Isolation and identification of the pyrethroid insecticide deltamethrin degrading bacteria from insects

Many studies have showed that the pesticide residues in the environment increase day by day because of their continuous use. Pesticides can degrade chemically, physically and biologically. Biodegradation is an eco-friendly, inexpensive and highly effective approach compared to other methods. Bacteria are the most commonly used biological agents in biodegradation studies. Widespread use of pyrethroid pesticides such as deltamethrin causes pollution of environment. A total of 14 bacterial isolates were isolated from insects (Poecilimon tauricola, Locusta migratoria, Gryllus bimaculatus and Forficula auricularia) living in pesticide contaminated environments. These bacterial isolates were identified and characterized as Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Bacillus atrophaeus, Acinetobacter lwoffii, Rhodococcus coprophilus, Brevundimonas vesicularis, Pseudomonas syringae, Yersinia frederiksenii, Bacillus licheniformis, Enterobacter intermedius and Serratia marcescens based on biochemical and morphological properties and fatty acid profiles. As a result, these bacterial isolates can be used for the remove of deltamethrin at various environments.


Introduction
Natural or synthetic pesticides (organochlorine, organophosphate, carbamate, pyrethroids) are widely used to control unwanted pests. Pyrethroids account for about one-fifth of the global agrochemical market. Pyrethroids have potent neurotoxic activity against insects and low toxicity to animals. With the permanent use of pyrethroid worldwide, its residue has become a problem to animals, including humans. Pyrethroid insecticides, e.g., cyphenothrin, fenvalerate, esfenvalerate, deltamethrin, cypermethrin, cyhalothrin, fluvarinate, tralomethrin, cycloprothrin, acrinathrinallethrin, imiprothrin, permethrin and fenpropathrin are used in agriculture, animal health, home, and garden pest control throughout the world (Cycoń and Piotrowska-Seget, 2016;Zhang et al., 2016;Hao et al., 2018).
Pesticides are degraded into simpler and often less toxic chemicals in various ways such as chemical reactions, photodegradation and biodegradation. Biodegradation is an environment friendly, cheap and high efficiency approach compared to other methods. Bacteria and fungi with high enzyme (transferases, isomerases, ligases and hydrolases especially esterases, peroxidases and oxygenases) activity are used in biodegradation studies (Ortiz-Hernández et al., 2013;Ozdal et al., 2017).
Deltamethrin (C22H19Br2NO3) is a broad-spectrum insecticide belonging to pyrethroids (Figure 1). Deltamethrin is widely used in agriculture because of its low cost, persistence, stability and low toxicity to mammals. It is used for the control of pests such as mosquitoes, cockroaches, flies, ants and fleas due to effective at very low concentrations (Hao et al., 2018;Lu et al., 2019) Figure 1. Chemical structure of deltamethrin Microflora in the digestive tract of insect species is being investigated. The nutrient-rich digestive tract of insects is an appropriate growth environment for these microorganisms. The bacterial flora in the digestive tract of the insect has a very variable and broad enzymatic potential. Insect gut bacterial isolates have been demonstrated to break down many compounds such as pesticide (Ozdal et al., 2016a, b). Insect intestines provide a suitable medium for gene transfer between bacteria. Microorganisms can adapt to new environments by acquiring different features with horizontal gene transfer, conjugative plasmid and simple mutations to different environmental conditions Ramakrishnan et al., 2019). In this context, it is highly possible to isolate pesticide resistant microorganisms from insect intestines.
In many insect groups, resistance to pesticides occurs as a result of the use of pesticides. The intestinal flora of insects, which are observed to be resistant to pesticides, is very rich in bacteria that can be used in the biodegradation of pesticides. The purpose of this study was to isolate the bacteria capable of degrading deltamethrin from different insects.

Chemicals
Deltamethrin and other chemicals used in the study were of analytical purity and were obtained from Sigma and the media were obtained from Merck and Difco. subculture was repeated under the same culture conditions, and then an aliquot (0.2 ml) from each culture was applied to solid deltamethrin-MSM for isolation of single colonies. Colonies of different character were isolated by transferring to Tryptic Soy Agar plates and stored on slant agar at + 4 ° C.

