Laboratory evaluation of the contact irritancy of a clothianidin solo formulation vs. clothianidin-deltamethrin mixture formulations for indoor residual spraying against pyrethroid-resistant Anopheles gambiae sensu lato.

BACKGROUND: Clothianidin-based indoor residual spraying (IRS) formulations have become available for malaria control as either solo formulations of clothianidin or a mixture of clothianidin with the pyrethroid deltamethrin. While both formulations have been successfully used for malaria control, studies investigating the effect of the pyrethroid in IRS mixtures may help improve our understanding for development of future IRS products. It has been speculated that the irritant effect of the pyrethroid in the mixture formulation may result in shorter mosquito contact times with the treated walls potentially leading to a lower impact. METHODS: We compared contact irritancy expressed as the number of mosquito take-offs from cement surfaces treated with an IRS formulation containing clothianidin alone (SumiShield® 50WG) to clothianidin-deltamethrin mixture IRS formulations against pyrethroid-resistant Anopheles gambiae sensu lato under controlled laboratory conditions using a modified version of the World Health Organisation cone bioassay. To control for the pyrethroid, comparison was made with a deltamethrin-only formulation. Both commercial and generic non-commercial mixture formulations of clothianidin and deltamethrin were tested. RESULTS: The clothianidin solo formulation did not show significant contact irritancy relative to the untreated control (3.5 take-offs vs. 3.1 take-offs, p = 0.614) while all deltamethrin-containing IRS induced significant irritant effects. The number of take-offs compared to the clothianidin solo formulation (3.5) was significantly higher with the commercial clothianidin-deltamethrin mixture (6.1, p = 0.001), generic clothianidin-deltamethrin mixture (7.0, p < 0.001), and deltamethrin-only (8.2, p < 0.001) formulations. The commercial clothianidin-deltamethrin mixture induced similar contact irritancy as the generic clothianidin-deltamethrin mixture (6.1 take-offs vs. 7.0 take-offs, p = 0.263) and deltamethrin-only IRS (6.1 take-offs vs. 8.2, p = 0.071), showing that the irritant effect in the mixture was attributable to its deltamethrin component. CONCLUSIONS: This study provides evidence that the enhanced contact irritancy of the pyrethroid in clothianidin-deltamethrin IRS mixtures can shorten mosquito contact times with treated walls compared to the clothianidin solo formulation. Further trials are needed to directly compare the efficacy of these formulation types under field conditions and establish the impact of this enhanced contact irritancy on the performance of IRS mixture formulations containing pyrethroids.

miRNA Dynamics for Pest Management: Implications in Insecticide Resistance.

Utilizing chemical agents in pest management in modern agricultural practices has been the predominant approach since the advent of synthetic insecticides. However, insecticide resistance is an emerging issue, as pest populations evolve to survive exposure to chemicals that were once effective in controlling them, underlining the need for advanced and innovative approaches to managing pests. In insects, microRNAs (miRNAs) serve as key regulators of a wide range of biological functions, characterized by their dynamic expression patterns and the ability to target genes. Recent studies are increasingly attributed to the significance of miRNAs in contributing to the evolution of insecticide resistance in numerous insect species. Abundant miRNAs have been discovered in insects using RNA sequencing and transcriptome analysis and are known to play vital roles in regulation at both the transcriptional and post-transcriptional levels. Globally, there is growing research interest in the characterization and application of miRNAs, especially for their potential role in managing insecticide resistance. This review focuses on how miRNAs contribute to regulating insecticide resistance across various insect species. Furthermore, we discuss the gain and loss of functions of miRNAs and the techniques for delivering miRNAs into the insect system. The review emphasizes the application of miRNA-based strategies to studying their role in diminishing insecticide resistance, offering a more efficient and lasting approach to insect management.

Transcriptomic and Metatranscriptomic Analyses Provide New Insights into the Response of the Pea Aphid Acyrthosiphon pisum (Hemiptera: Aphididae) to Acetamiprid.

