Role of Mosquito Microbiome in Insecticide Resistance

Abstract

The gut microbiota of insects is one of the unexplored areas. The association with these microbiomes plays a vital role in supporting their survival and combat with ecological challenges. Mosquito is one of the focal attention insects among the Arthopods, being the vector of many pathogenic diseases including dengue and malaria. A variety of strategies have been designed and implemented to fight against these vectors including obnoxious use of insecticides. Indiscriminate use of insecticides has led to development of resistance against broad range of insecticides. Crucial role of bacteria in insecticide resistance has been under discussion. Many studies focus on the insecticide resistance due to gut microbiome. Thus, the role of gut microbiome is an important area for designing new vector control strategies and their role in improvement of a healthy environment.

1. Introduction

The medical importance of mosquito can be estimated from this fact that almost 300–500 million people are affected from malaria annually, from which 1 million people lost their lives with the maximum numbers of mortalities in infants and young children. The region mostly affected by malaria is Sub-Saharan African region. In the recent years, dengue virus has expanded its range to 50–100 million population annually with thousands of mortalities due to severe form, i.e., dengue hemorrhage fever. In the past few decades, a new endemic emerged in East Africa and America by West Nile virus named Chikungunya, which caused many deaths in the region [1]. Mosquitoes (family Culicidae) have great medical importance due to their property as vector for medically important diseases of human. The wide range of disease spread is due to the dual property of mosquito as it can be biological vector as well as can be the mechanical vector [2, 3]. It is known that half of the world population is at risk of getting mosquito vectored disease such as Malaria, Dengue, Chikungunya, West Nile Virus, and Japanese encephalitis [4]. According a World Health Organization (WHO) report published in 2010, about 247 million world’s population became ill due to mosquito and around 1 million people got the disease in 2008. The worldwide distribution of mosquitoes is misinterpreted only in tropical and subtropical environments. But to a certain extent, it is not true as mosquito can cause annoyance or can also spread pathogens or viruses in temperate latitudes [4, 5, 6].

Current studies suggested the progress in the overall global malaria control, and it is estimated that 2 million more cases of malaria have appeared in 2017 as compared with 2016. The number of malaria cases is increasing within the region of the Americas [7]. Similarly, reports of resistance to insecticides increase over time [7, 8]. This fact constitutes a great challenge for malaria vector control programs [9]. The fundamental mechanisms of insecticide resistance in malaria vectors are not clearly identified and recognized. However, the following four basic mechanisms underlying insecticide resistance in mosquitoes were described [10].

1. The modification of the cuticle.

2. Amplified detoxification of the insecticides.

3. Insensitivity of the sites of target of the insecticides.

4. Behavioral avoidance of insecticides.

There are still substantial gaps available to young researchers, particularly in high-dose insecticide resistance in the mosquito population. The increased use of genomic methods has encouraged the study of many facets of mosquito biology, such as the microbiota of mosquito populations, which is related to insecticide resistance [10]. Just like other organisms, mosquito is also hosting various types of microbes, and these microbes are basically acquired during their immature developmental stages, such as from the habitat in which mosquitoes are breeding and also from the food source of mosquitoes from where these mosquitoes take their food [11]. Different ways of obtaining microorganisms have been reported, such as microbes obtained from the environment and/or the food supply, the transmission of the bacteria from the female at the time of egg laying through the transovarian mechanism [12], and transmission from the young stage to the adult stage [13]. These microbes have multiple roles in the mosquito, among which some are also known to the metabolizing nature against the insecticides [14, 15, 16] and vigorously change as per the physiology of the host [11, 17]. Hence, the microbiota of the mosquitoes has the capabilities to contribute toward the detoxification of the insecticides and increase the resistance in the host. It is the same phenomenon that has been reported previously in agricultural pests [18, 19, 20, 21, 22, 23].

2. Habitat of mosquitoes

Mosquitoes breed almost in every water place such as rivers, swamps, lakes, clean water, large or small water bodies even in permanent and temporary water bodies; this is due to their adaptation mechanism. This leads to the conclusion that there is hardly any water body that didn’t lend itself a breeding site for mosquito. In temporary flooded areas, the areas near rivers and lake with the water flow fluctuations, flood waters, mosquitoes such as Aedes vexans or Ochlerotatus sticticus have developed such adaptation that allows them to breed and their ability to fly in places even far from their breeding sites [24].

Some species of Ochlerotatus have also adapted to breed in the harsh environments such as snow-melt, swampy woodlands; these mosquitoes include Ochlerotatus communis, Ochlerotatus cataphylla, Ochlerotatus cantans, Ochlerotatus hexodontus, and Ochlerotatus punctor. There are some species of mosquitoes that encounter conditions and make them ideal for growth. In the flood plains along coastal areas, the environment contains large amount of salt, thus in these areas, the Halophilous species, which prefer salt water or brackish habitats, for example Ochlerotatus taeniorhynchus, Ochlerotatus sollicitans, Ochlerotatus vigilax, Ochlerotatus caspius, Ochlerotatus detritus, are in focus in large numbers [24].

