Dero digitata, a freshwater oligochaete worm found worldwide, serves as the definitive host for the myxozoan Henneguya ictaluri, the cause of proliferative gill disease (PGD) in ictalurid catfish. The study objectives were to describe pathologic changes associated with developing actinospores within D. digitata from a commercial catfish pond during an active PGD outbreak. Specific in situ hybridization probes identified H. ictaluri, Henneguya exilis, and Raabeia-type TGR 2014 actinospore development in pure infections associated with intestinal epithelial hypertrophy, hyperplasia, and vacuolation. Results support findings from other myxozoans demonstrating clonal expansion of myxozoan populations occurs through asexual reproduction of the oligochaete host.

Abstract Freshwater molluscs are hosts for diverse metazoan symbionts. However, apart from the digenean helminths, symbionts of molluscs are underreported worldwide. Therefore, this study focused on the diversity of oligochaetes, leeches, mites, insect larvae and nematodes associated with gastropods and bivalves from Lake Victoria, East Africa. Overall, 1,633 mollusc specimens representing 14 species were sampled from the Kenyan part of the lake. Each host specimen was examined to determine co‐occurrence of symbionts (including digeneans) and other metazoan invertebrates, and their microhabitat preferences on/in the hosts. In addition, prevalence and abundance of symbionts were compared for molluscs obtained from an unpolluted site, and sites that are eutrophic and polluted with heavy metals. In total, 33 species were recovered from the molluscs: six oligochaetes, four insects, two mites, two nematodes, one leech and 18 digeneans. The nematode Daubaylia potomaca, mite Unionicola macani, leech Batracobdelloides sp., oligochaetes Chaetogaster limnaei, Dero digitata, Aulophorus africanus, Allonais paraguayensis and Ophidonais serpentina, and gorgoderid digenean larvae are new records for Lake Victoria. Moreover, the genus Bratislavia (Oligochaeta) is reported for the first time in Africa. Our results show that parasitic mites and leeches were absent from mollusc specimens that harboured chironomids. Likewise, there was no co‐occurrence of C. limnaei, D. potomaca and digeneans, suggesting that antagonistic interactions occur between the symbionts. Although co‐occurrence of mites and digeneans in individual mollusc hosts was common, the symbionts occupied different microhabitats. This study shows that prevalence and intensities of U. macani and C. limnaei, were significantly higher in hosts from the unpolluted site, compared with the polluted sites. What is more, D. potomaca and trichopteran larvae, were recorded only from the unpolluted site. The distribution of the chironomid Kiefferulus chloronatus suggests their preference for sites that are rich in organic matter and their ability to tolerate pollution. Information on antagonistic interactions between symbionts might be applicable in biocontrol, especially of digenean species that have veterinary or medical importance. Finally, this study shows a potential for the use of U. macani, C. limnaei, D. potomaca and insect larvae as discriminators of the quality of aquatic habitats.

Abstract Dero digitata is the definitive oligochaete host of Henneguya ictaluri (Cnidaria: Myxobolidae), the causative agent of proliferative gill disease in U.S. farm-raised Channel Catfish Ictalurus punctatus and hybrid catfish (Channel Catfish × Blue Catfish I. furcatus). Several studies were performed to identify optimal conditions for culturing D. digitata in the laboratory for experimental life cycle studies. The first experiments investigated the effects of temperature on D. digitata population growth. Five different growth studies were conducted, each consisting of three different temperature treatments ranging from 7°C to 32°C. Cultures were held under different temperature regimes, and D. digitata numbers were determined after 2 weeks. Dero digitata populations reared at 21–32°C had significantly higher population growth than those maintained at lower temperatures. Additional experiments investigated the effects of supplemental diets on laboratory-reared D. digitata populations. Treatments consisted of autoclaved water that was collected from production ponds with substantial algal growth or a reservoir pond with a negligible algal bloom and maintained at ideal temperatures determined from the temperature trials (~26°C). Cultures were supplemented with three different commercially available feeds designed for bottom-feeding ornamental fish. Significantly larger numbers and greater reproductive rates, taken as an indirect measure of population fitness, were observed in D. digitata cultured in autoclaved pond water supplemented with algal wafers containing 29.0% minimum (min.) crude protein, 7.0% min. crude fat, 6.0% maximum (max.) crude fiber, 9.0% max. moisture, and 0.8% min. phosphorus. A final experiment showed that weekly water exchanges resulted in greater population growth of D. digitata relative to culture in nonexchanged water. This work optimizes the laboratory propagation of D. digitata and will facilitate life cycle studies and controlled experiments establishing developmental timelines and host–parasite interactions of H. ictaluri, similar to other myxozoan parasites.

