Culture of commercially obtained Lumbricus terrestris L.: Implications for sub-lethal ecotoxicological testing

The use of commercially purchased or field-collected earthworms of unknown age, exposure or pre-treatment in sub-lethal ecotoxicological studies is questionable. In this study, adult (clitellate) Lumbricus terrestris, obtained from 5 commercial suppliers in the UK and also field collected, were kept under controlled environmental conditions (15 °C and 24 h darkness) in a sterilised loam soil and fed horse manure. Survival, biomass and cocoon production was monitored every 4 weeks over 1 y. Marked differences were recorded in survival rates (ranging from 40-100% after 40 weeks) and cocoon production (ranging from 15.1 to 32.2 worm⁻¹ y⁻¹) between treatments. Biomass in all treatments (mean mass 4.32-5.61 g at the outset) increased with time to week 20 (maximum 6.7 g) and then declined steadily (3.23-4.7 g at week 52). This pattern was also observed in cocoon production and was considered to be a function of an initial period of acclimation (0-12 weeks) followed by a period of high production (12-36 weeks) under optimal conditions and then fatigue (36-52 weeks) caused by reproductive exhaustion. Results suggest that earthworm origin may influence the validity and reproducibility of sub-lethal ecotoxicological studies and where applicable laboratory-reared earthworms of known age and history are recommended as test subjects.     

Does the acephaline gregarine Monocystis sp. modify the surface behaviour of its earthworm host Lumbricus terrestris?

The earthworm Lumbricus terrestris has relatively low dispersal rates. This can represent a challenge for the internal gregarine parasite Monocystis, the dispersal of which often depends on the host. Mating of Monocystis can also be restricted to parasite stages within the current host individual. Adaptations, including host behavioural modification, that improve parasite transmission and simultaneously avoid inbreeding within a single host individual should be favoured by selection. We used a correlative approach to investigate the question of parasitic manipulation in this Gregarine-Lumbricid system. We observed mature earthworms (n=24) for 3 weeks while recording various measures of activity (surface activity, burrowing activity, surfacing delay, number of surface visits) and correlated parasite load with these activity measures. We found a significant positive correlation between surface activity and midden (or cast) production, which suggests a simple method for assessing the overall activity of local earthworm populations. However, we found no evidence of behavioural manipulation of any measure of host activity. We discuss these results in the context of inbreeding depression and host-parasite coevolution.     

High genetic diversity and heterogeneous parasite load in the earthworm Lumbricus terrestris on a German meadow

In parasite-host dynamics, parasites exert frequency-dependent selection on their hosts by favouring rare alleles that may confer resistance against infection. Therefore host populations that suffer strong parasite stress should maintain higher levels of genetic variability. We studied the Lumbricus terrestris-Monocystis sp. host-parasite system at a microgeographical scale. Using three polymorphic microsatellite loci on one large earthworm population sampled at 26 different sites (281 genotypes), we tested the relationship between parasite load and genetic variation in natural samples of the common earthworm L. terrestris. Our analysis yielded the following: (1) parasite load varied significantly across sites in this population; (2) there was no consistent evidence for heterozygote deficiency (observed heterozygosities ranged between 0.74 and 0.87), indicating a low level of inbreeding; (3) there was no significant genetic structuring among sample sites; (4) we could not identify a significant association between parasite load and population genetic diversity; (5) there was considerable population differentiation (15.17%) between our German samples and a Canadian L. terrestris reference population. Our study provides insight into the population genetics of one of the most economically important soil organisms on a microgeographic scale.     