The Anopheles larvae developed an associative link with mosquito species in every habitat such as freshwater, salt water, edges of streams, rice fields, mangrove swamps, and grassy ditches or in temporary or permanent water bodies. Some species are known that prefer tree hollows as habitat; these are known as tree species, among which are Aedes cretinus, Ochlerotatus geniculatus, Orthopodomyia pulcripalpis, and Anopheles plumbeus. Some species of mosquitoes can also breed in small water bodies such as containers, rain water, water drums, tires, cemetery pots, or small clay pots; these species include Culex pipiens, Aedes aegypti (Stegomyia aegypti), Aedes albopictus (Stegomyia albopicta), orOchlerotatus japonicas [4, 24]. These adaptations helped mosquitoes to change their habitat, such as Asian Tiger Mosquitoes Aedes albopictus that originally found in the tropical regions, but during this climate change, they brought evolution in them as they became photoperiodic sensitive. When the days are shorter, the photoperiodic sensitive female lays different eggs as it lays eggs in longer days. The eggs laid in shorter days are inactive and hatch themselves in suitable seasons, which ensures the species survival in the winter [4].

Medically important mosquitoes are responsible for transporting different valuable pathogens such as viruses, bacteria, and parasites that mostly produce lethal diseases such as Malaria, Dengue, Yellow Fever, Chikungunya fever and Encephalitis. The process of pathogen transmission can be in two ways: (i) mechanical vector (e.g., Myxomatosis in rabbits is caused by Myxoma virus); (ii) biological vector. The latest one is more complicated due to following reasons: (a) It associates in necessary rather obligatory period of replication by the parasite in host. (b) Pathogen’s development. (c) Parasitic containment by vector insect. The pathogens that are vectored by insects are one of the most leading causes of the pandemics and epidemics; it is also one of the leading causes of declining and fall of empires, for example, Roman Empire and Greece Empire. The malarial case study in the Roman Empire is best example of fall of Empire. The malaria was a big issue in latter days, and the Roman marshy places were notorious for the “Malaria” (bad air). The blood-sucking mosquitoes make them capable of attaining pathogens from one host, and this behavior makes them capable of passing it to other vertebrate hosts. The physiology of mosquitoes is applicable for the mechanism of transmitting the parasite from one host to other. When certain forms of blood stage parasites (gametocytes) are ingested by a female Anopheles mosquito during blood feeding, the gametes mate inside the gut of the female mosquito and beginning of a new life cycle occurs followed by growth and multiplication of parasite in the mosquito. After 10–18 days, a sporozoite form of the parasite is migrated to the salivary glands of the mosquito. When this Anopheles mosquito bites another human, sporozoites are injected into the blood of the human together with the saliva of mosquito. This sporozoite migrates to the liver and begins a new life cycle.

Thus, the infected mosquitoes carry the disease from one human to another human (acting as a “vector”), and infected humans transmit the falciparum parasite to the mosquitoes. Contrary to the human host, the mosquito vectors do not suffer despite the presence of the parasites.

The efficient vectors have a close association with their hosts, and they should have enough long-life span that it should be sufficient for them to make pathogen/parasite enable for the proliferation or to develop the infective stages in the vector. The successful parasite transmission is dependent on the multiple blood meals. If we investigate the stats of mortality and morbidity of vector-borne diseases, the mosquitoes are the most fatal vector to the humanity. The mosquitoes only threaten 3 billion people worldwide alone in subtropical or tropical areas and not only affect the human health but also the socioeconomic factors and political factors [25, 26, 27, 28].

3. Microbiota of mosquitoes

A mosquito’s gut microbiota contains prokaryotes and eukaryotic community. Mosquito gut microbiota is primarily acquired from the environment, its composition is highly dynamic, varying greatly with species, diet, stage of development of mosquitoes, and geography [29]. Sequencing of the 16S rRNA or18S rRNA hypervariable regions is used as a culture-independent tool for the study of mosquito microbiota composition [30]. Many of the mosquitoes are marine and terrestrial during their developmental periods as adults. Larvae primarily consume organic detritus, single-cell organisms, and small invertebrates, while adults of both sexes usually feed on extra floral nectarines. Results outlined in several recent studies suggest that adult mosquito gut microbiota may have both a positive and a negative effect on vector competency, referring to the capacity of females to obtain, retain, and transmit pathogen to vertebrates. Studies show that the microbes form colonies in mosquitoes, which influences their physiological and metabolic functions control. The mosquitoes have a community of microbes, which includes bacteria, algae, fungi, and viruses. These microbes live in close proximity causing the combined effect on the mosquito’s physiology and metabolic functions [31].

