Suborders Acotylea and Cotylea (Polycladida): Study on morphological, ecological and reproductive features of some representative species from Tunisian coasts (Mediterranean)

The aim of this work is to provide some important morphological, ecological and reproductive features of 8 polyclad species from Tunisian waters belonging to Acotylea: Echinoplana celerrima Haswell, 1907 , Leptoplana mediterranea (Bock, 1913) , Discocelis tigrina (Blanchard, 1847) and Imogine mediterranea (Galleni, 1976) and Cotylea: Thysanozoon brocchii (Risso, 1818) , Prosthiostomum siphunculus (Delle Chiaje, 1822), Yungia aurantiaca (Delle Chiaje, 1822) and Prostheceraeus moseleyi (Lang, 1884). New data on distribution of some species are added. Moreover, morphological data are provided for the first time in living specimens of D. tigrina . Based on our specimens, we confirm characterization of the two sub-orders Acotylea and Cotylea that have been already made in previous studies. Function of attachment organs in polyclads is discussed. On the other hand, data dealing with associated fauna are offered for all species. The two acotyleans E. celerrima and I. mediterranea were seen to cover their egg plates practicing thereby a parental care. This work could be a baseline for future taxonomic and behavioural investigations.


Introduction
Members of the order Polycladida are known to have a highly branched intestine (Hyman 1951). The subdivision of order in two suborders Acotylea and Cotylea was made by Lang (1884) based on the absence and presence of true ventral sucker behind female genital pore. The word "cotyl'' means sucker (Newman & Cannon 2003). The true sucker is a protuberance consisting of modified epithelium covering a very thin basement membrane and a thick muscular lamella (Prudhoe 1985). The almost 800 described polyclad species (Martín-Durán & Egger 2012) are divided into 450 acotyleans and 350 cotyleans. For identification of polyclads species, external morphology is not sufficient; study of reproductive system is needed (Faubel 1983(Faubel , 1984Prudhoe 1985). The acotyleans families (e.g.: Stylochidae, Planoceridae) are known to be voracious predators of barnacles (Branscomb 1976), and mussels (Galleni et al. 1980;Gammoudi et al. 2017a in press), rock oysters, pearl oysters and giant clams (Newman & Cannon 1993), scallops (Heasman et al. 1998) and edible oysters (Littlewood & Marsbe 1990;Chintala & Kennedy 1993;Jennings & Newman formed by a narrow vagina formed by three section, external vagina or female atrium, median vagina or shell chamber where open several shell glands and internal vagina where are connected separately the two uteri. Ovaries are dispersed mainly in the ventral parenchyma between digestive ramifications. Histological sections of female gonads show a ventral germinative zone containing young oocytes and dorsal growing zone containing growing oocytes (Fig. 3A).
Reproductive behaviour. During animal culture, specimens were seen to cover their egg plates with their bodies. Even after being moved to other area of the same container, they returned again to cover their eggs.

Leptoplana mediterranea (Bock, 1913) Gammoudi et al, 2012
Morphological features. The dorsal surface is dull in coloration, yellowish to brownish. Tentacular eyes and cerebral eyes form two dark clusters (Fig. 1B). Marginal eyes and tentacles are lacking. Pharynx is ruffled and opens in a mouth located almost in the half of ventral surface. A genital pit typical for the genus Leptoplana is located between the two genital pores. It is a kind of wall ventral depression. In histological sections, it appears as epidermal invagination devoid of muscle layers. Reproductive features. The whole copulatory apparatus is located in the posterior part of body. Male copulatory apparatus is provided with interpolated tubular prostatic vesicle with a ventral diverticulum. This vesicle communicates with oval shaped seminal vesicle that receives a common sperm duct (Fig. 3B). The female apparatus is formed by a vagina that extends dorsally then caudally.
Developmental model. The development is of direct type and the juveniles possess twelve eyes distributed in two groups: four anterior pairs and two posterior pairs. The body is flattened dorsoventrally and completely ciliated (Fig. 5A).

