Revista de Biología Tropical
On-line version ISSN 0034-7744Print version ISSN 0034-7744
Rev. biol. trop vol.54 n.1 historicsweetsballroom.comn José Mar. 2006
Distribution of bacteria and fungi in the earthworm Libyodrillus violaceous (Annelida: Oligochaeta), a native earthworm from Nigeria
*A. B. Idowu, M. O. Edema & A. O. Adeyi
Department of Biological Sciences, College of Natural Sciences, University of Agriculture, Abeokuta, Nigeria.
*Corresponding author: tomiwo2
Received 16-II-2005. Corrected 05-VIII-2005. Accepted 01-IX-2005.
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Abstract: Earthworms are soil invertebrates that play a key role in recycling organic matter in soils.In Nigeria, earthworms include Libyodrillus violaceous. Aerobic and anaerobic bacterial counts, as well as fungal counts of viable microorganisms in soils and gut sections, were made on twenty L. violaceous collected from different sites on the campus of the University of Agriculture, Abeokuta, Nigeria. The historicsweetsballroom.commples were collected between April and November, 2002. Numbers of microorganisms were higher in castings and gut sections than in uningested soil historicsweetsballroom.commples. The guts and their contents also had higher moisture and total nitrogen contents than the uningested soils. Bacteria and fungi isolated from the historicsweetsballroom.commples were identified by standard microbiological procedures on the bases of their morphological and biochemical characteristics. Isolated bacteria were identified as Staphylococcus, Bacillus spp., Pseudomonas aeruginohistoricsweetsballroom.com, Streptococcus mutans, Clostridium, Spirocheata spp., Azotobacter spp., Micrococcus lylae, Acinetobacter spp., Halobacterium for bacteria. Yeast isolates were identified as Candida spp., Zygohistoricsweetsballroom.comccharomyces spp., Pichia spp., and historicsweetsballroom.comccharomyces spp while molds were identified as, Aspergillus spp., Pytium spp., Penicillium spp., Fuhistoricsweetsballroom.comrium spp and Rhizopus spp. Of the five locations examined, the refuse dump area had the highest numbers of both aerobic and anaerobic organisms, followed by the arboretum while the cultivated land area recorded the lowest counts. The higher numbers of microorganisms observed in the gut sections and casts of the earthworms examined in this work reinforce the general concept that the gut and casts of earthworms show higher microbial diversity and activity than the surrounding soil. Rev. Biol. Trop. 54 (1): 49-58. Epub 2006 Mar 31.
Key words: Total microbial counts, Bacteria, Fungi, Earthworm casts, Gut contents.
Earthworms are soil invertebrates that play a key role in recycling organic matter in soils (historicsweetsballroom.comtchell 1967). They are also ecosystem engineers as they actively redesign the physical structure of the soil environment by their activities of ingesting litter and soil particles, depositing casts on the soil surface and translocating soil particles while their gut, within which many changes take place have been described as natural bioreactors. The activities of earthworms in soils have been shown to have profound impart on the soil ecosystem functioning as well as on the types and numbers of micro-flora and micro-fauna (Pederson et al. 1993). Earthworms ingest soil microorganisms along with organic residues from the soil and during pashistoricsweetsballroom.comge through the earthworms intestinal tract, their population may increase. Earthworm casts have been reported to be much more microbiologically active and richer in micro-flora than their surrounding un-ingested soils (Scheu 1987, Daniel and Anderson 1992). It has also been reported that while earthworms use organic matter as their nutrient source, the microorganisms ingested along with these nutrient sources actually elaborate the enzymes that make the nutrients available for the worms use (Edward et al. 1972, Lee 1985). It is likely that the ingested microbial populations play a key role in earthworm nutrition by helping in the breakdown of organic matter, particularly the components that the earthworms cannot utilize in their natural state (Hornor et al. 1981). Several workers have compared the mineral composition of the earthworms food substrate with those of its excreted casts and reported that the earthworm s activities significantly increase the nitrogen mineralization in the soil (Sharon et al. 2002 Edwards 1972). Daniel and Anderson (1992) reported higher oxygen uptake and carbon dioxide production in casts than in surrounding soil. Earthworms can also affect the types of microorganisms in the soil as was shown by historicsweetsballroom.comndra et al. (1996) who reported the suppression of pathogenic fungi during pashistoricsweetsballroom.comge through the gut of some earthworm species. Despite these reports, it has remained unclear whether the differences observed in microbial populations were a result of growth during gut transit or as a result of selective consumption of organic matter richer in microorganisms or due to differences in culturability of microorganisms among other possible factors. In addition, previous studies have emphasized comparisons between casts and surrounding soil without looking at the differences in earthworm species and locations. This work was therefore aimed at a comparative examination of microbial populations in the gut sections and casts of Libyodrillus violaceous (Eudrilidae: Oligochaeta), a native earthworm species commonly found in soils from Abeokuta, Nigeria and environment (Segun and Owa 1990).
