A new species of Gorgoderina (Digenea: Gorgoderidae) from Rana berlandieri in Los Tuxtlas tropical rainforest, Mexico, with the elucidation of its life cycle.

Species in the genus Gorgoderina Looss, 1902 are parasites of the urinary bladder of amphibians and include around 50 species described globally. Molecular data on species of the genus are scarce, as is the information of their life-cycle patterns. During a survey on the genetic characterization of the frog trematodes in the tropical rain forest of Los Tuxtlas, in the Gulf of Mexico slope of Mexico, specimens of two morphotypes of Gorgoderina were sampled from the Rio Grande leopard frog, Rana berlandieri. One of them represented an undescribed species which is described herein as Gorgoderina rosamondae n. sp., whereas the other one was morphologically very similar to an apparently widely distributed North American species, G. attenuata, which has been previously reported in the same geographical area. Specimens of both morphotypes were sequenced for two nuclear and one mitochondrial genes. Phylogenetic trees corroborated the distinction of the new species, and data on the internal transcribed spacer 2 revealed genetic differences between G. attenuata sequenced from frogs in USA and specimens of Gorgoderina sp. from Los Tuxtlas, indicating the possibility that they also represent an undescribed species. COI sequences showed high genetic divergence values between the new species and Gorgoderina sp. from Los Tuxtlas (8.63-9.99%). Additionally, COI sequences of the larval forms (sporocyst, cercariae and metacercariae) sampled in the same locality from their first and second intermediate hosts (Pisidium sp. and Agriogomphus tumens, respectively) showed conspecificity, and the 3 host life-cycle of the new species was elucidated.

Morphological and molecular characterization of larval digenean trematodes (Parvatrema: Gymnophallidae) and their pathological effects on the clam Leukoma thaca (=Protothaca thaca) (Bivalvia:Veneridae) (Molina, 1782) from northern Chile.

Trematodes are one of the largest taxa of mollusk parasites. The clam Leukoma thaca is an economically exploited bivalve found along the south-eastern Pacific coast of Peru and Chile. This bivalve is parasitized by various unidentified larval stages of digeneans in the mantle, gonads and digestive gland. The aims of this study were to determine and describe the different larval stages of the digeneans based on morphological characteristics, to identify them at the species level by performing molecular analyses, and to evaluate pathologies associated with the parasites of this clam. Individuals of L. thaca were collected in San Jorge Bay (23°S), Chile, between November 2018 and February 2019. Morphological description was carried out using in vivo and fixed specimens, and analyses including histological and scanning electron microscopy were performed. Individuals were also isolated for molecular analysis using nuclear ribosomal internal transcribed spacer 1 (ITS1), including partial subunit 18S rDNA (18S) and small subunit 5.8S gene (5.8S). Morphological characteristics indicated that the metacercaria larval stage belongs to the family Gymnophallidae, genus Parvatrema, which was supported by molecular analysis. Molecular results revealed that metacercaria, sporocysts and cercaria stages found in this clam belong to the same species of Parvatrema (genetic distance 0%), evidencing that this species uses L. thaca as the first and second intermediate host. Pathologies examined in the host were similar in nature to those reported in other gymnophallids in bivalves, but high prevalence of cercariae (20%) in gonads suggested an important castrator effect on the host.

Fine structure of the nervous system of Cercaria parvicaudata Stunkard & Shaw, 1931 (Digenea, Renicolidae).