Characterising myxozoan taxa parasitising fish hosts in catfish aquaculture ponds is crucial to understanding myxozoan community dynamics in these diverse and complex ecological systems. This work investigated the myxozoan fauna of the western mosquitofish, Gambusia affinis, a common, incidental species found in catfish aquaculture ponds in the southeastern United States. 598 fish were sampled in May of 2018 and 2019 from the pond facility of the Thad Cochran National Warmwater Aquaculture Center in Stoneville, Mississippi, USA. Fish were examined microscopically using wet mount preparations of fresh tissue and histology for myxozoans. 18S rRNA gene sequences were amplified from myxospores obtained at necropsy. Updated morphologic, histologic, and 18S rRNA gene sequence features are provided for Henneguya gambusi, Myxobolus pharyngeus, and Myxidium phyllium. Two potentially novel myxozoans were observed during this survey, an undocumented Myxobolus sp. associated with chondrolysis of bones throughout the body and a putative Myxobilatus sp. observed histologically in the renal tubules, ureters, and urinary bladder. However, inadequate samples were obtained for proper species descriptions. Lastly, the life cycle of M. pharyngeus, which is thought to utilize the oligochaete worm Dero digitata as their definitive host, was putatively confirmed by 18S rRNA sequence matching to actinospore stages from oligochaetes in catfish ponds in Mississippi. This work provides novel and expanded morphologic, histologic, molecular and biologic data of five myxozoan parasites of G. affinis, expanding our knowledge of myxozoan diversity in catfish aquaculture ponds.

A survey of aquatic Oligochaeta was conducted at Thiruvallur, Chennai, Kanchipuram, Villupuram and Nilgiris districts of Tamil Nadu from October 2015 to June 2016. Eleven taxa were recorded from a total of 922 specimens examined, eight of them were identified to species level. Five naidids identified during the present study: Branchiodrilus semperi, Dero dorsalis, Dero digitata, Dero indica and Dero zeylanica, and six tubificid taxa Branchiura sowerbyi, Tubificidae sp. 1, Tubificidae sp. 2, Tubificidae sp. 3, Limnodrilus hoffmeisteri and L. udekemianus. Dero dorsalis constitute the first report for Kanchipuram district. A DNA barcode sequence (GenBank accession no. MF125273) of the commercially important Limnodrilus hoffmeisteri, commonly known as ‘Tubifex worms’ in Chennai, was obtained and compared with other published COI sequences from that morphospecies from around the world.

Exposure and effects of sediment-spiked fludioxonil on macroinvertebrates and zooplankton in outdoor aquatic microcosms

Abstract Proliferative gill disease (PGD) in catfish is caused by the myxozoan Henneguya ictaluri. The complex life cycle requires Dero digitata as the oligochaete host. Efforts to control PGD by eradicating D. digitata have been unsuccessful. Smallmouth buffalo, Ictiobus bubalus, (SMB) are opportunistic bottom feeders and a putative option for controlling D. digitata. In 2011, 15 ponds (0.4 ha) were stocked with 5000 channel catfish, Ictalurus punctatus; 7 of these 15 ponds were also stocked with 300 SMB fingerlings. There were no differences in benthic invertebrate numbers or water quality variables between ponds with or without SMB. At harvest, there were no differences in percent survival, total weight, or catfish feed conversion ratio. In the second year, 18 ponds (0.4 ha) were stocked with 6000 channel catfish. Half the ponds were also stocked with 300 SMB. Sentinel fish were used to estimate disease severity, and pond water was collected for molecular estimation of H. ictaluri actinospore concentrations. Similar to the first year, there were no differences between treatments in any variable tested, including PGD severity in sentinel fish and parasite concentrations in pond water. Under these study conditions, presence of SMB did not have a measureable effect on PGD incidence, parasite density, or overall catfish production.