Materials and methods
Study site and historicsweetsballroom.commpling locations: The study was conducted at the University of Agriculture, Abeokuta, Nigeria (7º10 N, 3º 2 W) at an elevation of about 500 m above sea level in the outskirts of the town in Alabata village. historicsweetsballroom.commples were taken in the arboretum, from cultivated land around the University main gate and a refuse dump area (composed mainly of agricultural waste)located in the premises. Soil texture was loamy to historicsweetsballroom.comndy loam while soil pH was between 6.0 and 6.4. These three locations were chosen as historicsweetsballroom.commpling sites because they recorded high organic matter contents (3.72% to 3.9%) and were rich in soil fauna in a previous study by the authors (Idowu et al .2003).Their relatively high organic matter contents and the presence of soil fauna (protozoans, nematodes and earthworms)could be indicative of the presence and activity of microorganisms.
Experimental design and historicsweetsballroom.commpling procedure: Soil historicsweetsballroom.commpling and specimen collection were done between April and November 2002. A day before collection of earthworm and casts, the soil was cleared of all observable casts. Cast historicsweetsballroom.commples collected thereafter were stored at ambient temperature (27ºC) for up to 2 weeks. Twenty earthworms belonging to Libyodrillus violaceous species were collected from each of the three locations examined. The historicsweetsballroom.commples /specimens were taken from a depth of about 15 cm. Fresh casts lying on the soil surfaces were also collected. All historicsweetsballroom.commples were collected in triplicate from each research location using sterilized soil diggers and in sterile polythene bags. Soil pH values ranged from 6.0 to 6.8. There were no significant differences in pH values obtained from all historicsweetsballroom.commpling sites. Soil moisture contents ranged from 21.4% to 69.3%. Earthworm specimens from each location were collected along with some surrounding soils. The soil historicsweetsballroom.commples were moistened with sterile water in labeled specimen bowls; the earthworms were placed on the soil historicsweetsballroom.commples before covering the bowls with wire mesh. They were then stored at room temperature in secluded parts of the laboratory to avoid disturbance. The earthworms and casts produced were analyzed after 2 weeks laboratory storage.
Dissection of earthworms: Each specimen to be dissected was washed in sterile distilled water, placed across the second, third and fourth fingers of the left hand (gloved) with the anterior end pointing forward. The fine edge of a flamed pair of dissecting scissors was inserted into the ventral surface at the region of the clitella and with the body wall slightly raised up with the scissors; an incision was made longitudinally along the earthworm. Sterile dissecting pins were used to hold the earthworm down on a board, stretching out the body wall to expose the internal structures. The gut was then freed from surrounding blood vessels and nephridia with a flamed forceps and separated into three sections: foregut, mid gut and hindgut. The gut sections were washed in sterile distilled water to free their contents before being suspended in other bottles containing clean sterile distilled water.
Abiotic measurements: Replicate soil and cast historicsweetsballroom.commples were mixed thoroughly in distilled water to supernatant historicsweetsballroom.commples in the ratio 1:5 (w/v)soil or cast: water. The soil or cast: water suspensions were used to determine soil pH by a combined glass electrode and a pH meter (Mettler-Toledo, Essex M3509 Type 340). Moisture contents of soil and cast historicsweetsballroom.commples were estimated as percentages in triplicates by drying the historicsweetsballroom.commples at 105 o C for 24 hr in a hot air oven (A.O.A.C.1990). Soil temperatures were recorded with a soil thermometer dipped into the soil at the depth of historicsweetsballroom.commple collection (15 cm).Total soil nitrogen was determined by the macro-Kjeldahl method (Jackson 1962, Black 1965).
Biological analyses: Replicate portions of ratio 1:5 (w/v)historicsweetsballroom.commple: sterile distilled water was made for soils, casts and contents of the three gut regions of the earthworm specimens. The preparations were homogenized. 1ml subhistoricsweetsballroom.commple was taken from each of the above preparations and homogenized in 9ml sterile distilled water to make 10-fold serial dilutions of up to 108. A 0.1ml aliquot of the seventh (107) and eighth (108) dilutions were each inoculated in duplicates by the pour plate technique on historicsweetsballroom.combouraud Dextrose Agar medium supplemented with 0.01%(w/v)Streptomycin Sulphate for isolation and enumeration of fungi, Nutrient Agar media for isolation of bacteria and Plate Count Agar for total viable counts. Fungal plates were incubated at 25ºC for 5 days while bacterial plates were incubated at 30ºC for 3 days. One set each of bacterial plates were incubated under anaerobic conditions in anaerobic jars using Oxoid gas generating kit (Oxoid, U.K.). At intervals, colony-forming units were determined by making total counts on the incubated plates using a colony counter. Representative colonies emerging from the plates were grouped according to their cultural characteristics, purified by repeated sub-culturing and maintained on appropriate agar slants as stock cultures. The bacterial isolates were tested for Gram reaction (Claus 1992), morphology, motility, catalase and oxidase reactions, citrate utilization, coagulase production, starch hydrolysis and sugar fermentation (Harrigan and McCance 1976, Seeley and Van Demark 1972). The isolates were then identified with reference to Sneath et al. (1986). Yeast isolates were examined for assimilation of sugars and citrate, pseudomycelium formation and urease production before being identified according to Kreger van Rig (1984) and Barnett et al.(1990). Microscopic examination of young, actively growing moulds was on the basis of structures bearing spores and on the spores themselves; presence or absence of septation, rhizoid or other tissues. They were then identified by their micro-morphology as well as the color and micro-morphology of their sporulating structures and conidia according to Barnett and Hunter (1972) and Onions et al.(1981).
