Effects of circadian clock disruption on gene expression and biological processes in Aedes aegypti.

BACKGROUND: This study explores the impact of disrupting the circadian clock through a Cycle gene knockout (KO) on the transcriptome of Aedes aegypti mosquitoes. The investigation aims to uncover the resulting alterations in gene expression patterns and physiological processes. RESULTS: Transcriptome analysis was conducted on Cyc knockout (AeCyc-/-) and wild-type mosquitoes at four time points in a light-dark cycle. The study identified system-driven genes that exhibit rhythmic expression independently of the core clock machinery. Cyc disruption led to altered expression of essential clock genes, affecting metabolic processes, signaling pathways, stimulus responses and immune responses. Notably, gene ontology enrichment of odorant binding proteins, indicating the clock's role in sensory perception. The absence of Cyc also impacted various regulation of metabolic and cell cycle processes was observed in all time points. CONCLUSIONS: The intricate circadian regulation in Ae. aegypti encompasses both core clock-driven and system-driven genes. The KO of Cyc gene instigated extensive gene expression changes, impacting various processes, thereby potentially affecting cellular and metabolic functions, immune responses, and sensory perception. The circadian clock's multifaceted involvement in diverse biological processes, along with its role in the mosquito's daily rhythms, forms a nexus that influences the vector's capacity to transmit diseases. These insights shed light on the circadian clock's role in shaping mosquito biology and behavior, opening new avenues for innovative disease control strategies.

RNA Taste Is Conserved in Dipteran Insects.

BACKGROUND: Ribonucleosides and RNA are an underappreciated nutrient group essential during Drosophila larval development and growth. Detection of these nutrients requires at least one of the 6 closely related taste receptors encoded by the Gr28 genes, one of the most conserved insect taste receptor subfamilies. OBJECTIVES: We investigated whether blow fly larvae and mosquito larvae, which shared the last ancestor with Drosophila about 65 and 260 million years ago, respectively, can taste RNA and ribose. We also tested whether the Gr28 homologous genes of the mosquitoes Aedes aegypti and Anopheles gambiae can sense these nutrients when expressed in transgenic Drosophila larvae. METHODS: Taste preference in blow flies was examined by adapting a 2-choice preference assay that has been well-established for Drosophila larvae. For the mosquito Aedes aegypti, we developed a new 2-choice preference assay that accommodates the aquatic environment of these insect larvae. Finally, we identified Gr28 homologs in these species and expressed them in Drosophila melanogaster to determine their potential function as RNA receptors. RESULTS: Larvae of the blow fly Cochliomyia macellaria and Lucilia cuprina are strongly attracted to RNA (0.5 mg/mL) in the 2-choice feeding assays (P < 0.05). Similarly, the mosquito Aedes aegypti larvae showed a strong preference for RNA (2.5 mg/mL) in an aquatic 2-choice feeding assay. Moreover, when Gr28 homologs of Aedes or Anopheles mosquitoes are expressed in appetitive taste neurons of Drosophila melanogaster larvae lacking their Gr28 genes, preference for RNA (0.5 mg/mL) and ribose (0.1 M) is rescued (P < 0.05). CONCLUSIONS: The appetitive taste for RNA and ribonucleosides in insects emerged about 260 million years ago, the time mosquitoes and fruit flies diverged from their last common ancestor. Like sugar receptors, receptors for RNA have been highly conserved during insect evolution, suggesting that RNA is a critical nutrient for fast-growing insect larvae.

Potential key genes involved in metabolic resistance to malathion in the southern house mosquito, Culex quinquefasciatus, and functional validation of CYP325BC1 and CYP9M12 as candidate genes using RNA interference.

