Non-Susceptibility of Earthworm Eisenia fetida to the Rhabditid Nematode Phasmarhabditis hermaphrodita, a Biocontrol Agent of Slugs

The rhabditid nematode Phasmarhabditis hermaphrodita is a lethal parasite for slugs that is sold commercially in Europe under the trade name Nemaslug™. We evaluated the effects of P. hermaphrodita on the earthworm Eisenia fetida (Savigny). Adults of E. fetida were exposed in one-liter glass beakers to P. hermaphrodita at three concentrations (1×, 10× and 50× of the field recommended rate of 3×10⁹ billion nematodes/ha) during a 14-day period in an artificial soil substrate. The average body weight, burrowing behavior, mortality and other clinical signals of the earthworms were recorded at 0, 7, and 14 days after exposure to the treatments. In addition, injured earthworms (posterior ends removed) were exposed to the 10× field recommended rate of the commercial formulation. Neither intact nor injured E. fetida showed susceptibility to the slug-parasitic nematode P. hermaphrodita during the 14 days of exposure even at concentrations 10 and 50 times higher than the label dose. However, the worms in the attenuated control (autoclaved formulation) had higher mortality and lost less weight compared to the other treatments. Under the conditions of the test, we conclude that the use of the commercially available strain of P. hermaphrodita is safe to E. fetida.     

Susceptibility of North American Native and Non-native Slugs (Mollusca: Gastropoda) to Phasmarhabditis hermaphrodita (Nematoda: Rhabditidae)

Phasmarhabditis hermaphrodita caused significant mortality of the two native species, Deroceras laeve and Leidyula floridana, and of one introduced species D. reticulatum, but not of the other three introduced species, Arion hortensis, A. subfuscus and Limax maximus. Even the juvenile stages of A. subfuscus and L. maximus showed no mortality in nematode treatments. However, treatments with nematodes resulted in rapid and strong feeding inhibition in all six species. Surviving slugs resumed feeding when fresh food was provided. This study expands the host range of P. hermaphrodita to include a new family Vaginulidae and demonstrates the inability of P. hermaphrodita to cause mortality of A. hortensis, A. subfuscus and L. maximus. These three species use feeding suspension as an evasive behavior to escape nematode infection.     

Structure of a herbivore–parasitoid community: Are parasitoids shared by different herbivore guilds?

Plant–herbivore–parasitoid interactions are a common occurrence in terrestrial food webs. Few parasitoids are thought to be shared by host insects of different feeding guilds because different parasitism strategies are required to use hosts of different feeding types. However, this assumption has rarely been tested using data from nature. To clarify whether parasitoids are shared among host guilds, I examined the structure of parasitoid communities on herbivore guilds associated with two Rhododendron species (Ericaceae) in a temperate secondary forest in central Japan. Leaf- and flower-feeding insects were collected from Rhododendron reticulatum and Rhododendron macrosepalum shrubs and reared in the laboratory for 3 years from April 1999 to March 2002. In total, 79 species of holometabolous herbivores (Lepidoptera, Diptera, Coleoptera, and Hymenoptera) were recorded, with 62 species on R. reticulatum and 51 species on R. macrosepalum. A total of 81 parasitoid species (Hymenoptera and Diptera) was recorded from the sampled herbivores, with 48 species from those on R. reticulatum and 50 species from those on R. macrosepalum. In total, 36 herbivore species were parasitised by 1–18 parasitoid species per host species, although the number of parasitoid species was strongly affected by sample size. Parasitoids that had two or more host species frequently attacked herbivore species from different families or on different host plants, whereas they did not attack species from different herbivore guilds; no parasitoids were shared between external feeders and rollers. Therefore, my results support the hypothesis that few parasitoids are shared among herbivores of different feeding guilds.     