The effect of feeding behavior on Hg accumulation in the ecophysiologically different earthworms Lumbricus terrestris and Octolaseon cyaneum: A microcosm experiment

 A soil microcosm experiment was performed to assess the uptake of Hg from various Hg-spiked food sources (soil, leaf litter and root litter of Trifolium alexandrinum) by two earthworm species, Lumbricus terrestris (anecic) and Octolaseon cyaneum (endogeic). Treatments were applied in which one of the three food sources was Hg spiked and the other two were not. Additional treatments in which all or none of the food sources were Hg spiked were used as controls. Uptake of Hg from soil into tissues of both earthworm species was significantly higher than uptake of Hg from leaf litter or root litter, indicating that soil may be the most important pool for the uptake of Hg into earthworms. In addition, the anecic L. terrestris significantly accumulated Hg from all Hg-spiked food sources (leaf litter, root litter and soil), whereas the endogeic O. cyaneum took up Hg mainly from soil particles. Interestingly, there was no further increase in Hg in L. terrestris when all food sources were Hg spiked compared to the single Hg-spiked sources. This may be attributed to the relatively high Hg content in the soil, which may have influenced the feeding behavior of the earthworms, although their biomass did not significantly decline. We suggest that, in addition to the physiological differences, feeding behavior may also play a role in the contrasting uptake of Hg by the two earthworm species.     

Food quality affects production of Lumbricus terrestris (L.) under controlled environmental conditions

Birch leaves and horse manure were used to determine the effects of food quality on growth and reproduction of laboratory-reared Lumbricus terrestris. Animals grew to maturity within 6 months but attained a significantly (p < 0.001) larger adult size with manure (6.17 g) versus leaves (4.20 g). Cocoon production by recently-mated adults maintained in isolation, fed with birch leaves or horse manure, resulted in 4.53 and 3.84 cocoons ind.⁻¹ month⁻¹ respectively, with an initial hatchability of 86%, falling to zero after 18 months. Re-mating of these known individuals permitted long term monitoring of reproductive output (to 30 months). For the whole experimental period, overall hatchability of the 2010 cocoons produced was 44.4%. Median incubation time of those cocoons that hatched within accepted norms (less than 5 months at 15 °C) was 103 days and was not influenced by adult food type. A proportion (35.5%) of cocoons took in excess of 12 months to hatch. Adult mortality was minimal (25%) during the long term experiment but abnormal cocoon production was recorded after 2 years. Overall results demonstrate that food quality can have a significant influence on somatic and reproductive production of L. terrestris and these data may aid construction of production models for this earthworm in ecosystems with contrasting food quality.     

Field investigations of Lumbricus terrestris spatial distribution and dispersal through monitoring of manipulated, enclosed plots

A field experiment in managed woodland was set up to examine the effects of manipulated population density and resource availability on spatial distribution and dispersal of the anecic earthworm Lumbricus terrestris. Experiments over 2 years, made use of 1 m² field enclosures with associated trapping units to assess emigration rates at control and enhanced L. terrestris densities and different levels of leaf litter availability. Densities were manipulated twice; at the outset and again after 1 year when visually tagged animals obtained from 2 origins were introduced. Population density had a significant effect on dispersal (p < 0.01, p < 0.05 in Year 1 and Year 2 respectively) with more captures (pro rata) at the higher density compared with controls over the experimental period. Food availability only had a significant effect during the initial week of the experiment. L. terrestris midden arrangement was found to be regular across 1 m² plots and regularity increased with an increase in midden number. Mean (±S.E.) midden number was 30.34 ± 0.77 m⁻² and 28.06 ± 0.5 m⁻², during the first and second year of the experiment respectively and this was unaffected by additions. Inter-midden distance was recorded at 0.13 ± 0.0014 m. Results suggest that L. terrestris dispersal can be affected by population density and resource availability.     