Acetamiprid is a broad-spectrum neonicotinoid insecticide used in agriculture to control aphids. While recent studies have documented resistance to acetamiprid in several aphid species, the underlying mechanisms are still not fully understood. In this study, we analyzed the transcriptome and metatranscriptome of a laboratory strain of the pea aphid, Acyrthosiphon pisum (Harris, 1776), with reduced susceptibility to acetamiprid after nine generations of exposure to identify candidate genes and the microbiome involved in the adaptation process. Sequencing of the transcriptome of both selected (RS) and non-selected (SS) strains allowed the identification of 14,858 genes and 4938 new transcripts. Most of the differentially expressed genes were associated with catalytic activities and metabolic pathways involving carbon and fatty acids. Specifically, alcohol-forming fatty acyl-CoA reductase (FAR) and acyl-CoA synthetase (ACSF2), both involved in the synthesis of epidermal wax layer components, were significantly upregulated in RS, suggesting that adaptation to acetamiprid involves the synthesis of a thicker protective layer. Metatranscriptomic analyses revealed subtle shifts in the microbiome of RS. These results contribute to a deeper understanding of acetamiprid adaptation by the pea aphid and provide new insights for aphid control strategies.

Mapping malaria vectors and insecticide resistance in a high-endemic district of Haryana, India: implications for vector control strategies.

BACKGROUND: Achieving effective control and elimination of malaria in endemic regions necessitates a comprehensive understanding of local mosquito species responsible for malaria transmission and their susceptibility to insecticides. METHODS: The study was conducted in the highly malaria prone Ujina Primary Health Center of Nuh (Mewat) district of Haryana state of India. Monthly entomological surveys were carried out for adult mosquito collections via indoor resting collections, light trap collections, and pyrethrum spray collections. Larvae were also collected from different breeding sites prevalent in the region. Insecticide resistance bioassay, vector incrimination, blood meal analysis was done with the collected vector mosquitoes. RESULTS: A total of 34,974 adult Anopheles mosquitoes were caught during the survey period, out of which Anopheles subpictus was predominant (54.7%). Among vectors, Anopheles stephensi was predominant (15.5%) followed by Anopheles culicifacies (10.1%). The Human Blood Index (HBI) in the case of An. culicifacies and An. stephensi was 6.66 and 9.09, respectively. Vector incrimination results revealed Plasmodium vivax positivity rate of 1.6% for An. culicifacies. Both the vector species were found resistant to DDT, malathion and deltamethrin. CONCLUSION: The emergence of insecticide resistance in both vector species, compromises the effectiveness of commonly used public health insecticides. Consequently, the implementation of robust insecticide resistance management strategies becomes imperative. To effectively tackle the malaria transmission, a significant shift in vector control strategies is warranted, with careful consideration and adaptation to address specific challenges encountered in malaria elimination efforts.

A Framework for Unsupervised Profiling of Malaria Vectors' Insecticide Resistance Using Machine Learning Technique.

Background: There is a need to identify different insecticide resistance profiles that represent circumscription-encapsulation of knowledge about malaria vectors' insecticide resistance to increase our understanding of malaria vectors' insecticide resistance dynamics. Methods: Data used in this study are part of the aggregation of over 20,000 mosquito collections done between 1957 and 2018. We applied two data preprocessing steps. We developed three clustering machine learning models based on the K-means algorithm with three selected datasets. The elbow method was used to fine-tune the hyperparameters. We used the silhouette score to assess the clustering results produced by each of the three models. The proposed framework incorporates continuous learning, allowing the machine learning models to learn continuously. Results: For the first model, the optimal number of clusters (profiles) k was 17. For the second model, we found four profiles. For the third model, the optimal number of profiles was 7. Discussion: We found that the insecticide resistance profiles have dynamic resistance levels with respect to the insecticide component, species component, location component, and time component. This profiling task provided knowledge about the evolution of malaria vectors' insecticide resistance in the African continent by encapsulating the information on the complex interaction between the different dimensions of malaria vectors' insecticide resistance into different profiles. Policy makers can use the knowledge about the different profiles found from the analysis of available insecticide resistance monitoring data (through profiling) by using our proposed approach to set up malaria vector control strategies that consider the locations, species present in those locations, and potentially efficient insecticides.

Copy number variants underlie the major selective sweeps in insecticide resistance genes in Anopheles arabiensis from Tanzania.