The biology of free-living and parasitic Platyhelminthes is diverse. Taking into account the widespread prevalence of parasitic flatworms, Digenea is the least studied group regarding the fine structure of nervous system especially of the cercarial life stage. Here, we present a description of the fine structure of central nervous system (CNS) and two types of uniciliate sensory papillae of xiphidiocercaria Cercaria parvicaudata (Microphalloidea, Renicolidae). The present study documents that C. parvicaudata has a complex nervous system that includes a well-developed ganglion with a cortex of perikarya and glia-like sheaths, myelin-like structures within one of the dorsal nerve cords and four types of polarized synapses between neurites. Different types of neurons in the CNS could not be distinguished on ultrastructural level due to high similarity in their fine structure. Shared polarized synapses with high electron density of presynaptic components are numerous in the neuropile and nerve cords of this larva. Within the larval body, we detected specialized "support" processes that relate to different tissues. Some "support" processes are also closely related to the nervous system of C. parvicaudata, where they are considered as glia-like structures. In this case, the fine structure of glia-like "support" cells of C. parvicaudata differs from those described as glia-like cells in adult flatworms. We suggest a wide prevalence of glia-like cells among cercariae, as well as the fact that glia-like structures in digenean nervous systems can develop from various nonneuronal tissues.

Xiphidiocercariae from naturally infected Lymnaea columella (Mollusca, Gastropoda) in urban area: morphology and ultrastructure of the larvae and histological changes in the mollusc host / Xifidiocercárias obtidas em Lymnaea columella (Mollusca, Gastropoda) naturalmente infectadas em área urbana: morfologia e ultraestrutura da larva e alterações histológicas do molusco hospedeiro

Abstract Urban agriculture is very common in the cities, as Rio de Janeiro and adjacencies, being important as economic activity. However, this practice can create appropriate conditions for establishment and maintenance of life cycle of many parasites. Lymneid snails can act as intermediate hosts of many trematodes species, including those that infect humans and wild and domestic. In the present study, the snail Lymnaea columella was collected from an urban watercress plantations and the morphology of cercariae released by them was analyzed by light and scanning electron microscopy. The specimens were identified as xiphidiocercariae, belonging to the Cercariae Ornatae group, of the order Plagiorchiiformes. Histological examination of the digestive gland of the infected snails revealed the presence of developing sporocysts, with hemocitary infiltration and metaplasia in the epithelial tissue.

Resumo Agricultura urbana é muito comum nas cidades, como Rio de Janeiro e adjacências, sendo uma importante atividade econômica de subsistência. No entanto, esta prática pode criar condições adequadas para o estabelecimento e manutenção do ciclo de vida de muitos parasitos. Os moluscos limneídeos podem atuar como hospedeiros intermédios de muitas espécies de trematódeos, incluindo aqueles que infectam humanos e animais selvagens e domésticos. No presente estudo, o molusco Lymnaea columella foi coletado de plantações urbanas de agrião e a morfologia das cercárias liberadas por eles foi analisada por microscopia eletrônica de varredura leve e de varredura. As larvas foram identificadas como xifidiocercárias, pertencentes ao grupo das Cercariae Ornatae, Ordem Plagiorchiiformes. Análises histológicas da glândula digestiva dos moluscos infectados evidenciaram esporocistos, com formação de granuloma e metaplasia do tecido epitelial.

Xiphidiocercariae from naturally infected Lymnaea columella (Mollusca, Gastropoda) in urban area: morphology and ultrastructure of the larvae and histological changes in the mollusc host.

Urban agriculture is very common in the cities, as Rio de Janeiro and adjacencies, being important as economic activity. However, this practice can create appropriate conditions for establishment and maintenance of life cycle of many parasites. Lymneid snails can act as intermediate hosts of many trematodes species, including those that infect humans and wild and domestic. In the present study, the snail Lymnaea columella was collected from an urban watercress plantations and the morphology of cercariae released by them was analyzed by light and scanning electron microscopy. The specimens were identified as xiphidiocercariae, belonging to the Cercariae Ornatae group, of the order Plagiorchiiformes. Histological examination of the digestive gland of the infected snails revealed the presence of developing sporocysts, with hemocitary infiltration and metaplasia in the epithelial tissue.

Reproduction of trematodes in the molluscan host: an ultrastructural study of the germinal mass and brood cavity in daughter rediae of Tristriata anatis Belopolskaia, 1953 (Digenea: Notocotylidae).

