Autofluorescence in eleocytes of some earthworm species

Immunocompetent cells of earthworms, coelomocytes, comprise adherent amoebocytes and granular eleocytes (chloragocytes). Both cell populations can be expelled via dorsal pores of adult earthworms by exposure to an electric current (4.5 V) for 1 min. Analysis by phase contrast/fluorescence microscopy and flow cytometry demonstrated that eleocyte population of several species exhibits a strong autofluorescence. A high percentage (11-35%) of autofluorescent eleocytes was recorded in Allolobophora chlorotica, Dendrodrilus rubidus, Eisenia fetida, and Octolasion sp. (O. cyaneum, O. tyrtaeum tyrtaeum and O. tyrtaeum lacteum). In contrast, autofluorescent coelomocytes were exceptionally scarce (less than 1%) in representative Aporrectodea sp. (A. caliginosa and A. longa) and Lumbricus sp. (L. castaneus, L. festivus, L. rubellus, L. terrestris). Thus, this paper for the first time describes profound intrinsic fluorescence of eleocytes in some--but not all--earthworm species. The function (if any) and inter-species differences of the autofluorescent coelomocytes still remain elusive.     

Distribution, abundance and species associations of earthworms (Lumbricidae) in a reclaimed peat soil in Ireland

Fifteen species of earthworm were collected from a reclaimed fen peat soil used for rough hay in County Offaly, Ireland. The most common species were Aporrectodea tuberculata, Allolobophora chlorotica, Eisenia rosea and Octolasion tyrtaeum. A. tuberculata, A. chlorotica and E. rosea formed a significant association. The total numbers of earthworms collected by hand-sorting reached a maximum of 197.1 m⁻². Compared with hand-sorting, formalin-expulsion was an inefficient sampling method for most species. Soil moisture varied within the study area. Total densities and biomasses of the earthworms were greater where the soil was drier. A. tuberculata, A. turgida, Satchellius mammale and the pink form of A. chlorotica were more common in the dry soils whilst O. tyrtaeum, Lumbricus rubellus and the green form of A. chlorotica were more common in the wet soils.     

Earthworm community structure and diversity in experimental agricultural watersheds in Northeastern Ohio

Earthworms are known to have an important impact on soil fertility but much remains to be known about the factors that influence earthworm abundance and species diversity in agricultural soils and the impact of earthworm diversity on soil processes in those soils. We have studied factors that influence earthworm community structure and biodiversity in experimental agricultural watersheds at the North Appalachian Experimental Watershed near Coshocton, Ohio. We sampled earthworm communities in seven such watersheds from 1990 to 1992. Six earthworm species were present:Aporrectodea caliginosa, A. trapezoides, A. tuberculata, Lumbricus rubellus, Lumbricus terrestris and Octolasion tyrtaeum. The total earthworm biomass ranged from 2 to 32 g m^-2 and population levels ranged from 10 to 350 worms m^-2. Earthworm community structure and diversity differed among watersheds and was influenced by cropping patterns, geographic location and tillage. The greatest earthworm diversity and highest earthworm population levels occurred in a no-tillage watershed and a watershed that had previously been in ryegrass and long-term no-till. High earthworm populations were also observed in a chisel-plowed watershed. Watersheds that had been managed identically for 50 years had much different earthworm communities, indicating that factors other than management were important in determining species composition. The three watersheds with the lowest populations and diversity were adjacent to one another at one end of the station, indicating that at this site geographic location had the predominant influence on earthworm communities. A severe drought in 1991 greatly reduced earthworm populations and biomass. However, earthworm species differed in their response to drought withLumbricus rubellus appearing to be the most drought-sensitive andAporrectodea spp. the most drought-tolerant.     

