Mehrez Gammoudi

A transcriptomic-phylogenomic analysis of the evolutionary relationships of flatworms

Summary The interrelationships of the flatworms (phylum Platyhelminthes) are poorly resolved despite decades of morphological and molecular phylogenetic studies [1, 2]. The earliest-branching clades (Catenulida, Macrostomorpha, and Polycladida) share spiral cleavage and entolecithal eggs with other lophotrochozoans. Lecithoepitheliata have primitive spiral cleavage but derived ectolecithal eggs. Other orders (Rhabdocoela, Proseriata, Tricladida and relatives, and Bothrioplanida) all have derived ectolecithal eggs but have uncertain affinities to one another. The orders of parasitic Neodermata emerge from an uncertain position from within these ectolecithal classes. To tackle these problems, we have sequenced transcriptomes from 18 flatworms and 5 other metazoan groups. The addition of published data produces an alignment of >107,000 amino acids with less than 28% missing data from 27 flatworm taxa in 11 orders covering all major clades. Our phylogenetic analyses show that Platyhelminthes consist of the two clades Catenulida and Rhabditophora. Within Rhabditophora, we show the earliest-emerging branch is Macrostomorpha, not Polycladida. We show Lecithoepitheliata are not members of Neoophora but are sister group of Polycladida, implying independent origins of the ectolecithal eggs found in Lecithoepitheliata and Neoophora. We resolve Rhabdocoela as the most basally branching euneoophoran taxon. Tricladida, Bothrioplanida, and Neodermata constitute a group that appears to have lost both spiral cleavage and centrosomes. We identify Bothrioplanida as the long-sought closest free-living sister group of the parasitic Neodermata. Among parasitic orders, we show that Cestoda are closer to Trematoda than to Monogenea, rejecting the concept of the Cercomeromorpha. Our results have important implications for understanding the evolution of this major phylum.

Insemination and embryonic development of some Mediterranean polyclad flatworms

We document the specific patterns of egg plates, the numbers and size of embryos per cocoon and the major stages of the spiralian embryonic development in four Mediterranean polyclads. These include the two cotylean species Prosthiostomum siphunculus (Delle Chiaje, 1828) Lang, 1884 and Thysanozoon brocchii (Risso, 1818) Grube, 1840, both with Müllers larvae, the directly developing acotylean Echinoplana celerrima Haswell, 1907 as well as the acotylean Imogine mediterranea (Galleni, 1976) JenningsandNewman, 1996 with Göttes larva. The duration of embryonic development in three polyclads was tested at different temperatures, suggesting a strong decrease in developmental time with higher temperature (17°C instead of 15°C). In P. siphunculus, we also controlled salinity and light conditions, observing slightly faster development at salinity of 3.5%, and no major effect of light on development. In polyclads, insemination can occur by true copulation, dermal impregnation or hypodermic insemination. We used live observations and sagittal serial sections to detect sperm in tissues of the recipient animal. We found that there is no clear relationship between the presence or absence of a penis or Langs vesicle and the mode of insemination, which can vary considerably even between species of the same genus.

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.

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.

Annotations on marine and freshwater leeches (Annelida, Clitellata, Hirudinea) from North Africa

The purpose of this study is to provide information on the leech fauna of North Africa. This overview is based primarily on collected material, data from the literature and material in museum collections. A total of 37 species belonging to 6 families and 19 genera are presently known. Among these species, eight represent new records for North African countries. This report includes the first records for Tunisia of one freshwater leech, Helobdella stagnalis (Linneaus, 1758), and one marine leech, Trachelobdella lubrica (Grube, 1840). Four taxa are reported as new for Algeria: two marine species (Pontobdella muricata Linnaeus, 1758 and Branchellion torpedinis Savigny, 1822) and two freshwater subspecies (Dina punctata punctata Johansson, 1927 and Dina punctata maroccana Nesemann & Neubert, 1994). Pontobdella muricata is reported for the first time from Morocco and Alboglossiphonia iberica Jueg, 2008, previously known only from the Iberian Peninsula, is now reported from Morocco.

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.