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A new myxosporean parasite, Ortholinea nupchi n. sp. (Myxozoa; Bivalvulida), was isolated from the urinary bladder of the olive flounder Paralichthys olivaceus cultured on Jeju Island, Korea. Mature spores were subspherical in the valvular and apical views and ellipsoidal in the sutural view. The spores measured 7.6 ± 0.5 µm in length, 6.7 ± 0.3 µm in thickness, and 7.3 ± 0.5 µm in width. Two pyriform polar capsules measured 3.2 ± 0.1 µm in length and 2.7 ± 0.1 µm in width and were located at the same level at the anterior half of the myxospores. The suture line was straight in the middle of the spores, and the surface ridges ranged between five and seven, forming an intricate pattern. The result of the 18S rDNA comparison showed ≤ 93.0% similarity with other Ortholinea species. The phylogenetic tree demonstrated that O. nupchi n. sp. was closest to O. auratae and clustered with oligochaete-infecting myxosporeans (OIM) having urinary system infection tropism. Based on the comparison of environmental and host factors in the phylogenetic groups of the OIM clade, we propose that the infection of O. nupchi n. sp. originated from marine oligochaetes.

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Urbanization and population growth demand the construction of structures to facilitate the need for space, and old infrastructures must make space for new ones leading to demolition and concrete debris. In addition to demolition, aging and weather are factors leading to concrete deterioration and, thus, a new challenge as an environmental pollutant. Studies on how concrete debris and leachate affect biota in the environment are limited. The present study aimed to understand the effects of leachate from various sizes of concrete debris on the three oligochaete species Enchytraeus crypticus, Tubifex, and Lumbriculus variegatus. Acute toxicity testing was carried out to determine the adverse effects over time. The oligochaetes' survival was monitored as well as the activity of the biotransformation enzyme glutathione S-transferase and the antioxidative enzyme catalase as indicators of the oxidative stress status. Leachate from the smallest concrete particle size (<1 mm) was found to be the most toxic as it caused, on average, 6-fold increased oligochaete mortality compared to the larger pieces (2-5 cm) after 96 h of exposure, potentially due to the larger surface area facilitating the release of toxicants. Substrate buffered the toxic effect of the leachate with 42 ± 12% fewer mortalities and reduced adverse effects on the enzymes. Of the three oligochaetes, E. crypticus was the most resilient to the concrete leachate. The study is the first to investigate the effects of concrete leachate on oligochaetes.

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Bacillidium spp. exclusively infect oligochaetes and these microsporidian pathogens are typically characterized by their rod-shaped spores. Seven Bacillidium spp. are presently reported from different organs of oligochaetes. Here, we describe two new Bacillidium species, Bacillidium sinensis n. sp. and Bacillidium branchilis n. sp., from coelomocytes of Branchiura sowerbyi. This is the first report of Bacillidium spp. in oligochaetes from China. Both species of Bacillidium elicit the formations of opaque xenoma-like lesions in coelomocytes of the host. A diplokaryotic nucleus occurs in all life stages of these two new Bacillidium species. Mature spores of B. sinensis are 15.9 ± 0.6 (14.7-17.1) µm long (average ± standard error, range, n = 50) and 2.5 ± 0.1 (2.3-2.7) µm wide in fresh preparations. A new type of exospore (sixteen-layered exospore) is discovered from B. sinensis n. sp. which is distinctly different from B. branchilis n. sp., and other Bacillidium spp. (double-layered exospore) reported previously. These two Bacillidium species are morphologically distinguished from each other and all Bacillidium spp. described previously in terms of hosts, infection sites, spore size, spore wall or polar filament thickness. BLASTn searches indicated that these two new microsporidian parasites are surprisingly most similar to Janacekia tainanus (94.76% for B. sinensis and 90. 2% for B. branchilis) isolated from the fat body of midge larva (Kiefferulus tainanus). Phylogenetic analysis demonstrates that the two novel taxons cluster with J. debaisieuxi, J. tainanus, and Bacillidium sp. within the Jirovecia-Bacillidium-Janacekia clade. Other available 18S rRNA gene sequences for microsporidia that infect oligochaetes include J. sinensis, B. vesiculoformis, Neoflabelliforma aurantiae, and Bacillidium sp., but these do not form a single cluster with B. sinensis and B. branchilis, but are instead dispersed through the clade. Based on the ultrastructural features and molecular characteristics, two new species within the genus Bacillidium, B. sinensis n. sp. and B. branchilis n. sp., are designated.