3.1 Composition of gut microbiota of mosquitoes

Most of the microbiota in the gut of the mosquitoes is demonstrated as being predominantly Gram-negative of facultative nature, which actually belongs to four different phyla (Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria). These phyla are the most prominent members of the growing insect community, obtaining them primarily from the microenvironments in which they develop. With this, insects can easily proliferate on a regular basis. In addition, multiple members of the bacterial community are known to be part of the gut community and have also been successfully extracted and cultured in mosquitoes. Mosquito’s viruses have also been categorized in studies as a part of microbiome. Multiple genera belonging to Flaviviridae family are part of mosquitoes’ community, which sometimes have the activity of a pathogen in vertebrates. By comparison, the absence of bacteriophages in published research indicates that either viruses infecting bacteria in the intestine are underrepresented, or that few bacteriophages infect bacteria in the intestine of the mosquito [30].

3.2 Gut microbiota acquisition by larvae mosquito

Some species obtain intestine microbiota directly from their parents while others obtain their intestinal microbiota primarily from the environment. Three lines of evidence indicate that growing generation of mosquitoes reacquires the gut microbiota mainly from the environment. Second, laboratory experiments indicate that the mosquito larvae hatch and in their intestine left without bacteria. Second, gut-community composition studies suggest that the majority of microbes found in larvae correlate with those found in their aquatic environment. Third, mosquitoes host highly variable gut communities that it is not expected that the congeners acquired those communities directly. Studies indicate that adult mosquito reproductive tracts contain multiple species of bacteria and some of these bacteria are on the surface of laid female eggs; the majority of these bacteria are acquired of the mosquitoes environment. This can lead to the larvae to develop such microbes that can be ingest directly as eggshell fragments at hatching or inoculation of the aquatic environment in which larvae live. Many species of mosquitoes harbor intracellular bacteria that spread vertically such as bacteria of the genus Wolbachia. Additionally, these species are not part of the extracellular microbial population, which is the gut microbiota. Cultural studies initially indicated that mosquito larvae remove their gut microbiota at metamorphosis in a meconium having adults with little gut microbes. Such results have indicated that adults develop a gut microbiota by immersing water from the larval environment and/or feeding on resources such as extrafloral nectarines. However, studies of the composition of the gut bacteria community provide clear evidence that larvae of mosquitoes of genus Aedes and Anopheles transfer a proportion of their gut microbiota to adults. However, subsequently, the adult gut microbiota may change by consuming microbe containing water, nectar, or other food sources. Vertebrate blood generally contains a few bacteria, but some experiments indicate that the intake of a blood meal changes the composition of the gut microbiota persistently to transiently through alterations in redox status or metabolism. Infection by various vector-borne pathogens can also affect gut microbiota composition through unknown mechanisms [30].

3.3 Microbial variation in gut

As a holobiont, mosquito undergoes a metamorphic transformation from larval stage to adult stage. Microbial mosquito residents (P. falciparum) and their larvae refer to the microbial communities that colonize within the target organism (humans). In the adult mosquito, the larvae-associated microflora is replaced by a new set of microbes. This microbiota variation is due to significant changes in the host mosquito that are associated with the environment and feeding habits. This microbial cleaning and acquisition process is termed gut sterilization. Mosquitoes mainly consume bacteria and planktons as nutritious resources during their larval stage. This paves the initial stage of invasion of bacteria that contributes to the inhabitants. Among the microbes, the bacteria colonize more in the midgut than in the reproductive organs and salivary glands [32, 33, 34]. Later during adult stages, mosquitoes begin to feed on nectar and blood, which triggers the proliferation of some types of microbes and the decline of the other bacteria. Thus, the host diet and its developmental stage play a crucial role in shaping the gut microbiome [35]. Mosquitoes then begin feeding on nectar and blood during adult stages, it then regulates some types of the proliferation of different microbes and decline of some other bacteria. Therefore, the host diet and its level of growth play a key vital role in the structure of gut microbiome [36]. In the gut of mosquitoes, resident communities can vary from microscopic dominant bacteria to even Protista members. This resident consortium can be changed by the influx of new microbes from their natural habitat. Mosquitoes such as Anopheles, Aedes, and Culex normally lay eggs in water that contains bacteria [35]. Aquatic plants affect the microbial populations of mosquitoes providing microbes to larvae, and many of them are often transferred trans-steadily to adult gut [37, 38, 39]. These microbes have a significant impact on the characteristics of mosquito life such as fecundity, reproduction vector competency, and immunity. As per previous earlier studies, the general bacterial flora in mosquitoes includes Gram-negative phylum Proteobacteria (Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria) phylum Bacteroidetes, Gram-positive phylum Firmicutes including Clostridia, Actinomycetes, Spirochetes, and other species. Naturally, a bacterial community in mosquito gut can reduce the development of Plasmodium, a human parasite due to the presence of Gram-negative bacteria. The outer membrane of the cell wall in these Gram-negative bacteria contains lipopolysaccharides, which acts as a physical barrier for harmful agents such as hydrogen peroxide, etc. [40], while Gram-positive bacteria have no such barrier. Furthermore, different Gram-negative bacteria have varying effects against Plasmodium. These bacteria produce some metabolites that protect the mosquito. Plasmodium, for example, is found to be effective against prodigiosin of red pigment produced by Gram-negative bacteria. The mechanism for this is the upregulation of antimicrobial peptide encoding immune genes (AMP) and a protein containing thioester, which has an antiparasitic effect.