To keep ahead of the evolution of resistance to insecticides in mosquitoes, national malaria control programmes must make use of a range of insecticides, both old and new, while monitoring resistance mechanisms. Knowledge of the mechanisms of resistance remains limited in Anopheles arabiensis, which in many parts of Africa is of increasing importance because it is apparently less susceptible to many indoor control interventions. Furthermore, comparatively little is known in general about resistance to non-pyrethroid insecticides such as pirimiphos-methyl (PM), which are crucial for effective control in the context of resistance to pyrethroids. We performed a genome-wide association study to determine the molecular mechanisms of resistance to deltamethrin (commonly used in bednets) and PM, in An. arabiensis from two regions in Tanzania. Genomic regions of positive selection in these populations were largely driven by copy number variants (CNVs) in gene families involved in resistance to these two insecticides. We found evidence of a new gene cluster involved in resistance to PM, identifying a strong selective sweep tied to a CNV in the Coeae2g-Coeae6g cluster of carboxylesterase genes. Using complementary data from An. coluzzii in Ghana, we show that copy number at this locus is significantly associated with PM resistance. Similarly, for deltamethrin, resistance was strongly associated with a novel CNV allele in the Cyp6aa / Cyp6p cluster. Against this background of metabolic resistance, target site resistance was very rare or absent for both insecticides. Mutations in the pyrethroid target site Vgsc were at very low frequency in Tanzania, yet combining these samples with three An. arabiensis individuals from West Africa revealed a startling diversity of evolutionary origins of target site resistance, with up to 5 independent origins of Vgsc-995 mutations found within just 8 haplotypes. Thus, despite having been first recorded over 10 years ago, Vgsc resistance mutations in Tanzanian An. arabiensis have remained at stable low frequencies. Overall, our results provide a new copy number marker for monitoring resistance to PM in malaria mosquitoes, and reveal the complex picture of resistance patterns in An. arabiensis.

A CASE REPORT ON PRODUCT ROTATION TO MANAGE SEVERE LYSINIBACILLUS SPHAERICUS RESISTANCE IN CULEX PIPIENS FROM SALT LAKE CITY, UTAH.

The Salt Lake City Mosquito Abatement District (SLCMAD) detected a 20,000-fold resistance to Lysinibacillus sphaericus (Lsph) in Culex pipiens occurring in catch basins of Salt Lake City during 2016. In response, SLCMAD suspended use of Lsph and rotated use of spinosyn and s-methoprene products for the next three years. At the end of the third year, Lsph was evaluated again and efficacy similar to susceptible colony strains. During the second year of Lsph use, technicians observed lack of control of larvae at some urban sites. Bioassays performed during 2021 showed recurrence of some resistance to Lsph to varying degrees across SLCMAD urban areas. The rapidity with which resistant phenotypes reemerged clarifies that SLCMAD cannot in the near future rely on repeated use of Lsph, even after suspending use for three years and using within-season product rotations. Prior reports in other research groups have found long-term selection to Lsph, as is the case at SLCMAD, to not regress in spite of halting use of the products. However, our findings offer some optimism that regression may be relatively quick. More operational review is needed, and future work should characterize resistance alleles in field populations. Collectively, there is a lack of concrete data supporting the prevailing assumptions from adjacent industries that were adopted into mosquito abatement. We provide this short note as additional guidance for mosquito and vector control districts weighing options to remediate Lsph resistance.

Strong resistance to ß-cyfluthrin in a strain of the beetle Alphitobius diaperinus: a de novo transcriptome analysis.

The lesser mealworm, Alphitobius diaperinus, is an invasive tenebrionid beetle and a vector of pathogens. Due to the emergence of insecticide resistance and consequent outbreaks that generate significant phytosanitary and energy costs for poultry farmers, it has become a major insect pest worldwide. To better understand the molecular mechanisms behind this resistance, we studied a strain of A. diaperinus from a poultry house in Brittany that was found to be highly resistant to the ß-cyfluthrin. The strain survived ß-cyfluthrin exposures corresponding to more than 100 times the recommended dose. We used a comparative de novo RNA-Seq approach to explore genes expression in resistant versus sensitive strains. Our de novo transcriptomic analyses showed that responses to ß-cyfluthrin likely involved a whole set of resistance mechanisms. Genes related to detoxification, metabolic resistance, cuticular hydrocarbon biosynthesis and proteolysis were found to be constitutively overexpressed in the resistant compared to the sensitive strain. Follow-up enzymatic assays confirmed that the resistant strain exhibited high basal activities for detoxification enzymes such as cytochrome P450 monooxygenase and glutathione-S-transferase. The in-depth analysis of differentially expressed genes suggests the involvement of complex regulation of signaling pathways. Detailed knowledge of these resistance mechanisms is essential for the establishment of effective pest control.

