Novel method for the study of the population dynamics of a genetically modified microorganism in the gut of the earthworm Lumbricus terrestris

Summary A laboratory microcosm study was used to investigate the survival and population dynamics of genetically modified microorganisms (GMM) in the gut of Lumbricus terrestris. Three methods of axenic earthworm production were investigated. An antibiotic mixture of streptomycin and cycloheximide was introduced either passively, mixed with sterile soil or cellulose, or actively, by teflon catheter. Worms treated by all methods lost weight but this was least for the catheter method which was also the only method to produce axenic earthworms. Axenic earthworms were used to determine the effect of competition with indigenous gut bacteria on ingested GMM. The GMM used was Pseudomonas fluorescens, strain 10586/FAC510, with chromosomally inserted Lux genes for bioluminescence, and chromosomal resistance to rifampicin. The bacteria were grown up to the mid-exponential phase before inoculation into earthworms. Bacteria in faecal material were enumerated by dilution plate counting using selective agar. The GMM were re-isolated from the casts of both antibiotic-treated and untreated earthworms. Lower concentrations of GMM and higher concentrations of indigenous bacteria in the casts of untreated compared to antibiotic-treated earthworms suggested that competition is a fundamental control on population dynamics of the introduced bacterial inocula ingested by earthworms.The catheter method, developed in this study, is proposed as a technique to contribute to the risk assessment of environmental release of GMM.     

Gut load and food-retention time in the earthworms Lumbricus festivus and L. castaneus: A field study

Summary Lumbricus festivus and L. castaneus consume dung. In the field, below cow pats, their gut loads were about 0.15 and 0.14 g dry weight g^-1 ash-free dry weight of worm, respectively, but in free soil the loads were higher, about 0.21 and 0.19 g g^-1 ash-free dry weight of worm. The gut contents of dung were lighter than the total ingested material, at about 0.10 and 0.07 g dry weight g^-1 ash-free dry weight of worm, respectively. Field experiments showed that the retention time of dung ranged from >9 to 15 h for L. festivus, and from >3 to 6 h for L. castaneus. The experiments also indicated that L. festivus exploited 20- and 36-day-old dung in different ways, since the gut load was lower in those worms consuming 20-day-old dung than in those consuming 36- to 40-day-old dung. On the basis of these results the calculated consumption rate for L. festivus is 0.08 g dung day^-1 g live weight of worm^-1, and for L. castaneus 0.15 g dung day^-1, with retention times assumed to be at maximum, 15 h, for L. festivus and 6 h for L. castaneus. These calculations indicate that our field population of worms (75 g m^-2) consumes 10–15 t dung ha^-1 180 days^-1, corresponding to the amount of dung produced by 2–3 dairy cows.     

Changes in respiration rate and some physicochemical properties of soil during gut transit through Allolobophora molleri (Lumbricidae,Oligochaeta)

Summary Some physicochemical and biological measurements were carried out on the gut content and casts of Allolobophora molleri, an earthworm which lives in humid soils of Northern Spain. In the anterior part of the gut, water (22% of moist weight of soil) and soluble organic matter (27.4%) had been added to the ingested soil and pH had increased from 5.75 to 7.0. The amount of water-soluble compounds decreased sharply in the middle and posterior parts of the gut and were hardly detectable in control soil or casts. The average O₂ consumption, measured at 28° and 21°C, indicated respectively 2.75-to 12-fold increases in microbial respiration in the gut content compared to the non-ingested soil. These results extend the hypothesis of a mutualistic digestion in earthworms previously proposed for tropical endogeic species.     

Earthworms of different functional groups affect the fate of the Bt-toxin Cry1Ab from transgenic maize in soil

