Microbial Environment Affects Innate Immunity in Two Closely Related Earthworm Species Eisenia andrei and Eisenia fetida

Survival of earthworms in the environment depends on their ability to recognize and eliminate potential pathogens. This work is aimed to compare the innate defense mechanisms of two closely related earthworm species, Eisenia andrei and Eisenia fetida, that inhabit substantially different ecological niches. While E. andrei lives in a compost and manure, E. fetida can be found in the litter layer in forests. Therefore, the influence of environment-specific microbiota on the immune response of both species was followed. Firstly, a reliable method to discern between E. andrei and E. fetida based on species-specific primers for cytochrome c oxidase I (COI) and stringent PCR conditions was developed. Secondly, to analyze the immunological profile in both earthworm species, the activity and expression of lysozyme, pattern recognition protein CCF, and antimicrobial proteins with hemolytic function, fetidin and lysenins, have been assessed. Whereas, CCF and lysozyme showed only slight differences in the expression and activity, fetidin/lysenins expression as well as the hemolytic activity was considerably higher in E. andrei as compared to E. fetida. The expression of fetidin/lysenins in E. fetida was not affected upon the challenge with compost microbiota, suggesting more substantial changes in the regulation of the gene expression. Genomic DNA analyses revealed significantly higher level of fetidin/lysenins (determined using universal primer pairs) in E. andrei compared to E. fetida. It can be hypothesized that E. andrei colonizing compost as a new habitat acquired an evolutionary selection advantage resulting in a higher expression of antimicrobial proteins.     

Eisenia fetida (Oligochaeta, Lumbricidae) Activates Fungal Growth, Triggering Cellulose Decomposition During Vermicomposting

Cellulose is the most abundant polymer in nature and constitutes a large pool of carbon for microorganisms, the main agents responsible for soil organic matter decomposition. Cellulolysis occurs as the result of the combined action of fungi and bacteria with different requirements. Earthworms influence decomposition indirectly by affecting microbial population structure and dynamics and also directly because the guts of some species possess cellulolytic activity. Here we assess whether the earthworm Eisenia fetida (Savigny 1826) digests cellulose directly (i.e., with its associated gut microbiota) and also whether the effects of E. fetida on microbial biomass and activity lead to a change in the equilibrium between fungi and bacteria. By enhancing fungal communities, E. fetida would presumably trigger more efficient cellulose decomposition. To evaluate the role of E. fetida in cellulose decomposition, we carried out an experiment in which pig slurry, a microbial-rich substrate, was treated in small-scale vermireactors with and without earthworms. The presence of earthworms in vermireactors significantly increased the rate of cellulose decomposition (0.43 and 0.26% cellulose loss day⁻¹, with and without earthworms, respectively). However, the direct contribution of E. fetida to degradation of cellulose was not significant, although its presence increased microbial biomass (C_mic) and enzyme activity (cellulase and β-glucosidase). Surprisingly, as fungi may be part of the diet of earthworms, the activity of E. fetida triggered fungal growth during vermicomposting. We suggest that this activation is a key step leading to more intense and efficient cellulolysis during vermicomposting of organic wastes.     

Species-specific effects of epigeic earthworms on microbial community structure during first stages of decomposition of organic matter

Background

Epigeic earthworms are key organisms in organic matter decomposition because of the interactions they establish with microorganisms. The earthworm species and the quality and/or substrate availability are expected to be major factors influencing the outcome of these interactions. Here we tested whether and to what extent the epigeic earthworms Eisenia andrei, Eisenia fetida and Perionyx excavatus, widely used in vermicomposting, are capable of altering the microbiological properties of fresh organic matter in the short-term. We also questioned if the earthworm-induced modifications to the microbial communities are dependent on the type of substrate ingested.

Methodology/Principal Findings

To address these questions we determined the microbial community structure (phospholipid fatty acid profiles) and microbial activity (basal respiration and microbial growth rates) of three types of animal manure (cow, horse and rabbit) that differed in microbial composition, after being processed by each species of earthworm for one month. No differences were found between earthworm-worked samples with regards to microbial community structure, irrespective of type of manure, which suggests the existence of a bottleneck effect of worm digestion on microbial populations of the original material consumed. Moreover, in mesocosms containing cow manure the presence of E. andrei resulted not only in a decrease in bacterial and fungal biomass, but also in a reduced bacterial growth rate and total microbial activity, while no such reduction was found with E. fetida and P. excavatus.