Quantification of the slug parasitic nematode Phasmarhabditis hermaphrodita from soil samples using real time qPCR

Phasmarhabditis hermaphrodita is a nematode parasite that infects and kills several species of slugs. The nematode is produced commercially as a biological control agent for slug pests of agriculture and horticulture. Given the difficulties of distinguishing this species from other nematode species in soil samples, very little is known about its natural ecology or its behaviour and persistence following application for biological control. Here we describe a method to quantify P. hermaphrodita in soil samples based on real time PCR. We designed primers and a dual labelled fluorescent probe that can be used to quantify numbers of P. hermaphrodita and which is capable of distinguishing this species from the morphologically identical Phasmarhabditis neopapillosa. We compared different methods whereby the entire nematode community is extracted prior to DNA extraction, and three methods to extract DNA directly from soil samples. Both nematode extraction and DNA extraction from large (10 g) samples of soil gave reliable estimates of nematode numbers, but methods which extracted DNA from small (1 g or less) soil samples substantially underestimated numbers. However, direct extraction of DNA from soils may overestimate numbers of live nematodes as DNA from dead nematodes was found to persist in soil for at least 6 days. The technique could be modified for detection and quantification of all soil borne parasitic nematodes.     

Studies of the behaviour of Artioposhia triangulata (Platyhelminthes; Tricladida), a predator of earthworms

A significant negative correlation between the numbers of earthworms and the flatworm was demonstrated in fromalin-sampled lawns. Their distributions, recorded by a transect from a shrub border where the flatworm is suspected to have been introduced, indicated that predation may lead to earthworm extinction. Two different vermarium designs demonstrated a gradation of earthworm vulmerability to the flatworm due to depth within the soil and burrow width rather than perference for specific earthworm species. A strong correlation between the level of earth worm predation and flatworm movement implied active hunting. A predation rate was found to be 0.67 of an earthworm per flatworm per week with a metabolic conversion rate of 10%.     

江阴港口外来船舶压载舱沉积物中甲藻包囊种类及组成

为了探究外来船舶压载舱沉积物中甲藻休眠包囊的种类组成及外来甲藻入侵风险, 本文根据包囊及其萌发细胞的形态特征辅以分子生物学信息对江阴港5艘外来船舶压载舱沉积物中的活体甲藻休眠包囊的种类进行鉴定和分析。共鉴定出甲藻休眠包囊29种(不含3种未鉴定种), 包括膝沟藻类、钙甲藻类、裸甲藻类、翼甲藻类以及原多甲藻类, 活体休眠包囊密度介于0.73-44.3 cysts/g DW。其中塔玛亚历山大藻复合种(Alexandrium tamarense species complex)、网状原角藻(Protoceratium reticulatum)、具刺膝沟藻(Gonyaulax spinifera)等有毒有害甲藻的包囊在取样的5艘船舶中均有发现。此外还发现了多种中国近海未报道的甲藻包囊种类, 其中一种经过分子手段确定为异常亚历山大藻(Alexandrium insuetum)包囊, 萌发和培养实验表明该种包囊能够在中国近海萌发并增殖, 有潜在的入侵风险和暴发赤潮的可能性。     

Structure of 45,46,47-trinorhomoyessotoxin, a new yessotoxin analog, from Protoceratium reticulatum which represents the first detection of a homoyessotoxin analog in Japan

Contamination of yessotoxin (YTX) and its analogs in shellfish has occurred worldwide and has seriously damaged shellfish industries. One of the sources of YTX has been identified the dinoflagellate Protoceratium reticulatum. A new analog of YTX, 45,46,47-trinorhomoYTX, was isolated from cultures of the dinoflagellate P. reticulatum collected at Yamada Bay, Iwate in Japan. Its structure was determined by analysis of MS and NMR experiments. This is the first isolation and confirmation of a homoYTX analog in Japan.     

