Updated inventory and distribution of free-living flatworms from Tunisian waters.

Records of free-living flatworms (turbellarians) from Tunisian waters are scattered. Based on new material and published accounts, an annotated checklist of free-living flatworm species from Tunisian waters is provided. A total of 29 species is recorded, including 18 species with new material and 11 species only from literature records. For each species, information on systematics, habitats and distribution is supplied, together with taxonomic or biological remarks. Three species, the acotylean polyclads Cestoplana rubrocinta Lang, 1884 and Comoplana agilis (Lang, 1884) as well as the marine triclad Cercyra hastata Schmidt, 1861 are recorded for the first time in Tunisia. The controversial occurrence of the leptoplanid polyclad Leptoplana tremellaris in the Mediterranean is supported with histological sections and embryological data. The proseriate Monocelis fusca Örsted, 1843 previously reported from Tunisia is considered to be a misidentification.By presenting a checklist of the recorded species, this work summarizes our current knowledge of the turbellarian fauna diversity in Tunisia, providing baseline data for future biogeographical, ecological, behavioral and evolutionary investigations.

Surface ultrastructure and internal properties of the Erpobdella testacea (Hirudinae, Arynchobdellidae) cocoon membrane.

The Erpobdella testacea cocoon membrane is studied for the first time by transmission and scanning electron microscopy. It has an ovoid form, displays a cambered dorsal side in which various micro-organisms are attached and a flattened ventral side. Symmetrically positioned, 2 opercula occur at the distal ends of the cocoon. The internal ultrastructure reveals fibrils (17.5nm) packed in layers forming C, S, bow shaped, parallel and stippling patterns lines, interrupted by Polygon-shaped cavities (1.8µm). Transverse sections show that each fibril presents an external dark part (6nm) and a central hole approximately 5.06nm in diameter. These features are discussed with bibliographic data signalized for other species. Phylogenetic and functional significations of the cocoon wall structure in leeches are suggested.

Ultrastructure of the ovary and oogenesis in the flatworm Prosthiostomum siphunculus (Polycladida, Cotylea).

Based on light and electron microscopy observations, oogenesis in the cotylean polyclad Prosthiostomum siphunculus was investigated for the first time. The numerous ovarian follicles are dispersed essentially in the dorsal parenchyma. In the follicles, a ventral germinative zone with undifferentiated germs cells of different sizes and a dorsal growth zone with larger growing and abortive oocytes are present. The oogenesis could be subdivided into four stages: (1) Oogonia with a dark nucleus and a dark, ribosome-rich cytoplasm. (2) Early oocyte stage, represented by relatively small cells (10 µm in diameter), a cytoplasm showing some mitochondria and some endoplasmic reticula. (3) Previtellogenic stage, with a decrease of the nucleo-cytoplasmic ratio due to the remarkable increase in ooplasm volume. Immature eggshell globules are observed. (4) Vitellogenic stage, including early vitellogenic ovarian stage, in which a second type of globule (inclusion globule) is formed, and a late vitellogenic uterine stage, in which the inclusion globules are not present anymore. The mature eggshell globules form a peripheral layer under the cell membrane. Eggshell and inclusion globules were analyzed with electron energy loss spectroscopy, electron spectroscopic imaging, protease treatment, and with periodic acid thiocarbohydracide silver proteinate to detect polysaccharides. Chromatoid bodies are present in all four stages. For the first time in a flatworm, we provide evidence that accessory cells, forming a tunica around the ovarian follicles, are epithelial or epithelium-like and likely contribute nutrients for the growth of the oocytes.

Ultrastructure of spermatogenesis and mature spermatozoa in the flatworm Prosthiostomum siphunculus (Polycladida, Cotylea).

This is the first study investigating spermatogenesis and spermatozoan ultrastructure in the polyclad flatworm Prosthiostomum siphunculus. The testes are numerous and scattered as follicles ventrally between the digestive ramifications. Each follicle contains the different stages of sperm differentiation. Spermatocytes and spermatids derive from a spermatogonium and the spermatids remain connected by intercellular bridges. Chromatoid bodies are present in the cytoplasm of spermatogonia up to spermatids. During early spermiogenesis, a differentiation zone appears in the distal part of spermatids. A ring of microtubules extends along the entire sperm shaft just beneath the cell membrane. An intercentriolar body is present and gives rise to two axonemes, each with a 9 + "1" micro-tubular pattern. Development of the spermatid leads to cell elongation and formation of a filiform, mature spermatozoon with two free flagella and with cortical microtubules along the sperm shaft. The flagella exit the sperm shaft at different levels, a finding common for acotyleans, but so far unique for cotylean polyclads. The Golgi complex produces numerous electron-dense bodies of two types and of different sizes. These bodies are located around a perinuclear row of mitochondria. The elongated nucleus extends almost along the entire sperm body. The nucleus is wide in the proximal part and becomes narrow going towards the distal end. Thread-like chromatin mixed with electron-dense intranuclear spindle-shaped bodies are present throughout nucleus. The general sperm ultrastructure, the presence of intranuclear bodies and a second type of cytoplasmic electron-dense bodies may provide characters useful for phylogenetic analysis.