Results and Discussion
There is a close relationship between bacteria and other living things. Therefore, insect microflora enables us to find new and biotechnological microorganisms. Serratia marcescens MO-1 isolated from grasshopper (Poecilimon tauricola) has both chitinase activity (Okay et al., 2013) and the ability to produce prodigiosin pigment (Kurbanoglu et al., 2015) which has antimicrobial and anticancer properties. Also, Pseudomonas aeruginosa OG1 isolated from cockroaches (Blatta orientalis) can produce pyocyanin, a pigment of biotechnological importance (Ozdal, 2019). Insects can change their ecological and physiological properties thanks to symbiotic bacteria . Kikuchi et al., (2012) indicated that bacteria of the genus Burkholderia develop resistance against the fenitrothion (organophosphate pesticide) in the bean bug (Riptortus pedestris). Chlorpyrifos and fipronil resistant strains of diamond back moth (Plutella xylostella) have higher levels of Lactobacillales, Pseudomonadales and Xanthomonadales compared to susceptible insects (Xia et al., 2013). Stenotrophomonas maltophilia OG-2, isolated from the intestine of the cockroach (Blatta orientalis), can degrade both α-endosulfan (Ozdal et al., 2017) and synthetic pyrethroid α-cypermethrin (Gur et al., 2014).
In this study, insects belonging to Orthoptera and Dermaptera were collected from the areas where insecticides were used ( Table  1). As a result of isolations, 14 bacteria were isolated on solid medium containing deltamethrin. Table 1 lists the insect species and isolate groups from which the isolates were obtained. A total of 14 bacterial isolates were isolated from medium containing deltamethrin based on visible colony differences. Among all these 14 deltamethrin degrading bacterial isolates, 3 were Gram-positive rods and 11 were Gram-negative rods. Total 2 isolates indicated positive results for endospore. All the isolates were catalase positive. Of these isolates, 3 were oxidase positive, 10 were motile and 3 were urease positive (Table 2).  According to the results of MIS analysis, fatty acid profiles of the isolates are summarized in Table 3. As a result of MIS analysis, 8 different genera and 13 different species of bacterial isolates were identified. Isolates based on morphological, biochemical and fatty acid data were identified as Acinetobacter lwoffii (DLM4), Pseudomonas aeruginosa (DPT5, DBG2), Stenotrophomonas maltophilia (DFA2), Bacillus licheniformis (DFA1), DLM1), Bacillus atrophaeus (DPT1), Pseudomonas syringae (DPT2), Yersinia frederiksenii (DLM2), Enterobacter intermedius (DPT3), Serratia marcescens (DPT4) and Flavimonas oryzihabitans (DLM3). The genera of the isolated bacteria were mainly identified as Pseudomonas and Bacillus. The highest bacterial diversity was observed in Poecilimon tauricola (5) and Locusta migratoria (4).

Table 4 Deltamethrin degrading microorganisms isolated from different environments
Many different bacteria have been isolated and characterized with their ability to degradation various pesticides. In previous studies, bacteria capable of degrading deltamethrin were mostly isolated from agricultural areas where intensive pesticides were used. However, the potential of these microorganisms to degrade deltamethrin has been confirmed for some bacteria of the genera Acinetobacter, Bacillus, Brevibacillus, Pseudomonas, Serratia, Rhodococcus (Table 4). Song et al., (2015) studied the deltamethrin biodegradation with Pseudomonas aeruginosa JQ-41 strain isolated from the pyrethroid contaminated soil. In another study, Acinetobacter calcoaceticus MCm5 was used in biodegradation of deltamethrin (Akbar et al., 2015a Zhan et al., 2018 in this study. When Table 4 is analyzed, it is seen that the bacteria used in deltamethrin degradation are generally isolated from soil and sludge. All bacteria obtained from this study were isolated from insect flora. In addition, it has been determined that new species may be effective in deltamethrin biodegradation.
The bacteria isolated in this study can undoubtedly be used in biodegradation studies. As seen in Table 5, different strains of the species isolated in this study have been reported to have been used for the degradation of many different pesticides. It has been determined that insects are important source for the isolation of bacteria that break down pesticides.

Conlusion
Strains of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Bacillus atrophaeus, Acinetobacter lwoffii, Rhodococcus coprophilus, Brevundimonas vesicularis, Pseudomonas syringae, Yersinia frederiksenii, Bacillus licheniformis, Enterobacter intermedius and Serratia marcescens, able to use deltamethrin as the only carbon source, were isolated from Poecilimon tauricola, Locusta migratoria, Gryllus bimaculatus and Forficula auricularia. Pesticide resistant insect microbiota has been shown to be a rich source for isolation of microbes that can degradation pesticides and a promising tool for biotechnological discovery in bioemediation programs. In order to find new biocatalysts in the degradation of pesticides, isolation can be made from insects that can live in pesticide environments. As a result, it can be said that isolated deltamethrin degrading microorganisms can be used in the treatment studies in the dirty areas of this insecticide. However, optimization studies are also needed to make biodegradation highly efficient and feasible.