Prey preference and gregarious attacks by the invasive flatworm Platydemus manokwari

The flatworm Platydemus manokwari (Tricladida: Rhynchodemidae) preys on various species of land snail, and its introduction to areas outside of its native range is thought to have caused the extinction of native land snails on several Pacific islands. Platydemus manokwari occurs in areas where land snails have been absent since its invasion, suggesting that the flatworm can prey on animals other than land snails. To identify the alternative prey and prey preferences of P. manokwari, I examined the feeding ecology of P. manokwari under field and laboratory conditions. Individuals were observed attacking live earthworms in a forest where land snails are already extinct, on Chichijima, Ogasawara (Bonin) Islands, Japan. I also observed them attacking earthworms and a species of isopod and land snail in the lab. To prey on the worm, similar to other earthworm-eating flatworms, P. manokwari wrapped itself around the prey and fed on it by inserting its pharynx into the earthworm’s body. Large earthworms were able to escape P. manokwari attacks by autotomy, but the autotomized body parts were eaten. Several P. manokwari individuals together attacked earthworms that were larger than themselves. The laboratory experiments showed that such gregarious attacks increased predation success on both large earthworms and land snails. The flatworms also attacked the isopods, although the predation rate was low. These results demonstrate that P. manokwari is a polyphagous predator of slow-moving soil invertebrates (land snails, slugs, earthworms, and isopods) and that invasion by P. manokwari may directly and indirectly impact native soil fauna.     

Drought stress in rice (Oryza sativa L.) is enhanced in the presence of the compacting earthworm Millsonia anomala

Earthworms increase growth of most plant species through a number of poorly investigated mechanisms. We tested the hypothesis that earthworm modifications of soil structure and the resulting changes in water availability to plants explain this positive effect. Addition of endogeic earthworms Millsonia anomala induced a 40% increase in shoot biomass production and a 13% increase in CO₂ assimilation rate of well watered rice plants grown in pots. Conversely, when plants were subjected to water deficit, presence of earthworms had no effect on shoot biomass production and a negative impact on CO₂ assimilation rate (−21%). Early stomatal closure in presence of earthworms indicated lower water availability. The hypothesis that earthworms improve plant biomass production through soil physical structure modification was thus rejected. Three hypotheses were tested to explain this decrease in water availability: (i) a decrease in soil water retention capacity, (ii) an increase in evaporation from the soil or/and (iii) an increase in plant transpiration. Results showed that earthworms significantly reduced soil water retention capacity by more than 6%, but had no effect on evaporation rate. Water losses through transpiration were greater in the presence of earthworms when the soil was maintained at field capacity, but this was not the case under drought conditions. This experiment showed that the endogeic compacting earthworm M. anomala significantly increased plant photosynthesis by an undetermined mechanism under well-watered conditions. However, photosynthesis was reduced under drought conditions due to reduced soil water retention capacity.     

海南岛蚯蚓物种组成及其系统发育分析

海南岛独特的地理位置和复杂的古地理事件使其成为我国生物多样性的热点地区。已有的海南岛蚯蚓资料显示, 该岛的蚯蚓区系十分特殊, 且与大陆地区蚯蚓存在扩散与迁移。然而, 当海南岛与周围大陆断开形成独立岛屿后, 海南岛蚯蚓如何为适应海南岛的环境而逐渐形成现在岛内的分布与区系, 仍然是一个值得研究和探讨的问题。因此, 本研究在海南岛全面调查和采集蚯蚓标本, 整理海南岛地区的蚯蚓物种组成及其地理分布特征, 并联合5个线粒体基因COⅠ、COⅡ、ND1、12S rRNA和16S rRNA构建海南岛蚯蚓的分子系统发育树, 推测其分化时间和祖先分布区域, 探讨海南岛蚯蚓在岛内的分化与扩散过程。研究结果表明: (1)海南岛共有蚯蚓6科9属122种, 巨蚓科蚯蚓为优势科, 且全部为环毛类蚯蚓, 其中103种为海南岛特有种。蚯蚓物种数沿海拔呈先增加后减少的趋势, 在800‒1,000 m最大; (2)海南岛环毛类蚯蚓不同水平的遗传距离与我国巨蚓科蚯蚓的遗传距离区间基本一致。物种水平上, 美丽远盲蚓(Amynthas scitulus*)与纬向远盲蚓(A. zonarius)的基因遗传距离最小。在亚种水平, 保宁腔蚓指名亚种(Metaphire magna magna)和保宁腔蚓小型亚种(M. magna minuscula)的遗传距离均接近整体的物种水平。在种群水平, 等毛远盲蚓(A. homosetus)不同种群间遗传距离均接近整体的亚种水平; (3)海南岛环毛类蚯蚓可划分为7个类群, 其祖先于68.26 Ma开始分化, 可能起源于吊罗山。在新生代, 7个类群均得到较大发展。通过对海南岛蚯蚓组成及系统发育的梳理, 不仅为我国蚯蚓物种多样性研究提供基础资料, 也为岛屿蚯蚓物种系统发育关系分析提供科学参考。     

