Isolation and identification of microbiota from Egyptian common insect pests invading economically important crops

 

Waill A. Elkhateeb¹, Marwa O. ELnahas², Kareem M. Mousa³, Adel A. Ahmed⁴,
Abdu Galib ALKolaibe⁵, Ghoson M. Daba⁶

¹Chemistry of Natural and Microbial Products Department, Pharmaceutical Industries Division, National Research Centre, Dokki, Giza, 12622, Egypt.

²Chemistry of Natural and Microbial Products Department, Pharmaceutical Industries Division, National Research Centre, Dokki, Giza, 12622, Egypt.

³Economic Entomology Department, Faculty of Agriculture, Kafrelsheikh University, Kafr El–Sheikh 33–516, Egypt.

⁴Pests & Plant Protection Department., National Research Centre, Dokki, Giza, Egypt.

⁵Microbiology Department, Faculty of science, Taiz University, Taiz, Yemen.

⁶Chemistry of Natural and Microbial Products Department, Pharmaceutical Industries Division, National Research Centre, Dokki, Giza, 12622, Egypt.

 

ABSTRACT:

Insects bodies contain multiple distinctive microbial colonies which play key role in the metabolism, development and health of an insect. In the current study bacterial and fungal microbiota were isolated from larval feces and adult wings of two major insect pests in Egypt, the Cotton leaf worm Spodoptera littoralis and the black cutworm Agrotis ipsilon. Isolated pathogens were identified and characterized according to the scientific described methods in such cases. Two different genera of bacteria (Bacillus & Serratia) were isolated from larval feces of both insects while isolated fungus were Paecilomyces variotii and Absidia corymbifera from the feces of A. ipsilon larvae, while Rhozopus stolonifer and Penicillium chrysogenum were isolated from adults wings of the same insect. The fungus Aspergillus flavus, Aspergillus niger and Mucor circinilloides were isolated from feces of S. littoralis larvae. Numerous species of bacteria and fungi had been documented as a bio-agent against many insect pests, and most of them exhibited disruption in life-cycle of different species of Lepidopterous pests. The obtained data sheds light on microbial colonies associated with two major insect pests, however further studies are required to involve the isolated microbiota in the biological control programs of insect pests.

 

 

1. INTRODUCTION:

Insects are considered the most diverse animal clade, it is successfully adopted with conceivable type of habitats in its number of species and biomass ^1-3. Amongst those insect pests, the black cutworm Agrotis ipsilon (Hufnagel) and The Egyptian cotton leaf worm Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) are two major polyphagous insect pests that hampering many agricultural crops, and widely distributed in numerous countries worldwide^4-7, cause considerable losses in crop production and quality in many areas worldwide.

 

The fitness and health of an insect, to a large extent, defined by its associated microbiome^8,9. Some microbiome can augment host longevity and productivity, while others can cause diseases and death, so it may be effective in some insect pest control programs^10,11. Multiple factors including the surrounding environment¹², developmental stage^{1, 13} and diet¹⁴ can alter the content of insect microbiome. The presence of microbiota in the gut of different organisms help in degrading and digestion process of complex organic matter. Such microorganisms are involved in detoxification of food poisonous components^[15,16]. Moreover, those microbiomes have important contributions in protecting against parasites or pathogens^17-19. In general, the different roles played by microbiota are vital for survival of different organisms. Hence, studying the microbial flora associated with different organisms is of great importance, mainly those isolated from harmful insect pests causing serious economic losses. It was found that the isolated microbiota changes according to the insect’s developmental stage which suggests that different physical/chemical factors control these symbiotic interactions.

 

The current study monitored the bacterial and fungal composition associated with the intestinal tracts of A. ipsilon and S. littoralis larvae compared with bacterial and fungal composition existing on the wing scales of their adult moths, depending on the particular insect developmental stage hence the living environment and feeding behavior, we focused on the divergence in the bacterial and fungal composition of mature and immature stages of A. ipsilon and S. littoralis.

 

2. MATERIALS AND METHODS:

2.2 Insect samples:

In this study, laboratory strains of two important insect pests, Egyptian cotton leaf worm Spodoptera littoralis and black cutworm Agrotis ipsilon were obtained from a settled colony maintained on castor leaves diet, with no history of exposure to microbial or chemical insecticides.  Both larvae and moth of mentioned insects were reared for multiple generations at the laboratory of Pests and Plant Protection Department, National Research Centre, Giza, Egypt.

 

2.3 Isolation and identification of microbiota from surface and faeces of S. littoralis and A. ipsilon:

2.3.1 Media:

Two different media, Nutrient agar, and de Man, Rogosa, Sharpe medium (MERCK, Germany) (MRS) agar were used to isolate bacterial flora from the surface and fresh faeces of the adult insects. On the other hand, potato dextrose agar was used to isolate fungal isolates (Sigma, Germany).

