Microbial Diversity in the Gut of Cashew Stem Girdler, Analeptes trifasciata Fabricius (Coleoptera: Cerambycidae), in Ibadan, Nigeria.

The cashew stem girdler, Analeptes trifasciata, is a major insect pest of cashew in Nigeria causing economic damage in cashew plantations even at low density. In this study, newly emerged adults of A. trifasciata reared from field-infested cashew stems were collected from the rearing cages, sexed, and dissected to reveal the internal structures of the insects. The gut was excised and separated into the foregut, midgut, and hindgut. The dissected gut compartments were blotted dry by sandwiching in sterile Whatman No. 1 (150 mm) filter paper for a minute. The inoculated gut parts showed the presence of eight fungi flora, namely, Aspergillus repens, Trichoderma spp., Fusarium verticillioides, Lasiodiplodia theobromae, yeast, Aspergillus niger, Fusarium spp., and Rhizopus stolonifer. The frequencies of occurrence of bacteria in the gut compartments of A. trifasciata were Enterobacter spp.: 83.33%; Escherichia coli and Streptococcus spp.: 55.56% each; Staphylococcus spp.: 44.44%; Klebsiella pneumonia: 50% and Salmonella shigella: 11.11%, while each of Serratia marceascea, Pseudomonas spp., and Micrococcus lutea had 5.56% occurrence. The occurrence of mycoflora and microbiota species varied in the gut compartments of A. trifasciata, indicating the role of these microorganisms in metabolic and other bioprocesses of A. trifasciata during digestion and synthesis of complex food substances from the cashew stem substrate. This study would provide basic information for enzymatic studies of A. trifasciata with a view to developing an integrated pest management (IPM) protocol for managing the pest in cashew plantations.

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,2–4 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),5–7 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.2–4,6–13

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

Laboratory rearing of A. trifasciata

Adult emergents of A. trifasciata were obtained from a culture from the fallen infested host plant (cashew stem) samples. As described by Asogwa et al,1 field-collected cashew stems with signs of infestation and egg laying spots of A. trifasciata were collected from cashew plots in Cocoa Research Institute of Nigeria, Ibadan, and cultured in rearing cages (62 cm length × 62 cm breadth × 115 cm height) at ambient tropical temperature (27 ± 2°C) and relative humidity (70 ± 5%). The infested cashew stems were left in the cages undisturbed until adults started emerging at about 55–60 days postcollection into the rearing cages in the Entomology Section, CRIN, Ibadan. Fresh cashew stems with no signs of infestation were supplied weekly as food substrate and laying site for newly emerged A. trifasciata adults.

Dissection of A. trifasciata

Newly emerged adults (1–3-day old) A. trifasciata (three males and three females) that had been feeding on the cashew stems immediately after emerging as adults were collected from the rearing cages, sexed, surface sterilized in 70% alcohol for one minute and rinsed in sterile water, and dissected to reveal the internal structures of the insects. Under a computer-connected Celestron USB Microscope (20×–800× magnification), the gut was excised and dissected in sterile distilled water using sterile scalpel and forceps into three parts (foregut, midgut, and hindgut) following the description by Chapman et al19 for each sex to have the following: foregut male (FGM1–3), midgut male (MGM1–3), hindgut male (HGM1–3), foregut female (FGF1–3), midgut female (MGF1–3), and hindgut female (HGF1–3). The dissected parts were kept separately in sample bottles containing deionized water before inoculation on growth media. The experiment was replicated three times per sex.

Experiment 1: culture and isolation of the mycoflora species

Each of the dissected parts was blotted dry by sandwiching in sterile Whatman No. 1 (150 mm) filter paper for one minute. The potato dextrose agar (PDA) was routinely prepared in the laboratory, sterilized at 121°C for 15 minutes, allowed to cool to about 45°C on the bench, and poured into Petri dishes. The gut parts of A. trifasciata were aseptically inoculated into the acidified PDA in Petri dishes in triplicates and incubated at 28 ± 2°C for five to seven days for the isolation of mycoflora in the gut. The mixed culture growth was subcultured to obtain pure cultures of incidence organisms. The microbial identifications were made on the observed morphological features and characteristics of the isolates with reference to published identification manuals such as Barnett and Barnett and Hunter among others.

