Effects of Seasonal Changes on the Microflora In the Hindgut of Wood-Eating Termites
The bacterial population in the hindgut of the higher
wood-eating termite, Amitermes evuncifer Silvestri was estimated
at both dry and wet seasons. The total bacterial counts in the soldier
and worker termites were 1.46Â±0.26x106 and 2.51Â±0.31x106
cfu mL-1 during the wet season; while it was 5.30Â±1.1x104
and 5.8Â±0.9x104 cfu mL-1 during the dry season,
respectively. There was no significant difference in the total bacterial
count in the hindgut of the worker and soldier termites in both seasons.
The total bacterial count in the dry season was significantly lower (pâ‰¤0.05)
than the population during the wet season. The bacterial species were
identified to be Bacillus subtilis, B. cereus, Micrococcus
luteus, Streptococcus sp. and Serratia marcescens.
Termites are social insects having three distinct castes, the reproductive
(queen and kings), the soldiers and workers (Malaka, 1996; Roger et
al., 2006). The workers are involved in the building and maintenance
of the nests. They do all the foraging and care for the eggs and young
as well as caring for the queen. The soldiers are defenders of the nests
(Malaka, 1996). Gut microbes are very important for termite vitality and
much of the termite nutrition is derived from products of microbial metabolism.
Production of acetate, propionates and other organic acids during microbial
fermentation of carbohydrates in the hindgut serves as important oxidizable
energy source for termites (Todaka et al., 2007).
Until recently, hydrolysis and utilization of cellulose in amount sufficient
to produce usable energy to an organism were thought to be carried out
exclusively by microorganisms. It appears that some animal species including
termites, nematodes and crayfish produce their own cellulases, which differ
substantially from those of their indigenous micro flora (Bera-Maillet
et al., 2000; Watanabe and Tokuda, 2001; Khademi et al.,
2002). Wood-eating termites have caused substantial economic loses as
a result of their destruction of wood edifices. Their nefarious activities
are more pronounced during the wet/ rainy season. New frontiers are conquered
and more nests are established during the wet season. There are two seasons
in Nigeria; the rainy/wet and the dry/harmattan, which are characterized
by different ambient temperature, amount of rainfall, relative humidity
and intensity of sunshine. These environmental factors might affect both
the population and types of microorganisms in the hindgut of these insects.
This study reports the effects of different seasons (dry and wet) on
the population of the wood-eating termite, A. evuncifer Silvestri.
The microbial load in the hindgut of workers and soldier termites were
MATERIALS AND METHODS
Geographical Parameters of the Study Area
The amount of total rainfall was monitored using a rainguage (Casela,
London), while the temperature was determined with a dry bulb thermometer
(Model 88982C Casela, London). The relative humidity of the study area
was monitored with a whirling hygrometer (Model T8563/1, Casela, London).
Collection of Termites
Wood-eating termites were collected during the dry (December to March)
and rainy (April to November) season from infested planks and bamboo in
the University of Ado-Ekiti campus and environs. Termite-infested wood
containing both the worker and soldier termites were moistened with distilled
water and kept in plastic boxes prior to processing. Termite samples were
identified as Amitermes evuncifer of the order Isoptera family
Termidae at the Department of Zoology, University of Ado-Ekiti, Nigeria.
Dissection of Termite Samples
Termites were surface-sterilized in 10% (v/v) sodium hypochlorite
and distilled water. Thereafter, they were rinsed in two changes of sterile
distilled water. Each of them was aseptically dissected to remove the
hindgut, which was then placed in 10 mL sterile ringers solution containing
the following (g L-1): NaCl, 10.0; KCl, 0.42; CaCl2.2H2O,
0.48 and NaHCO3, 0.20. The gut sample was then crushed with
a sterile glass rod to release the intestinal contents. Serial dilution
of gut sample was carried out in sterile dilution blanks of distilled
water and plated on Nutrient Agar (NA) plates and Potato Dextrose Agar
(PDA) amended with penicillin (Ako-Nai et al., 1992). The replicate
plates were incubated at 37Â°C for 2 days (NA) to 5 days (PDA), respectively.
