Larvicidal Activity of Carbon Black against the Yellow Fever Mosquito Aedes aegypti.

The yellow fever mosquito Aedes aegypti is one of the deadliest animals on the planet because it transmits several medically important arboviruses, including Zika, chikungunya, dengue, and yellow fever. Carbon-based nanoparticles (CNPs) derived from natural sources have previously been shown to have toxic effects on mosquito larvae and offer a potential alternative to chemical insecticides such as pyrethroids, for which mosquitoes have evolved resistance. However, CNPs derived from industrial sources, such as carbon black, have not previously been evaluated as larvicides. Here, we evaluate the effects of a commercially-available carbon black, EMPEROR® 1800 (E1800), on mortality and development of pyrethroid-susceptible (PS) and pyrethroid-resistant (PR) strains of Ae. aegypti. We found that E1800 exhibited concentration-dependent mortality against 1st instar larvae of both strains within the first 120 h after exposure, but after this period, surviving larvae did not show delays in their development to adults. Physical characterization of E1800 suspensions suggests that they form primary particles of ~30 nm in diameter that fuse into fundamental aggregates of ~170 nm in diameter. Notably, larvae treated with E1800 showed internal accumulation of E1800 in the gut and external accumulation on the respiratory siphon, anal papillae, and setae, suggesting a physical mode of toxic action. Taken together, our results suggest that E1800 has potential use as a larvicide with a novel mode of action for controlling PS and PR mosquitoes.

Comparative and functional genomics of the ABC transporter superfamily across arthropods.

BACKGROUND: The ATP-binding cassette (ABC) transporter superfamily is comprised predominantly of proteins which directly utilize energy from ATP to move molecules across the plasma membrane. Although they have been the subject of frequent investigation across many taxa, arthropod ABCs have been less well studied. While the manual annotation of ABC transporters has been performed in many arthropods, there has so far been no systematic comparison of the superfamily within this order using the increasing number of sequenced genomes. Furthermore, functional work on these genes is limited. RESULTS: Here, we developed a standardized pipeline to annotate ABCs from predicted proteomes and used it to perform comparative genomics on ABC families across arthropod lineages. Using Kruskal-Wallis tests and the Computational Analysis of gene Family Evolution (CAFE), we were able to observe significant expansions of the ABC-B full transporters (P-glycoproteins) in Lepidoptera and the ABC-H transporters in Hemiptera. RNA-sequencing of epithelia tissues in the Lepidoptera Helicoverpa armigera showed that the 7 P-glycoprotein paralogues differ substantially in their tissue distribution, suggesting a spatial division of labor. It also seems that functional redundancy is a feature of these transporters as RNAi knockdown showed that most transporters are dispensable with the exception of the highly conserved gene Snu, which is probably due to its role in cuticular formation. CONCLUSIONS: We have performed an annotation of the ABC superfamily across > 150 arthropod species for which good quality protein annotations exist. Our findings highlight specific expansions of ABC transporter families which suggest evolutionary adaptation. Future work will be able to use this analysis as a resource to provide a better understanding of the ABC superfamily in arthropods.

Stop the crop: Insights into the insecticidal mode of action of cinnamodial against mosquitoes.

Cinnamodial (CDIAL) is a drimane sesquiterpene dialdehyde found in the bark of Malagasy medicinal plants (Cinnamosma species; family Canellaceae). We previously demonstrated that CDIAL was insecticidal, antifeedant, and repellent against Aedes aegypti mosquitoes. The goal of the present study was to generate insights into the insecticidal mode of action for CDIAL, which is presently unknown. We evaluated the effects of CDIAL on the contractility of the ventral diverticulum (crop) isolated from adult female Ae. aegypti. The crop is a food storage organ surrounded by visceral muscle that spontaneously contracts in vitro. We found that CDIAL completely inhibited spontaneous contractions of the crop as well as those stimulated by the agonist 5-hydroxytryptamine. Several derivatives of CDIAL with known insecticidal activity also inhibited crop contractions. Morphometric analyses of crops suggested that CDIAL induced a tetanic paralysis that was dependent on extracellular Ca2+ and inhibited by Gd3+, a non-specific blocker of plasma membrane Ca2+ channels. Screening of numerous pharmacological agents revealed that a Ca2+ ionophore (A23187) was the only compound other than CDIAL to completely inhibit crop contractions via a tetanic paralysis. Taken together, our results suggest that CDIAL induces a tetanic paralysis of the crop by elevating intracellular Ca2+ through the activation of plasma membrane Ca2+ channels, which may explain the insecticidal effects of CDIAL against mosquitoes. Our pharmacological screening experiments also revealed the presence of two regulatory pathways in mosquito crop contractility not previously described: an inhibitory glutamatergic pathway and a stimulatory octopaminergic pathway. The latter pathway was also completely inhibited by CDIAL.