The symbiotic microbes are beneficial for the host in several ways. These require nutritional supplementation, strengthening of the digestive system, and tolerance to environmental perturbation and prevention against parasites. The Anopheline gut microbiome is strongly influenced by microbes suspended in its natural habitat. This has been proved by gut analysis of mosquito larvae by Howland [41], who dissected over 1000 larvae of eight species, in where they identified algae in the gut community and associated them to abundance in the food. She concluded that the abundance of algae in the larval food is correlated with algal abundance in the habitats. Also, this has been shown in another study on Anopheles quadrimaculatus larvae, a common vector of malaria in the Eastern United States [42] wherein the elimination of algae from a small pond with copper sulfate demonstrated its absence in their food. However, after recolonization the same pond, algal cells were again observed in the larval gut.

Researchers have been identified 98 genera of bacteria in the Anopheles mosquitoes, the most common being Pseudomonas, Aeromonas, Asaia, Comamonas, Elizabethkingia, Enterobacter, Klebsiella, Pantoea, and Serratia. Likewise, Gram-negative bacteria also predominate in Aedes spp. The Anopheline gut is dominated by resident bacteria of genus Pantoea and Asaia. These bacteria have shown stable association with Anopheline mosquitoes during different life stages. Pantoea, natural mosquito symbiont, can cross-colonize several mosquito species and is readily transformed and cultured. This property of Pantoea has been proposed for paratransgenic applications [43, 44]. Asaia acts as an immunomodulator by producing antimicrobial peptides that interfere with the course of infection particularly its invasion to epithelial tissues and salivary gland [36]. Recent research on two Anopheles species An. gambiae and An. coluzzii from Ghana [45] compared the midgut microbiota of mosquitoes during rainy and dry seasons from urban and rural breeding sites using 454 pyrosequencing. The data suggested that An. gambiae and An. coluzzi do not differ significantly in their gut microenvironment. Shewanellaceae family was observed in both the species. Bacterial families Enterobacteriaceae and Aeromonadaceae were also associated with Anopheles mosquitoes. The only difference observed was among An. gambiae collected from the different breeding site during summer. Aeromonas, Shewanella, and Thorsellia were other bacterial genera with variation in abundance according to the breeding sites. This indicates that larval breeding site has a significant impact on the adult mosquito midgut composition. The presence of Enterobacter and Serratia strain in Anopheles mosquito gut has an antiparasitic effect on mosquito. Enterobacteriaceae that survived during the rainy season is found to be more in number than that of during the dry season. Two members of this family include Enterobacter species and Thorsellia anopheles. This Gram-negative Enterobacter can directly act on Plasmodium falciparum and hinders the development of the parasite. T. anophelis was the dominant species in the midgut of An. gambiae. This symbiotic association with host mosquito vector attributes to its high tolerance for mosquito midgut alkalinity. Serratia marcescens HB3, isolated from laboratory-reared An. stephensi mosquitoes, inhibits Plasmodium development within the mosquito midgut by interrupting ookinete invasion through the midgut epithelial cells. Phenotypic variation at the cellular and structural levels was observed and directly correlated with the ability to induce resistance against Plasmodium invasion [46]. The prevailing environmental conditions have a great influence on the gut microbiome and host-vector competence. One parameter is the influence of chemicals in regulating the bacterial community in mosquito gut. For example, Pseudomonas aeruginosa boost the larval development of Culex quinquefasciatus in phosphate-rich medium [47].

A part of the mosquito gut microbiota is eukaryotic microorganism such as fungi. Its position as commensal, mutualist, or pathogenic in preserving the ecological balance of mosquitoes is inevitable. During the metamorphic transition, mosquitoes are exposed to fungi in the form of mosquito larvae in water, or by ingestion of fungi in sugar meals, or physical contact with conidia (adult mosquitoes) [48]. Filamentous fungi and yeast are the common fungal isolates present in the midgut and other tissues of mosquitoes. A filamentous fungus comprises some species of Aspergillus and Penicillium as pathogenic forms and some genera of fungi such as Beauveria and Metarhizium as entomopathogenic forms [49]. Different genera of yeast such as Candida, Pichia, and Wickerhamomyces have been identified in Aedes and Anopheles mosquitoes through culture-dependent and culture-independent methods. Earlier explorations in mosquito fungi diversity were based on these types of the culture-dependent method. For example, a yeast strain Wickerhamomyces anomalus has been reported in the midgut and reproductive organ of An. stephensi, a primary vector of malaria [50]. Recently, with the advent of high-throughput sequencing (HTS) technique, the knowledge about mosquito mycobiome has widened [51]. This HTS approach has been used to analyze the mycobiome formation in Ae. triseriatus, from the Japanese E. The series documented the presence of 21 distinct taxonomic fungal operating units (OTUs), of which 15 were identified by both parties. Ascomycota phylum is the major fungal taxa among these two Aedes species. Although the existence of mycobiome in mosquito is evident, the tripartite connection between vector, pathogen, and fungus is less known. Hence, there are enough evidences of the fungi present in mosquitoes. These eukaryotic organisms are responsible for the masking of many signals in the organisms.