Tissue distribution and characterization of cholinesterase activity in six earthworm species

To validate cholinesterase activity as a biomarker of pesticide exposure, we characterized the tissue distribution (whole body, nervous tissue and crop/gizzard), activity at two seasons of cholinesterase in six different species of earthworms collected in an unpolluted field: Lumbricus terrestris, Lumbricus castaneus, Aporrectodea nocturna, Aporrectodea caliginosa, Allolobophora chlorotica and Aporrectodea rosea. The major part of total cholinesterase activity was found in the nervous tissue while activity in crop/gizzard was weak. The level of the total cholinesterase activity was stable for each species considered throughout the year (spring and autumn). Lumbricus species exhibited three-fold higher specific activity than the others (0.086+/-0.015 U mg(-1) and 0.235+/-0.036 U mg(-1) for Allolobophora or Aporrectodea, and Lumbricus species respectively). This stability of the base level makes cholinesterase activity a useful biomarker for monitoring effects of pesticide under natural conditions. Cholinesterase activity was characterized using different substrates and inhibitors. It seems likely that the cholinesterases are acetylcholinesterases in most species investigated as they preferentially hydrolyzed acetylthiocholine and were inhibited by eserine, but not by tetraisopropyl pyrophoramide (iso-OMPA). Characterization of cholinesterase from Allolobophora chlorotica is uncertain and it cannot be classified as a true AChE.     

Opening a can of worms: unprecedented sympatric cryptic diversity within British lumbricid earthworms

Earthworms play a major role in many aspects of soil fertility, food web ecology and ecosystem functioning, and hence are frequently the subjects of, for example, ecological and toxicological research. Our aim was to examine the genetic structure of common earthworm species, to identify cryptic lineages or species that may be distinct ecotypes or biotypes (and hence confound current research based upon morphotypes) and to try to explain the massive cryptic diversity that eventually emerged. We demonstrated that species such as Allolobophora chlorotica, Aporrectodea longa, Aporrectodea rosea and Lumbricus rubellus all comprise highly divergent lineages with species-level divergence at the mitochondrial cytochrome oxidase I (COI) gene. In Allo. chlorotica alone, we found 55 haplotypes for COI, with 35 of these being found in pink and 20 in green morph worms. There were no cases of the two colour morphs sharing COI haplotypes. Phylogenetic analyses of mitochondrial COI and 16S genes showed the presence of five highly divergent lineages, suggesting the presence of multiple cryptic species within Allo. chlorotica. There was no clear geographical pattern to lineage distribution and many populations were polymorphic for both mitochondrial DNA lineage and colour morph. Amplified fragment length polymorphism results, based on two primer combinations, were broadly congruent with mitochondrial DNA results with one significant exception. Despite showing over 14% divergence at COI, amplified fragment length polymorphism markers showed that the two green morph lineages may be interbreeding and therefore represent a single taxon. The cryptic diversity revealed by these results has profound consequences for all areas of earthworm research.     

Tracking and predation on earthworms by the invasive terrestrial planarian Bipalium adventitium (Tricladida, Platyhelminthes)

The potential ecological impact of exotic terrestrial planarians will be determined in part by their sensory abilities and predatory behavior. It has been suggested that these flatworms may only encounter their earthworm prey by chance, hence restricting the breadth of species they will feed upon and the number of microhabitats in which predator-prey interactions occur. We hypothesized that those flatworms that have already successfully invaded North America (genus Bipalium) actually detect and follow chemical trails of earthworms and possess the behavioral repertoire needed to feed on the prey in a range of microhabitats. We examined: (1) the tendency of Bipalium adventitium to follow chemical trails left by injured and un-injured earthworms; (2) the behavioral repertoire and predatory success of B. adventitium feeding on three earthworm species in subterranean tunnels; and (3) the response of flatworms to the reportedly defensive secretions of the earthworm Eisenia fetida in tunnels. B. adventitium detected and followed trails of earthworm mucus and secretions left by injured and un-injured earthworms. Flatworms followed trails on a range of substrates and pursued and captured three species of earthworms in subterranean tunnels, including individuals many times their mass. Although most behavior exhibited during underground attacks was similar to that reported for surface encounters, the flatworms also behaved in ways that blocked earthworm escape from tunnels. The flatworms were less successful at preying on E. fetida than on Lumbricus rubellus and Lumbricus terrestris in underground tunnels and showed some aversion to the secretions from E. fetida.     

