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The interrelationship between genetic resources and traditional knowledge under the Convention on Biological Diversity (CBD) has seldom been discussed. However, this interrelationship affects the a...

Aeolosoma viride, a globally distributed freshwater annelid, has a semitransparent appearance with 10 to 12 segments, about 2 to 3 mm in length. It is easy to raise and handle in laboratory conditions. Due to its robust regenerative capacity and applicability of various molecular tools including EdU labeling, whole-mount in situ hybridization (WISH), and RNA interference (RNAi), it rises as a promising model for studying whole-body regeneration.

Although apoptosis has been widely observed during the regenerative process, the mechanisms by which it is regulated and its roles in regeneration remained unclear. In this study, we introduced Aeolosoma viride, a fresh water annelid with an extraordinary regenerative ability as our model organism to study the functions and regulations of apoptotic caspases. Here we showed that major events of apoptosis were detected near the wounded area and showed spatial correlation with the expression patterns of caspase gene namely Avi-caspase X and two apoptosis regulators namely Avi-Bax and Avi-Bcl-xL. Next, we investigated how Avi-caspase X gene expression and apoptosis influence regeneration following head amputation. RNA interference of Avi-caspase X reduced the amounts of apoptotic cells, as well as the percentage of successful regeneration, suggesting a critical role for apoptosis in anterior regeneration of A. viride. In addition, we also discovered that the expression of apoptotic caspases was regulated by the canonical Wnt signaling pathway. Together, our study showed that caspase dependent apoptosis was critical to the anterior regeneration of A. viride, and could be regulated by the canonical Wnt signaling pathway.

Annelids are regenerative animals, but the underlying mechanisms await to be discovered. Because Wnt pathway is involved in animal regeneration to varying extents, we used Aeolosoma viride to interrogate whether and how this pathway plays a role in annelid anterior regeneration. We found that the expression of wnt4, β-catenin and nuclear-localized β-catenin protein were up-regulated during blastemal formation and down-regulated as anterior structures gradually reformed. Consistent with potential Wnt activities in the blastema, treatments with either Wnt pathway activator (azakenpaullone) or inhibitor (XAV939) inhibited head regeneration, which further supports a role of Wnt pathway during anterior regeneration. Detailed tissue-level examines demonstrated that wound closure and blastemal cell proliferation were impaired by over-activating the pathway, and that neuronal and musculature differentiation were affected under Wnt inhibition. Combined, gene expression and chemical inhibitor data suggest the presence of dynamic Wnt activities at different anterior regeneration stages: an initial low activity may be required for wound closure, and the following activation may signal blastemal formation and cell differentiation. In a nutshell, we propose that the canonical Wnt signaling regulates blastemal cellular responses during annelid regeneration.

Regeneration has long been the focus of scientific interest for its potential to restore lost, damaged, or aged tissues and organs. A wide range of regenerative studies have been conducted on different vertebrate and invertebrate model organisms. Annelids are known for their regenerative capacities, and because of their relatively complex organ systems, they are an ideal organism for regeneration study. Our present work focused on the freshwater annelid Aeolosoma viride, an asexually reproducing annelid capable of regenerating both anteriorly and posteriorly. Even though regenerative ability has been documented in this animal in previous studies, detailed characterization of the process is still unavailable. The objective of this study was to evaluate the regenerative ability of A. viride. We described the sequential morphological events during the process of regeneration, such as wound healing and the formation of blastema, mouth, and pygidium. In order to clarify the capacity and type of regeneration, we conducted a series of observations and experiments using a cell proliferation assay. Massive proliferation and the absence of cell migration indicated that the animal regenerates primarily through epimorphosis. Our study of the epimorphic regenerative process of A. viride provides a clearer picture of the evolutionary origin of regeneration in annelids.

Aging is a complex process involving declines in various cellular and physical functionalities, including regenerative ability. Telomere maintenance is thought to be necessary for regeneration, and telomere attrition is one mechanism that contributes to aging. However, it is unclear if aging affects regeneration owing to deterioration of telomeric maintenance. We introduce Aeolosoma viride—a freshwater annelid with strong regenerative abilities—as a new model for studying the effects of aging on telomere functions and regeneration. We show that the anterior regenerative ability of A. viride declines with age. We characterized the A. viride telomere sequence as being composed of TTAGGG repeats and identifyied the telomerase gene Avi-tert. In adult A. viride, telomerase was constantly active and telomere lengths were similar among different body sections and stably maintained with age. Notably, we found that regeneration did not result in telomere shortening at regenerating sites. Moreover, transient up-regulation of Avi-tert expression and telomerase activity was observed at regenerating sites, which might promote telomere lengthening to counteract telomere erosion resulting from cell proliferation. Our study suggests that although aging affects A. viride regeneration independent of steady-state telomere length, timely regulation of telomerase functions is critical for the regeneration process in A. viride.