Identification of uncultured bacteria tightly associated with the intestine of the earthworm Lumbricus rubellus (Lumbricidae; Oligochaeta)

The bacteria associated with the intestine and casts of the earthworm Lumbricus rubellus Hoffmeister, 1843, were examined by direct counts, culturability studies, 16S rRNA gene clone libraries, and fluorescent in situ hybridization (FISH). A significant fraction, 24-47%, of the total numbers of prokaryotes remaining in the intestine after casting were tightly associated with the intestinal wall. Bacterial 16S rRNA gene clone libraries constructed from washed earthworm intestinal tissue suggested that the bacterial community was dominated by a few phylotypes that were either absent from, or in low abundance, in the casts. The specific phylotypes present depended on the date of sampling and included representatives of the Acidobacteria, Firmicutes, β-Proteobacteria, and one phylogenetically deep, unclassified group. Juvenile earthworms subsequently collected contained three of the four phylotypes observed in the intestine clone libraries. The Firmicutes phylotype was examined by FISH and was found to be a short rod that represented only a small fraction of the total population of the juvenile samples. These results suggested that the microbial community tightly associated with the intestine was dominated by a small number of phylotypes and that this association was opportunistic rather than obligate.     

Experiments on the origin of C in the calcium carbonate granules produced by the earthworm Lumbricus terrestris

Granules of calcium carbonate up to 2.5 mm in size have been found in archaeological soils and sediments for many years. Recent work has shown conclusively that these granules are derived from the calciferous glands of earthworms and most commonly (in Northern European soils) from Lumbricus terrestris. Valuable pedological and archaeological information could be obtained from a radiocarbon date if the ¹⁴C in the granule carbonate reflects the true age of formation. Preliminary information on the possible sources of carbon in the granule calcite was obtained by a series of simple experiments growing worms in artificial substrates made up of materials with marked differences in δ¹³C. The results showed that carbon in the granule calcite comes from the dietary inputs, but may be coming partly from atmospheric CO₂ and also partly from old soil carbon. Little appears to come from the soil calcium carbonate. Most of the results imply that a fractionation of around +12‰ is occurring between the carbon ingested by the worm and the carbon output in the granules. This is comparable to (but slightly higher than) the widely quoted non-biological value (ca. +10‰) of total kinetic fractionation between CO₂-CaCO₃.     

Abundance of the earthworm Lumbricus terrestris in relation to subsurface drainage pattern on a sandy clay field

Subsurface drainage induces systematic spatial variability in soil properties which may be reflected in the abundance and distribution of soil organisms. We compared the population density of the deep burrowing earthworm Lumbricus terrestris L. above and between tile subdrains in 41 sample pairs on an eight hectare grass field. Above the drains the median number of  individuals was twice as high and their total fresh mass five times as high as between the drains (4.5 vs. 2.1 individuals m^-2  and 9.6 vs. 1.9 g m^-2, respectively). The mean difference (above drain – between drains) was 2.5 individuals m^-2  (95% CI = 1.0 to 4.0) and 6.6 grams m^-2 (95% CI = 3.6 to 9.6). The relatively larger difference in fresh mass was due to a high proportion of adult individuals above drains. One likely explanation for the pattern of abundance is that the lowered water table level near the drains provides an environment beneficial for the population growth of L. terrestris. Due to the role of L. terrestris burrows as flow paths of percolating water the observations may have implications on subdrain function.     

Genetic and ecological impacts of heavy metal and flooding stress on the earthworm Lumbricus rubellus in floodplains of the Rhine river

In the highly polluted river Rhine system, earthworms face environmental stress resulting from flooding and elevated heavy metal concentrations in the floodplain soil. Previous field studies have revealed adaptation to flooding for the earthworm species Lumbricus rubellus as this species matures at a lower weight in floodplain sites with a high frequency of flooding compared to less frequently flooded sites. Also heavy metals have effects on L. rubellus and heavy metals are influencing the genetic composition of this species. In this study, it was tested whether flooding and heavy metals had an impact on the genetic composition of L. rubellus living in floodplains along the river Rhine system. Earthworms were sampled at three sites previously studied along the river to assess earthworm diversity, biomass, density, and individual weight, and developmental status of L. rubellus. The genetic variation by means of isozymes was studied for 175 individuals. The results showed lowest density of L. rubellus adult and sub-adult life-stages and lowest individual weight of these life-stages at the frequent inundated site. The genetic composition, however, showed no effects of flooding on the genetic composition, but effects of heavy metals could not be ruled out.     

