Allolobophora Chlorotica Research Articles

The effects of earthworm species on organic matter transformations and soil microbial communities are only partially related to their bioturbation activity

Earthworms are pivotal in shaping soil ecological processes through their bioturbation activity and organic matter consumption. Earthworm species are known to have different impacts on soil structure, but only a small number of species have been studied so far, and few studies have examined how earthworms simultaneously affect soil functions. Here, we measured the impact of different earthworm species on soil structure (bioturbation function), carbon (C) and nitrogen (N) dynamics and the microbial community (organic matter transformation function), while exploring the links between these functions, across distinct soil compartments (surface casts, below-ground drilosphere, and bulk soil). Six earthworm species (Lumbricus terrestris, Allolobophora chlorotica, Octolasion cyaneum, Octodrilus complanatus, Aporrectodea caliginosa meridionalis and Microscolex dubius) of different ecological categories and functional groups were incubated in soil cores with soil and alfalfa litter for 6 weeks. Our results on the bioturbation function illustrated a great diversity of behaviors and confirmed the relevance of a functional classification based on bioturbation metrics. The main microbial hotspots were surface casts, whose characteristics allowed to distinguish two groups of species. Octod. complanatus, L. terrestris and M. dubius induced high humidity (respectively, +57, +48, +74%), high C (respectively, 19.9, 24.8, 33.2 g kg⁻1 dry soil) and N (respectively, 2, 2.3, 3.2 g kg⁻1 dry soil) content and microbial community selection, promoting C and N mineralization. The three other species had a lower impact. The below-ground drilosphere only showed specific characteristics in the case of L. terrestris. The effects of the studied species on the organic matter transformation function did not align with their bioturbation activities nor with their ecological category. These findings show that the contribution of earthworms to C and N turnover is only partially dependent on their bioturbation effects and suggest the usefulness of developing distinct functional groups based on the specific soil functions under consideration.

Study on the Effect of Foliar Fertilizers on the Growth and Yield of V108 Heat-Tolerant Cucumber Variety

This study evaluates the efficacy of various foliar fertilizers on the growth and yield of the heat-tolerant cucumber variety TV108. The experimental design included six different foliar fertilizers and a control, with applications made weekly at a concentration of 1 mL per liter of water. Results showed that all foliar treatments significantly enhanced plant height compared to the control. Among these, Treatment T4 (ATONIK foliar fertilizer) demonstrated the most pronounced effect, achieving a main stem height of 216.57 cm by the 70th day, which was significantly higher than other treatments and the control. Treatment T4 also excelled in increasing stem diameter, with a final measurement of 1.48 cm, and in promoting both male and female flower formation. The fruit set rate was highest in Treatment T4, reaching 28.67% on day 56 and maintaining at 8.67% by day 70. This treatment also proved most effective in reducing the incidence of key diseases and pests, including powdery mildew, white mold, green worms, and yellow beetles. Regarding yield, Treatment T4 produced the highest individual fruit yield (2.32 kg/plant), actual yield (33.8 tons/ha), and theoretical yield (84.91 tons/ha). Additionally, it enhanced fruit quality, as indicated by the largest fruit length (15.22 cm), diameter (4.13 cm), and the highest Brix value (3.50%). These results substantiate that Treatment T4 is the most effective foliar fertilizer for enhancing both growth and quality of heat-tolerant cucumbers under experimental conditions.

Aporrectodea caliginosa life history traits are improved by positive earthworm interaction and organic matter addition

