Degradation and detoxification of endosulfan isomers by a defined co-culture of two Bacillus strains

The degradation of alpha and beta isomers of endosulfan by a two-member bacterial co-culture was studied. Results were similar whether the two isomers were present individually or together, as in technical endosulfan. The degradation of both isomers was accompanied by the formation of endosulfan diol and endosulfan lactone. Accumulation of the metabolite, endosulfan sulfate was, however, not observed during the reaction with either of the isomers. The microbial degradation of endosulfan isomers was also accompanied by a decrease in its toxicity to the test organism Tubifex tubifex Müller.     

A comparison of two tubificid oligochaete species as candidates for sublethal bioassay tests relevant to subtropical and tropical regions

Two tubificid oligochaetes, Branchiura sowerbyi and Tubifex tubifex, were evaluated as potential test organisms for sediment bioassays. We attempt to reproduce the sediment bioassay proposed by Reynoldson et al. (1991) using his strain of Tubifex tubifex and his reference sediment and to compare this technique using Branchiura sowerbyi. This species was chosen because it is more common and dominant in tropical and subtropical environments than Tubifex tubifex. Data on survival and reproduction were obtained for both species, and growth estimates were obtained for B. sowerbyi. The sublethal bioassay with T. tubifex confirmed earlier estimates of a test duration of 4 weeks at 22.5 °C. B. sowerbyi cultures can produce usable estimates in 21 days at 30 °C.     

The Tubifex tubifex assay for the determination of acute toxicity

An express (3-minute) test for acute toxicity determination by using the oligochaete annelid, Tubifex tubifex, is described. The EC50(Tubifex tubifex) [EC50(Tt)] for movement inhibition was calculated by using a concentration-response dependence. The reproducibility of the test was checked over several years and by several workers. Its applicability is limited to compounds which are soluble in water. The calculated EC50(Tt) indices correlate with LC50 values determined by using the fish, Pimephales promelas (96-hour assay), and with ICG50 values determined by using the ciliate, Tetrahymena pyriformis (48-hour assay) with high statistical significance (r = 0.822, n = 35, and r = 0.927, n = 80, respectively). The correlation between the EC50(Tt) indices and rat oral LD50 values (48-hour assay) was r = 0.519 (n = 67). The correlation within organic compounds was closer (r = 0.635, n = 60) than with the heterogeneous series of chemicals. A similar trend was noticed for the correlation with mouse oral LD50 values (r = 0.479, n = 56) with the heterogeneous series of chemicals, as compared that with the series without inorganic salts (r = 0.605, n = 42), and similarly with mouse intraperitoneal LD50 values, where r = 0.543 (n = 50) with the heterogeneous series of chemicals and r = 0.893 (n = 33) with the series of organic chemicals.     

Prey location and prey choice by the freshwater leech Erpobdella octoculata using foraging kairomones

1. The freshwater leech, Erpobdella octoculata , is a generalist predator feeding on prey organisms such as Tubifex spp., Chironomus spp. and Asellus aquaticus. Using different experimental designs, we studied the use of foraging kairomones by the leeches for prey location.
2. Leeches were attracted to living as well as to freshly killed larvae of Chironomus sp., to Tubifex sp., and to A. aquaticus offered in Petri dishes covered with gauze. The leeches also reacted to an extract of macerated Tubifex sp. presented in agar.
3. Using ion exchange chromatography, the presence of histidine and glutamic acid was demonstrated in water contaminated by living larvae of Chironomus sp. and Tubifex sp. Agar blocks containing a synthetic mixture of these compounds at concentrations above 5 mg mL⁻¹ were attractive to the leeches.
4. Choice tests showed that leeches preferred chemical signals from Tubifex sp. over larvae of Chironomus sp. No difference was found between chemicals from Tubifex sp. and A. aquaticus , and A. aquaticus and larvae of Chironomus sp.
5. The results demonstrate that E. octoculata uses specific foraging kairomones in searching for prey and indicate that amino acids serve as foraging kairomones.     

