**5.3 Identification of actinobacteria**

Different physical, chemical and molecular methods are available for identification of actinobacteria species. Generally, actinobacteria are identified in the petriplate based on the aerial and substrate mycelia, melanin pigments, pigment production, elevation and surface of each culture on the media [119]. Kelly [120] designated the colony arrangement of the different types of actinobacteria. Sporulation arrangement and Spores structures of the actinobacteria species were examined microscopically [121]. To check the cultural characterization of actinobacteria, different strains were streaked in different optimized growth media. Physiological and biochemical characters were done using the streaking of the culture in different media gelatin agar plates (gelatin hydrolysis) and starch agar plates (starch hydrolysis and sodium chloride resistance) etc. Isolated actinobacteria were streaked in the Petri plates and incubated at different temperatures for 7 days to check the optimal temperature for maximum growth through visual analysis [122]. Gel-diffusion and Fluorescent antibody (FA) procedures were used to identify *Actinomyces* species [123]. *Actinomyces israelii* was identified by the method of Slack et al., [124]. Spores' arrangements and Mycelium of the actinobacteria were carried out using scanning electron microscope (SEM). For genetic level identification, 16s rRNA was used for different actinobacteria [125].

#### **5.4 Secondary metabolites from actinobacteria for mosquito control**

The secondary metabolites isolated from actinobacteria are highly toxic to mosquitoes and have low toxicity to nontarget organisms. It is a good source for eco-friendly control of immature stages of mosquitoes [96]. Metabolites from actinobacteria were tested against mosquito life stages: three actinobacteria were reported to have ovicidal activity and 35 strains of actinobacteria had larvicidal activity; two *Streptomyces* sp. and one *Paecilomyces* sp. showed potent activity against tested mosquitoes. Aqueous solutions of actinobacteria presented potent larvicidal activity [126]. Karthik et al., [11] isolated the extract of *S. gedanensis* and tested it against the larvae of *Cx. gelidus* and *Cx.tritaeniorhynchus*. The results exhibited promising activity with LC50 values of 108.08 ppm and 609.15 ppm. Crude extracts of *S. gedanensis* and *S. roseiscleroticus* also revealed repellent activity at 1,000 ppm against *Cx. tritaeniorhynchus* and *Cx. gelidus*. Govindarajan et al., [127] reported that four *Streptomyces* sp. (A14, A21, A49 and A63) revealed potent larvicidal activity. Tanvir et al., [128] isolated twenty-one endophytic actinobacteria from plants. Among them, 10 actinobacteria species exposed strong larvicidal activity. Kekuda et al., [129] isolated extract from *Streptomyces* sp. and it showed 100% larvicidal activity against *Ae. aegypti* mosquito larvae after 24 h of treatment. Dhanasekaran et al., [130] reported that 35 actinobacteria isolated from different samples exhibited good activity against mosquitoes. Deepika et al., [131] reported 100% larval mortality for extract from *Streptomyces* sp. against *Cx. quinquefasciatus*. The marine actinobacterium (LK1) was isolated and crude extract was purified using reversed-phase high-pressure liquid chromatography. The extract presented good larvicidal activity against *An. stephensi* and *Cx. tritaeniorhynchus* with LC50 values of 31.82 ppm and 26.62 ppm, respectively, at tested concentrations [132]. Seven *Streptomyces* sp. isolated from marine sediments of South China produced siderophores, which acted as biocontrol agents and inhibited the growth of *Vibrio* spp. [133]. Gomes et al., [134] reported that five *Streptomyces* spp. were very efficient signifying their potential as biocontrol agents. Dhanasekaran et al., [135] isolated some actinobacteria and tested them for insecticidal activity. Totally four isolates showed strong larvicidal (100%) activity against larvae of *Anopheles* mosquito. Marine actinobacteria extracts had larvicidal, repellent and ovicidal activity against *Culex gelidus* and *Culex tritaeniorhynchus* [11].

Anwar et al., [136] collected different soil samples from various sites in salt range of Kalar Kahar, Pakistan and isolated 41 actinobacteria cultures. Among them, three actinobacteria: *Streptomyces minutiscleroticus*, *Streptomyces rochei* and *Streptomyces phaeoluteigrisseus* presented 100% larvicidal activity against *Cx. quinquefasciatus*. Vijayakumar et al., [137] tested the actinobacteria extract in different concentrations. The results presented that the isolates CC11 and SH22 (20%), CC110 and SH23 (16%), SH15 (12%), CC19 and S22 (8%), and S21 (4%) had good activity at 3 h against *Anopheles* mosquito. Filtrates of *Streptomyces citreofluorescens* presented good activity against *A. stephensi* and *Cx. quinquefasciatus* with LC50 values of 122.6 and 60.0 μl/ml, respectively [113]. Sanjenbam and Kannabiran, [138] isolated *Streptomyces* sp. VITPK9 from soil sample and tested for mosquitocidal activity. Ethyl Acetate extract gave good mortality against *Cx. tritaeniorhynchus, An. subpictus* and *Cx. gelidus* with LC50 values of 489.21, 831.78, and151.29 at 1000 ppm concentration.