Measuring local genetic variation in permethrin-resistant head lice (Phthiraptera: Pediculidae) from Buenos Aires, Argentina.

The cosmopolitan ectoparasite human head louse, Pediculus humanus capitis (De Geer)(Phthiraptera:Pediculidae), affects mostly school-aged children, with infestations reported every year mainly due to louse resistance to pyrethroids. One of the main resistance mechanisms of pyrethroids is the target site insensitivity (kdr), which is caused by single-nucleotide point mutations (SNPs) located in the voltage-sensitive sodium channel gene. In this study, we analyzed individual head lice toxicologically via the description of their susceptibility profile to permethrin and genetically through the genotypification of their kdr alleles as well as nuclear microsatellite loci. Lice were collected from 4 schools in the city of Buenos Aires, Argentina. The resistance ratios varied from 33.3% to 71.4%, with a frequency of the T917I kdr mutation of 87.31% and with 83.6% of the head lice being homozygous resistant to pyrethroids. Microsatellite data indicated that all the louse school populations had genotype proportions that deviated from Hardy-Weinberg expectations, with FIS > 0 reflecting a deficit of heterozygotes. Bottleneck analysis suggested that all louse school populations underwent a recent reduction in population sizes, while 3 of the 4 schools had gene flow values around 1, indicating ongoing gene flow among those schools. Our study suggests that school louse populations in the city of Buenos Aires may form a metapopulation, where each school represents a small population that undergoes extinction and recolonization processes under strong permethrin selection. This is the first multilevel analysis integrating toxicological, kdr-genotyping, and microsatellite data in human louse populations.

Toxicity of isocycloseram, an isoxazoline insecticide, against laboratory and field-collected German cockroaches (Blattodea: Ectobiidae).

Isocycloseram is a new insecticide in the isoxazoline class that targets insect GABA-gated chloride channels. In this study, we evaluated a cockroach gel bait formulation containing 1% isocycloseram against a susceptible strain (UCR) and 5 field-collected strains (WM, RG386, Ryan, CDR, and SY) of the German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae), and compared it with several commercial insecticide baits in the laboratory. Using the Ebeling choice box method, we also tested a residual deposit of an SC formulation of isocycloseram against the UCR, RG386, and Ryan strains. The isocycloseram bait was among the fastest-performing treatments against adult males (mean survival time: 0.9-2.7 days) and mixed stages and sexes (mean survival time: 1.4-5.4 days) across all strains. Secondary transfer effects of the bait were demonstrated in the UCR strain by exposing new adult males to individuals killed by direct bait treatment. Physiological resistance was not detected in the WM, CDR, and RG386 strains with topical treatment of a diagnostic dose (3× LD95) of isocycloseram developed using the UCR strain. However, topical assays revealed resistance ratios (RR50) of 1.6 and 3.0× in the Ryan and SY strains, respectively. The performance of a 0.05% isocycloseram residual application against the Ryan strain was improved with the addition of piperonyl butoxide.

Transcriptomic analysis of Anopheles gambiae from Benin reveals overexpression of salivary and cuticular proteins associated with cross-resistance to pyrethroids and organophosphates.

