DNA barcoding reveals Mexican diversity within the freshwater leech genus Helobdella (Annelida: Glossiphoniidae)

We investigated the genetic distances and taxonomic status among species of Helobdella, a genus of non-blood-feeding leeches, based on mitochondrial cytochrome c oxidase subunit I sequences. Sampling included 20 specimens representing nine nominal species collected in 11 states in Mexico as well as previously published sequences of different species of Helobdella from several places. A neighbor-joining tree, as well as identification of diagnostic nucleotides, was used to suggest the presence of seven species of Helobdella in Mexico including potentially two undescribed forms.     

A serological investigation of the diet of Helobdella stagnalis (Hirudinea: Glossiphoniidae) in British lakes

The food of Helobdella stagnalis living in unproductive and productive British lakes has been determined using the precipitin test. Antisera were produced against each of ten potential prey groups. Correction factors were applied to basic data to compensate for differences in leech size and seasonal changes in field temperatures. In both types of lake, Oligochaeta followed by Chironomidae were most heavily utilized, whilst Trichoptera, Cladocera and Tricladida were least favoured. More Mollusca were eaten in productive than unproductive lakes, reflecting availability. In productive lakes, Asellus and Amphipoda together constituted the same proportion (about 25%) of the overall diet as Ephemeroptera and Plecoptera in unproductive lakes. Diet did not vary much during the course of a year, though diversity was lower in winter. Feeding activity varied seasonally being lowest in winter. Prey category utilization by small, medium and large leeches did not differ significantly.     

Egg-feeding in the freshwater piscicolid leech Cystobranchus virginicus (Annelida, Hirudinea)

Abstract. Leeches are common terrestrial and aquatic annelids, parasitizing or feeding on a wide range of host taxa. Here we report evidence for an unusual feeding behavior of egg‐feeding in the piscicolid leech Cystobranchus virginicus. We identified distended specimens of C. virginicus in the nests of at least 4 different fish species: Campostoma anomalum, Moxostoma carinatum, Moxostoma sp. (either M. anisurum and/or M. breviceps), and Nocomis leptocephalus. We collected a total of 41 leeches from the nests of these host species and documented at least 1 leech in 19 of 55 nests (35%), with many sites containing multiple leeches. Individuals of C. virginicus were not identified feeding on any of the 41 adult specimens of Moxostoma spp. or the 635 adult specimens of Nocomis leptocephalus examined, and were never found in the absence of active host spawning (26 sites). These results are consistent with individuals of C. virginicus being an opportunistic or possibly even an obligate egg‐feeder, potentially timing their own reproductive activities with the spawning of their fish hosts. The current distribution of C. virginicus has been expanded to include North Carolina, South Carolina, and Tennessee. The potential for leech species to induce mortality in developing fish eggs could be a concern for fish conservation and merits further investigation.     

The Golden Gate Leech Helobdella californica (Hirudinea: Glossiphoniidae): Occurrence and DNA‐Based Taxonomy of a Species Restricted to San Francisco

Leeches of the genus Helobdella are small brooding annelids that inhabit lakes and streams on every continent, notably in South America. The type species, H. stagnalis L. 1758, occurs in Europe and North America. Here I provide novel observations on the occurrence, morphology, and parental care patterns of the related H. californica, a taxon described in 1988, based on specimens collected in Stow Lake, Golden Gate Park, San Francisco. In 2007, the original H. californica population no longer existed, possibly due to eutrophication of this popular “duck pond”. However, in other, cleaner lakes of the Golden Gate Park dense, stable populations of H. californica were discovered. Between 2007 and 2010 adult individuals were investigated in the laboratory with respect to their pigment patterns and feeding behaviour. The leeches suck the red, haemoglobin‐rich haemolymph from insect larvae (Chironomus sp.) and other small aquatic invertebrates and feed their young attached to their ventral surface. A typical feeding episode is described and documented. In addition, a neighbour‐joining analysis was performed based on a newly acquired DNA sequence of part of the mitochondrial gene cytochrome c oxidase subunit I (CO‐I) for H. californica, combined with other, related leech species. These molecular data corroborate that the “Golden Gate leech” is a separate species and not a colour variant of H. stagnalis. Since, over the past 25 years, H. californica has only been found in the freshwater ecosystems of the Golden Gate Park, it is concluded that this rare leech is a species restricted to San Francisco. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Genome‐wide search for leech antiplatelet proteins in the non‐blood‐feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

The genome of the non‐blood‐feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen‐stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross‐validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e‐values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal‐peptide toward the N‐terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeria‐derived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP‐tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non‐blood‐feeding leech has a hematophagous ancestry.     

