A Review of Sperm Ultrastructural Characters in the Opecoelidae (Digenea) and Their Phylogenetic Implications, with New Data on Peracreadium characis, a Parasite of Diplodus puntazzo in Tunisia.

The spermatozoon ultrastructure of Peracreadium characis (Stossich, 1886) (Digenea: Opecoelidae), an intestinal parasite of the sheephead bream Diplodus puntazzo (Walbaum, 1792) (Sparidae), is described by means of transmission electron microscopy (TEM). The mature spermatozoon possesses two axonemes of the 9+'1' trepaxonematan pattern, an anterior electron-dense material, two mitochondria, a nucleus and parallel cortical microtubules distributed in two bundles. The absence of external ornamentation of the plasma membrane and spine-like bodies are the noteworthy characters that distinguish the spermatozoon of P. characis from those of most opecoelids. In fact, only Helicometra fasciata lacks external ornamentation in the spermatozoon. A comparative study with the remaining opecoelids described so far reveals similarities in the ultrastructural organization of their sperm cells. In addition, the current data on sperm ultrastructure in species of the recognized opecoelid subfamilies are compared, namely the Hamacreadiinae, Helicometrinae, Opecoelinae, Opistholebetinae and Plagioporinae.

Spermatological Characterization of the Cestode Meggittina gerbilli (Cyclophyllidea: Catenotaeniidae), a Parasite of Gerbils, Gerbillus gerbillus and Gerbillus campestris (Rodentia: Muridae) in Tunisia.

Ultrastructural characters of spermiogenesis and the mature spermatozoon of the cestode Meggittina gerbilli (Cyclophyllidea: Catenotaeniidae), a parasite of the Lesser Egyptian gerbil (Gerbillus gerbillus) and the North African gerbil (Gerbillus campestris) (Rodentia: Muridae) in the Djebel Dahar (South of Tunisia), were studied using transmission electron microscopy. The spermiogenesis of M. gerbilli is of Bâ and Marchand's type III, which is mainly characterized by a proximodistal fusion of a single flagellum with a cytoplasmic extension. In this catenotaeniid, the proximal fusion is preceded by a 90° rotation of the flagellum. The spermatozoon is a Levron et al. type VI, which presents a single axoneme with the 9 + '1' trepaxonematan pattern, a periaxonemal sheath, two crest-like bodies, twisted cortical microtubules, and a spiraled nucleus. The obtained results show similarities with the remaining studied catenotaeniids, namely Catenotaenia pusilla and Skrjabinotaenia lobata. The results are compared and discussed according to several characteristics found in the catenotaeniids and other studied cyclophyllideans.

Ultrasound guidance versus anatomical landmarks for subclavian vein catheterization: a prospective study.

BACKGROUND: Several clinical practice guidelines strongly support the use of ultrasound guidance (USG) for internal jugular vein catheterization. The level of evidence concerning the use of USG for subclavian vein (SCV) cannulation remains low. AIM: To compare the effectiveness and safety of USG and anatomical landmarks approaches for cannulation of SCV. METHODS: This was a prospective randomized study. Patients aged over 18 years old who requiring elective central venous catheterization were included. Non-inclusion criteria were thrombosis of the vein or major coagulopathy. All catheterizations were performed by two anaesthesiology residents. Patients were randomized into two groups: ultrasound guidance group (US group) and anatomical landmarks (LM group). The main outcome was the success rate. The secondary outcomes were the first attempt success rate and the incidence of complications. RESULTS: Seventy patients were included (35 in each group). The success rate was higher in US group compared to LM group without statistical significance (100% vs 85.7%; p=0.054). The first attempt success rate was significantly higher in the US group (82.9% vs. 40%; p <10-3). The incidence of mechanical complications was significantly lower in the US group compared to LM group (5.7% vs. 37.1%; p=0,001). CONCLUSION: according to our study, US guidance for SCV catheterization seems to be an interesting alternative to anatomical landmarks approaches.

Ultrastructural and molecular study of the microsporidian Toguebayea baccigeri n. gen., n. sp., a hyperparasite of the digenean trematode Bacciger israelensis (Faustulidae), a parasite of Boops boops (Teleostei, Sparidae).

