ABSTRACT Synthetic chemical insecticides are widely used in population control of pests. Aedes aegypti is the primary vector of dengue, Zika, chikungunya and yellow fever to humans, and has proven resistance to chemical insecticides. As an alternative vector control method, the ethanolic extract of the leaves of Piper peltatum L. (Piperaceae) showed larvicidal activity against Ae. aegypti. Despite the wide medicinal use of this plant, the biological activity of its isolated constituents remains unexplored. In this sense, we isolated, identified and evaluated the larvicidal activity of 4-nerolidylcatechol (4-NC) from P. peltatum against Ae. aegypti, Culex quinquefasciatus and Anopheles darlingi, focusing on the larvicidal, adulticidal and genotoxic potential of 4-NC on Ae. aegypti. Larvae were captured in the city of Manaus, Amazonas state, Brazil. 4-NC was isolated from the extract of the leaves of P. peltatum via silica gel chromatography. This was identified using nuclear magnetic resonance spectroscopy and tested in Artemia franciscana (6.25, 12.5, 25, 50, 100 and 200 µg/mL). In the toxicity bioassay, Ae. aegypti larvae were exposed to 30, 50, 70, 90, and 110 µg/mL of 4-NC, while Cx. quinquefasciatus and An. darlingi were exposed to 6.25, 12.5, 25, 50 and 100 µg/mL. Ae. aegypti larvae were also subjected to 40 and 60 µg/mL of 4-NC (genotoxicity bioassay), and adult females to 62.5 to 1,000 µg/mL (adulticidal bioassay). The results of the 4-NC toxicity assays showed that there was 100% mortality in larvae of Ar. franciscana at the concentration of 200 µg/mL, with an LC50 of 8.0 μg/mL. In the larvae of Ae. aegypti, mortality was 100%, with an LC50 of 62 μg/mL. In larvae of Cx. quinquefasciatus, 97% mortality occurred, with an LC50 of 52.3 μg/mL, and in An. darlingi larvae there was an 83% mortality rate, with an LC50 of 55.8 μg/mL. In adults of Ae. aegypti, however, there was no adulticidal activity. In the larvae of Ae. aegypti, the genotoxic effect of 4-NC (40 and 60 µg/mL) showed significant frequency (p < 0.05) of cellular abnormalities (micronuclei, budding and nuclear bridges) of interphasic nuclei of neuroblasts and oocytes in relation to the negative control. This result may be associated with a decrease in oviposition of females, which was observed in two generations. We can confirm that 4-NC has larvicidal activity against Ae. aegypti, Cx. quinquefasciatus and An. darlingi. Although it does not present adulticidal activity in Ae. aegypti, it reduced the oviposition of females. Therefore, 4-NC seems to be a strong candidate for the development of an alternative method for the control of these mosquitoes in the immature phase. pests dengue Zika humans L Piperaceae (Piperaceae Ae plant unexplored sense 4nerolidylcatechol nerolidylcatechol 4 4NC NC (4-NC P Manaus state Brazil chromatography 625 6 25 (6.25 125 12 5 12.5 10 20 µgmL . µg mL bioassay 30 70 90 11 4NC, NC, Cx An 6.25 genotoxicity , bioassay) 62. 1000 1 000 1,00 bioassay. Ar LC LC5 80 8 0 8. μgmL μg μg/mL 97 occurred 523 52 3 52. 83 rate 558 55 55. however (4 p 0.05 005 05 micronuclei, micronuclei (micronuclei bridges generations Therefore phase 2 (6.2 12. 7 9 6.2 00 1,0 ( 0.0 (6. 6. 1, 0. (6

ABSTRACT Background: Mansonia mosquitoes transmit arboviruses to humans. This study describes the karyotypes and C-banding of Mansonia humeralis, Mansonia titillans, Mansonia pseudotitillans, and Mansonia indubitans. Methods: From the 202 larvae, the brain ganglia were dissected (n=120) for the preparation of slides. Twenty slides with well-distended chromosomes for each species (10 for karyotyping and 10 for C-banding) were selected for further study. Results: The haploid genome and the average lengths of the chromosomal arms differed in relation to the centromere between species, and intraspecific differences also occurred in the distribution of the C-bands. Conclusions: These results are useful for better understanding of the chromosomal variability of Mansonia mosquitoes. Background humans Cbanding C banding humeralis titillans pseudotitillans indubitans Methods 20 larvae n=120 n120 n 120 (n=120 welldistended well distended (1 1 Results Cbands. Cbands bands. bands C-bands Conclusions 2 n=12 n12 12 (n=12 ( n=1 n1 (n=1 n= (n= (n

ABSTRACT Background: Malaria is a global health problem and is transmitted by the Anopheles species. Due to the epidemiological importance of the genus, studies on biological, phylogenetic, and evolutionary aspects have contributed to the understanding of adaptation, vector capacity, and resistance to insecticides. The latter may result from different causes such as mutations in the gene that encodes the sodium channel (NaV). Methods: In this study, the NaV subunit I scaffold of 17 anopheline species was used to infer phylogenetic relationships of the genus Anopheles using Bayesian inference. The evolutionary phylogenetic tree of the NaV gene was aligned in the AliView program and analyzed utilizing Bayesian inference, using the software MrBayes. Results: The anophelines were grouped into five well-supported clusters: 1 - Anopheles darlingi and Anopheles albimanus; 2 - Anopheles sinensis and Anopheles atroparvus; 3 - Anopheles dirus; 4 - Anopheles minimus, Anopheles culicifacies, Anopheles funestus, Anopheles maculatus, and Anopheles stephensi; and 5 - Anopheles christyi, Anopheles epiroticus, Anopheles merus, Anopheles melas, Anopheles gambiae, Anopheles coluzzii, and Anopheles arabiensis. Conclusions: The topology confirms the phylogenetic relationships proposed in studies based on the genome of some anophelines and reflects the current taxonomy of the genus, which suggests that NaV undergoes selection pressure during the evolution of the species. These data are useful tools for inferring their ability to resist insecticides and also help in better understanding the evolutionary processes of the genus Anopheles.