This study describes the fine structure of the germinal mass in daughter rediae of Tristriata anatis. The germinal mass consists of undifferentiated cells, germinal cells and supporting cells and contains numerous cercarial embryos up to tail bud stage. Supporting cells and their outgrowths form a tight meshwork of the germinal mass. In its basal part, this meshwork serves as scaffolding for undifferentiated and germinal cells, naked cell aggregates and early germinal balls. More mature embryos are located apically. The hypertrophied supporting tissue appears to be involved in an intensive transport of substances, as indicated by abundant gap junctions between cell outgrowths and numerous pinocytotic vesicles and microtubules in their cytoplasm. Germinal cells contain annulate lamellae and the nuage, typical organelles of animal oocytes. In young rediae containing embryonic cercariae at the tail bud stage, the supporting tissue starts to degenerate in the apical part of the germinal mass, and a primordial brood cavity emerges though it develops fully only in mature rediae containing late embryonic cercariae. An unusual feature of the germinal mass in T. anatis rediae is an enhancement of the embryo brooding function. At the same time, the performance of this function by the brood cavity is reduced. This is the first time such a redistribution of the embryo brooding function between the germinal mass and the brood cavity has been reported.

Egg-Hatching Mechanism of Human Liver Fluke, Opisthorchis viverrini: A Role For Leucine Aminopeptidases From the Snail Host, Bithynia siamensis goniomphalos.

The human liver fluke Opisthorchis viverrini (Platyhelminthes, Trematoda, Digenea) uses snails of the genus Bithynia as first intermediate host. Peculiarly among trematodes, the eggs of O. viverrini hatch within the digestive tract of its snail host. It remains uncertain whether hatching in this species is mediated through mechanical fracture of the eggshell or by digestion with specific digestive enzymes. This study aimed to characterize enzymes with specific inhibitors and factors involved in the hatching activity of O. viverrini eggs. For measuring egg hatching in vivo, 50 O. viverrini mature eggs were fed to individual Bithynia siamensis goniomphalos snails at various temperature conditions for 24 hr. Ex vivo, mature eggs were incubated with crude snail extract and commercial leucine aminopeptidase (LAP). Egg-hatching of O. viverrini was temperature dependent, with optimal hatching occurring at 24-28 C, with a peak of hatching of 93.54% in vivo and 30.55% ex vivo occurring at these temperatures. Ex vivo hatching rates increased to 45.87% under anaerobic conditions at 28 C. Some 22.70% and 16.21% of heat-killed eggs also hatched within the snail digestive tract and snail extract, respectively, indicating that host molecules are involved in the hatching response. Most eggs hatch in the anterior regions of the digestive tract. Hatching was completely inhibited in the presence of bestatin, an inhibitor of LAP, but not in the presence of phosphatase inhibitors. Bestatin inhibition of hatching was reversible. Finally, egg hatching could be induced by addition of a porcine LAP. The results indicate that this digenean utilizes both LAP of the snail host and movement of miracidia for hatching.

Histological description of Cercaria etgesii Shchenkov, 2017 daughter sporocysts (Trematoda: Pleurogenidae): compartmentalization of the brood cavity and germinal mass morphology.

Morphology of the daughter sporocysts of Cercaria etgesii Shchenkov, 2017 (Pleurogenidae) has been described according to conventional histological techniques and subsequent 3D reconstruction with Bitplanelmaris® software. Novel data were obtained concerning the spatial organization of the brood cavity of Pleurogenidae daughter sporocysts. It was shown that the laminated structures of the endocyst are lining the brood cavity and carry out its compartmentalisation by formation of the chambers around developing and disintegrating embryos of cercariae. First description of the germinal mass of the Pleurogenidae daughter sporocysts is given. No mitoses in germinal mass were evident although apparent production of the cercariae embryos by the sporocysts take place. The analogous principle of daughter sporocysts morphofunctional organisation was noted for the studied representatives of closely related taxa of trematodes: Pleurogenidae (C. etgesii) and Microphallidae (Microphallus 'pygmaeus' group).