Molecular phylogeny of tubificid oligochaetes with special emphasis on Tubifex tubifex (Tubificidae)

Tubifex tubifex is a cosmopolitan freshwater oligochaete whose presence has been studied as a health indicator of the aquatic environment and as a host for several myxozoan parasites of fish. Unfortunately, current morphological criteria used to distinguish Tubifex spp. (Tubificidae) are inadequate. We therefore developed mitochondrial 16S ribosomal DNA markers to examine phylogenetic relationships among aquatic oligochaetes and to distinguish species of Tubifex that might serve as hosts for a particular myxozoan parasite, Myxobolus cerebralis. Our phylogenetic analyses of oligochaetes based on a 378-bp segment yielded one most parsimonious tree with three major groups that corresponded to the families Lumbricidae, Sparganophilidae, and Tubificidae. T. tubifex and T. ignotus formed a monophyletic assemblage, and a sister relationship between the genera Tubifex and Limnodrilus was strongly supported. A second analysis of the relationship within the genus Tubifex identified six genetically distinct lineages of T. tubifex from North America and Europe that were separated by genetic distances comparable to those found for "well-defined" species of Limnodrilus. Therefore, the existence of several morphologically indistinguishable, thus cryptic, species of Tubifex in North America and Europe is suggested.     

Cyst formation of Tubifex tubifex (Müller) — an adaptation to survive food deficiency and drought

Adult individuals of Tubifex tubifex (Müller) (Oligochaeta, Tubificidae) could be induced to form cysts in the laboratory. Hiding in cysts, Tubifex tubifex survived a five-month drought period in the field. Encysted Tubifex tubifex had a lower super-cooling-point than mobile individuals. Food shortage is believed to be the main factor in cyst formation.     

A field test of a sediment bioassay with the oligochaete worm Tubifex tubifex (Müller, 1774)

A sediment toxicity test using reproduction in the tubificid oligochaete Tubifex tubifex was tested in 11 geographic locations in the Laurentian Great Lakes where sediments have previously been documented as being contaminated. Samples were taken from both potentially contaminated and clean sites in each of the 11 areas. Using test endpoints such as survival and growth, results from 72 samples were classified using cluster and ordination techniques. Six response groups were identified and four groups of toxic sites determined. Toxic effects ranged from mortality of adult worms to reduced reproduction. Using correlation and discriminant analysis there was good evidence that high concentrations of metals such as copper and lead were responsible for the observed effects at some sites.     

Amino acid sequence of the monomer subunit of the giant extracellular hemoglobin of the aquatic oligochaete, Tubifex tubifex

The extracellular hemoglobin of the aquatic oligochaete Tubifex tubifex consists of four subunits: a monomer of 16.5 kDa, a disulfide-bonded trimer of about 50 kDa and at least two subunits of about 30 kDa. The complete amino acid sequence of the monomeric subunit was determined: it consists of 141 amino acid residues and has a molecular mass of 16,286 Da including a heme group. 39 residues (28%) were found to be identical with those in the corresponding positions in the monomeric globin chains from Lumbricus terrestris, Pheretima sieboldi, and Tylorrhynchus heterochaetus. Tubifex and Lumbricus are most similar, with 75 amino acid identities (53%). There are eight invariant residues amongst these monomeric globins and the intracellular monomeric globin of Glycera and the human beta-globin. The monomeric globin from Tubifex aligns best with those of group A, globins which have a Cys in their second position and an invariant Lys-Val-Lys at positions 9-11 [Gotoh et al. (1987) Biochem. J. 241, 441-445]. The two cysteine residues, at positions 2 and 131, appear to be disulfide-bonded.     

Some aquatic oligochaetes of the Nilgiris,South India

Eight species of aquatic oligochaetes, belonging to the families Naididae and Tubificidae, are reported, of which six species are new to the Nigiris. Tubifex tubifex known from Coonoor is reported again from this area.     

What is Tubifex tubifex (O. F. Müller) (Oligochaeta,Tubificidae)?