A new microsporidian Toguebayea baccigeri n. gen., n. sp., hyperparasite of Bacciger israelensis (Digenea, Faustulidae), parasite of Boops boops (Teleostei, Sparidae) is described by means of transmission electron microscopy. The phylogenetic analysis, based on the SSU rDNA gene, places the new species in the clade containing mainly crustacean-infecting microsporidia of the genus Cucumispora, within superclade V (Marinosporidia) sensu Vossbrinck et al., 2014. Mature spores of T. baccigeri are ovoid, uninucleated and measure 2.5 × 1.4 µm. The number of coils of the polar tube is 8-10. The polaroplast is composed of an external lamellar part and an internal vesicular or granular part. The main differences that distinguish the new genus and new species from the closely related microsporidia include hyperparasitism in a digenean host infecting a marine fish, the geographic distribution (coast of Tunisia), presence of one nucleus at all developmental stages, disporoblastic sporogony, and the absence of sporophorous vacuoles.

TITLE: Étude ultrastructurale et moléculaire de la microsporidie Toguebayea baccigeri n. gen., n. sp., hyperparasite du trématode digène Bacciger israelensis (Faustulidae), parasite de Boops boops (Teleostei, Sparidae). ABSTRACT: Une nouvelle microsporidie Toguebayea baccigeri n. gen., n. sp., hyperparasite de Bacciger israelensis (Digenea, Faustulidae), parasite de Boops boops (Teleostei, Sparidae) est décrite en microscopie électronique à transmission. L'analyse phylogénétique, basée sur l'étude du gène SSU rDNA, place la nouvelle espèce dans le clade qui contient notamment les microsporidies parasites de crustacés du genre Cucumispora, dans le superclade V (Marinosporidia) sensu Vossbrinck et al, 2014. Les spores mûres de T. baccigeri sont ovoïdes, uninucléées et mesurent 2,5 × 1,4 µm. Le nombre de tours de spire décrits par le tube polaire est de 8 à 10. Le polaroplaste est composé d'une partie externe lamellaire et d'une partie interne vésiculeuse ou granuleuse. Les principales différences entre le nouveau genre et la nouvelle espèce et les microsporidies les plus proches sont : l'hyperparasitisme d'un digène parasite d'un poisson marin, la localisation géographique (côtes de la Tunisie), et certains aspects du développement, notamment la présence d'un seul noyau à tous les stades de développement, la sporogonie disporoblastique et l'absence de vacuoles sporophores.

Spermatological characters in the Lepocreadioidea, with first data on Holorchis pycnoporus (Aephnidiogenidae), a parasite of the Striped seabream Lithognathus mormyrus (Sparidae) from the Gulf of Gabes (Tunisia).

The ultrastructural characteristics of the mature spermatozoon of Holorchis pycnoporus (Digenea, Lepocreadioidea, Aephnidiogenidae) are described by means of transmission electron microscopy (TEM). Live worms were collected from the digestive tract of the Striped seabream Lithognathus mormyrus (Teleostei, Sparidae), off the Gulf of Gabès at La Chebba (Tunisia). The ultrastructural study reveals that the male gamete of H. pycnoporus is a filiform cell tapered at both extremities and exhibiting the type III of the digenean spermatozoon proposed by Bakhoum et al. (2017a), characterized by the presence of (1) two axonemes with the 9 + '1' pattern of the Trepaxonemata, (2) external ornamentation of the plasma membrane located in a posterior part of the anterior region of the spermatozoon and associated with cortical microtubules, (3) two bundles of parallel cortical microtubules with maximum number located in the middle part of the spermatozoon, and (4) generally two mitochondria. Moreover, H. pycnoporus shares a set of ultrastructural characteristics with the studied Aephnidiogenidae such as: (1) two 9+'1' axonemes of different lengths, (2) an anterior electron-dense material, (3) mitochondrion/a, (4) an external ornamentation of the plasma membrane associated with cortical microtubules, and (5) two bundles of parallel cortical microtubules with their maximum number (around 24 microtubules) located in the middle or posterior part of the spermatozoon. In the Aephnidiogenidae, the mature spermatozoon exhibits a similar ultrastructural pattern. Some differences are observed, particularly the location of maximum number of cortical microtubules and the number of mitochondria. The presence of the anterolateral electron-dense material is the major particularity in species belonging to the Lepocreadioidea. This anterior dense material could be a synapomorphy for the superfamily and an ultrastructural argument supporting the monophyletic status of the Lepocreadioidea (Bray and Cribb, 2012).

Sperm characters in the Hemiuridae (Digenea): first data on Aphanurus stossichii (Aphanurinae) and Ectenurus lepidus (Dinurinae).