Ventral concavity and musculature arrangement in notocotylid maritae (Digenea: Notocotylidae).

Maritae of family Notocotylidae have no ventral sucker, an organ that serves for attachment in many other digeneans. Instead, attachment is accomplished through the ventral concavity which is formed by the whole body. Musculature providing such an attachment was previously described only in part, and specific patterns of body-wall and internal muscle arrangement in notocotylids have never been studied before. In this paper we describe muscle system organization in maritae of three notocotylid species by means of fluorescent actin staining and confocal microscopy. A very special U-shaped pattern of body-wall longitudinal muscle fibres is found to support the ventral concavity. Together with very prominent dorsoventral musculature this pattern must play a key role in actions of ventral concavity. Other details concerning the muscle system are provided, including the musculature of ventral protrusions, oral sucker, walls of digestive tract and reproductive organs. We discuss functionality of the musculature and also make comparisons with cercariae of this family.

Musculature arrangement and locomotion in notocotylid cercariae (Digenea: Notocotylidae) from mud snail Ecrobia ventrosa (Montagu, 1803).

Cercariae of digenean family Notocotylidae are characterized by a set of morphological traits which make them easily distinguishable from any other. One of the key features is absence of ventral sucker. This affects basic ways of locomotion and attachment. To understand how these functions are fulfilled we studied musculature arrangement in cercariae of two species by means of fluorescent-phalloidin staining and confocal microscopy. We used Cercaria Notocotylidae sp. No. 11 and 12 Deblock, 1980 from mud snails Ecrobia (=Hydrobia) ventrosa. Information on gross morphology (especially body-tail junction) and basic behavioural patterns of these cercariae is also updated. Major special features of musculature are associated with the ventral concavity: extreme development of dorsoventral muscle fibres and formation of annular arrangement of longitudinal muscle fibres on the ventral side. Additional body-wall and internal muscle bundles in the anterior region are also specific for notocotylid cercariae and seem to play important role in twisting movements during substratum testing. Musculature of dorsal adhesive pockets, oral sucker and tail is also described. These results are discussed in relation to observed locomotory patterns.

Stephanoprora chasanensis n. sp. (Digenea: Echinochasmidae): Morphology, life cycle, and molecular data.

Echinostomatoidea is a large, globally distributed and heterogeneous group of hermaphroditic digeneans that parasite, as adults, vertebrate hosts of all classes. Species of this group have received attention from researchers as they can cause diseases in wildlife and humans. Here we describe the biological and molecular phylogenetic characteristics of Stephanoprora chasanensis n. sp. (Digenea: Echinochasmidae). The life cycle of this fluke was experimentally completed by the use of hosts, i.e. Stenothyra recondite Lindholm, 1929 snail (the 1st intermediate), Rhynchocypris percnurus mantschuricus (Berg, 1907) freshwater fish (the 2nd intermediate) and Gallus gallus chicken (the definitive host). In the adult worms, vitelline follicles were distributed anteriorly to the mid-level of the ventral sucker in our specimens whereas they did not reach the level of anterior testis in other species of Stephanoprora previously reported. Phylogenetic analysis based on 28S rDNA revealed that Stephanoprora and Echinochasmus with 20-22 collar spines grouped together in a single cluster. In addition, we showed that Stephanoprora chasanensis n. sp. was closely related to Echinochasmus milvi Yamaguti, 1939. Cercariae of these two echinostomes commonly have a long tail.

Developmental stages of fish blood flukes, Cardicola forsteri and Cardicola opisthorchis (Trematoda: Aporocotylidae), in their polychaete intermediate hosts collected at Pacific bluefin tuna culture sites in Japan.