 

2.3.2 Isolation:

The body of the adult insect was cut into small pieces and laid under aseptic conditions on the surface of dried plates containing different agar media. On the other hand, faeces of larvae were suspended in sterile distilled water, and ten-fold dilutions were prepared from this solution. Appropriate dilutions were inoculated and mixed with agar media before solidification, then poured into plates. After that, plates were incubated for 48h at 37±2°C in case of bacterial isolates^20,21, and 7 days at 30±2°C for isolation of fungi²². Resulting colonies were re-plated and purified on suitable agar media plates till pure colonies were obtained²³. Resulting isolates were stored at -80°C in suitable broth media with 15% (v/v) glycerol as frozen stock.

 

2.4 Identification of bacterial isolates:

Bacterial isolates obtained during our investigation were identified on the genera level. The identification was conducted according to colony morphology, catalase reaction, Gram staining, and spore formation²⁴. For catalase reaction, each isolate was tested by placing a drop of 3% hydrogen peroxide solution on 24h old vegetative cells. Immediate formation of bubbles indicates the presence of catalase in the cells^[25]. For more confirmation, biochemical tests were conducted as described previously²⁶.

 

2.5 Identification of fungal isolates:

Morphological and microscopic identification of the isolated fungi during our investigation was carried out using the features described by Domsch et al.,²⁷, and Moubasher²⁸. Also, identification of the isolated fungi was reviewed and compared with the same species deposited in Assiut University Mycological Center (AUMC).

 

3. RESULTS AND DISCUSSION:

Insects are accompanied by a huge variety of other organisms and this is very important factor affecting the ecosystem. Microorganisms are among the other organisms that are found in close association with insects which in turn greatly affect their evolution and ecology. There are many kinds of microbes that can associate insects either transiently or permanently, such as: bacteria, fungi, viruses, archaea, etc. Of which some are considered to be beneficial to insects and other are harmful^{29, 30}. As it was mentioned that some microbes could be beneficial to many insects where they provide a protection to them, where they help them to tolerate with various unfavorable abiotic or/and biotic factors. This benefit- relationship has been proved in aphids, where Buchnera endosymbionts resulted in a mutation that control the thermal tolerance of their hosts (pea aphid), and eventually positively influencing their performance at lower temperatures^31-33. Spodoptera littoralis (the Egyptian cotton leaf worm) and Agrotis ipsilon (the black cutworm) gain a lot of attention due to their negative effect on economy³⁴. Thus, many studies related to the biological control of these two insects as well as studying the effect of different microbes that could be beneficial or harmful to those insects were done. Thus, this article aims to study the various microbes associated with these two important insect pests.The symbiosis between insects and bacteria is an important key that illustrates the presence of an association between some insects and the commensal bacteria found on the insects or inside their gut^35-37. The bacterial communities inhabiting some insects are affected by a various factors, including: pH, temperature, life stage, host environment and phylogeny as well as the diet^{14, 38}. Recently, many studies have reported that the microbiota might be affected by host phylogeny and/or by diet. Understanding the insect- bacterial community might help in improving pest controlling methods that specifically target the economically important insects. This includes studying the insect bacterial community, detecting the symbionts and finally, find new methods to use symbionts in order to control insect pests. An important step is to monitor the symbionts found inside the insect pest as well as monitor their various transmission routes, that is because the insects-bacterial community is usually unstable^39,40. In this study two bacterial genera were isolated from larval feces of Egyptian cotton leaf worm (Spodoptera littoralis) and black cut worm (Agrotis ipsilon) (Tables 1, Figure 1).

 

Table (1): Bacteria isolated from Spodoptera littoralis and Agrotis ipsilon

 

The bacteria isolated from the two insects was classified in two different genera which are Bacillus and Serratia.

 

Although some studies indicated that some Bacillus species are considered pathogenic to many insects and show various insecticidal effects^[41,42]. Other found that Bacillus species could be beneficial to many insect pests. Some of the Bacillus species have been reported as alkaloid degraders⁴³. Such degrading ability might be so crucial to insects as they can overcome various defense mechanisms exerted by the plant on which the insect feed. Moreover, Bacillus cereus showed its ability to metabolize indoxacarb, due to the presence of a carboxylesterases, and hence it can survive this insecticide⁴⁴.

 

On the other side, another pathogenic species belonging to the genus Serratia have been isolated from many insects⁴⁵. These species might be considered as bio-control agents against various insects⁴⁶. Among these species S. marcescens is well known pathogenic species that are able to produce some hydrolytic enzymes, many of these enzymes are found to be toxins ²⁰. S. marcescens belongs to the family Enterobacteriaceae that is known to be gram-negative, facultative anaerobic rods. This bacterium is characterized by producing a specific pink or red pigment. Although S. marcescens is insect pathogen, sometimes it is not considered to be pathogenic when it is present in small amount in the insect digestive tract. However, once this species reaches the hemocoel, it starts to  multiply very fast and leads to death within 1–3 days⁴⁷. The studies also showed that S. marcescens Rb2 isolated from Rhynchites bacchus (L.) exerted about 73% mortal effect towards R. bacchus larvae⁴⁸.

