Three new species of Carcinonemertes (Nemertea, Carcinonemertidae) from the southeastern coast of Brazil

Three new species of Carcinonemertes from the southeastern coast of Brazil are described: Carcinonemertes divae new species, Carcinonemertes caissarum new species and Carcinonemertes sebastianensis new species. They were found, respectively, associated with the crabs Libinia spinosa, Hepatus pudibundus and Menippe nodifrons, each a newly recorded host for Carcinonemertes. Characters not previously used to describe members of the family Carcinonemertidae, such as distance from ovaries to tip of head, distance from brain to tip of head and distance from stylet to tip of head are included in the descriptions of the new species and are discussed. The locations of these new carcinonemertid worms in their respective hosts are presented in detail and a novelty regarding the infestation site is registered.


Introduction
Nemertean worms of the family Carcinonemertidae are symbiotic egg predators of many decapod crustaceans. Owing to their life cycle, intimacy and use of chemically mediated cues from their hosts, their biology is effectively akin to parasitism; their ecological impact, however, is that of a predator because they kill individual embryos (Kuris 1993;Torchin et al. 1996).
Crabs were transported to the nearby laboratory facilities at the Centro de Biologia Marinha, Universidade de São Paulo, São Sebastião, where they were kept alive in tanks with a flow-through seawater system until dissection. Identification of crabs followed Melo (1996).
The exoskeleton surface and the arthrodial membranes of the crabs were macroscopically examined for nemerteans. The dorsal carapace was removed to expose the branchial chambers. Gills and pleopods were removed with the aid of forceps and were examined under a dissecting microscope.
The nemerteans were collected from crabs and placed in Petri dishes filled with seawater until the moment of taking measurements, pictures and notes on characters. Nemerteans were relaxed in a 1:1 solution of 7.5% MgCl 2 (prepared with bottled drinking water) and seawater for 15-30 min, after which, length and width of body were determined with the aid of an ocular micrometer in a dissecting microscope. Measurements of internal features were made with the aid of an ocular micrometer in a compound microscope after covering the worms with a coverslip. Photomicrographs were made with a Canon PowerShot A10 digital camera. Holotype and paratypes of each new species are deposited at the Museu de Zoologia da Universidade de São Paulo, Brazil (Abbreviation: MZUSP). Some mucus sheaths produced by Carcinonemertes sebastianensis n. sp were prepared for scanning electron microscopy to obtain more details on their morphology. The mucus sheaths were cleaned in a 1:1 solution of 7.5% MgCl2 (prepared with bottled drinking water) and seawater and were fixed in a 10% seawater formalin solution. Mucus sheaths were dehydrated in a series of graded ethanol (50, 70, 95, 100 and 100% for 10 min each), dried by the critical point method with CO 2 and coated with gold, then examined with a JEOL 6400 Visions scanning electron microscope.

Systematics
Family Carcinonemertidae Sumner, Osburn & Cole, 1913 Diagnosis Modified by Shields et al. (1989) from Humes (1942): Monostiliferous hoplonemerteans living as symbionts (egg predators) on the gills, under the abdomen, on the apodemes, and axillae, and in or on the egg masses of decapod crustaceans. Short proboscis, reaching scarcely beyond the posterior end of the muscular portion of the esophagus. Lateral nerves lie internal to the well-developed submuscular glands. Cephalic glands well developed, with cephalic muscle fibers present. Cerebral organs lacking. Takakura's duct system present in males. Internal fertilization and oviparity occur commonly. In most species, adult worms occupy, at least temporarily, mucus sheaths secreted and attached to the setae on the pleopods and hairs of endopodites of ovigerous decapods. Embryos hatch as hoplonemertean larvae.

Diagnosis
From Coe (1902) and amended here (italic): Nemerteans living as symbionts (egg predators) on various species of Crustacea. Proboscis but little developed, very small in size, and extremely short, without lateral pouches of reserve stylets, but armed with central stylet and basis only; anterior proboscis very short, without distinct muscular layers, without distinct nerves, and without a thickened glandular epithelium. Cerebral organs lacking. Two ocelli. Cephalic glands massively developed. Usually oviparous, though fertilization often takes place internally.