Ultrastructural study of spermatogenesis and sperm in the African medicinal leech Hirudo troctina Johnson, 1816 (Annelida, Hirudinida).

This paper presents the process of spermatogenesis in the leech Hirudo troctina Johnson, 1816 using light, fluorescent and transmission electron microscopy. At the onset of spermatogenesis in testes, the pear-shaped spermatogonia divide mitotically without full cytokinesis and as a result isogenic groups are formed (clusters, clones) with 2, 4, 8, 16, 32, 64, 128 spermatogonia and, finally, 256 primary spermatocytes occur. The final meiotic divisions of spermatocytes give rise to clones with 1024 spermatids. There are hundreds of developing germ-line clones in each testis. In each clone, the male germ cells divide in full synchrony and they are in the same phase of spermatogenesis. During complex spermiogenesis each spermatid becomes a filiform spermatozoon with a helicoid nucleus, which is characterized by the presence of a long acrosome with two regions - anterior and posterior, which are followed by a helicoid nucleus, a midpiece with only one mitochondrion and a long flagellum. Our results were compared to those on other clitellate annelids that have been studied to date, especially to sperm formation in Hirudo medicinalis Linnaeus, 1785. Only minor differences were found in the length and the diameter of different organelles and the number of spermatids in germ-line clones.

Evolutionary history of Chaetognatha inferred from molecular and morphological data: a case study for body plan simplification.

BACKGROUND: Chaetognatha are a phylum of marine carnivorous animals which includes more than 130 extant species. The internal systematics of this group have been intensively debated since it was discovered in the 18(th) century. While they can be traced back to the earlier Cambrian, they are an extraordinarily homogeneous phylum at the morphological level - a fascinating characteristic that puzzled many a scientist who has tried to clarify their taxonomy. Recent studies which have attempted to reconstruct a phylogeny using molecular data have relied on single gene analyses and a somewhat restricted taxon sampling. Here, we present the first large scale phylogenetic study of Chaetognatha based on a combined analysis of nearly the complete ribosomal RNA (rRNA) genes. We use this analysis to infer the evolution of some morphological characters. This work includes 36 extant species, mainly obtained from Tara Oceans Expedition 2009/2012, that represent 16 genera and 6 of the 9 extant families. RESULTS: Cladistic and phenetic analysis of morphological characters, geometric morphometrics and molecular small subunit (SSU rRNA) and large subunit (LSU rRNA) ribosomal genes phylogenies provided new insights into the relationships and the evolutionary history of Chaetognatha. We propose the following clade structure for the phylum: (((Sagittidae, Krohnittidae), Spadellidae), (Eukrohniidae, Heterokrohniidae)), with the Pterosagittidae included in the Sagittidae. The clade (Sagittidae, Krohnittidae) constitutes the monophyletic order of Aphragmophora. Molecular analyses showed that the Phragmophora are paraphyletic. The Ctenodontina/Flabellodontina and Syngonata/Chorismogonata hypotheses are invalidated on the basis of both morphological and molecular data. This new phylogeny also includes resurrected and modified genera within Sagittidae. CONCLUSIONS: The distribution of some morphological characters traditionally used in systematics and for species diagnosis suggests that the diversity in Chaetognatha was produced through a process of mosaic evolution. Moreover, chaetognaths have mostly evolved by simplification of their body plan and their history shows numerous convergent events of losses and reversions. The main morphological novelty observed is the acquisition of a second pair of lateral fins in Sagittidae, which represents an adaptation to the holoplanktonic niche.

Fine structure of the spermatozoon of Perinereis macropus (Claparède, 1870) (polychaeta, Nereidae).