Electron-microscopic observations of normal coelomocytes from the earthworm,Lumbricus terrestris

Summary Coelomocytes of the earthworm, Lumbricus terrestris, were studied by transmission electron microscopy. Four morphological cell types are distinguishable: lymphocytic coelomocytes, granulocytic coelomocytes, eleocytes (chloragogen cells), and inclusion-containing coelomocytes. Within these major categories, several distinct cell types differ and may represent developmental stages. The two types of lymphocytic coelomocytes are small with central nuclei and scanty cytoplasms. Two types of granulocytic coelomocytes differ greatly in shape and content; both have small dark-staining granules that resemble lysosomes. Electrocytes, derived from chloragogen tissue, contain a variety of granules, inclusions and vacuoles. Inclusion-containing coelomocytes appear as two types which may be immature and mature forms. Although these cells resemble those that have been referred to as erythroid cells in other invertebrates, the large inclusion bodies are apparently unrelated to hemoglobin; they can undergo morphologic transformation and be extruded by exocytosis. This information on lymphocytic, granulocytic and inclusion-containing coelomocytes is crucial to understanding more about cellular immunity in the earthworm.D.S.L. is supported by USPHS Training Grant AI-00453-05. E.A.S., D.H.M. and E.L.C. are supported by USPHS Grant HD-09333-03     

Prospects of using the electrophoresis technique for identification of the taxonomic status of earthworms (Lumbricidae)

The polyacrylamide gel electrophoresis technique was used for comparison of common protein of body wall tissues in five species of Lumbricidae (Eisenia nordenskioldi, E. fetida, Lumbricus rubellus, Aporrectodea caliginosa, A. longa). The statistic processing of indexes of electrophoretic similarity revealed three levels of similarity: intraspecies, interspecies, and intergenus. It was discovered that the similarity of E. nordenskioldi and E. fetida proteins corresponded to the intergenus level. The use of this method for determination of the earthworm’s taxonomic status is discussed.     

The interaction of Staphylococcal protein A with free coelomocytes of annelids.

The Staphylococcal protein A (SpA) binding protein was detected on the surface of annelid coelomocytes. The flow cytometric analysis revealed that 50% coelomocytes of Lumbricus terrestris react with SpA, a figure six times higher than the number of positive coelomocytes found in Eisenia foetida.     

Eisenia fetida中蚯蚓纤溶酶P-Ⅲ组分序列多态性

为了探讨蚯蚓纤溶酶P—Ⅲ组分序列多态性及其与蚓种关系。从Eisenia fetida中提取总RNA,利用P-Ⅲ组分序列两端的保守性设计引物,通过RT—PCR获得P—Ⅲ组分基因,并构建该组分质粒（pUC18）文库,经PCR筛选后进行序列测定及分析。在原已获得的Efp-Ⅲ-1和Efp-Ⅲ-3的基础上,利用中间引物从文库中筛选到Efp-Ⅲ-2基因。序列分析表明,该基因编码序列为720bp,编码239个氨基酸,与GenBank报道的点Efp-Ⅲ-2五条序列整体相似性高达99．02％。通过序列比对,发现Efb-Ⅲ-1、2和3具有完全相同的结构域,差异位点零星分布在序列中间;Lumbricus rubellus的六条序列同样具有Eisenia fetida这个特点,并无明显差异。序列比对证明它们是蚯蚓体内P-Ⅲ组分基因多态性的表现,而且在Lumbricidae科中与蚓种无关。     

Earthworm populations in conventional and integrated farming systems in the LIFE Project (SW England) in 1990–2000