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A new microsporidian parasite, Naidispora caidianensis n. gen. n. sp. was found in coelomocytes of oligochaete Branchiura sowerbyi Beddard, 1892 from Wuhan city, Hubei Province, China. Opaque, hypertrophied coelomocytes (0.17-0.24 mm in diameter) depicted clinical signs of infection. Electron microscopy revealed a microsporidian with monokaryotic life stages. Rounded uninucleate meronts subsequently transformed into multinucleate merogonial plasmodia with masses of electron-dense projections on their plasma membrane. Electron-dense sporogonial plasmodia separated into uninucleate sporonts through rosette-like budding, and further developed into sporoblasts, enclosed by a sporophorous vesicle. Uninucleate mature spores were pyriform, measured 4.1 ± 0.1 (3.9-4.3) µm × 2.1 ± 0.07 (1.9-2.2) µm (average ± SE, range, n = 50), and contained a mushroom-like anchoring disk, bipartite polaroplast, electron-lucent posterior vacuole, trilaminar spore wall and 12-14 turns of an isofilar polar filament arranged in 2-3 ranks. The 1542 bp sequence from B. soweryi showed the highest similarity (below 80 %) with Nematocenator marisprofundi (accession number: JX463178). SSU rRNA gene-based phylogenetic analysis demonstrated that the novel taxon formed an independent clade from known microsporidian parasites. Based on the ultrastructural features and SSU rRNA gene sequence, we propose the establishment of a novel genus (Naidispora n. gen.) and species (Naidispora caidianensis n. sp.) to contain this parasite.

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Exploring the effectiveness of DNA barcoding in species identification is a prerequisite for biodiversity conservation and environmental monitoring. Aquatic oligochaetes could serve as excellent indicators in aquatic monitoring programmes. However, few studies have examined the effectiveness of DNA barcoding in these specific organisms. The mitochondrial cytochrome C oxidase (COI) gene of 83 specimens belonging to 40 species of 18 genera were sequenced in this study. The results showed that there was a barcode gap between species of Naididae and the intraspecific genetic distances of each species were smaller than interspecific genetic distances. The classification results of ABGD (Automatic Barcode Gap Discovery) were consistent with those of morphological identification, except for Tubifextubifex and Lumbriculusvariegatus. All species were successfully distinguished in the phylogenetic tree, based on the ITS2 region, which was coincident with the morphological result. Our results provided evidence that DNA barcoding can be used as an effective and convenient tool for species identification of the family Naididae and even for other aquatic oligochaetes.

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The use of copper oxide (CuO) NPs results in the release of these particles into the aquatic environment. Here, the particles settle out and accumulate in the sediment. However, little is known about the biodynamics of sediment-associated NPs in benthic organisms. We compared the toxicity and biodynamics of CuO NPs (7 nm) and dissolved Cu (CuCl2) in the sediment-dwelling oligochaete, Tubifex tubifex, to gain insights into the relative importance of metal form (CuCl2 vs CuO NPs) and exposure route (water vs sediment). Isotopically enriched 65Cu was used as a tracer to distinguish background from newly accumulated 65Cu in worms. For each exposure route, we conducted three experiments: one uptake, one elimination, and one longer-term net accumulation experiment to parameterize uptake and elimination of 65CuCl2 and 65CuO NPs in T. tubifex. 65Cu accumulation was detected for both 65CuCl2 and 65CuO NPs regardless of whether T. tubifex were exposed in sediment- or water-only setups. Water exposures to 65CuCl2 resulted in tail trauma whereas limited effects were seen for sediment exposures or exposures to 65CuO NPs via either exposure route. Uptake rate constants and accumulation of 65Cu in T. tubifex were higher following 65CuCl2 exposure than 65CuO NPs, in water, but not in sediment. Thus, the relative importance of exposure route and Cu form for uptake dynamics is not straightforward suggesting that findings on bioaccumulation and toxicity in water exposures cannot be directly extrapolated to sediment.