The fate of the insecticidal Cry1Ab protein from crop residues (leaves and roots) of the transgenic maize variety MON810 was studied in the presence and absence of two earthworm species (Lumbricus terrestris, Aporrectodea caliginosa; separate incubations) in soil microcosms. The recombinant Cry1Ab protein was quantified using a highly sensitive ELISA. Control microcosms received corresponding non-transgenic plant material. All earthworms survived in the microcosms over a period of 5 weeks, irrespective of whether they received MON810 or non-transgenic plant material. Weight loss was observed for both earthworm species, independent of the plant material or transgenic modification. A strong decline of immunoreactive Cry1Ab in plant residues (mean initial concentration approx. 5000 ng g⁻¹) of MON810 was observed in all treatments, but in microcosms with earthworms this decline was significantly higher with less than 10% of the initial Cry1Ab concentration remaining after 5 weeks. Cry1Ab concentrations in casts were only 0.1% of those found in remaining plant material of the respective microcosms. No immunoreactive Cry1Ab proteins were found in earthworm tissues (threshold of detection: 0.58 ng g⁻¹ fresh weight). No further decline was found for Cry1Ab concentrations in casts of A. caliginosa during a subsequent period of 3 months of incubation in bulk soil (<0.1 ng g⁻¹) after removal of the earthworms from the microcosms, while in casts of L. terrestris the concentration decreased from 0.4 to below 0.1 ng g⁻¹. In conclusion, this study demonstrates that earthworms enhance the decline of immunoreactive Cry1Ab proteins from maize residues.     

Inorganic and organic phosphorus pools in earthworm casts (Glossoscolecidae) and a Brazilian rainforest Oxisol

We compared differences in soil phosphorus fractions between large earthworm casts (Family Glossoscolecidae) and surrounding soils, i.e., Oxisols in 10 year-old upland agroforestry system (AGR), pasture (PAS), and secondary forest (SEC) in the Central Brazilian Amazon. AGR and PAS both received low-input fertilization and SEC received no fertilization. We found that earthworm casts had higher levels of organic hydroxide P than surrounding soils, whereas fertilization increased inorganic hydroxide P. Inorganic P was increased by fertilization, and organic P was increased by earthworm gut passage and/or selection of ingested materials, which increased available P (sum of resin and bicarbonate fractions) and moderately available P (sum of hydroxide and dilute acid fractions), and P fertilizer application and land-use increased available P. The use of a modified sequential P fractionation produced fewer differences between earthworm casts and soils than were expected. We suggest the use of a condensed extraction procedure with three fractions (Available P, Moderately Available P, and Resistant P) that provide an ecologically based understanding of the P availability in soil. Earthworm casts were estimated to constitute 41.0, 38.2, and 26.0 kg ha⁻¹ of total available P stocks (sum of resin and bicarbonate fractions) in the agroforestry system, pasture, and secondary forest, respectively.     

The effect of earthworms (Lumbricidae) on nitrogenase activity in soil

Summary Nitrogenase activity associated with earthworms, their faeces and activity in soil was measured by the acetylene reduction technique. A clear increase in nitrogenase activity was found in field-deposited casts of Aporrectodea caliginosa in comparison with surrounding soil, although potential nitrogenase activity was significantly higher in soil than in casts. Nitrogenase activity associated directly with earthworms (Lumbricus rubellus ) was detected, indicating the presence of active N₂-fixing bacteria on the body surface and/or in the gut. Laboratory experiments showed that nitrogenase activity in the casts of L. rubellus was higher than in unmodified soil, and that nitrogenase activity in soil was significantly increased by the burrowing and feeding activity of these worms. This paper discusses the possible causes of these earthworm effects on soil nitrogenase activity and some methodological problems of determining the nitrogenase activity.     

Survival of bacteria introduced into soil by means of transport by Lumbricus rubellus

Four strains of bacteria, Rhizobium leguminosarum biovar trifolii, Pseudomonas fluorescens, Pseudomonas cepacia, and Flavobacterium sp., were introduced into loamy sand and then transported by earthworms of the species Lumbricus rubellus to uninoculated soil. Cell densities recovered from the earthworm gut and casts (both expressed per gram dry material) were significantly lower (up to 3 log units) than cell densities recovered from the inoculated soil. Total bacterial counts in casts were similar to those in the inoculated soil. In casts excreted into a sterile environment numbers of colony-forming units (CFU) increased, suggesting a favourable environment for growth. In casts excreted in a non-sterile environment, cell densities of introduced strains decreased. Casts therefore did not offer the introduced bacteria a protective micro-environment for survival in the bulk soil. Transport by worms of R. leguminosarum biovar trifolii and of P. fluorescens appeared to occur mostly by means of cast production; with the Flavobacterium sp. and P. cepacia a large proportion of the cells was possibly transported on the skin of earthworms.     

