applied soil ecology 39 (2008) 127–132

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Changes in density of nematodes, protozoa and total coliforms after transit through the gut of four epigeic ()

Fernando Monroy *, Manuel Aira, Jorge Domı´nguez

Departamento de Ecoloxı´a e Bioloxı´a , Universidade de Vigo, Lagoas-Marcosende, E-36310 Vigo, Spain article info abstract

Article history: Epigeic earthworms are detritivorous organisms that live and feed in the soil litter layer. Received 17 April 2007 These earthworms exert important effects on the presence of decomposer micro-organisms Received in revised form and their microbial grazers, which lead to changes in the rate of decomposition of the 21 November 2007 organic matter. To assess the effect of the transit through the gut of epigeic earthworms on Accepted 29 November 2007 the decomposer organisms, we analysed the gut contents of four epigeic species reared under laboratory conditions, with pig slurry as food source. The numbers of nematodes, protozoa (flagellates, testate and naked amoebae) and total coliforms in the hindgut of the Keywords: species, , Eisenia andrei, Lumbricus rubellus and eugeniae, were Gut associated processes compared with the numbers of the same organisms in the pig slurry. No nematodes were Decomposer community found after transit through the gut of the earthworms and there was a decrease between 3 À1 Eisenia fetida 85% and 98% in the numbers of total coliforms of the pig slurry (723 Æ 125 Â 10 CFU g dw). E. andrei The effect on the numbers of protozoa depended on both groups of protozoa and species of Lumbricus rubellus earthworm considered. The numbers of flagellates were greater in the gut samples of E. 3 À1 Eudrilus eugeniae fetida and E. andrei (363 Æ 46 Â 10 MPN g dw) than in the ones of L. rubellus and E. eugeniae (105 Æ 26 Â 103 MPN gÀ1 dw). The density of testate amoebae was not affected by the transit through the gut of earthworms, and the numbers of naked amoebae were greater in the gut samples of L. rubellus (2500 Æ 780 Â 103 MPN gÀ1 dw) than in the fresh pig slurry (755 Æ 311 Â 103 MPN gÀ1 dw). The results indicate that short-time effects associated with the digestive activity in the earthworm gut play an important role in the changes that epigeic species exert on the decomposer community. # 2007 Elsevier B.V. All rights reserved.

1. Introduction matter (Lavelle and Spain, 2001). Microbial communities also support a large number of soil invertebrates, which in turn Although it is well known that soil decomposer organisms have an important regulatory effect on the microbial popula- interact in a complex way, most studies addressing these tions (Edwards, 2000). The way that soil invertebrates interact interactions focus on the relationships between soil fauna and with soil micro-organisms largely depends on their size and micro-organisms (fungi and bacteria) (Bardgett, 2005). Micro- morphology (Swift et al., 1979). Microfauna (2–100 mm body organisms are directly responsible for the decomposition width) such as nematodes and protozoa, are restricted to films process, and show a high degree of specialization and display a of water and water-filled pores, and feed directly on fungi and large number of enzymes for the breakdown of the organic bacteria; meso- and macro-fauna (0.1–2 mm and 2–20 mm

* Corresponding author. Tel.: +34 986 812 593; fax: +34 986 812 556. E-mail address: [email protected] (F. Monroy). 0929-1393/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2007.11.011 128 applied soil ecology 39 (2008) 127–132