Colonies on plates were counted and multiplied with the dilution factor
to determine the total microbial count. The mean of the total count was
calculated and expressed as colony forming units per milliliter (cfu mL-1).
Cultural characteristic of bacterial isolates on agar plates were recorded.
Pure cultures of the isolates were obtained on agar plates obtained by
sub-culturing serially onto sterile Nutrient agar plates.
Characterization and Identification of Bacterial Isolates
Pure cultures of the isolates were identified on the basis of their
cultural, morphological and physiological characteristics by reference
to Holt et al. (1994) and Barrow and Feltham (1993). The tests
performed include Gram-stain, spore-stain, motility test, catalase test,
coagulase, urease production, nitrate reduction, methyl red, Voges-Proskauer,
oxidative/fermentative test, gelatin hydrolysis, oxygen requirement, growth
at different salt concentration and utilization of carbon-sources.
Physiological Studies of Microorganisms in the Hindgut of Amitermes
Bacterial isolates were screened for their cellulolytic and amylolytic
Determination of Cellulolytic Activities
The medium was Skinner cellulose medium B (Skinner, 1960), supplemented
with either 1% (w/v) Whatman powdered cellulose or 1% carboxymethyl cellulose
(CMC). The Skinner medium without cellulose served as control. Each of
the isolates were streaked on plates containing carboxymethyl cellulose
and incubated at 35Â°C for 48 h, while those containing cellulose powder
were incubated at 27Â°C for 5 days. Culture plates were flooded with
1% Congo red, followed by 2M NaCl; and observed for zone of clearing by
cellulose or carboxymethyl cellulose-degrading bacteria (Paul et al.,
Determination of Amylolytic Activity
Alpha amylase production was tested by inoculating the bacterial culture
on starch agar (Nutrient agar plus 1% soluble starch). Plates were incubated
at 35Â°C for 3 days, after which they were flooded with iodine solution
(0.3% I2 (w/v) in 3% KI (w/v)). Amylase activity was indicated
by a clear/brown zone surrounding the colony.
Analysis of variance of data obtained was carried out using the Statistical
Package for Social Sciences (SPSS version 11.0).
The amount of total rainfall in the study area was about 1500 mm in September
and October. During wet season (April to November), the relative humidity
ranged between 85 and 90%, while it ranged between 50 and 55% during the
dry season (December to March). The temperature during the wet season
ranged from 23 and 26Â°C, while it ranged between 27 and 32Â°C during
the dry season. The annual mean temperature was approximately 27Â°C.
The bacterial count in the hindgut of A. evuncifer is shown in
Table 1. The total bacterial counts in soldier and worker
termites were 1.46Â±0.26x106 and 2.51Â±0.31x106
cfu mL-1, respectively during the wet season. During the dry
season, the bacterial counts were 5.30Â±1.1x104 and 5.80Â±0.9x104
cfu mL-1 in the soldier and worker termites respectively. The
total microbial count during the wet season was significantly higher than
the dry season (pâ‰¤0.05). However, there were no significant differences
in the total bacterial counts in the gut of the worker and soldier termites
in both seasons.
The characterization of bacterial isolates revealed that five different
species of bacteria were isolated in the hindgut of A. evuncifer
during the wet season. The bacteria were identified as Micrococcus
luteus, Bacillus subtilis, B. cereus, Streptococcus
sp. and Serratia marcescens. However, only Micrococcus
luteus, Bacillus subtilis and Streptococcus sp. were
isolated during the dry season (Table 1). The rate of
occurrence of the bacterial isolates is shown in Table 2.