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Myxobolus cerebralis (Hofer, 1903), the etiological agent of salmonid whirling disease, reportedly matures in only the oligochaete 'Tubifex tubifex'. The concept of 'T. tubifex' is problematic because it is renowned as a species complex (or having 'strains'), and many sequences ascribed to this taxon in GenBank are misidentified or indicate several cryptic species. These facts cast doubt on the long-held notion that M. cerebralis is strictly host-specific to the single definitive host, T. tubifex. Herein, as part of an ongoing regional whirling disease monitoring project, oligochaetes (452 specimens) were collected from 31 riverine sites in western North Carolina (August through September 2015) and screened for infection by M. cerebralis. The species-specific nested PCR for M. cerebralis was positive for 8 oligochaete specimens from the French Broad River Basin (Mill Creek and Watauga River) and New River Basin (Big Horse Creek). We individually barcoded these M. cerebralis-positive oligochaete specimens using cytochrome oxidase 1 (CO1) primers and then conducted a Bayesian inference phylogenetic analysis. We identified 2 oligochaete genotypes: one sister to a clade comprising Limnodrilus udekemianus (Haplotaxida: Naididae) and another sister to Limnodrilus hoffmeisteri. This is the first detection of M. cerebralis from an oligochaete in the SE USA and the first detection of M. cerebralis from an oligochaete other than T. tubifex. These results suggest that other non-T. tubifex definitive hosts can harbor the pathogen and should be considered in the context of fish hatchery biosecurity and monitoring wild trout streams for M. cerebralis and whirling disease in the southeastern USA.

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Despite that silver (Ag) is among the most studied nanomaterials (NM) in environmental species and Ag's embryotoxicity is well known, there are no studies on Ag NMs embryotoxicity in soil invertebrates. Previous Full Life Cycle (FLC) studies in Enchytraeus crypticus, a standard soil invertebrate, showed that Ag materials decreased hatching success, which was confirmed to be a hatching delay effect for silver nitrate (AgNO3) and mortality for Ag NM300K. In the present study, we aimed to investigate if the impact of Ag takes place during the embryonic development, using histology and immunohistochemistry. E. crypticus cocoons were exposed to a range of concentrations of Ag NM300K (0-10-20-60-115 mg Ag/kg) and AgNO3 (0-20-45-60-96 mg Ag/kg) in LUFA 2.2 soil, in an embryotoxicity test, being sampled at days 1, 2, 3 and 6 (3, 4, 5 and 7 days after cocoon laying). Measured endpoints included the number of embryonic structures, expression of transferrin receptor (TfR) and L type calcium channels (LTCC) through histological and immunohistochemistry analysis, respectively. Results confirmed that Ag materials affected the embryonic development, specifically at the blastula stage (day 3). The expression and localization of TfR in E. crypticus was shown in the teloblasts cells, although this transcytosis mechanism was not activated. Ag affected calcium (Ca) metabolism during embryonic development: for AgNO3, LTCC was initially activated, compensating the impact, for Ag NM300K, LTCC was not activated, hence no Ca balance, with irreversible consequences, i.e. terminated embryonic development. An Adverse Outcome Pathway was drafted, integrating the mechanisms here discovered with previous knowledge.