BACKGROUND: Insecticide resistance (IR) is one of the major threats to malaria vector control programs in endemic countries. However, the mechanisms underlying IR are poorly understood. Thus, investigating gene expression patterns related to IR can offer important insights into the molecular basis of IR in mosquitoes. In this study, RNA-Seq was used to characterize gene expression in Anopheles gambiae surviving exposure to pyrethroids (deltamethrin, alphacypermethrin) and an organophosphate (pirimiphos-methyl). RESULTS: Larvae of An. gambiae s.s. collected from Bassila and Djougou in Benin were reared to adulthood and phenotyped for IR using a modified CDC intensity bottle bioassay. The results showed that mosquitoes from Djougou were more resistant to pyrethroids (5X deltamethrin: 51.7% mortality; 2X alphacypermethrin: 47.4%) than Bassila (1X deltamethrin: 70.7%; 1X alphacypermethrin: 77.7%), while the latter were more resistant to pirimiphos-methyl (1.5X: 48.3% in Bassila and 1X: 21.5% in Djougou). RNA-seq was then conducted on resistant mosquitoes, non-exposed mosquitoes from the same locations and the laboratory-susceptible An. gambiae s.s. Kisumu strain. The results showed overexpression of detoxification genes, including cytochrome P450s (CYP12F2, CYP12F3, CYP4H15, CYP4H17, CYP6Z3, CYP9K1, CYP4G16, and CYP4D17), carboxylesterase genes (COEJHE5E, COE22933) and glutathione S-transferases (GSTE2 and GSTMS3) in all three resistant mosquito groups analyzed. Genes encoding cuticular proteins (CPR130, CPR10, CPR15, CPR16, CPR127, CPAP3-C, CPAP3-B, and CPR76) were also overexpressed in all the resistant groups, indicating their potential role in cross resistance in An. gambiae. Salivary gland protein genes related to 'salivary cysteine-rich peptide' and 'salivary secreted mucin 3' were also over-expressed and shared across all resistant groups. CONCLUSION: Our results suggest that in addition to metabolic enzymes, cuticular and salivary gland proteins could play an important role in cross-resistance to multiple classes of insecticides in Benin. These genes warrant further investigation to validate their functional role in An. gambiae resistance to insecticides.

Nicotinamide deficiency promotes imidacloprid resistance via activation of ROS/CncC signaling pathway-mediated UGT detoxification in Nilaparvata lugens.

Metabolic alternation is a typical characteristic of insecticide resistance in insects. However, mechanisms underlying metabolic alternation and how altered metabolism in turn affects insecticide resistance are largely unknown. Here, we report that nicotinamide levels are decreased in the imidacloprid-resistant strain of Nilaparvata lugens, may due to reduced abundance of the symbiotic bacteria Arsenophonus. Importantly, the low levels of nicotinamide promote imidacloprid resistance via metabolic detoxification alternation, including elevations in UDP-glycosyltransferase enzymatic activity and enhancements in UGT386B2-mediated metabolism capability. Mechanistically, nicotinamide suppresses transcriptional regulatory activities of cap 'n' collar isoform C (CncC) and its partner small muscle aponeurosis fibromatosis isoform K (MafK) by scavenging the reactive oxygen species (ROS) and blocking the DNA binding domain of MafK. In imidacloprid-resistant N. lugens, nicotinamide deficiency re-activates the ROS/CncC signaling pathway to provoke UGT386B2 overexpression, thereby promoting imidacloprid detoxification. Thus, nicotinamide metabolism represents a promising target to counteract imidacloprid resistance in N. lugens.

Is Anopheles gambiae (sensu stricto), the principal malaria vector in Africa prone to resistance development against new insecticides? Outcomes from laboratory exposure of An. gambiae (s.s.) to sub-lethal concentrations of chlorfenapyr and clothianidin.

Indiscriminate use of pesticides in the public health and agriculture sectors has contributed to the development of resistance in malaria vectors following exposure to sub-lethal concentrations. To preserve the efficacy of vector control tools and prevent resistance from spreading, early resistance detection is urgently needed to inform management strategies. The introduction of new insecticides for controlling malaria vectors such as clothianidin and chlorfenapyr requires research to identify early markers of resistance which could be used in routine surveillance. This study investigated phenotypic resistance of Anopheles gambiae (sensu stricto) Muleba-Kis strain using both WHO bottle and tube assays following chlorfenapyr, clothianidin, and alpha-cypermethrin selection against larvae and adults under laboratory conditions. High mortality rates were recorded for both chlorfenapyr-selected mosquitoes that were consistently maintained for 10 generations (24-h mortality of 92-100% and 72-h mortality of 98-100% for selected larvae; and 24-h mortality of 95-100% and 72-h mortality of 98-100% for selected adults). Selection with clothianidin at larval and adult stages showed a wide range of mortality (18-91%) compared to unselected progeny where mortality was approximately 99%. On the contrary, mosquitoes selected with alpha-cypermethrin from the adult selection maintained low mortality (28% at Generation 2 and 23% at Generation 4) against discrimination concentration compared to unselected progeny where average mortality was 51%. The observed resistance in the clothianidin-selected mosquitoes needs further investigation to determine the underlying resistance mechanism against this insecticide class. Additionally, further investigation is recommended to develop molecular markers for observed clothianidin phenotypic resistance.