Abstract INTRODUCTION: Semi-synthetic dillapiole compounds derived from Piper aduncum essential oil are used as alternative insecticides to control insecticide-resistant Aedes aegypti. Thus, we aimed to evaluate the genotoxic effects of semi-synthetic isodillapiole on the nuclei of neuroblasts (larvae) and oocytes (females) and the mean oviposition rates of the females over four generations (G1, G2, G3, and G4) of Ae. aegypti. METHODS: Larvae were captured in the city of Manaus, Amazonas state, Brazil, and exposed to isodillapiole in bioassays (20, 40, and 60 µg/mL) and a negative control (0.05% DMSO in tap water) for 4 h. The cerebral ganglia were extracted from the larvae and oocytes from the adult females to prepare slides for cytogenetic analysis. Breeding pairs were established and eggs counts were quantified taken after the bioassays. RESULTS: The analysis of 20,000 interphase nuclei of neuroblasts and oocytes indicated significant genotoxicity (micronuclei, budding, polynucleated cells, and other malformations) compared to that of the control. Metaphasic and anaphasic nuclei presented chromosomal breaks; however, no significant variation and damage was observed in the negative control. A significant reduction in mean oviposition rates was also recorded following exposure to isodillapiole over the four generations (G1, G2, G3, and G4). CONCLUSIONS: The toxic and genotoxic effects of isodillapiole on Ae. aegypti were caused by reduced oviposition in the females and nuclear abnormalities over the four generations of the trials. Further studies are required, rather than our in vitro assays, to verify the efficacy of exposure to this compound for controlling Ae. aegypti.

Fluorescence in situ hybridization of Anopheles darlingi and A. nuneztovari demonstrated nucleolar organizer region activity at the end of the fourth larval instar, when the nucleolar organizer regions underwent gradual condensation. The heteromorphic sex chromosomes showed intraindividual size variation in the rDNA blocks located in the pericentromeric region and this coincided with the location of constitutive heterochromatin (C-banding).

Pela técnica do bandamento C detectou-se variação de marcação dos blocos heterocromáticos dos cromossomos de A. darlingi e A. nuneztovari de Manaus, Amazonas, e de Macapá, Amapá, Brasil. Os cromossomos sexuais de ambas as espécies mostraram duas formas de cromossomos X e o Y foi totalmente heterocromático. No cromossomo X1 de A. darlingi a marcação atingiu 1/3 e no cromossomo X2 foi apenas na região centromérica. Nos autossomos de ambas as espécies as marcações foram constantes nas regiões centroméricas, e o cromossomo II de A. darlingi mostrou um bloco heterocromático em um dos braços. A. nuneztovari mostrou polimorfismo de tamanho para o cromossomo X, tendo o X maior (X1) três blocos e o menor (X2) dois blocos heterocromáticos. Fêmeas homozigotas (X2X2) não foram detectadas nas duas localidades. Em machos de A. darlingi foram encontrados os cromossomos X1 e X2, enquanto que em machos de A. nuneztovari somente o cromossomo X1 foi detectado. Apenas variação intraespecífica de blocos heterocromáticos nos cromossomos X e nos autossomos foi registrada nas duas populações de ambas as espécies estudadas em cada localidade.

C-banding was used to study the variations in heterochromatic block markings in chromosomes of Anopheles darlingi and A. nuneztovari from Manaus, State of Amazonas, and Macapá, State of Amapá, Brazil. Both species had two differently shaped X chromosomes and a Y chromosome that was entirely heterochromatic. The X1 chromosome of A. darlingi had markings that extended 1/3 of the total length whereas in the X2 chromosome the markings were located around the centromeric region. The markings on autosomal chromosomes were concentrated in the centromeric region in both species, with a heterochromatic block in one arm of chromosome II of A. darlingi. A. nuneztovari had three heterochromatic blocks in chromosome X1 (longer) and two blocks in X2 (shorter). X2X2 females were not detected in either species. The X1 and X2 chromosomes of males were found in A. darlingi, whereas in A. nuneztovari only the X1 chromosome was detected. Only intraspecific variation was found in heterochromatic block markings in the sex chromosomes and autosomes in the two populations of both species at each location.

Metaphase karyotypes of Anopheles (Nyssorhynchus) darlingi and Anopheles (N.) nuneztovari from Manaus and Highway BR-174 (Manaus-Boa Vista), State of Amazonas, and Macapá, State of Amapá, Brazil, showed 2n = 6. They consisted of a pair of metacentric (chromosome II) and a pair of submetacentric (chromosome III) autosomes as well as sex chromosomes X and Y. In the sex chromosomes, the X was acrocentric in A. darlingi and submetacentric in A. nuneztovari. The Y chromosome was pointed in both species. The chromosomes of A. nuneztovari were larger than those of A. darlingi except for the Y chromosome. A. nuneztovari showed size polymorphism in the X chromosome. Somatic pairing and a strong constriction around the centromeric region in chromosomes II and III were exhibited in both species. The secondary constriction was only detected in chromosome II of A. darlingi from the Manaus population and chromosome III from the Macapá population.