Earthworm primers for DNA-based gut content analysis and their cross-reactivity in a multi-species system

Specific primers can be used in polymerase chain reactions (PCR) to amplify prey DNA from the gut content of generalist predators at high specificity and sensitivity. A prerequisite for applying this approach to field studies, however, is to confirm that primers are actually targeting specific prey species or prey groups and do not produce false positive results by amplifying DNA either from predator species or from the wide range of potential alternative prey found under natural conditions. Here, we report on a new group-specific primer pair for earthworms designed from cytochrome oxidase c subunit 1 (COI) sequences of 11 earthworm species found in Central Europe that can be used to detect consumption of earthworms by invertebrate predators. Besides inter-specific also considerable intra-specific variation was found for COI sequences among most of the earthworm species. We, therefore, combined a universal forward primer with an earthworm-specific reverse primer which amplified a 523 bp product from all 11 species tested. Earthworm DNA amplification was also successful in the presence of excess DNA of a predator species. The primer pair was tested against 82 non-target invertebrate species commonly found in the same habitats, including potential prey for generalist predators and predators themselves. The earthworm primer was highly specific: only one of the non-target species showed a product of similar length as the earthworms, whereas PCR with 12 non-target species produced amplicons whose length differed from that of earthworms. We conclude that the new primer will be a useful tool to investigate the role earthworms play as a food resource in soil food-webs. Moreover, we suggest that future studies utilizing DNA-based approaches for prey detection should select non-target species for cross-reactivity tests according to their abundance and importance rather than choosing representatives of taxonomic units; this will help validate the results achieved using species- or group-specific primers and guarantee their meaningful ecological interpretation.     

Two major evolutionary lineages revealed by molecular phylogeny in the parthenogenetic collembola species Folsomia candida

In order to measure the genetic variability and determine the evolutionary relationships among strains of the parthenogenetic “standard” springtail Folsomia candida, we used Random Amplified Polymorphic DNA (RAPD-PCR) markers and determined the nucleotide sequence of the 18S and 28S rDNA genes. Both types of molecular characters were found to be polymorphic. We obtained phylogenetic trees using Direct Optimization in the dynamic homology paradigm. The trees were polarized with Isotoma viridis as an outgroup. All the trees based on one or the other type of molecular characters or based on all characters pooled together, support the hypothesis of an early divergence of two distinct lineages among the 11 strains of F. candida under study. Our results also suggest that these lineages differ in their rate of evolution and mode of diversification. The geographical origin of the studied strains was examined but we found no clear relation between the phylogenetic relationships and probable geographical origins. The early divergence of several lineages in this species should be taken into account when comparing studies on genetically different strains of this model organism. RAPD-PCR typing is an easy and efficient tool for doing such a task.     

6个地方鸡种线粒体 COⅠ基因的DNA条形码研究

本研究利用线粒体细胞色素C氧化酶I (cytochrome c oxidase ⅠCOⅠ)这一特定基因的特定区段来做DNA条形编码的基础，选择其中二段序列（Bar1 Bar2）设计2个引物对6个中国地方鸡种的线粒体DNA进行扩增，以对其进行种品鉴定。本研究选择的2段序列中，以Bar1序列648bp（712位点到1359位点）多态性最高，突变位点最多，为24个，品种间平均多态性为3.860%，6个鸡种在此序列中有各自不同的特异位点，NJ聚类分析6个品种基本被聚为不同的类别，与形态学分类基本一致，该COⅠ基因的Bar1序列用于这些品种鉴定是可行的；而Bar2序列进行序列多态性分析，由于其多态性低，大多品种没有其特异位点，NJ聚类分析6个品种基本被聚为不同的类别，与形态学分类基本一致，不利于品种鉴定。从本研究结果看，利用COⅠ基因的DNA条形编码（DNA Barcodes）进行不同鸡品种鉴定具有方便、快捷、经济、准确等优点，结合传统形态学鉴定手段将会揭示更深层次的遗传多样性和系统进化关系, 经过进一步改进是用于品种鉴定的优良工具。     

Molecular phylogenetic relationships of puffer fish inferred from partial sequences of cytochrome b gene and restriction fragment length polymorphism analysis

Phylogenetic relationships among puffer fish were investigated by comparing cytochrome b gene sequences and restriction endonuclease assays of 16 species from Taiwan. DNA was prepared for sequencing by PCR. No variation in sequences was detected among individuals within each species. Direct estimates of mitochondrial cytochrome b gene sequence divergence among 16 puffer fish were from 3.41 to 31.78%. Different restriction patterns were found among 16 puffer fish with 10 restriction endonucleases, whereas no variation in patterns was detected among individuals within each species. The polymorphisms obtained by RFLP have provided a new set of genetic markers for the accurate identification of sibling puffer species. It is the first molecularly based study of puffer diversity and sheds light on the evolution and taxonomy of this major puffer fish family.     