Micrococcus had the highest occurrence of 100%, followed by B.
subtilis which had 87.5% in both worker and soldier termites, respectively
during the wet season. However the occurrence rate of B. subtilis
during the dry season was 35 and 45% in the hindgut of soldier and worker
termites, respectively. All the isolates in this study were amylolytic,
while 73.7% of the Bacillus species was cellulolytic. None of the
isolate showed cellulolytic activity on Whatman cellulose agar medium
|| Total load (cfu mL-1) of bacteria in the
hindgut of Amitermes evuncifer during the wet and dry seasons
|Cfu = Colony forming unit
|| Rate of occurrence of bacterial isolates
||Enzymatic (amylase and cellulase) activities of bacteria
isolated from the hindgut of A. evuncifer
|*CMC = Carboxymethyl cellulose
This study has shown that the hindgut of worker termites were more populated
with bacteria than the hindgut of the soldier caste, both during the wet
and the dry seasons. This observation is in agreement with the findings
of Amund et al. (1986), who suggested that the high bacterial counts
in workers could be attributed to the division of labour in the termite
colonies. The workers are more active because they are responsible for
the building of forage galleries nest and also take care of the young
ones (Malaka, 1996). The high total bacterial population during the wet
season may be due to the more pronounced activity of the termites during
the wet season in which they build nest. The various environmental factors
viz temperature, relative humidity and amount of rainfall characterizing
the different season could be favorable to termite proliferation and the
multiplication of the hindgut micro flora during the wet season. Four
genera of bacteria were encountered in the hindgut of the wood-eating
termite, A. evuncifer and they were identified to be species belonging
to the genera Serratia, Micrococcus, Bacillus and
Streptococcus. Some other researchers have reported the presence
of these genera of bacteria in the gut of termites (Amund et al.,
1986; Ohkuma and Kudo, 1996; SchÃ¤ffer et al., 1996). Micrococcus
luteus had the highest incidence rate in both workers and soldier
The involvement of the bacterial flora of the termite gut in the degradative
activities, particularly of cellulose, which forms the bulk of the termite
diet, was investigated in this study. Bacillus sp. were found to
constitute the major celluloytic bacteria in the hindgut of A. evuncifer
The occurrence of M. luteus and other non-cellulolytic organism
may indicate the preponderance of degradative bye-products of cellulolytic
activities in the hindgut. Such degraded compounds could readily be utilized
to enhance the proliferation of non-cellulolytic microbes in the hindgut.
Lee et al. (2002) reported that in nature, cellulose utilization
is carried out by multiple cellulolytic species coexisting with each other
and with many non-cellulolytic species. While cellulolytic species compete
for cellulose, both cellulolytic and non-cellulolytic species compete
for the products of cellulose hydrolysis (Fondevilla and Dehority, 1996).
All the strains of Bacillus species were cellulolytic on carboxymethyl
cellulose agar medium; none of them hydrolyzed Whatman cellulose powder.
This may be due to the difference in the structures of the two cellulose
substrates. It has been reported that the hydrolysis of amorphous cellulose
is much faster than the crystalline forms of cellulose (Beguin, 1990).
However, Serratia marcescens hydrolysed both the crystalline and
non-crystalline forms of cellulose. Cellulose degradation requires the
presence of a complex of enzymes namely exo-Î²-1, 4 glucanase, endo-Î²-1,
4 glucanase (cellobiohydrolase) and Î²-D-glucoside glucohydrolase
(Î² glucosidase) (Beguin and Aubert, 1994).The detection of amylolytic
activity in all the bacterial isolates might be due to the fact that amylase
is a constitutive enzyme in many microorganisms (Pandey et al.,
Even though it has been well documented that the microflora in the hindgut
of A. evuncifer, could degrade cellulose, further research is needed
to determine the presence of endogenous cellulase gene in the hindgut
of Amitermes evuncifer and also to investigate its relationship
with the cellulase gene of the bacterial isolates in the hindgut of A.
Ako-Nai, K.A., O. Adejuyigbe, T.O. Adewumi and O.O. Lawal, 1992. Sources of intra-operative bacteria colonization of clean surgical wound and subsequent post-operative wound infection in a Nigerian hospital. East Afr. Med. J., 69: 500-507.
