Time of Day-Specific Changes in Metabolic Detoxification and Insecticide Tolerance in the House Fly, Musca domestica L.

Both insects and mammals all exhibit a daily fluctuation of susceptibility to chemicals at different times of the day. However, this phenomenon has not been further studied in the house fly (Musca domestica L.) and a better understanding of the house fly on chronobiology should be useful for controlling this widespread disease vector. Here we explored diel time-of-day variations in insecticide susceptibility, enzyme activities, and xenobiotic-metabolizing enzyme gene expressions. The house fly was most tolerant to beta-cypermethrin in the late photophase at Zeitgeber time (ZT) 8 and 12 [i.e., 8 and 12 h after light is present in the light-dark cycle (LD)]. The activities of cytochrome P450, GST, and CarE enzymes were determined in the house flies collected at various time, indicating that rhythms occur in P450 and CarE activities. Subsequently, we observed diel rhythmic expression levels of detoxifying genes, and CYP6D1 and MdαE7 displayed similar expression patterns with enzyme activities in LD conditions, respectively. No diel rhythm was observed for CYP6D3 expression. These data demonstrated a diel rhythm of metabolic detoxification enzymes and insecticide susceptibility in M. domestica. In the future, the time-of-day insecticide efficacy could be considered into the management of the house fly.

Interactions Between Ephedra sinica and Prunus armeniaca: From Stereoselectivity to Deamination as a Metabolic Detoxification Mechanism of Amygdalin

Mahuang–Xingren (MX, Ephedra sinica Stapf-Prunus armeniaca L.) is a classic herb pair used in traditional Chinese medicine. This combined preparation reduces the toxicity of Xingren through the stereoselective metabolism of its main active ingredient amygdalin. However, whether stereoselectivity is important in the pharmacokinetic properties of amygdalin either in the traditional decoction or in the dispensing granules is unclear. Amygdalin is hydrolyzed to its metabolite, prunasin, which produces hydrogen cyanide by degradation of the cyano group. A comprehensive study of the metabolic pathway of amygdalin is essential to better understand the detoxification process. In this article, the potential detoxification pathway of MX is further discussed with regard to herb interactions. In this study, the pharmacokinetic parameters and metabolism of amygdalin and prunasin were investigated by comparing the traditional decoction and the dispensing granule preparations. In addition, several potential metabolites were characterized in an incubation system with rat liver microsomes or gut microbial enzymes. The combination of Xingren with Mahuang reduces exposure to D-amygdalin in vivo and contributes to its detoxification, a process that can be further facilitated in the traditional decoction. From the in vitro co-incubation model, 15 metabolites were identified and classified into cyanogenesis and non-cyanogenesis metabolic pathways, and of these, 10 metabolites were described for the first time. The level of detoxified metabolites in the MX traditional decoction was higher than that in the dispensing granules. The metabolism of amygdalin by the gut microbial enzymes occurred more rapidly than that by the rat liver microsomes. These results indicated that combined boiling both herbs during the preparation of the traditional decoction may induce several chemical changes that will influence drug metabolism in vivo. The gut microbiota may play a critical role in amygdalin metabolism. In conclusion, detoxification of MX may result 1) during the preparation of the decoction, in the boiling phase, and 2) from the metabolic pathways activated in vivo. Stereoselective pharmacokinetics and deamination metabolism have been proposed as the detoxification pathway underlying the compatibility of MX. Metabolic detoxification of amygdalin was quite different between the two combinations, which indicates that the MX decoctions should not be completely replaced by their dispensing granules.

Expansive and Diverse Phenotypic Landscape of Field Aedes aegypti (Diptera: Culicidae) Larvae with Differential Susceptibility to Temephos: Beyond Metabolic Detoxification