The RAPD evidence for the phylogenetic relationship of the closely related species of cultivated apple

The phylogenetic relationship of cultivated apple and its closely related species is still not clear in the taxonomy of genus Malus. To try to find new evidence for the origin and evolution of the cultivated apple, random amplified polymorphic DNA (RAPD) markers of 14 taxa of Malus, among which a reference species (M. toringoides) and six presumably ancestral species of cultivated apple in the genus were investigated. The RAPD data obtained were used to construct both unrooted and rooted trees using TREECON software package. The result showed in our rooted tree that M. sieversii from the Xinjiang Autonomous Region of China is the species which is most closely related to the cultivated apple, M. domesticacv. `Golden Delicious'. The phylogenetic relationships among the species studied are discussed.     

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.     

Quantitative image analysis of earthworm-mediated soil displacement

The way soil is disrupted and deformed by earthworm movement is hard to quantify non-destructively. Two anecic earthworm species, Aporrectodea longa (Ude) and Lumbricus terrestris L., were compared in their effect on the soil around them as they burrow. Image analysis (particle image velocimetry, PIV) was used successfully to quantify the distance and direction of soil particle displacement by earthworm locomotion giving a unique insight into their effect on the soil structure development. The data collected from both species using PIV show a decline in soil displacement at increasing distance from the earthworm’s body. The quantity of soil being displaced was more to the sides of the earthworms (radially) than in front of the prostomium (axially). Also, L. terrestris displaced more soil than A. longa both axially and radially. The findings from this study suggest that PIV image analysis is a viable tool for examining soil displacement by earthworms and the method used has the potential to be applied to other soil organisms.     

Earthworms influenced by reduced tillage,conventional tillage and energy forest in Swedish agricultural field experiments

Abstract

We compared earthworm density, depth distribution and species composition in three soil cultivation experiments including the treatments ploughless tillage and mouldboard ploughing. Sampling was done in September 2005 and for one experiment also in 1994. By yearly sampling 1995–2005, earthworms in an energy forest of Salix viminalis were compared with those in an adjacent arable field. Sampling method was digging of soil blocks and hand sorting and formalin sampling in one cultivation experiment. Both methods were used in the energy forest and arable land comparison.

In two soil cultivation experiments, highest abundances or biomass were found in ploughless tillage. Earthworm density was higher in the upper 10 cm, especially in the ploughless tillage. Earthworm density was significantly higher in the energy forest than in the arable field. Formalin sampling revealed c. 36% of the earthworm numbers found by digging in the energy forest and gave almost no earthworms in the arable field. In all treatments with soil cultivation, species living and feeding in the rhizosphere and soil dominated. One such species, Allolobophora chlorotica, was more abundant under mouldboard ploughing than ploughless tillage. Lumbricus terrestris, browsing on the surface and producing deep vertical burrows, was more common in the ploughless tillage. Species living and feeding close to the soil surface were almost only found in the energy forest, which had not been soil cultivated since 1984.

The findings support earlier studies pointing out possibilities to encourage earthworms by reduced soil cultivation. This is one of the first published studies that followed earthworm populations in an energy forest plantation during several years. Explanation of earthworm reactions to management and environmental impacts should be done with consideration of the ecology of species or species groups. Earthworm sampling by formalin must always be interpreted with caution and calibrated by digging and hand-sorting sampling.     