Earthworm species interact with each other in soils, but these interactions are poorly understood. Moreover, these key soil organisms are influenced by abiotic soil components such as organic matter. Here, we investigated the influence of Allolobophora chlorotica and Aporrectodea nocturna, two earthworm species from different ecological categories, on the incorporation of organic matter, reproduction and weight change of the endogeic Aporrectodea caliginosa. Two different types of organic matter i) a compost and ii) a fresh refined organic material, both from green waste, were used. Earthworm parameters were monitored during an 8-weeks laboratory experiment using a vineyard soil in order to identify positive and negative interactions between species. Irrespective of species, earthworms interacted preferentially with smaller particles, more decomposed and with a lower C/N ratio. For an equivalent earthworm biomass, similar amount of green compost was incorporated by A. caliginosa and A. nocturna. However, A. chlorotica did not bury this material. The green compost increased the reproduction rate of A. caliginosa when associated to A. chlorotica. Moreover, the association with the epi-anecic A. nocturna increased the reproduction rate of A. caliginosa with the addition of refined organic matter (fresh material). Furthermore, in both earthworm associations, the weight loss of A. caliginosa was reduced by the addition of green compost to the soil surface. These results highlight the importance of earthworm interactions in maintaining populations, and emphasized the need of field studies to confirm these interactions, particularly in the context of soil fertility where organic amendments are often applied.

Effect of an organophosphorus insecticide, soil texture and earthworm species on the turnover of soil, gut and cast microbiota during the earthworm's gut transit

The growing usage of plant protection products in agroecosystems questions their unintentional deleterious effects on non-target organisms such as earthworms and their related ecological function. The aim of this study was to investigate the turnover of soil microbiota during the gut transit of two endogeic earthworms (Aporrectodea caliginosa and Allolobophora chlorotica) and how it is possibly affected by the organophosphorus insecticide ethyl-parathion (Pt). We measured prokaryotic and micro-eukaryotic community composition and abundances using two different soil types in the continuum of bulk soil, gut and casts samples collected during and after one week exposure to Pt. The presence of earthworm and Pt had limited effects on the soil microbiota, although Pt altered temporarily soil microbial abundances in the presence of earthworms, not in their absence. The earthworms’ gut harbored a remarkably low prokaryotic diversity, dominated by two molecular Operational Taxonomic Units (mOTUs) affiliated to Rhodococcus and Pseudarthrobacter. Contrastingly, gut micro-eukaryotic communities were maintained at diversity levels similar to that of the soil, yet Pt augmented their diversity and changed their composition. Fresh casts collected out of the soil revealed evident variation of abundance, diversity and community composition according to the soil origin and the earthworm species. In particular, following gut transit casts were enriched with Bacteroidia and γ-Proteobacteria while depleted of Acidobacteria. Exposure to Pt increased the diversity and relative abundance of depleted mOTUs, which paves the way for future investigations on the role of Pt in rising microbial consumption due to an over-excitation of gastrointestinal motility.

Tracking earthworm fluxes at the interface between tree rows and crop habitats in a Mediterranean alley cropping field

Alley cropping is the combination of tree rows and crop alleys. The tree row is covered by an understory vegetation strip (UVS), providing a beneficial habitat for many soil fauna, which could disperse through spillover to the crop alleys. However, such movements have never been directly studied. Our experiment investigated earthworm fluxes in the tree row vicinity using a trap technique, in a Mediterranean agroforestry alley cropping field cultivated with peas and planted with walnut trees. We assessed earthworm density at different distances from the UVS (0 m, 0.3 m, 1 m and 6 m) by hand sorting soil monoliths (25*25*30 cm) in spring 2019, at the start and the end of a two-month experiment. During this period, we detected earthworm fluxes by placing directional traps at 30 cm from the UVS border. Traps consisted of three glued plastic walls placed vertically in the soil. They delimited a soil block of 25*25 cm by 20 cm depth and were open on one side. More epigeic earthworms were found in the UVS and up to 30 cm from the UVS border than in the middle of the crop alley. By contrast, the earthworm Allolobophora chlorotica presented a homogeneous distribution in the plot. Trapped earthworms were mostly of the All. chlorotica species, and 1.6 times more earthworms were found in traps open towards the crop alley than in traps open towards the UVS. These results suggest that in spring, earthworms are moving more from the crop alley towards the UVS than in the other direction, probably using the tree row and its vicinity as a refuge against adverse summer conditions in the crop alley.