Etymology
The species name is a noun in the genitive singular and honors Dr Diva D. Corrêa, a Brazilian nemertean specialist, formerly from the Universidade de São Paulo, who dedicated almost 40 years of her life to the study of nemerteans and greatly contributed to the knowledge of this phylum.

Description
The description is based on living adults and one larva. The latter was obtained from crabs collected at Anchovas Beach. Measurements are given as mean¡SE (range, number of specimens observed).
Female. Body color varied from translucent white to orange; gut orange; gonads translucent white. Two eyes, black, irregular, circular or elliptical; elliptical eyes the most common shape. Found free among egg mass of host or in a filiform, ornamented mucus sheath attached to the pleopods. Lapilli homogeneously distributed on mucus sheath and 4¡1 mm (3-5 mm; n52) in height. Anterior end of body rounded or pointed ( Figure 1A); posterior end pointed. Dimensions of relaxed worms 2.6¡0. Male. Body color cream; gut orange; gonads translucent white. Two eyes, black, irregular, circular or elliptical; elliptical eyes the most common shape. Found free among egg mass of host or in a filiform, ornamented mucus sheath attached to the pleopods. Lapilli homogeneously distributed on mucus sheath and 4 mm (n51) in height. Anterior end of body rounded or pointed ( Figure 1D), posterior end pointed ( Figure 1E).  Figure 1E) easily visible under stereomicroscope in the majority of adult male worms.

Infestation site
Adult worms were found in the host's egg mass ( Figure 1F) (ovigerous females with eggs in initial, intermediate and final stages of development). Immature worms were found on the abdomen of juvenile male crabs; on the abdomen and at the arthrodial membrane of pereopods of juvenile female crabs; at the base of pereopods, on the ventral and dorsal sides of the abdomen of adult male crabs; on the ventral and dorsal sides of the abdomen of nonovigerous adult female crabs; at the base of pleopods, on the abdomen and on the eggs of ovigerous females; at the base of pleopods and on the ventral side of the abdomen of postovigerous females.

Diagnosis
Body color varies from translucent white to cream; male with a red spot at posterior end. Two eyes, black. Anterior end of body rounded, posterior end rounded or truncated (males). Worms 1.2-11.0 mm long. Accessory stylets absent. Ovaries arranged in one row on each side of intestine. Takakura's duct present. Ornamented and filiform mucus sheath may be present in adult worms.

Material examined
Nine females and eleven males were examined.

Etymology
The species name is a noun in the genitive plural and is in recognition of the human communities known as ''caiçaras'' (a tupi-guarani word), which are found in the localities where the crabs were collected. The people of these communities depend mainly on fisheries; therefore, this name honors the fishermen who helped us collect, Rogério dos Santos Jú nior and his father, Rogério dos Santos.

Description
The description is based on living adults. Measurements are given in mean¡SE (range, number of specimens observed).
Female. Body color varied from translucent white to cream; gut varied from orange to brownish; gonads translucent white. Two eyes, black, round or cup-shaped; round eyes the most common shape. Found free among egg mass of host or in a filiform, ornamented mucus sheath (Figure 2A). Lapilli 7¡0 mm (7-8 mm; n52) in height, larger at center of sheath and smaller and scarce at extremities. Anterior and posterior end of body rounded. Dimensions of relaxed worms 5.5¡1.0 mm (2.6-11.0 mm; n57) long and 282¡20 mm ( Male. Body color cream with a red spot at the posterior end; gut varied from orange to brownish; gonads translucent white. Two eyes, black, irregular, circular or cup-shaped. Found free among egg mass of host or in a filiform, ornamented mucus sheath. Lapilli 4 mm (n51) in height. Anterior end of body rounded, posterior end rounded ( Figure 2B)

Infestation site
Adult worms were found in the host's egg mass (ovigerous females with eggs in initial, intermediate and final stages of development). Immature worms were found on the ventral side of the abdomen of adult male crabs; on the abdomen, at the gonopores, at the base of pleopods and encysted on the setae of pleopods ( Figure 2D) of non-ovigerous adult female crabs; encysted on the setae of pleopods of ovigerous females; on the abdomen, at the gonopores, on the arthrodial membrane of pleopods, and encysted on the setae of pleopods of post-ovigerous females. Takakura's duct present. Ornamented and filiform mucus sheath may be present in adult worms.