A review of the fairly muddled conditions in the early history of Tubifex tubifex (O. F. Müller, 1774) is given. The identity of Müller's (1774) Lumbricus tubifex is discussed, analytical examinations of samples from the most likely type-locality of the species are accounted for, and a neo type has been selected. For comparison, other "Tubifex" specimens, both European and American, are likewise analysed and a scrutiny of the literature concerning the species and its synonyms is undertaken. The survey results in a clearer picture of T. tubifex and a revised view of the qualifying characters on the specific level. Diagnoses of the species T. tubifex (O. F. Müller) and T. fissidens sp.n., Kernall, MS, 1914, are given.     

Screening the Toxicity of Ni,Cd, Cu,Ivermectin, and Imidacloprid in a Short-Term Automated Behavioral Toxicity Test with Tubifex tubifex (Müller 1774) (Oligochaeta)

A new automated online toxicity test for screening of short-term effects of chemicals is presented using the freshwater oligochaete Tubifex tubifex in the Multispecies Freshwater Biomonitor? (MFB). Survival and locomotory behavior of the worms were observed during 24 h of exposure to metals (Cd, Cu, Ni), pesticides (Imidacloprid), and pharmaceuticals (Ivermectin). The LC₅₀ values revealed increasing toxicity in the following order: Ni (> 100 mg/l) < Cu (15.2 mg/l) < Cd (4.9 mg/l) < Ivermectin (1.8 mg/l) < Imidacloprid (0.3 mg/l). The EC₅₀ for locomotion showed a similar order of increasing toxicity: Ni (86 mg/l) < Cu (3.8 mg/l) < Ivermectin (2.0 mg/l) < Cd (1.1 mg/l) < Imidacloprid (0.09 mg/l). Toxicity was dependent on both concentration and exposure time. This could be demonstrated in 3d response models and proven in the statistical analysis showing a significant interaction term (C × T) for the experiments with Cu and Ni. T. tubifex proved to be very tolerant, but even then behavioral responses were more sensitive than mortality for Cu, Cd, and Imidacloprid.     

The effect of cohabitation of Tubifex tubifex (Oligochaeta: Tubificidae) populations on infections to Myxobolus cerebralis (Myxozoa: Myxobolidae)

The competitive interactions between susceptible and resistant Tubifex tubifex (Oligochaeta: Tubificidae) exposed to Myxobolus cerebralis (Myxozoa: Myxobolidae) infections were investigated in two laboratory trials. Competition was assessed by the total parasite production over the course of the trials in mixed and pure cultures of M. cerebralis exposed worms, and by the genetic analyses of worms from the control and experimental groups at the beginning and end of the experiments. Mixed cultures of resistant and susceptible worms showed a 70% reduction in production of parasites released when compared with pure cultures of susceptible worms. In studies with laboratory and field-collected oligochaetes the mixed cultures at the end of the cohabitation experiments were dominated by resistant Tubifex from lineage V (HB strain) this strain of Tubifex has a competitive advantage over worms from other lineages. The results of this study suggest that certain species of Tubifex may be dead-end hosts to M. cerebralis by absorbing or inactivating the parasite and may also show greater survival compared to susceptible oligochaetes in certain whirling disease enzootic habitats.     

Growth, fat content and fatty acid profile of South American catfish, surubim (Pseudoplatystoma fasciatum) juveniles fed live, commercial and formulated diets

South American catfish, barred surubim ( Pseudoplatystoma fasciatum ) juveniles (117.6 ± 11.8 mg individual weight; 28.3 ± 2.5 mm total length) were fed various diets: one live ( Tubifex worms), two commercial (Aglo Norse and Bio Kyowa), and one semi-purified formulated diet (75% peptide based protein) over a 2-week period. Fish fed the Aglo Norse diet showed the highest growth performance, but cannibalism also was very high (42%). Fish fed peptide based formulated diet demonstrated the lowest growth rate, with no cannibalism. The highest survival was achieved with fish fed Tubifex worms (100%). Lipid level in the whole body of the fish fed four different experimental diets did not differ significantly, averaging 3.6 ± 0.7%. Fatty acid composition of neutral and phospholipid fractions of whole body lipids of fish reflected the fatty acid composition of the diets. The high level of 20:4 n -6 in Tubifex worms resulted in a high level of this fatty acid in the tissue of fish fed this diet. It remains uncertain how high survival and no cannibalism is related to dietary lipids/fatty acids. In all cases, the increasing ratio of n -3 HUFA (highly unsaturated fatty acids)/ n -6 HUFA in phospholipid fractions suggested the elongation and desaturation of 18:3 n -3 to 22:6 n -3 via 20:5 n -3. Moreover, in respect to the 20:4 n -6 levels in the diets, an increase in the concentration of this fatty acid in phospholipid fraction suggests that South American catfish can transform linoleate into arachidonate.     