Dilute formalin was used to sample earthworm populations in a field experiment comparing conventional (CFS) and integrated (IFS) farming systems, at Long Ashton in SW England during 1990–2000. Crops in the CFS were established by ploughing. Those in the IFS treatments were established by non‐inversion tillage: in the first 5 yrs using a Dutzi cultivator or by direct drilling (IFS‐1); from 1994 onwards using either a Vaderstad cultivator (IFS‐2) or a Dutzi cultivator (IFS‐3). These treatments had little or no effect on earthworm populations over the first 3 yrs of the experiment, but after this time total earthworm numbers and biomass were generally greater in the IFS treatments than in the CFS. Individual earthworm species (determined from 1995 onwards) differed in their response to the different farming systems. Numbers of Allolobophora chlorotica, Lumbricus festivus, L. rubellus and L. terrestris averaged over all years were significantly greater in IFS‐3 than in CFS, with densities in IFS‐2 nearly always intermediate and not significantly different from either extreme. Average treatment differences for other species were not significant, although in most years numbers of Aporrectodea longa, Lumbricus castaneus and Octolasion spp. were greatest in IFS‐3, with IFS‐2 similar to or less than CFS. There were no consistent differences between treatments for Aporrectodea caliginosa and A. rosea. Tillage method and the number of crop establishment passes were probably the main factors affecting earthworm populations, although the abundance and distribution of some species may also have been influenced by the amount of soil organic matter. During the period 1995–2000,13 species of earthworms were recorded from the site with A. caliginosa and Al. chlorotica the dominant species. These and other species which normally live in temporary burrows close to the soil surface formed about 80% of the total earthworms extracted, with the larger deep burrowing species, such as A. longa and L. terrestris, representing <20% of the total catch. Species diversity was consistently greater in the Dutzi‐based integrated system (IFS‐3) than in either the Vaderstad‐based integrated system (IFS‐2) or the conventional system (CFS). It is suggested that knowledge of the species composition of earthworm communities is essential in order to properly anticipate the likely impact of modified farming practices on earthworm populations. Careful consideration of the specific machine(s) used for tillage operations may also be required.     

Glutathione S-transferases in earthworms (Lumbricidae).

Glutathione S-transferase activity (EC 2.5.1.18) was demonstrated in six species of earthworms of the family Lumbricidae: Eisenia foetida, Lumbricus terrestris, Lumbricus rebellus, Allolobophora longa, Allolobophora caliginosa and Allolobophora chlorotica. Considerable activity was obtained with 1-chlorl-2,4-dinitrobenzene and low activity with 3,4-dichloro-1-nitrobenzene, but no enzymic reaction was detectable with sulphobromophthalein 1,2-epoxy-3-(p-nitrophenoxy)propane of trans-4-phenylbut-3-en-2-one as substrates. Enzyme prepartations from L. rubellus and A. longa were the most active, whereas A. chlorotica gave the lowest activity. The ratio of the activities obtained with 1-chloro-2,4-dinitrobenzene and 3,4-cichloro-1-nitrobenzene was very different in the various species, but no phylogenetic pattern was evident. Isoelectric focusing gave rise to various activity peaks as measured with 1-chloro-2,4-dinitrobenzene as a substrate, and the activity profiles of the species examined appeared to follow a taxonomic pattern. The activity of Allolobophora had the highest peak in the alkaline region, whereas that of Lumbricus had the highest peak in the acid region. Eisenia showed a very complex activity profile, with the highest peak ne pH 7. As determined by an enzymic assay, all the species contained glutathione, on an average about 0.5 mumol/g wet wt. Conjugation with glutathione catalysed by glutathione S-transferases may consequently be an important detoxification mechanism in earthworms.     