Structure and diversity of earthworm communities in long-term irrigated soils with raw effluent and treated wastewater

Abstract This study was conducted in two natural meadows: first, soils were irrigated with raw wastewater (SIRWW) and in the second, soils were irrigated with treated wastewater (SITWW). Earthworms were sampled in eight soil blocks spaced 10 m apart at each site. Earthworm community was characterized and compared using density, biomass, composition, structure, species richness, and diversity parameters. At both meadows, 459 earthworm individuals from two families and seven species were collected. The highest earthworm density and species richness were recorded at SIRWW. Nicodrilus caligenus was the most abundant species. Most of earthworm community parameters decreased significantly at SITWW. Only two species (N. caligenus and Octodrilus complanatus) were common between the two grasslands. Among the seven species identified at both meadows, four (Allolobophora longa, Eisenia foetida, Allolobophora rosea, Allolobophora chlorotica) were exclusively present in SIRWW, whereas a single species (Amynthas sp.) was characterized in SITWW. Three ecological earthworm groups (epigeic, endogeic, and anectic) were represented in SIRWW, with the dominance of endogeics. Further studies are needed to quantify pollution in this soils and the accumulation of pollutant load in earthworms. It is also important to highlight the relationship between the abundance and diversity of earthworms in these two ecosystems with soil biological activity.

Earthworm populations and diversity under annual and perennial wheat in a North to South gradient in Western Europe

The challenge to sustain food security while halting the loss of biodiversity and soil quality might be achieved by a transformation in agriculture from high-input management of annual crops to a more nature-based solution introducing perennial cropping systems. This study analysed earthworm communities (numbers, biomass, ecological categories) and diversity over two years, from annual wheat and perennial intermediate wheatgrass (IWG, Thinopyrum intermedium, Kernza®) within the EU-Biodiversa project NAPERDIV from Southern to Northern Europe. Study sites in France, Belgium and Sweden represented diverse soil, climatic and plant growth conditions. In total, 16 species were identified with IWG in France having the highest (13) and annual wheat in Belgium and Sweden the lowest (7) species numbers. Improved biodiversity under perennial wheat was indicated by alpha-diversity indices (Simpson index, Shannon-Weaver index, Evenness). Earthworm abundance and biomass were generally significantly higher in IWG across the three sites (GLMM model). The overall mean earthworm number under IWG was 424.7 No. m−2 compared to 164.7 No. m−2 for annual wheat. Mean earthworm biomass under IWG was 83.7 g m−2 relative to 45.9 g m−2 under annual wheat, respectively. Remarkably, mean number of juvenile earthworms was several times higher on IWG sites relative to the annual comparatives. Moreover, endogeic and epigeic earthworms were supported on the IWG plots. Beta diversity (Sorensen coefficient) emphasised highest similarity between Belgium and Sweden and lowest between France and Sweden, indicating a possible South to North distribution within Western Europe. The Canonical Correspondence Analysis showed discrete clusters for study sites and species distribution (including the subtypes of Allolobophora chlorotica) in relation to soil parameters (pH, soil texture, TOC, TN, WHC, C–N ratio). The CCA additionally discriminated between annual and perennial plots in France. In summary, earthworm communities were more diverse under IWG and seemed to follow a South to North gradient.

Endogeic Earthworms Avoid Soil Mimicking Metal Pollution Levels in Urban Parks

In response to long-lasting high levels of metallic trace elements (MTEs) in urban soils, we expect soil invertebrates inhabiting urban environments to have evolved detection and avoidance and/or tolerance mechanisms to MTE pollution. In this study, I used artificial soils with concentrations of lead, zinc, copper, chromium and nickel that reflect pollution levels in the soils of Parisian parks. Using choice experiments, I compared habitat preference (i.e., the occurrence of individuals in the polluted vs. unpolluted soil) and health status (i.e., body mass maintenance, mobility, mortality) between three species of endogeic earthworms—Aporrectodea caliginosa, Aporrectodea icterica and Allolobophora chlorotica—originating either from urban or rural grasslands. This study highlights a clear avoidance of MTE-polluted soils in all three species, as well as MTE-induced health impairments, especially in A. chlorotica. Interestingly, earthworm response to MTE exposure only slightly differed between earthworms of urban and rural origin, suggesting the absence of widespread acclimatization or adaptation mechanisms to MTE pollution in cities. As a consequence, MTE pollution is expected to significantly shape earthworm spatial distribution in both urban and rural environments and, as a consequence, affect ecosystem functioning.

