Marine Leech Anticoagulant Diversity and Evolution.

Leeches (Annelida: Hirudinea) possess powerful salivary anticoagulants and, accordingly, are frequently employed in modern, authoritative medicine. Members of the almost exclusively marine family Piscicolidae account for 20% of leech species diversity, and they feed on host groups (e.g., sharks) not encountered by their freshwater and terrestrial counterparts. Moreover, some species of Ozobranchidae feed on endangered marine turtles and have been implicated as potential vectors for the tumor-associated turtle herpesvirus. In spite of their ecological importance and unique host associations, there is a distinct paucity of data regarding the salivary transcriptomes of either of these families. Using next-generation sequencing, we profiled transcribed, putative anticoagulants and other salivary bioactive compounds that have previously been linked to blood feeding from 7 piscicolid species (3 elasmobranch feeders; 4 non-cartilaginous fish feeders) and 1 ozobranchid species (2 samples). In total, 149 putative anticoagulants and bioactive loci were discovered in varying constellations throughout the different samples. The putative anticoagulants showed a broad spectrum of described antagonistic pathways, such as inhibition of factor Xa and platelet aggregation, which likely have similar bioactive roles in marine fish and turtles. A transcript with homology to ohanin, originally isolated from king cobras, was found in Cystobranchus vividus but is otherwise unknown from leeches. Estimation of selection pressures for the putative anticoagulants recovered evidence for both positive and purifying selection along several isolated branches in the gene trees, and positive selection was also estimated for a few select codons in a variety of marine species. Similarly, phylogenetic analyses of the amino acid sequences for several anticoagulants indicated divergent evolution.

Barbronia weberi (Clitellata, Hirudinida, Salifidae) has ovary cords of the Erpobdella type.

The organization of the ovaries in representative of the Salifidae (Hirudinida, Erpobdelliformes) was studied at the ultrastructural level for the first time. Like in other leeches, the ovaries of Barbronia weberi are composed of an outer envelope (i.e., an ovisac made up of two coelomic epithelia, muscle cells, and connective tissue) and several internal units, which are broadly similar to the ovary cords found in representatives of the Erpobdellidae. There are usually 6-8 ovary cords that are twisted or cambered with a narrow apical part and a broader, irregularly shaped distal end in each ovisac of B. weberi. Each ovary cord is built from somatic and germ-line cells and the latter tend to form multicellular cysts that are equipped with a central cytoplasmic core (cytophore). There are two morphologically different subpopulations of germ-line cells: oocytes and more numerous nurse cells. Growing oocytes form protuberances on the ovary cord surface and eventually detach from the cord and float freely in the ovisac lumen, whereas the other components of germ-line cysts (i.e., nurse cells and cytophore) degenerate. It should be pointed out that there is a prominent gradient of germ-cell development along the long axis of the cord. The somatic cells form the ovary cord envelope (the so-called spongiosa cells) and also penetrate the spaces between germ-line cells. Both kinds of the somatic cells, that is, those forming the cord envelope and the somatic cells that are associated with oocytes (follicular cells) have a well-developed system of intercellular channels. Additionally, one prominent somatic cell, the apical cell, was found at the apical tip of each ovary cord. Because the aforementioned features of ovary cords found in B. weberi are very similar (with a few minor exceptions) to the ovary cords that have been described in Erpobdella octoculata and E. johanssoni, we propose the term "ovary cords of the Erpobdella type" for them. Our results support a close phylogenetic relationship between Salifidae and Erpobdellidae.

Biodiversity and ecology of Hirudinea (Annelida) from the Natural Reserve of Isla Martín García, Río de la Plata, Argentina / A Biodiversidade e a Ecologia de Hirudinea (Annelida) da Reserva Natural da Ilha Martín García, na Argentina

The Island of Martin Garcia lies at the confluence of the Uruguay and Paraná Rivers (upper Río de la Plata). This island is an outcrop of the crystalline basement. Due to basalt exploitation the island exhibits several ponds covered by carpets of free-floating macrophytes. Seven major environmental variables were measured: water and air temperature, percentage of oxygen saturation, dissolved oxygen, electrical conductivity, total dissolved solids and pH. Eleven leech species were found, Helobdella striata, H. diploides, H. adiastola and H. hyalina were new records. UPGMA clustering of species based on their occurrence in different ecological conditions revealed three main species groups. Canonical Correspondence Analysis (CCA) explained 97.2 percent of the correlation between species and environmental variables. H. triserialis shows the widest range of tolerance, H. hyalina shows positive relationships to conductivity; H. diploides shows a high correlation with dissolved oxygen, H. striata, H. lineata, and S. similis are negatively correlated with water temperature, and H. simplex is positively correlated with pH. Relationships between the species richness (S) and the sampling sites were negatively correlated with water temperature and positively correlated with dissolved oxygen. Leech biodiversity from the water bodies of Martín García Island, shows a great diversity of species and a wide plasticity regarding the characteristics of the environmental factors considered.