Peat amendment and production of different crop plants affect earthworm populations in field soil

A field experiment was conducted to study the effects of peat amendment and crop production system on earthworms. The experiment was established on a field previously cultivated with oats and with silt as the main soil type. Perennial crops strawberry, timothy and caraway, and annual crops rye, turnip rape, buckwheat, onion and fiddleneck were cultivated with conventional methods. All the crops were grown with and without soil amendment with peat. Earthworms were sampled twice: 4 and 28 months after establishment of the experiment. In the former case part of the experimental plots were soil sampled and hand sorted for estimation of earthworms. In the latter case all experimental plots were sampled and both soil sampling and mustard extraction was carried out. Soil organic carbon and microbial biomass was measured at 14 and 28 months. Peat increased the abundance of juvenile Aporrectodea caliginosa by 74% in three growing seasons, but had no effect on adult numbers. Lumbricus terrestris numbers were not increased by peat treatment. Three season cultivation of caraway favoured both A. caliginosa and L. terrestris. An equal abundance of A. caliginosa was also found in plots cultivated with turnip rape and fiddleneck. Total earthworm and especially A. caliginosa numbers were very small in plastic-mulched strawberry beds. This was mainly attributed to repeated use of the insecticide endosulfan. With the strawberry plots omitted there was a significant correlation between soil microbial N measured at 14 months and juvenile Aporrectodea spp. and Lumbricus spp. numbers measured at 28 months. Adult earthworm numbers were not associated with either soil organic C or microbial biomass.     

Cropping history and peat amendment-induced changes in strawberry field earthworm abundance and microbial biomass

Common agricultural practices, e.g. soil tillage and organic amendment, may affect field earthworm communities considerably. However, there is little data to show how long the changes persist after a certain action. The effect of peat, commonly used in Finland to improve the horticultural soil structure, on key soil organisms is also largely unknown. Earthworm abundance and microbial biomass were studied in a strawberry field experiment (soil type silty clay) with a history of different crops (strawberry, timothy, caraway, rye, turnip rape, fiddleneck, onion and buckwheat) and peat treatments. Sampling was carried out after three years of perennial cropping of strawberry. Half of the area was peat-amended twice three years apart. The earthworm community consisted mainly of Aporrectodea caliginosa and Lumbricus terrestris. Soil peat amendment almost doubled the number of endogeic A. caliginosa, but had no effect on the anecic L. terrestris. The effect of cropping history on earthworms diminished after three years of strawberry cropping. Only the positive effect of caraway on juvenile Lumbricus spp. was detectable three years after its cropping had been finished. However, some crops had secondary effects on the earthworm distribution without significant influence on their numbers while they were grown, e.g. high numbers of A. caliginosa were recorded from soil with a history of timothy ley. The effect of strawberry cropping was contradictory: six years of continuous strawberry cropping decreased the number of the anecic L. terrestris, but during the last three years on strawberry, the proportion of L. terrestris increased from 6% to 40% in the experimental area with a concomitant great drop in the number of A. caliginosa. The role of different agricultural practices (no tillage, mulching, inter-row grass cover and pesticides) is discussed. The crop-induced changes persisted in the microbial biomass for three years (onion cropping reduced microbial biomass C), but soil amendment had no effect on microbes. The abundance of A. caliginosa was associated with soil organic C, but not with soil microbial biomass.     

Growth of Lumbricus terrestris and Aporrectodea caliginosa in an acid forest soil, and their effects on enchytraeid populations and soil properties

A laboratory experiment was carried out to test the hypothesis that the earthworms Lumbricus terrestris and Aporrectodea caliginosa are able to maintain their populations and reproduce in the acid forest soil of a deciduous forest where no lumbricids were found in the field. The experiment was conducted in 45-l containers in which layers of mineral subsoil, humus and organic topsoil collected from the site were established. Both species survived and at least L. terrestris reproduced during the 60 weeks’ incubation. Burrows and middens of L. terrestris were recorded and quantities of litter were consumed. The presence of lumbricids increased the organic matter content of humus, reduced the acidity of the topsoil and humus layers, and suppressed the population of the enchytraeid Cognettia sphagnetorum. A dense population of Enchytraeus albidus was found in L. terrestris middens. It is concluded that edaphic factors do not explain the absence of earthworms, but isolation from cultural landscapes and lack of opportunity to colonize the site from the surroundings is the decisive factor.     