Conclusions/Significance

Our results point to the species of earthworm with its associated gut microbiota as a strong determinant of the process shaping the structure of microbial communities in the short-term. This must nonetheless be weighed against the fact that further knowledge is necessary to evaluate whether the changes in the composition of microbiota in response to the earthworm species is accompanied by a change in the microbial community diversity and/or function.     

Aged Eisenia fetida earthworms exhibit decreased reproductive success

The effect of age on male and female reproductive activities was examined in 1–6-year-old age-matched populations of the simultaneous hermaphroditic earthworm, Eisenia fetida, (Annelida; Oligochaeta). As earthworm ages increase from 3 to 6 years, cocoon and hatchling production gradually decreased. Sperm counts from 2-year-old and 5-year-old earthworms did not differ. Old earthworms successfully inseminated young virgin earthworms. Thus, female functions associated with successful cocoon production diminish before the male function. These results indicate that E. fetida earthworms spend up to one-third of their adult life with limited reproductive capabilities.     

Cadmium and Lead Accumulation in the Earthworm Eisenia fetida (Savigny) and its Impact on Cholinesterase and Metabolic Pathway Enzyme Activity

The uptake of cadmium and lead was studied in the earthworm Eisenia fetida (Annelida: Oligochaeta) using an artificial soil exposure. Although cadmium and lead are bioconcentrated in Eisenia fetida tissue, bioaccumulation is not shown for concentrations below 100 ppm for lead, individuals eliminating as much metal as they ingest or the interactions between lead and organic matter in our substratum reduce the bioavailability of lead at low concentration. The cholinesterase activity was not inhibited when individuals were exposed for 8 weeks to either 8 or 80 ppm of cadmium or 100 or 2,000 ppm of lead. Results are different from those reported in another species Eisenia fetida andrei (= E. andrei) showing an inhibitory effect of lead on ChE activity; thus, differences in cholinesterase inhibition reflects the existence of two separate species. No effect of cadmium and lead on the activity of esterases, malate dehydrogenase, phosphoglucomutase and glutamate oxalate transferase was found in our experimental conditions, but we observed the disappearance of the fast moving band after electrophoretic separation for phosphoglucose isomerase.     

Mortality of the earthworm Eisenia fetida (Savigny) presented to the terrestrial planarian Ártioposthia triangulata (Dendy) (Tricladida: Terricola)

Ártioposthia triangulata is a terrestrial planarian that feeds on earthworms. Up to 76% of field collected A. triangulata fed on Eisenia fetida earthworms at 10°C in the laboratory. In one experiment, the feeding rate declined from 63% of earthworms attacked to 36% and 34% in the first, second and third feeding opportunities over a week. The average number of E. fetida eaten was 1.4 per planarian per week. The gain in weight of individual A. triangulata was significantly related to the amount of earthworm tissue lost during the feeding process. Over a one week period, the amount of earthworm tissue lost was related to the total amount presented to the planarians but not to their size. Frequency of attack was, however, related to planarian size. The results are discussed in relation to an hypothetical population of earthworms in pasture. It is concluded that the results support the contention that A. triangulata could seriously deplete earthworm populations.     

Earthworms newly from Mongolia (Oligochaeta,?Lumbricidae,Eisenia)

Two new megadrile earthworms from the steppes, the first species wholly from Outer Mongolia, are ascribed to the partially parthenogenetic Eisenia nordenskioldi (Eisen, 1879) species-complex. Taxonomic justification of sympatric Eisenia nordenskioldi mongol and Eisenia nordenskioldi onon ssp. n. are supported by mtDNA COI barcodes. The unreliability of molecular differentiation based on voucher names compared to definitive types is again demonstrated, as pertains to the ultimate Eisenia andrei Bouché, 1972 synonym of the Eisenia fetida (Savigny, 1826) sibling species-complex composed of more than a dozen prior names. Similar species described from Northeast China [formerly Manchuria] and North Korea are briefly considered, albeit they are intermittently held in synonymy of cosmopolitan Aporrectodea rosea (Savigny, 1826) along with many other taxa including some exotic lumbricids initially found in India. Japanese and North American lumbricids are also mentioned. Distributions are discussed and an annotated checklist of all nine Siberian/sub-arctic Eisenia nordenskioldi ssp. is appended.     