Variation in the Chemical Sensitivity of Earthworms from Field Populations to Imidacloprid and Copper.

The chemical risk of pesticides for nontarget soil macroorganisms has mainly been assessed using the compost earthworm Eisenia fetida. However, E. fetida does not occur in agroecosystems, and it is generally less sensitive than other earthworm species. Thus, the extrapolation of its response to pesticides to other earthworm species may lead to uncertainties in risk assessment. Because toxicity data for other earthworms are scarce, we assessed the chemical sensitivity of five species (Allolobophora chlorotica, Aporrectodea caliginosa, Aporrectodea longa, Aporrectodea rosea, and Lumbricus rubellus) from different habitats (forests, wetlands, and grasslands), as well as E. fetida, to imidacloprid and copper in single-species acute toxicity tests. In addition, we examined the relationship between earthworm traits (ecotype and weight), habitat characteristics (ecosystem type and soil pH), and chemical sensitivity. The lower limits of the hazardous concentration affecting 5% (HC5) of species were 178.99 and 0.32 mg active ingredient/kg dry weight for copper and imidacloprid, respectively. Some concentrations that have been measured in European agroecosystems for both pesticides were above the HC5s, indicating toxic risks for these organisms. Furthermore, soil pH from the sampling habitat played a significant role, with earthworms sampled from extremely acidic soils being less sensitive to copper than earthworms from neutral soils. In addition, endogeic earthworms were more sensitive to imidacloprid than epigeic earthworms. This may translate to changes in soil functions such as bioturbation, which is mainly carried out by endogeic earthworms. Our results suggest that risk assessment should include a wider range of earthworms covering different habitats and ecosystem functions to achieve a better protection of the biological functions carried out by these key soil organisms. Environ Toxicol Chem 2023;42:939-947. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Role of soil texture and earthworm casts on the restoration of soil enzyme activities after exposure to an organophosphorus insecticide

Pesticides exert important effects on the soil fauna and health. However, little is known about the interactions of soil, microorganisms and earthworms in the presence of pesticides and about their respective roles in the soil biological activity. The aim of this study was to evaluate the effect of the soil type on enzyme activities, measured in bulk soil and in casts of two earthworm species, after exposure to the organophosphorus pesticide parathion. To this aim, two endogeic earthworm species (Apporectodea caliginosa and Allolobophora chlorotica) were cross-acclimated in two different soil textures (each representing the most favorable soil environment for that species). Enzyme activities were measured as a soil quality indicator in samples of bulk soil (collected at day 4 and day 7 of exposure to parathion) and in earthworm casts (collected at day 7). A short exposure (4 days) to parathion significantly (ANOVA, p < 0.001) inhibited carboxylesterases (25–43 % of inhibition) and alkaline phosphatase (∼23 % of inhibition). At day 7 of exposure, parathion impact on the overall soil enzyme activities mainly depended on the soil texture. Indeed, activity inhibition was higher (ANOVA, p < 0.001) in silt-clay soil (decrease by 37 %) than in silt-loamy soil (decrease by ∼18 %). Conversely, parathion effect was not influenced by earthworm presence/absence and earthworm species. However, after soil exposure to parathion, earthworms (both species) improved enzyme activity restoration in their casts.

EFFECT OF NITROGEN ON GROWTH AND YIELD OF OKRA VARIETIES (Abelmoschus esculentus L.) GROWN IN THUA THIEN HUE

The experiments were conducted in a three-replicate split-plot design of 2 × 4 treatments, with variety as the main plot and nitrogen fertilizer application as the sub-plot. Four nitrogen rates: 60, 90, 120, and 150 kg/ha and two varieties: the local variety and the F1 variety, were investigated. The results show that nitrogen significantly affects the stock diameter at 37-day planting, fruit diameter, length, number per plant, weight, theoretical yield, and actual yields of both varieties. However, the interaction between fertilizer and variety is insignificant for all parameters. Nitrogen fertilizer at 150 kg/ha increases the plant height, the number of leaves per plant, and the yield of both varieties, whereas the 60 kg/ha rate shows the lowest value of these traits. More diamondback moths and Green worms are found on the F1 variety than that on the local variety. Nitrogen does not affect the pests that harm the plants of the F1 variety. It is recommended to apply 150 kg/ha of nitrogen for okra grown in the Winter-Spring season.