The present work provides the first ultrastructural analysis of spermatozoa of two digeneans (Aphanurus stossichii (Monticelli, 1891) and Ectenurus lepidus Looss, 1907) belonging to the unexplored subfamilies of the Hemiuridae, namely, the Aphanurinae and the Dinurinae. In March 2019, these hemiurids were collected respectively from the digestive tract of the bogue Boops boops (Teleostei, Sparidae) and the Atlantic horse mackerel Trachurus trachurus (Teleostei, Carangidae) captured in the coastal zone of the Mediterranean Sea, off La Chebba (Tunisia). The ultrastructural study reveals that both spermatozoa exhibit the Bakhoum et al.'s type II of the digenean sperm cells characterized by the presence of two 9+'1' axonemes, an external ornamentation of the plasma membrane not associated with cortical microtubules and located in the anterior part of the spermatozoon, a single bundle of cortical microtubules, the maximum number of cortical microtubules located in a middle part of the sperm cell, and one mitochondrion. Moreover, they share several ultrastructural features with the studied spermatozoa of Hemiuridae such as the presence of two axonemes with the 9+'1' trepaxonematan pattern, a reduced number of parallel cortical microtubules organized into one field with their maximum number located in the median (A. stossichii) or posterior (E. lepidus) part of the spermatozoon, an external ornamentation of the plasma membrane in the anterior part of the spermatozoon, one mitochondrion, a nucleus, and a small amount of glycogen granules. However, the two studied hemiurids could be distinguished by the morphology of the anterior and posterior spermatozoon extremities and the presence of mitochondrial matrix granules in A. stossichii.

Ultrastructural organisation of the spermatozoon of Allopodocotyle tunisiensis Derbel and Neifar, 2009 (Digenea, Opecoelidae), an intestinal parasite of Solea aegyptiaca Chabanaud, 1927 (Teleostei, Soleidae).

The ultrastructure of the spermatozoon of Allopodocotyle tunisiensis (Digenea, Opecoelidae), an intestinal parasite of Solea aegyptiaca (Teleostei, Soleidae), is described by transmission electron microscopy (TEM). The mature spermatozoon is a filiform cell that exhibits two axonemes of different length with the 9+'1' pattern of trepaxonematan Platyhelminthes. In the anterior spermatozoon extremity, cortical microtubules are absent. They appear after the disappearance of an anterior electron-dense material, being initially in a continuous and submembranous layer. They surround only partially the sperm cell. Later, these cortical microtubules are distributed into two bundles. Additionally, the spermatozoon of A. tunisiensis shows two mitochondria, a nucleus, an external ornamentation of the plasma membrane, spine-like bodies, and a large amount of glycogen granules. According to the location of the external ornamentation, A. tunisiensis presents a Quilichini et al.'s type 2 spermatozoon. With respect to the posterior extremity, the sperm cell of A. tunisiensis corresponds to the Quilichini et al.'s opecoelid type. The morphology of the first mitochondrion with a U-shaped posterior extremity is described for the first time in a digenean spermatozoon.

Functional ultrastructure and cytochemistry of vitellogenesis and mature vitellocytes of the digenean Cainocreadium labracis (Dujardin, 1845), parasite of Dicentrarchus labrax (L., 1758).

Vitellogenesis and vitellocytes of Cainocreadium labracis were studied by transmission electron microscopy (TEM) and TEM cytochemistry. Four developmental stages were distinguished during vitellogenesis: (I) stem cell of high nucleo-cytoplasmic ratio; (II) early differentiation with chief activity focused on the beginning of protein synthesis and shell globule formation; (III) advanced differentiation with rapid intensification of protein synthesis, progressive fusion of single shell globules into large globule clusters, and formation of unsaturated lipid droplets surrounded by ß-glycogen particles; and (IV) mature vitellocyte. Early vitellogenesis with vitellocyte maturation consists of: (1) increase in cell volume; (2) increased development of large, parallel cisternae of GER with production of proteinaceous granules; (3) development of small Golgi complexes that package granules; and (4) within vacuoles, progressive enlargement of proteinaceous granules into shell globule clusters formed during vitellogenesis. Three types of inclusions accumulate in large amounts in mature vitelline cells: (1) shell globule clusters, important component in the formation of egg shell; (2) numerous unsaturated lipid droplets. Though fewer, there are also diphasic droplets consisting of saturated and unsaturated lipids in the same droplet, and (3) a relatively small amount of ß-glycogen particles, usually surround a few groups of lipid droplets. The ß-glycogen and lipid droplets are nutritive reserves for embryogenesis. General pattern and functional ultrastructure of vitellogenesis greatly resemble those observed in some lower cestodes, such as bothriocephalideans and diphyllobothrideans. Variations and differences in the amount of lipids and of glycogen during vitellogenesis in lower cestodes and other trematodes are compared and discussed.