Trophic transfer of fatty acids from gut microbiota to the earthworm Lumbricus terrestris L

The diet of earthworms includes soil organic matter, soil microbes and other microfauna, but the relative contribution of these dietary components to earthworm nutrition is not well known. Analysis of fatty acid (FA) profiles can reveal trophic relationships in soil food webs, leading to a better understanding of the energy and nutrient flows from microbiota to earthworms. The objective of this study was to determine the origin of FAs assimilated by the earthworm Lumbricus terrestris L. We analysed the pattern of FAs in: (i) the bulk soil, (ii) soil in the earthworm gut, (iii) the absorptive tissue of the earthworm gut wall, and (iv) the muscular layers of the earthworm body wall. Multivariate analyses performed on the FA profiles suggest that the microbial community in the earthworm gut differs from that in bulk soil. Diverse bacterial and fungal derived FAs, which earthworms cannot synthesize, were found in the earthworm gut wall and body wall, and in the neutral lipids (storage lipids) of the gut wall. The major compounds isolated were 20:4ω6, 20:5ω3 and 18:2ω6, followed by the monoenoic 18:1ω7 and 18:1ω9c, and the saturated 18:0. The microbial FA assemblage in the gut wall resembled the gut soil more than the bulk soil, and the body wall of L. terrestris showed the same microbial derived FA pattern as the gut wall, although at reduced concentrations. We propose the existence of a specific microbial community in the earthworm gut that provides FAs to the earthworm. It appears that L. terrestris may derive more of its energy and nutrients from gut specific microbiota than from microbiota already present in the ingested soil, based on the trophic relationships revealed through FA analysis.     

Genetic structure among earthworms (Lumbricus terrestris L.) from different sampling sites in western Germany based on random amplified polymorphic DNA

Earthworms are being used as bio-indicators to assess terrestrial pollution. However, it is often not known whether their populations possess a uniform genetic structure, which would allow comparison of residues or biological properties of earthworms from different sampling locations. In order to investigate this point, random amplified polymorphic DNA (RAPD) variation was surveyed in earthworms (Lumbricus terrestris) from five different sampling sites in Germany. Forty oligonucleotide RAPD primers (10 base pairs in length) were screened, three of which produced high polymorphic band patterns. A total of 61 DNA fragments were detected in 90 individuals of L. terrestris from five sampling sites with 49 (80.3%) RAPD markers being polymorphic. The genetic similarities within (band sharing rates between 0.756 and 0.795) and among the L. terrestris populations (0.635) were similar even at widely separate locations. Inter-population variation in the RAPD pattern for all five earthworm populations accounted for 37.9% of the total variation, while intra-population variation for three adjacent Saarland populations accounted for only 18.0% of the total variation. Principal component analysis (PCA) and the genetic distances of the populations confirm these results. Twenty-four percent of the genetic distance is caused by geographical isolation as shown by a test for isolation by distance. These results show that L. terrestris fulfils the genetic qualifications for a bio-indicator particularly at closely located sampling sites. However, the results also suggest that earthworm studies of widely separated locations should include genetic characterisation of the earthworm samples.     