There are more than 200 species of Henneguya described from fish. Of these, only three life cycles have been determined, identifying the actinospore and myxospore stages from their respective hosts. Two of these life cycles involve the channel catfish (Ictalurus punctatus) and the freshwater oligochaete Dero digitata. Herein, we molecularly confirm the life cycle of a previously undescribed Henneguya sp. by matching 18S ribosomal RNA (rRNA) gene sequence of the myxospore stage from channel catfish with the previously described actinospore stage (Aurantiactinomyxon mississippiensis) from D. digitata. Gill tissue from naturally infected channel catfish contained pseudocysts restricted to the apical end of the primary lamellae. Myxospores were morphologically consistent with Henneguya spp. from ictalurid fishes in North America. The spores measured 48.8 ± 4.8 μm (range = 40.7-61.6 μm) in total spore length. The lanceolate spore body was 17.1 ± 1.0 μm (14.4-19.3 μm) in length and 5.0 ± 0.3 μm (4.5-5.5 μm) in width. The two polar capsules were 6.2 ± 0.4 μm (5.8-7.0 μm) long and 5.0 ± 0.3 μm (4.5-5.5 μm) wide. The polar capsule contained eight to nine coils in the polar filament. The two caudal processes were of equal length, measuring 31.0 ± 4.1 μm (22.9-40.6 μm). The 1980-bp 18S rRNA gene sequence obtained from two excised cysts shared 99.4% similarity (100% coverage) to the published sequence of A. mississippiensis, an actinospore previously described from D. digitata. The sequence similarity between the myxospore from channel catfish and actinospore from D. digitata suggests that they are conspecific, representing alternate life stages of Henneguya mississippiensis n. sp.

In the southeastern USA, the channel catfish Ictalurus punctatus is a host to at least eight different species of myxozoan parasites belonging to the genus Henneguya, four of which have been characterized molecularly using sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. However, only two of these have confirmed life cycles that involve the oligochaete Dero digitata as the definitive host. During a health screening of farm-raised channel catfish, several fish presented with deformed primary lamellae. Lamellae harbored large, nodular, white pseudocysts 1.25 mm in diameter, and upon rupturing, these pseudocysts released Henneguya myxospores, with a typical lanceolate-shaped spore body, measuring 17.1 ± 1.0 μm (mean ± SD; range = 15.0-19.3 μm) in length and 4.8 ± 0.4 μm (3.7-5.6 μm) in width. Pyriform-shaped polar capsules were 5.8 ± 0.3 μm in length (5.1-6.4 μm) and 1.7 ± 0.1 μm (1.4-1.9 μm) in width. The two caudal processes were 40.0 ± 5.1 μm in length (29.5-50.0 μm) with a spore length of 57.2 ± 4.7 (46.8-66.8 μm). The contiguous SSU rRNA gene sequence obtained from myxospores of five excised cysts did not match any Henneguya sp. in GenBank. The greatest sequence homology (91% over 1,900 bp) was with Henneguya pellis, associated with blister-like lesions on the skin of blue catfish Ictalurus furcatus. Based on the unique combination of pseudocyst and myxospore morphology, tissue location, host, and SSU rRNA gene sequence data, we report this isolate to be a previously unreported species, Henneguya bulbosus sp. nov.

The present study was carried out to evaluate the distribution and diversity of oligochaete fauna in selected ponds of Thiruvananthapuram district in Kerala, South India. The sediment samples were collected from three ponds seasonally during the period December 2006 to November 2008. In the study, 10 oligochaete species which belong to 8 genera were identified in three selected ponds. These include Dero digitata, Dero nivea, Dero obtusa, Pristina longiseta, Aulophorus furcatus, Stylaria fossularis, Chaetogaster spp., Aeolosoma spp., Tubifex tubifex and Limnodrilus hoffmeisteri. Tubifex tubifex and Limnodrilus hoffmeisteri are the pollution-indicator oligochaete species identified in the fresh water ponds, which reveals that the studied ponds are subjected to pollution.