Semi-synthetic cinnamodial analogues: Structural insights into the insecticidal and antifeedant activities of drimane sesquiterpenes against the mosquito Aedes aegypti.

The Aedes aegypti mosquito serves as a major vector for viral diseases, such as dengue, chikungunya, and Zika, which are spreading across the globe and threatening public health. In addition to increased vector transmission, the prevalence of insecticide-resistant mosquitoes is also on the rise, thus solidifying the need for new, safe and effective insecticides to control mosquito populations. We recently discovered that cinnamodial, a unique drimane sesquiterpene dialdehyde of the Malagasy medicinal plant Cinnamosma fragrans, exhibited significant larval and adult toxicity to Ae. aegypti and was more efficacious than DEET-the gold standard for insect repellents-at repelling adult female Ae. aegypti from blood feeding. In this study several semi-synthetic analogues of cinnamodial were prepared to probe the structure-activity relationship (SAR) for larvicidal, adulticidal and antifeedant activity against Ae. aegypti. Initial efforts were focused on modification of the dialdehyde functionality to produce more stable active analogues and to understand the importance of the 1,4-dialdehyde and the α,ß-unsaturated carbonyl in the observed bioactivity of cinnamodial against mosquitoes. This study represents the first investigation into the SAR of cinnamodial as an insecticide and antifeedant against the medically important Ae. aegypti mosquito.

Heterologous Expression of Aedes aegypti Cation Chloride Cotransporter 2 (aeCCC2) in Xenopus laevis Oocytes Induces an Enigmatic Na⁺/Li⁺ Conductance.

The yellow fever mosquito Aedes aegypti possesses three genes encoding putative Na⁺-coupled cation chloride cotransporters (CCCs): aeNKCC1, aeCCC2, and aeCCC3. To date, none of the aeCCCs have been functionally characterized. Here we expressed aeCCC2 heterologously in Xenopus oocytes and measured the uptake of Li⁺ (a tracer for Na⁺) and Rb⁺ (a tracer for K⁺). Compared to control (H2O-injected) oocytes, the aeCCC2-expressing oocytes exhibited significantly greater uptake of Li⁺, but not Rb⁺. However, the uptake of Li⁺ was neither Cl--dependent nor inhibited by thiazide, loop diuretics, or amiloride, suggesting unconventional CCC activity. To determine if the Li⁺-uptake was mediated by a conductive pathway, we performed two-electrode voltage clamping (TEVC) on the oocytes. The aeCCC2 oocytes were characterized by an enhanced conductance for Li⁺ and Na⁺, but not K⁺, compared to control oocytes. It remains to be determined whether aeCCC2 directly mediates the Na⁺/Li⁺ conductance or whether heterologous expression of aeCCC2 stimulates an endogenous cation channel in the oocyte plasma membrane.

Cap 'n' collar C regulates genes responsible for imidacloprid resistance in the Colorado potato beetle, Leptinotarsa decemlineata.

The Colorado potato beetle (CPB), Leptinotarsa decemlineata developed resistance to imidacloprid after exposure to this insecticide for multiple generations. Our previous studies showed that xenobiotic transcription factor, cap 'n' collar isoform C (CncC) regulates the expression of multiple cytochrome P450 genes, which play essential roles in resistance to plant allelochemicals and insecticides. In this study, we sought to obtain a comprehensive picture of the genes regulated by CncC in imidacloprid-resistant CPB. We performed sequencing of RNA isolated from imidacloprid-resistant CPB treated with dsRNA targeting CncC or gene coding for green fluorescent protein (control). Comparative transcriptome analysis showed that CncC regulated the expression of 1798 genes, out of which 1499 genes were downregulated in CncC knockdown beetles. Interestingly, expression of 79% of imidacloprid induced P450 genes requires CncC. We performed quantitative real-time PCR to verify the reduction in the expression of 20 genes including those coding for detoxification enzymes (P450s, glutathione S-transferases, and esterases) and ABC transporters. The genes coding for ABC transporters are induced in insecticide resistant CPB and require CncC for their expression. Knockdown of genes coding for ABC transporters simultaneously or individually caused an increase in imidacloprid-induced mortality in resistant beetles confirming their contribution to insecticide resistance. These studies identified CncC as a transcription factor involved in regulation of genes responsible for imidacloprid resistance. Small molecule inhibitors of CncC or suppression of CncC by RNAi could provide effective synergists for pest control or management of insecticide resistance.