#### **5.5 Compounds from actinobacteria for mosquito control**

Pure compounds isolated from actinobacteria like faerifungin, tetranectin, avermectins, flavonoids and macrotetrolides were found to be toxic against immature stages of vector mosquito and other insect pests. Actinobacteria like *Streptomyces* sp., *Streptomyces griseus*, *Streptomyces avermitilis*, *Streptosporangium albidum* and *Streptomyces aureus* produce these kinds of toxic metabolites that kill mosquitoes. Different types of genera were found to be producing toxic metabolites against mosquitoes; they are Actinomadura, Sreptoverticillium, Actinoplanes, Micropolyspora, Nocardiopsis, Thermomonospora, Oerskonia, Micromonospora, and Chainia [139–143].

Three new alpha class milbemycins (named milbemycins alpha28, alpha29, and alpha30)were isolated from *Streptomyces bingchenggensis*. They exhibited potent acaricidal and nematocidal activities [144]. Ichthyomycin, a compound isolated from *Streptomyces* sp. (strain 1107) was checked against larvae of *Culex pipiens autogenicus*, and the results exhibited that mortality of larvae was concentration-dependent [143]. Deepika et al., [131] isolated *Streptomyces* sp. VITDDK3 which produced the compound (2S,5R,6R)-2-hydroxy-3,5,6-trimethyloctan-4-one and which was tested for acaricidal and larvicidal activities against blood-sucking parasites. A compound trioxacarcin A (2a) and D (2d), isolated from the extract of *Streptomyces* sp. (B8652), influenced particularly high antiplasmodial activity against *Plasmodium falciparum* [145]. Prumycin, isolated from *Streptomyces* sp. showed antimalarial activity against drug-resistant Plasmodia [146]. Actinobacteria like *Streptomyces spinosa* have been reported to have a high level of activity against phytophagous insects and insects impacting public and animal health [147]. Metacycloprodigiosin, bafilomycin A1, and spectinabilin, isolated from *Streptomyces spectabilis* (BCC 4785) showed strong in vitro activity against *P. falciparum* [148]. The compound, salinosporamide A, isolated from marine actinobacteria, *Salinispora tropica*, presented strong inhibitory activity against Plasmodium growth [149]. Isolation of 10 new nine-membered bislactones, splenocins A–J (1–10) from organic extract of *Streptomyces* species (strain CNQ431) presented potent biological activities [150].

**110** The compounds tetranectin, avermectins, faerifungin, and macrotetrolides were isolated from *Streptomyces aureus*, *S. avermitilis*, *Streptomyces albidum* and *Streptomyces griseus* which showed insecticidal activity [139–141, 143]. Salinosporamide A and Depsipeptides derived from actinobacteria were used as antimalarial compounds [149, 151]. Avermectin family of 16-membered macrocyclic lac Streptomycestones isolated from *Streptomyces avermectinius* had antihelminthic activity [152]. Saurav et al. [153] isolated the pure compound, 5-(2, 4-dimethylbenzyl) pyrrolidin-2-one, from *Streptomyces* VITSVK5 sp. which exhibited strong activity against *R. (B.) microplus*, *An. stephensi*, and *Cx. tritaeniorhynchus*. Faeriefungin, a polyol polyene macrolide

*Metabolites from Actinobacteria for Mosquito Control DOI: http://dx.doi.org/10.5772/intechopen.106885*

lactone was isolated from the mycelium of *S. griseusvar. autotrophicus*. It showed 100% larval mortality of *A. aegypti* [154]. The compound aculeximycin, isolated from the *Streptosporangium albidum*, exhibited strong larvicidal activity against mosquito larvae as well as antimicrobial activities [126].

The compounds 5-azidomethyl-3-(2-ethoxy carbonyl-ethyl)-4-ethoxycarbonylmethyl-1H-pyrrole-2-carboxylic acid, ethyl ester (18.2%) 2; and akuammilan-16-carboxylic acid, 17-(acetyloxy)-10-methoxy, methyl ester (16R) (53.3%) were isolated from *Streptomyces* VITSTK7 sp. which had mosquitocidal activity [155]. Antonio et al., [156] reported that spinosad is a mixture of two tetracyclic macrolides produced during the fermentation of soil actinobacteria, and it was used for controlling dengue vector, *A. aegypti*.