Distribution, prevalence and intensity of earthworm populations in arable land and grassland in Scotland

The earthworms of arable and pasture fields from 100 randomly chosen arable farms were identified and counted and their relationship with soil factors examined. Thirteen species of earthworm were recorded including Lumbricus friendi from Scotland for the first time. The most prevalent and numerous species were Aporrectodea longa, Aporrectodea caliginosa and Lumbricus terrestris. All earthworm species had cosmopolitan distributions apart from Aporrectodea nocturna and Lumbricus festivus which were confined to southern/central Scotland. Four soil characteristics (% moisture, % sand, % organic matter and pH) shared no relationship with species recorded but tillage may have had a detrimental effect on species composition and size of population. It is suggested that the magnitudes of the earthworm populations found, particularly in permanent pasture, were important in maintaining soil structure and fertility, and the spread of the New Zealand flatworm (Artioposthia triangulata), an obligate predator of earthworms, could have a considerable impact on agricultural productivity and wildlife in Scotland.     

两种生态类型蚯蚓几种消化酶活性比较研究

蚯蚓在有机残体转化和土壤养分循环中起着重要的作用,为明确不同生态类型蚯蚓的食性及其消化有机物质的能力,测定了表居型蚯蚓赤爱胜蚓（Eisenia fetida）和上食下居型蚯蚓威廉环毛蚓（Pheretima guillemi）肠道内纤维素酶、蛋白酶、酸性磷酸酶和碱性磷酸酶的活性;同时还对威廉环毛蚓排泄物中蛋白酶、磷酸酶以及CO2呼吸强度与原土进行了比较,结果表明,赤爱胜蚓肠道内纤维素酶活性远远高于威廉环毛蚓,而蛋白酶和酸性及碱性磷酸酶活性显著低于威廉环毛蚓;两种蝗 蚓肠道消化酶活化的差异与赤爱胜蚓直接以植物残体为食,而威廉环毛蚓以半分解的有机残体上的微生物为食有关。根据研究结果,提出了饲养环毛 时要注意增加饵料中微生物量的观点。     

Lumbricid earthworm invasion in the Carpathian Mountains and some other sites in Romania

Recent field research shows that the ubiquitous Lumbricus terrestris and Octolasion lacteum (the large parthenogenetic form) have invaded new habitats in Romania, replacing mostly endemic or rare species of the former earthworm communities which were recorded 20–30 years ago. Seven different cases of earthworm invasion are currently described. Invader species have larger ecological tolerances as compared with the endemic species.     

STUDIES ON THE RELATIONSHIPS BETWEEN EARTHWORMS AND SOIL FERTILITY: I. BIOLOGICAL STUDIES IN THE FIELD

Changes in the seasonal activity of several species of earthworms have been followed in a permanent pasture field for 18 months, 1945–6. The two soil conditions which chiefly determine activity are temperature and moisture. Other factors are the occurrence of an obligatory diapause in the two species Allolobophora nocturna and A. longa , and changes in population in A. chlorotica and Lumbricus terrestris.
Soil temperature and soil moisture also determined the weight of soil thrown up in the form of wormcasts during autumn, winter and spring. It is suggested that only two species, A. nocturna and A. longa , are responsible for wormcasts and that the other four common species present play little or no part in this activity.
At Rothamsted it was found that the previous agricultural history of the field is an important factor in determining the fauna. Old permanent pasture is characterized by a high percentage of A. nocturna and a rather lower percentage of A. caliginosa. Ploughing old permanent pasture and reseeding to grass after 1 or 2 years arable reduces the proportions of A. nocturna and A. caliginosa and increases that of Eisenia rosea. Arable fields have A. chlorotica as the dominant species and pasture fields 2–7 years grass after many years of arable fanning still show a high percentage of A. chlorotica and a low proportion of A. nocturna.
A survey of the Carse of Stirling, Scotland, showed that soil type is also an important factor in determining the earthworm fauna. A. caliginosa was the dominant species on the three soil types studied, but the subdominant species varied. On clay soil, A. longa was subdominant; on loam, A. longa and L. rubellus ; on sandy soil, A. longa, L. rubellus and A. chlorotica. The dominant species of pasture land at Rothamsted, A. nocturna , was not found at Stirling.     