A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

Cap n collar transcription factor regulates multiple genes coding for proteins involved in insecticide detoxification in the red flour beetle, Tribolium castaneum.

In invertebrates, a heterodimer of xenobiotic transcription factors, cap n collar C isoform (CncC) and muscle aponeurosis fibromatosis (Maf) mediate cellular defense. In insects, these proteins regulate expression of genes involved in insecticide detoxification. In the current study, we performed sequencing of cDNA copied from RNA isolated from Tribolium castaneum pyrethroid resistant strain (QTC279) beetles injected with CncC or green fluorescence protein (GFP, control) dsRNA. Differential expression analysis of sequences identified 662 genes that showed a decrease and 91 genes that showed an increase in expression (p value ≤ 0.01 and log2 fold change of ≥ 1.5) in CncC knockdown insects when compared to their expression in control insects. We selected a subset of 27 downregulated genes and verified their differential expression using qRT-PCR. This subset of 27 genes included 21 genes with a predicted function in xenobiotic detoxification. RNAi and insecticide bioassays were employed to study the function of six of these genes coding for CYP4G7, CYP4G14, GST-1 and four ABC transporters, ABCA-UB, ABCA-A1 and ABCA-A1L and ABCA-9B involved in all three phases of insecticide detoxification. These data suggest that CncC regulates genes coding for proteins involved in detoxification of insecticides.

Transcription factor cap n collar C regulates multiple cytochrome P450 genes conferring adaptation to potato plant allelochemicals and resistance to imidacloprid in Leptinotarsa decemlineata (Say).

Colorado potato beetle (CPB), Leptinotarsa decemlineata is a notorious pest of potato. Co-evolution with Solanaceae plants containing high levels of toxins (glycoalkaloids) helped this insect to develop an efficient detoxification system and resist almost every chemical insecticide introduced for its control. Even though the cross-resistance between plant allelochemicals and insecticides is well acknowledged, the underlying molecular mechanisms are not understood. Here, we investigated the molecular mechanisms involved in detoxification of potato plant allelochemicals and imidacloprid resistance in the field-collected CPB. Our results showed that the imidacloprid-resistant beetles employ metabolic detoxification of both potato plant allelochemicals and imidacloprid by upregulation of common cytochrome P450 genes. RNAi aided knockdown identified four cytochromes P450 genes (CYP6BJa/b, CYP6BJ1v1, CYP9Z25, and CYP9Z29) that are required for defense against both natural and synthetic chemicals. These P450 genes are regulated by the xenobiotic transcription factors Cap n Collar C, CncC and muscle aponeurosis fibromatosis, Maf. Studies on the CYP9Z25 promoter using the luciferase reporter assay identified two binding sites (i.e. GCAGAAT and GTACTGA) for CncC and Maf. Overall, these data showed that CPB employs the metabolic resistance mediated through xenobiotic transcription factors CncC and Maf to regulate multiple P450 genes and detoxify both imidacloprid and potato plant allelochemicals.

Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects.

RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects.

Transcription factors, CncC and Maf, regulate expression of CYP6BQ genes responsible for deltamethrin resistance in Tribolium castaneum.

Insecticide resistance is a global problem that presents an ongoing challenge to control insects that destroy crops, trees and transmit diseases. Dramatic progress has been made during the last decade on identification of insecticide resistance-associated genes. In one of the most common resistance mechanisms, insects acquire resistance by increasing the levels of their detoxification enzymes especially the cytochrome P450 monooxygenases (P450's). Previous studies in our laboratory showed that the pyrethroid resistance in QTC279 strain of Tribolium castaneum is achieved through constitutive overexpression of the P450 gene CYP6BQ9 by 200-fold higher in the resistant strain as compared to that in the susceptible strain. RNAi-aided knockdown in the expression of probable genes that regulate P450 gene expression in QTC279 identified cap 'n' collar C (CncC) and muscle aponeurosis fibromatosis (Maf) family transcription factors as the key regulator of these genes, CncC and Maf regulate expression of multiple genes in the CYP6BQ cluster. Studies on the promoters of these genes using reporter assays identified binding sites that mediate CncC and Maf regulation of CYP6BQ gene expression.