Microbial community indigenous to the earthworm Eisenia foetida

 Microbial populations of the earthworm Eisenia foetida, their casts and farmyard manure (FYM) in which E. foetida had lived were enumerated with the dilution plate method using several media. The microbial community of earthworms appeared to be similar to that of the FYM in which they had lived, while that of earthworms starved in distilled water for 24 h greatly differed from that of the FYM. The community of the starved earthworms consisted exclusively (more than 90%) of fast growers, which were defined as those that formed their colonies within 2 days, unlike that of the FYM (10–30%). About 30 isolates were randomly chosen and some physiological properties were examined. The community of starved earthworms consisted exclusively of Gram-negative, oxidase-positive, fermentative bacteria, whereas bacteria from the FYM were much more diverse and there were no fermentative bacteria in the FYM. The predominant bacteria species of the starved earthworms were identified as Aeromonas hydrophila and considered to be indigenous to the earthworm E. foetida. Received: 18 June 1999     

Effects of earthworm activity and P‐solubilizing bacteria on P availability in soil

The effects of phosphate solubilizing bacteria (Bacillus megateriumi) (PSB) and earthworms (Pheretima guillelmi and Eisenia fetida) on phosphorus (P) turnover and transformation in soil were investigated in a laboratory experiment lasting for 21 days. The treatments included soil + P. guillelmi (SW1), soil + E. fetida (SW2), soil + B. megaterium (SB), soil + P. guillelmi + B. megaterium (SBW1), soil + E. fetida + B. megaterium (SBW2), and the control with sterilized soil (control) only. The results showed that the number of B. megaterium. was enhanced in all treatments especially those with earthworms added when compared with the control. Activities of acid phosphatase (pH = 6.5) increased in all treatments, especially in the presence of P. guillelmi with or without PSB (64 % and 38 %, respectively). Significant increases in both inorganic P and water‐soluble P were observed in treatments involving B. megaterium. and earthworms when compared with the control. Inoculation of both earthworms and PSB had significant effects on microbial growth, enzymatic activity, and thus enhanced the release of available P. The dual inoculation of earthworms and bacteria further accelerated P transformation. Different performances observed for the earthworm species were probably due to their different feeding habits and physiology.     

用ITS序列识别牧草种质资源

DNA条形码技术能够快速、准确地识别物种,对于开展基础性的分类学研究和应用性的生物多样性研究极为重要。本文以广西畜牧研究所牧草种质资源圃的8个物种共22个品种为材料,进行植物条形码ITS测序。PCR结果显示ITS的扩增成功率达到100%,同源性的分析表明各物种中的ITS序列变异显著,共找到99个可以用来区分物种的变异位点,系统进化树的结果表明利用ITS序列进行物种水平鉴定成功率达100%,能够作为DNA条形码识别植物物种。     

No evidence of facilitation between invasive Rhamnus cathartica (European buckthorn) and invasive earthworms in west central Minnesota

Biological invaders are seen as a real and growing threat to biodiversity, and there is much interest in the role that positive interactions among invasive species may play in promoting further invasions. Both the invasive tree Rhamnus cathartica (European buckthorn) and invasive Eurasian earthworms are reported to alter forest dynamics across large parts of the northern portion of the eastern deciduous forest of North America. Some evidence suggests that the two invaders may be aided by mutual facilitation, and it has recently been hypothesized that interactions between the two organisms may form the basis of a larger multi-species “invasional meltdown.” We examined covariance among R. cathartica and earthworm distributions over a seven year period at six forested sites near the prairie-forest border in west central Minnesota and found no evidence to support facilitation. In fact, the correlation between R. cathartica and earthworm abundance and biomass was weak: 95% confidence intervals for Kendall's tau correlation coefficients in some cases indicated a negative association among the two organism types, often suggesting no association, and never allowing for a positive association greater than τ = 0.38. Individual earthworm species varied in their correlation with R. cathartica, but most correlations were negative and none were substantially positive. We thus suggest that the relationship between these invaders may be more complicated than previously thought and question whether earthworms and R. cathartica can be seen as the foundation of an invasional meltdown.     

Schwermetallbelastung von Aporrectodea caliginosa (Oligochaeta,Lumbricidae) im Raum Kiel

Heavy metal content in Aporrectodea caliginosa (Oligochaeta, Lumbricidae) in the city of Kiel (Germany, Schleswig-Holstein) In November 1995 the content of lead, cadmium, copper and zinc in earthworms of the Aporrectodea caliginosa species was investigated at 28 sites of the Kiel area, as well as at one reference site (Belauer See, ca. 20 kilometres south of Kiel), and compared with the heavy metal content in the soil taken from all those sites. As for the lead and copper content, a positive correlation could be established between the earthworms and the soil. However, the lead content in the earthworms did not depend only on its amount in the soil, but also on soil pH and calcium content. Their copper content decreased exponentially as weight increased. The content of lead, cadmium and copper in A. caliginosa suggested an influence of urban soil pollution on the heavy metal content in earthworms. According to the grouping of heavy metal content in the earthworms, which was done in the course of the research work, the city of Kiel ranges among a minor to a medium degree of pollution caused by the investigated heavy metals.     

