Species-specific oxylipins and the effects of ontogeny and predation on their emission from freshwater snails.

Chemical cues play important roles in mediating ecological interactions. Oxylipins, oxygenated metabolites of fatty acids, are one signalling molecule type that influences the physiology and function of species, suggesting their broader significance in chemical communication within aquatic systems. Yet, our current understanding of their function is restricted taxonomically and contextually making it difficult to infer their ecological significance. Snails and leeches are ubiquitous in freshwater ecosystems worldwide, yet little is known about their oxylipin profiles and the factors that cause their profiles to change. As snails and leeches differ taxonomically and represent different trophic groups, we postulated oxylipin profile differences. For snails, we hypothesized that ontogeny (non-reproductive vs reproductive) and predation (non-infested vs leech-infested) would affect oxylipin profiles. Oxylipins were characterized from water conditioned with the snail Planorbella duryi and leech Helobdella lineata, and included three treatment types (snails, leeches, and leech-infested snails) with the snails consisting of three size classes: small (5-6 mm, non-reproductive) and medium and large (13-14 and 19-20 mm, reproductive). The two species differed in the composition of their oxylipin profiles both in diversity and amounts. Further, ontogeny and predation affected the diversity of oxylipins emitted by snails. Our experimental profiles of oxylipins show that chemical cues within freshwater systems vary depending upon the species emitting the signals, the developmental stage of the species, as well as from ecological interactions such as predation. We also identified some candidates, like 9-HETE and PGE2, that could be explored more directly for their physiological and ecological roles in freshwater systems.

The genome of medicinal leech (Whitmania pigra) and comparative genomic study for exploration of bioactive ingredients.

BACKGROUND: Leeches are classic annelids that have a huge diversity and are closely related to people, especially medicinal leeches. Medicinal leeches have been widely utilized in medicine based on the pharmacological activities of their bioactive ingredients. Comparative genomic study of these leeches enables us to understand the difference among medicinal leeches and other leeches and facilitates the discovery of bioactive ingredients. RESULTS: In this study, we reported the genome of Whitmania pigra and compared it with Hirudo medicinalis and Helobdella robusta. The assembled genome size of W. pigra is 177 Mbp, close to the estimated genome size. Approximately about 23% of the genome was repetitive. A total of 26,743 protein-coding genes were subsequently predicted. W. pigra have 12346 (46%) and 10295 (38%) orthologous genes with H. medicinalis and H. robusta, respectively. About 20 and 24% genes in W. pigra showed syntenic arrangement with H. medicinalis and H. robusta, respectively, revealed by gene synteny analysis. Furthermore, W. pigra, H. medicinalis and H. robusta expanded different gene families enriched in different biological processes. By inspecting genome distribution and gene structure of hirudin, we identified a new hirudin gene g17108 (hirudin_2) with different cysteine patterns. Finally, we systematically explored and compared the active substances in the genomes of three leech species. The results showed that W. pigra and H. medicinalis exceed H. robusta in both kinds and gene number of active molecules. CONCLUSIONS: This study reported the genome of W. pigra and compared it with other two leeches, which provides an important genome resource and new insight into the exploration and development of bioactive molecules of medicinal leeches.

A tale of two leeches: Toward the understanding of the evolution and development of behavioral neural circuits.

In the animal kingdom, behavioral traits encompass a broad spectrum of biological phenotypes that have critical roles in adaptive evolution, but an EvoDevo approach has not been broadly used to study behavior evolution. Here, we propose that, by integrating two leech model systems, each of which has already attained some success in its respective field, it is possible to take on behavioral traits with an EvoDevo approach. We first identify the developmental changes that may theoretically lead to behavioral evolution and explain why an EvoDevo study of behavior is challenging. Next, we discuss the pros and cons of the two leech model species, Hirudo, a classic model for invertebrate neurobiology, and Helobdella, an emerging model for clitellate developmental biology, as models for behavioral EvoDevo research. Given the limitations of each leech system, neither is particularly strong for behavioral EvoDevo. However, the two leech systems are complementary in their technical accessibilities, and they do exhibit some behavioral similarities and differences. By studying them in parallel and together with additional leech species such as Haementeria, it is possible to explore the different levels of behavioral development and evolution.

Behavioral analysis of substrate texture preference in a leech, Helobdella austinensis.

Leeches in the wild are often found on smooth surfaces, such as vegetation, smooth rocks or human artifacts such as bottles and cans, thus exhibiting what appears to be a "substrate texture preference". Here, we have reproduced this behavior under controlled circumstances, by allowing leeches to step about freely on a range of silicon carbide substrates (sandpaper). To begin to understand the neural mechanisms underlying this texture preference behavior, we have determined relevant parameters of leech behavior both on uniform substrates of varying textures, and in a behavior choice paradigm in which the leech is confronted with a choice between rougher and smoother substrate textures at each step. We tested two non-exclusive mechanisms which could produce substrate texture preference: (1) a Differential Diffusion mechanism, in which a leech is more likely to stop moving on a smooth surface than on a rough one, and (2) a Smoothness Selection mechanism, in which a leech is more likely to attach its front sucker (prerequisite for taking a step) to a smooth surface than to a rough one. We propose that both mechanisms contribute to the texture preference exhibited by leeches.

