**3.12 Whole-genome sequence analysis**

With the emergence of the field of genomics techniques in the last decade, the studies about the insecticide resistance have been revolutionized. With the help of the WGS analysis of the mosquitoes, mainly *Ae. aegypti*, *An. gambiae*, *Cx. quinquefasciatus*, and the *An. darlingi*, is one of the major achievements, which have boosted the development of the high-throughput analysis through the genomic studies. Also have enhanced the knowledge of the basic and most critical biological processes, which are responsible for this resistance of the insecticides in the mosquitoes. Furthermore, these high-throughput techniques guarantee the most novel and innovative approaches for the control of the mosquitoes as the vector and hence reducing the

#### *Role of Mosquito Microbiome in Insecticide Resistance DOI: http://dx.doi.org/10.5772/intechopen.104265*

mosquito borne-disease on the global scale. The collective data on the EST known as expressed sequence tags and some of the most very known and easily accessible techniques such as NGS (Next-Generation Sequencing), oligonucleotide microarray, applied quantitative trait loci analysis, and suppression subtractive hybridization have the most significant impact on the studies related to the expression. These expression analysis has a very significant role in making a new perspective of the role of the genes in insecticide resistance on the genomic level. These high-throughput techniques allow the researchers to study the mechanism of the insecticide resistance on the whole-genome level. Also very highly complex biological pathways have been developed with the help of the whole-genome investigation of the mosquitoes. With the help of the analysis of the genome, we have found enough knowledge on the complexities of the presence of the genes inside the genome of mosquitoes, which in turn detoxifies the insecticides in the mosquito populations. Some examples are 31 GSTs, 51 esterases, and 111 P450s sequences of genes in the mosquito belonging to *An. gambiae*. Also, 26 GSTs, 49 esterases, and 160 P450 sequences of genes in the mosquito belonging to *Ae. aegypti* [89]. And 35 GSTs, 71 esterases, and 204 P450 genes sequences in the *Cx. quinquefasciatus*. Lastly, 30 GSTs, 20 esterases, and 89 of P450s sequences of genes in *An. Darlingi* [98–105].

For itself, the cover interaction/expression relationship among the detoxification at the metabolic level and the multiple of the genes involved in detoxification have been shown in multiple genera of mosquitoes. There is evidence in which the DDT and pyrethroids resistance include genes such as GSTs genes, P450 genes which were overexpressing in the species of *Anopheles gambiae*, *Ae. Aegypti*, and *Anopheles funestus*. This has been explored in species resistant to DDT and pyrethroids, including multiple P450 and GST genes that are overexpressed or that interact in DDT/pyrethroidresistant in *An. gambiae*, pyrethroid-resistant *An. funestus*, pyrethroid-resistant, *Ae. aegypti*. Also, multiple P450 genes that are overexpressed in DDT and pyrethroidresistant *Ae. aegypti* and pyrethroid-resistant *An. gambiae*. Collectively, with the help of these explorations, it is widely accepted that there are various genes that are regulating and interacting in the mechanism of the resistance in the mosquitoes. With high-throughput technologies, the researchers understand the expressing genes involved in insecticide resistance. With the help of novel technique of SSH/cDNA, Liu et al. discovered 22 new genes, which were overexpressing in the *Cx. quinquefasciatus* for the pyrethroid resistance. The genes for P450 were 2 in number, for EST genes, 20 new genes were described and all of these genes were responsible for the transduction of the signal in insecticides resistance. Likewise, another high-throughput technique known as EST/cDNA microarray analysis has been revealing the overexpression of the genes responsible for the DDT resistance. Some of these genes belong to those species not already studied and they were directly involved in the mechanism of the resistance. Some of these genes encoding calcium/sodium, peptidases and lipid/ carbohydrates metabolism. The genes involved in the detoxification with the help of metabolism and some other genes, which are identified newly, have been proven to have a very significant role in the resistance against insecticides, and the relationship among the phenotype of resistance and the overexpression of the genes, thought to have the most significant role, is yet not clear [106–117].

Numerous strategies have been used for the validation of the overexpression of the genes and the resistance phenotype, to analyze the exact phenomenon of the resistance in the mosquitoes. These strategies include the in vitro protein metabolism assay, in vivo silencing of genes with the help of the RNAi techniques and also the modeling, these techniques are opted as they can fill up the gap between the

conventional proteomics and genomics and the novel area of the field named as functional genomics. The in vitro functional studies and the in silico presentation functional validation are being done for the confirmation of the theory that overexpressed genes are involved in the metabolization of the insecticides in the mosquitoes or not, this is very important to determine as it will narrow down the number and names of genes, which are actually involved in the insecticide resistance. Mitchell et al. have performed a functional study on the DDTs metabolism with the help of the *An. gambiae* P450 reductase and recombinant CYP6M2. Same studies have also been done for the assessment of the abilities of the recombinant CYP6M2 from the mosquitoes *An. gambiae* is used for the metabolism of pyrethroids and the *An. funestus* have the recombinant CYP6P9a and CYP6P9b. In an insect-baculovirus expression system, CYP6Z1 of *An. gambiae* and CYP6P7 and CYP6AA3 in *An. minimus* are capable of metabolizing DDT and pyrethroids, respectively. In silico 3-D homology modeling and molecular docking of metabolic enzyme substrate interactions are new and effective tools for understanding the relationship between protein structures and substrates, which can provide reasonable explanations for substrate specificities and differences in metabolism. Six regions of P450 proteins, designated substrate-recognition sites (SRS1 6; 46), contribute to the function of P450s, with SRS1, SRS4, SRS5, and SRS6 involved in the formation of catalytic sites and SRS2 and SRS3 participating in substrate access channel configuration. With this new computer modeling system to complement highly complex functional metabolism studies, researchers can now confidently state that several mosquito P450s, including CYP6Z1, CYP6AA3, CYP6P7, and CYP6M2, are important in insecticide resistance. This approach explains both how the molecular structures (proteins and chemicals) interact and how changes in the insect's metabolism are caused by allelic variation [118–121].

### **3.13 Metagenomics expression libraries**

On the basis of functional genes, metagenomics libraries are made by the help cloning vectors and the gene expressions are observed by functional assays. These gene expressions are then stored in metagenomics databases to help the researcher to access the previously unknown/uncharacterized genes. Furthermore, the characteristics of functional gene such as enzyme activities are expressed with a proficient vector. Heterologous expression of a gene in the host cells is impeded by various steps such as transcription, translation, and posttranslational process or maturation. Few metagenomics expression data of genes, which are isolated from the functional expression library technique, are listed in **Table 1**.
