Microbiology of the Hindgut and Survival of Amitermes evuncifer (Silvestri) on Some Nigerian Woods

MATERIALS AND METHODS

Collection of Materials
Wood-eating termites were collected from infested planks and bamboo in the University of Ado-Ekiti campus, Ado-Ekiti. Termite infested wood containing both the termite workers and soldiers was moistened with distilled water and kept in plastic boxes prior to processing. Termite samples collected were identified as Amitermes evuncifer of the order Isoptera, family Termitidae at the Department of Zoology, University of Ado-Ekiti, Nigeria.

Wood shavings of five tree species were collected from Bashiri Sawmill in Ado-Ekiti. These species are; Milicia excelsa (Welw) C.C. Berg, Mansonia altissima A. Chev, Khaya grandifolila C.D.C., Brachystegia eurycoma Harms and Terminalia superba Engl and Diels.

Termite Survival Tests
Survival of Amitermes evuncifer on five separate shavings of heartwood was measured. Ten grams of wood shavings were placed on sand in a plastic container (12x11 cm). Each container was filled with 500 g of sand and moistened with 50 mL of water to maintain a relative humidity near saturation (Carter et al., 1978; Duryea et al., 1999). The sand (plus substrate) was placed in a chamber while forty termites were added to each container and their conditions were checked daily for 1 week. The control experiment consists of a dish with no substrate (wood shaving), having forty termites, which was set up to determine if termites needed to eat during the experimental period. The survival rate was determined by the number of live termites found in each treatment after the 7 day period.

Assessment of Microflora in the Hindgut of A. evuncifer
Termite samples were taken on daily basis from each of the separate wood shavings and analyzed to determine the bacterial load in the gut samples. Each termite was surface sterilized by rinsing in 10% (v/v) sodium hypochlorite solution and then sterile distilled water. Each of the termites was dissected aseptically to remove the hindgut, which was then placed in 10 mL sterile Ringer’s solution containing the following chemicals (g L^-1): NaCl, 10.0; KCl, 0.42; CaCl₂.2H₂O, 0.48 and NaHCO₃, 0.20 (Amund et al., 1986). The gut sample was then crushed using a sterile glass rod to release the intestinal contents.

Serially diluted gut suspension was plated on Nutrient agar plates in replicates and incubated at 37°C for 48 h. Colonies on plates were counted to determine the total colony forming units of bacteria (cfu) count. Pure cultures of the isolates were obtained by sub culturing serially onto Nutrient agar plates.

Characterization and Identification of Bacterial Isolates
Pure cultures of the isolates were identified based on their cultural, morphological and biochemical characteristics in accordance with the taxonomic scheme of Barrow and Feltham (1999).

Determination of Moisture Content
Moisture content was determined by the AOAC (1990) method. Ten grams of wood shaving of the samples were dried at 50°C to constant weight in a hot air oven (Gallenkamp). The moisture content (%) was calculated using the formula below;

Where,

Determination of Wood Density
Wood disk samples were cut from each wood type. The samples were weighed with a weighing balance and the dimensions were also measured using a vernier caliper. The density was calculated using the formula below;

Where,

Determination of pH
Ten grams each of the wood shaving samples were mixed with 100 mL of sterile distilled water in 250 mL conical flask, shaken and allowed to stand for 3 h. The pH of the mixture was read with a pH meter. The mean of triplicate determinations were calculated.

Statistical Analysis
Analysis of Variance (ANOVA) and regression analysis of data obtained were carried out using SPSS (Statistical Package for Social Sciences) version 11.0 software.

RESULTS AND DISCUSSION

The moisture content, density and the pH of wood samples investigated are shown in Table 1. The moisture content ranged from 10 to 18%, while the pH value ranged from 5.52 to 5.88. The density ranged from 409 to 758 kg m^-3. At the end of the seven days no-choice food experiment, Khaya grandifoliola and Milicia excelsa were left with 5 and 7.5% live termites, while Terminalia superba had 22.5% surviving termites. Survival on Brachystegia enrycoma and Mansonia altissima were 0%, respectively (Fig. 1). Termites consumed 3.5% of the Terminalia superba, 1.5% of Khaya grandifoliola and 1.2% of Milicia excelsa.