body width, respectively) are detritivores that ingest soil and Table 1 – Physicochemical characteristics of the fresh pig decaying materials together with micro-organisms (Lavelle slurry used and Spain, 2001). Nematodes are the most abundant metazo- Moisture content (%) 85 Æ 2.0 ans in soils (Dindal, 1990), and together with protozoa Organic matter content (%) 86 Æ 1.0 pH 8.3 Æ 1.0 constitute the main consumers of soil bacteria (Ekelund and Electrical conductivity (mS cmÀ1) 0.25 Æ 0.01 Rønn, 1994). These microbial grazers have an important Total nitrogen (mg gÀ1 dw) 24 Æ 2 regulatory effect on the decomposition process, since they + À1 N-NH4 (mgg dw) 2400 Æ 100 À À1 affect microbial activity and increase nutrient mineralization N-NO3 (mgg dw) 250 Æ 50 (Ingham et al., 1985; Bamforth, 1988). Total carbon (mg gÀ1 dw) 455 Æ 60 À1 Some larger , such as earthworms, can simulta- Dissolved organic carbon (mg g dw) 11.1 Æ 0.1 neously affect soil micro-organisms and their microbial dw: the dry weight. grazers through their feeding, burrowing and casting activities (Brown et al., 2000). The relative importance of each of these activities on the soil community depends on the ecological 2.2. Mesocosms features of the earthworm species under consideration (Lavelle and Spain, 2001). Epigeic earthworms (i.e. those that For each of the four earthworm species, we set up five feed and live in the litter layer) increase decomposition rates mesocosms consisting of plastic containers (3 L), which were and strongly affect populations of other litter inhabiting filled to three-fourth of their capacity with sieved (>2 mm) and organisms (Domı´nguez et al., 2003; McLean and Parkinson, moistened (80% moisture content) vermiculite. We chose 2000a,b). Nevertheless, little is known about whether and to vermiculite as a substrate because it is chemically inert and what extent these changes are due to direct effects of does not contain any nutrients, which thus obliges the earthworms on the decaying material (i.e. transformations earthworms to ingest the pig slurry provided. Each of the of the ingested material during passage through the gut) or to mesocosms was inoculated with either 50 mature earthworms indirect effects related to their casting and burrowing (E. fetida, E. andrei) or 20 mature specimens (L. rubellus, E. activities (Brown et al., 2000; Domı´nguez, 2004). eugeniae). A plastic mesh (1 cm pore size) was placed over the The first step of the interactions between these animals surface of the vermiculite and the pig slurry (ca. 200 g, fresh and the rest of the soil community are the short-term changes weight) was placed on top of the mesh, to avoid mixing the pig associated with digestion of the organic material in earth- slurry with the vermiculite and to facilitate removal of worms’ guts (Domı´nguez, 2004). In the present study we tested consumed material. Mesocosms were checked twice weekly, the hypothesis that digestion of organic material by epigeic the pig slurry replaced and the vermiculite washed to prevent earthworms has negative effects on the density of both the earthworms ingesting casts. The mesocosms were main- microfauna and micro-organisms, and that those effects are tained at a constant temperature (20 8C) in a scientific incubator. earthworm species-specific. In order to test this hypothesis, the densities of protozoans, nematodes and total coliforms in 2.3. Sampling method the gut contents of four epigeic earthworm species were quantified and compared with the densities of the same After 1 week in the mesocosms, several mature earthworms of organisms in the food source supplied to the earthworms. similar weight were removed from each mesocosm, washed three times with sterile distilled water, and the gut contents released by gently pressing the bodies of intact worms, with 2. Materials and methods tweezers, from the last third to the posterior end (Bonkowski and Schaefer, 1997). In doing this, we assumed that the gut 2.1. Earthworm species and breeding culture content corresponding to the final section of the intestine (hindgut) was obtained (Horn et al., 2003). Gut contents from Four earthworm species belonging to two different families earthworms belonging to the same species and mesocosms were used in the study. Specimens of Eisenia fetida (Savigny, were pooled to obtain samples that weighed approximately 1826), Eisenia andrei Bouche´,1972andLumbricus rubellus 50 mg (fresh weight). Five specimens of E. eugeniae and 15 each Hoffmeister, 1843 (family Lumbricidae) were obtained by of E. fetida, E. andrei and L. rubellus were required to achieve the sampling (hand-sorting method) a manure heap and a pig- appropriate weight of sample. breeding farm near the University of Vigo, Spain. Specimens of Eudrilus eugeniae Kinberg, 1867 (family ) were obtained 2.4. Microfaunal and microbial analysis from a commercial supplier (Minhobox, Brazil). Earthworms of all four species were reared in the laboratory, with pig slurry as Numbers of three different groups of protozoa (flagellates, the substrate, for 2 months before the start of the experiment. naked and testate amoebae) were estimated by the MPN Fresh pig slurry, obtained from a pig-breeding farm near the method (Darbyshire et al., 1974). For this, 30–60 mg of gut University of Vigo, NW Spain, was homogenized in a slurry pit, content from each earthworm species and 1 g of pig slurry thenstoredinsealedplasticcontainersandkeptat5 8Cuntiluse. (n = 5) were suspended in NBAS (1:100) (Griffiths, 1989). The solid fraction (15% dry weight) of the pig slurry was used to Twofold dilution series with NBAS were set up in microtitre avoid the worms cominginto contact with the percolates,which plates (8 Â 12 wells) that were incubated at 15 8C in dark may be harmful to earthworms. The main physicochemical conditions. The plates were checked with an inverted characteristics of the pig slurry are shown in Table 1. microscope (200Â, Olympus CK2) after 4 days for the presence applied soil ecology 39 (2008) 127–132 129

