was to analyze the effectiveness of different control agents of Aedes aegypti and Aedes albopictus associated with ovitraps under laboratory and field conditions. Five treatments were used: grass infusion + Bacillus thuringiensis israelensis (gI + Bti), grass infusion + Saccharopolyspora spinosa (gI + Ss), grass infusion + Pyriproxyfen (gI + P), distilled water + Toxorhynchites haemorrhoidalis (dW + Th), and grass infusion (gI) (control). The highest mean number of eggs of both species were obtained with grass infusion in the laboratory. Among control agents, the lowest mean of A. aegypti eggs occurred with gI + Ss and the lowest mean of A. albopictus eggs occurred with dW + Th. There was no difference between treatments in A. aegypti (P = 0.4320) and A. albopictus (P = 0.7179). In the field, the highest mean number of eggs for both species were obtained with gI + Ss, and the lowest values were obtained with gI + P (P = 0.0124). The treatments can be applied to both the surveillance and the control, but ovitraps with biological larvicide Bti were more effective and safer considering the number of eggs laid and selectivity of pathogens for mosquitoes.

Monitoramento e controle de Aedes aegypti (Linnaeus, 1762) e Aedes albopictus (Skuse, 1984) com uso de ovitrampas / Monitoring and control of Aedes aegypti (Linnaeus, 1762) and Aedes albopictus (Skuse, 1984) with use of ovitraps

Aedes aegypti (Linnaeus, 1762) é o principal vetor do vírus do dengue, e coloca sob risco metade da população mundial. É um mosquito antropofílico e veicula também os vírus Chikungunya e Zika vírus. Aedes albopictus (Skuse, 1894) é abundante em áreas com vegetação próximas a residências e pode transmitir os vírus Chikungunya e febre amarela. Estratégias de monitoramento e controle desses vetores são necessários para redução da circulação viral. Estimar a variação populacional de Ae. aegypti e Ae. albopictus em relação à urbanização e aferir relações de sobreposição e competição desses vetores com o uso de ovitrampas foram os objetivos deste estudo. A amostragem foi realizada em 10 locais no campus da Universidade Estadual de Londrina, entre abril e outubro de 2015. A ovitrampa foi elaborada com pote plástico escuro, contendo no seu interior água, uma palheta, mistura atrativa e temephós. Os ovos coletados nas palhetas foram submetidos a eclosão para identificação das espécies por meio das larvas. Coletou-se 46.879 ovos de Aedes e abril apresentou a maior densidade de ovos (IDO=49,0). A temperatura apresentou forte correlação (r=0,88) com a atividade de oviposição, entre abril a julho. Aedes aegypti predominou em áreas urbanas nos diferentes locais. Os resultados obtidos comprovam que a ovitrampa é uma ferramenta eficaz para monitorar e auxiliar em estratégias de controle da população destes vetores.

Aedes aegypti (Linnaeus, 1762) is the main vector virus of dengue, and puts at risk half of the world population. It is an anthropophilic mosquito and carries the Chikungunya and Zika viruses. Aedes albopictus (Skuse, 1894) is abundant in vegetated areas near residences and can transmit Chikungunya virus and yellow fever. Strategies for monitoring and controlling these vectors are necessary to reduce viral circulation. Estimate the population variation of Ae. aegypti and Ae. albopictus in relation to urbanization and to verify relations of overlap and competition of these vectors with the use of ovitraps were the objective of this work. The research was carried out in 10 sites in the campus of the State University of Londrina, from April to October of 2015. The ovitrap was elaborated with dark plastic pot, containing in its interior water, a palette, attractive mixture and temephós. The eggs collected on the palettes were stimulated to hatch to identify the species through the larvae. We collected 46,879 Aedes eggs and April had the highest egg density (IDO=49.0). The temperature had a strong correlation with to oviposition activity between April and July (r=0.88). Aedes aegypti predominated in different sites in the urban areas. The results obtained prove that ovitrap is an effective tool to monitor and assist strategies to control the population of these vectors.

Larvicidal Activity against Aedes aegypti and Chemical Characterization of the Inflorescences of Tagetes patula.

The crude acetone extract (CAE) of defatted inflorescences of Tagetes patula was partitioned into five semipurified fractions: n-hexane (HF), dichloromethane (DF), ethyl acetate (EAF), n-butanol (BF), and aqueous (AQF). BF was fractionated by reversed-phase polyamide column chromatography, obtaining 34 subfractions, which were subjected to HSCCC, where patuletin and patulitrin were isolated. CAE and the fractions BF, EAF, DF, and AQF were analyzed by LC-DAD-MS, and patuletin and patulitrin were determined as the major substances in EAF and BF, respectively. BF was also analyzed by HPLC and capillary electrophoresis (CE), and patulitrin was again determined to be the main substance in this fraction. CAE and the semipurified fractions (750, 500, 300, 100, and 50 mg/L) were assayed for larvicidal activity against Aedes aegypti, with mortality rate expressed as percentage. All fractions except AQF showed insecticidal activity after 24 h exposure of larvae to the highest concentration. However, EAF showed the highest activity with more than 50% reduction in larval population at 50 mg/L. The insecticidal activity observed with EAF might have been due to the higher concentration of patuletin present in this fraction.