The mature spermatozoa of Perinereis macropus were investigated by transmission electron microscopy. The spermatozoon is composed with a large anterior part (head), a short middle piece and a long flagellum. The head contains a large acrosomal complex with a convex acrosomal vesicle, a subacrosomal space, a fibrillar crown and an acrosomal rod which penetrates into the nucleus invagination. The later is U shaped (in longitudinal section). The short middle piece contains about nine to eleven mitochondria and a centriole associated with the flagellum. This centriole, slightly eccentric to the sperm axis, anchors to the plasma membrane by nine satellite rays of the pericentriolar complex. The axoneme has a "9+2″ arrangement of microtubules. In cross section, the flagellar membrane extends in two lateral protuberances, aligned with the axis formed by the two central microtubules of the axoneme. The spermatozoon of P. macropus conforms to the primitive type with an acrosomal extension. Nevertheless, the acrosome complex ultrastructure shows noticeable modifications from the basic form. This finding agrees with the previously observed reproductive pattern (broadcast spawning - free-swimming larvae), and may be helpful to classify the sperm type of P. macropus.

Distribution pattern and activity of mitochondria during oocyte growth and maturation in the ascidian Styela plicata.

The process of oocyte maturation is underlined by a redistribution of cellular organelles, among which mitochondria play a functional role for the acquisition of fertilization and developmental competence. In this paper, we applied electron and confocal microscopy by using DIOC6 and JC-1 stain to evaluate mitochondria distribution pattern and activity during different stages of oocyte growth in the ascidian Styela plicata. Three categories of oocytes at the germinal vesicle stage underlying the vitellogenic process were characterized on the basis of size, pigmentation and accessory cells. Mitochondria were spread throughout the cytoplasm at the smallest oocyte stage and gradually migrated to the periphery of the subcortical cytoplasm at the intermediate stage. At the fully grown oocyte stage, mitochondria were aggregated in the subcortical cytoplasm. This pattern of polarized mitochondria distribution correlates significantly with an increase in mitochondria potential and activity. In this paper we discuss the relationship of mitochondria to the acquisition of oocyte developmental competence.

Erpobdellid leeches (Annelida, Clitellata, Hirudinida) from Tunisia: new records with the description of a new Trocheta species.

Up to now in Tunisia, freshwater Hirudinida are represented by two mainly haematophagous families: Hirudinidae and Glossiphonidae, and a predatory one: the family Erpobdellidae. The present study provides new information on the diversity and taxonomy of erpobdellid leeches. Identification was based, in addition to morphological data, on the length of sperm ducts and the lengths of ovisacs in relation to the neurosomite (ns) and on the shape and size of the male atrium. Five taxons are found. Two subspecies are reported for the first time in the country: Dina punctata punctata Johansson, 1927 and Dina punctata maroccana Nesemann and Neubert, 1994. Tunisian populations of two species, Erpobdella testacea (Savigny, 1820) and Trocheta africana Nesemann and Neubert, 1994, are described, with records of new localities. The new Trocheta tunisiana n. sp. is discovered and described in detail. Trocheta species live in springs in elevated areas while Erpobdella seem to prefer low altitude reservoirs. A comprehensive comparison of the three genera is presented. The disparity between the actual systematics and phylogeny is discussed. This study gives also a detailed distribution of the five species in the north of Tunisia with notes on ecological preference of the genus Dina. Finally a key for the determination of freshwater erpobdellid species from Tunisia is proposed.

An ultrastructural study of the ovary cord organization and oogenesis in Erpobdella johanssoni (Annelida, Clitellata: Hirudinida).

The aims of the present study were to analyze the ovary cord structure and oogenesis in Erpobdella johanssoni under light, fluorescent and transmission electron microscopy and to compare the obtained results with other clitellate annelids, especially with other arhynchobdellid leeches. Each of the paired ovaries is composed of the ovary wall (ovisac) and several (7-8) short, cone-shaped ovary cords. The ovary cords are of the "Erpobdella" type, i.e. they are short and polarized and five zones containing germ cells at consecutives stages of their development can be distinguished along their long axis. One, huge somatic cell (the apical cell), oogonia and premeiotic germ cells occur at the tip of the apical part of the ovary cord - zone I. Below, in zone II germ cells enter meiosis, whereas in zone III only a few cells continue meiosis and gather nutrients (oocytes), while the rest become nurse cells. In zone IV, huge vitellogenic oocytes form protuberances on the surface of the cord, and degenerating germ cells were observed at the base of the ovary cord (zones IV and V). The germline cells form syncytial cysts in zones I-III. The germline cysts have broadly the same architecture as in the ovaries of all of the clitellate annelids that have been described to date. Each germ cell in a cyst has only one cytoplasmic bridge connecting it to the common cytoplasmic mass - the cytophore. The cytophore is poorly developed, and it has the form of thin, long cytoplasmic strands. The presence of two categories of germ cells suggests a meroistic mode of oogenesis. The germline cysts are closely associated with somatic, follicular cells. There are two subpopulations of follicular cells: one envelops the growing oocytes, while the second is distributed between other germ cells. The entire ovary cord is additionally enveloped by a layer of somatic cells with a spongy appearance - the spongiosa cells. A characteristic feature of vitellogenic oocytes is the condensation of the chromosomes into a karyosome. Fully grown oocytes are excluded from the ovary cords and float freely in the ovisac lumen.