The life history and production of the leech Helobdella stagnalis (Hirudinea:Glossiphonidae) in the River Ely, South Wales

SUMMARY. A population of the glossiphonid leech Helobdella stagnalis was studied over a period of 16 months in the organically polluted River Ely, South Wales. There were two generations each year. The overwintering population released young (the spring-brood) in June. The spring-brood in turn produced their own offspring (summer-brood) in August.
Seasonal patterns of mortality were related to flow, temperature and life-cycle stage. Individual growth rates were dependent upon both temperature and life-cycle stage.
Annual population production (P) was 7.56 g wet wt m^-2, mean annual population biomass (B) was 1.43 g wet wt m^-2 and the annual P/B ratio was 5.29.     

Social group size, potential sperm competition and reproductive investment in a hermaphroditic leech, Helobdella papillornata (Euhirudinea: Glossiphoniidae)

Social group size may affect the potential for sperm competition, and this in turn may favour ontogenetic adjustments in testicular mass according to the likely requirements for sperm and spermatophore production. In a number of comparative analyses of testis mass among vertebrate species that differ in mating system or social organization, increasing potential for sperm competition is associated with larger testis size. Intraspecific phenotypic plasticity should be able to produce the same pattern if social group size is heterogenous and reflects differing degrees of average sperm competition, but this intraspecific effect is less well studied. We tested the effect of social groups on both male and female investment in the simultaneously hermaphroditic leech, Helobdella papillornata. Leeches were placed in groups of one, two, four or eight. Sexual investment at the onset of reproductive maturity was quantified as the total testisac volume for male function and total egg volume for female function. We found that testisac volume (statistically adjusted for body size) showed a significant increase with increasing group size. Total egg volume (also adjusted for body size) was unaffected by group size. Our findings indicate adaptive developmental plasticity in male gonad investment in response to the potential for sperm competition.     

Structure of the vector tissue in piscicolid leeches (Annelida, Hirudinea, Rhynchobdellida, Piscicolidae)

Hypodermic insemination occurs in piscicolid leeches (Hirudinea, Rhynchobdellida, Piscicolidae). The spermatophore is implanted in a specialized region of the leech body, the copulatory area. Just beneath the copulatory area, there is a specialized connective tissue (vector tissue) that is considered to guide the sperm toward the ovaries. In this study, we show that the vector tissue in the four species of the genus Piscicola is composed of a mass of cells located directly beneath the copulatory area, and two thin strands extend toward the ovaries. The ultrastructure of the vector tissue has been described for the first time. Four cell types were identified, constructing the vector tissue. The envelope of this tissue is made up of extracellular fibrous matrix and two types of cells: vesicular and flat envelope cells, which are embedded within the matrix. The rest of the tissue is formed of granular and plasmatic cells. Both of these last cell types have prominent cytoplasmic projections, filled with a filamentous material. However, only granular cells have numerous small electron-dense granules in their cytoplasm. The vector tissue was described prior, during and following copulation. Sperm passes within free spaces between the granular and plasmatic cells. Characteristic vector tissue cells also occur within the ovary wall and inside the ovary lumen. This supports earlier data, which postulated that the vector tissue appears to be an outgrowth of the ovary wall.     

Oogenesis in the leech Glossiphonia heteroclita (Annelida, Hirudinea, Glossiphoniidae) II. Vitellogenesis, follicle cell structure and egg shell formation

By the end of previtellogenesis, the oocytes of Glossiphonia heteroclita gradually protrude into the ovary cavity. As a result they lose contact with the ovary cord (which begins to degenerate) and float freely within the hemocoelomic fluid. The oocyte's ooplasm is rich in numerous well-developed Golgi complexes showing high secretory activity, normal and transforming mitochondria, cisternae of rER and vast amounts of ribosomes. The transforming mitochondria become small lipid droplets as vitellogenesis progresses. The oolemma forms microvilli, numerous coated pits and vesicles occur at the base of the microvilli, and the first yolk spheres appear in the peripheral ooplasm. A mixed mechanism of vitellogenesis is suggested. The eggs are covered by a thin vitelline envelope with microvilli projecting through it. The envelope is formed by the oocyte. The vitelline envelope is produced by exocytosis of vesicles containing two kinds of material, one of which is electron-dense and seems not to participate in envelope formation. The cortical ooplasm of fully grown oocytes contains many cytoskeletal elements (F-actin) and numerous membrane-bound vesicles filled with stratified content. Those vesicles probably are cortical granules. The follicle cells surrounding growing oocytes have the following features: (1) they do not lie on a basal lamina; (2) their plasma membrane folds deeply, forming invaginations which eventually seem to form channels throughout their cytoplasm; (3) the plasma membrane facing the ovary lumen is lined with a layer of dense material; and (4) the plasma membrane facing the oocyte forms thin projections which intermingle with the oocyte microvilli. In late oogenesis, the follicle cells detach from the oocytes and degenerate in the ovary lumen.     