Discocelis tigrina (Blanchard, 1847) Lang, 1884
Morphological features. The body is oval shaped with a posterior end more pointed than the anterior. The length of specimens can reach 25 mm. Dorsal face is cream to white. Many small spots of brown color are present throughout the dorsal surface and are more concentrated on the median line. Tentacles are lacking. Tentacular and cerebral eyes form two black clusters (Fig. 1C). Marginal eyes are distributed along the two anterior thirds of body. Observation of whitish transparent ventral face shows a huge ruffled pharynx (Fig.  4A).

Reproductive features.
A pair of uteri and a pair of vasa deferentia are visible by transparency in living specimens extending on either side of the pharynx (Fig. 4A). A common genital pore located in the second fourth of body. Pyriform prostatoid organs provided with muscular wall are seen also by transparency. The female genital apparatus is provided with a horse-shoes shaped Lang's vesicle (Fig. 4A).
Histological sections show the common antrum, the prostatoid organs and the vagina (Fig. 4B). The diagrammatic reconstruction of copulatory apparatus show the male apparatus formed by the penis and prostatoid organs projected in a common antrum. The genital pore is common. The female apparatus is formed by vagina and Lang's vesicle (Fig. 4C) Associated fauna. They were found essentially among oysters Crassostrea gigas (Thunberg, 1793). It is likely that the flatworms are feeding on the bivalves.

Imogine mediterranea (Galleni, 1976) Jennings & Newman, 1996
Morphological features. The body is relatively thick and fleshy. The dorsal face is white containing several brown spots. It is almost the only species that could be handly collected without using a paint brush. Two retractile nuchal tentacles are present (Fig. 1D). Tentacular and cerebral eyes are present. Marginal eyes form a row along the entire body margin. The pharynx is voluminous and opens in a mouth located in the half of body.

Reproductive features.
The copulatory apparatus is located in the last fourth of body. The sperm ducts open into a tripartite anchor shaped seminal vesicle connected to a free prostatic vesicle by a prostatic canal. The ejaculatory duct is narrow and leads to a male genital pore located 0.5 mm anterior to the female pore. The latter leads to a vagina provided with epithelial folds.
Developmental study. The development is indirect and a four-lobed Götte's larva hatchs. It is provided with 3 eyes (Fig. 5B).

Yungia aurantiaca (Delle Chiaje, 1822) Lang, 1884
Morphological features. The length reaches 45 mm. The body is oval shaped. The colour of the dorsal face is orange with presence of white spots dispersed throughout its entirety. Two well-developed marginal tentacles are present (Fig. 1E). The ventral surface is whitish and shows the mouth, the male and female gonopores and the very developed true sucker. The cerebral eyes form two semicircular spots. The tentacular eyes are also present but they are more spaced than the cerebral.
Reproductive features. The male copulatory apparatus show a free prostatic vesicle and elongated seminal vesicle. The vagina is provided with a cement pouch. The two gonopores are separated. A true well developed sucker is present behind female genital pore. It is formed by a protuberance armed with muscular layer and modified epithelium.
According to our observation, the animal used its sucker to attach itself to a substrate fighting against strong action of waves.

Thysanozoon brocchii (Risso, 1818) Grube, 1840
Morphological features. Body is oval. Length can reach 40 mm. Two prominent marginal tentacles are present (Fig. 1F). The pharynx is ruffled and located in the anterior half of body. Dorsal face is provided with papillae (Fig. 1F) and ventral one is smooth. A well developed sucker is located behind the mouth and genital pores. According to our observation, as in Yungia aurantiaca, the animal used this structure to attach itself to a substrate fighting against strong action of waves.