Ultrastructure of spermatogenesis and mature spermatozoon of the freshwater planarian Schmidtea mediterranea (Platyhelminthes, Paludicola).

The aim of this study was to characterize for the first time spermatogenesis and spermiogenesis in the freshwater planarian Schmidtea mediterranea using both light and electron microscopy. Starting from the border towards the testis lumen we found types I and II spermatogonia, clusters of primary and secondary spermatocytes, spermatids and free spermatozoa. Light microscope observations show that type I spermatogonia have a large and pale nucleus whereas type II spermatogonia are significantly smaller than the one of type I, and show a darker and central bulky nucleus. At the ultrastructure level, both type I and type II spermatogonia are characterized by a wide nucleus with scanty cytoplasm containing free ribosomes, mitochondria and a dense chromatoid body whereas endoplasmic reticulum and Golgi complex were not observed. The cytoplasm of primary and secondary spermatocytes displays numerous free ribosomes and many endoplasmic reticulum cisternae and Golgi complexes, suggesting that the development of these organelles during spermatogenesis might contribute to the synthesis of hormones and proteins such as testosterone, transcription factors and tubulin. Mature spermatozoa structure closely resembles those of other freshwater triclads with a nucleus, a single fused mitochondrion, a row of cortical microtubules and a pair of flagella conforming to the 9+'1' microtubule pattern described for other Platyhelminthes.

[An ultrastructural study of oogenesis in the planarian Schmidtea mediterranea (Platyhelminthe, Paludicola)]. / Étude ultrastructurale de l'ovogenèse chez la planaire Schmidtea mediterranea (Plathelminthe, Paludicole).

The ovary of the freshwater planarian Schmidtea mediterranea has been studied for the first time using both light and electron microscopy methods. The ultrastructure of the ovary revealed two types of cells: accessory cells and germinal cells at various stages of differentiation, distributed along a maturation axis. Initially, oogonia underwent cytoplasm growth due to the development of organelles, such as endoplasmic reticulum, Golgi complex, and mitochondria, which are all involved in the production of cytoplasmic inclusions or yolk globules. It is shown that the chromatoid body and fibrogranular aggregates may participate in the synthesis of vitelline inclusions. When completely mature, the oocytes have become larger, due to the accumulation of nutritive inclusions, which are round in shape and have a paracrystalline structure. These inclusions are interpreted as being yolk globules and may represent a kind of nutritive material for the developing embryo. These ultrastructural features of the ovary agree with the available phylogenetic tree, based on morphological and karyological characters that considers Schmidtea group as a genus and not a subgenus. The presence of sperm between the oocytes suggests that fertilization may occur within the ovary, representing an uncommon condition within the Triclads, in which fertilization usually takes places outside of the ovaries.

[Oogenesis of Perinereis macropus Claparede, 1870 (Annelida, Polychaeta) in the Gulf of Gabes (Tunisia)]. / Ovogenèse de Perinereis macropus Claparède 1870 (Annélide, Polychète) dans le golfe de Gabès (Tunisie).

The polychaete Perinereis macropus (Claparède, 1870) (Nereididae) is present in the Gulf of Gabes, but its reproductive biology is unknown. An intensive study was conducted from August 2004 to July 2005 to characterize the life cycle of a population in the mouth of wadi Ferd in Gabes. The examination of sexual products allows us to describe the morphological oocytes aspects at different stages. The results show that P. macropus has an asynchronous oogenesis. Moreover, the biometric study of oocytes growth allows us to clarify the female sexual cycle. The female maturity occurs in April and the mature oocyte diameter is approximately 250+/-32.67 microm. However, spawning occurs in May, when the seawater temperature starts rising. We propose that the reproductive season stretches from March to June in the Gulf of Gabes.