Acidovorax-like symbionts in the nephridia of earthworms

Dense accumulations of bacteria in the excretory organs, nephridia, were first described more than 75 years ago in members of the annelid family Lumbricidae (earthworms). These nephridial symbionts were assumed to play a role in the degradation of proteins in the excretory fluid for nitrogen recycling. In the present study, the phylogenetic affiliation of the nephridial bacteria of the earthworms Lumbricus terrestris, Aporrectodea tuberculata, Octolasion lacteum and Eisenia foetida was resolved. The 16S rRNA gene sequences of the symbionts formed a monophyletic cluster within the genus Acidovorax. Similarity between symbiont sequences from different host species was 95.5-97.6%, whereas similarity was> 99% between symbiont sequences from individuals of the same species. Densely packed bacteria were detected in the ampulla of the nephridia by fluorescence in situ hybridization (FISH) using Acidovorax-specific oligonucleotide probes. No other bacterial cells could be found by FISH, although a few sequences other than Acidovorax had been found by PCR and cloning. These results suggest that the Acidovorax-earthworm symbiosis is a stable, host-specific association that has evolved from a common bacterial ancestor. Given the close phylogenetic relationship of the symbionts to proteolytic, free-living Acidovorax species, they may indeed play a role in protein degradation during nitrogen excretion by earthworms.     

Decomposition of kitchen waste amended with cow manure using an epigeic species (Eisenia fetida) and an anecic species (Lampito mauritii)

An epigeic (surface dweller) earthworm species Eisenia fetida and an anecic (deep burrower) earthworm species Lampito mauritii have been tested for decomposition of kitchen waste plus cow dung. Chemical analyses of worm-worked substrates by both species showed g/kg increases in nitrogen, phosphorus and potassium and decreases in C/N and C/P ratios after 150 days of vermicomposting. However, organic carbon matter showed reduction in their amounts for 3-4 months and afterwards slightly increased up to 150 days. E. fetida produced 0.27%, 156%, 41% and 38% increases in organic carbon, nitrogen, phosphorus, and potassium as well as 61% and 29% decreases in C/N and C/P ratios as compared to control after 150 days of earthworm inoculation. In contrast, L. mauritii produced 14%, 102%, 33% and 42% increases in organic carbon, nitrogen, phosphorus and potassium as well as 43% and 14% decreases in C/N and C/P ratios as compared to control after 150 days of earthworm activity. There was moderate mineralization and faster decomposition by E. fetida in comparison to moderate mineralization and moderate decomposition by L. mauritii. The average numbers of cocoons and adults produced were greater by E. fetida than by L. mauritii after 150 days. These results indicate E. fetida may be a better adapted species for decomposition of kitchen waste plus cow dung under tropical conditions.     

Effects of nickel, zinc, and lead-contaminated soil on burrowing rate and coelomocytes of the earthworm, Allolobophora chlorotica

We have shown previously that stubby worms Allolobophora chlorotica are sensitive to environmental stress, including metal-polluted soil. In order to discern the mechanisms of this sensitivity, adult (clitellate) Al. chlorotica were exposed in the laboratory to soil samples soaked with water (control) or Ni (1 and 2 mg/kg), Zn (1.25 and 2.5 g/kg) or Pb (5 and 10 g/kg) chlorides. Worms avoided contact with metal contaminants by prolonging burrowing time in metal-soaked samples, especially in the case of lead. Higher concentrations of the investigated metals were lethal for worms. During a 3 week exposure to lower metal concentrations, nickel and lead readily accumulated in the bodies of worms while zinc was efficiently regulated. However, body weights and numbers of non-invasively retrieved free coelomocytes (consisting of amoebocytes and riboflavin-loaded eleocytes) were significantly lower only in zinc-exposed worms. We assume that zinc regulation in worm bodies is more energy-demanding than nickel or lead bioaccumulation, thus this might be responsible for inhibition of the body gain and diminution of immunocompetent cells in zinc-exposed earthworms. Alternatively, missing free coelomocytes may actually be involved in Zn trafficking and removal through nephridia and/or in the formation of multicellular brown bodies, since metal can unbalance host/bacteria relationships.