Regeneration has long attracted scientists for its potential to restore lost, damaged or aged tissues and organs. A wide range of studies have conducted on different model organisms on both cellular and molecular levels. Current evidences suggest that a variety of regenerative strategies are developed and used by different species, and their regenerative strategies are highly correlated to their reproductive methods. Our present work focused on the freshwater annelid Aeolosoma viride, which reproduces by paratonic fission and is capable of complete regeneration. We found out that A. viride can regenerate both anterior and posterior end, even with only 3 segments remained. This process is characterized by epimorphosis that involves large amount of cell proliferation which drives the formation of blastema. Cell proliferation and regeneration successful ratio were significantly decreased when treated with microtubule inhibitor taxol or Avi-tubulin dsRNA, which confirmed that cell proliferation served as a key event during regeneration. Together, our data described the regenerative processes of A. viride, which includes high level of cell proliferation and the formation of blastema. Furthermore, our findings demonstrated A. viride as a potential model for the study of regeneration.

Within minutes of removal from the California mussel, Mytilus californianus hemocytes become sticky for one another and for foreign surfaces. We sought to understand the cell surface changes responsible for this altered state. Hemocyte aggregation and adhesion assays were used in experiments in which a variety of reagents potentially capable of interfering with aggregation were screened. Caffeine, nor-ethylmaleimide, cytochalasin B, and EDTA were completely or partially inhibitory towards aggregation and adhesion. However, RGD-containing peptides, glycosaminoglycans, protease inhibitors, heparin, or poly-L-lysine were without effect. Low temperature (4°C) slowed hemocyte adhesion and hemocyte cohesion. Based on the findings, it appears that (1) Mytilus hemocyte aggregation, in vitro, is a two-step process that requires metabolic energy and divalent cations (calcium and magnesium), and is temperature-sensitive; and (2) Mytilus hemocyte adhesion and hemocyte aggregation are two associated but different cell behaviors.

Comparison of HBV recover ratio by BBAP, Velvet, SOAPdenovo, and Genovo assembly of full and partial D2_1 data sets. Figure S2: Correlation between assembled scaffold length and scaffold degeneracy for all 12 data sets. Figure S3: (a) Nucleotide sequence of the R1 scaffold. (b) Schematic alignment of the R1 scaffold, HBV X gene and HBV precore/core gene. Figure S4: Schematic diagram of the T1 scaffold and its corresponding HBV genome regions. Figure S5: Schematic diagram of the T6 scaffold and its corresponding HBV genome and Sanger reference sequence regions. Figure S6: Alignment of Sanger (SR) and partial D2_1 data set assembled scaffolds reference assembled (PDR) scaffolds to the Sanger reference sequence. Figure S7: Diversity profile of D2_1 HBV quasispecies according to assembly results of partial data set reference assembly of the full data set. Figure S8: Schematic diagram of two Genovo assembled scaffolds with identified HBV structural variants and its corresponding HBV genome regions. (PDF 489 kb)

A. Variant sequences and human genome sequences. B. Diversity profile of D2_1 HBV quasispecies. C. Structure variation by Genovo. D. Determining optimal size of partial data set. (DOC 67 kb)

Ultraviolet (UV) radiation leads to photooxidation in various organisms. Our previous study demonstrated that ultraviolet B (UV-B) radiation is lethal for particular species of earthworms, but the mechanisms responsible for the lethality are unclear. In our current study, we investigated that ultraviolet light causes photooxidative damage and reduces antioxidant responses in the earthworm Amynthas gracilis . Intact earthworms and skin/muscle tissue extracts were exposed to UV-B radiation for in vivo and in vitro studies. Both in vitro and in vivo results showed that the products of photooxidative damage, MDA and H 2 O 2 , increased after UV-B exposure. Glutathione peroxidase (GPx) and catalase were inhibited immediately after exposure to high doses (3000 J/m 2 ) of UV-B radiation in vivo. Catalase activity was increased following a low UV-B dose (500 J/m 2 ) in vivo, but decreased in response to all dosage levels in vitro. These data indicate that a relationship exists between UV-B induced damage and photooxidation and also that catalase and GPx act as important antioxidants to prevent photooxidation. According to these data, A. gracilis exhibits high sensitivity to environmental levels of UV-B. Therefore, A. gracilis represents a sensitive and cost-effective model organism for investigations of UV-radiation damage and environmental UV stress.

Four new species of terrestrial earthworms belonging to the genus Amynthas were collected on the islands of Kinmen and Lieyu, Taiwan from March to November, 2008. They are Amynthas kinmenensis sp. nov. , Amynthas wuhumontis sp. nov. , Amynthas wujhouensis sp. nov. , and Amynthas taiwumontis sp. nov. Amynthas kinmenensis sp. nov. is quadrithecal and is the most abundant earthworm widely distributed on the main island of Kinmen. It has numerous small genital papillae and is closely related to Amynthas polyglandularis (Tsai, 1964) from northern Taiwan. Amynthas wuhumontis sp. nov. is sexthecal and is distributed only in areas around Mt. Wuhu and Mt. Taiwu in east Kinmen. It has male pores each surrounded by three genital papillae: one anterior, one posterior and one medial. Amynthas wujhouensis sp. nov. and Amynthas taiwumontis sp. nov. are octothecal. The former has a sporadic distribution in Kinmen while the latter was only found in areas around Mt. Taiwu. Amynthas wujhouensis sp. nov. has a pair of large genital papillae closely adjacent to the crescent or semicircular shaped male porophores in XVIII. Amynthas taiwumontis sp. nov. has simple male pore structure and no genital papillae or genital markings. DNA barcodes (the 5’ end sequences of the mitochondrial cytochrome c oxidase subunit 1 gene) from type specimens and other materials of the first three species are also reported.