Spatiotemporal Expression of Anticoagulation Factor Antistasin in Freshwater Leeches.

Antistasin, which was originally discovered in the salivary glands of the Mexican leech Haementeria officinalis, was newly isolated from Helobdella austinensis. To confirm the temporal expression of antistasin during embryogenesis, we carried out semi-quantitative RT-PCR. Hau-antistasin1 was uniquely expressed at stage 4 of the cleavage and was strongly expressed in the late stages of organogenesis, as were other antistasin members. In order to confirm the spatial expression of antistasin, we performed fluorescence in situ hybridization in the late stages of organogenesis. The expression of each antistasin in the proboscis showed a similar pattern and varied in expression in the body. In addition, the spatial expression of antistasin orthologs in different leeches showed the possibility of different function across leech species. Hau-antistasin1 was expressed in the same region as hedgehog, which is a known mediator of signal transduction pathway. Hau-antistasin1 is probably a downstream target of Hedgehog signaling, involved in segment polarity signal pathway.

Leeches Predate on Fast-Escaping and Entangling Blackworms by Spiral Entombment.

We investigate how the Helobdella spp. freshwater leeches capture and consume Lumbriculus variegatus blackworms despite the blackworm's ultrafast helical swimming escape reflex and ability to form large tangled 'blobs'. We describe our discovery of a unique spiral 'entombment' strategy used by these leeches to overcome the blackworms' active and collective defenses. Unlike their approach to less reactive and solitary prey like mollusks, where leeches simply attach and suck, Helobdella leeches employ this spiral entombment strategy specifically adapted for blackworms. Our findings highlight the complex interactions between predator and prey in freshwater ecosystems, providing insights into ecological adaptability and predator-prey dynamics.

Glossiphoniid leeches as a touchstone for studies of development in clitellate annelids.

My goals in this chapter are to share my enthusiasm for studying the biology of leeches, to place this work in context by presenting my rationale for studying non-traditional biological models in general, and to sample just three of the questions that intrigue me in leech biology, namely segmentation, genome evolution and neuronal fate specification. I first became excited about the idea of using leeches as a subject of investigation as an undergraduate in 1970 and have been engaged in this work since I arrived at Berkeley as a postdoc in 1976, intending to study leech neurobiology. Both my research interests and the rationale for the work have expanded greatly since then. What follows is a fragmentary personal and historical account-the interested reader may find more comprehensive treatments elsewhere (Kuo et al., 2020; Shankland & Savage, 1997; Shain, 2009; Weisblat & Huang, 2001; Weisblat & Kuo, 2009, 2014; Weisblat & Winchell, 2020).

Leeches from Mexico City, remnants of the ancient lake.

Genetic barcodes (partial sequences of the mitochondrial cytochrome c oxidase subunit I) were generated for freshwater leeches that inhabit the Mexico Basin, upon which Mexico City and its metropolitan area have developed. Once a continuous lake, the basin has passed through continuous events of artificial desiccation in the last 500 years so that it is currently conformed by a few and highly modified and polluted isolated freshwater bodies. Six species of leeches from three families were collected in five localities. Current sequence databases were able to determine five of the six species collected for this study with the only exception of Haemopis caballeroi, for which no sequence data are available in public repositories. Taxonomic assignment of cocoons was possible via comparison of barcode sequences. We discuss the presence of a population of Erpobdella ochoterenai in Tecocomulco Lake that bares high genetic divergence from its conspecifics, which may indicate it is an undescribed species.

Broad geographic sampling and DNA barcoding do not support the presence of Helobdella stagnalis (Linnaeus, 1758) (Clitellata: Glossiphoniidae) in North America.

The description of Helobdella stagnalis (Linnaeus, 1758) has emphasized the presence of a nuchal, chitinous scute located on the dorsal surface in the first third of the body as the diagnostic character for the species. Historically, identifications of species of Helobdella have relied heavily on this character and, as a result, Helobdella stagnalis has been reported from an inordinately broad geographic range, including Europe, Asia, Africa, North America, and South America. In addition to a few earlier investigations, a recent analysis showed that great genetic distances (orders of magnitude greater than previous estimations of intraspecific divergence in leeches) are present between scute-bearing specimens identified as H. stagnalis from Europe and North America, implying that H. stagnalis does not occur in North America. The present study expands the geographic boundaries of taxon sampling for both European and North American taxa, and re-examines the phylogenetic relationships and cytochrome c oxidase subunit I (COI) variation within scute-bearing species of the genus Helobdella. Our analyses include specimens putatively identified as "Helobdella stagnalis" from Sweden, Norway, Iceland, England, France, Italy, Slovenia, Turkey, Russia, and Iran, as well as numerous localities covering Canada and the USA. Our results corroborate previous studies in that European and west Asian specimens form a clade, including the neotype, which is separate from North American taxa. To alleviate future taxonomic confusion, we redescribe H. stagnalis and designate a neotype from the inferred type locality. The designation of a neotype stabilizes the taxonomy of scute-bearing leeches of the genus Helobdella and enables us to definitively correct erroneous identifications reported in previous studies. We also note that at least four lineages of scute-bearing, North American species of Helobdella lack formal descriptions.