Experiment 2: culture and isolation of the microbiota species

The routinely prepared nutrient agar and MacConkey agar were used for the bacteriological assay of the gut of A. trifasciata. The gut parts were separately inoculated into the media and incubated at 37°C for 24 hours. The colonies were counted, occurrence of the associated bacteria was recorded, and gram staining was performed.

Data analysis

Data collected were rated in percentage and frequency for occurrence, while colonies were counted visually to determine the incidence using standard pathological procedures.

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.

GUTS OF Analeptes trifasciata	FUNGI COLONY COUNT	MYCOFLORA	INCIDENCE
FGM1	3	Aspergillus repens, Trichoderma spp., Fusarium spp.	1, 1, 1
FGM2	3	Trichoderma spp.	3
FGM3	3	Trichoderma spp.	3
MGM1	1	Fusarium verticillioides	1
MGM2	1	Rhizopus stolonifer	1
MGM3	2	Trichoderma spp.	2
HGM1	3	A. repens, Lasiodiplodia theobromae	2, 1
HGM2	1	L. theobromae	1
HGM3	1	Trichoderma spp.	1
FGF1	3	A. niger, L. theobromae	2, 1
FGF2	3	Yeast	3
FGF3	3	Yeast	3
MGF1	2	Yeast, L. theobromae	1, 1
MGF2	3	Yeast, L. theobromae	2, 1
MGF3	3	L. theobromae	3
HGF1	3	L. theobromae	3
HGF2	3	Trichoderma spp.	3
HGF3	3	Trichoderma spp.	3

Abbreviations: FGM1–3, foregut male: 1–3; MGM1–3, midgut male: 1–3; HGM1–3, hindgut male: 1–3; FGF1–3, foregut female: 1–3; MGF1–3, midgut female: 1–3; HGF1–3, hindgut female: 1–3.

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 (MGM1–3), 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 MGM1–3, Klebsiella pneumoniae was absent in FGM1–3 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.

GUTS OF Analeptes trifasciata	Escheria coli	Klebsiella pneumonia	Enterobacter spp.	Serratia marceascea	Salmonella shigella	Pseudomonas spp.	Staphylococcus spp.	Micrococcus lutea	Streptococcus spp.
FGM1	−	−	−	−	−	−	+	−	+
FGM2	+	−	+	+	−	−	+	−	−
FGM3	−	−	−	−	−	−	−	−	+
MGM1	−	+	+	−	−	−	−	−	−
MGM2	−	+	+	−	−	−	−	−	−
MGM3	−	+	+	−	−	−	−	−	−
HGM1	+	+	+	−	−	−	+	−	+
HGM2	+	+	+	−	−	−	+	+	+
HGM3	−	+	+	−	−	−	+	−	+
FGF1	+	−	+	−	−	+	−	−	−
FGF2	−	+	+	−	−	−	−	−	−
FGF3	+	+	+	−	−	−	−	−	−
MGF1	+	−	+	−	+	−	−	−	−
MGF2	+	+	+	−	−	−	+	−	+
MGF3	+	−	−	−	−	−	+	−	+
HGF1	−	−	+	−	−	−	−	−	+
HGF2	+	−	+	−	−	−	−	−	+
HGF3	+	−	+	−	+	−	+	−	+
% occurrence	55.56	50	83.33	5.56	11.11	5.56	44.44	5.56	55.56

Abbreviations: FGM, foregut male; MGM, midgut male; HGM, hindgut male; FGF, foregut female; MGF, midgut female; HGF, hindgut female.

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 reports4,20 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.2,4,21–23 Other fungi species in the fore-gut 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 insect2,4,24 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.27–30 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 studies7,16,29 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.