Microbial activity and nutrient dynamics in earthworm casts (Lumbricidae)

Summary Microbial respiration, microbial biomass and nutrient requirements of the microflora (C, N, P) were studied in the food substrate (soil taken from the upper 3 cm of the mineral soil of a beech wood on limestone), the burrow walls and the casts of the earthworm Aporrectodea caliginosa (Savigny). The passage of the soil through the gut caused an increase in soil microbial respiration of about 90% over a 4-week period. Microbial biomass was increased only in freshly deposited casts and decreased in aging faeces to a level about 10% lower than in soil. Microbial respiration of the burrow walls was only increased over a shorter period (about 2 weeks). The microflora of the soil and the burrow walls was limited by P, whereas in earthworm casts, microbial growth was limited by the amount of available C. In aging faeces the P requirement of the microflora increased and approached that of the soil. Immobilization of phosphate in earthworm casts is probably caused by mainly abiotic processes. C mineralization by soil microflora fertilized with glucose and P was limited by N, except in freshly deposited casts. Ammonium, not nitrate, was responsible for this process. N dynamics in earthworm casts are discussed.     

Organic resources and earthworms affect phosphorus availability to sorghum after phosphate rock addition in semi-arid West Africa

A field experiment was laid out in Burkina Faso (West Africa) on an Eutric Cambisol to investigate the interaction of organic resource quality and phosphate rock on crop yield and to assess the contribution of earthworms (Millsonia inermis Michaelsen) to P availability after phosphate rock application. Organic resources of different quality were applied at a dose equivalent to 40 kg N ha^–1 with or without phosphate rock from Kodjari (Burkina Faso) at a dose equivalent to 25 kg P ha^–1, and were compared with control and single phosphate rock treatments in a factorial complete block design with four replicates. Sorghum (Sorghum bicolor L. Moench) variety SARIASSO 14 was grown. Sheep dung had the highest impact on earthworm casting intensity followed by maize straw. Combining organic resources with phosphate rock reduced earthworm casting activities compared to a single application of organic resources or phosphate rock. Addition of phosphate rock to maize straw reduced P availability in earthworm casts whereas combining sheep dung or compost with phosphate rock increased P availability. The contribution of earthworms to Kodjari phosphate rock solubilisation mainly occurred through their casts, as the available P content of casts was 4 times higher than that of the surrounding soil.     

Short- and long-term effects of the endogeic earthworm Millsonia anomala (Omodeo) (Megascolecidæ, Oligochæta) of tropical savannas,on soil organic matter

Summary This study aimed to establish the effects of Millsonia anomala, a tropical geophagous earthworm common in the humid savannas of Lamto (Ivory Coast), on soil organic matter dynamics over different time scales under laboratory conditions. The texture of casts produced by the worms fed on a shrub savanna soil was not significantly different from that of the soil, which showed that M. anomala ingested soil particles without selection. Physical fractionation of soil organic matter showed that the coarse organic fraction (250–2000 m) was depleted by 25–30% in fresh casts compared to the control noningested soil; this was mainly due to a fragmentation of coarse organic debris. Incubation of casts and a 2-mm sieved control soil under laboratory conditions for more than 1 year showed that the C mineralisation rate was almost four times lower in the casts (3% year^-1) than in the control soil (11% year^-1). We therefore concluded that on a long time scale M. anomala populations may significantly reduce the decomposition rate of soil organic matter in Lamto savannas.     

Changes in potassium availability and other soil properties due to soil ingestion by earthworms

An incubation experiment was conducted to study the changes that occur in potassium availability and other soil properties with ingestion of soil by earthworms. Two soils were used. Raumai soil with high non-exchangeable K and Milson soil with low non-exchangeable K were incubated with two species of earthworm, Aporrectodea caliginosa and Lumbricus rubellus, for 8 weeks. The casts and soil samples were analysed for exchangeable K, Ca, Mg, Na, and H, pH, organic C, and texture. The results indicated that in Raumai soil, the exchangeable K levels of the casts of both earthworm species were significantly higher than for the control soil, the effect being more marked for L. rubellus than for A. caliginosa. In Milson soil, the exchangeable K levels were significantly lower in the casts of both types of earthworm than in the control soil. The nitric acid-extractable K of the soil and casts was not markedly different for either soil type, but available non-exchangeable K values were significantly higher for the casts of L. rubellus from Milson soil than for the noningested Milson soil. In Raumai soil, the exchangeable Ca was higher in the casts of L. rubellus, exchangeable Mg and H were reduced, and exchangeable Na did not change markedly in the cast compared to the control soil. For Milson soil, the casts contained lower exchangeable Ca and H but higher Na and Mg than the control. The casts of both species of earthworm had significantly higher pH values for both soil types. There was no marked difference in the organic C content of the control soil and cast samples for Milson but a reduction in the casts of A. caliginosa for the Raumai soil. Finer fractions increased in the casts of both earthworm species in both soil types.     