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Naphthalene sulfonic acids (NSAs) are used extensively in industrial applications as dispersants in dyes, rubbers, and pesticides, and as anti-corrosive agents in coatings, gels, and sealants. This study examined the toxicity of three NSA congeners, barium dinonylnaphthalene sulfonate (BaDNS), calcium dinonylnaphthalene sulfonate (CaDNS), and dinonylnaphthalene disulfonic acid (DNDS), to two benthic species, Tubifex tubifex and Hyalella azteca. Two substrates with different levels of organic carbon (sediment [2%] and sand [0%]) were used in toxicity tests. Juvenile production was the most sensitive endpoint for T. tubifex: the 28-d EC50s were <18.2, 22.2, and 64.0 µg/g dw in sand and 281.3, 361.6, and 218.9 µg/g dw in sediment for BaDNS, CaDNS, and DNDS, respectively. The 28-d LC50s for H. azteca were similar among compounds: 115.3, 82.1, and 49.0 µg/g dry weight (dw) in sand, and 627.3, 757.9, and >188.5 µg/g dw in sediment, for BaDNS, CaDNS, and DNDS, respectively. However, when LC50s were estimated based on concentrations of NSAs measured in overlying water (which can be an important route of exposure for H. azteca), BaDNS and CaDNS were 3-4 orders of magnitude more toxic than DNDS. The NSAs examined were >3-fold more toxic when present in substrates with no organic carbon (e.g., sand) for all H. azteca endpoints where LC/EC50s could be calculated and for sublethal endpoints for T. tubifex. The organic carbon content of the sediment appears to have acted as a sink and reduced NSA toxicity by decreasing bioavailability. Environmental sediment samples were collected from 12 river sites across southern Ontario. The maximum concentration of CaDNS observed in sediment collected from this region was 2.8 µg/g dw in sediment with 2% organic carbon; 100-fold lower than the lowest EC10 in the current study.

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The presence of the oligochaete species Quistadrilus multisetosus (Smith, 1900) originating from North America has been mentioned for several decades in Europe, the Middle East and Russia. Its distribution and abundance in Europe is still unknown but it can be considered as potentially invasive. This species was recently discovered in Lake Geneva (Switzerland/France) and three other Swiss lakes. The aims of the present work are to report its repartition and abundance in Lake Geneva, to study its ecology and to determine its invasive potential in this lake. We also provide an identification key for correctly differentiating Q. multisetosus from the closely related species Spirosperma ferox Eisen, 1879 and Embolocephalus velutinus (Grube, 1879), and study the phylogenetic position of Q. multisetosus within several Tubificinae lineages based on the cytochrome c oxidase (COI) marker. Twenty-eight sites have been monitored since 2009 in Lake Geneva. In several sites, the COI sequence corresponding to this species was also searched for in sediment samples using high-throughput sequencing. In addition, we examined specimens collected in this lake before 2009 likely to belong to Q. multisetosus and to have been misidentified. We found that Q. multisetosus was only present in the lake downstream of a wastewater treatment plant and a combined sewer overflow in the Vidy Bay (near Lausanne) and at a site located nearby. These results confirmed the high tolerance of this species to organic matter pollution. Q. multisetosus was already present in this location in 1974 (misidentified as Spirosperma ferox), which suggests that Q. multisetosus has a limited capacity to disseminate in this lake. However, we recommend continuing monitoring its presence in Lake Geneva in the future, especially in the context of warming of waters that could contribute to the expansion of this species.

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We report a new microsporidium Jirovecia sinensis sp. n. from a freshwater oligochaete, Branchiura sowerbyi collected in Hongze city, Jiangsu province, East China. Numerous whitish hypertrophied coelomocytes of 0.33-0.59 mm in diameter indicated infection. Transmission electron microscopy observations revealed that all developmental stages were diplokaryotic. The earliest life stages observed were meronts that were in direct contact with host cytoplasm, accumulated peripherally in the hypertrophied coelomocytes and connected with host cytoplasm through many pinocytotic canals. Mature spores are rod-shaped with a blunt end, measuring 17.0 ± 0.1 (14.9-18.5) µm long and 2.0 ± 0.2 (1.7-2.2) µm wide. The most conspicuous character of the novel microsporidian parasite is the tail-like posterior prolongations, with a length of 29.6-40.8 µm. Mature spores have a manubrium with a diameter of 447-485 nm which consist of six density-discontinuous concentric circles. Spores possess a collar-shaped anchoring disk and a bipartite polarplast with an anterior lamellar region and a posterior tubular section. SSU rDNA-based phylogenetic analysis indicated with high support values that the new species clustered with two Bacillidium species (B. vesiculoformis and Bacillidium sp.) infecting the freshwater oligochaetes and Janacekia debaisieuxi infecting the insect Simulium maculatum. Based on the ultrastructural features and molecular characteristics, a new species in the genus Jirovecia, Jirovecia sinensis sp. n., is designated.