Short-term responses of two contrasting species of earthworms in an agricultural soil amended with coal fly-ash

With the renewed interest in the use of coal fly-ash for amendment of agricultural soils in Australia, we assessed how earthworms, as indicators of soil health, responded to this ameliorant. We assessed survival, weight, burrowing and elemental concentrations for earthworms of a native unnamed Megascolecid species and of exotic Aporrectodea trapezoides in intact soil cores treated with an alkaline fly-ash at rates equivalent to 0, 5 and 25 t/ha over 6 weeks. Fly-ash did not affect survival, growth, number of burrows created or phosphorus solubilisation. Transfer of the earthworms to the new environment having vastly different pH from where they were collected, and possibly overcrowding, caused mortality in the soil cores for all treatments. A. trapezoides that had smaller individuals suffered mortality of 12% compared with 23% for the larger earthworms of Megascolecids. Earthworms of Megascolecids each increased their weight by 0.24g (25% of their original weight) while those of A. trapezoides lost 0.18g each (21% of their original weight). The difference in growth between the two earthworms was associated with grazing habit and probably with the large difference in the pH between source soil and that of the core soil. Megascolecids appeared to minimize grazing on ash-tainted soil and so ingested less Zn, which was more abundant in the fly-ash than in the soil, compared with A. trapezoides that had elevated concentration of this metal. Extractable P in the soil was increased with both species of earthworms, more so with the exotic species that solubilized 11% more P than the native Megascolecids. The benign influence of fly-ash on survival and growth of worms was associated with the pH of soil remaining unchanged during the six weeks of incubation.     

Study of clonally propagated cassumunar ginger (<Emphasis Type="Italic">Zingiber montanum</Emphasis> (Koenig) Link ex Dietr.) and its relation of wild <Emphasis Type="Italic">Zingiber</Emphasis> species from Thailand revealed by RAPD markers

The genetic relatedness among 51 accessions, 14 species of the genus Zingiber and genetic variability of a clonally propagated species, Zingiber montanum (Koenig) Link ex Dietr., from Thailand were studied using random amplified polymorphic DNA (RAPD) profiles. Twenty-nine random primers gave reproducible amplification banding patterns of 607 polymorphic bands out of 611 scored bands accounting for 99.40% polymorphism across the genotypes. Jaccard’s coefficient of similarity varied from 0.119 to 0.970, indicative of distant genetic relatedness among the genotype studied. UPGMA clustering indicated eight distinct clusters of Zingiber, with a high cophenetic correlation (r = 1.00) value. Genetic variability in Z. montanum was exhibited by the collections from six regions of Thailand. High molecular variance (87%) within collection regions of Z. montanum accessions was displayed by AMOVA and also explained the significant divergence among the sample from six collection regions. Our results indicate that RAPD technique is useful for detecting the genetic relatedness within and among species of Zingiber and that high diversity exists in the clonally propagated species, Z. montanum.     

Earthworm effects on selected physical and chemical properties of soil aggregates

Summary Some physical and chemical properties of 1-to 2-mm aggregates obtained from casts and the burrow-wall material of the earthworm species Lumbricus terrestris, Aporrectodea longa, and Aporrectodea caliginosa were determined in order to show the effects of earthworms on the stabilization of soil aggregates. The results were compared with those of the natural soil from the Ap horizon of a Parabraunerde (Luvisol, FAO). Both the tensile strength and the water stability of aggregates from casts and burrow-wall material were reduced compared with those of the natural aggregates but were increased compared with those of remoulded aggregates. These results indicate that to a great extent existing bonds are destroyed by earthworm ingestion. Nevertheless, earthworm activities are advantageous for the stabilization of reformed aggregates. The coarse sand fraction is reduced by selective ingestion by earthworms. The organic C content is increased by 4.1–21.0% for burrow-wall material and by 21.2–43.0% for casts. The carbonate content of aggregates from casts and burrow-wall material of L. terrestris was reduced by more than 50%, while that of A. longa showed no noticeable changes and that of A. caliginosa was increased by more than 60%. The total content of polysaccharides was increased by 35–87% for casts and by 33–46% for the burrow-wall material of all earthworm species. The most frequently detected monosaccharides were glucose, galactose, and glucosamine. L. terrestris appeared to have the strongest effect on the interparticle bonding of the reformed aggregates, measured both as tensile strength and water stability, followed by A. longa and A. caliginosa.     