Farming of Pacific bluefin tuna (PBT), Thunnus orientalis, is a rapidly growing industry in Japan. Aporocotylid blood flukes of the genus Cardicola comprising C. orientalis, C. opisthorchis and C. forsteri are parasites of economic importance for PBT farming. Recently, terebellid polychaetes have been identified as the intermediate hosts for all these parasites. We collected infected polychaetes, Terebella sp., the intermediate host of C. opisthorchis, from ropes and floats attached to tuna cages in Tsushima, Nagasaki Prefecture, Japan. Also, Neoamphitrite vigintipes (formerly as Amphitrite sp. sensu Shirakashi et al., 2016), the intermediate host of C. forsteri, were collected from culture cages in Kushimoto, Wakayama Prefecture, Japan. The terebellid intermediate hosts harbored the sporocysts and cercariae in their body cavity. Developmental stages of these blood flukes were molecularly identified using species specific PCR primers. In this paper, we describe the cercaria and sporocyst stages of C. opisthorchis and C. forsteri and compare their morphological characteristics among three Cardicola blood flukes infecting PBT. We also discuss phylogenetic relations of the six genera of the terebellid intermediate hosts (Artacama, Lanassa, Longicarpus, Terebella, Nicolea and Neoamphitrite) of blood flukes infecting marine fishes, based on their morphological characters.

Discovery of intermediate hosts for two species of blood flukes Cardicola orientalis and Cardicola forsteri (Trematoda: Aporocotylidae) infecting Pacific bluefin tuna in Japan.

Fish blood flukes (Aporocotylidae) are important pathogens of farmed finfish around the world. Among them, Cardicola spp. infecting farmed tuna are considered to be serious threats to tuna farming and have received tremendous attention. We conducted periodical samplings at a tuna farming site in Japan between January and May, 2015 to determine the life cycle of Cardicola spp. We collected over 4700 terebellid polychaetes from ropes, floats and frames of tuna culture cages and found nearly 400 infected worms. Sporocysts and cercariae found in Nicolea gracilibranchis were genetically identified as Cardicola orientalis by 28S and ITS2 ribosomal DNA sequences. This was the first discovery of the intermediate host for this parasite species. Infection prevalence and the abundance of N. gracilibranchis significantly varied between sampling points and the highest number of infected terebellids were collected from ropes. We also demonstrated morphologically and molecularly that asexual stages found in a single Amphitrite sp. (Terebellidae) and adult worms isolated from farmed juvenile tuna were Cardicola forsteri. This is the first report of C. forsteri in Pacific bluefin tuna (PBT) Thunnus orientalis in Japan. Our results demonstrated that all three species of Cardicola orientalis, C. forsteri and Cardicola opisthorchis exist in Japanese farmed PBTs and that they all use terebellid polychaetes as the intermediate hosts.

New species of Proterometra (Digenea: Azygiidae) and its life cycle in the Chickasawhay River, Mississippi, USA, with supplemental observations of Proterometra autraini.

We describe Proterometra ariasae n. sp. based upon cercariae shed from a freshwater snail, Pleurocera sp., and adults infecting the buccal cavity of longear sunfish, Lepomis megalotis, captured from the Chickasawhay River, Mississippi, USA. We also provide supplemental observations of cercarial and adult paratypes of Proterometra autraini from the Au Train River, Michigan, USA. Sequence data for the ribosomal internal transcribed spacer 2 (ITS2) from adults and cercariae of the new species were identical. Adults of the new species differ from congeners by having (i) a markedly large body, (ii) a proportionally large oral sucker, (iii) ovoid testes, (iv) a strongly muscular and laterally expanded pars prostatica, (v) a uterus that is extensively convoluted between the ovary and ventral sucker (vi) and a vitellarium as long as the caeca and extending posteriad beyond the anterior margin of the testes. Cercariae of the new species differ from those of its congeners by having (i) a tail stem that is shorter than 10mm and that lacks a medial constriction, (ii) obcordate furcae that are wider than long, (iii) mamillae distributed throughout the anterior tail stem only, and (v) a proportionally small distome that has relatively few uterine eggs and remains withdrawn in the anterior tail stem region in actively swimming cercariae. This is the first report of Proterometra from Mississippi, the second description to employ morphology and sequence data to elucidate a life cycle for Proterometra, and the third species of Proterometra from an intermediate host not assigned to Elimia.