Reproductive features.
This species have two male copulatory apparatus, indeed there exists a pair of seminal vesicles, a pair of vesicles prostatic, a pair of ejaculatory ducts and a pair of male gonopores. Thee latters are located on the same horizontal line. Both of copulatory apparatus are functional as sperm is seen inside both seminal vesicles and vasa deferentia. The prostatic vesicles are of free-type. Each ejaculatory duct leads to a penis papilla armed with a stylet. The testes are usually ventral. The ovaries are located in dorsal parenchyma. Both uteri are filled with eggs. The vagina is oriented anteriorly and is provided with uterine ramifications. A widening of vertical section of vagina surrounded by glands is noted, this is the cement pouch characteristic representatives of the Suborder Cotylea.
Histological sections show a dorsal germinative zone containing young oocytes and ventral growing zone containing growing oocytes (Fig. 3B).

Prostheceraeus moseleyi Lang, 1884
Morphological features. Its general colour is milky white, greyish, speckled with white spots and numerous small dark spots, violet or black slightly more elongated in the middle of the body. The edge of the worm is a bit corrugated and bordered with orange-brown and yellow. Two marginal tentacles are present (Fig. 1G). Two clusters of cerebral eyes are present. Marginal tentacular eyes occur. The ventral surface shows clearly a well developed true sucker characteristic of the suborder Cotylea. The mouth lies in the anterior hind of pharyngeal cavity.

Reproductive features.
Like all cotyleans, the copulatory apparatus is oriented posteriorly. The two vasa deferentia open separately in an elongated seminal vesicle. The ejaculatory canal communicates with a free prostatic vesicle via a short prostatic canal. The prostatic vesicle is provided with a smooth glandular epithelium. It is surrounded by an inner layer of longitudinal fibers muscle and a thicker outer of circular fibers. The ejaculatory canal leads to an unarmed penis papilla. The latter opens at the outside by a gonopore located at the last third of animal body.
The vagina shows a cement pouch characteristic of the Cotylea suborder. It extends vertically and then anteriorly to form the internal vagina. The internal part of vagina is provided with small vesicles serving for storage of eggs called uterine vesicles.

Prosthiostomum siphunculus (Delle Chiaje, 1822) Lang, 1884
Morphological features. The body is elongated rounded anteriorly and pointed posteriorly. The length can reach 28 mm. Dorsal face is smooth and shows clearly the tubular pharynx. The tentacles are lacking (Fig.  1H). Marginal, cerebral and tentacular eyes are present. Ventral face is transparent and shows tubular pharynx, vasa deferentia and uteri. A developed true sucker is present behind genital pores (Fig. 3C).

Reproductive features.
Testes are mainly ventral and ovaries are dispersed dorsally. A pair of free prostatic vesicles of free-type is present. The unique seminal vesicle receives the two prostatic canals and communicates with the ejaculatory duct which leads to a penis papillae armed with a stylet. The two gonopores are separated. The vagina shows a widening, the cement pouch and then curve dorsally where it receives the uteri.
Developmental model. The development is indirect and a ciliated Müller's larva hatchs (Fig. 5C, D). It is provided with eight lobes.
Associated fauna. Specimens were found to be associated with the sea urchin Paracentrotus lividus (Lamarck, 1816).

