Introduction

A myriad of insect pests attack the cashew plant, Anacardium occidentale (leaf, stem, flower, and fruit–-the pseudo apple), on the field, and prominent among them is the cashew stem girdler, Analeptes trifasciata. This pest causes economic damage to the cashew plant via some of its biological activities (especially feeding and reproduction) on the cashew stem, and it poses serious threat to sustainable cashew production in Nigeria if uncontrolled. The pest is widely distributed in most of the cashew-producing states in Nigeria, and despite being a low-density insect, its damage profile on cashew stem is enormous.¹

Generational success of insects has depended, in part, on the vast relationships with beneficial microorganisms that aid digestion of nutrient-poor diets and recalcitrant food components; protect from predator, parasites, and pathogens; aid the inter- and intraspecific communication,^2–34 and govern mating and reproductive systems.⁵ Similarly, insect guts contain symbiotic protozoans, mycoflora, and/or microbiota populations specialized for performing different functions in insect bioactivities, including nutrient provision (amino acids),^5–67 lignocellulose digestion,^8,9 protection against parasites,^4,7,8 fermentation, and other pertinently prominent roles in the digestive tract of an insect, where they may serve as key mediators of the varied lifestyle of insect host.^{2–34,6–78910111213}

Microbial colonization depends on the physicochemical conditions of the gut compartments, which could be exhibited in varied pH, availability of particular substrates, and redox potential.⁴ Insect guts with large microbial communities have actively high microbial metabolism that determines the condition of different compartments of the gut. For instance, in scarab beetle, Pachnoda ephippiata, there was abundant but varied microbial fermentation products such as acetate, formate, and lactate in the midgut and hindgut of the insect, signifying the specialized activities of microbial communities in the gut compartment of the insect.¹⁴

Since A. trifasciata is a cashew stem girdler, a wood-feeding insect at larval and adult stages, it is believed that it will harbor gut microbial species, especially fungi and bacteria, that are involved in cellulose degradation during metabolism as reported earlier on pine engraver Ips pini,¹⁰ wood-boring beetles–-Anoplophora glabripennis and Saperda vestita,¹⁵–-cricket,¹⁶ termites and cockroaches,¹⁷ and Bombyx mori.¹⁸ However, the microbial communities existing in the gut of this pest (A. trifasciata) are not yet known. The objective of this study was to provide basic and preliminary information on the population diversity of the microbes in the gut of A. trifasciata in Nigeria.

In a bid to control A. trifasciata, chemical control option had been the main viable strategy presently adopted by farmers in Nigeria. This control option has attendant shortcomings such as resurgence of the insect pest 24 hours after the knockdown effect of the available chemicals at the manufacturer's recommended dosage. With the usage of higher concentrations above manufacturers' dosage rate, more insecticides are being used by farmers to control this pest at an extra cost, resulting in other risk factors such as killing of nontarget organisms and also environmental pollution through pesticide residues. Therefore, this study preliminarily explored the bioecology of A. trifasciata via the assessment of the microbial species diversity in the gut of the pest with a view to identifying some potential antagonist microorganism(s) that can alter and/or disrupt the bioactivities of symbiotic microbes in the gut of A. trifasciata, especially during metabolism as an environment-friendly control option.

Materials and Methods

Results

Experiment 1: mycoflora diversity in the gut parts of A. trifasciata

The inoculated gut parts showed the presence of eight fungi flora, namely, Aspergillus repens, Trichoderma spp., Fusarium verticillioides, Lasiodplodia theobromae, yeast, Aspergillus niger, Fusarium spp., and Rhizopus stolonifer (Table 1). Each of the gut parts of A. trifasciata had two or more mycoflora present, each of HGF and MGF had two fungi, and both had L. theobromae and either of Trichoderma spp. or yeast, but three fungi were recorded in each of FGM, HGM, FGF, and MGM. A. repens was common to FGM and HGM, only MGM had F. verticillioides and R. stolonifer, and yeast was common to MGF and FGF.

Table 1.

Incidence, fungi colony count, and the mycoflora species in the gut of A. trifasciata.

The assay of the fungi present in the gut of Analeptes showed that L. theobromae was present in four (HGM, HGF, MGF, and FGF) of the six dissected gut parts and likewise was Trichoderma spp. cultured from four others (FGM, HGM, HGF, and MGM). Fusarium spp. and A. niger were only found in FGM and FGF, respectively. The combination of the highest occurring fungi (L. theobromae and Trichoderma spp.) was recorded in HGM and HGF (Table 1).

Experiment 2: microbiota diversity in the gut parts of A. trifasciata

The bacterial assay of the gut of A. trifasciata is shown in Table 2, comprising nine bacterial genera. The occurrence of the bacteria varied in the gut parts, the Enterobacter spp. recorded 83.33%, Escherichia coli and Streptococcus spp. had 55.56%, while each of Serratia marceascea, Pseudomonas spp., and Micrococcus lutea had 5.56%. A comparison of the incidences of the nine bacterial species is shown in Figure 1. E. coli was present in either one or two replicates of all the gut parts of A. trifasciata except in the midgut of the male (MGM_1–3), Salmonella shigella was found in HGF₃ and MGF₁, and S. marceascea was present only in FGM₂. Both Streptococcus spp. and Staphylococcus spp. were absent in FGF₂ and MGM_1–3, Klebsiella pneumoniae was absent in FGM_1–3 and FGF₁, while Enterobacter spp. was found in all the gut parts. MGF region recorded the highest number of bacteria, followed by FGM, HGM, and HGF regions that recorded five bacterial isolates, but MGM region had only K. pneumonia and Enterobacter spp.; Staphylococcus and Streptococcus were commonly cultured from all gut parts except FGF and MGM, while E. coli, K. pneumonia, and Enterobacter spp. were isolated from HGM, MGF, and FGF regions, but E. coli and Enterobacter spp. occurred together in all gut parts, except one (Table 2).

