Isolation of 4-nerolidylcatechol from leaves of Piper peltatum L., and evaluation of larvicidal activity in mosquito vectors, with emphasis on Aedes aegypti (Diptera: Culicidae)

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.

Identification and validation of the first EST-SSR markers based on transcriptome of Anopheles darlingi, the primary transmitter of malaria in Brazil.

BACKGROUND: Anopheles darlingi is a monotypic species in terms of its morphological, genetic, and behavioral aspects and is the primary transmitter of human malaria (99%) in Brazil, especially in the Brazilian Amazon. In this pioneering study, 15 expressed sequence tag (EST)-simple sequence repeat (SSR) markers were obtained and characterized in samples from the municipality of São Gabriel da Cachoeira, Amazonas state, Brazil, with polymorphisms that can be used for further genetic research. METHODS AND RESULTS: The specimens (from egg to larval stage) collected were bred in the insectary at INPA (National Institute for Amazonian Research). The SSR repeats within the contigs of the A. darlingi EST banks were confirmed on the Vector Base site. DNA was extracted and amplified using polymerase chain reaction and then genotyped. Fifteen polymorphic SSR loci were identified and characterized. The number of alleles totaled 76 and ranged from 2 to 9. The observed heterozygosity varied between 0.026 and 0.769, the expected heterozygosity between 0.025 and 0.776, and the mean polymorphism information content was 0.468. Eight loci showed Hardy-Weinberg equilibrium (HWE) after Bonferroni correction (P: (5%) ≤ 0.0033). No linkage disequilibrium was found among the loci. CONCLUSIONS: The polymorphic SSRs of the loci have been shown to be efficient for investigation of the variability and genetic population structure of A. darlingi.

Study of karyotype and constitutive heterochromatin of Mansonia spp. (Diptera: Culicidae) in the area surrounding the Jirau Hydroelectric Dam, Rondônia, Brazil.

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.

Study of karyotype and constitutive heterochromatin of Mansonia spp. (Diptera: Culicidae) in the area surrounding the Jirau Hydroelectric Dam, Rondônia, Brazil

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.

Phylogenetic relationships of the supercontig of sodium channel subunit I (NaV) in 17 species of Anopheles (Diptera: Culicidae).

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.

Phylogenetic relationships of the supercontig of sodium channel subunit I (NaV) in 17 species of Anopheles (Diptera: Culicidae)

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.

Physical Mapping of the Anopheles (Nyssorhynchus) darlingi Genomic Scaffolds.

The genome assembly of Anopheles darlingi consists of 2221 scaffolds (N50 = 115,072 bp) and has a size spanning 136.94 Mbp. This assembly represents one of the smallest genomes among Anopheles species. Anopheles darlingi genomic DNA fragments of ~37 Kb were cloned, end-sequenced, and used as probes for fluorescence in situ hybridization (FISH) with salivary gland polytene chromosomes. In total, we mapped nine DNA probes to scaffolds and autosomal arms. Comparative analysis of the An. darlingi scaffolds with homologous sequences of the Anopheles albimanus and Anopheles gambiae genomes identified chromosomal rearrangements among these species. Our results confirmed that physical mapping is a useful tool for anchoring genome assemblies to mosquito chromosomes.

Genotoxic Effects of Semi-Synthetic Isodillapiole on Oviposition in Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae).

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.

Histopathological, cytotoxicological, and genotoxic effects of the semi-synthetic compound dillapiole n-butyl ether in Balb/C mice.