Maackiain in Cicer bijugum Rech. f. associated with resistance to Botrytis grey mould

Resistance in Cicer bijugum Rech. f. a wild relative of chickpea, to Botrytis grey mould (BGM), caused by Botrytis cinerea Pers., was shown to be associated with high concentrations of maackiain when compared to three susceptible species. The two BGM resistant accessions of C. bijugum contained between 200 and 300 μg maackiain g⁻¹ of foliage whereas the BGM susceptible species C. arietinum, C. echinospermum and C. reticulatum contained less than 70 μg g⁻¹. Furthermore, the concentration of maackiain increased to more than 400 μg g⁻¹ in the resistant wild species after being inoculated with the pathogen whereas no significant increase was recorded in the susceptible species. The germination of spores of Botrytis cinerea, treated with maackiain, was inhibited in a dose dependent manner; less than 10% of spores germinated when treated with 500 μg ml⁻¹. The data indicate that maackiain may be an important component in BGM resistance in the wild chickpea C. bijugum and that the resistance is enhanced in the presence of the pathogen.     

Lysosomal neutral red retention time as a biomarker of organophosphate exposure in the earthworm Aporrectodea caliginosa: laboratory and semi-field experiments

Aporrectodea caliginosa is the most common endogeic (topsoil) earthworm in New Zealand and, because of its habitat, is potentially vulnerable to surface-applied pesticides. Lysosomal damage to earthworms, which can be visualized by the use of the neutral red retention assay (NRRA) has been evaluated in this species as a biomarker of organophosphate exposure. Earthworms were exposed in the laboratory to sub-lethal concentrations of chlorpyrifos and diazinon. In a semi-field experiment, earthworms were placed in mesocosms in a field sprayed with these pesticides at the rate recommended for a vegetable crop. In the laboratory, the neutral red retention time (NRRT) was significantly reduced following exposure to both pesticides compared with controls. In the semi-field experiment, earthworm NRRT was significantly reduced by both pesticides. These experiments have shown that the NRRA is very sensitive to exposure to chlorpyrifos and diazinon even at field rates. It therefore shows promise as a potential biomarker of contamination of soil by organophosphates.     

蚯蚓在我国南方土壤修复中的应用

蚯蚓作为生物量最大的土壤动物, 对土壤生态系统和环境质量影响深远。本研究介绍了华南地区主要应用的皮质远盲蚓(Amynthas corticis)、毛利远盲蚓(A. morrisi)、壮伟远盲蚓(A. robustus)、参状远盲蚓(A. aspergillum)、南美岸蚓(Pontoscolex corethrurus)和赤子爱胜蚓(Eisenia fetida)的生态特征, 阐述了它们与土壤pH值、酶活性、金属富集和有效性改变、孔道和微团聚体形成之间的紧密关系: (1)蚯蚓生存的土壤酸碱性范围较广(pH为3.8-7.9), 其存活率与土壤类型、有机质含量和成分、土壤污染程度和蚯蚓种类相关; (2)肠道内、蚓粪和蚓触圈的酶活性分别表征了蚯蚓取食喜好、土壤养分循环及微生物种群特征; (3)蚯蚓能够富集不同种类的金属并改变其有效性, 这些变化具有蚓种间、金属种类间和土壤类型之间的差异; (4)蚯蚓活动及其生产的蚓粪能改变土体结构、产生孔道、影响土壤团聚体数量、大小和分布。蚯蚓的上述作用使其在解决中国南方红壤酸化、土壤金属污染、茶园土壤养分不平衡、高速公路建设临时用地土壤损毁等方面具有广阔的应用前景。目前, 由于华南远盲蚓的生理特征差异研究较少, 远盲蚓繁育技术的缺乏一定程度上限制了这些蚯蚓在中型和大型尺度下应用技术的研究和推广。有必要进一步挖掘蚯蚓在土壤修复中的潜力, 进行蚯蚓主导的相关技术研发, 深入探讨其影响机制。     