Mosquito acts as an exclusive host for a large group of viruses, which are insect-specific. A metagenomic approach was used to evaluate viral load in two genera of mosquitoes Aedes and Culex. The comparison presented a striking difference in the virome of mosquitoes, where in genus Aedes showed a low viral diversity and less abundance than Culex. This metagenomic approach led to the identification/discovery of different viral families in mosquitoes such as Bunyaviridae, Rhabdoviridae, Orthomyxoviridae, Flaviviridae, Mesoviridae, Reoviridae, unclassified Chuvirus, and Negevirus groups. Most resident virome acts as commensal microbe due to its inability to infect vertebrate cell lines, prolonged host infection, and vertical transmission [52, 53, 54].

3.4 Microbes influence on host vector property

Vectorial capacity is a quantitative measure of several factors such as cellular, biochemical, behavioral, immunological, genetic, and environmental parameters, which can influence vector density, longevity, and vector competence. All these factors are interrelated and can determine the pathogenicity and nonpathogenicity in mosquitoes. Acetobacteria, a dominant member of gut microflora, may interact directly or indirectly with invading pathogens. The indirect interaction is by activating innate immune response. Usually pattern recognition receptors (PRRSs) on the host cell recognize preserved surface determinants known as pathogen-associated molecular patterns (PAMPs) that are present/found in microbes exclusively. Such linking activates immune signaling mechanisms such as the road toll or the route to immune deficiency (IMD). A cascade of events leads to the degradation of IF ranging from transcription factor (Cactus), nuclear translocation of NF—ranging from transcription factors (Dif and Dorsal) to antimicrobial peptide (AMP) genes being expressed, in the toll cell signaling pathway. This AMP, produced in the fat body, is secreted into hemolymph where it directly kills the invading microorganism. Genetic research showed that the AMP gene expressions are mainly regulated through the toll pathway and the IMD pathway. The toll pathway is mainly activated by Gram-positive bacteria, human P. falciparum, and DENV. The development of Gram-negative bacteria stimulates the IMD pathway, which regulates the antibacterial peptide gene [55, 56, 57].

3.5 Applications of microbiome of insects

Microbiome study in the last few decades has led to an understanding of the potential microbial functions. The few examples of which are as follows:

3.5.1 Cellulose and xylan hydrolysis

Termites belong to an extremely successful class of organisms that degrade wood, and they are considered as the potential catalytic sources for efforts aimed to convert wood into biofuels. Researchers have reported the presence of a huge, diverse set of cellulose and xylan-hydrolyzing bacterial genes through the metagenomic and genomic analysis of the communities of bacteria residing in the hindgut of higher wood-feeding Nasutitermes species. They have identified a number of previously uncharacterized protein families. Thus, degradation of lignocellulose does not occur by a single enzyme but due to the interaction of many macromolecular complexes that lead to its degradation. These macromolecular complexes have been termed as cellulosomes and are partially known in several microbes. The cellulose degradation of termite was long thought to rely only on microbial gut symbionts. More recently, cellulase gene transcripts have been identified from the termite itself. Similarly, three xylanases genes have been discovered from lepidopteran intestinal tract samples, and one from termite sample. The digestome of the insect gut comprising microbial as well as termite coded enzymes acts together to bring out the complete digestion of lignocelluloses. Many microbes have been identified and play important roles in the conversion of wood into a biofuel, such as ethanol, because of its potential for at least partially replacing fossil fuels in transportation and thereby lowering greenhouse gas emissions.

3.5.2 Vitamin production

The sequencing of genome of Wigglesworthia sp., the mycetocyte symbiont of Glossina brevipalpis, has been done and the annotation has shown the presence of encoded genes for the formation of thiamin (Vitamin B1), pantothenate (Vitamin B5), riboflavin FAD (Vitamin B2), nicotinamide (Vitamin B3), pyridoxine (Vitamin B6), biotin (Vitamin B7), and folate (Vitamin B9).

3.5.3 Phenolics metabolism and nitrogen fixation

Insects can absorb the atmospheric nitrogen only through the symbiotic association with gut-associated bacteria because the ability to fix nitrogen is widely available among bacteria but apparently absent from all eukaryotes. Nitrogen-fixing Enterobacter species have been isolated from the southern pine beetle, which together with some fungal associates may concentrate nitrogen on developing larvae. Rahnella aquatilis, Klebsiella species, and Pantoea species were commonly found in southern pine beetle, and the pine beetle (Dendroctonus frontalis) larvae are known to fix nitrogen in other environments. Another important role might be detoxification of conifer defensive compounds, which consists primarily of monoterpenes, diterpenes, and phenolics groups known to be metabolized by bacteria.

3.5.4 Antibiotic resistance

It has been reported in a study that microbial community of gypsy moth midgut shelters genes of hitherto unknown antibiotic resistance. For example, a new group of enzymes beta lactamase was identified from midgut metagenome of gypsy moth. The genes encoding these enzymes were found to be responsible for creating antibiotic resistance in E. coli, showing that insects may play some role in propagating resistance genes against important antibiotics.