of naked amoebae and flagellates, and after 10 days for testate log transformed to fulfil the requirement of homogeneity of amoebae. The presence of protozoa was transformed to MPN variances. Because some of the protozoan cultures were lost by following the tables from Rowe et al. (1977). during the incubation process, post hoc comparisons were The nematodes in the pig slurry were extracted from 10 g of performed by Unequal N HSD test for flagellates and naked sample (fresh weight, n = 5) in modified Baermann funnels and amoebae, and by Tukey HSD test for testate amoebae and total live specimens were counted at a magnification of 140Â under coliforms. Values given throughout the text are means Æ S.E. a dissecting microscope (Hooper, 1986). The numbers of nematodes in the gut contents of the different earthworm species were counted directly in suspensions of the gut 3. Results content of each earthworm prepared in 1 mL of saline solution (n = 20). The nematodes were sorted in trophic groups (Yeates All the nematodes extracted from the pig slurry et al., 1993) using their oesophageal structure as an indicator (1450 Æ 110 individuals gÀ1 dw) were bacterivores. Passage of (Parmelee et al., 1995). the pig slurry through the gut of all the earthworm species The presence of total coliforms was estimated by the studied had a noteworthy effect on the presence of nema- modified membrane filtration technique (Clesceri et al., 1998; todes, since no live specimens were found in any of the gut EPA, 2000). Samples of 1 g of fresh pig slurry and 30–60 mg of content samples analysed (n = 20). The presence of nematodes gut content from the different earthworm species (n = 5) were was reduced to the appearance of a residual number of suspended at 1:100 in sterile saline. Aliquots of 100 mL from fragmented nematode cuticles in some of the samples. each sample were diluted in 100 mL of sterile saline and then The effect of the gut passage on the presence of protozoa filtered through membrane filters. The filters were incubated was less evident. In general there was no great difference 1 in Petri dishes with Chromocult Coliform Agar (Merk KGAA, between the numbers of flagellates, testate and naked Darmstad, Germany) for 24 h in an incubator chamber at 37 8C. amoebae in the pig slurry and in the gut contents of the four The presence of total coliforms was then estimated by earthworm species studied (Fig. 1). Nevertheless, the density counting the number of colony forming units (CFU), at 140Â of flagellates in the gut samples differed in the different under a dissecting microscope. earthworm species, and was three times higher in E. fetida and E. andrei than in E. eugeniae and L. rubellus (Fig. 1a). The testate 2.5. Statistical analyses amoebae were the least abundant group of protozoa in both the fresh pig slurry and the gut content samples (52.7 Æ 10.5 The data were analysed by the GLM procedure of the statistical and 50.9 Æ 9.7 Â 103 MPN gÀ1 dw, respectively), and there were package Statistica (vers. 7). Data (except for flagellates) were no significant differences in the densities of these organisms

Fig. 1 – Differences in the densities of protozoa and total coliforms in samples of pig slurry and gut contents from four species of epigeic earthworms (GLM test). Ef, Eisenia fetida; Ea, Eisenia andrei; Lr, Lumbricus rubellus; Ee, Eudrilus eugeniae. Means W S.E. Different letters indicate significant differences (graphs (a) and (c): unequal N HSD test; graphs (b) and (d): Tukey HSD test). 130 applied soil ecology 39 (2008) 127–132