A random survey of aquatic Oligochaeta was conducted in Chennai, Villupuram, Dindugal, Thiruvallur, and Sivagangai districts of Tamil Nadu from November 2010 to March 2012. Thirteen taxa were identified from a total of 644 aquatic oligochaetes examined from various qualitative samples of aquatic vegetation and sediments. Chennai district had 3 families: Naididae, Pristinidae and Tubificidae along with a polychaete. The naidids were most diverse with 5 species: Branchiodrilus semperi, Aulophorus furcatus, Dero digitata, Dero indica and Dero zeylanica, the pristinids were represented by Pristina brevichaeta and Pristina jenkinae and the tubificids by 2 species, Branchiura sowerbyi and Limnodrilus hoffmeisteri. The nine aquatic oligochaetes collected from Chennai were already reported from this region. Two naidids, Branchiodrilus semperi and D. indica, along with the tubificid Branchiura sowerbyi, were collected from Villupuram district and they constitute the first reports for that district. Limnodrilus udekemianus from Kodaikanal, Dindugal district constitutes the first report for that region of Tamil Nadu. Megadriles belonging to the family Octochaetidae (Dichogaster bolaui?) and unidentified Megascolecidae collected from Thiruvallur and Sivaganga districts respectively were found to occur in aquatic habitats.

Abstract Proliferative gill disease (PGD) is a major problem in cultured channel catfish, Ictalurus punctatus. This parasite requires Dero digitata to complete its life cycle. It is believed potassium permanganate disinfects ponds and reduces D. digitata populations, but this practice has not been verified experimentally. We evaluated potassium permanganate as a pond disinfectant to reduce D. digitata populations before stocking fish. In the first study, 2 L catfish pond mud and 18 L pond water were placed in each of 16 20‐L microcosms. Four microcosms were dosed at each of four potassium permanganate treatment levels (0, 10, 20, and 30 mg/L). After treatment, D. digitata present in the sample were counted. In a second study, benthic populations were compared in 10 0.04 ha ponds before and after treatment with 20 mg/L potassium permanganate. In the microcosm study, all treatment levels significantly (P < 0.05) reduced D. digitata populations relative to controls. However, in the field trial, there were no significant (P < 0.05) differences pre‐ and post‐treatment with potassium permanganate at 20 mg/L in Dero populations or total benthic organism populations. Although we believed such high levels of potassium permanganate would sterilize the pond, the results of the pond treatments in this study indicate otherwise.

Aquatic Oligochaetes community in the Iraqi southern marshes, Al- Hawiezah, Al-Chibayish and Al-Hammar, have been studied for the period from July 2006 to June 2007. Two sites within each marsh, in addition to a reference site located at Shatt Al-Garma, have been chosen for sample collection using Ekman dredge. The results revealed that the highest average annual density was 16800 individual / m2 recorded in Al-Hawiezah marsh, while in Al-Chibayish and al-Hammar marsh the density did not exceed 5111 individual/m2. In contrast, the density of Oligochaetes recorded in Shatt Al-Garma was about 10500 individual / m2. The highest monthly density was recorded in April 2007.The highest local percentage occurrence was 96% recorded in Al-Hawiezah marsh, while the highest average monthly percentage occurrence was recorded in January 2007.The study revealed the presence of 19 Oligochaetes species belonging to 14 genera and 4 families. Naididae was represented by 8 spp.; Tubuficidae 8 spp.; Lumbriculidae 2 spp. and Lumbricidae 1 spp. Tubificid worms formed about 73% of the total count while Naididae formed 15%, Lumbriculidae 8% and Lumbricidae 4%.Among Tubificidae, Limnodrilus was the most abundant genus composed about 35% of L. hoffmeisteri; 11.5% L. claparedianus; 8% L. profundicola and 4.7% L. maumeensis, followed by Tubifex tubifex 23%. Other species were Branchuira sowerbyi formed 15%; 2.8 and 2 % for Peloscolex velutinus and Aulodrilus pigueti respectively. Naididae worms comprised 8 species including 32% of Dero digitata, 24% of Nais variabilis, 14% of Amphichaeta sp. and 11% of each of D.obtuse and Pristina longiseta. However, D. dorsalis represented only 7.5% of the total Naididae. Few individuals of B. sowerbyi and Lumbriculus variegates, with two regenerated parts at the posterior end were also detected in some samples. This case was considered a rare observation within the population of these worms in this particular area.