Determination and quantification of Schistosoma mansoni cercarial emergence from Biomphalaria glabrata snails.

Living and fixed samples of Schistosoma mansoni -infected Biomphalaria glabrata snails were used to determine the relative contributions of different snail tissues to cercarial emergence (shedding). Three methods of observations were employed: (1) direct microscopical observations of shedding snails; (2) microscopic analysis of 5 µm serial sections (H&E stained) of actively shedding snails; and (3) scanning electron microscopic (SEM) observations of snails that were fixed while actively shedding. For this investigation, there were advantages and disadvantages to using each method. We confirmed the results of others that there were 3 tissues of the snail that contributed most prominently to cercarial release (mantle collar, pseudobranch, and headfoot). Based on histological analysis of cercarial accumulations in presumed shedding sites in these 3 tissues, 57% of the cercariae could be seen in the mantle collar, 30.6% in the pseudobranch, and 12.5% in the headfoot. Other anterior structures were involved to a much lesser extent. SEM observations clearly showed cercariae emerging either body first, tail first, or likely emerging en masse from blebs, especially from the mantle collar. These studies provide a more quantitative appraisal of the role the different anterior snail tissues play in cercarial emergence.

Localization of the cystogenous glands of Opisthorchis viverrini cercariae.

Opisthorchiasis in northeastern Thailand is an important etiology of cholangiocarcinoma. To form the infectious stage, free swimming cercariae penetrate cyprinid fish, shed their tails, and then secret a cystic substance to cover their larval stage to form metacercariae in the fish body. We determined the location of the cystogenous glands in Opisthorchis viverrini cercariae. The cercariae and metacercariae were obtained from the naturally infected snail host, Bithynia siamensis goniomphalos and from cyprinid fish, respectively. The cyst walls of the metacercariae were separated and used to immunize inbred male BALB/c mice to obtain cyst wall antibodies. The general characteristics of the O. viverrini cercariae and metacercariae were studied by hematoxylin and eosin (H&E) staining of sections. The location and ultrastructure of the cystogenous glands of cercariae were studied by immunoperoxidase, immunofluorescence and transmission electron microscopy. The structures and organelles of cercariae and metacercariae could be identified, but the cystogenous glands could not be detected in H&E sections. The immunoperoxidase and immunofluorescence sections revealed positive reactions for cystogenous glands predominanted in the lateral part of the cercariae and were clearly seen in the cyst wall of the metacercariae. The ultrastructure of the cystogenous glands contained semitranslucent electron dense oval shaped granules. If interference occurs during the formation of the cysts by fish immune response, the metacercariae may not develop to maturity. It may be easily digested or degraded by human stomach acid and pepsin. This may be an efficient method for control of O. viverrini infection which requires further detailed study.

Trematode reproduction in the molluscan host: an ultrastructural study of the germinal mass in the rediae of Himasthla elongata (Mehlis, 1831) (Digenea: Echinostomatidae).

The germinal mass in Himasthla elongata rediae was studied in detail using transmission electron microscopy. It was shown to be a specialized reproductive organ consisting of germinal cells at various maturation stages, supporting cells and stem cells. The germinal mass also contains early cercarial embryos emerging as a result of cleavage division of mature germinal cells. The stem cells that give rise to germinal cells have heterochromatin-rich nuclei with distinct nucleoli and scarce cytoplasm containing mainly free ribosomes and few mitochondria. The differentiating germinal cells undergo a growth, which is accompanied by an emergence of annulate lamellae and the nuage in their cytoplasm, a noticeable development of RER and Golgi apparatus and an increase in the number of mitochondria. The mitochondria form a large group at one of the cell poles. During differentiation, the nucleus and nucleolus of the germinal cell enlarge while the chromatin becomes gradually less condensed. The supporting tissue of the germinal mass is made up of cells connected by septate junctions. These supporting cells are distinctly different in cellular shape and nuclear ultrastructure. Their outgrowths form a tight meshwork housing stem cells, germinal cells and early cercarial embryos. The cytoplasm of the supporting cells in the mesh area is separated into fine parallel layers by labyrinthine narrow cavities communicating with the intercellular space. The supporting tissue contains differentiating and degenerating cells which indicates its renewal. The results of this ultrastructural study lend support to the hypothesis that the germinal cells of digeneans are germ line cells.