after the passage through the gut of the earthworms (Fig. 1b). only food source (Neuhauser et al., 1980), which supports the Naked amoebae were the most abundant protozoa in the pig idea that these organisms constitute an important part of the slurry (1.4 Æ 0.5Â 106 MPN gÀ1 dw). Passage through the gut of diet of earthworms (Bonkowski and Schaefer, 1997). E. fetida, E. andrei and E. eugeniae did not affect the density of The remarkable increase in the density of naked amoebae naked amoebae; however the number of amoebae in the gut after the transit through the gut of L. rubellus indicated that samples of L. rubellus was 3.4 times higher than in the pig earthworms could also have positive effects on protozoan slurry (Fig. 1c). populations. In organic matter-rich substrates like the pig Significantly lower numbers of total coliforms were slurry, earthworms could feed preferentially on organic C observed in the earthworm gut contents than in the fresh compounds than on micro-organisms (Aira et al., 2006); in pig slurry (Fig. 1d). Passage through the gut of earthworms some cases, this selective use of the food resources could decreased total coliforms by as much as 85% and this effect favour the populations of protozoa, which remain undigested was species-specific (Fig. 1d). in the earthworm gut. The observed differences among earthworms regarding the density of flagellates and naked amoebae in the gut samples seem to be associated with the 4. Discussion genus of earthworm considered. There were no differences between E. fetida and E. andrei, and the effects of these species Passage through the guts of epigeic earthworms affected the on flagellates were in direct contrast to the effects exerted by E. composition of the microfaunal community of pig slurry. The eugeniae and L. rubellus. These differences may be due to the observed reduction in numbers of nematodes is consistent morphological and physiological characteristics of the diges- with the results of Dash et al. (1980), who reported a large tive system of these earthworms. The oesophagus is longer in decrease in numbers of nematodes in a soil after passage lumbricids (E. fetida, E. andrei and L. rubellus) than in other through the gut of the tropical earthworm Lampito mauritii.In families (Edwards and Bohlen, 1996); the gut typhlosole and our study, the presence of nematode cuticles in the gut the gut enzymatic array vary with the species considered samples indicated that the nematodes were ingested by the (Edwards and Fletcher, 1988; Makeschin, 1997), and the gut earthworms; because of the high density of nematodes transit time is also species-specific (Dash et al., 1986; present in the pig slurry, these organisms may constitute an Hendriksen, 1991). Moreover, E. fetida and E. andrei are closely important part of the diet of the earthworm species under related species that differ morphologically only in their body study. The earthworms probably digest nematodes by the pigmentation (Domı´nguez et al., 2005), and thus close proteolytic activity of the enzymes present throughout their similarities in their digestive abilities should be expected. gut (Edwards and Fletcher, 1988), which would allow the As well as the effects on the microfauna, epigeic earth- earthworms to assimilate amino acids and other compounds worms also reduced the presence of total coliforms, which from the nematodes (Pokarzhevskii et al., 1997). There is no indicates that gut transit has an important effect on the direct evidence that earthworms can feed on nematodes, but microbiological composition of the pig slurry. Total coliforms application of nematicides has a negative effect on earthworm are characteristic of pig manures (Zhu, 2000), and Joergensen populations (McColl, 1984), which may suggest a trophic et al. (1998) showed that the presence of these bacteria was relationship between these organisms. Such a relationship related to the total microbial biomass in soil; nevertheless, would also explain the observed reduction in nematode the density of total coliforms was up to 60 times lower in the numbers in the presence of earthworms (Yeates, 1981; gut samples than in the fresh pig slurry, which suggests that Hyvonen et al., 1994; Ra¨ty and Huhta, 2003; Domı´nguez the effect on coliforms was stronger than on the rest of et al., 2003). micro-organisms. Although Aira et al. (2006) reported a In the present study, the density of protozoa in the pig significant decrease of 1.74-fold in the microbial biomass of slurry was not reduced after transit through the earthworm the pig slurry after passage through the gut of E. eugeniae, guts. Although there is some evidence of a negative effect of several authors found that the densities of bacteria, actino- earthworms on the density of protozoans in the materials that micetes and yeasts in earthworm faeces were equal or greater they ingest (Bonkowski and Schaefer, 1997; Cai et al., 2002), than in parent materials (Shaw and Pawluk, 1986; Scho¨ nhol- such reductions occurred only when the active forms of the zer et al., 1999; Wolter and Scheu, 1999). Earthworm digestion protozoa were considered and did not affect estimates of the can increase the availability of nutrients for micro-organisms, total population (Bonkowski and Schaefer, 1997). Soil proto- rising microbial numbers in casts (Parthasarathi and Ranga- zoan populations are formed by active and resistant forms nathan, 1999) and changing the microbial composition (Ekelund and Rønn, 1994); dilution culture techniques favour (Brown, 1995). There is increasing evidence that earthworms the presence of excysting individuals (Berthold and Palzen- have a specific gut microflora (Karsten and Drake, 1995; Horn berger, 1995; Stevik et al., 1998), and most-probable-number et al., 2005), and the decrease in total coliforms might be calculations probably underestimate the effect of the passage related to competitive interactions between coliforms and through the gut of the earthworms. The secretion of micro-organisms that are specific to the earthworm gut substances in the midgut of Lumbricus terrestris is associated (Brown and Mitchell, 1981). Moreover, the negative effect of with a decrease in the presence of protozoa (Piearce and the passage through the earthworm gut observed in enter- Phillips, 1980); lysis of protozoan cells entails the release of obacteria such as Serratia marcescens, Escherichia coli and organic compounds that can be assimilated by the earth- Salmonella enteridis (Day, 1950; Bru¨ sewitz, 1959; Brown and worms and that appear to be essential for their development Mitchell, 1981) suggests the occurrence of selective effects on (Miles, 1963). In fact, E. fetida can grow with protozoa as the the ingested micro-organisms. applied soil ecology 39 (2008) 127–132 131

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