Earthworms influence metal transfer from soil to snails

Most of the studies focusing on metal transfer from soil to biota ignore the possible non-trophic influence of an organism on pollutant transfer to other species. We hypothesised that an earthworm (Aporrectodea tuberculata) might modify the bioavailability of metals in soil and thus, their transfer to the snail Helix aspersa. Snails were exposed for 2 weeks to a multicontaminated field soil with or without earthworms, under controlled conditions. When exposed with earthworms, snails had higher concentrations of Cd, Cu and Zn than when they were exposed alone, while no difference was detected for Pb. For Cd only, the difference in snail bioaccumulation corresponded to an increase in its water-soluble fraction. Internal concentrations of metals in earthworms remained similar in the presence or absence of snails. Two non-exclusive possible mechanisms, including variations in bioavailable fractions and/or total accessible pools of metals, are proposed to explain how earthworms could modulate the transfer of metals from soil to snails. This work demonstrated that metal transfer from soil to one invertebrate species was influenced by another invertebrate. We conclude that the concept of intermediary species, usually used to describe interactions among species, should be extended to the interactions between biota and pollutants in non-biotic compartments.     

The influence of mineral and organic fertilisers on the growth of the endogeic earthworm Octolasion tyrtaeum (Savigny)

Endogeic earthworms play an important role in mobilisation and stabilisation of carbon and nitrogen in forest and arable soils. Soil organic matter is the major food resource for endogeic earthworms, but little is known about the size and origin of the organic matter pool on which the earthworms actually live. We measured changes in body mass of juvenile endogeic earthworms, Octolasion tyrtaeum (Savigny), in soils with different C and N contents resulting from different fertiliser treatments. The soil was taken from a long-term experiment (Statischer Düngungsversuch, Bad Lauchstädt, Germany). The treatments included (1) non-fertilised soil, (2) NPK fertilised soil, (3) farmyard manure fertilised soil and (4) NPK + farmyard manure fertilised soil. The soil was incubated in microcosms with and without one juvenile O. tyrtaeum for 80 days.Earthworm biomass decreased in non-fertilised soil by 48.6%, in NPK soil by 9.4%, but increased in farmyard manure soil by 19.7% and 42.8% (soil with additional NPK application). In farmyard manure treatments the biomass of bigger individuals decreased, but in smaller individuals it increased. In NPK fertilised soil without farmyard manure only small O. tyrtaeum increased in body mass, whereas in the non-fertilised soil all individuals decreased in body mass. Generally, soil respiration correlated positively with soil carbon content. Earthworms significantly increased soil respiration and nitrogen leaching and this was most pronounced in farmyard manure treatments. Microbial activity was generally higher in farmyard manure soil indicating that farmyard manure increases labile organic matter pools in soil. Also, biomass of earthworms and microorganisms was increased in farmyard manure soil. The presence of earthworms reduced microbial biomass, suggesting that earthworms feed on microorganisms or/and that earthworms and soil microorganisms competed for similar organic matter pools in soil. The results demonstrate that NPK fertilisation only is insufficient to sustain O. tyrtaeum, whereas long-term fertilisation with farmyard manure enables survival of endogeic species due to an increased pool of utilisable soil organic matter in arable soil.     

Associations between soil texture,soil water characteristics and earthworm populations in grassland

Abstract

The aim of the present study was to investigate the relationships between soil physical characteristics and earthworms in a regional-scale field study in Denmark. The earthworm populations along within-field gradients in soil texture were quantified at five field sites, representing dominant soil types of Denmark. Eleven earthworm species were found, but populations were mainly dominated by Aporrectodea tuberculata and A. longa. Despite considerable variation in soil parameters across the five study sites the results suggest that the biomass of anecic worms (or A. longa as a species) was not causally associated with the soil parameters studied. This indicates that there must be other causal factors associated with the abundance (and composition) of anecic worms that are not among the soil texture and structure parameters studied. On the other hand, soil texture (Coarse sand) was associated with the abundance of the dominant endogeic species, A. tuberculata, but not endogeic worms in general. It was hypothesized that anecic and endogeic earthworms might respond to local soil water characteristics rather than soil texture, but this hypothesis could not be confirmed with the present data.