Do earthworms increase grass biomass production and phosphorus uptake under field conditions?

The key nutrient phosphorus (P) binds strongly to reactive soil particles, which makes it poorly available for plant uptake. In the search for sustainable ways to overcome a resulting P shortage, it has been shown that earthworms can increase the pool of plant available P and enhance plant P uptake under controlled (greenhouse) conditions. To validate these findings under field conditions and to study the effect of earthworm community composition, we conducted a mesocosm-field experiment on grassland. Mesocosms containing a sandy soil with a low P-status and communities of five earthworm species common to the Netherlands (Lumbricus rubellus, Aporrectodea caliginosa, Allolobophora chlorotica, Lumbricus terrestris and Aporrectodea longa; monocultures, three- or five-species mixtures and controls without earthworms) were installed in a field. Aboveground biomass production and P uptake of Lolium perenne were monitored for over two years. Earthworm community composition varied between the start and the end of the experiment, but multiple linear regression on the final earthworm communities yielded strong indications that earthworms increased both biomass production (R2adj = 0.52, p < 0.001, n = 76) and P uptake (R2adj = 0.48, p < 0.001, n = 76). The species A. longa and L. terrestris were most influential for this earthworm effect. We did not observe any general relations between the number of earthworm species in a community and a P effect. Our results suggest that earthworms can indeed increase grass biomass production and P uptake on a low P soil in the field, and can thereby contribute to making the P nutrition of our agricultural systems more sustainable.

Gut microbiome reflect adaptation of earthworms to cave and surface environments

BackgroundCaves are special natural laboratories for most biota and the cave communities are unique. Establishing population in cave is accompanied with modifications in adaptability for most animals. To date, little is known about the survival mechanisms of soil animals in cave environments, albeit they play vital roles in most terrestrial ecosystems. Here, we investigated whether and how gut microbes would contribute to the adaptation of earthworms by comparing the gut microbiome of two earthworm species from the surface and caves.ResultsTwo dominant earthworm species inhabited caves, i.e., Allolobophora chlorotica and Aporrectodea rosea. Compared with the counterparts on the surface, A. rosea significantly decreased population in the cave, while A. chlorotica didn’t change. Microbial taxonomic and phylogenetic diversities between the earthworm gut and soil environment were asynchronic with functional diversity, with functional gene diversity been always higher in earthworm gut than in soil, but species richness and phylogenetic diversity lower. In addition, earthworm gut microbiome were characterized by higher rrn operon numbers and lower network complexity than soil microbiota.ConclusionsDifferent fitness of the two earthworm species in cave is likely to coincide with gut microbiota, suggesting interactions between host and gut microbiome are essential for soil animals in adapting to new environments. The functional gene diversity provided by gut microbiome is more important than taxonomic or phylogenetic diversity in regulating host adaptability. A stable and high-efficient gut microbiome, including microbiota and metabolism genes, encoded potential functions required by the animal hosts during the processes of adapting to and establishing in the cave environments. Our study also demonstrates how the applications of microbial functional traits analysis may advance our understanding of animal-microbe interactions that may aid animals to survive in extreme ecosystems.

High-resolution melting (HRM) curve analysis as a potential tool for the identification of earthworm species and haplotypes.