A comparative study of gut microbiota profiles of earthworms fed in three different substrates

The gut microbiome of earthworms has a complex interdependence with the host. When the soil minerals pass through earthworm’s gut, they may affect the gut microbiota. To gain insight into the response of gut microbiota to the passed minerals, we fed earthworm (Eisenia fetida) on nutrient-poor soil and ore powder, and used high throughput sequencing to characterize the earthworm intestinal microbial community to find evidence for a core bacterial community of the E. fetida. The results showed that earthworms’ gut maintained a core microbiome that appeared in all samples. These core microbiota may play a significant role in a species’ environmental interactions. The composition of intestinal microbiomes varied with substrates. The earthworm guts from two nutrient-poor substrates had similar microbial communities and they were different from nutrient-rich substrate. Proteobacteria and Bacteroidetes were more abundant in the gut of earthworms kept on a nutrient-poor substrate such as ore powder or mineral soil than in the gut of earthworms kept in organic-rich compost soil; some of these microorganisms may help earthworms survive in nutrient-poor substrates.     

Cold hardiness and geographic distribution of earthworms (Oligochaeta,Lumbricidae, Moniligastridae)

Cold hardiness of 12 species and 2 subspecies of earthworms from Northern Eurasia was studied. Supercooling temperatures, the water content and the thresholds of tolerated temperatures of worms and their cocoons were determined. The threshold values varied within ?1…?35°C for worms and within ?1…?196°C for cocoons. Earthworms of 4 species and 2 subspecies survived freezing. Cocoons of all species except Eisenia fetida possessed a protective dehydration mechanism which prevented their freezing. During wintering at subzero temperatures, earthworms lost up to 20% of water, cocoons up to 37%. Species of the same life form can overwinter at different phases and have different cold hardiness values. On the whole, epigeic and epi-endogeic species (except for Eisenia fetida) were more resistant to cold than endogeic ones. The following preliminary classification of earthworms according to their tolerance to negative temperatures is proposed: (1) both onthogenetic phases are tolerant; (2) only cocoons are tolerant; (3) both onthogenetic phases are intolerant. The geographic distribution of all the studied species (except for Eisenia nordenskioldi nordenskioldi) is partially or completely limited by insufficient resistance of the worms to negative temperatures. A significant cold hardiness of cocoons of most species is nonadaptive, since the worms hatched from the eggs in spring die without having enough time to reach maturity and to lay cocoons before the onset of subzero temperatures. Only 3 species (Eisenia nordenskioldi nordenskioldi, Eisenia atlavinyteae, and Dendrobaena octaedra) can live in permafrost regions; this is the main reason for a drastically reduced diversity of earthworm assemblages in eastern Siberia except for its southern, mountain parts. In general, the reasons for the impoverishment lie in the modern climatic conditions correlated with the ecophysiological capacities of earthworms.     

The Mixed Media Bag

This article provides basic information on how a common species of earthworm, Eisenia fetida, can be used in the biology classroom as well as a discussion of how to establish and care for a vermicompost bin. We discuss ideas for inquiry activities with the organism and provide a sample-guided inquiry that demonstrates how a long-term, theme-based unit involving earthworms can address a wide variety of content from the National Science Education standards.     

Size-assortative mating in the earthworm Eisenia fetida (Oligochaeta, Lumbricidae)

In many species of simultaneous hermaphrodites, body size correlates with fecundity, and larger partners are preferred to small ones. Since sperm exchange is usually reciprocal, small individuals may be rejected by larger partners resulting in size-assortative mating. We studied the mating patterns in a natural population of the simultaneous hermaphroditic earthworm Eisenia fetida (Oligochaeta, Lumbricidae). We found that size-assortative mating processes existed, with variance in body weight within pairs lower than between pairs in mating earthworms. This non-random mating pattern probably reveals the existence of mate selection in this species, which lives at elevated densities with high availability of potential mates.     