Identification of putative promoter elements for epsilon glutathione s-transferases genes associated with resistance to DDT in the malaria vector mosquito anopheles arabiensis.

The purpose of this study was to identify the putative regulatory elements in the promoter region of An. arabiensis strains which differed in susceptibility to DDT and compare with those identified in its sibling An. gambaie. Basal expression level of Epsilon class GSTs (Glutathione S - transferases) GSTe1 gene was 0.512 - 0.658 (95% CI) and GSTe2 0.672 - 1.204 (95% CI) in adults of DDT resistant KGB compared to 0.031 - 0.04 (95% CI) and 0.148 - 0.199 (95% CI) respectively in susceptible MAT strains of An. arabiensis. Induced mean expression of GSTe2 in larvae exposed to DDT for one hour was 0.901 - 1.172 (95% CI) in KGB and 0.475 - 0.724 (95% CI) in MAT strain. In present work, strain specific primers were used to amplify and sequenced the promoter regions of GSTe1 and GSTe2 in the KGB, MAT and field specimens. Computational analysis revealed presence of classical arthropod initiator sequence TCAGT and putative core promoter elements, GC, CAAT, TATA boxes. A typical TATA box was identified at 35 bp upstream Transcription Start Site (TSS) in GSTe1 but was absent in GSTe2. Several binding sites for regulatory elements downstream and multiple polymorphic sites were identified between strains. The role of these regulatory elements in transcription of these genes has not been determined. However, on comparison the 2 bp adenosine indel (insertion/deletion) which was essential in driving the promoter activity in An. gambiae was identified only DDT resistant KGB strain.

Three chemosensory proteins enriched in antennae and tarsi of Rhaphuma horsfieldi differentially contribute to the binding of insecticides.

Antennae and legs (primarily the tarsal segments) of insects are the foremost sensory organs that contact a diverse range of toxic chemicals including insecticides. Binding proteins expressed in the two tissues are potential molecular candidates serving as the binding and sequestering of insecticides, like chemosensory proteins (CSPs). Insect CSPs endowed with multiple roles have been suggested to participate in insecticide resistance, focusing mainly on moths, aphids and mosquitos. Yet, the molecular underpinnings underlying the interactions of cerambycid CSPs and insecticides remain unexplored. Here, we present binding properties of three antenna- and tarsus-enriched RhorCSPs (RhorCSP1, CSP2 and CSP3) in Rhaphuma horsfieldi to eight insecticide classes totaling 15 chemicals. From the transcriptome of this beetle, totally 16 CSP-coding genes were found, with seven full-length sequences. In phylogeny, these RhorCSPs were distributed dispersedly in different clades. Expression profiles revealed the abundant expression of RhorCSP1, CSP2 and CSP3 in antennae and tarsi, thus as representatives for studying the protein-insecticide interactions. Binding assays showed that the three RhorCSPs were tuned differentially to insecticides but exhibited the highest affinities with hexaflumuron, chlorpyrifos and rotenone (dissociation constants <13 µM). In particular, RhorCSP3 could interact strongly with 10 of tested insecticides, of which four residues (Tyr25, Phe42, Val65 and Phe68) contributed significantly to the binding of six, four, three and four ligands, respectively. Of these, the binding of four mutated RhorCSP3s to a botanical insecticide rotenone was significantly weakened compared to the wildtype protein. Furthermore, we also evidenced that RhorCSP3 was a broadly-tuned carrier protein in response to a wide variety of plant odorants outside insecticides. Altogether, our findings shed light on different binding mechanisms and odorant-tuning profiles of three RhorCSPs in R. horsfieldi and identify key residues of the RhorCSP3-insecticide interactions.

Behavioral responses and life history traits of Taiwanese and Indonesian populations of Aedes aegypti surviving deltamethrin-clothianidin treatment.