BackgroundEarthworm communities are an important component of soil biodiversity and contribute to a number of ecosystem functions such as soil-nutrient cycling. Taxonomic identification is an essential requirement to assess earthworm biodiversity and functionality. Although morphological identification of species is labour-intensive, it is the most commonly used method due to a lack of cost-efficient alternatives. Molecular approaches to identify earthworms at species and haplotype level such as DNA barcoding are gaining popularity in science but are rarely applied in practice. In contrast to barcoding, the differentiation of PCR products based on their thermal denaturation properties using high-resolution melting (HRM) curve analysis is a fast and cost-efficient molecular closed-tube, post-PCR tool that allows identification of taxa.MethodsWe developed a HRM curve assay to identify eight earthworm species common to agricultural soils in Central Europe (Allolobophora chlorotica, Aporrectodea caliginosa, Apo. limicola, Apo. longa, Apo. rosea, Lumbricus castaneus, L. rubellus, and L. terrestris). For this, a new primer pair targeting a 158-bp long subregion of the cytochrome c oxidase I (COI) gene was designed. Our HRM assay was further tested for the differentiation of COI haplotypes using 28 individuals of the earthworm species Allo. chlorotica. Furthermore, we developed a novel extraction method for DNA from earthworm tissue that is fast and requires minimal consumables and laboratory equipment.ResultsThe developed HRM curve assay allowed identifying all eight earthworm species. Performing the assay on 28 individuals of the earthworm species Allo. chlorotica enabled the distinction among different COI haplotypes. Furthermore, we successfully developed a rapid, robust, scalable, and inexpensive method for the extraction of earthworm DNA from fresh or frozen tissue.ConclusionsHRM curve analysis of COI genes has the potential to identify earthworm species and haplotypes and could complement morphological identification, especially for juvenile or damaged individuals. Our rapid and inexpensive DNA extraction method from earthworm tissue helps to reduce the costs of molecular analyses and thereby promote their application in practice.

Mitonuclear discordance and patterns of reproductive isolation in a complex of simultaneously hermaphroditic species, the Allolobophora chlorotica case study

Historical events of population fragmentation, expansion and admixture over geological time may result in complex patterns of reproductive isolation and may explain why, for some taxa, the study of mitochondrial (mt) and nuclear (nu) genetic data results in discordant evolutionary patterns. Complex patterns of taxonomic diversity were recently revealed in earthworms for which distribution is largely the result of paleogeographical events. Here, we investigated reproductive isolation patterns in a complex of cryptic species of earthworms in which discordant patterns between mt and nu genetic lineages were previously revealed, the Allolobophora chlorotica aggregate. Using four nu microsatellite markers and a fragment of the cytochrome c oxidase subunit I mt gene, we carried out a parentage analysis to investigate the mating patterns (i) between individuals belonging to two divergent mt lineages that cannot be distinguished with nu markers and (ii) between individuals belonging to lineages that are differentiated both at the mt and nu levels. Amongst the 157 field‐collected individuals, 66 adults were used in cross‐breeding experiments to form 22 trios based on their assignment to a mt lineage, and 453 obtained juveniles were genotyped. We showed that adults that mated with both their potential mates in the trio produced significantly more juveniles. In crosses between lineages that diverged exclusively at the mt level, a sex‐specific pattern of reproduction characteristic to each lineage was observed, suggesting a possible conflict of interest concerning the use of male/female function between mating partners. In crosses between lineages that diverged both at the mt and nu levels, a high production of cocoons was counterbalanced by a low hatching rate, suggesting a post‐zygotic reproductive isolation. Different degrees of reproductive isolation, from differential sex allocation to post‐zygotic isolation, were thus revealed. Lineages appear to be at different stages in the speciation process, which likely explain the observed opposite patterns of mitonuclear congruence.

Therapeutic Values of Earthworm Species Extract from Azad Kashmir as Anticoagulant, Antibacterial, and Antioxidant Agents.