Background The accuracy of metagenomic assembly is usually compromised by high levels of polymorphism due to divergent reads from the same genomic region recognized as different loci when sequenced and assembled together. A viral quasispecies is a group of abundant and diversified genetically related viruses found in a single carrier. Current mainstream assembly methods, such as Velvet and SOAPdenovo, were not originally intended for the assembly of such metagenomics data, and therefore demands for new methods to provide accurate and informative assembly results for metagenomic data. Results In this study, we present a hybrid method for assembling highly polymorphic data combining the partial de novo-reference assembly (PDR) strategy and the BLAST-based assembly pipeline (BBAP). The PDR strategy generates in situ reference sequences through de novo assembly of a randomly extracted partial data set which is subsequently used for the reference assembly for the full data set. BBAP employs a greedy algorithm to assemble polymorphic reads. We used 12 hepatitis B virus quasispecies NGS data sets from a previous study to assess and compare the performance of both PDR and BBAP. Analyses suggest the high polymorphism of a full metagenomic data set leads to fragmentized de novo assembly results, whereas the biased or limited representation of external reference sequences included fewer reads into the assembly with lower assembly accuracy and variation sensitivity. In comparison, the PDR generated in situ reference sequence incorporated more reads into the final PDR assembly of the full metagenomics data set along with greater accuracy and higher variation sensitivity. BBAP assembly results also suggest higher assembly efficiency and accuracy compared to other assembly methods. Additionally, BBAP assembly recovered HBV structural variants that were not observed amongst assembly results of other methods. Together, PDR/BBAP assembly results were significantly better than other compared methods. Conclusions Both PDR and BBAP independently increased the assembly efficiency and accuracy of highly polymorphic data, and assembly performances were further improved when used together. BBAP also provides nucleotide frequency information. Together, PDR and BBAP provide powerful tools for metagenomic data studies. Electronic supplementary material The online version of this article (doi:10.1186/s12859-017-1630-z) contains supplementary material, which is available to authorized users.

Parthenogenesis has been known in a variety of animal taxa and is commonly found in oligochaetes. Many pheretimoid earthworms in Megascolecidae have been known to be or suspected to be parthenogenetic, but there has not been any genetic investigation on them. Amynthas catenus and Amynthas hohuanmontis are earthworms belonging to the Pheretima complex of the family Megascolecidae. Both are endemic to Mt. Hohuan at an elevation of about 3000 m in central Taiwan, and both have spermathecae from three pairs in segments VI–VIII (sexthecal) to absence (athecal) and reproductive organs in different degrees of degeneration. Here we present the genetic evidence revealed by amplified fragment length polymorphism (AFLP) markers to confirm the parthenogenetic mode of reproduction in A. catenus and A. hohuanmontis . Sixty-two selective primer combinations were used to generate a total of 4593 and 4812 bands for A. catenus and A. hohuanmontis , respectively. We found that the two earthworms are automictic (meiotic) parthenogens with offsprings showing slightly reduced number of AFLP markers compared with their parents. There was no sperm in any of the spermathecae examined. Selection and reproductive success among different genotypes of the two earthworms will be an important topic for future investigation.

Metallothionein-2, a ubiquitous metal-inducible protein, is involved in direct metal sequestration and is associated with detoxification and metal accumulation in eukaryotic organisms. In this study, higher levels of cadmium (Cd) accumulation were detected in earthworms exposed to higher Cd concentrations or longer exposure times. However, the bio-concentration factor (BCF), derived from the ratio of the Cd levels in the earthworms to that in soil, indicated that earthworms could relatively efficiently remove Cd from soil with low Cd contamination levels. In addition, earthworm recombinant metallothionein-2 effectively eliminated oxidative DNA damage in vitro and was probably able to counteract DNA damage caused by Cd, as shown by the comet assay in earthworm coelomocytes. Because earthworm metallothionein-2 proteins were evaluated as possible biomarkers for heavy-metal pollution in soil, the metallothionein-2 genes of six Megascolecidae earthworms and one Glossoscolecidae earthworm were cloned and sequenced using mRNA differential display. Based on the resolved earthworm metallothionein-2 nucleotide sequences, quantitative PCR was used to investigate the metallothionein-2 transcription levels from three earthworm species (Metaphire posthuma, Polypheretima elongata and Pontoscolex corethrurus) in response to different Cd concentrations or exposure times. The result demonstrated that Cd treatments elevated metallothionein-2 transcription in a dose- and time-dependent manner. In conclusion, the induction of earthworm metallothionein-2 would not only provide cellular protection against Cd-induced oxidative stress but also increase Cd accumulation consistent with a potential role in bioremediation.