BACKGROUND: Indoor residual spraying (IRS) capitalizes on the natural behavior of mosquitoes because Aedes aegypti commonly seeks indoor resting sites after a blood meal. This behavior allows mosquitoes to be exposed to insecticide-treated surfaces and subsequently killed. Combinations of deltamethrin and clothianidin with different modes of action have shown promise in IRS, effectively targeting both susceptible and pyrethroid-resistant malaria vectors. However, the effects of this approach on Aedes mosquitoes remain unclear. The present study tested the effects of deltamethrin-clothianidin mixture treatment on behavioral responses and life history traits of Taiwanese and Indonesian populations of Ae. aegypti. METHODS: We adopted an excito-repellent approach to explore the behavioral responses of pyrethroid-resistant Ae. aegypti populations from Indonesia and Taiwan to a deltamethrin-clothianidin mixture used in contact irritancy and non-contact repellency treatments. We further evaluated the life history traits of surviving mosquitoes (i.e., delayed mortality after 7-day post-treatment, longevity, fecundity, and egg hatching) and investigated the potential transgenerational hormetic effects of insecticide exposure (i.e., development rate and survival of immatures and adult mosquitos). RESULTS: All tested field populations of Ae. aegypti displayed strong contact irritancy responses; the percentage of escape upon insecticide exposure ranged from 38.8% to 84.7%. However, repellent effects were limited, with the escape percentage ranging from 4.3% to 48.9%. We did not observe immediate knockdown or mortality after 24 h, and less than 15% of the mosquitoes exhibited delayed mortality after a 7-day exposure period. However, the carryover effects of insecticide exposure on the survival of immature mosquitoes resulted in approximately 25% higher immature mortality than that in the control. By contrast, we further documented stimulated survivor reproduction and accelerated transgenerational immature development resulting from the sublethal effects of the insecticide mixture. In particular, the number of eggs laid by treated (both treatments) female mosquitoes increased by at least 60% compared with that of eggs laid by control female mosquitoes. CONCLUSIONS: IRS with deltamethrin-clothianidin effectively deters Aedes mosquitoes from entering residential areas and thereby reduces mosquito bites. However, the application rate (deltamethrin: 25 mg/m2; clothianidin: 200 mg/m2) may be insufficient to effectively kill Aedes mosquitoes. Insecticide response appears to vary across mosquito species; their behavioral and physiological responses to sublethal doses have crucial implications for mosquito control programs.

Non-Coding RNAs Potentially Involved in Pyrethroid Resistance of Anopheles funestus Population in Western Kenya.

Backgrounds: The resurgence of Anopheles funestus, a dominant vector of human malaria in western Kenya was partly attributed to insecticide resistance. However, evidence on the molecular basis of pyrethroid resistance in western Kenya is limited. Noncoding RNAs (ncRNAs) form a vast class of RNAs that do not code for proteins and are ubiquitous in the insect genome. Here, we demonstrated that multiple ncRNAs could play a potential role in An. funestusresistance to pyrethroid in western Kenya. Materials and Methods: Anopheles funestus mosquitoes were sampled by aspiration methods in Bungoma, Teso, Siaya, Port Victoria and Kombewa in western Kenya. The F1 progenies were exposed to deltamethrin (0.05%), permethrin (0.75%), DDT (4%) and pirimiphos-methyl (0.25%) following WHO test guidelines. A synergist assay using piperonyl butoxide (PBO) (4%) was conducted to determine cytochrome P450s' role in pyrethroid resistance. RNA-seq was conducted on a combined pool of specimens that were resistant and unexposed, and the results were compared with those of the FANG susceptible strain. This approach aimed to uncover the molecular mechanisms underlying pyrethroid resistance. Results: Pyrethroid resistance was observed in all the sites with an average mortality rate of 57.6%. Port Victoria had the highest level of resistance to permethrin (MR=53%) and deltamethrin (MR=11%) pyrethroids. Teso had the lowest level of resistance to permethrin (MR=70%) and deltamethrin (MR=87%). Resistance to DDT was observed only in Kombewa (MR=89%) and Port Victoria (MR=85%). A full susceptibility to P-methyl (0.25%) was observed in all the sites. PBO synergist assay revealed high susceptibility (>98%) to the pyrethroids in all the sites except for Port Victoria (MR=96%, n=100). Whole transcriptomic analysis showed that most of the gene families associated with pyrethroid resistance comprised non-coding RNAs (67%), followed by imipenemase (10%), cytochrome P450s (6%), cuticular proteins (5%), olfactory proteins (4%), glutathione S-transferases (3%), UDP-glycosyltransferases (2%), ATP-binding cassettes (2%) and carboxylesterases(1%). Conclusions: This study unveils the molecular basis of insecticide resistance in An. funestus in western Kenya, highlighting for the first time the potential role of non-coding RNAs in pyrethroid resistance. Targeting non-coding RNAs for intervention development could help in insecticide resistance management.