Dillapiole n-butyl ether is a substance derived from dillapiole, which exhibits potential insecticidal effects on Aedes aegypti, the principal vector of the Dengue fever, Zika, and Chikungunya viruses, as well as Aedes albopictus, a vector of Dengue fever. As these mosquitoes are resistant to synthetic insecticides, dillapiole n-butyl ether may represent a valuable, plant-based alternative for their control. Dillapiole n-butyl ether has insecticidal and genotoxic effects on A. aegypti and A. albopictus, as shown by the reduction in clutch size and egg viability, and increased mortality rates, as well as a high frequency of micronuclei and chromosomal aberrations. However, the potential cytotoxic and genotoxic effects of this substance in mammals are still unknown. In Balb/C mice, structural changes were detected in hepatic, renal, and cardiac tissues, which were directly proportional to the concentration of the dose applied, in both genders. The induction of genotoxic, mutagenic, and cytotoxic effects was also observed at the highest concentrations (150 and 328 mg/kg). Further research will be necessary to better characterize the potential genotoxicity of this substance at lower concentrations, for the evaluation of the potential health risks related to its presence in environmental features, such as drinking water.

Genotoxic Effects of Semi-Synthetic Isodillapiole on Oviposition in Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae)

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.

Insect (Hexapoda) diversity in the oceanic archipelago of Fernando de Noronha, Brazil: updated taxonomic checklist and new records

ABSTRACT Hexapods, commonly known as insects, are a neglected taxonomic group in the Fernando de Noronha archipelago, with unanswered questions about their species richness and the ecological processes in which they are involved (e.g., colonization, introduction, establishment, and extinction). Herein, we provide an updated Hexapod checklist with current nomenclatural combinations. The entomofauna of the Fernando de Noronha archipelago is currently composed of 453 species in 21 orders. The orders, and their respective number of species, are: Blattaria (9), Coleoptera (118), Collembola (29), Dermaptera (3), Diplura (1), Diptera (134), Embioptera (1), Hemiptera (29), Hymenoptera (59), Isoptera (2), Lepidoptera (25), Mantodea (1), Neuroptera (3), Odonata (5), Orthoptera (11), Phasmatodea (1), Phthiraptera (6), Psocoptera (3), Siphonaptera (1), Thysanoptera (10), and Zygentoma (2). The archipelago has 263 new taxon records (family + genera + species). Thirty-eight species (3.39%) were described from local specimens and most of them are likely endemic species. This study more than doubles our knowledge (from the previous 190 records) of the entomofauna in this large Brazilian archipelago. This study also provides a baseline for studies on its conservation status and for implementing future environmental management programs.

Toxic effect and genotoxicity of the semisynthetic derivatives dillapiole ethyl ether and dillapiole n-butyl ether for control of Aedes albopictus (Diptera: Culicidae).

Two derivatives of dillapiole, dillapiole ethyl ether (1KL39-B) and butyl ether-n dillapiole (1KL43-C), were studied for their toxicity and genotoxicity against Aedes albopictus, to help develop new strategies for the control of this potential vector of dengue and other arboviruses, because it is resistant to synthetic insecticides. Eggs and larvae exposed to different concentrations of 1KL39-B (25, 30, 50, 70, and 80µg/mL) and of 1KL43-C (12.5, 20, 25, 30 and 40µg/mL) exhibited toxicity and susceptibility, with 100% mortality. The LC50 was 55.86±1.57µg/mL for 1KL39-B and 25.60±1.24µg/mL for 1KL43-C, while the LC90 was 70.12µg/mL for 1KL39-B and 41.51µg/mL for 1KL43-C. The gradual decrease in oviposition of the females of the G1 to G4 generations was proportional to the increase in concentrations of these compounds, which could be related to the cumulative effect of cell anomalies in neuroblasts and oocytes (P<0.05), including micronuclei, budding, multinucleated cells and nuclear bridges. These findings showed that both 1KL39-B and 1KL43-C can serve as potential alternatives in the control of A. albopictus.

Insecticidal and genotoxic potential of two semi-synthetic derivatives of dillapiole for the control of Aedes (Stegomyia) aegypti (Diptera: Culicidae).