Mitochondrial DNA reveals cryptic oligochaete species differing in cadmium resistance.

Species of the family Tubificidae represent a major faunal element in benthic freshwater communities throughout the world. Some of them are considered particularly tolerant of the influence of toxicants such as cadmium. One of the most abundant species, "Tubifex tubifex," is frequently used as an indicator of environmental pollution, despite considerable taxonomic problems caused by phenotypic plasticity and genetic heterogeneity. Our study provides a phylogeny of "T. tubifex" based on a segment of the mitochondrial 16S rDNA and presents a rapid PCR-based method of genotype screening which was then applied in cadmium toxicity studies on natural populations. Phylogenetic analysis identified five major mitochondrial lineages, some of them separated by large genetic distances (up to 13%) but morphologically indistinguishable, thus highly suggestive of the existence of cryptic species. All lineages were present at different frequencies in the European river populations studied, with a tendency of the more resistant lineages to occur at higher frequencies in the more tolerant populations. In fact, lineage-specific toxicity experiments showed that individuals of different mitochondrial lineages consistently varied in cadmium resistance, suggesting that in benthic oligochaetes, evolution seems to proceed predominantly through natural selection acting on physiological, rather than morphological, characters. In consequence, toxicological studies involving "T. tubifex" as a monitoring or test organism should allow for the possibility of genetic inhomogeneity of this mudworm group by combining both toxicological and genetic methods.     

Ultrastructural analysis of sperm flagella in two clitellates (Annelida), plasma membrane and periaxonemal area

The region between axonemes and plasma membrane in the sperm tails of the tubincid oligochaetes Tubifex tubifex and Monopylephorus limosus has been studied by means of thin sections of conventionally and tannic acid fixed material, and of freeze-fracture replicas. The main portion of the flagellum in both species showed prominent, regularly repeating bridges connecting doublets to plasma membrane. In correspondence to the doublets, characteristic double rows of intramembrane particles are present, with an arrangement reminiscent of the "zipper lines" described in other species. A well-developed cortical web with a honeycomb appearance underlies the plasma membrane. Glycogen granules are regularly arranged within the cells. An outstanding difference between the two species is to be found in the presence only in Monopylephorus of complex muff-like structures apparently formed by membrane particles and series of teeth embedded in the cortical web. Nothing similar has been found in Tubifex. This difference may be related to the fact that spermatozoa in Monopylephorus are not enclosed in spermatozeugmata as are those of Tubifex.     

Anionic and neutral glutathione S-transferase isoenzymes in the freshwater worm Tubifex tubifex (O.F.M.)

The anionic and the neutral glutathione S-transferase (GST) isoenzymes from the freshwater worm Tubifex tubifex (O.F.M.) were separated in one step by chromatofocusing on a Polybuffer exchanger 94 column, eluted with Polybuffer 74. Their pI values ranged between 4.12 and 6.98, and their molecular weight between 30 000 and 38 400. The apparent Km values towards glutathione and 1-chloro-2,4-dinitrobenzene were also determined. The high number of non-cationic GST isoenzymes is unusual. Tubifex worms seems well equipped for attacking environmental pollutants.     