Arboviruses including dengue, Zika, and chikungunya are amongst the most significant public health concerns worldwide. Arbovirus control relies on the use of insecticides to control the vector mosquito Aedes aegypti (Linnaeus), the success of which is threatened by widespread insecticide resistance. The work presented here profiled the gene expression of Ae. aegypti larvae from field populations of Ae. aegypti with differential susceptibility to temephos originating from two Colombian urban locations, Bello and Cúcuta, previously reported to have distinctive disease incidence, socioeconomics, and climate. We demonstrated that an exclusive field-to-lab (Ae. aegypti strain New Orleans) comparison generates an over estimation of differential gene expression (DGE) and that the inclusion of a geographically relevant field control yields a more discrete, and likely, more specific set of genes. The composition of the obtained DGE profiles is varied, with commonly reported resistance associated genes including detoxifying enzymes having only a small representation. We identify cuticle biosynthesis, ion exchange homeostasis, an extensive number of long noncoding RNAs, and chromatin modelling among the differentially expressed genes in field resistant Ae. aegypti larvae. It was also shown that temephos resistant larvae undertake further gene expression responses when temporarily exposed to temephos. The results from the sampling triangulation approach here contribute a discrete DGE profiling with reduced noise that permitted the observation of a greater gene diversity, increasing the number of potential targets for the control of insecticide resistant mosquitoes and widening our knowledge base on the complex phenotypic network of the Ae. aegypti response to insecticides.

Ecdysteroid kinase-like (EcKL) paralogs confer developmental tolerance to caffeine in Drosophila melanogaster

A unique aspect of metabolic detoxification in insects compared to other animals is the presence of xenobiotic phosphorylation, about which little is currently understood. Our previous work raised the hypothesis that members of the taxonomically restricted ecdysteroid kinase-like (EcKL) gene family encode the enzymes responsible for xenobiotic phosphorylation in the model insect Drosophila melanogaster (Diptera: Ephydroidea)—however, candidate detoxification genes identified in the EcKL family have yet to be functionally validated. Here, we test the hypothesis that EcKL genes in the rapidly evolving Dro5 clade are involved in the detoxification of plant and fungal toxins in D. melanogaster. The mining and reanalysis of existing data indicated multiple Dro5 genes are transcriptionally induced by the plant alkaloid caffeine and that adult caffeine susceptibility is associated with a novel naturally occurring indel in CG31370 (Dro5-8) in the Drosophila Genetic Reference Panel (DGRP). CRISPR-Cas9 mutagenesis of five Dro5 EcKLs substantially decreased developmental tolerance of caffeine, while individual overexpression of two of these genes—CG31300 (Dro5-1) and CG13659 (Dro5-7)—in detoxification-related tissues increased developmental tolerance. In addition, we found Dro5 loss-of-function animals also have decreased developmental tolerance of the fungal secondary metabolite kojic acid. Taken together, this work provides the first compelling functional evidence that EcKLs encode detoxification enzymes and suggests that EcKLs in the Dro5 clade are involved in the metabolism of multiple ecologically relevant toxins in D. melanogaster. We also propose a biochemical hypothesis for EcKL involvement in caffeine detoxification and highlight the many unknown aspects of caffeine metabolism in D. melanogaster and other insects.

An Overview of Meta-Analysis for the Importance of the Overexpression of Detoxification Genes in Insecticide Resistance

Insect-borne diseases, such as malaria, and plant pathogens, like the tobacco mosaic virus, are responsible for human deaths and poor crop yields in communities around the world. The use of insecticides has been one of the major tools in the insect pest control. However, the development of insecticide resistance has been a major problem in the control of insect pest populations that threaten the health of both humans and plants. The overexpression of detoxification genes is thought to be one of the major mechanisms through which pests develop resistance to insecticides. Hundreds of research papers have explored how overexpressed detoxification genes increase the resistance status of insects to an insecticide in recent years; this study is, for the first, a synthesis of these research data aimed at (1) examining the overexpression of metabolic detoxification genes and their involvement in increased levels of resistance to different insecticides in diverse insect species, including agriculturally and medically important insect species. While (2) seeking to determine if the overexpression detoxification genes are most responsible for insecticide resistance in insect pests in general. The strongly correlated with increased levels of insecticide resistance were observed in tested insects with the cytochrome P450 (CYP), glutathione-S-transferase (GST), and esterase gene superfamilies confirming that the overexpression of detoxification genes is indeed involved in insecticide metabolism of insects, including medical (e.g., mosquito and housefly) and agricultural (e.g., planthopper and caterpillar) insect pests.