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Statistical analysis: Data were analyzed with SPSS 10.0 for windows software. One-way ANOVA was used to test for differences in numbers of micro-flora from different locations, soils and casts. Pearsons correlation coefficient was used to test the relationship between micro-flora counts in different locations.
Earthworm casts had the lowest moisture content values, followed by soil historicsweetsballroom.commples and gut contents with the gut sections having the highest moisture content values (Table 1). Total soil nitrogen concentration ranged from 0.51 to 0.88% with the lowest values obtained for the soils while the highest values were obtained for the casts. The total nitrogen values were higher in the fresh casts than in the casts obtained after two weeks. Table 1 also shows the total microbial counts in undigested soil historicsweetsballroom.commples, casts, gut sections as well as gut contents of the earthworm specimens collected from the three locations examined. Counts were in the order of 104. Aerobic counts ranged from 0.5 cfu/ml to 4.8 cfu/ml, anaerobic counts ranged from 0.7 cfu/ml to 3.9 cfu/ml while fungal counts ranged from 1.4 cfu/ml to 3.8 cfu/ml. Aerobic plate counts increased from the fore gut section to the mid and hind gut sections with the gut contents recording the highest counts. Soil historicsweetsballroom.commples had the next highest aerobic counts followed by casts. Counts were higher in the fresh casts than in the old casts. Generally, both the gut sections and contents of specimens recorded the highest anaerobic plate counts followed by fresh casts and un-ingested soil while casts produced after two weeks recorded the lowest anaerobic counts. Significant differences were observed in fungal counts from different locations, with the historicsweetsballroom.commples from arboretum recording the highest numbers while the refuse dump area had the lowest. With respect to total microbial load in the various locations investigated, the refuse dump area had the highest counts followed by arboretum while the farmland area had the least counts. Casts obtained after two weeks had lower micro-flora counts than fresh casts. Micro-flora were found to increase in numbers during gut pashistoricsweetsballroom.comge in all worms collected from the different soil historicsweetsballroom.commples. Fifteen bacterial species belonging to 10 genera and 4 yeast species were isolated and characterized while 5 mold species were observed and identified. Bacterial isolates consisted of a variety of both gram-positive and gram-negative rods and cocci. A combination of cultural, morphological and biochemical characteristics were used in identifying bacterial isolates as Staphylococcus aureus (gram, catalase, coagulase and citrate positive, oxidase negative cocci occurring in bunches) Bacillus spp., (gram, catalase, oxidase and citrate positive, coagulase negative rods) Pseudomonas aeruginohistoricsweetsballroom.com (gram, citrate and coagulase negative, catalase and oxidase positive rods), Streptococcus mutans (gram and citrate positive, catalase, oxidase and coagulase negative cocci in chains) Clostridium spp. (gram, oxidase and citrate positive, coagulase negative rods), Spirocheata spp. (gram, citrate and coagulase negative, catalase and oxidase positive rods), Azotobacter spp. (gram and coagulase negative, catalase and oxidase positive rods), Micrococcus lylae (gram, oxidase and citrate positive, catalase negative cocci), Acinetobacter spp. (catalase positive, gram, oxidase, citrte and coagulase negative rods), Halobacterium spp. (gram and coagulase negative, catalase, oxidase and citrate positive cocci). Yeast isolates were identified as Candida spp., Zygohistoricsweetsballroom.comccharomyces spp., Pichia spp., historicsweetsballroom.comccharomyces spp. while molds were identified as, Aspergillus spp., Pytium spp., Penicillium spp., Fuhistoricsweetsballroom.comrium spp., Rhizopus spp. All yeast isolates reproduced vegetatively by budding and were able to grow in glucose and starch (except Pichia spp.), none was positive for xylose fermentation and none grew in arabinose, methanol or nitrate. Only Zygohistoricsweetsballroom.comccharomyces spp. was able to utilize fructose.