3.5.5 Signal mimics

Microbes produce metabolites with diverse chemical feature and biological activities. Signal molecules have been reported from the uncultured microbial world through insect gut metagenomics. A study applied the matrix screen to a metagenomic library constructed from the microorganism associated with midgut of gypsy moth. They have reported the identification of a metagenomic clone of gypsy moth midgut microbiomes that produce inducers of quorum sensing and that are chemically different from the earlier quorum sensing inducers. The clone harbored the gene coding for monooxygenase homologue that mediates a pathway of indole oxidation, which resulted in the production of a quorum-sensing compound.

Impact of microbiota on mosquitoes plays critical roles in many mosquito biology processes including feeding, digestion, matting and sexual reproduction, development, immune response, and refractory pathogeny [58].

3.6 Impact of microbiota on mosquito physiology

Scientists have compared transcriptome between septic and aseptic adult female mosquitoes fed various diets and observed that microbiota stimulates some genes involved in digestion and metabolic processes such as glycolysis, gluconeogenesis, and sugar transport. Midgut microbiota, most especially Enterobacter sp. in Ae. aegypti and Serratia sp., have hemolytic activity that can contribute to lysis of the red blood cells (RBCs) and hemoglobin release. Antibiotic treatment of female mosquitoes reduced RBC lysis and egg production within Ae. aegypti. Yet not every bacterium supports the growth of the eggs. Various bacterial genera have been used to construct adult mosquitoes that have evolved from gnotobiotic larvae. Tests were carried out on five bacteria (Aquitalea, Sphingobacterium, Chryseobacterium, Paenibacillus, and Comamonas) that helped with egg development in Ae. aegypti. It was observed that Ae. atropalpus only was helped by Comamonas in the development of eggs [58].

3.7 Metabolic detoxification of insecticides

Three major metabolic gene families are being involved in the mechanism of the detoxification of insecticides in mosquitoes: esterases, cytochrome P450s (P450s), and the S-transferases (GSTs) glutathione. Cytochrome P450s are among those genes families that have the most significant role in both biochemical and the physiological functions of the living organisms. Cytochrome P450s are the most critical and significant to detoxify and also to activate the endogenous compounds as well as the xenobiotics [59]. The largest quantity of the exogenous as well as the endogenous compounds in the metabolic detoxification and the excretion are GSTs, which are dimeric proteins having the property of the solubilization [60, 61, 62]. An important property of the GSTs and the P450s is the upregulation at the transcriptional level, which in turn results in the formation of excessive production of proteins; hence, excessive enzymatic activity is being done. Moreover, it also increases the detoxification of the insecticides and toxins of plants with oxidation of compounds, in the insects, and this further leads to the tolerance of these chemical compounds. It was also stated that the production of the resistance against the insecticides required that genes encoding P450s be amplified/duplicated. A large number of organisms have a variety of esterase enzymes being a heterogeneous community of enzymes. The overproduction of these enzymes has been studied extensively as the amplification, and non-frequent overexpression of the genes of esterase enzymes has been proven to have increased detoxifying protein production [63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73].

Researchers have done the comparison of the toxicity level with or without the synergists and conclude that these enzymes are related to detoxification mechanisms in resistance development. In same way, research on resistance to pyrethroids in various species of mosquitoes strongly supports the importance of mitochondrial detoxification in insecticide resistance. Nonetheless, the findings of synergistic studies must be interpreted with caution: while in many cases the use of synergists can correctly indicate the role of detoxification proteins in insecticide resistance, in some cases synergists may be imperfect inhibitors for some of the detoxification enzymes induced by the resistance. Further work is required to support the synergistic study’s findings. Metabolic enzyme activity assays are alternative and separate diagnostic tool for detecting the possible involvement of a metabolic enzyme in resistance is to assess elevated levels of enzyme activity and/or an increase in insecticidal metabolism. For permithrin-resistant Cx, the metabolism of permithrin to 4-hydroxypermethrin by microsomal P450 monooxygenases was stated to be significantly greater. Quinquefasciatus mosquitoes than vulnerable counterparts thereof [74, 75, 76, 77, 78, 79, 80]. Elevated levels of cytochrome P450 monooxygenase, esterase, or GST activities in insecticide-resistant mosquitoes of several species have also been reported; these include An. albimanus, An. gambiae, An. stephensi, An. funestus, Ae. aegypti, An. culicifacies, Anopheles annularis [81, 82, 83, 84, 85]. Although these types of measurements suggest the critical function of metabolic enzymes in the production of resistance. No specific evidence is available to determine the performance of the metabolic enzymes. Initiation of individual metabolic gene/protein characterization cloning, sequencing of partial or full-length individual metabolic genes, purification of individual metabolic proteins, and screening of resistant mosquito-resistant cDNA libraries have helped to understand the molecular basis of metabolic-detoxification-mediated resistance. This area has been extensively studied in the 20 years since the publication of the first report on 17 partial CYP4 gene sequences from An. albimanus. After this, it was followed by the first full-length CYP6E1 sequence from Cx. quinquefasciatus, which was calculated using techniques for reaction of the polymerase chain. The availability of individual partial or complete sequences has allowed researchers to identify gene expression and amplification and protein expression. These findings reveal significant details about the metabolic enzyme characteristics associated with increased metabolic detoxification of insecticides in resistant mosquitoes via transcriptional up regulation/DNA amplification. Several P450 genes, including P450 genes from deltamethrin-resistant Anopheles minimus, pyrethroid-resistant, have been individually reported to be overexpressed in resistant mosquito species/strains. Funestus, Cx-resistant to permithrin Quinquefasciatus and Cx-resistant to deltamethrin Pippiens pallens. Along with same methodologies, the overregulation of the GST genes especially GSTE-2 was being identified in the mosquito resistant to the DDT, which is An. gambiae. And, the findings suggest that the overproduction of the esterase genes can help the amplification of GST genes in the mosquito Cx. quinquefasciatus. Certainly, there are a lot of studies that suggest the duplication and amplification of the detoxifying genes. It is very critical in the insecticide resistance phenomenon related to bacteria, since not a single gene analysis shows the complete complexity of this process. It is not clear that how so many of these genes are responsible for the detoxification directly or indirectly in mosquitoes species and what are the methods with which these genes are upregulated. There is no pathway that is completely showing the role in insecticide resistance [86, 87, 88, 89, 90, 91, 92].

