**4. Microbes as insecticides**

Eradication of insect pest population through pesticides derived from microorganisms is highly effective and generally has benefits over synthetic insecticides. The metabolites derived from microbes are host specific and there is no detrimental effect on the non-target organism and surrounding environs. Around the world, only 5% of fungi and 0.1% of bacteria have been described [64]. Different types of microorganisms like fungi, bacteria, nematodes and viruses are biologically toxic to insect pests [65, 66]. Microorganisms from different sediments are important sources of bioactive components for antibiotics; many bioactive secondary metabolites are used for biotechnology and pharmacological studies [67].

Larvicides derived from microbes, especially bacteria have been used to eradicate mosquito population for the past few years. Bacteria like *Bacillus sphaericus* are widely used for potential biolarvicide in mosquito control programmes worldwide; it exhibited effective larvicidal activity against larvae of several mosquito species. Commercial larvicides from active strain of *B. sphaericus* are used to control various types of insects which act as vectors [68–70]. Toxins Bin and Mtxs are produced during the sporulation and vegetative stages of *B. sphaericus*, and some of the toxic strains have been extensively used for controlling the populations of mosquito [71]. Larvicides developed from *B. sphaericus* against mosquitoes have led to development of resistance [72]. *B. sphaericus* biolarvicide is limited in India due to the resistance development in the target mosquito. In the early stage, *An. stephensi* had developed resistance against *B. sphaericus* [73, 74].

Metabolites from *Bacillus thuringiensis var. israelensis* were toxic to the larvae and pupae of *Cx. quinquefasciatus* [75]. *Bacillus thuringiensis*is naturally present in the soil and normally it is used as a pest-control microorganism. Different types of *Bacillus thuringiensis* have been used to control insect pests. It is the only insecticide extensively used in all parts of the world. δ-endotoxin produced from *Bacillus thuringiensis* is toxic to various insect species. The toxin initiates growth of a lytic protein in the midgut epithelial membrane, which leads to cell lysis, termination of feeding, and leads to death of the larva. They produce two different types of toxin proteins such as Cry and Cyst proteins [76–78]. *Bacillus sphaericus* and *Bacillus thuringiensis* var. *israelensis* H-14, *Bacillus amyloliquefaciens* and *Bacillus amyloliquefaciens* were highly effective against different species of mosquitoes [79–82]. Secondary metabolites derived from various types of fungal species like *Beauveria bassiana*, *Chrysosporium tropicum*, *Aspergillus niger*, *Cochliobolus lunatus*, *Fusarium oxysporum*, *Chrysosporium lobatum*, *Trichophyton ajelloi*, *Fusarium moniliforme*, *Trichophyton mentagrophytes*, *Paecilomyces carneus*, *Paecilomyces marquandii, Isaria fumosorosea, Metarhizium anisopliae, Penicillium* sp., *Paecilomyces lilacinus* and *Evlachovaea kintrischic* are also used to control the immature stages of mosquito population [83–89]. In recent years, the research on microscopic organisms has increased to identify microbial agents for various biological uses.

## **5. Actinobacteria for mosquito control**

Actinobacteria are a group of filamentous bacteria; they are Gram-positive, dwelling in the soil, marine, and some of them are endophytic; they produce a large number of secondary metabolites. In the pharmaceutical industries, 75–80% of antibiotics are derived from microbes like actinobacteria [90–92]. Microorganisms present in different environments serve as an important natural resource for novel antibiotics, antitumor agents, and other therapeutic substances. Antibiotics such as erythromycin, vancomycin, and streptomycin are used for various pharmacological purposes [93–95]. The molecules isolated from microbes like actinobacteria are extremely toxic to insects like mosquitoes and have low toxicity to other beneficial organisms and environment. The use of secondary metabolites from actinobacteria may be a good approach for environment-friendly insect pest management [96].

#### **5.1 Isolation of actinobacteria**

Actinobacteria are present in different habitats, and they have a capacity to produce a large amount of various active secondary metabolites. Between the diversity of microbes, actinobacteria produce an enormous amount of secondary metabolites which have been used for various biological and biotechnological activities like anticancer drugs, antibiotics, pesticides, immunosuppressors and enzyme inhibitors [97–100]. Isolation of actinobacteria from different environments like cold, halophilic and alkaliphilic is possible; some of them present in high temperatures are called extremophiles. More than 61% of the secondary metabolites are isolated from actinobacteria genus *Streptomyces*; some of the metabolites have been isolated from rare actinobacteria. The samples collected from extreme environments — particularly in places which are isolated from human dwellings yield good results [101–104]. Generally, microorganism is isolated using serial membrane filter technique, dilution method and direct inoculation technique [105]. The collected samples are spread on different types of media used for actinobacteria isolation, such as actinomycetes isolation agar (AIA), humic acid vitamin agar (HVA), Starch casein agar, Glycerol-asparagine agar, Bennet's agar (BA) medium, Gause`s No.1 medium, Complex HV Agar, HV agar, humic acid vitamin agar, ZSSE (Zhang' Starch Soil Extract Agar, Kuster's agar, inorganic salt starch agar, starch nitrate agar, glycerol glycine agar, chitin agar, soil extracts agar and Glycerol-asparagine agar etc. [106–108]. The endophytic actinobacteria are isolated using the recommended method of Otoguro et al., [109]. Based on the colony morphology, the actinobacteria are selected and purified on ISP-2 (International *Streptomyces* project medium No. 2) for further bioactive studies [110].

#### **5.2 Pre-treatment and selection of actinbacteria**

Samples collected from different places are pre-treated using different procedures to remove the fungi, bacteria and other unwanted microbes. Pre-treatments of the collected samples encourage or enrich the growth of the actinobacteria, especially rare actinobacteria. In one of the pre-treatments, CaCo3 was used to treat the soil samples to decrease the number of other unwanted bacteria, and it allowed the excess actinobacteria spores cells to survive [111]. In Physico-chemical treatment, the soil sample (1 g) was suspended in 10 ml of normal saline, and the sample was heated for 1 h at 120°C to increase and encourage the growth of the actinobacteria [112]. Samples were treated with 1.5% phenol for 30 min at 30°C by the recommended method by Hayakawa et al., [112]. To decrease the growth of other unwanted microbes, the growth media were added with nalidixic acid (100 mg/l) and ketoconazole (30 mg/l) [113]. To increase the number of actinobacteria, the soil sample is treated with peptone (6%) and sodium lauryl sulphate (0.05%) at 50°C for 10 min [114]. Soil samples are added to 10 ml of sterilized distilled (Wet heat) water and heated in water bath at 30–50°C for 2–6 min and allowed to cool before serial dilution; without distilled water samples are heated (Dry heat) in hot air oven at 50–70°C [112, 115]. In other treatment, the soil sample is added to sterile water and centrifuged at 10,000 rpm for 30 min and used for isolation of actinobacteria [116]. Soil samples are treated with sodium dodecyl sulphate as per the prescribed method by Janaki et al., [117]. Sample is also treated in microwave oven as per the recommended method by Bulina et al., [118].