Aims Current research aimed to explore the therapeutic values of different earthworms as antibacterial, anticoagulant, and antioxidant agents. Methods Ten different earthworms, i.e., Amynthas corticis, Amynthas gracilis, Pheretima posthuma, Eisenia fetida, Aporrectodea rosea, Allolobophora chlorotica, Aporrectodea trapezoides, Polypheretima elongata, Aporrectodea caliginosa, and Pheretima hawayana, were collected and screened for biological activities. Antibacterial effect analysis of earthworm species was done against fourteen bacterial pathogens, i.e., Escherichia coli, Serratia marcescens, Streptococcus pyogenes, Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa (1), Salmonella typhimurium, Shigella flexneri, Enterobacter amnigenus, Serratia odorifera, Pseudomonas aeruginosa (2), Staphylococcus warneri, and Lactobacillus curvatus, via agar well diffusion, crystal violet, MTT, agar disc diffusion, and direct bioautography assays. Antioxidant potential was evaluated through ABTS and DPPH assays. Lipolytic, proteolytic, and amylolytic assays were done for lipase, protease, and amylase enzymes confirmation. In vitro anticoagulant effects were examined in the blood samples by measuring prothrombin time. Results Results revealed that all earthworm extracts showed the inhibition of all tested bacterial pathogens except P. aeruginosa (1), P. aeruginosa (2), S. warneri, and L. curvatus. The maximum zone of inhibition of E. coli was recorded as 14.66 ± 0.57 mm by A. corticis, 25.0 ± 0.0 mm by P. posthuma, 20.0 ± 0.0 mm by E. fetida, and 20.0 ± 0.0 mm by A. trapezoid. Cell proliferation, biofilm inhibition, the synergistic effect of extracts along with antibiotics, and direct bioautography supported the results of agar well diffusion assay. Similarly, P. hawayana, A. corticis, A. caliginosa, and A. trapezoids increase the prothrombin time more efficiently compared to other earthworms. A. corticis, A. gracilis, A. rosea, A. chlorotica, P. elongata, and A. trapezoides showed maximum DPPH scavenging potential effect. Conclusions The coelomic fluid of earthworms possessed several bioactive compounds/enzymes/antioxidants that play an important role in the bacterial inhibition and act as anticoagulant agents. Therefore, the development of new therapeutic drugs from invertebrates could be effective and potential for the prevention of the emergence of multidrug-resistant bacteria.

Assessing the efficacy of antibiotic treatment to produce earthworms with a suppressed microbiome

Earthworms are an integral part of soil ecosystems, especially for their role in soil functions such as organic matter (OM) decomposition and nutrient cycling. Earthworms and microorganisms are interdependent, and a considerable portion of the contribution earthworms make to influencing OM fate is through interactions with microorganisms. However, the importance of the earthworm-associated microbiome is not fully understood, because it is difficult to separate the direct influence of the earthworms from the indirect influence of their microbiome. Here, we evaluated an antibiotic-based procedure to suppress the microbiome of individuals of ecologically-contrasting earthworm species (Eisenia fetida, Lumbricus terrestris, Allolobophora chlorotica) as the first step towards soil studies aimed at understanding the importance of the earthworm microbiome for host health and function. Individual earthworms were exposed to antibiotics: cycloheximide (150 μg ml−1), ampicillin (100 μg ml−1), ciprofloxacin (50 μg ml−1), nalidixic acid (50 μg ml−1), and gentamicin (50 μg ml−1) either singly or in a cocktail via culture (96 h) in a semi-solid agar carrier. Compared to the non-antibiotic treated control, the cocktail (for all three species) and ciprofloxacin (for E. fetida and A. chlorotica) treatments significantly reduced (P < 0.05) culturable microbial abundance on nutrient agar and potato dextrose agar. The microbial counts were reduced to below detection (<50 CFU individual−1) for E. fetida and A. chlorotica receiving the cocktail. Illumina 16S rDNA amplicon sequence analysis of culturable L. terrestris -associated bacteria showed that antibiotic treatment influenced community composition revealing putative sensitive (Comomonas, Kosakonia and Sphingobacterium) and insensitive (Aeromonas, Pseudochrobactrum) taxa. Overall, we report a rapid, with minimal earthworm-handling, process of creating suppressed-microbiome E. fetida, A. chlorotica and L. terrestris as a tool to be used in future ecological studies of earthworm microbial interactions affecting host health and function.