### **6. Spinosad as a microbial pesticide**

Insect control metabolite spinosad was isolated from soil bacterium *Saccharopolyspora spinosa*. It exhibited high toxicity to the insect pest compared to the formerly developed chemical insecticides. Environmental Protection Agency of the United States gave permission to use Spinosad against various insect pests [157, 158]. It is a combination of two tetracyclic macrolide neurotoxins, spinosyns A and D. Insecticide from spinosad targets the nervous system of pest which contains nicotinic acetyl-choline and GABA receptors leading to immobilization and death. Due to its specific toxicity and its favourable nontarget organism and ecological profile, spinosad is considered by IPM practitioners as a significant new-generation pesticide [159]. The pesticide developed from spinosad is currently used against different insect orders like dipteran, lepidopteran, thysanopteran, and some coleopteran. Recently, research reports have recognized that spinosads are used to control several important mosquito species which act as vectors [160, 161]. Some of the insect orders reported that spinosad acts as stomach poison with direct contact poison and it is most active against Diptera, Lepidoptera, some Coleoptera, ants, termites and thrips [162]. Spinosad presented effective controlling of the population, particularly during the development in immature aquatic stages of mosquito vectors such as *Ae. albopictus*, *Ae. gambiae*,*Ae. aegypti*, *Ae. pseudopunctipennis*, *Cx. pipiens*, *Ae.albimanus*and *Cx. quinquefasciatus* [163]. Spinosad has been found to eradicate mosquito population all over the world. Eradication of mosquitoes from water jars in Thailand, Mexico cemetery water containers, septic tanks in Turkey, field microcosms in California, flooded fields in Egypt, street drains, cesspits, and disused wells in India, plots in Florida, water tanks in India, basins in Connecticut USA has been reported [56, 147, 164–173].

#### **7. Future prospects of actinobacteria for mosquito control**

In this chapter, we have stated that actinobacteria are used to control mosquito population in immature stages like egg, larvae, pupae and mature adults. Insecticides from secondary metabolites derived from actinobacteria are an important component in controlling vector-borne diseases by controlling the population of mosquitoes. Identification of the compound present in the secondary metabolites paves the way to preparing an effective insecticide to control insect pests, especially mosquito. Metabolites from actinobacteria show species-specific target activity and nontoxicity to other animals and humans. The feasibility of pesticides in field application is considered as the most consistent agent for controlling immature stages of mosquitoes. Preparation of mosquito coil, cream, repellent and evaporator from isolated compounds of actinobacteria give great impact on mosquito population

control. Only a limited number of research have been done on the actinobacteria to control mosquitoes. In future, researchers should focus on actinobacteria to identify novel compounds to effectively control mosquitoes. An efficient mosquitocide prepared from mixing of different compounds eluted from actinobacteria acts as a best alternative to synthetic insecticides to control mosquito-borne diseases without adverse residual effects. Government and private sectors should give priority to these kinds of research to promote mosquito control programmes.

## **8. Conclusion**

Pesticides from actinobacteria are reliable mosquito control agents; they are in wider use in field applications to control various insect populations. Several research report that compounds from actinobacteria exhibit promising activity against mosquito population. Insecticides from natural resources like actinobacteria

#### **Figure 1.**

*Some of the compounds isolated from actinobacteria for mosquito control. (A) Cyclopentanepropanoic acid, 3,5-bis(acetyloxy)-2-[3-(methoxyimino) octyl], methyl ester [2]; (B) 5-azidomethyl-3-(2-ethoxycarbonylethyl)-4-ethoxycarbonylmethyl-1Hpyrrole- 2-carboxylic acid, ethyl ester [2]; (C) akuammilan-16-carboxylic acid, 17-(acetyloxy)-10-methoxy, methyl ester [2]; (D) DEHP [80]; (E) (Z)-1-((1-hydroxypenta-2,4 dien-1-yl)oxy)anthracene-9,10-dione [20]; (F) (2S,5R,6R)-2-hydroxy-3,5,6-trimethyloctan-4-one [53]; (G) 5-(2,4-Dimethylbenzyl)pyrrolidin-2-one [123]; and (H) Antimycin [64].*

metabolites are easily produced in large quantities without disturbing other animals and ecosystems. In future, more research should focus on the isolation of compounds from actinobacteria with significant mosquito control metabolites from various natural sources such as desert, forest, marine and mangrove environments to control vector populations (**Figure 1** and **Table 1**).



#### **Table 1.**

*Some of the actinobacteria species used for mosquito control.*