Correlation between the activity of digestive enzymes and nonself recognition in the gut of Eisenia andrei earthworms

Earthworms Eisenia andrei, similarly to other invertebrates, rely on innate defense mechanisms based on the capability to recognize and respond to nonself. Here, we show a correlation between the expression of CCF, a crucial pattern-recognition receptor, and lysozyme, with enzyme activities in the gut of E. andrei earthworms following a microbial challenge. These data suggest that enzyme activities important for the release and recognition of molecular patterns by pattern-recognition molecules, as well as enzymes involved in effector pathways, are modulated during the microbial challenge. In particular, protease, laminarinase, and glucosaminidase activities were increased in parallel to up-regulated CCF and lysozyme expression.     

A New Case of Consensual Decision: Collective Movement in Earthworms

Collective movements are reported for many species from microorganisms to humans. But except for a few soil‐inhabiting species, intra‐specific interactions in soil are poorly studied. Some intra‐specific interactions occur in earthworms. Most of them are negative, concerning parameters like the rate of survival, maturation, food ingestion or growth. Virtually nothing is known about collective movement in earthworms that represent the dominant biomass of the soil. This study, the first one on annelids, highlights a consensual decision phenomenon based only on contact between followers. Using an olfactometer set‐up and modelling, we show that earthworms Eisenia fetida influence each other to select a common direction during their migration. Experiments in a binary choice test showed that contacts between individuals are responsible for collective movement. This coordination in movement could allow earthworms to benefit from forming clusters. The resulting local higher densities, enhancing individual survival and favouring the cooperation, may be at the origin of Allee effects reported for these species.     

An enzyme from the earthworm Eisenia fetida is not only a protease but also a deoxyribonuclease

The earthworm enzyme Eisenia fetida Protease-III-1 (EfP-III-1) is known as a trypsin-like protease which is localized in the alimentary canal of the earthworm. Here, we show that EfP-III-1 also acts as a novel deoxyribonuclease. Unlike most DNases, this earthworm enzyme recognizes 5′-phosphate dsDNA (5′P DNA) and degrades it without sequence specificity, but does not recognize 5′OH DNA. As is the case for most DNases, Mg²⁺ was observed to markedly enhance the DNase activity of EfP-III-1. Whether the earthworm enzyme functioned as a DNase or as a protease depended on the pH values of the enzyme solution. The protein acted as a protease under alkaline conditions whereas it exhibited DNase activity under acid conditions. At pH 7.0, the enzyme could work as either a DNase or a protease. Given the complex living environment of the earthworm, this dual function of EfP-III-1 may play an important role in the alimentary digestion of the earthworm.     

Coprophilous streptomycetes and fungi—Food sources for enchytraeid worms (Enchytraeidae)

Food selection experiments demonstrated thatEnchytraeus crypticus (Oligochaeta, Enchytraeidae) was attracted byStreptomyces species and microscopic fungi in vermiculture substrates and in the gut content ofEisenia andrei earthworms. Consumption of spores and/or mycelia of attractive strains influenced markedly the proliferation ofE. crypticus. There was a 74-fold increase in the numbers of enchytraeids fed on the mixture ofAspergillus flavus andVerticillium tenerum mycelia or on mycelium of one strain ofStreptomyces in reproduction tests. Lower rates of increase ofE. crypticus (50-fold or less) were observed in variants whereV. tenerum or mixtures of fungi and streptomycetes were offered as food. We showed a potential importance of microbial populations in vermicultures and indicated that their regulation may provide a way to increase the productivity of such systems.     

Changes in microbial community structure and function during vermicomposting of pig slurry

Most studies investigating the effects of earthworms on microorganisms have focused on the changes before and after vermicomposting rather than those that occur throughout the process. In the present study, we designed continuous feeding reactors in which new layers of pig slurry (1.5 and 3 kg) were added sequentially to form an age gradient inside the reactors in order to evaluate the impact of the earthworm species Eisenia fetida on microbial community structure and function. The activity of earthworms greatly reduced the bacterial and fungal biomass and microbial diversity relative to the control values. However, the pronounced presence of earthworms in the younger layers stimulated microbial activity and as such increased carbon mineralization probably due to the fact that the microorganisms may have been less resource-limited as a result of earthworm activity, as indicated by the ratio of monounsaturated to saturated PLFAs.     