3.8 Methods to study microbiota of insects

Insect’s gut descends from the mouth to anus and is one of the largest organs in insect body. The major microbiome community is present in the insects’ gut. Thus, it is very important to carefully isolate the insects’ gut microbiota. For this purpose, no specific technique has been standardized up till now. Firstly, we have to carefully disinfect the insects’ body by a disinfecting buffer and make dissection to obtain the complete gut. The insects’ gut can be separated into three parts, i.e., fore gut, mid gut, and hind gut. After the collection of each part, it is treated with extraction buffer, and metagenomics DNA extraction is made. Cell lysis is a critical step in metagenomics DNA extraction; thus it is carried out with the help of gentle means such as lysis enzymes. The gut cells are lysed and the remaining gut microbial cellular community is washed. For this purpose, mechanical lysis can also be made like homogenization, bead beating, and shocks to attain complete lysis [93].

3.9 Cultivation of obtained microbiome on the culture

The obtained gut sample is then suspended in saline, phosphate buffers, and then serially diluted to get cultured on the suitable growth medium. The culturing plates are then kept in incubator for 48 h. After this the morphological characteristics are carried for the characterization of bacterial colonies with at least three dilutions. Subsequently, enzyme activities are studied by gene coding for enzymes are cloned and DNA is sequenced for genomic libraries. The cultivated bacteria are then obtained and then used for the DNA extraction. Subsequently, enzyme activities are studied by gene coding for enzymes are cloned and DNA is sequenced for genomic libraries [94].

3.10 Accessing total genome of microbiota

It is not yet universally accepted literature published for the extraction of metagenomics DNA from insects. The major goal is to access unbiased microbial genome of whole communities along with the contamination and degradation of the genome should be taken under consideration. In the DNA isolation the sheering or DNA damage should be taken with care so that the DNA with high molecular weight can be obtained, which can then be used to create DNA libraries through BAC vectors. The DNA should be free from downstream of the applications such as cloning and PCR so, for this purpose no macromolecules should be attached to DNA [95].

3.11 Specified gene enrichment in DNA

Genes are the functional units, they control the phenotypes of a particular organism. For the quest of specific function, gene enrichment technique is used, which in return increases the efficiency of cloning prospective and also leads to the discovery of uncharacterized genes from a microbial community. The typical methods for the enrichment are to control the environment of the community by exposing them to pressure, temperature, pH, light, or electric shock. This in return controls the phenotype of the genes. The enrichment techniques include suppressive subtractive hybridization phage display and affinity capture [96, 97].

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 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, 99, 100, 101, 102, 103, 104, 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/pyrethroid-resistant in An. gambiae, pyrethroid-resistant An. funestus, pyrethroid-resistant, Ae. aegypti. Also, multiple P450 genes that are overexpressed in DDT and pyrethroid-resistant 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, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 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, 119, 120, 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.

Sr. no.	Insect source	Enzyme/gene	Potential application	Reference
1.	Reticulitermes flavipes	RfBGluc-1 beta-glucosidase	Digestion of lignocellulose	[122]
2.	Rotschildia lebaeu (Lepidoptera)	Xylanase	Degradation of xylane	[123]
3.	Termites (Nasutitermitidae)	Endo-1, 4-xylanase	Degradation of xylane	[124]
4.	Nasutitermes ephratae	Glycosyl hydrolase	Digestion of lignocellulose	[125]

Table 1.

Examples of insect’s source with their enzymes and genes isolated by the metagenomics functional expression analysis.