BACKGROUND: Metabolic detoxification is one of the major mechanisms contributing to the development of resistance in mosquitoes, including the southern house mosquito, Culex quinquefasciatus. The three major detoxification supergene families, cytochrome P450s, glutathione S-transferases and general esterases, have been demonstrated to play an important role in metabolic resistance. In this study, we performed differential gene expression analysis based on high-throughput transcriptome sequencing on samples from four experimental groups to give insight into key genes involved in metabolic resistance to malathion in Cx. quinquefasciatus. We conducted a whole transcriptome analysis of field captured wild Cx. quinquefasciatus from Harris County (WI), Texas and a malathion susceptible laboratory-maintained Sebring colony (CO) to investigate metabolic insecticide resistance. Field captured mosquitoes were also phenotypically classified into the malathion resistant and malathion susceptible groups following a mortality response measure conducted using a Centers for Disease Control and Prevention (CDC) bottle assay. The live (MR) and dead (MS) specimens from the bottle assay, along with an unselected WI sample and a CO sample were processed for total RNA extraction and subjected to whole-transcriptome sequencing. RESULTS: We demonstrated that the genes coding for detoxification enzymes, particularly cytochrome P450s, were highly up-regulated in the MR group compared to the MS group with similar up-regulation observed in the WI group compared to the CO group. A total of 1,438 genes were differentially expressed in comparison between MR and MS group, including 614 up-regulated genes and 824 down-regulated genes. Additionally, 1,871 genes were differentially expressed in comparison between WI and CO group, including 1,083 up-regulated genes and 788 down-regulated genes. Further analysis on differentially expressed genes from three major detoxification supergene families in both comparisons resulted in 16 detoxification genes as candidates potentially associated with metabolic resistance to malathion. Knockdown of CYP325BC1 and CYP9M12 using RNA interference on the laboratory-maintained Sebring strain significantly increased the mortality of Cx. quinquefasciatus after exposure to malathion. CONCLUSION: We generated substantial transcriptomic evidence on metabolic detoxification of malathion in Cx. quinquefasciatus. We also validated the functional roles of two candidate P450 genes identified through DGE analysis. Our results are the first to demonstrate that knockdown of CYP325BC1 and CYP9M12 both significantly increased malathion susceptibility in Cx. quinquefasciatus, indicating involvement of these two genes in metabolic resistance to malathion.

Gamma radiation induced changes in expression of heat shock proteins (Hsc70 and Hsp83) in the dengue vector Aedes aegypti (L.).

We aimed to assess the effect of gamma radiation on the expression of heat shock proteins Hsc70 and Hsp83 in Aedes aegypti. Adult males were irradiated with 50Gy of gamma radiation, and changes in the expression of proteins in SDS-PAGE gel bands corresponding to molecular weights ~60-75kDa and ~80-95kDa were analyzed at two different time points 6 and 12-hour post-irradiation, using a temporal mass spectrometry based semi-quantitative analysis. A 2-3-fold increase was observed in both proteins Hsc70 and Hsp83, at both time points. In addition, the experiment also revealed the overexpression of several other molecules such as Arginine Kinase - known to be upregulated in certain insects during stress, Esterase B1- implicated in insecticide resistance, and also down-regulation of the 26S proteasome non-ATPase regulatory subunit 1 and ubiquitin-activating enzyme E1 - both known to be involved in ubiquitin-mediated protein degradation. The results taken together with existing data on Hsp83 and Hsc70, indicate that these proteins may enhance the survival of Ae. aegypti following gamma radiation and could serve as molecular markers for the detection of radiation-induced stress.

Impact of disabled circadian clock on yellow fever mosquito Aedes aegypti fitness and behaviors.

Like other insects, Aedes aegypti displays strong daily patterns in host seeking and mating. Much of these behaviors are believed to be under the control of a circadian clock, an endogenous timekeeping mechanism relying on transcriptional/translational negative feedback loops that drive rhythmic physiology and behavior. To examine the connection between the circadian clock and various Ae. aegypti behaviors, we knocked out the core clock gene cycle using CRISPR/Cas9. We found that the rhythmic pattern and intensity of mRNA expression of seven circadian genes, including AeCyc-/-, were altered across the day/night cycle as well as in constant darkness conditions. We further show that the mutant CYC protein is incapable of forming a dimer with CLK to stimulate per expression and that the endogenous clock is disabled in AeCyc-/- mosquitoes. AeCyc-/- do not display the bimodal locomotor activity pattern of wild type, have a significantly reduced response to host odor, reduced egg hatching rates, delayed embryonic development and reduced adult survival and mating success. Surprisingly however, the propensity to blood feed in AeCyc-/- females is significantly higher than in wildtype females. Together with other recent work on the circadian clock control of key aspects of mosquito biology, our data on how cycle KO affects mosquito behavior and fitness provides a basis for further work into the pathways that connect the mosquito endogenous clock to its vector competence.

Evaluation of gamma radiation-induced DNA damage in Aedes aegypti using the comet assay.

The study was undertaken to evaluate gamma radiation-induced DNA damage in Aedes aegypti. The comet assay was employed to demonstrate the extent of DNA damage produced in adult male A. aegypti exposed to seven different doses of gamma radiation, ranging from 1 Gy to 50 Gy. DNA damage was measured as the percentage of comet tail DNA. A significant linear increase in DNA damage was observed in all samples; the extent of damage being proportional to the dose of gamma radiation the organism received, except in those treated with 1 Gy. The highest amount of DNA damage was noticed at 1 h postirradiation, which decreased gradually with time, that is, at 3, 6 and 12 h postirradiation. This may indicate repair of the damaged DNA and/or loss of heavily damaged cells as the postirradiation time increased. The comet assay serves as a sensitive and rapid technique to detect gamma radiation-induced DNA damage in A. aegypti. This could be used as a potential biomarker for environmental risk assessment.