Material examined
Four females, six males, 12 eggs and one larva were examined. Holotype: male, from the egg mass of Menippe nodifrons; type locality: Figueira

Etymology
The species name is an adjective in feminine singular and is in recognition of the locality where the host crab was found, São Sebastião.

Description
The description is based on living adults, eggs and larva. Measurements are given in mean¡SE (range, number of specimens observed).
Male. Body color translucent white; gut yellow; gonads translucent white. Two eyes, brown, circular or elliptical; elliptical eyes the most common shape. Found free among  Larva. Body ciliated with anterior and posterior ciliary tufts. Body shape ovoid. Two eyes. Body length 92 mm (n51). Body width 62 mm (n51).

Infestation site
Adult worms were found in the host's egg mass (ovigerous females with eggs in initial, intermediate and final stages of development). All immature worms were found encysted in a mucus sheath without ornamentation in the following sites: on the ventral side of the abdomen, on the setae that follow the border of the abdomen, on the central axis of pleopods ( Figure 5B), and on the setae of pleopods of non-ovigerous adult female crabs; on the ventral side of the abdomen, on the central axis of pleopods, and on the setae of pleopods of ovigerous females; at the ventral side of the abdomen, on the central axis of pleopods, and on the setae of pleopods of post-ovigerous females. No immature worms were found on male crabs.

Discussion
The three species described here are gonochoric, do not present accessory stylets and, thereby, conform to the diagnosis of Carcinonemertidae provided by Humes (1942) and modified by Shields et al. (1989) and to the diagnosis of Carcinonemertes provided by Coe (1902). The genus Ovicides is characterized by being hermaphroditic and by having two accessory stylets (Shields 2001). Carcinonemertes divae n. sp., C. caissarum n. sp. and C. sebastianensis n. sp. can be distinguished from each other by color of the body, shape of the posterior region of body, eye length, and by distance between the first ovary and tip of head (Table I).
Carcinonemertes pinnotheridophila is found on the floor and wall of branchial chambers as well as on eggs of its hosts (McDermott and Gibson 1993). Juvenile worms of C. c. carcinophila, C. c. imminuta and C. mitsukurii are found between the gills of their hosts (Humes 1942;Shields 1992;Santos and Bueno 2001; Table II). None of the three species described here was found in the branchial chamber or between the gills of their hosts.
The three species described here also can be distinguished from C. pinnotheridophila by the presence of eyes in the adult worms, shape of the egg mucus sheath and by the number of rows of ovaries on each side of the intestine (McDermott and Gibson 1993) (Table II). Carcinonemertes coei also has two rows of ovaries on each side of the intestine (Humes 1942; Table III).
Carcinonemertes divae n. sp., C. caissarum n. sp. and C. sebastianensis n. sp. can be distinguished from C. c. carcinophila by body length (Humes 1942), and from C. c. imminuta by body length and by eye width (Humes 1942; Table II).
Adult worms of C. errans do not have a mucus sheath (Wickham 1978), whereas adult worms of C. regicides have a mucus sheath with no lapilli (Shields et al. 1989). The adult worms of the three species described here have a mucus sheath with lapilli, which are, according to Humes (1942), irregularly shaped, raised concretions, present on the surface of the sheath. In addition, C. errans can be distinguished from C. divae n. sp., C. caissarum n. sp. and C. sebastianensis n. sp. by diaphragm length and by stylet basis length (personal Table I. Morphological and ecological differences that distinguish the species Carcinonemertes divae n. sp., Carcinonemertes caissarum n. sp. and Carcinonemertes sebastianensis n. sp. (measurements in mm). observation; Table III). The stylet basis in C. regicides is 38-44 mm long (Shields et al. 1989; Table III).