Amund, O.O., O.S. Yakubu and S.L.O. Malaka, 1986. A study of bacteria from the digestive system of two advanced termites (Isoptera, Termitidae) in Nigeria. Nig. J. Biol. Sci., 1: 19-24.
Barrow, G.I. and R.K.A. Feltham, 1993. Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd Edn., Cambridge University Press, Cambridge, UK., Pages: 331.
Beguin, P. and J.P. Aubert, 1994. The biological degradation of cellulose. FEMS Microbiol. Rev., 13: 25-58.
Beguin, P., 1990. Molecular biology of cellulose degradation. Ann. Rev. Microbiol., 44: 219-248.
Bera-Maillet, C.L., P. Arthaud and M.N. Rosso, 2000. Biochemical characterization of MI-ENGI a family 5 endoglucanase secreted by the root-knot nematode Meladogyne incognita. Eur. J. Biochem., 267: 3255-3263.
CrossRef | PubMed |
Fondevilla, M. and B.A. Dehority, 1996. Interaction between Fibrobacter succinogenes, Prevotella ruminicola and Ruminococcus flavefaciences in the digestion of cellulose from forages. J. Anim. Sci., 74: 678-684.
Direct Link |
Holt, J.G., N.R. Kreig, P.H.A. Sneath, J.T. Staley and S.T. Williams, 1994. Bergey's Manual of Determinative Bacteriology. 9th Edn., Lippincott Williams and Wilkins, Baltimore, USA., ISBN-13: 9780683006032, Pages: 787.
Khademi, S., L.A. Guarino, H. Watanabe, G. Tokuda and E.F. Meyer, 2002. Structure of an endoglucanase from termite Nasutitermes takasagoensis. Acta Cryst., D58: 653-659.
Direct Link |
Lynd, L.R., P.J. Weimer, W.H. van Zyl and I.S. Pretorius, 2002. Microbial cellulose utilization: Fundamentals and biotechnology. Microbiol. Mol. Biol. Rev., 66: 506-577.
CrossRef | PubMed | Direct Link |
Malaka, S.L.O., 1996. Termites in West Africa. 1st Edn., University of Lagos Press, USA, ISBN: 978-017-144-4, pp: 165.
Ohkuma, M. and T. Kudo, 1996. Phylogenetic diversity of the intestinal bacteria community in the termite Reticulitermes speratus. Applied Environ. Microbiol., 62: 461-468.
Pandey, A., P. Nigam, C.R. Soccol, V.T. Soccol, D. Singh and R. Mohan, 2000. Advances in microbial amylases. Biotechnol. Applied Biochem., 31: 135-152.
PubMed | Direct Link |
Paul, J., S. Saxena and A. Varma, 1993. Ultrastructural studies of the termite Odontermes obesus gut microflora and its cellulolytic properties. World J. Microbiol. Biotechnol., 9: 108-112.
Roger, E.G., V. Harry and J.G. Grady, 2006. Subterranean Termites. Texas Agricultural Extension Service. The Texas A and M University System.
Schafer, A., R. Konrad, T. Kuhnigk, P. Kampfer, H. Hertel and H. Konig, 1996. Hemicellulose-degradating bacteria and yeasts from the termite gut. J. Applied Microbiol., 80: 471-478.
CrossRef | Direct Link |
Skinner, F.A., 1960. The Isolation of Soil Clostridia. In: Isolation of Anaerobes. Society of Applied Bacteriology Technical Series 5. Shapton, P. and R.G. Board (Eds.). Academic Press, New York, pp: 57-80.
Todaka, N., S. Moriya, K. Saita, T. Hondo and I. Kiuchi et al., 2007. Environmental cDNA analysis of-the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus. FEMS Microbiol. Ecol., 59: 592-599.
Direct Link |
Watanabe, H. and G. Tokuda, 2001. Animal cellulases. Cell Mol. Life Sci., 58: 1167-1178.