Amynthas catenus Tsai et al., 2001 is a terrestrial earthworm belonging to the Pheretima complex of the family Megascolecidae. It is endemic to Mt. Hohuan of central Taiwan at elevations of 2985–3004 m. Its reproductive organs showed various stages of degeneration in the present study. The spermathecae varied from being absent (athecal) to three pairs in VI–VIII (sexthecal), and the prostate glands from a large, symmetrical pair to complete absence. For the 44 specimens examined from May 2008 to May 2010, there were 47.7% athecals, 36.4% sexthecals and 15.9% intermediates. Results of a flow cytometric analysis showed that there were three DNA ploidy levels – diploid, triploid and tetraploid (2n, 3n, and 4n) – for the athecals but only diploid for the sexthecals. The intermediates consisted of diploids and triploids. The diploids had 2n = 112 and the triploids 3n = 168. The haploid chromosome number of A. catenus was 56. Spermatogenesis occurred throughout the year with the highest activity in summer for both athecals and sexthecals, but the number of mature ova peaked in summer for the athecals and in spring for the sexthecals. There was no significant difference in mean number of mature ova produced between the athecals and the sexthecals, but the sexthecals had significantly higher spermatogenetic activity than the athecals. For all specimens examined, sperm were present in testes and seminal vesicles but not in spermathecae, an indication of parthenogenesis in the species.

Although prey-detecting and searching abilities of predatory leeches of rhynchobdellid or the Erpobdelliformes of arhynchobdellid species have been studied in the past, hirudiniformes leeches are rarely mentioned. In this study, we investigated the chemosensory ability for prey-detecting and searching in Whitmania laevis, a hirudiniformes species that mainly preys on freshwater snails, and examined if such ability aided in their prey selection. Five sympatric snail species, i.e., apple snail Pomacea canaliculata, thiarid snail Thiara tuberculata, viviparid snail Sinotaia quadrata, ear pond snail Radix auricularia swinhoei and tadpole snail Physa acuta were used as prey. Our results showed that W. laevis has the chemosensory ability to detect the waterborne odors of snails. However, they follow the snails by their mucus trails, and not by the odor that the snails leave in the water. Of these five snail species, W. laevis only followed the trails of the thiarid snails, ear pond snails and tadpole snails, and did not show a different response to the trails produced by snails of different sizes. Our results suggest that W. laevis can use waterborne odors to detect the existence of prey. They rely on mucus trails to follow their preferred prey, but do not distinguish between snails of a preferred size by their mucus trails. In addition, when following the trail of a preferred snail, W. laevis exhibits a newly described searching behavior, i.e., head tapping, and may use it to locate a snail trail and increase its probability of finding the trail-laying snail nearby.

Summary 1. Optimal foraging theory assumes that a predator preys selectively to maximize fitness gain. The associated model assesses predator’s fitness gain by estimating prey profitability in terms of appropriate foraging costs and benefits. While model developing, unsuccessful foraging was included in the model because it is inevitable and costly for a predator. However, it has rarely been taken into account in previous studies, nor has the consumption success been included in the estimation of prey profitability. Thus, although such model may successfully predict prey selection of many predators, it is inadequate in explaining prey selection of predators with highly variable consumption success. 2. In this study, we tested the prediction of optimal foraging model by accounting for consumption success in prey profitability for the prey selection of Whitmania laevis (Hirudinida: Hirudinidae), a shell-invading leech with highly variable consumption success in different prey. Five snail species, including three operculated species and two non-operculated species, were used as prey. 3. Our results showed that W. laevis exhibits prey-size selection. Leeches consumed more medium-sized snails among operculated species and more large-sized snails among non-operculated species, and the selection fitted the prediction of prey profitability by accounting for consumption success. Leeches also practiced prey-species selection, but such selectivity was confined to non-operculated species rather than snails with higher profitability. 4. Our study showed that when the consumption success was incorporated into the estimation of prey profitability, the optimal foraging model could, as predicting the prey-size selection of W. laevis, predict the prey selection of a predator with highly variable consumption success, which has been poorly predicted previously. This could also be the case for other predators with variable consumption success in future studies.

The effects of cadmium (Cd) contamination in soil onto the bacterial communities of the guts pooled from ten Metaphire posthuma were addressed during 14 days' incubation. We found that about 50% of Cd (5mg/kg, dry weight soil) in the contaminated soil was bio-accumulated into the earthworms. DNA was extracted from the guts of M. posthuma and their dwelling soil irrespective of Cd treatment for the analysis of the bacterial communities of guts in M. posthuma and in soil by PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis). A distinctive cluster of bacterial communities of the guts in the earthworm with and without Cd treatment using the analysis of unweighted pair-group method using arithmetic averages (UPGMA) was observed, indicating that the bacterial community of guts could be changed by Cd. However, no differences in the bacterial communities in soil irrespective of Cd treatment were observed, which could be resulted from the bioremediation of Cd by earthworms leading to insignificant effect of Cd on bacterial communities in soil. For the sequencing of some of the dominant bands in the DGGE profile, Bradyrhizobium japonicum, Stenotrophomonas sp. D2, and Labrys, sp. CC-BB4, whose sequences display an identity of more than 97% using blast program against a known sequence in the GeneBank database and Ribosomal database, were identified. Collectively, our results showed that earthworm treatment can decrease the concentrations of Cd in soil, and Cd cause a shift in the bacterial communities in the guts of M. posthuma. The application of M. posthuma for Cd bioremediation would be desired.

In this study, two new species of Helobdella leeches, Helobdella octatestisaca n. sp. and Helobdella melananus n. sp., and a newly recorded species, Helobdella europaea, are reported. Morphological characters and DNA barcode analysis were used to identify the three new species. The differences between these species and other morphologically congeneric species were also compared. In addition, the potential ecological impact of these species was discussed and a checklist of the hirudinea fauna in Taiwan, including eight species in Family Glossiphoniidae, four in Hirudinidae, two in Haemopidae, two in Haemadipsidae, and two in Salifidae, was provided.