Effect of gamma radiation on life history traits of Aedes aegypti (L.).

Aedes aegypti is an important vector for Dengue and Dengue hemorrhagic fever. Considering its medical importance and its relevance as a model system, this study was undertaken to evaluate the impact of different doses of gamma radiation for three generations of A. aegypti. Two to three days old virgin males of A. aegypti were irradiated with 15 doses of gamma radiation, ranging from 1 to 50 Gy and were immediately mass mated with the same aged virgin females. Observations were made for changes on their life history traits, particularly fecundity, hatchability, adult emergence, sex ratio and longevity, for three generations. Adult males exposed 30, 35, 40, 45 and 50 Gy doses showed a significant decrease in fecundity in F0 generations. While hatchability was observed to have decreased with increasing radiation doses from 3 Gy onwards in the F1 generation, samples irradiated with 30, 35, 40, 45 and 50 Gy maintained significant decline in hatchability in their succeeding generations, F2 and F3 also. Similarly, a decline was observed in adult emergence from 3 Gy onwards in all three generations. A male favoring sex ratio distortion was observed at the doses of 35, 40, 45 and 50 Gy in all three generations. Following exposure to 4 Gy, parental males and the resultant progeny showed increased longevity by 10.56 and 8.66 days respectively. Similarly, the F1 generations of samples irradiated with 30, 35 and 40 Gy exhibited an increase in longevity by 7.16, 7.44 and 6.64 days respectively. Dose response curve for fertility among the three generations was drawn and presented. The effect of radiological exposure on the life history traits of A. aegypti varies with dose for the three generations studied. These results have potential implications in mutational studies and risk assessment and also contribute to a better understanding towards employment of the sterile insect technique in A. aegypti, plausibly paving the way to an effective mosquito genetic control program.

Inheritance Pattern of Temephos Resistance, an Organophosphate Insecticide, in Aedes aegypti (L.).

The present paper reports the mode of inheritance of resistance in laboratory induced temephos resistant and susceptible strains of Ae. aegypti. Homozygous resistant and susceptible strains of Ae. aegypti were generated by selective inbreeding at a diagnostic dose of 0.02 mg/L of temephos. Genetic crosses were carried out between these strains to determine the inheritance pattern of temephos resistance. The log-dosage probit mortality relationships and degree of dominance (D) were calculated. The dosage-mortality (d-m) line of the F 1 generation was nearer to the resistant parent than the susceptible one. The "D" value was calculated as 0.15 indicating that the temephos resistant gene is incompletely dominant. The d-m lines of the F 2 generation and progeny from the backcross exhibited clear plateaus of mortality across a range of doses indicating that temephos resistance is controlled by a single gene. Comparison of the mortality data with the theoretical expectations using the χ (2) test revealed no significant difference, confirming a monogenic pattern of inheritance. In conclusion, the study provides evidence that the temephos resistance in Ae. aegypti follows an incompletely dominant and monogenic mode of inheritance.

Insecticide susceptibility status in three medically important species of mosquitoes, Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus, from Bruhat Bengaluru Mahanagara Palike, Karnataka, India.

BACKGROUND: Development of resistance among mosquito vectors of diseases is of growing concern in many countries of the subtropics, including much of India. The distributions of susceptibility/resistance to insecticides are scarcely recorded and are unknown in many parts of India. This manuscript reports the susceptibility status among strains of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus collected from different geographical areas of Bruhat Bengaluru Mahanagara Palike (BBMP), Karnataka, to six conventionally used insecticides. RESULTS: An. stephensi, Ae. aegypti and Cx. quinquefasciatus from different geographic areas showed susceptibility towards temephos. However, susceptibility was meagre for most of the other insecticides used in the study. Strong resistance to propoxur was reported in Ullala (RR(90) = 467.1) and Bannerghatta (RR(90) = 12,265) populations of An. stephensi and Ae. aegypti respectively. RR(90) ≥ 12,830 was recorded as higher in the Yelahanka (YLK) population of Cx. quinquefasciatus to neem. Significant differences at LC(50) and LC(90) concentrations were observed against various insecticides in different mosquito populations. Resistance was observed against some of the larvicides for the diagnostic dose recommended by WHO. CONCLUSION: The results form baseline data on the susceptibility status of three medically important mosquito vectors of diseases to commonly used insecticides in the field at present emphasise the importance of carrying out more effective and planned vector control measures.