Table 1:	Physicochemical properties of wood samples
Averages within a column followed by the same letter(s) are not significantly different, as gauged by Duncan multiple range test

Fig. 1:	Survival of A. evuncifer on wood shaving during a seven-day no-choice food experiment

Fig. 2:	Wood consumption rate (%) by termites during a seven day no-choice food experiment

However only about 0.5% of Brachystegia eurycoma and 1% of Mansonia altissima was consumed by A. evuncifer (Fig. 2). There was a high degree of correlation (R = 0.96; R² = 0.922) between the amount of wood consumed and survival of A. evuncifer on the different wood samples (Fig. 3). There was a low level of inverse correlation between the wood density and the wood consumption rate (Fig. 4). The regression analysis of the physicochemical properties and wood consumption rate is shown in Table 2. There was a gradual decrease in the microbial load from the onset of the experiment to the end. There was a decrease from 10⁴ to 10² cfu mL^-1 in the bacterial population of the hindgut of the termites (Table 3). The bacterial isolates recovered from the gut include Micrococcus luteus, Serratia marcescens, Bacillus sp. and Streptococcus sp. However only Bacillus sp. and Micrococcus luteus persisted till the end of the experiment (Table 4).

Fig. 3:	Correlation curve of wood consumed and termite survival

Fig. 4:	Correlation curve of wood consumed and wood density

Table 2:	Regression analysis of physicochemical properties, survival of termites and wood consumed as dependent variable

Table 3:	Microbial load (cfu mL-1) in the hindgut of A. evuncifer during a seven-day no-choice food experiment
cfu = Colony forming units

Table 4:	Bacterial isolates recovered from hindgut of A. evuncifer during a 7-day no-choice food experiment
+ = Present; - = Absent

The study showed that Brachystegia eurycoma and Mansonia altissima were more resistant to termite infestation than the other three species used in this study. The survival rate of termites on these two woods was 0% for Brachystegia eurycoma (within 48 h) and Mansonia altissima (within 72 h). The termites’ samples died within 48 h of exposure to Brachystegia eurycoma. Termites without food starved and also died within 48 h. The high mortality rate observed in termites fed with B. eurycoma and M. altissima may be due to starvation. Among the wood species T. superba was more consumed by A. evuncifer than other wood types, indicating that the former is more susceptible to termite attack. The wood density was inversely correlated with the rate of consumption of the different wood species. This may explain why T. superba with a density of 409 kg m^-3 was the highest consumed wood. Bultman et al. (1979) reported a general inverse relationship between the hardness of wood and the amount of damage it received from Coptotermes formosanus with the lighter’ softer woods being more heavily damaged than heavier harder woods. Waller et al. (1990), however reported that the wood densities of pine and mahogany (0.52 and 0.73 g cm^-3), respectively did not affect the amount of wood consumed by C. formosanus. Peralta et al. (2004) also reported that wood consumption rates were not correlated significantly with their wood densities; however there was a tendency for softwoods to be more consumed by subterranean termites than the woods of intermediate hardness. The factors affecting wood consumption by termites are multifaceted and complexly linked together (Peralta et al., 2004). Factors affecting wood consumption by termites include wood species and hardness, presence of toxic substances, feeding inhibitors or deterrents, presence or absence of fungi and degree of fungal decay, moisture content of wood and soil (Nagnan and Clement, 1990; Scheffrahn, 1991; Grace, 1997). It has being well documented that in both laboratory and field tests that some antitermic chemical compounds found in woods like terpenoids and quinines acted as a natural repellent for termites (Carter et al., 1978; Scheffrahn, 1991; Grace, 1997).

There was a gradual decline in the microbial load in the hindgut of the termite A. evuncifer maintained on different wood shavings during the seven-day old no-choice food experiment (Table 2). This may be due to the change in the environmental conditions from the natural habitat to the laboratory environment resulting into reduction in the activity of the termites. The amount of wood consumed by the termites correlated with survival rate. However there were no correlations with pH and moisture content (R = 0.5), while the correlation with density of wood was negative.

CONCLUSIONS

Amitermes evuncifer Silvestri consumed more of Terminalia superba than Brachystegia eurycoma and the other wood species tested in this study, indicating that the former is more susceptible to termite attack. Wood consumption correlated with the survival of A. evuncifer, while the wood densities also appeared to influence to some extent the rate of wood consumption. Some other factors such as the chemical components and extractives in the wood may be responsible for the differential rate of wood consumption and termite survival. The present investigation has revealed that B. eurycoma and M. altissima are more resistant to termite attack. Further studies on the effects of the application of the purified extracts of these woods on susceptible woods, will provide additional information on the possibility of using wood extracts as alternative control measure to termite infestation.