Aquatic Oligochaetes community in the Iraqi southern marshes, Al- Hawiezah, Al-Chibayish and Al-Hammar, have been studied for the period from July 2006 to June 2007. Two sites within each marsh, in addition to a reference site located at Shatt Al-Garma, have been chosen for sample collection using Ekman dredge. The results revealed that the highest average annual density was 16800 individual / m2 recorded in Al-Hawiezah marsh, while in Al-Chibayish and al-Hammar marsh the density did not exceed 5111 individual/m2. In contrast, the density of Oligochaetes recorded in Shatt Al-Garma was about 10500 individual / m2. The highest monthly density was recorded in April 2007.The highest local percentage occurrence was 96% recorded in Al-Hawiezah marsh, while the highest average monthly percentage occurrence was recorded in January 2007.The study revealed the presence of 19 Oligochaetes species belonging to 14 genera and 4 families. Naididae was represented by 8 spp.; Tubuficidae 8 spp.; Lumbriculidae 2 spp. and Lumbricidae 1 spp. Tubificid worms formed about 73% of the total count while Naididae formed 15%, Lumbriculidae 8% and Lumbricidae 4%.Among Tubificidae, Limnodrilus was the most abundant genus composed about 35% of L. hoffmeisteri; 11.5% L. claparedianus; 8% L. profundicola and 4.7% L. maumeensis, followed by Tubifex tubifex 23%. Other species were Branchuira sowerbyi formed 15%; 2.8 and 2 % for Peloscolex velutinus and Aulodrilus pigueti respectively. Naididae worms comprised 8 species including 32% of Dero digitata, 24% of Nais variabilis, 14% of Amphichaeta sp. and 11% of each of D.obtuse and Pristina longiseta. However, D. dorsalis represented only 7.5% of the total Naididae. Few individuals of B. sowerbyi and Lumbriculus variegates, with two regenerated parts at the posterior end were also detected in some samples. This case was considered a rare observation within the population of these worms in this particular area.

Abstract Proliferative gill disease (PGD) in channel catfish Ictalurus punctatus is caused by the myxozoan parasite Henneguya ictaluri, which uses the freshwater oligochaete Dero digitata as an alternate host. Controlled studies on the transmission of PGD require sustainable D. digitata mass cultures. We developed methods to grow and sustain large D. digitata populations and compared population growth in aerated versus nonaerated containers. White paper towels (2–3‐cm squares) were placed into six 10.7‐L dish pans into which 4 L of autoclaved pond water from commercial channel catfish ponds were then added along with 0.1 g of fish food. Each of the six dish pans was initially stocked with 100 D. digitata and maintained at 22–25°C; three pans received aeration, and three did not. All worms were counted and returned to their respective pans once each week for 5 weeks. To compensate for evaporative loss, autoclaved pond water was added to the pans weekly, and pond water and paper towels were completely changed after 4 weeks. Over 5 weeks, the mean number of worms per pan increased significantly more in the nonaerated pans than in the aerated pans. We have maintained D. digitata mass cultures in our laboratory for over a year, starting with two pans containing 100 worms each. Through routine exchanges of pond water and paper towel squares, these populations have expanded to 16 pans, each supporting 3,000–6,000 worms. We have also removed thousands of worms periodically to give to other researchers or for research in our laboratory. Using methods described here, researchers can maintain D. digitata mass cultures and predict population numbers that will be available at given times for studies on myxozoan life cycles and oligochaete control.Received March 12, 2010; accepted May 24, 2010