A Ilha Martín García encontra-se na afluência dos Rios Uruguay e Paraná (no Rio de la Plata superior) e constitui um afloramento do maciço cristalino de Brasília. Por causa da exploração do basalto, formaram-se lacunas que apresentam tapetes de vegetação flutuantes e macrófitas enraizadas. Mediram-se sete variáveis físico-químicas da água: temperatura do ar e da água, porcentagem de saturação de oxigênio, oxigênio dissolvido, condutividade elétrica, sólidos totais dissolvidos e pH. Encontraram-se onze espécies de sanguessuga, como Helobdella striata, H. diploides, H. adiastola e H. hyalina que constituem novos registros para a ilha. A análise de agrupamento baseado na ocorrência das espécies em condições ecológicas diferentes revelou três grupos principais. A análise canônica da correspondência sugere que a distribuição das espécies relaciona-se às variáveis ambientais consideradas, 97,2 por cento da correlação entre as espécies e as variáveis se distribui no eixo dois do diagrama de ordenamento. As variáveis com maior flutuação foram a condutividade e o pH. H. triserialis foi a espécie mais tolerante às variáveis ambientais; H. hyalina se relacionou positivamente com a condutividade; H. diploides teve alta correlação com o oxigênio dissolvido; H. striata, H. lineata e Semiscolex similis relacionaram-se negativamente com a temperatura da água e H. simplex, positivamente com o pH. As relações entre a riqueza de espécies (S) e os lugares de amostras foram negativas com a temperatura da água e positivas com o oxigênio dissolvido. Os corpos da água da Ilha Martín García mostram uma grande biodiversidade de espécies de sanguessuga, assim como uma ampla ductilidade com relação aos fatores ambientais considerados.

Sanguijuelas (Hirudinea: Glossiphoniidae) asociadas a Cryphiops caementarius (Decapoda: Palaemonidae) del Río Limarí, Chile / Leeches (Hirudinea: Glossiphoniidae) associated to Cryphiops caementarius (Decapoda: Palaemonidae) of the Limarí River, Chile

Two leech species were found parasitizing the shrimp Cryphiops caementarius from Limarí river, Chile. These ectoparasites can act as population regulators or development inhibitors in their host; however the biological characteristics of this host-parasite interaction are unknown. We analyze the inter-specific differences of the parasitism and its relation with host size and sex using quantitative descriptors. Abundance, prevalence, intensity and range were estimated with respect to host size and sex. The leeches belong to two species of Glossiphonidae: Helobdella triserialis and H. duplicata. Forty-seven percent of the hosts were parasitized by one or both leech species. H. triserialis was found only in the pleopod of the hosts with the highest prevalence values (42%), intensity (2.9) and range (1-11). H. duplicata was found only in the branchial filaments with 7 % prevalence, an intensity of 1.7 and a range of 1-4. The host's total length and sex can explain the variation in leech intensity and infection prevalence. However, when only the host's sex is considered, prevalence only increased with size in female hosts. Morphological characteristics of the females could explain the greater H. triserialis intensity in the base of the pleopod and would be related to a greater abdomen development.

Higher level relationships of leeches (Annelida: Clitellata: Euhirudinea) based on morphology and gene sequences.

The evolutionary patterns of divergence of seven euhirudinean families were investigated by cladistic analysis of 33 euhirudinean species. Oligochaetes, Acanthobdella peledina, and branchiobdellidans were included as outgroup taxa. Cladistic analysis employed 1.8 kb of nuclear 18S ribosomal DNA and 651 bp of mitochondrial cytochrome c oxidase subunit I in addition to morphological data. The use of two molecular data sets, one nuclear gene and one mitochondrial gene, as well as morphological data combined historical information evolving under a variety of different constraints and therefore was less susceptible to the biases that could confound the use of only one type of data. Results suggest that the nuclear 18S rDNA gene yields a meaningful historical signal for determining higher level relationships. The more rapidly evolving CO-I gene was informative for recent or local areas of the evolutionary hypothesis, such as within-family relationships. Analyses combining all data from the three character sets yielded one most-parsimonious tree. Most of the higher taxa in recent leech systematics were well corroborated in the resulting topology. However, these results suggested paraphyly of the order Rhynchobdellida, which contradicts the presence of a proboscis as a synapomorphy. The medicinal leech family Hirudinidae was polyphyletic because Haemadipsidae and Haemopidae each have a hirudinid ancestor. In addition, all but one of the genera within the family Erpobdellidae must be either abandoned or renamed. Unusual findings included compelling evidence of historical plasticity in bloodfeeding behavior, having been lost at least four times in the course of euhirudinean evolution. Biogeographic patterns supported a New World origin for Arhynchobdellida.

Cystobranchus meyeri sp. n. (Hirudinea: Piscicolidae) from Catostomus commersoni Lacépède in North America.

Cystobranchus meyeri sp. n. (Hirudinea: Piscicolidae) is described from the white sucker, Catostomus commersoni Lacépède (Osteichthyes: Catostomidae) from the Mohawk River drainage of eastern New York. Size, details of internal anatomy, and the presence of 8 ocelli on the posterior sucker distinguish it from the 3 other species in North America. A table compares the members of this genus and a key to the species is included. Problems of classification, host records, and host specificity are discussed.