Feeding activity of the earthworm Eisenia andrei in artificial soil

Quantitative information on the feeding activity of earthworms is scarce but this information is valuable in many eco(toxico)logical studies. In this study, the feeding activity of the compost worm Eisenia andrei is examined in artificial soil (OECD medium), with and without a high-quality food source (cow manure), and at two temperatures (10 and 20 °C). Methods are provided to estimate the most important parameters: gut load, selection of organic matter (OM), digestion efficiency, compaction, gut retention time, and fraction of manure in the diet. Lanthanides (Lu and Tm) were successfully used as inert markers in soil and manure, and we applied Bayesian statistics to analyse the data and fully capture the compounded uncertainty in the parameter estimates. Results show that the compost worm does not feed on soil indiscriminately but is able to select an OM-enriched diet from apparently homogeneous OECD medium. When manure is present on the soil surface, approximately three-quarters of the diet still consists of soil particles. The gut load of the worms was approximately 10% (dwt gut/wwt empty worm), varying little with the treatments. Unfortunately, the digestion efficiency could only be reliably estimated at 20 °C, and was approximately 40%. Temperature clearly affected feeding as a 10° temperature decrease nearly doubled the gut retention time (from 2.9 to 5.5 h), which corresponds to a two-fold decrease in feeding rate. The present data may be used to interpret toxicity and accumulation studies with E. andrei in OECD medium. However, care must be taken, as it seems possible that feeding is influenced by the size of the worm and subtle differences in experimental set-up.     

Automated observation and analysis of earthworm surface behaviour under experimental habitat quality and availability conditions

Currently, the driving factors of active earthworm dispersal across the soil surface are not sufficiently understood, and distances traversed by individual earthworms have rarely been quantified. Research progress has been hampered by the lack of adequate observation methods as well as fast, objective and quantitative measurements of nocturnal earthworm behaviour.In this work, we report on the potential of a new, automated method using infrared-sensitive webcams and computer image analysis. Nightly surface activities of Lumbricus terrestris L. were monitored quantitatively while manipulating levels of disturbance, burrow availability and congener presence in standard observation units.The automated observation system proved to be simple and inexpensive to build, provided reliable quantitative measures of locomotive behaviour without animal disturbance, and considerably reduced human workload and bias.Waterlogging of the burrow zone stimulated surface activities around and away from the home burrow as compared to habitat disturbance by pesticide application and vibration. However, dispersing earthworms never settled in prefabricated burrows. Surface activity of individuals subjected to waterlogged conditions was influenced by the availability of alternative habitat. Minimal habitat disturbance (vibration) and the presence of conspecific individuals resulted in an increased amount of burrow-anchored, possibly sexually oriented, behaviour, but reciprocal burrow visits and mating were not observed. Pesticide application did not result in dispersal, increased foraging activities or sexual attraction attempts during this short-term study.The presence, extent and mechanisms of the remote assessment of important cues in the surroundings of the home burrow by L. terrestris need further research. Infrared monitoring opens new research avenues of earthworm surface behaviour studies (e.g. density-dependent dispersal, habitat selection, (re)colonization, invasion).     

Effects of heavy metals on the litter consumption by the earthworm Lumbricus rubellus in field soils

Aim of this study was to determine effects of heavy metals on litter consumption by the earthworm Lumbricus rubellus in National Park the “Brabantsche Biesbosch”, the Netherlands. Adult L. rubellus were collected from 12 polluted and from one unpolluted field site. Earthworms collected at the unpolluted site were kept in their native soil and in soil from each of the 12 Biesbosch sites. Earthworms collected in the Biesbosch were kept in their native soils. Non-polluted poplar (Populus sp.) litter was offered as a food source and litter consumption and earthworm biomass were determined after 54 days. Cd, Cu and Zn concentrations were determined in soil, pore water and 0.01 M CaCl₂ extracts of the soil and in earthworms. In spite of low available metal concentrations in the polluted soils, Cd, Cu and Zn concentrations in L. rubellus were increased. The litter consumption rate per biomass was positively related to internal Cd and Zn concentrations of earthworms collected from the Biesbosch and kept in native soil. A possible explanation is an increased demand for energy, needed for the regulation and detoxification of heavy metals. Litter consumption per biomass of earthworms from the reference site and kept in the polluted Biesbosch soils, was not related to any of the determined soil characteristics and metal concentrations.