In this study we report the sequences of MT-2 cDNA from two species of Megascoleidae earthworms, Metaphire posthuma and Polypheretima elongata, by mRNA differential display after exposure of the organisms to cadmium. Complementary (c)DNA was verified as the MT-2 gene by the characteristics of its predicted translation product, namely a high cysteine content, conserved CXC motifs, and a molecular weight of around 8 kDa. Amino acid sequence alignment revealed a conserved TKCCG in the cloned MT-2 of both megascolecid earthworms instead of the corresponding conserved TQCCG found in lumbricid earthworms. The cDNAs corresponding to the two megascolecid MT-2 genes were expressed, and the MT-2 proteins were purified for biochemical characterization. The binding of Cu2+ exhibited monophasic kinetics and those of Zn2+ and Cd2+ biphasic kinetics. The proteins bound more tightly to Cd2+ than to Zn2+ and more tightly still to Cu2+. Zn-MT and apo-MT were the most effective at scavenging free radicals, followed by Cd-MT. In conclusion, MT-2s from M. posthuma and P. elongata showed unique sequence features compared to those of lumbricid earthworms. These earthworms could be used to evaluate heavy-metal pollution in soil due to the inducible MT-2 by cadmium exposure.

Summary This paper re-evaluated the use of DNA barcodes in earthworm species identification by re-analyzing sequence data for the mitochondrial cytochrome c oxidase subunit 1 (COI) gene. This analysis unveiled and confirmed taxonomic inconsistencies which significantly affect data interpretation. When considering synonymy and misidentification in published records, our results revealed no shared COI haplotypes between morphologically distinct species and higher interspecific than intraspecific divergence in most cases, with interspecific and intraspecific distances averaging 18.7% and 1.3% respectively. However, a few earthworm species endemic to Taiwan have deep intraspecific divergences which may represent potential cases of cryptic diversity, although incomplete lineage sorting cannot be ruled out without further study. We recognize the potential of DNA barcoding for earthworm taxonomy, but have identified several issues regarding the evolution of the COI gene in these organisms which remain to be further elucidated.

Two species of earthworms were used to unravel why some earthworm species crawl out of the soil at night after heavy rain. Specimens of Amynthas gracilis, which show this behavior, were found to have poor tolerance to water immersion and a diurnal rhythm of oxygen consumption, using more oxygen at night than during the day. The other species, Pontoscolex corethrurus, survived longer under water and was never observed to crawl out of the soil after heavy rain; its oxygen consumption was not only lower than that of A. gracilis but also lacked a diurnal rhythm. Accordingly, we suggest that earthworms have at least two types of physical strategies to deal with water immersion and attendant oxygen depletion of the soil. The first is represented by A. gracilis; they crawl out of the waterlogged soil, especially at night when their oxygen consumption increases. The other strategy, shown by P. corethrurus, allows the earthworms to survive at a lower concentration of oxygen due to lower consumption; these worms can therefore remain longer in oxygen-poor conditions, and never crawl out of the soil after heavy rain.

SUMMARYThe purpose of this study was to investigate the adverse effects of ultraviolet (UV) radiation on earthworms. Earthworms that crawl out of the soil may die within a few hours after sunrise. This study shows that UV exposure can be lethal. In general, UV-B had a stronger damaging effect than UV-A. Different species of earthworms had different tolerances to UV exposure. In this study, Pontoscolex corethrurus showed the highest tolerance of the three tested species to UV radiation, while Amynthas graciliswas the most sensitive. UV radiation induced both acute and chronic responses. The acute response, which occurred immediately on or after UV exposure, was characterized by the appearance of abnormally strong muscle contractions,including S-shaped movements and jumping behavior, possibly caused by bad coordination between the circular and longitudinal muscles. The chronic response included damage to the skin and muscle cells, which resulted in a high mortality rate. Oxygen consumption by A. gracilis was significantly decreased after exposure to UV-A or UV-B. Since the circulation in earthworms is mediated by muscle contraction and the skin is the main organ of respiration, it is reasonable to expect that abnormal muscle contraction and a damaged epithelium could cause suffocation. Because of their sensitive responses, we propose that some earthworms, such as A. gracilis,could serve as a new model for studying UV-induced photodamage.

Terrestrial megadrile earthworms variously reported from Taiwan including Lanyu Island (Botel Tobago) number approximately 70 species with 29 of these, or 40%, non-natives for which 69% are Asiatic Megascolecidae and 21% are Lumbricidae of Holarctic origin. An additional 27 unnamed species have been identified from Ilan county north-eastern Taiwan by Chen et al. (2003) that, if published, would bring the total to around 100 known species. The checklist is also provided with the confirmation and new records of the exotic lumbricids Eisenia fetida (Savigny) and Eiseniella tetraedra (Savigny).