Characters
Carcinonemertes australiensis can be distinguished from C. divae n. sp., C. caissarum n. sp. and C. sebastianensis n. sp. by stylet basis length, stylet basis width and by stylet length (Campbell et al. 1989; Table III).
The stylet basis is 10-12 mm wide (Gibson and Jones 1990) in C. humesi and 14 mm (Shields and Kuris 1990) in C. wickhami. The latter also differs from C. divae n. sp., C. caissarum n. sp. and C. sebastianensis n. sp. in diaphragm length, stylet basis length, stylet length and in stylet:basis ratio (Shields and Kuris 1990; Table IV). Stylet:basis ratio in C. humesi varies from 0.219 to 0.267 (Gibson and Jones 1990).
Carcinonemertes divae n. sp., C. caissarum n. sp. and C. sebastianensis n. sp. can be distinguished from C. epialti by diaphragm width (personal observation; Table IV). Carcinonemertes epialti differs from C. divae new species in the alignment of proboscis and in Table III. Morphological and ecological differences that distinguish the species Carcinonemertes coei, Carcinonemertes errans, Carcinonemertes regicides and Carcinonemertes australiensis from the species described here (measurements in mm).

Characters
C  the eye length (Coe 1902; Table IV). The eye is 8-15 mm long in C. caissarum n. sp. and 10-18 mm long in C. sebastianensis n. sp. Carcinonemertes epialti also can be distinguished from C. caissarum n. sp. by stylet length (personal observation) and from C. sebastianensis new species by the distance between the eyes and tip of head (personal observation; Table IV). Juvenile worms of some nemerteans of the genus Carcinonemertes were previously found on the pleopods of their hosts (at the base or on the arthrodial membranes). A novelty regarding the infestation site by nemerteans of the genus Carcinonemertes is registered in the present study for C. sebastianensis n. sp., whose juveniles can be found encysted on setae of pleopods (a site also used by juveniles of C. caissarum n. sp.) or on the axis of pleopods, or even on setae that follow the border of the abdomen of the hosts.
In addition to the diagnostics mentioned above, the three new species differ from other carcinonemertids in that they infest, respectively, L. spinosa, H. pudibundus and M. nodifrons, all registered here for the first time as hosts for nemerteans of the genus Carcinonemertes.
Specimens of H. epheliticus and M. mercenaria collected at Grand Isle, Louisiana, USA, were registered by Humes (1942) as hosts for C. c. imminuta. Humes (1942) found juvenile worms on gills of H. epheliticus, which makes us believe he could be right about the identification of these specimens of Carcinonemertes. Nevertheless, the following facts suggest that there is a chance the worms found in H. epheliticus and M. mercenaria were misidentified as C. c. imminuta: (1) only one worm was found on the gill of H. epheliticus from Louisiana (Humes 1942); this infestation could be casual, not a regular pattern; (2) an adult male of H. epheliticus was collected in Florida and none of the 90 juvenile worms infesting the crab was found on gills (personal observation); (3) Humes (1942) did not find any worms on the gills of M. mercenaria and stated that ''in the non-portunid species infested at Grand Isle, the worms were all minute, though were sexually mature''; it seems that the worms found in H. epheliticus and in M. mercenaria by Humes (1942) were smaller than C. c. imminuta.
There are some factors that can lead to misidentifications among the species belonging to the family Carcinonemertidae: (1) the small size of worms, (2) morphological simplification apparently resulting from their parasite-like life style, and (3) the consequent morphological ambiguity of structures and morphological similarity between species. For example, the structure referenced as anterior proboscis chamber in some publications (Shields et al. 1989;Shields and Kuris 1990) appears to us to be the proboscis diaphragm and that is how Table IV. Morphological and ecological differences that distinguish the species Carcinonemertes wickhami, Carcinonemertes humesi and Carcinonemertes epialti from the species described here (measurements in mm).
C. sebastianensis n. sp. we treat it. Similarly, the region designated as the stylet bulb, appears to include the diaphragm in a number of publications (Gibson 1972;Campbell et al. 1989;Gibson and Jones 1990;McDermott and Gibson 1993); we distinguish between the two. The stifling morphological homogeneity of Carcinonemertes species led us to search for other, relatively practical characters, such as distance from first gonad to tip of head, distance from brain to tip of head and distance from stylet to tip of head. Distance from first gonad to tip of head enabled us to distinguish among the three species described here. We believe that information, such as infestation site and infestation according to sex and maturity of host, may also provide valuable diagnostic and systematic data. With this information in hand from previously described species it may be possible to conduct phylogenetic analyses with useful results that will lead to understanding of the diversification and host relationships of this potentially damaging group of nemerteans.