Bioaccumulation of PCBs and their hydroxy and sulfonated metabolites in earthworms: Comparing lab and field results

Sulfonated and hydroxy-sulfonated PCBs were recently discovered by our group as new PCB soil contaminants, constituting about 1% of their parent compounds in soil. Here we investigate for the first time the bioaccumulation of these metabolites as well as hydroxy-PCBs and native PCBs in earthworms. A sequence of three experiments, at increasing complexity and ecological realism, were performed with four different earthworm species (Eisenia foetida Savigny, Lumbricus terrestris L, Allolobophora chlorotica Savigny and Aporrectodea caliginosa Savigny) exposed to contaminated soils. The first experiment confirmed that when exposing earthworms to soil contaminated with a single hexa-chlorinated congener (PCB 155), no formation of polar metabolites in earthworms could be detected. This allowed to plan the following two experiments, using a soil from a PCB contaminated site and rich in relatively high levels (10–130 μg kg−1) of hydroxy-, sulfonated-, and hydroxy-sulfonated-PCBs. Bioaccumulation factors (BAFs) and bioconcentration factors (BCFs) were then obtained in the second and third experiments, to compare the accumulation behavior of these chemicals in laboratory and natural conditions. Regressions between BAF/BCF and Log Kow/Log D, produced a variety of results, being generally significant between BCF and PCBs and not significant in the other cases. In general, the metabolites accumulated in earthworms with detectable concentrations in their tissues (8–115 μg kg−1), although sulfonated and hydroxy-sulfonated PCBs showed BAF and BCF values lower (up to two orders of magnitude) than those calculated for the parent PCBs, given their lower lipophilicity.

Antifungal Activity of Earthworm Coelomic Fluid Obtained from Eisenia andrei, Dendrobaena veneta and Allolobophora chlorotica on Six Species of Phytopathogenic Fungi

The functioning of soil ecosystems greatly depends on the interactions occurring between soil biota communities. It is well known that earthworms are an important soil component that substantially affects its function, including their meaningful impact on the development of different phytopathogenic soil fungi. Phytopathogenic fungi are responsible for crop disease and cause great economic damage. It has previously been established that earthworms’ coelomic fluid can suppress the growth of phytopathogenic fungi, but the exact molecular mechanism is unknown. The present study aimed at broadening the proof of this observed phenomenon by investigating the effects of the coelomic fluid extract of three different earthworm species (Eisenia andrei, Dendrobaena veneta and Allolobophora chlorotica) on the growth of six different phytopathogenic fungi species (Berkeleyomyces basicola, Fusarium culmorum, Globisporangium irregulare, Rhizoctonia solani, Macrophomina phaseolina, and Sclerotinia sclerotiorum). Coelomic fluid extract was obtained by electrostimulation or usage of extraction buffer (only in case of A. chlorotica) and prepared in three different concentrations by diluting the obtained coelomic fluid with physiological saline. The coelomic fluid extract of the three investigated earthworm species had an inhibitory effect on the growth of all six phytopathogenic fungi species. The greatest inhibitory effect was achieved with the E. andrei coelomic fluid extract reducing the growth of R. solani fungi. The findings of this research confirm the antifungal activity of coelomic fluid obtained from earthworm species belonging to different ecological categories and may be of potential use in crop protection against phytopathogenic fungi.

A method to assess glyphosate, glufosinate and aminomethylphosphonic acid in soil and earthworms

A new sensitive and selective analytical methodology to quantify glyphosate (GLY), aminomethylphosphonic acid (AMPA), and glufosinate (GLU) in both soil and earthworms (Allolobophora chlorotica) was developed. The extraction and purification methods were optimized. The samples were extracted with various aqueous solutions (HNO3, H2O, KOH and borate buffer) and derivatized with 9-Fluorenylmethyl chloroformate (FMOCCl). To optimize the extraction step, a method to remove the excess FMOCCl was applied based on liquid-liquid extraction with diethyl ether. The purification of derivatized extracts was carried out using XLB solid phase extraction (SPE) cartridges before internal standard quantification by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The elution step was optimized to obtain the best recoveries possible, which was with acidic methanol (1% formic acid) (67% for GLY, 70% for GLU and 65% for AMPA). The extraction and purification method followed by analysis of the two herbicides and AMPA in soils using LC/MS/MS determined limit of quantification (LOQ) values of 0.030 μg g − 1 for GLY, 0.025 μg g − 1 for AMPA and 0.020 µg g − 1 for GLU . For earthworms, LOQ were 0.23 μg g − 1 for GLY, 0.20 μg g − 1 for AMPA and 0.12 μg g − 1 for GLU. .The developed method was applied to determine these compounds in natural soils and earthworms.