Characteristics of earthworm casts affecting herbicide sorption and movement

Various physical and chemical characteristics of earthworm casts collected from a laboratory incubation and a field experiment were examined in relation to their effect on the sorption and the movement of three ¹⁴C-labelled ionic herbicides: atrazine, 2,4-dichlorophenoxyacetic acid, and metsulforon methyl. The earthworm casts contained higher levels of fine fractions and total and soluble C. This is attributed to the grinding action of the earthworm gut and selective feeding on zones with higher organic matter and fine size fractions. The earthworm casts had a higher pH than the source soil, resulting in a higher number of surface negative charges. The earthworm casts sorbed higher amounts of herbicides than the source soil, mainly due to the increases in the amount of organic C and fine size fractions. The incrased sorption of herbicides by the casts resulted in decreased leaching.     

Using the spermosphere model technique to describe the dominant nitrogen-fixing microflora associated with wetland rice in two Egyptian soils

Summary This study is an attempt to describe the dominant N₂-fixing microflora associated with the roots of wetland rice. Rice cultivar Giza 171 was grown in a phytotron on two alluvial Egyptian soils for 8 days, a stage when the nitrogenase activity of undisturbed plants reached a level of 245 × 10^–6 mol C₂H₄ h^–1 g^–1 dry weight of leaf. The roots and rhizosphere soils were then used for counting and isolating dominant diazotrophs. Counts and initial enrichment steps were carried out on a selective medium made of an axenic rice plantlet, the spermosphere model, incubated under 1 % acetylene. The counts were very high, exceeding 10⁸ bacteria g^–1 dry weight of rhizosphere soil. Enterobacteriaceae were dominant; most isolates were Enterobacter cloacae belonging to different biotypes in the two soils. Enterobacter agglomerans, Citrobacter freundii and Klebsiella planticola were also present as members of the dominant microflora. Azospirillum brasilense and Azospirillum lipoferum were present as well, but less abundant.     

Symbiosis with bacteria enhances the use of chitin by the springtail,Folsomia candida (Collembola)

Summary The relationship between Folsomia candida and chitin-degrading microorganisms was studied. On chitin agar, 10¹⁰ bacteria were isolated per g faeces, and 3.8×10¹¹ bacteria per g gut contents, 1/3 of them showing a clear (chitin-free) zone around the colony. The most abundant chitin-degrading bacteria were Xanthomonas maltophilia and Curtobacterium sp. To determine the bacterial contribution in the use of chitin by F. candida, a feeding experiment was carried out. F. candida were fed with chitin, either amended with or without tetracycline as an inhibitor of bacteria. When tetracycline was omitted the biomass of F. candida was increased compared to those fed chitin with tetracycline. However, this result was observed only when the food replacement intervals were long enough to allow bacterial colonization before ingestion of the food. In a food-selection experiment, a preference for chitin colonized with microorganisms as opposed to sterile chitin was found. The results indicate that a mutualistic symbiosis of F. candida with chitinolytic microorganisms is likely to enhance chitin degradation. This relationship is not only intra-intestinal but also involves an extra-intestinal phase.Dedicated to the late Prof. Dr. W. Kühnelt     

Prospects for control of Pythium damping-off of lettuce with Trichoderma,Gliocladium and Enterobacter spp.

Summary Damping-off of lettuce (Lactuca sativa) caused by Pythium ultimum was studied in pots containing a non-sterile potting mix in the glasshouse. Fifty P. ultimum sporangia g^-1 compost reduced the plant stand to 15% and shoot dry weight to 18%, but this reduction was totally prevented by applying Trichoderma harzianum at 2×10⁵ viable propagules g^-1 potting mix. Gliocladium virens and Enterobacter cloacae also alleviated the damping-off. E. cloacae did not adversely affect the action of the fungal antagonists. The co-existence of the bacterial and fungal antagonists was revealed on the root surface and inner surface of the testa by scanning electron microscopy, indicating the compatibility of the biocontrol agents.     