Reproductive Behaviour and Parental Care of Helobdella striata (Hirudinea,Glossiphoniidae): a Leech that Feeds its Young

The leech Helobdella striata Kutschera (1985) is a protandrous hermaphrodite. It copulates by hypodermic injection: a spermatophore is attached to the skin of the partner and sperm injected through it. During their male phase, the leeches copulate repeatedly and with different partners. In the female phase, 3 to 7 cocoons, each containing 2 to 20 eggs, are produced from the female gonopore and attached to the ventral side of the parent. The larvae which hatch from these eggs cling to the ventral side of the parent. After they have used up their yolk supply and have developed into juvenile leeches they remain attached to the parent for a further three weeks. Helobdella striata capture Tubifex worms and give them to the young they carry. This access to food allows the juveniles to grow from a size of 1 mm to 6 mm length while being carried and protected by the parent. H. striata reared in isolation produce young; at present it is not known whether they reproduce by parthenogenesis or by self-fertilization. The probable phylogenetic development of parental care patterns in the Hirudinea is reviewed.     

Surface topology and structural integrity of the Theromyzon tessulatum (Annelida: Hirudinea: Glossiphoniidae) cocoon

Cocoons secreted by the aquatic leech Theromyzon tessulatum comprise a tubular, membranous ovoid, sealed at each end by a glue‐like substance, called an operculum. Scanning electron microscopy showed surface features of the T. tessulatum cocoon that included a circuitous bulge, cups that conformed to the shape of embryos, relief folds that radiated from opercula, and asymmetric distributions of protuberances on the upper aspect of the cocoon surface. The structural integrity of the T. tessulatum cocoon was assessed after exposure to a variety of denaturing conditions (e.g., extreme heat, detergents, acids). Although both the fibrous cocoon membrane and opercula were strikingly resilient, the membrane/operculum boundary appeared to be the weakest structural component of the cocoon, consistent with its functional role as an escape hatch for juvenile leeches. The operculum itself was more sensitive to denaturation than the cocoon membrane, and thus was probably the source of a major protein component isolated from the T. tessulatum cocoon (i.e., Tcp; Theromyzon cocoon protein). J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Dystrophin-associated proteins in obliquely striated muscle of the leech Pontobdella muricata (Annelida, Hirudinea)

The distribution of dystrophin-associated proteins (-dystroglycan, -, -, - and -sarcoglycan, -syntrophin and sarcospan) were studied in obliquely striated muscle of the leech Pontobdella muricata. Western blot analysis and immunohistochemical electron microscopy, using various polyclonal antibodies, were employed. Western blot analysis of all of these antibodies showed a single band, with approximately the same molecular weights as similar proteins detected in vertebrate muscles. The immunoelectron microscopy study confirmed specific immunogold labelling in the membrane of muscle cells. Since all dystrophin complex components have similar molecular weights and the same localisation in leech as in vertebrate skeletal muscle, we assume that these proteins have similar properties in leech and vertebrate muscle. The presence of these molecules in annelid muscles, together with a short version of dystrophin (previously described as IDLp-140) is of particular interest since phylogenetic and functional studies on this material could help to shed new light on the role and function of this complex in the muscle membrane.     

Bacterial endosymbionts of Placobdella (Annelida: Hirudinea: Glossiphoniidae): phylogeny,genetic distance,and vertical transmission

Blood-feeding leeches of the genus Placobdella have acquired intracellular alphaproteobacterial endosymbionts of the genus Reichenowia that potentially aid in the production of B vitamins, thereby ameliorating the lack of these essential nutrients in the diet of the hosts. For Placobdella associates, little is still known about the diversity, genetic makeup, and the mode of transmission of bacteria between leeches. We aimed to (i) place the bacterial symbionts in a phylogenetic context, (ii) compare patterns of cladogenesis between the bacteria and hosts to search for evidence of co-speciation, and (iii) explore the mode of bacterial transmission between leech parent and offspring. DNA sequencing of the bacterial 16S rDNA and 23S rDNA loci suggests that, whereas Reichenowia forms a monophyletic group within the alphaproteobacterial family Rhizobiaceae, no evidence for co-speciation between hosts and bacteria can be traced. Attempts at DNA amplification for ovarial tissues were negative for a range of species, but two 16S rDNA sequences retrieved from the testisacs of P. rugosa showed very high similarity with Reichenowia. Although we cannot rule out that this may be a contamination, or a different, potentially free-living species of bacteria, our results may indicate that Reichenowia is transferred from leech parent to offspring via the testisacs.