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We describe, morphologically and molecularly, a new actinosporean from the intestinal epithelium of the freshwater oligochaete Ilyodrilus templetoni in the upper estuary of the River Minho, Northern Portugal. Mature actinospores resembled both echinactinomyxon and raabeia types, emphasizing the previously known lack of a clear boundary between these 2 collective groups. Historically, raabeia and echinactinomyxon types have been differentiated solely based on the shape of the valvular processes being curved or straight, respectively. Our observations, however, show that this morphological character is too variable for distinguishing between these 2 collective groups, since the actinospores of the raabeia described here displayed valvular processes that could either be straight, downward or upward curved. Several similar cases can be found in the available literature. Considering this overlap in actinospore morphology, we propose that echinactinomyxon be deemed invalid and its types be included in raabeia, as the latter constitutes the older of the 2 groups. Known echinactinomyxon types, however, should not be renamed as raabeia, as this would create unnecessary confusion. Accordingly, a more comprehensive definition of the raabeia collective group is provided. Phylogenetic analyses revealed polyphyletic clustering of raabeia/echinactinomyxon types among members of the myxosporean suborders Variisporina and Platysporina, reiterating the lack of agreement between actinosporean morphotypes and myxosporean genera. The new type described here specifically clusters within the Paramyxidium clade, alongside other SSU rDNA sequences of raabeia, echinactinomyxon, aurantiactinomyxon and synactinomyxon. Considering that most Paramyxidium spp. parasitize Anguilla anguilla (Linnaeus, 1758), future myxozoan surveys in the River Minho should include this species.

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An aurantiactinomyxon type is described from the marine naidid Tubificoides pseudogaster (Dahl, 1960), collected from the lower estuary of a Northern Portuguese River. This type constitutes the first of its collective group to be reported from Portugal, and only the fourth described from a marine oligochaete worldwide. Extensive morphological comparisons of new aurantiactinomyxon isolates to all known types without available molecular data are proposed to be unnecessary, given the artificiality of the usage of morphological criteria for actinosporean differentiation and the apparent strict host specificity of the group. Recognition of naidid oligochaetes as the hosts of choice for marine types of aurantiactinomyxon and other collective groups, suggests that the family Naididae played a preponderant role in the myxosporean colonization of estuarine communities. Molecular analyses of the type in study further infer its involvement in the life cycle of Paramyxidium giardi (Cépède, 1906) Freeman and Kristmundsson, 2018, a species that infects the kidney of European eel Anguilla anguilla (Linnaeus, 1758) and that has been reported globally, including from Portuguese waters. The low intraspecific difference registered in relation to Icelandic isolates of P. giardi (0.6%) is hypothesized to result from the emergence of genotypically different subspecies due to geographic isolation.

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We investigated the involvement of oligochaetes in the life cycles of fresh water myxozoan parasites in Brazil. In a fish farm in the State of Mato Grosso do Sul, we examined 192 oligochaetes and found that two (1%) released Aurantiactinomyxon type actinospores. We identified infected oligochaetes by morphology: both were Pristina synclites, from family Naididae. This is the first report of the involvement of this species in the life cycle of myxozoans. Small-subunit ribosomal DNA sequences of Aurantiactinomyxon type 1 (1882 nt) and Aurantiactinomyxon type 2 (1900 nt) did not match any previously sequenced myxozoan in the NCBI database, with the highest BLAST search similarities of 83% with Myxobolus batalhensis MF361090 and 93% with Henneguya maculosus KF296344, respectively, and the two aurantiactinomyxons were only 75% similar to each other (over ~ 1900 bases). Phylogenetic analyses showed that Aurantiactinomyxon type 1 had closest affinities with myxozoans from fish hosts in Order Characiformes, and Aurantiactinomyxon type 2 had affinities with myxozoans from fish of Order Siluriformes.