Discussion
In this work, 8 polyclads species belonging to the two suborders Acotylea and Cotylea were described. Some anatomical data were shown for the first time such as Lang's vesicle and accessory prostatic organs known as prostatoid organs in living specimens of Discocelis tigrina.
New data dealing with distribution of polyclad species in Tunisian waters were provided. D. tigrina and Echinoplana celerrima were signalized respectively from the coasts of Hergla and Monastir for the first time. Monastir station is considered hitherto to be the most southern Tunisian site of polyclads. It has to note that the south eastern coasts of Tunisia still be completely unexplored.
On the other hand, during this study it was possible to detect some differences in morphology, developmental models of acotyleans and cotyleans and then to confirm the observation already made by Lang (1884), Laidlaw (1903), Marcus & Marcus (1966) and more extensively by Faubel (1983Faubel ( , 1984 and Prudhoe (1985).
The main morphological difference between the two suborders is the presence of true sucker 'cotyl' behind female genital pore in Cotylea. With respect to the function of this sucker, it is not yet known (Prudhoe, 1985). According to our observation, the animal used this structure to attach itself to a substrate fighting against strong action of waves. The same behavior was described by Jokiel & Townsley (1974) in Prosthiostomum sp. The author concluded that the ability to withstand a high rate of water movement probably is an adaptation of the worm to wave turbulence as organ of attachment; the animal uses this organ to be attached to a substrate. On the other hand, Jennings (1957) mentioned that the cotylean Cycloporus papillosus was seen attached to the surface of the colony of Botryllus schlosseri by its ventral sucker and extract whole zooids. The same behaviour was signalized by Jokiel & Townsley (1974) in Prosthiostomum sp. which attaches to the coral Montipora verrucosa. Therefore, the true sucker is an organ that can be used also for feeding. The true sucker of cotyleans differs from others pseudosuckers of acotyleans mainly by being armed with muscular layers and then by its localization. It differs from genital pit described in species of genus Leptoplana, L. mediterranea and corrugated surface of E. celerrima which are located between the two genital pores. These two structures are probably implied in copulation process (Gammoudi et al. 2012a) In some Cestoplanidae a sucker could be present behind female genital pore but in the hind most end of the body contrary to cotyleans provided with a sucker located more or less in the middle region of the body (Prudhoe, 1985).
Study of development showed a direct development in L. mediteranea (acotylean) with a twelve eyed juvenile (Fig. 5A) and indirect development in both Imogine mediterranea (acotylean) and Prosthiostomum siphunculus (cotylean) with Götte's larva (Fig. 5B) and Müller's larva (Fig. 5C, D) respectively. Gammoudi et al. (2012b) deduced that the influence of the temperature on the speed of development is connected with the mode of development: in directly developing polyclads, temperature changes have less pronounced effects on developmental time than in indirectly developing polyclads.
At ultrastructural level and based on bibliographic data, studies of male and female reproductive system has shown a dorsal disposition of ovarian germinative zone in the acotyleans Pleioplana atomata and E. celerrima (Liana & Litvaitis 2009;Gammoudi et al. 2016a) whereas this zone is ventral in the cotyleans P. siphunculus (Gammoudi et al. 2016b). The same applies to Hallez (1911) who showed a ventral position of germinative zone in the cotyleans Enterogonimus aureus, Laidlawia trigonopora and Stylochoides albus. Here in our work we confirm this disposition using histological section of ovaries, dorsal position of germinative zone in the acotylean Echinoplana celerrima and ventral in the cotylean Thysanozoon brocchii (Fig. 3A, B). It seems that disposition of ovarian germinative and growing zone is typical for each suborders. On the other hand, ultrastructural investigations of male gamete revealed that in Acotylea, nucleus of spermatozoon is located in posterior part of shaft and flagella are attached to the sperm shaft (Liana & Litvaitis 2007;Liana & Litvaitis 2010a, 2010b whereas in Cotylea, spermatozoon possess elongated nucleus extending almost along the entire sperm body. Flagella are free and are not in close contact with the sperm shaft (Liana & Litvaitis 2007;Gammoudi et al. 2016c;Liana & Litvaitis 2010b).
In this study, we showed that E. celerrima and I. mediterranea covered their egg plates. This parental care has been described previously In E. celerrima (Johnston & Lee 2008;Rawlinson et al. 2008), and in I. mediterranea by Galleni et al. (1980). This strategy has been also shown in other acotyleans (Pearse & Wharton 1938;Rzhepishevskjj 1979;Murina et al. 1995;Merory & Newman 2005;Rwalinson et al. 2008). Rawlinson et al. (2008) considered that this strategy may be used as protection against potential predators.