Metabolic Pathways for S-Metolachlor Detoxification Differ Between Tolerant Corn and Multiple-Resistant Waterhemp

Herbicide resistance in weeds can be conferred by target-site and/or non-target-site mechanisms, such as rapid metabolic detoxification. Resistance to the very-long-chain fatty acid (VLCFA)-inhibiting herbicide, S-metolachlor, in multiple-herbicide resistant populations (CHR and SIR) of waterhemp (Amaranthus tuberculatus) is conferred by rapid metabolism compared with sensitive populations. However, enzymatic pathways for S-metolachlor metabolism in waterhemp are unknown. Enzyme assays using S-metolachlor were developed to determine specific activities of glutathione S-transferases (GSTs) and cytochrome P450 monooxygenases (P450s) from CHR and SIR seedlings to compare with tolerant corn and sensitive waterhemp (WUS). GST activities were greater (~2-fold) in CHR and SIR compared to WUS, but much less than corn. In contrast, P450s in microsomal extracts from CHR and SIR formed O-demethylated S-metolachlor, and their NADPH-dependent specific activities were greater (>20-fold) than corn or WUS. Metabolite profiles of S-metolachlor generated via untargeted and targeted liquid chromatography-mass spectrometry from CHR and SIR differed from WUS, with greater relative abundances of O-demethylated S-metolachlor and O-demethylated S-metolachlor-glutathione conjugates formed by CHR and SIR. In summary, our results demonstrate S-metolachlor metabolism in resistant waterhemp involves Phase I and Phase II metabolic activities acting in concert, but the initial O-demethylation reaction confers resistance.

Toxin breakdown does not preclude the potential for defensive toxin use in a fruit fly

Animals that ingest toxins can themselves become toxic or unpalatable to predators and parasites. However, most animals rapidly eliminate toxins to survive toxin ingestion. It is therefore unclear how species transition from susceptibility and toxin elimination to tolerance and accumulation as chemical defense emerges. Studies of chemical defense have generally focused on species that display active toxin sequestration and target-site insensitivity mutations that permit survival without necessitating toxin metabolism. Here we investigate whether animals that presumably rely on toxin elimination for survival can also utilize ingested toxins for defense. We use the A4 and A3 Drosophila melanogaster fly strains from the Drosophila Synthetic Population Resource (DSPR), which respectively possess elevated and reduced metabolic toxin resistance. We find that ingesting nicotine increased the survival of A4 but not of A3 flies against Leptopilina heterotoma wasp parasitism. Further, we find that despite possessing enhanced toxin clearance mechanisms, A4 flies accrued more nicotine than A3 individuals. Our results suggest that enhanced metabolic detoxification can allow for greater toxin intake by offsetting the cost of toxin ingestion. Passive toxin accumulation that accompanies increased toxin intake may underlie the early origins of chemical defense.

Multiplex Real-Time PCR for genotyping Glutathione S-Transferase deletion polymorphisms v1

Glutathione S-transferases (GSTs) are enzymes that act in the conjugation of reactive metabolites to the reduced glutathione. The GSTM1 and GSTT1 deletion polymorphisms promote a detriment in the metabolic detoxification process of xenobiotics, due to present a non-functional null allele, causing oxidative stress, may be related to the pathophysiology of various diseases. Genetic association case-control studies usually determine whether a statistical association exists between the disease trait and the genetic marker, such as GST deletion polymorphism. Therefore, techniques to genotyping these polymorphisms through multiplex PCR are highly desired and of wider interest. This method allows for the detection of genotypes for genetic association studies in molecular pathology and it is more cost-effective than hybridization probes.

Dengue fever and insecticide resistance in Aedes mosquitoes in Southeast Asia: a review

Dengue fever is the most important mosquito-borne viral disease in Southeast Asia. Insecticides remain the most effective vector control approach for Aedes mosquitoes. Four main classes of insecticides are widely used for mosquito control: organochlorines, organophosphates, pyrethroids and carbamates. Here, we review the distribution of dengue fever from 2000 to 2020 and its associated mortality in Southeast Asian countries, and we gather evidence on the trend of insecticide resistance and its distribution in these countries since 2000, summarising the mechanisms involved. The prevalence of resistance to these insecticides is increasing in Southeast Asia, and the mechanisms of resistance are reported to be associated with target site mutations, metabolic detoxification, reduced penetration of insecticides via the mosquito cuticle and behavioural changes of mosquitoes. Continuous monitoring of the status of resistance and searching for alternative control measures will be critical for minimising any unpredicted outbreaks and improving public health. This review also provides improved insights into the specific use of insecticides for effective control of mosquitoes in these dengue endemic countries.