In this study, the situation with imported aquatic invertebrates via the live aquarium ornamental trade in Taiwan was investigated from March 2004 to February 2005. The scientific names, native geographic regions, amount, and trade pathways of imported invertebrates were recorded. Among the 108 imported invertebrate species recorded in this survey, 13 are freshwater species, while the others are marine ones, and most of them were from tropical zones. Through the assessments of invasive potential including the imported amount, the similarity of the native geographic regions to Taiwan, and its invasion record, three species from the class Anthozoa (Cribrinopsis crassa, Condylactis passiflora, and Alcyonium palmatum), one from the class Scyphozoa (Phyllorhiza punctata), three from the order Decapoda (Procambarus clarkia, Cherax destructor, and Neocaridina sp.), one from the class Bivalvia (Lima scabra), and two from the class Gastropoda (Pomacea bridge and Indoplanorbis exustus) were regarded as having high invasive potentials.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both low-molecular-weight lysophospholipid (LPL) ligands which are recognized by the Edg family of G protein-coupled receptors (GPCRs). In endothelial cells, these two ligands activate Edg receptors resulting in cell proliferation and cell migration. Interleukin-8 (IL-8) is a C-X-C chemokine and acts as a chemoattractant of neutrophils, whereas monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine and functions mainly as a chemoattractant of monocytes/macrophages. Both factors are secreted from endothelial cells and have been implicated in the processes leading to atherosclerosis. We examined the effects of LPLs on the expression of IL-8 and MCP-1, key regulators of leukocyte recruitment in human umbilical cord vein endothelial cells (HUVECs). Work illustrated in this article showed that LPA and S1P enhanced IL-8 and MCP-1 mRNA expressions, and protein secretions in dose- and time-dependent fashions. Maximal mRNA expression appeared at 16 hr post-ligand treatment. Using prior treatments with chemical inhibitors, LPLs enhanced IL-8 and MCP-1 expressions through a Gi-, Rho-, and NFkappaB-dependent mechanism. In a chemotaxis assay system, LPL treatments of endothelial cells enhanced monocyte recruitment through upregulating IL-8 and MCP-1 protein secretions. Pre-incubation with AF12198, an IL-1 receptor antagonist or IL-1 functional blocking antibody both suppressed the enhanced effects elicited by LPLs of IL-8 and MCP-1 mRNA expressions in HUVECs. These results suggest that LPLs released by activated platelets might enhance the IL-8- and MCP-1-dependent chemoattraction of monocytes toward the endothelium through an IL-1-dependent mechanism, which may play an important role in facilitating wound-healing and inflammation processes.

Summary In this study the synonymy of Amynthas formosae (Michaelsen, 1922) and A. yuhsii (Tsai, 1964) are reevaluated and the intraspecific phylogeography between them is studied. The morphological comparison and phylogenetic analysis based on cytochrome c oxidase subunit I suggest that A. formosae and A. yuhsii are not synonyms and both of them belong to Metaphire. The phylogeographic analysis suggests that the formation of mountains and rivers during the uplift of the Western Foothills in Taiwan comprised the vicariance events responsible for the intraspecific genetic differentiation between these two species. & 2005 Elsevier GmbH. All rights reserved.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both low-molecular-weight lysophospholipids, which promote cell proliferation, migration, and invasion via interaction with a family of specific G protein-coupled receptors. Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes, which are involved in degradation of the extracellular matrix and play critical roles in endothelial cell migration and matrix remodeling during angiogenesis. Among these MMPs, MMP-2 is known to trigger cell migration. In our present study, we examined the effects of LPA and S1P on MMP-2 expression in human endothelial cells. We showed that LPA and S1P enhanced MMP-2 expression in mRNA, protein levels, and also enzymatic activity of cells of the EAhy926 human endothelial cell line. The enhancement effects occurred in concentration- and time-dependent manners. Results from real-time PCR, Western blots, and substrate gels indicated that these enhancement effects were mediated through MAPK kinase/ERK-, nuclear factor-κB-, and calcium influx-dependent pathways. Furthermore, we show that endothelial cell invasion of the gel was enhanced by lysophospholipids, and the induction could be prevented by an MMP inhibitor, GM6001. These observations suggest that LPA and S1P may play important roles in endothelial cell invasion by regulating the expression of MMP-2.

The complete sequence of the mitochondrial gene encoding NADH dehydrogenase subunit 1 (ND1) from a megascolecid earthworm, Amynthas gracilis (Kinberg, 1867), is published in this study. The gene is 923 bp in size, encoding a polypeptide of 307 amino acid residues. Its nucleotide sequence and amino acid sequence are 71.8% and 77.7% identical to those of the European earthworm Lumbricus terrestris ND1 gene, respectively. In addition, the GC contents among some published annelid ND1 full sequences were compared, and the relationships among these sequences were reconstructed using a phylogenetic analysis. Accordingly, we believe that a 3000-bp sequence comprising the ND1 and the CO1 genes and the genes used by Jamieson et al. (2002) can be a good candidate to be used in the phylogenetic analysis to unravel the phylogeny of the Megascolecidae.

This paper described a new species of earthworm from southern Taiwan Metaphire feijani. This species belongs to the octothecate stephensoni-species group within the genus Metaphire. The morphological comparison of species within this group revealed the identity of this new species.