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Bisphenol A (BPA) is a component of polycarbonate plastics and a near ubiquitous environmental endocrine disrupting chemical. Bisphenol S (BPS), a substitute of BPA, is also hormonally active. This study examines the effects of aqueous exposure to BPA and BPS on the freshwater annelids Lumbriculus variegatus, a keystone species in shallow water ecosystems. Both BPA and BPS, at both low dose (10-9 M) and high dose (10-6 M), retarded the initial phase of body regrowth after cutting/fragmentation, which is the main mode of reproduction of L. variegatus. Both acute and five day exposure to BPA and BPS increased pulse rate of the dorsal blood vessel. For all the measured endpoints, the effects of BPA and BPS were nearly indistinguishable. These results indicate that BPA and BPS have similar and significant effects on the physiology of L. variegatus. These findings have implication for the potential impact of these bisphenols on invertebrates in the ecosystem.

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Substituted phenylamine antioxidants (SPAs) are additives in a variety of commercial polymers (e.g., lubricants, plastics, etc.). Based on their physicochemical properties, if SPAs were to enter an aquatic system, they would likely partition into sediment and have the capacity to bioaccumulate in biota. This study investigated the potential of four sediment-associated SPAs, diphenylamine (DPA), N-phenyl-1-naphthalene (PNA), N-(1,3-dimethylbutyl)-N'-phenyl-1,4-phenylenediamine (DPPDA), and 4,4'-methylene-bis[N-sec-butylaniline] (MBA) to accumulate in the tissues of freshwater mussels (Lampsilis siliquoidea) and oligochaete worms (Tubifex tubifex). Mussels and worms were exposed to sediment spiked with individual SPAs for 28 d. The concentration of SPAs was measured in the gill, gonad, and remaining viscera of the mussels and entire body of the worms. The majority of biota-sediment accumulation factors (28-d BSAFs) for the different tissues of mussels were < 1. The highest concentrations of SPAs were consistently observed in the gill tissue of mussels relative to the gonad and viscera. The 28-d BSAFs for DPPDA and MBA for worms were < 1, and for DPA and PNA, they ranged from 0.38-2.13 and 1.54-33.24, respectively. The higher 28-d BSAFs observed for worms compared to mussels were likely because worms are endobenthic and feed on sediment-associated organic matter. PNA and DPPDA have similar octanol-water partition coefficients (Kow) but greater 28-d BSAFs were observed for PNA compared to DPPDA for both species. This observation provides evidence that biota may be able to metabolize and/or excrete SPAs with similar physicochemical properties at considerably different rates. The 28-d BSAFs observed for sediment-associated SPAs are lower than those typically required for a chemical to be classified as bioaccumulative.

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As compared to their temperate counterparts, few toxicity tests have been conducted so far into the evaluation of the sensitivity of indigenous tropical species to pesticides. Especially mixture toxicity assessments appear to be scarce. To contribute to increase our knowledge in this arena, we evaluated the acute toxicity of diuron and carbofuran and their mixtures to the neotropical oligochaetes Allonais inaequalis and Dero furcatus, and the ostracod Strandesia trispinosa. Tests were performed with both the pure active ingredients, as well as their formulated products. The toxicity of the latter to the three test organisms was generally greater than that of the pure active ingredients, although absolute differences were rather small. The sensitivity of the indigenous species was slightly greater than temperate test species from the same taxonomic groups. The concentration addition conceptual model best described the results of the mixture toxicity data. Derived deviations of this model appeared to be dependent on the test organism and as to whether the pesticides were applied as active ingredients or their commercial products. Reported field concentrations of the two pesticides indicate risks to freshwater biota, especially if they are both present. The test species used in the present study are concluded to be suitable candidates as surrogate test organisms in local pesticide risk evaluations.