Description of a new synactinomyxon type from the River Sousa, Portugal

Actinospore infection of oligochaetes collected from the mud of 2 freshwater biotopes in Portugal was studied. Using the 'cell-well plate method', a new synactinomyxon type was found in 2 specimens (1.3%) of the examined Tubifex tubifex oligochaetes from the River Sousa north of Porto, Portugal. In Criodrilus lacuum and Dero digitata specimens collected from the same river, no actinosporeans were released during the 12 wk observation period. Infected oligochaetes were only found immediately post-collection, and no further actinosporean release was recorded in Tubifex specimens kept alive for several weeks. Actinospore infection showed high intensity in oligochaetes in both positive cases. No actinosporean stages of myxosporeans have as yet been described from Portugal. On the basis of spore morphology and 18S rDNA sequence data, the synactinomyxon type presented in this paper differs from those already known and described in the literature.     

Sediment toxicity in lakes along the river Kolbäcksån,central Sweden

The River Kolbäcksån system is located in a historical mining and steel works district of central Sweden. Ten years ago, intensive limnological studies indicated that the sediments in many of the lakes of this system were contaminated with metals and oil (grease). More recently a very high toxicity was found in the sediments from some of these lakes in tests with Daphnia magna and Tubifex tubifex. The objective of this study was to determine the toxicity to Daphnia magna of surficial sediments from representative locations (N = 39) within this system of lakes and to look for possible correlations with concentrations of metals (Cd, Cr, Cu, Hg, Ni, Pb, Zn) and oil (total) analysed in parallel samples. These results were also compared with spiked sediment toxicity data generated for these metals and with the bottom fauna at 8 of the 39 locations. Among the metals analyzed, only Cd, Pb and Zn were correlated with whole sediment toxicity. Only for Zn and Cr did the maximum concentrations approach those that were toxic in the spiked sediments. Correlations using single and combined metal contamination and toxicity (additive models) explained 16% of the sediment toxicity (r ² = 0.16; N = 39). Significant correlations with the bottom fauna in situ (gross abundance, biomass and species richness) were found for whole sediment toxicity, but not for metal contamination (N = 8). The use of the sediment quality triad approach for assessment of sediment quality is exemplified with a numerical normalization procedure for the latter 8 sites.     

Cyclic Changes in Shape of A Non-Nucleate Egg Fragment of Tubifex (Annelida, Oligochaeta)

A non-nucleate fragment separated from the fertilized Tubifex egg at metaphase of the second meiosis showed temporary surface deformation at 3–3.5 hr intervals, i.e. , synchronously with the onset of formation of the second polar body and early cleavages in control eggs. From the two-cell stage on, the periodicity of the surface activity in the non-nucleate fragment was found to be synchronous with the cleavage cycles of the CD-cell and its descendants, but not with those of the AB-cell. This surface deformation was completely inhibited by cytochalasin B (50 μg/ml). Electron microscopy shows that microfilaments are present exclusively in the cortical layer of the deforming fragments. Cycloheximide-treated egg fragments commenced surface deformation after a delay of 1–2 hrs; pulse-treatment indicated that the surface deformation requires proteins synthesized specifically during the period of the previous surface deformation. These results are discussed in relation to the nucleus-independent cytoplasmic rhythm and asynchronous cleavage of Tubifex eggs.     

Expression of <Emphasis Type="Italic">hunchback</Emphasis> protein in a subset of ectodermal teloblasts of the oligochaete annelid <Emphasis Type="Italic">Tubifex</Emphasis>

The early expression patterns of hunchback protein (T-hb protein) were examined in the oligochaete Tubifex, using an antibody raised to the LZF2 protein in leech. This antibody recognizes a 60-kDa polypeptide in the Tubifex embryo. Before teloblastogenesis, T-hb protein is expressed in every cleavage-stage blastomere. At the completion of teloblastogenesis, the only cells expressing T-hb are a fraction of the micromere-derived epithelial cells. During gastrulation, nuclear T-hb is seen in spreading micromere-derived epithelial cells and also in a subset of ectodermal teloblasts. Comparisons of these results with those from other annelids suggest that hb expression in the early cleavage blastomeres and the micromere-derived epithelium are features highly conserved among annelids. In contrast, hb expression in teloblasts appears to be an innovation evolved in the oligochaetes.