Insights into the development of Notocotylus attenuatus (Digenea: Notocotylidae) in Lymnaea stagnalis: from mother sporocyst to cercariae.

Notocotylus attenuatus (Digenea: Notocotylidae) is a monostome fluke parasitizing the intestinal caeca of waterfowl that uses an injection apparatus to infect its intermediate snail host. Morphology of the invading larva (a sporocyst), and the intramolluscan larval development of this fluke have not been characterized extensively. In this study, experimental infections of Lymnaea stagnalis using N. attenuatus eggs resulted in the development of sporocysts containing one germ ball or mother redia between 12 and 21 days post exposure (p.e.) within the hepatopancreas. Independent mother rediae and developing daughter rediae were present between day 25 and day 42 p.e. Cercariae, within the body of rediae, were detected 42 days p.e. The development of daughter rediae and cercariae started posteriorly in the body of parent redia and these larvae migrated anteriorly during development towards the birth pore. A cercaria was also observed emerging from the birth pore and released cercariae maturated further within the snail hepatopancreas prior to leaving the snail. The intramolluscan development was completed 45 days p.e. when the first fully formed cercariae were shed into the outer environment. These data detail the fascinating post-embryonic development of N. attenuatus and highlight the intricate nature of larval transitions within its snail host.

[On the validity of the genus Otidilepis Yamaguti, 1959 (Cestoda: Hymenolepididae) and the classification of the rostellar hooks of its type species, O. tetraciS (Cholodkowsky, 1906)].

The type material on Otidilepis tetracis (Cholodkowsky, 1906), the type species of the genus Otidilepis Yamaguti, 1959 is reinvestigated. The validity of the genus Otidilepis is confirmed. It is clearly distinguished from the genus Hispaniolepis Lopez Neyra, 1942 by the form and dimensions of the rostellar hooks as well as by the proportions of the attachment organs of the scolex (diameter of suckers: width of the rostellum). "Orthodiorchoid" type based on the hooks of D. acuminata (Clerc, 1902), type species of the genus Diorchis Clerc, 1903, is introduced. The former "diorchoid" type, based on the hooks of D. americana Ransom, 1909, is renamed in "americanoid". Rostellar hooks of O. tetracis belong to the americanoid type.

Maritrema orensense and Maritrema bonaerense (Digenea: Microphallidae): descriptions, life cycles, and comparative morphometric analyses.

We elucidate the life cycle of Maritrema orensense for the first time and experimentally confirm that of the sympatric Maritrema bonaerense. In Argentinean estuaries, both species parasitize the cochliopid snail Heleobia australis as first intermediate host, the grapsid crabs Neohelice granulata and Cyrtograpsus angulatus as second intermediate hosts, and gulls as definitive hosts. Here, we describe the daughter sporocysts and cercariae of M. orensense and redescribe these stages for M. bonaerense. Sporocysts of M. orensense are shorter, with fewer developed cercariae than M. bonaerense. The cercariae of M. orensense have longer, larger, and more undulating cephalic glands than M. bonaerense. We redescribe metacercariae and adults of both species and compare them with the previous descriptions. Intestinal ceca length, vitellaria shape and extension, and egg size are the most relevant characteristics in metacercariae and adults for differentiating the species. Hence, the detailed morphological description and comparative analyses of morphometrics obtained from natural and experimental infections permit clear differentiation of M. orensense and M. bonaerense at each life stage.