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Myzobdella lugubris is a commensal leech on crustaceans and a parasite to fishes, surviving predominantly in brackish waters throughout North America. Specimens in this study were collected within the tidal zone of the Delaware River basin (New Jersey and Pennsylvania). To compare regional M. lugubris specimens, defined characters were scored after dorsal and ventral dissections, and phylogenetic relationships were resolved using cytochrome c oxidase subunit 1 (CO1), 12S ribosomal RNA (rDNA) and 18S rDNA gene fragments. Variance between regional populations was low, suggesting recent dispersal events and/or strong evolutionary constraints. The reproductive biology of M. lugubris was explored by quantitative analysis of secreted cocoons. Specimens produced 32.67 ± 4.50 cocoons with fertilization ratios of 88.1% and hatching times of 48 ± 7 days at 17°C under laboratory conditions. At 22°C, 46 ± 28 cocoons were produced with fertilization ratios of 70.27% and hatching times of 28 ± 5 days. Surprisingly, each cocoon supported only one embryo, which is unusual among oligochaetes.

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Azo dyes are synthetic compounds used as industrial colorants, and some are predicted to be inherently toxic, bioaccumulative, and/or persistent based upon their chemical composition. This study addresses data gaps in current research which include the need to evaluate the toxicity of hydrophobic azo dyes to benthic invertebrates. The toxicity of a solvent dye, Sudan Red G (SRG), and two disperse dyes, Disperse Yellow 7 (DY7) and Disperse Orange 13 (DO13), to Hexagenia spp. and Tubifex tubifex was assessed in spiked-sediment exposures. The dye compounds appeared to degrade readily in the equilibrium and exposure periods, suggesting a limited persistence of the parent compounds in the environment under test conditions. Although azo dye degradation products could not be reliably quantified, one was detected in DY7 sediment samples that elicited toxic effects to Hexagenia and Tubifex, providing evidence that DY7 degrades. Hexagenia survival and growth endpoints responded with similar sensitivity to the dyes, but DY7 was the most toxic, with a 21-day IC25 (concentration associated with 25% inhibition) for growth of 9.6 µg/g. Comparatively, Tubifex reproduction was the most sensitive endpoint for all dyes with 28-day IC25s for young production ranging from 1.3 to 11.8 µg/g. At sublethal concentrations, toxic effects to Tubifex differed between dyes: the solvent dye exerted an effect primarily on gametogenesis (cocoon production), while disperse dyes, most notably DY7, caused effects on embryogenesis (development of worm inside the cocoon). This study indicates that there could be potential hazard to oligochaetes based on the observed effect concentrations, but given the lack of environmental measurements, the risk of these compounds is unknown. Further research is required to determine if degradation products were formed in all dye samples and whether toxicity was caused by the parent molecules, which have limited persistence under test conditions, or by their degradation products. To avoid underestimating toxicity, this study stresses the need to use an infaunal deposit feeder such as the oligochaete Tubifex in sediment toxicity assessments where highly hydrophobic compounds are present.

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We have cloned and characterized the expression of a novel maternal gene festina lente (designated Ttu-fel) from the clitellate annelid Tubifex tubifex. Northern blot analyses have shown that Ttu-fel mRNA is approximately 8 kbp in length and that its expression is restricted to oocytes undergoing maturation division and early embryos up to 22-cell stage. Maternal transcripts of Ttu-fel are first detected in oocytes in the ovary of young adults (ca. 40 days after hatching); its expression continues in growing oocytes in the ovisac. Ttu-fel mRNA is distributed broadly throughout the egg undergoing maturation divisions. During the process of ooplasmic segregation that results in the pole plasm formation, Ttu-fel mRNA becomes concentrated to the animal and vegetal poles. The RNA in the animal hemisphere is distributed in a gradient with highest concentration in the cortical region. During the first two cleavages, Ttu-fel mRNA is segregated to CD cell then to D cell; it is subsequently inherited by the three D quadtrant micromeres, 1d, 2d and 3d. Around the time of transition to 22-cell stage, Ttu-fel mRNA becomes undetectable throughout the embryo.

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