Helobdella blinni sp. n. (Hirudinida, Glossiphoniidae) a new species inhabiting Montezuma Well, Arizona, USA.

A new leech species Helobdella blinnisp. n., is described from Montezuma Well, an isolated travertine spring mound located in central Arizona, USA. In its native habitat, Helobdella blinni had been previously identified as Helobdella stagnalis (Linnaeus, 1758), which was later reclassified to Helobdella modesta (Verrill, 1872). Similar to the European Helobdella stagnalis and North American Helobdella modesta, Helobdella blinni has six pairs of testisacs, five pairs of smooth crop caecae, one lobed pair of posteriorly-directed crop caecae, one pair of eyes, a nuchal scute, and diffuse salivary glands. However, the pigmentation of this new species ranges from light to dark brown, unlike Helobdella modesta which tends to be light grey in color. Also, Helobdella modesta produces a clutch of 12--35 pink eggs, whereas Helobdella blinni produces smaller clutches of white eggs (7-14, 0.5 ± 0.15 mm, N = 7) and consequently broods fewer young (1-14, 7 ± 3.3 mm, N = 97). Helobdella blinni are also able to breed year-round due to the constant warm water conditions in Montezuma Well. Their breeding season is not restricted by seasonal temperatures. These species are morphologically similar, however, comparing the COI mtDNA sequences of Helobdella blinni with sequences from nearby populations of Helobdella modesta and other Helobdella species from GenBank indicate that Helobdella blinni is genetically distinct from these other Helobdella populations.

Leeches of the genus Helobdella as model organisms for Evo-Devo studies.

Model organisms are important tools in modern biology and have been used elucidate mechanism underlying processes, such as development, heredity, neuronal signaling, and phototropism, to name but a few. In this context, the use of model organisms is predicated on uncovering evolutionarily conserved features of biological processes in the expectation that the findings will be applicable to organisms that are either inaccessible or intractable for direct experimentation. For the most part, particular species have been adapted as model organisms because they can be easily reared and manipulated in the laboratory. In contrast, a major goal in the field of evolutionary developmental biology (Evo-Devo) is to identify and elucidate the differences in developmental processes among species associated with the dramatic range of body plans among organisms, and how these differences have emerged over time in various branches of phylogeny. At first glance then, it would appear that the concept of model organisms for Evo-Devo is oxymoronic. In fact, however, laboratory-compatible, experimentally tractable species are of great use for Evo-Devo, subject to the condition that the ensemble of models investigated should reflect the range of taxonomic diversity, and for this purpose glossiphoniid leeches are useful. Four decades ago (1975), leeches of the species-rich genus Helobdella (Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae) were collected in Stow Lake, Golden Gate Park, San Francisco, CA (USA). These and other Helobdella species may be taken as Evo-Devo models of leeches, clitellate annelids, and the super-phylum Lophotrochozoa. Here we depict/discuss the biology/taxonomy of these Evo-Devo systems, and the challenges of identifying species within Helobdella. In addition, we document that H. austinensis has been established as a new model organism that can easily be cultivated in the laboratory. Finally, we provide an updated scheme illustrating the unique germ line/soma-differentiation during early development and speculate on the mechanisms of sympatric speciation in this group of aquatic annelids.

DNA barcodes reveal the presence of the introduced freshwater leech Helobdella europaea in Spain.

Abstract We report the finding of the freshwater leech Helobdella europaea in Spain for the first time. Three leech specimens were found attached to the European pond turtle Emys orbicularis. Helobdella europaea is not a blood feeder and, like all members of the genus, feeds on the hemolymph of aquatic invertebrates including snails and worms. Despite the fact that the original geographical distribution or source population of this species is unknown, the close relationship between H. europaea and leeches of the "triserialis" series (sensu Sawyer, 1986) suggests a New World origin. Given its ability to invade and persist in new environments, this leech has been described as a new species by local taxonomists resulting in some nomenclatural problems. The presence of this introduced organism in Spain may represent serious obstacles to the current efforts to preserve endemic fauna and the potential negative impacts of this species in European environments should be investigated.

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.