     

Structure of the germinal vesicle during oogenesis in leech Glossiphonia heteroclita (Annelida, Hirudinea, Rhynchobdellida)

Oogenesis in the glossiphoniid leech Glossiphonia heteroclita (Hirudinea, Rhynchobdellida) is nutrimental, i.e., the growing oocyte is supported by specialized germline cells, the nurse cells. The main function of the nurse cells is to provide oocytes with cell organelles and RNAs (mainly rRNA). However, in studied leech species, irrespective of the nutrimental mode of oogenesis, the germinal vesicle (GV = oocyte nucleus) seems to be very active in rRNA production. As shown in the present study, during early previtellogenesis in the GV the meiotic chromosomes and prominent primary nucleoli occur. In late previtellogenesis the chromosomes condense and occupy a limited space of nucleoplasm in close vicinity to primary nucleolus, forming a karyosome. At the onset of vitellogenesis several prominent extrachromosomal DNA bodies appear in close association with the karyosome. At the same time, the primary nucleolus is no longer visible in the GV. As vitellogenesis proceeds the extrachromosomal DNA bodies undergo fragmentation and numerous spherical, RNA- and AgNOR-positive inclusions occur in the nucleoplasm. They are regarded as multiple nucleoli. Finally, in late oogenesis numerous accessory nuclei are formed in close proximity to the nuclear envelope. They usually contain one dense body, morphologically similar to multiple nucleoli. The amplification of rDNA genes, the occurrence of extrachromosomal DNA bodies, as well as the presence of multiple nucleoli and accessory nuclei are described for the first time in the phylum Annelida.     

Fever in the leech,Nephelopsis obscura (Annelida)

Summary Leeches were placed in a 0.8 m linear temperature gradient from 0°C to 40°C. The temperature selected by the leeches was recorded over 30 min intervals; series of animals then received an injection of either pure sterile water,Escherichia coli endotoxin (LPS) at doses of 0.05, 0.25 and 10 g·g^–1, alcohol, or prostaglandin E₁ (PGE₁) at doses of 2 and 4 g·g^–1. After a latency of about 90 min, the leeches developed a dose-dependent fever in response to LPS. Maximal preferred temperature was near 29.6±1.4 whereas the control was 20.5±0.6°C. PGE₁ also evoked dose-dependent fevers of the same magnitude as LPS (30.0±1.0°C), but without latency. Another series of leeches received an injection of LPS of 10 g·g^–1 while in water containing 1.6 mg acetaminophen per 20 l water. Acetaminophen prevented the development of LPS fever. It is concluded that annelids respond to infection with a behavioral fever analogous to that of vertebrates.     

Observations on the growth and development of the duck leech, Theromyzon tessulatum (Hirudinea: Glossiphoniidae), as a function of feeding

Theromyzon tessulatum were reared individually from hatching until they attained sexual maturity. They were fed at different seasons and cultured at one of two temperature regimes. A series of weight measurements enabled growth curves to be constructed. Observations were also made on the digestive processes. In keeping with at least one other sanguinuous glossiphoniid, a restricted and constant number of blood meals precede reproduction and the hungry and sated stages show contrasting and distinctive behaviour. A notable feature is the great increase in weight after feeding resulting from the absorption of water and its incorporation into the body tissues. It probably enters through the body surface. The role of microorganisms within epithelial cells of certain crop caecae, was also considered.     