The effects of two semi-synthetic dillapiole derivatives, ethyl-ether dillapiole and n-butyl ether dillapiole, on eggs and larvae of Aedes aegypti were studied in view of the need for expansion and renovation of strategic action to control this mosquito - the vector of Dengue virus -, which currently shows a high resistance to chemical insecticides. Eggs and third-instar larvae of A. aegypti that had been exposed to different concentrations of these two compounds showed toxicity and susceptibility, with 100% mortality. Classical cytogenetic assays showed genotoxicity caused by the two compounds in A. aegypti from the cumulative effect of nuclear abnormalities, indicating that these derivatives may be potential alternatives to control A. aegypti.

Phylogenetic analysis of the GST family in Anopheles (Nyssorhynchus) darlingi.

Anopheles darlingi Root, 1926 and Anopheles gambiae (Diptera: Culicidae) are the most important human malaria vectors in South America and Africa, respectively. The two species are estimated to have diverged 100 million years ago. Studies on the phylogenetics and evolution of gene sequences, such as glutathione S-transferase (GST) in disease-transmitting mosquitoes are scarce. The sigma class GST (KC890767) from the transcriptome of An. darlingi captured in the Brazilian Amazon was studied by in silico hybridization, and mapped to chromosome 3 of An. gambiae. The sigma class GST of An. darlingi was used for phylogenetic analyses to understand the GST base composition of the most recent common ancestor between An. darlingi, Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus. The GST (KC890767) of An. darlingi was studied to generate the main divergence branches using a Neighbor-Joining and bootstrapping approaches to confirm confidence levels on the tree nodes that separate the An. darlingi and other mosquito species. The results showed divergence between An. gambiae, Ae. Aegypti, Cx. quinquefasciatus, and Phlebotomus papatasi as outgroup, and the homology relationship between sigma class GST of An. darlingi and GSTS1_1 gene of An. gambiae was valuable for phylogenetic and evolutionary studies.

Karyotype of Brazilian Anopheles albitarsis sensu lato (Diptera:Culicidae).

Anopheles (Nyssorhynchus) albitarsis sensu lato is an important malaria vector in Brazil, especially in the Brazilian Amazon region. Chromosome preparations of fourth-instar larvae of A. albitarsis from Iranduba and Coari (AM) and Ilha Comprida (SP) were analyzed for karyotype determination and to improve cytogenetic identification of this species. Anopheles albitarsis possesses 2n = 6 chromosomes, with two pairs (submetacentric and metacentric) of autosomes and one pair of sex chromosomes, with X-Y dimorphism. The sex pair is homomorphic and acrocentric in females and heteromorphic in males, with a punctiform Y chromosome. Somatic pairing was detected in the prometaphase and metaphase chromosomes of the three A. albitarsis populations. Apparently, sex chromosome evolution in the Culicidae does not function as does evolution in the Culicidae, since it occurs in the subfamily Anophelinae, which possesses heteromorphic sex chromosomes and is regarded as primitive, based on several criteria. These karyotype data on the albitarsis complex reinforce the hypothesis that sex chromosome evolution in the subfamily Anophelinae is conserved, and the variation revealed in the mean size of chromosomes in three populations indicates that selective pressure in these populations is occurring only at a genetic level.

The physical gene Hsp70 map on polytene chromosomes of Anopheles darlingi from the Brazilian Amazon.

In situ hybridization was used to determine the physical location of the Hsp70 genes in salivary polytene chromosomes of Anopheles darlingi from Manaus and Macapá, Brazil, and to assess the usefulness of the Hsp70 locus as a genetic marker in A. darlingi populations. In both populations, the double markings corresponding to the Hsp70-12A and Hsp70-14A genes were located on the right arm of chromosome 2. The Hsp70 locus was considered to be an excellent marker for studying chromosomal evolution and relationships among A. darlingi populations.

Location of ribosomal genes in the chromosomes of Anopheles darlingi and Anopheles nuneztovari (Diptera, Culicidae) from the Brazilian Amazon.

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).

Location of ribosomal genes in the chromosomes of Anopheles darlingi and Anopheles nuneztovari (Diptera, Culicidae) from the Brazilian Amazon

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)