Ultrastructure of the spermatozoon of Macvicaria obovata (Digenea: Opecoelidae), a parasite of Sparus aurata(Pisces: Teleostei) from the Gulf of Gabès, Mediterranean Sea.

The ultrastructural organization of the spermatozoon of the digenean Macvicaria obovata (Opecoelidae) is described by transmission electron microscopy. Alive digeneans were collected from the digestive tract of Sparus aurata (Teleostei, Sparidae), caught from the Gulf of Gabès in Chebba, Tunisia (Eastern Mediterranean Sea). The male gamete of M. obovata is a filiform cell, tapered at both extremities and exhibits typical characters such as two axonemes of different lengths showing the 9+'1' trepaxonematan pattern, a nucleus, mitochondria, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. Cortical microtubules are absent in the anterior spermatozoon extremity and appear after the disappearance of the electron-dense material. The first mitochondrion is of moniliform type and it is associated with the external ornamentation of the plasma membrane and spine-like bodies. The second mitochondrion is more posteriorly located, reaching the nuclear region. The present study provides new data on the mature male gamete of M. obovata that may be useful for the understanding of digenean relationships and phylogenetic studies.

Total mercury and selenium concentrations in Sarpa salpa and Balistes capriscus and in their respective Digenean endoparasites Robphildollfusium fractum and Neoapocreadium chabaudi from Tunisia.

The present study reports the levels of mercury and selenium in Sarpa salpa and Balistes capriscus collected along the coast of Mahdia and Sfax (Tunisia). The systems constituted by S. salpa and Robphildollfusium fractum and by B. capriscus and Neoapocreadium chabaudi were tested as potential bioindicators to monitor environmental Hg pollution in marine ecosystems. Mercury and selenium concentrations were assessed in kidney, liver and muscle of 51 S. salpa and of 45 B. capriscus as well as in their respective endoparasites R. fractum and N. chabaudi. The Se:Hg molar ratios were evaluated for both species across the study areas. Surprisingly, the Se:Hg molar ratio in B. capriscus muscle from Mahdia is significantly lower than in Sfax. Our results indicate that some parasites may also be implicated in the amount of Se and Hg available in tissues and therefore contribute to oscillations of the Se:Hg molar ratios. In the model involving the carnivorous species (B. capriscus), the 5.1-times higher levels of mercury in N. chabaudi than in B. capriscus muscle in Sfax enable this fluke to be a sensitive biomonitoring tool for Hg pollution. The present results confirm that the habitual consumption of S. salpa should not suppose any potential health risk for Tunisian people. On the other hand, the consumption of B. capriscus may be of concern and further monitoring is advisable, since the Hg average concentration in Mahdia was above the maximum allowed Hg concentration in the edible portion of fish fixed by the European Union.

Ultrastructural characters of the spermatozoon of the digenean Hypocreadium caputvadum Kacem et al., 2011 (Lepocreadioidea: Lepocreadiidae), an intestinal parasite of Balistes capriscus in Tunisia.

The ultrastructural organization of the spermatozoon of the digenean Hypocreadium caputvadum (Lepocreadioidea: Lepocreadiidae) is described. Live digeneans were collected from Balistes capriscus (Teleostei: Balistidae) from the Gulf of Gabès, Tunisia (Eastern Mediterranean Sea). The mature spermatozoon of H. caputvadum shows several ultrastructural characters such as two axonemes of different lengths exhibiting the classical 9+"1" trepaxonematan pattern, a nucleus, two mitochondria, granules of glycogen, external ornamentation of the plasma membrane and two bundles of parallel cortical microtubules. Moreover, in the anterior extremity, the second axoneme is partly surrounded by a discontinuous and submembranous layer of electron-dense material. Our study provides new data on the spermatozoon of H. caputvadum in order to improve the understanding of phylogenetic relationships in the Digenea, particularly in the superfamily Lepocreadioidea. In this context, the electron-dense material surrounding one of the axonemes in the anterior spermatozoon extremity constitutes the unique distinguishing ultrastructural character of lepocreadioideans, and it is present in spermatozoa of lepocreadiids, aephnidiogenids and gyliauchenids.