Abstract There is recent interest in using smallmouth buffalo Ictiobus bubalus and treatment with potassium permanganate (KMnO4) to control Dero digitata, the aquatic oligochaete host involved in the life cycle of proliferative gill disease. To complement those investigations, we determined the changes in water quality, phytoplankton, and zooplankton resulting from KMnO4 treatment and stocking of smallmouth buffalo into nursery ponds for channel catfish Ictalurus punctatus; we did not evaluate the attendant control effectiveness on Dero spp. Stocking buffalo at a rate of 75/ha in nursery ponds did not cause changes in water quality, nutrients, or zooplankton concentrations. Ponds treated with KMnO4 (applied at 57 g/ha when partially filled to about 30 cm deep) contained higher total dissolved solids (230 mg/L) than untreated ponds (204 mg/L). Phytoplankton and zooplankton concentrations in the KMnO4 ponds increased 2−5 weeks after application compared with the control ponds. The observed changes in KMnO4 ponds may be related to nutrient regeneration through oxidation by KMnO4 or may indicate that potassium is a limiting nutrient for phytoplankton in these ponds. The increase in green algae, diatoms, and copepods would be beneficial to catfish fry culture because these algae blooms are desirable over cyanobacteria blooms and copepods are a preferred food resource of catfish fry.

The freshwater oligochaete, Tubifex tubifex, is a common resident of organic-rich sediments worldwide. Although it is a familiar species to fish enthusiasts, toxicologists and parasitologists, T. tubifex often confounds definitive identification due to the similarity that immature specimens bear to several other common oligochaetes, and given the degree of plasticity of key morphological characters due to environmental conditions and/or age of specimen. To solve this identity crisis, we used a polymerase chain reaction based molecular approach and developed a T. tubifex specific assay with primers that amplify a 192 bp fragment of the internal transcribed spacer region 1 ribosomal DNA. We tested these primers on four T. tubifex mitochondrial genotypes, and on other oligochaete species from nine genera. The primers amplified all specimens identified morphologically as T. tubifex. They did not amplify any other species, including morphologically similar worms possessing hair chaetae (Dero digitata, Ilyodrilus templetoni, Tubifex ignotus or Rhyacodrilus spp.) or other oligochaetes often found with T. tubifex (Lumbriculus variegates, Limnodrilus hoffmeisteri, Stylodrilus heringianus or Trichodrilus sp.). This technique should remove the uncertainties all too often associated with identification of T. tubifex.

Abstract With the use of a polymerase chain reaction assay previously developed for Henneguya ictaluri, the myxozoan parasite associated with proliferative gill disease (PGD), a new technique was developed to detect the actinospore stages in pond water samples and in the aquatic oligochaete host Dero digitata. Samples of water and aquatic oligochaetes were collected from 40 commercial ponds containing channel catfish Ictalurus punctatus. Using a previously reported technique to detect H. ictaluri‐infected gills and this new technique, we found 35 out of the 40 ponds had H. ictaluri‐positive oligochaetes, 32 ponds had H. ictaluri‐positive fish, and 25 ponds had water testing positive for H. ictaluri actinospores. This new technique is not only less time‐consuming and labor‐intensive than current methods, it is also sensitive enough to detect the stages of this parasite in oligochaetes or in the water before channel catfish are infected. A detection method such as this one could be used to determine the time to stock ponds or restock ponds after a PGD outbreak has occurred.

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.

Abstract Ultrastructural analysis has revealed that metanephridia in Dero digitata arise from three nephroblast cells in the frontal epithelium of a septum suggesting its mesodermal origin. Each cell has a fixed developmental destination, one nephroblast cell produces the entire canal part and two cells give rise to the nephrostome. The nephroblast cell nearest to the body wall enlarges and proliferates a first set of canal cells, then one of the two proximally adjacent nephroblast cells differentiates into the envelope of the nephrostome generating the marginal cilia of the opening (mantle cell) and the second one transforms into the anteriormost cell of the funnel, producing a flame of cilia that beats into the canal lumen (flame cell). Thereafter, new canal cells appear, mainly by mitosis of the first cell, enlarging the body of the nephridium whose further differentiation was not analysed. Comparison with other clitellate species suggests a mantle cell (or some marginal cells) and a flame cell (or a central cell) to be special characters of the metanephridium in the stem species of the Clitellata and that, compared to many polychaete species, its early development assumes a special course by a precocious determination and arrangement of nephroblast cells, which, in both groups, probably originate from an identical mesodermal stem cell. Results further indicate that the nonclitellate Aeolosomatidae, by virtue of corresponding nephrostomata, are possibly closer related to the Clitellata.