The Metaphire formosae species group is a member of the Pheretima complex of the family Megascolecidae. It is composed of 12 nominal taxa, Metaphire bununa Tsai et al., 2000, Metaphire feijani ChangChen, 2004, Metaphire formosae (Michaelsen, 1922), Metaphire glareosa Tsai et al., 2000, Metaphire nanaoensis ChangChen, 2005, Metaphire paiwanna paiwanna Tsai et al., 2000, Metaphire paiwanna hengchunensis (James et al., 2005), Metaphire paiwanna liliumfordi Tsai et al., 2000, Metaphire tahanmonta ChangChen, 2005, Metaphire taiwanensis Tsai et al., 2004, Metaphire trutina Tsai et al., 2003, and Metaphire yuhsi (Tsai, 1964). In this study, we describe a new species, Metaphire tengjhihensis sp. nov., and two new subspecies, Metaphire nanaoensis truku ssp. nov. and Metaphire taiwanensis tsaii ssp. nov., belonging to this species group. DNA barcodes (partial sequences of the mitochondrial cytochrome c oxidase subunit 1, COI) from type specimens of M. feijani, M. tengjhihensis sp. nov., M. nanaoensis truku ssp. nov., M. tahanmonta and M. taiwanensis tsaii ssp. nov. have been deposited in GenBank in previous studies and are explicitly linked to the type specimens for the first time, enabling unambiguous identification using both morphology and DNA barcodes. Finally, we comment on the systematics of the M. formosae species group and suggest an integrative taxonomic approach that combines morphology and DNA barcodes for future descriptions of new species of Amynthas and Metaphire.

Four new species of terrestrial earthworms belonging to the genus Amynthas were collected on the islands of Kinmen and Lieyu, Taiwan from March to November, 2008. They are Amynthas kinmenensis sp. nov., Amynthas wuhumontis sp. nov., Amynthas wujhouensis sp. nov., and Amynthas taiwumontis sp. nov. Amynthas kinmenensis sp. nov. is quadrithecal and is the most abundant earthworm widely distributed on the main island of Kinmen. It has numerous small genital papillae and is closely related to Amynthas polyglandularis (Tsai, 1964) from northern Taiwan. Amynthas wuhumontis sp. nov. is sexthecal and is distributed only in areas around Mt. Wuhu and Mt. Taiwu in east Kinmen. It has male pores each surrounded by three genital papillae: one anterior, one posterior and one medial. Amynthas wujhouensis sp. nov. and Amynthas taiwumontis sp. nov. are octothecal. The former has a sporadic distribution in Kinmen while the latter was only found in areas around Mt. Taiwu. Amynthas wujhouensis sp. nov. has a pair of large genital papillae closely adjacent to the crescent or semicircular shaped male porophores in XVIII. Amynthas taiwumontis sp. nov. has simple male pore structure and no genital papillae or genital markings. DNA barcodes (the 5' end sequences of the mitochondrial cytochrome c oxidase subunit 1 gene) from type specimens and other materials of the first three species are also reported.

Forty-seven carbohydrates failed to inhibit the cell aggregation and substrate adhesion behaviors of Mytilus californianus hemocytes. Treatment of hemocytes with sodium periodate and with YV-acetyl glucosaminidase interfered with these behaviors. Trypsin slightly reduced adhesion, and had no effect on aggregation. Hyaluronidase and neuraminidase failed to inhibit either behavior, and slightly enhanced them. An inhibitory effect of caffeine was not altered by hyaluronidase of N-acetyl-glucosaminidase, but neuraminidase partially reversed this inhibition. Inhibition by the calcium channel blocker verapamil was reversed by hyaluronidase. Cell-cell and cell-substrate adhesion behaviors probably involve complex carbohydrates in molluscan hemocytes.

The earthworms of the Metaphire formosae species group distributed in Taiwan are members of the Pheretima complex within the Megascolecidae. In this study, the systematics and phylogeography of this species group were investigated using DNA sequences of mitochondrial cytochrome c oxidase subunit I (COI), 16S ribosomal (r)RNA, and NADH dehydrogenase subunit 1 (ND1). The results indicated that the 13 taxa of the M. formosae species group form a clade, including a cryptic species discovered in this study. In addition, Metaphire hengchunensis (James, S.W., Shih, H.-T., Chang, H.-W., 2005. Seven new species of Amynthas (Clitellata: Megascolecidae) and new earthworm records from Taiwan. J. Nat. Hist. 39, 1007-1028) should be regarded as a subspecies of Metaphire paiwanna Tsai, C.-F., Shen, H.-P., Tsai, S.-C., 2000a. Native and exotic species of terrestrial earthworm (Oligochaeta) in Taiwan with reference to Northeast Asia. Zool. Stud. 39, 285-294, Tsai, C.-F., Tsai, S.-C., Liaw, G.-J., 2000b. Two new species of pontandric pheretimoid earthworms belonging to the genus Metaphire (Megascolecidae: Oligochaeta) from Taiwan. J. Nat. Hist. 34, 1731-1741, and Metaphire bununa glareosa Tsai et al. 2000 should be elevated to specific status. Phylogeographical inferences showed that allopatric speciation occurred in this species group during the rapid uplift of the main island of Taiwan between 5.0 and 2.5 million years ago. Our analysis exposes non-monophyly within each of the genera Amynthas and Metaphire, and more generally within the Pheretima complex. Further revisions of this speciose complex are urgently needed.