Studies on the local dispersal of the New Zealand flatworm, a predator of earthworms, in Edinburgh between 1993 and 1999

The flatworm Artioposthia triangulata was found, from studies using weighted down plastic sheeting, to move predominantly through the soil rather than over it and to use earthworm burrows. Under compost‐filled plastic sacks the flatworm was most active at night although its numbers were similar to those during the day. The transfer of specimens into an area covered with weighted plastic sheeting had no lasting effect on their numbers. The flatworm was regularly removed over 6 years from under paving stones, sheets of newspaper and cardboard placed on the ground in a garden. The rise and fall of the numbers of the flatworm under this debris suggest a predator‐prey periodicity between the flatworm and earthworms of 3 years.     

The regeneration capacity of an earthworm, Eisenia fetida, in relation to the site of amputation along the body

It is well known that parts of earthworms can survive if they are cut off. Our aim was to link the regeneration capacity of an earthworm, Eisenia fetida (Oligochaeta, Annelida) with the site of the amputation, so we amputated earthworms at different body segment locations along the length of the body to examine the different survival rates and regeneration lengths of the anterior, posterior, and medial sections.
The greatest survival rates occurred for earthworms with the most body segments remaining after amputation. The anterior regeneration lengths were of two types. The lengths of regeneration of amputated from body segment 6/7 to further down the body posteriorly increased gradually (Type L_I). However, the regeneration lengths of earthworm which were amputated behind the 23rd segment, with less than a quarter of the total segments remaining, did not increase until the blastema and tail bud formation (Type L_II). These treatments were not completely regeneration. There were significant differences in both survival rates and lengths of regeneration lengths between immature earthworms and clitellate adult earthworms during the early stages of regeneration, but not at later stages of regeneration. The immature earthworms had a greater regeneration potential than clitellate adults amputated at the same segment. The survival rates of earthworms were correlated significantly with the number of body segments remaining after amputation, but not with the position of the amputation. The relationships between the survival rates and the numbers of remaining segments could be described by linear regressions. The anterior regeneration lengths were correlated with the position of the amputation, but not with the number of remaining segments; the posterior regeneration lengths, were not correlated with the number of segments remaining nor the amputation position. The anterior regeneration length was not related to the survival rates for all earthworm amputations after 30 days but was related in this way after 60 days.     

The regeneration capacity of an earthworm, Eisenia fetida, in relation to the site of amputation along the body

It is well known that parts of earthworms can survive if they are cut off. Our aim was to link the regeneration capacity of an earthworm, Eisenia fetida (Oligochaeta, Annelida) with the site of the amputation, so we amputated earthworms at different body segment locations along the length of the body to examine the different survival rates and regeneration lengths of the anterior, posterior, and medial sections.
The greatest survival rates occurred for earthworms with the most body segments remaining after amputation. The anterior regeneration lengths were of two types. The lengths of regeneration of amputated from body segment 6/7 to further down the body posteriorly increased gradually (Type L_I). However, the regeneration lengths of earthworm which were amputated behind the 23rd segment, with less than a quarter of the total segments remaining, did not increase until the blastema and tail bud formation (Type L_II). These treatments were not completely regeneration. There were significant differences in both survival rates and lengths of regeneration lengths between immature earthworms and clitellate adult earthworms during the early stages of regeneration, but not at later stages of regeneration. The immature earthworms had a greater regeneration potential than clitellate adults amputated at the same segment. The survival rates of earthworms were correlated significantly with the number of body segments remaining after amputation, but not with the position of the amputation. The relationships between the survival rates and the numbers of remaining segments could be described by linear regressions. The anterior regeneration lengths were correlated with the position of the amputation, but not with the number of remaining segments; the posterior regeneration lengths, were not correlated with the number of segments remaining nor the amputation position. The anterior regeneration length was not related to the survival rates for all earthworm amputations after 30 days but was related in this way after 60 days.     