Table 2.

Percentage occurrence of bacteria species in the gut compartments of A. trifasciata.

Figure 1.

Isolated bacteria species in the guts of A. trifasciata.

Discussion

This study has revealed the diversity in the mycoflora and microbiota communities existing within different gut compartments of A. trifasciata in Nigeria. From this study, only 17 microbial species were identified in the gut of A. trifasciata, and it consists of nine bacteria and eight fungal species. This is in consonance with earlier reports^4,20 that most insect guts contain relatively few microbial species as compared with mammalian guts. The foregut of male A. trifasciata consists mainly of Trichoderma species (78%), which is known to be parasitic to other fungi species, and this may suggest the protection role it plays in the foregut of A. trifasciata in order to maintain balance by killing other harmful fungi species ingested by the insect.^2,4,^21–2223 Other fungi species in the foregut of A. trifasciata include A. repens and Fusarium sp., but they are negligible in incidence rating with 11% each. The Aspergillus species (niger and repens) produce citric and gluconic acids,^{4,16,17,21,22} which might be playing significant roles in the digestion of food materials in the gut of A. trifasciata. This corroborates the possibility of ingestion of environmental fungal spores such as Aspergillus species by the insect that might be playing significant roles in some biological processes of the insect^2,4,24 and that there are antagonistic interactions between different gut microorganisms in insects.⁴

There was a reduction in the percentage occurrence of Trichoderma spp. in the midgut of male A. trifasciata from 77% to about 50% that occurred in the foregut. The midgut of male A. trifasciata had 25% each of F. verticillioides–-a fungus symbiont that is a source of detoxification enzyme²¹–-and R. stolonifer; whereas, the female midgut had mainly yeast (37.5%) and L. theobromae (62.5%). Similarly, reduction trend in Trichoderma sp. along the gut compartments of male A. trifasciata was repeated in the hindgut with 20% occurrence, while other species such as A. repens and L. theobromae had 40% occurrence in all the colonies. This suggests how the gut compartments of this insect harbor varied symbiotic mycofloral communities specific for different functions or roles.^22,25,26 However, there was no incidence of Trichoderma species in the foregut of female A. trifasciata, but A. niger, L. theobromae, and yeast had 22.22%, 11%, and 66.67%, respectively. This suggests the varied digestion processes in the sexes of A. trifasciata, indicating different nutritional requirements for other bioactivities such as mating, egg formation, and laying.^27–282930 The hindguts of male and female A. trifasciata presented varied fungi species with no regular pattern. For instance, the HGM had Trichoderma spp. (20%), L. theobromae (40%), and A. repens (40%), while HGF had only L. theobromae (33.33%) and Trichoderma sp. (66.67%). The occurrence of 40% A. repens in the HGM suggests incomplete digestion of lignocellulose ingested by male A. trifasciata in the midgut compartment and that further digestion and/or reabsorption of food materials via citric and gluconic acid production might be taking place in the HGM; whereas the HGF had no A. repens, suggesting complete digestion of food materials in the MGF. Presence of L. theobromae in HGM and HGF indicates the introduction of the pathogen into the environment (soil) via its excreta and might be indicative for the spread of L. theobromae that is implicated for infections in plant materials.

The species richness of bacteria in the gut compartments of A. trifasciata was low with E. coli existing in the representative samples of FGM, HGM, FGF, MGF, and HGF except in MGM where E. coli was not isolated. The absence of E. coli in MGM might be a factor responsible for the presence or absence of some symbiotic fungi in the gut compartments of the insect as recorded in this study. This corroborates earlier studies^7,16,29 that bacterial mutualists in insect guts play important roles in protecting the host from other potentially harmful microbes and may as well engage in opportunistically harmful interactions with the host. The FGM and HGF recorded 0% occurrence of K. pneumoniae, whereas K. pneumoniae was isolated from other gut compartments of A. trifasciata. Some species identified in the gut compartment of A. trifasciata from this study had been identified in other studies and/or organisms (adults or developmental stages) and include the following: Pseudomonas sp. and Streptococcus sp. in Ips pini;¹⁰ Enterobacter sp., Staphylococcus spp., and Streptococcus spp. in Agrilus planipennis;⁵ S. marceascea and Enterobacter sp. in Longitarsus flea beetle, and Microbacterium sp. in M. lutea.⁷ All these gut microbial species of A. trifasciata play significant roles in its ability to digest the food substrate (cashew stem) containing complex compounds, and this needs further investigations to ascertain the specific role and/or function of the microbiota and mycoflora communities in the gut compartments of cashew stem girdler.

Conclusion

Identification of the 17 gut microbial populations (nine bacteria and eight fungi species) of A. trifasciata forms the basis for investigating the general and specific functions of the fungal and bacterial species associated with the insect, in relation to its generational success in terms of food digestion and utilization, secretion of enzymes, and its reproductive capacity. This information can be very useful in developing entomopathogenic organisms as control options in managing this economic pest of cashew.

Author Contributions

Conceived and designed the experiment and cultured and dissected the insects: AVO. Isolated and identified associated microorganisms in the gut of the insect: DOA. Contributed the reagents: AVO and DOA. Wrote the paper: AVO and DOA. Both authors reviewed and approved of the final manuscript.