Nitrogen mineralization and reorganization in casts of the geophagous tropical earthworm Pontoscolex corethrurus (Glossoscolecidae)

Summary Mineral N concentrations ranged from 133.1 to 167.8 g g^-1 dry soil in fresh casts of the endogeic earthworm Pontoscolex corethrurus fed on an Amazonian Ultisol; this was approximately five times the concentration in non-ingested soil. Most of this N was in the form of NH _inf4 ^sup+ . N also accumulated in microbial biomass, which increased from a control value of 10.5–11.3 to 67.5–74.1 g g^-1 in fresh casts. During a 16-day incubation, part of the NH _inf4 ^sup+ -N was nitrified and/or transferred to the microbial biomass. Total labile N (i.e., mineral+biomas N) decreased sharply at first (ca. 50% in the first 12 h), and then more slowly. The exact fate of this N (microbial metabolites, denitrification, or volatilization) is not known. After 16 days, the overall N content of the casts was still 28% higher than that of the control soil. Incubation of the soil before ingestion by the earthworms significantly increased the production of NH _inf4 ^sup+ in casts. We calculate that in a humid tropical pasture, 50–100 kg mineral N may be produced annually in earthworm casts. Part of this N may be conserved in the compact structure of the cast where the cast is not in close contact with plant roots.     

Microbial populations,enzyme activities and nitrogen-phosphorus-potassium enrichment in earthworm casts and in the surrounding soil of a pineapple plantation

Summary Total populations of bacteria and fungi, dehydrogenase activity (as a measure of total potential microbial activity), and urease and phosphatase activities were determined in earthworm casts and surrounding laterite soils planted to pineapple. The casts contained higher microbial populations and enzyme activities than the soil. Except for fungal populations, statistically significant (P = 0.05) increases were found in all other parameters. Microbial populations and enzyme activities showed similar temporal trends with higher values in spring and summer and lower values in winter. The earthworm casts contained higher amounts of N, P, K and organic C than the soil (P = 0.05). Selective feeding by earthworms on organically rich substrates, which break down during passage through the gut, is likely to be responsible for the higher microbial populations and greater enzyme activity in the casts.     

Effect of earthworm casts on protein synthesis in radish (Raphanus sativum) and lettuce (Lactuga sativa) seedlings

Summary The protein-synthesizing capacity of 3-day-old seedlings of radish and lettuce grown in the presence of earthworm casts was investigated using L-¹⁴-C-leucine incorporation. The results showed that earthworm casts increased protein synthesis by 24% for lettuce and 32% for radish, althought no significant differences in protein content were evident.     

Mucus excretion and carbon turnover of endogeic earthworms

Summary Mucus excretion of endogeic earthworms, by the body surface to burrow walls and by the intestine to casts, was investigated using uniformly ¹⁴C-labelled adult or subadult specimens of Octolasion lacteum (Örley) in laboratory incubations in soil from a beechwood on limestone. The daily loss of C due to mucus excretion from the body surface and in casts was calculated as 0.2 and 0.5% of total animal C, respectively. The C loss due to mucus excretion by subadult or adult individuals of O. lacteum is assumed to account for 63% of total C losses (including mucus excretion and respiration) of the earthworms. In a second experiment we studied the incorporation of ¹⁴C from labelled soil, again from a beechwood on limestone, into the tissue of the endogeic earthworm species Aporrectodea caliginosa (Savigny). The results of this experiment indicate the existence of two C pools, one more labile and one more stable, in earthworms. It is assumed that the C investment for respiration and mucus excretion is derived from the labile C pool of endogeic earthworms.     

Potential for higher rates of denitrification in earthworm casts than in the surrounding soil

Summary Earthworms (Lumbricus terrestris L.) were cultured in the laboratory and fed on lucerne (Medicago sativa L.). Denitrification rates in the surface casts and the surrounding soil were quantified using C₂H₂-inhibition of nitrous oxide reductase. The investigation also included determination of the N₂O-formation by nitrification as well as CO₂-formation as a measure of respiration. The denitrification rates of wet earthworm casts were found to be significantly higher than those occurring in wet samples from the soil. The low N₂O-formation observed seemed to be due to denitrification. Respiration was higher in casts, indicating higher oxygen demand which resulted in more anaerobic conditions. The energy supply was probably better in casts compared with the surrounding soil.