Three new species of octothecate Metaphire pheretimoid earthworms are described from Taiwan. They have close phylogenetic relationships with five described species, Metaphire paiwanna, Metaphire bununa, Metaphire riukiuensis, Metaphire taiwanensis, and Amynthas formosae. These eight species are all octothecate. They have similar body size, colour, prostate glands and caeca, and the seven species belonging to the genus Metaphire have C‐shaped male pores. Excluding M. riukiuensis, which has been found only in the Ryukyu Islands, the other seven species are endemic species in Taiwan. Each of them has a distinct geographical distribution. Accordingly, we inferred that the ancestor of these eight species, including M. riukiuensis, came to Taiwan from the continent of East Asia during the Pleistocene. A few of their descendants went northward to the Ryukyu Islands and were isolated. The others remained in Taiwan, dispersed, and were isolated by geographical barriers such as mountains and rivers. Subsequent allo...

Different species of earthworms show distinct patterns of oxygen consumption pattern. Amynthas gracilis has a diurnal rhythm of oxygen consumption, consuming more oxygen at night, whether being incubated at 20, 25, or 30°C. The higher oxygen consumption of A. gracilis is directly related to its behavior, as it shows higher activity at night. In contrast, Pontoscolex corethrurus showed no obvious diurnal rhythms of oxygen consumption or behavior. In addition, its oxygen demand is not related to temperature. A. gracilis has higher oxygen consumption than P. corethrurus at 20 and 30°C. The difference in temperature adaptation of oxygen consumption between these two species may be a strategy by which earthworms adapt to different temperatures. This might explain why A. gracilis disperses on the soil surface at night after a rain, in contrast to P. corethrurus for which such behavior was not been observed. J. Exp. Zool. 301A:737–744, 2004. © 2004 Wiley Liss, Inc.

The Metaphire formosae species group is a member of the Pheretima complex of the family Megascolecidae. It is composed of 12 nominal taxa, Metaphire bununa Tsai et al., 2000, Metaphire feijani Chang & Chen, 2004, Metaphire formosae (Michaelsen, 1922), Metaphire glareosa Tsai et al., 2000, Metaphire nanaoensis Chang & Chen, 2005, Metaphire paiwanna paiwanna Tsai et al., 2000, Metaphire paiwanna hengchunensis (James et al., 2005), Metaphire paiwanna liliumfordi Tsai et al., 2000, Metaphire tahanmonta Chang & Chen, 2005, Metaphire taiwanensis Tsai et al., 2004, Metaphire trutina Tsai et al., 2003, and Metaphire yuhsi (Tsai, 1964). In this study, we describe a new species, Metaphire tengjhihensis sp. nov., and two new subspecies, Metaphire nanaoensis truku ssp. nov. and Metaphire taiwanensis tsaii ssp. nov., belonging to this species group. DNA barcodes (partial sequences of the mitochondrial cytochrome c oxidase subunit 1, COI) from type specimens of M. feijani, M. tengjhihensis sp. nov., M. nanaoensis truku ssp. nov., M. tahanmonta and M. taiwanensis tsaii ssp. nov. have been deposited in GenBank in previous studies and are explicitly linked to the type specimens for the first time, enabling unambiguous identification using both morphology and DNA barcodes. Finally, we comment on the systematics of the M. formosae species group and suggest an integrative taxonomic approach that combines morphology and DNA barcodes for future descriptions of new species of Amynthas and Metaphire.

On removal from the animal, molluscan hemocytes change from an aggregation-incompetent state to one in which they aggregate and stick avidly to foreign surfaces. Aggregation and substratum adhesion behaviors of Mytilus californianus (California mussel) hemocytes are inhibited reversibly and non-lethally by caffeine. We investigated the mechanistic basis of this inhibition by means of assays that determined the abilities of several compounds to compete or synergize with caffeine in achieving its effect. Adenosine and the adenosine receptor agonists 2-chloro-adenosine or 5′-N-ethylcarboxamido-adenosine reversed caffeine inhibition. In contrast, isobutyl-methylxanthine (both an adenosine receptor antagonist and an inhibitor of phosphodiesterase) and the adenosine receptor antagonists cyclo-hexyladenosine and R-N6-phenyl-isopropyladenosine synergized with caffeine to inhibit hemocyte adhesion. Caffeine treatment reduced intracellular cAMP. Alterations in cAMP concentrations in response to caffeine with or without adenosine and adenosine analogues reflected alterations in adhesion behavior in the same drugs. R-N6-phenyl-isopropyladenosine plus caffeine yielded intracellular CAMP levels lower than those in cells treated with caffeine alone. However, both cAMP level and hemocyte adhesion increased when adenosine or 5′-N-ethylcarboxamido-adenosine replaced R-N6-phenyl-isopropyladenosine. This mollusc's hemocytes appear to express adenosine receptors that modulate phagocyte behavioral responses by altering second messenger transduction.

Body size and colouration are two characters commonly used in the taxonomy of many animal taxa. However, they are seldom used by earthworm taxonomists because they are subject to environmental influences and tend to vary intraspecifically. In the present study, DNA sequences of the mitochondrial COI gene are used to evaluate whether specimens of the megascolecid earthworm Amynthas wulinensis Tsai, Shen & Tsai, 2001 that differ in body size and/or colouration belong to different genetic lineages. Phylogenetic analyses and morphological comparisons indicate that A. wulinensis in the previous broad sense is a species complex composed of three species differing in body size, colouration, and genital markings. Consequently, two new species, Amynthas lini and Amynthas meishanensis, are described. Taxonomic affinities of the A. wulinensis species complex are discussed, as is the feasibility of using body size and colouration in earthworm taxonomy.