Effects of deltamethrin exposure on the cytochrome P450 monooxygenases of Aedes albopictus (Skuse) larvae from a dengue-endemic region of northern part of West Bengal, India.

Aedes albopictus is highly prevalent in the northern part of West Bengal and is considered to be responsible for the recent dengue outbreaks in this region. Control of this vector is largely relied on the use of synthetic pyrethroids, which can lead to the development of resistance. In the present study, larvae of three wild Ae. albopictus populations from the dengue-endemic regions were screened for deltamethrin resistance, and the role of cytochrome P450 monooxygenases (CYPs) was investigated in deltamethrin exposed and unexposed larvae. Two populations were incipient resistant, and one population was completely resistant against deltamethrin. Monooxygenase titration assay revealed the involvement of CYPs in deltamethrin resistance along with an induction effect of deltamethrin exposure. Gene expression studies revealed differential expression of five CYP6 family genes, CYP6A8, CYP6P12, CYP6A14, CYP6N3 and CYP6N6, with high constitutive expression of CYP6A8 and CYP6P12 in all the populations before and after deltamethrin exposure. From these findings, it was evident that CYPs play an important role in the development of deltamethrin resistance in the Ae. albopictus populations in this region.

Heat shock proteins, thermotolerance, and insecticide resistance in mosquitoes.

Mosquitoes transmit pathogens that pose a threat to millions of people globally. Unfortunately, widespread insecticide resistance makes it difficult to control these public health pests. General mechanisms of resistance, such as target site mutations or increased metabolic activity, are well established. However, many questions regarding the dynamics of these adaptations in the context of developmental and environmental conditions require additional exploration. One aspect of resistance that deserves further study is the role of heat shock proteins (HSPs) in insecticide tolerance. Studies show that mosquitoes experiencing heat stress before insecticide exposure demonstrate decreased mortality. This is similar to the observed reciprocal reduction in mortality in mosquitoes exposed to insecticide prior to heat stress. The environmental shifts associated with climate change will result in mosquitoes occupying environments with higher ambient temperatures, which could enhance existing insecticide resistance phenotypes. This physiological relationship adds a new dimension to the problem of insecticide resistance and further complicates the challenges that vector control and public health personnel face. This article reviews studies illustrating the relationship between insecticide resistance and HSPs or hsp genes as well as the intersection of thermotolerance and insecticide resistance. Further study of HSPs and insecticide resistance could lead to a deeper understanding of how environmental factors modulate the physiology of these important disease vectors to prepare for changing climatic conditions and the development of novel strategies to prevent vector-borne disease transmission.

Using RNA interference targeting a nicotinic acetylcholine receptor subunit to counteract insecticide accommodation mechanisms: example of the ß1 subunit in the imidacloprid-accommodated American cockroach, Periplaneta americana.

Insecticide accommodation and resistance are limiting factors to the much-needed increase in agricultural production. Various physiological and cellular modifications, such as the changes of insecticide molecular targets, have been linked to these events. Thus, a previous study demonstrated that the imidacloprid accommodation set up by the cockroach Periplaneta americana after an exposure to a sublethal dose of this insecticide involves functional alterations of two nicotinic acetylcholine receptor (nAChR) subtypes. As RNA interference (RNAi) is one of the most promising strategies for controlling pest insects, we evaluated, in this study, the use of RNAi that targets the ß1 nAChR subunit to counteract the imidacloprid accommodation phenomenon in cockroaches. Interestingly, we showed that ingestion of dsRNA-ß1 increased the sensitivity to imidacloprid of accommodated cockroaches. Thus, we have demonstrated for the first time that RNAi that targets an nAChR subunit can counteract the accommodation mechanism to insecticide targeting nAChRs set up by an insect.