3.14 Metagenomic analysis of microbiomes

16S rRNA sequencing became the standard and normal method of determining the structure of a human microbiome population. The V1V3 and V3V5 regions of the hypervariable 16S rRNA gene help to distinguish the taxonomic structure of different bacterial species. To study the composition of microbiota, researchers categorize this gene into Operational Taxonomic Unit (OTU). Sanger sequencing was the primary instrument for sampling the entire amplicon range (16S rDNA). However, people discovered that species diversity can be classified utilizing shorter DNA stretches with higher sequence coverage and thus the developments of NGS, i.e., Roche 454 pyrosequencing, Illumina, and Ion Torrent sequencing are also used for the meta-genomic sequencing. Numerous analytical methods for studying the 16S rRNA sequences of microbes were also developed later to better understand their biology in the microbials. Nonetheless, even though we have strong coverage and longer sequencing reads using 16S rRNA sequencing, it would still be challenging to access the genomic details of low-abundance species. Therefore, recent work has moved to the use of high-throughput data techniques to develop both the qualitative and quantitative microbiome DNA information, mRNA transcripts, metabolites, and microbial community proteins. Metagenomic methods will help give a more detailed functional view of microorganisms and their functions within the microbiome. Shotgun metagenomic sequencing was the first step in this direction in which the whole genomic DNA of human/environmental bacteria samples were analyzed with a view to identifying all species and recognizing the microbe’s gene function potential. Another example is the HMP Unified Metabolic Analysis Network (HUMAnN), which performs metabolic and functional metagenomic data reconstructions [126]. This technique was performed on 102 individuals at seven key locations in the human body, namely diarrhea, dorsal tongue, and anterior nares. For various sites, they established the main metabolic pathways, genes, and functional modules that were distinct across individuals. Glycosaminoglycan degradation, phosphate and amino acid transport within this microbiota have been shown to be more involved in the vaginal microbiome; these methods have also been applied for insect’s microbiome. Computational modeling strategies such as metabolic genome scale models (GEMs) have been developed to integrate and interpret data for research purpose based on the increased experimental data produced by the high-throughput strategies. Throughout recent years, meta-omics results are used on a genome scale throughout tandem with metabolic models (GEMs). The genome size of metabolic models and metagenomic data were taken as feedback by using MAMBO (Metabolomic Analysis of Metagenomes using fBa and Optimization). The use of in vitro, ex vivo, and in situ laboratory evidence with in silico models serves as an outstanding testing tool for the discovery in human microbiomes of the elusive microbial microbe–microbe and microbe–host relationships that suggest major therapeutic progresses. Each of the respective omic data types provides useful knowledge in characterizing the organism’s working, and certain data types are incorporated more directly into the modeling formalism than others. For example, Vanee et al. used a proteomics-derived model to describe the Thermobifida fusca microbe’s metabolism functionalities where the growth rates seen in experimental and silico results were almost similar [127].

3.15 Homology-based analysis of metagenome sequenced DNA

Compared with functional/expression analysis, homology-based metagenomics are more precise as they target the gene on the basis of the data present and existing conserved genomics databases. Sequence-based screening methods depend on the existing conserved sequences and hence, may not help to identify brand new nonhomologous enzymes [128]. The sequence-based search combined with powerful bioinformatics tools has led to a higher rate of identification of novel genes than function-based methods do. Bioinformatics tools for sequence mining have been developed, based not only on homology of the primary sequence but also on the predicted protein structures. Gene function can be predicted with the improvement of the protein sorting and modeling tools, the putative active sites. Some tools of gene finding such as MetaGene has been used in order to predict 90% of shotgun sequences [129]. Many recent publications identify metagenome sequence databases that look for genes and enzymes that would be useful for commercial development in prospecting. For example, 71 million base pairs of sequence data were created by sequencing a metagenome library of hindgut microbiota from the largest family of wood-feeding termites. By detecting complete domains using global alignment, over 700 homologous domains of the glycoside hydrolase catalytic site corresponding to 45 different carbohydrate active enzyme families were identified, including a rich diversity of putative cellulases and hemicellulases [130].

3.16 Insecticide resistance

Numerous studies have shown that the individual mosquito species are involved in multiple mechanisms of resistance. In particular, two mechanisms increased metabolic detoxification of insecticides and reduced target protein sensitivity, which is the most critical target of insecticide. The insensitivity of the target site has been studied very extensively and has been accepted due to its extreme importance. The relationship between the genes related to the resistance on the regulation level of genes has provided with a very excellent example showing that how precisely these resistances develop in the insects. In the coding region, the overexpression and the amplification of mutant result in the structural differences inside the proteins and are linked with the resistance of the insecticides in the populations of mosquitoes. The overexpression at the transcriptional level of these genes shows resistance to the insecticides in mosquitoes. Collectively it is very easy for the researchers to conclude that these resistances are not only being transmitted from one generation to the other, but also it is being regulated at gene level. It is not yet clear which genes are directly or indirectly involved in the resistance and also how many are involved in the phenomenon [131, 132, 133, 134, 135, 136, 137].