Ultrastructure of the anterior salivary gland cells of the giant leech, Haementeria ghilianii (Annelida, Hirudinea)

The giant anterior salivary gland cells from the large mammalian blood-sucking, glossiphoniid leech, Haementeria ghilianii, can be subdivided into three morphologically and functionally distinct regions: 1) a soma, responsible for the synthesis and storage of secretory products; 2) a long cell process, responsible for the storage and intracellular transport of the secretory vesicles; and 3) the site of exocytosis at the process terminal. The giant somata are densely packed with secretory vesicles. Deep plasmalemmal invaginations invade the soma and form an extensive system of extracellular lacunae. The rough endoplasmic reticulum (ER) and the Golgi apparatus are organized in the cell periphery, near the highly branched nucleus, and along the lacunae. The somata taper into long processes extending over several centimeters to the proboscis tip. These contain secretory vesicles through their whole length. In the process periphery, the vesicles are completely ensheathed by a concentric subplasmalemmal smooth ER cisterna. This originates deeply within the soma and extends through the whole cell process to its terminal. The ER provides support for up to several hundred longitudinally oriented microtubules. Secretion occurs at the very tip of the cell processes, each of which terminates at the proboscis tip at the base of a cuticular pore. We found synapses close to the sites of exocytosis, providing morphological evidence for neuronal control of secretion.     

Cell dynamics during cocoon secretion in the aquatic leech, Theromyzon tessulatum (Annelida: Clitellata: Glossiphoniidae)

One distinguishing feature of clitellate annelids is the presence of specialized segments comprising the clitellum, whose primary function is to secrete a cocoon. Using histological analyses, we have documented cell types (I-V) and cellular processes associated with cocoon secretion in the aquatic leech, Theromyzon tessulatum. Our data indicate that the bulk of the cocoon's biomass arises from precursor cells of a single type that hypertrophy and proliferate ∼1 week prior to egg laying, and then differentiate into either of two cell types (i.e., Type II or Type III) depending on their position within the clitellum. Type II cells are concentrated along the lateral edges and venter of the clitellum and secrete alcian blue-staining granules that form opercula (i.e., glue-like material that seals both cocoon ends), while Type III cells populate the dorsal midline and secrete azocarmine-staining granules that build the cocoon wall. Both cell types occupy spaces between deep muscle layers and extend long-neck tubules to the surface epithelium as they fill with granules a few days prior to egg laying. Other cell types appear to make minor contributions to the cocoon (e.g., Type I, Type IV) or have supporting or signaling roles (e.g., Type V). Our observations suggest that post-translational modification (i.e., glycosylation) of the same core protein(s) distinguishes the granules of Type II/III cells, and that the default state of the Type II/III precursor may be evolutionarily linked to secretory cells in basal polychaetes.     

The life-cycle,ecology and host specificity of the freshwater leech Alboglossiphonia polypompholyx (Glossiphoniidae) in Egypt

Alboglossiphonia polypompholyx spends most of its life cycle endoparasitic in the mantle cavity of the snail Bulinus truncatus — possibly its only host. Adult A. polypompholyx leaves the snail about one month before the commencement of egg-laying. Hatching occurred after about 15 d and after brooding on the venter of the parent for 7–10 d, the young briefly become free-living before entering the mantle cavity of B. truncatus. Within the mantle cavity, A. polypompholyx feeds and grows for 16–20 weeks before leaving the snail as adults. In the field and in laboratory experiments, adult leeches left the snails in August/September and January with cocoons produced in October/November and February/March, respectively. Free-living adult leeches do not feed and die 1–2 months after the cessation of brooding.     

Presence of invertebrate dystrophin-like products in obliquely striated muscle of the leech, Pontobdella muricata (Annelida, Hirudinea)

Dystrophin is a 427-kDa cytoskeletal protein, which occurs in scant amounts in vertebrate muscle and nerve cells. No previous references to dystrophin or associated proteins in invertebrates at the protein level have been found, while two recent studies investigated the presence of genes encoding proteins homologous to dystrophin in sea urchin and other invertebrates such as Drosophila melanogaster. In this study, the possible presence and distribution of dystrophin-like proteins were studied in different invertebrate muscle cell types and species through Western blot analysis and light and electron microscope immunohistochemistry using a panel of antibodies whose specificities have been determined in vertebrates. Crude protein extracts of leech Pontobdella muricata were analysed by Western blotting. The revealed protein band, with 140kDa molecular weight, was related to dystrophin, utrophin or dystrophin-related protein-2 (DRP2) according to the specificities of the antibodies used to detect them. The immunofluorescence study showed positive immunoreactions in obliquely striated muscle of this hyrudinean. The immunoelectron microscopy study confirmed specific immunogold labelling beneath the sarcolemma of muscle cells. We thus assume that this protein is an invertebrate dystrophin-like product that is referred to as IDLp140. The potential functions of this invertebrate dystrophin-like protein in invertebrate muscles are discussed relative to previous data in vertebrate tissues.