The parasitic nematode Phasmarhabditis hermaphrodita defends its slug host from being predated or scavenged by manipulating host spatial behaviour

Infective stages of commercially used molluscicidal rhabditide nematodes Phasmarhabditis hermaphrodita contain bacterial symbionts which kill their host by septicaemia. The nematodes feed on the multiplying bacteria and entire host tissue, develop and repeatedly reproduce. Invertebrate cadavers are rapidly (from minutes to hours) removed by scavengers. However nematodes need days to complete their life cycle inside the host.

The post mortem locations of slugs killed by six different treatments (three types of molluscicides, a simulation of unsuccessful predation and two P. hermaphrodita nematode treatments) were compared.

In comparison to other pathogenic states, significantly more slugs killed by the nematodes died within the soil, where the scavenging pressure is weaker than on the soil surface (where most of the slugs died regardless treatment). We suggest that this is an outcome of behavioural manipulation, which prevent the parasites from being predated or scavenged together with their host until the nematodes complete development inside the host cadaver.     

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.     

A spatio-temporal analysis of a New Zealand flatworn (Arthurdendyus triangulatus) population in western Scotland

The New Zealand flatworm (Arthurdendyus triangulatus), which is an obligate predator of native earthworms, is an alien species to the British Isles and is widely distributed in Scotland. What little is known about its biology under field conditions is mainly from Northern Ireland. Samples taken from single sampling dates have shown A. triangulatus to have an aggregated distribution. To determine the spatio‐temporal distribution of A. triangulatus, a grass field in western Scotland was intensively sampled over a 16‐month period. Data indicate an increase in flatworm numbers and seasonal trends in body weight, the appearance of egg capsules and hatchlings. Results also showed that spatially, the flatworms were aggregated, but this was a transient phenomenon over the period of the experiment. The distribution of egg capsules in the study area was strongly aggregated and related to the appearance of hatchlings. Flatworms may aggregate in areas where soil moisture is optimal for the survival of the New Zealand flatworm.     

The impact of the ‘New Zealand flatworm’, Arthurdendyus triangulatus, on earthworm populations in the field

The ‘New Zealand flatworm’, Arthurdendyus triangulatus, is a native of the South Island of New Zealand, which has established in the UK, Ireland and the Faroe Islands. In its introduced range, it is a predator of lumbricid earthworms. To assess the impact of A. triangulatus on earthworm species, flatworm distributions were manipulated into ‘high’, control and ‘low’ densities within a replicated field experiment. Earthworm biomass in the ‘high’ flatworm density treatment was significantly lower than the control or ‘low’ treatments. This was due to a reduction in the anecic species Lumbricus terrestris and, to a lesser extent, Aporrectodea longa. There was little evidence of negative effects on other earthworm species, with even a weakly positive relationship between flatworm density and epigeic biomass. Principal components analysis showed a clear separation of anecic species from A. triangulatus, but the epigeic species Lumbricus festivus and Lumbricus rubellus grouped with A. triangulatus, suggesting that they could be benefitting from reduced intraguild competition. Flatworm densities of 0.8 per m², comparable to natural infestations in grassland, were predicted to give a reduction in total earthworm biomass of c. 20 %. The bulk of this was comprised of a reduction in anecic species biomass. In particular, it is considered that A. triangulatus poses a serious risk to L. terrestris populations, with implications for soil functioning and indigenous earthworm-feeding wildlife.