Verminephrobacter eiseniae type IV pili and flagella are required to colonize earthworm nephridia

The bacterial symbiont Verminephrobacter eiseniae colonizes nephridia, the excretory organs, of the lumbricid earthworm Eisenia fetida. E. fetida transfers V. eisenia into the egg capsule albumin during capsule formation and V. eiseniae cells migrate into the earthworm nephridia during embryogenesis, where they bind and persist. In order to characterize the mechanistic basis of selective tissue colonization, methods for site-directed mutagenesis and colonization competence were developed and used to evaluate the consequences of individual gene disruptions. Using these newly developed tools, two distinct modes of bacterial motility were shown to be required for V. eiseniae colonization of nascent earthworm nephridia. Flagella and type IV pili mutants lacked motility in culture and were not able to colonize embryonic earthworms, indicating that both twitching and flagellar motility are required for entrance into the nephridia.     

Effects of Soil Nutrient Heterogeneity and Earthworms on Aboveground Biomass of Experimental Plant Communities

Soil nutrients are commonly heterogeneously distributed and earthworms are one of the most common soil organisms. While effects of both soil nutrient heterogeneity and earthworms have been well studied, their interactive effect on plant community productivity has rarely been tested. In a greenhouse experiment, we constructed experimental plant communities by sowing seed mixtures of four grasses, two legumes and two forbs in either a heterogeneous soil consisting of low and high nutrient soil patches or a homogeneous soil where the low and high nutrient soil patches were evenly mixed. The earthworm Eisenia fetida was either added to these soils or not. Aboveground biomass of the whole communities, grasses and legumes did not differ between the homogeneous and heterogeneous soils or between the soils with and without earthworms. However, soil nutrient heterogeneity reduced aboveground biomass of forbs, and such an effect did not interact with earthworms. In response to soil heterogeneity and earthworms, biomass ratio of the three functional groups showed similar patterns as that of their biomass. At the patch level, aboveground biomass of the whole community, grasses and legumes were greater in the high than in the low nutrient soil patches within the heterogeneous soil. A similar pattern was found for the forbs, but this was only true in the absence of earthworms. Our results suggest that soil nutrient heterogeneity and earthworms may not influence aboveground biomass of plant communities, despite the fact that they may modify the growth of certain plant functional groups within the community.     

1H NMR and GC/MS metabolomics of earthworm responses to sub-lethal DDT and endosulfan exposure

The metabolic response of the earthworm Eisenia fetida to two pesticides, dichlorodiphenyltrichloroethane (DDT) and endosulfan, was characterized in contact tests using proton nuclear magnetic resonance (¹H NMR) and principal component analysis (PCA). PCA loading plots suggested that maltose, leucine and alanine were important metabolites contributing to the differences in dosed and control earthworms for both compounds at doses of 0.5, 1.0 and 2.0 μg/cm². Gas chromatography/mass spectrometry (GC/MS) was used to quantify the metabolites identified in E. fetida and determine if the changes in maltose, leucine and alanine following exposure to DDT and endosulfan (at 0.5 and 1.0 μg/cm²) were reproducible and greater than the natural variability. Quantification by GC/MS suggested that maltose was not a reliable biomarker since it both increased and decreased in earthworms exposed to DDT and increased by just 3% with exposure to endosulfan. Leucine was not stable with the GC/MS derivitization method used in this study and could not be confirmed as a reliable biomarker. However, alanine consistently increased for both DDT and endosulfan exposed E. fetida. Alanine showed considerable variability in control earthworms (±41.6%), yet the variability in alanine to glycine ratios was just ±10.5%. Increases in the alanine to glycine ratio were statistically significant at the P = 0.05 level for the 1.0 μg/cm² DDT dose and both the 0.5 and 1.0 μg/cm² endosulfan doses, suggesting that deviations from the normal homeostatic ratio of 1.5 for alanine to glycine is a potential biomarker of DDT and endosulfan exposure warranting further study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Environmental Metabolomics Special Issue of Metabolomics.