 

Figure 1. Isolated bacteria from Spodoptera littoralis and Agrotis ipsilon (a) Serratia Sp., (b) Bacillus Sp. (Photos was taken by Dr. Waill Elkhateeb).

 

Table (2): Fungi isolated from Spodoptera littoralis and Agrotis ipsilon

 

3.2 Fungi isolated from insects:

Similar to bacteria, fungi (including yeast and molds) play a vital role in nutrients provision and host defenses regulation. The insects-fungi relationship has been studied via investigating pathogenic invasions as well as vector biology^[49]. Additionally, some symbiosis relationships have been identified between fungi and some insects such as mound-building termites and ants, where fungi play a vital role in digesting wood and providing food^[50]. The interesting relationships between insects and fungi had been studied since 1950s^[51].

 

In this study seven fungal species were isolated from the adults of Egyptian cotton leaf worm (Spodoptera littoralis) and back cut worm (Agrotis ipsilon) (Tables 2, Figure 2).

 

Recently, more interest has been directed towards the application of entomopathogenic fungi as biocontrol agents for many insect pests^[52]. The entomopathogenic fungi specifically infect insects or arthropods killing^[53-55]. The majority of the fungi are plants nonpathogenic, and almost non-toxic to animals and humans^[56]. nearly, 60% of insect diseases are resulted from these fungi^[57]. Although these fungi show many advantages compared to other chemical and biological products, entomopathogenic fungi still underutilized^[58]. One of the fugal isolates in this study is Rhozopus stolonifera. The effect of Rhozopus on pupation and adult emergence percentages was studied by El-Hawary,^[59] who found that various formulation products of such fungi were able to delay the pupation and adults emergence percentage of Spodoptera littoralis. Moreover, the mode of action of Rhizopus sp. as pest biocontrol was explained, where the studies showed that this fungal species exhibits some hydrolytic enzyme activity such as lipase, chitinases, and protease. One of the most crucial mechanisms of insect fungal infection that ends with mortality is the catalyzing ability exerted by specific enzymes^[60].

 

Another fungus that was isolated in this study from Agrotis ipsilon is Paecilomyces variotii. Some studies reported that Paecilomyces sp. greatly decreased the adult emergence percentage. Also, there was a great decline in the pupation percentage to be 2.5% at concentration of 0.5x10^9,59. Additionally, it was able to penetrate the insect cuticle and cause proliferation in diamondback moth hemolymph⁶¹.

 

Figure 2. Isolated fungi from Spodoptera littoralis and Agrotis ipsilon (a) Paecilomyces variotii, (b) Mucor circinilloides, (c) Penicillium chrysogenum, (d) Aspergillus niger (e) Aspergillus flavus (f) Rhozopus stolonifer (g) Absidia corymbifera. (Photos wastaken by Dr. Waill Elkhateeb).

 

Moreover, Aspergillus flavus was also isolated from Spodoptera littoralis. The association of A. flavus with a huge variety of insects was also reported where it showed its existence with Heliothis zea (corn earworm)⁶² and Ostrinia nubilalis (the European corn borer larvae)⁶³. It was found that 30% of the total fungal isolates got from corn earworms and European corn borer as well as other species were belonging to the group of A. flavus⁶⁴. 

 

Another fungus that was isolated from Spodoptera littoralis in during this study was Mucor circinilloides. The previous studies found that the partially purified crude extract of the protein isolated from Mucor circinelloides has shown chitin deacetylase activity^[65]. Chitinase gain a lot of importance due to its ability to lyse the insect cuticle^66-68. Penicillium chrysogenum and Absidia corymbifera were also isolated during this study from adult wings and larval feces of Agrotis ipsilon respectively. Generally, all kinds of relations between insects and other microorganisms are continuously attracting scientists attention especially where mushrooms^[69-77] or actinomycetes are involved^78-79.

 

4. CONCLUSION:

Long time use of synthetic chemical agents to control insect pests resulted several negative points against non-target organisms, users and environment in general in addition to increase of target pest resistance. To avoid such problems, many researches searching intensively for more safe alternatives such as, natural enemis, natural products toxic to the pests and different types of pathogens associated with the pests or isolated from surrounded area to get more successful results. In this study, some bacterial and fungal isolates were isolated from mature and immature stages of two lepidopterous insect pests (Spodoptera littoralis and Agrotis ipsilon). Isolates obtained can be used in integrated pest management (IPM) programs to protect several field crops against insect infestation especially from order lepidoptera as stated in many previous studies.   

 

5. ACKNOWLEDGMENT:

Not applicable

 

6. CONFLICT OF INTEREST:

The authors declare there is no conflict of interest.

 

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Accepted on 24.02.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(11):5825-5830.