COPULATION OF Opisthogonimus fonsecai RUIZ & LEÃO , 1942 ( TREMATODA , DIGENEA , PLAGIORCHIIDAE ) , PARASITE OF Bothrops moojeni HOGE , 1966 ( SERPENTES , VIPERIDAE

Copulation in Opisthogonimus fonsecai Ruiz & Leao, 1942 (Trematoda, Digenea, Plagiorchiidae), parasite of Bothrops moojeni Hoge, 1966 (Serpentes, Viperiade), was reported. Data on the histology of the cirrus and metraterm of the copulants were presented. Morphology of the cirrus was also examined by scanning electron microscopy.


INTRODUCTION
Most digenean trematode species are hermaphrodites.Literature reports that selffertilization may happen but it is uncommon, since a number of flukes usually occur in the same host.Reproduction of some of these helminths involves copulation, which happens when the cirrus of one fluke is inserted into the terminal portion of the other fluke's uterus (metraterm) (14).
Studies on the mating behavior of adult digenean trematodes have been carried out with some species of the families Philophthalmidae (5,6,7), Echinostomidae (9,10), and Paramphistomidae (11).In these reports, self and cross-insemination patterns were observed by using transplantation and autoradiography techniques.Besides, Sey et al. (13) reported the copulation of Lasiotocus sp. by scanning electron microscopy (SEM).
The copulatory organ of most digenean is a cirrus.Frequently, it has circular and longitudinal muscles in its walls and presents spines (4).The metraterm is muscular to varying degrees in different forms and may serve as a vagina (4).
Although some reports about trematodes reproduction are available in literature, histological and SEM data on the copulation of trematode parasites are scarce.Thus, the objective of the present study was to report copulation in Opisthogonimus fonsecai (Trematoda, Digenea, Plagiorchiidae).

MATERIAL AND METHODS
The trematodes were collected from the mouth of a Bothrops moojeni (Serpentes, Viperidae) specimen from Porto Primavera, São Paulo State, and fixed in cold AFA solution without compression (2).Some trematodes were stained with carmine and mounted in Permount resin.The O. fonsecai (Figure 1) species was identified (1,12,15).Among the collected helminths, three pairs of copulants were found (Figure 2A).One of them was studied by SEM, and the other two were analyzed after histology.For the histological study, fixed helminths were embedded in historesin (2hydroxyethyl methacrylate); 4-µm transverse serial sections were prepared, stained with hematoxylin-eosin and examined by using a computerized image analysis system (Qwin Lite 2.5 -Leica).

RESULTS
In each of the three cases of copulation observed, only one of the copulants had its cirrus introduced into the metraterm of the other fluke.Trematodes that were receiving the cirrus had their own intromittent organ everted in one case and withdrawn in the other two cases.The trematodes were parallel or diagonal to one another.In two cases, the copulants had similar size, and in the third, they were slightly different (Figure 2A).
In one case histologically examined, the two trematodes had the genital atrium at a similar level; and in the transverse histological sections, it was observed that the cirrus of one specimen was introduced into the metraterm of the other (Figure 2B).The longest cirrus observed measured 636 µm.The extruded extent of the cirrus was 414 µm, but only 121 µm was introduced into the other fluke's metraterm (Figure 2C).
The widest cirrus measured 134 µm in diameter; and a constriction, which resulted in a decrease of the cirrus diameter to 42 µm, was observed at the genital pore of the trematode that was receiving the cirrus (Figure 2C).The cirrus presented a canal (ejaculatory duct) with a lumen of 6-7 µm in diameter.This duct was covered with a mucous epithelium of 8-11.5 µm thickness and two layers of smooth musculature: a circular one, with 1-1.5 µm thickness, and a longitudinal one, with 1-3 µm thickness.
Externally, the cirrus was covered with a tegument of 9.5-12 µm thickness.Under this tegument, there was a basal lamina of 0.5-1 µm thickness and two layers of smooth musculature: a circular one, with 2.5-3 µm thickness, and a longitudinal one, with 1-2 µm thickness.Internally, there was a parenchyma and also glands.The cirrus had no spines (Figure 2D).
In the other case histologically studied, the cirrus was in a diagonal position; and after the analysis of all histological serial sections, it was observed that only a small portion of the cirrus' extremity was introduced into the metraterm of the other fluke (Figure 2E).In this second case, we obtained a good section of the metraterm with a great amount of spermatozoa in the lumen (Figures 2E-F).This metraterm was surrounded by a large number of glands (Figure 2E-F) and measured 288 µm long and 125.5 µm wide.The lumen of this structure was 58 µm in diameter.There was a mucous epithelium of 8.5-15 µm thickness; a basal lamina of 1-2.5 µm thickness; and two layers of smooth muscle: a circular one, with strong muscle fibers of 15.5-21 x 1-1.5 µm thickness, and a longitudinal one of 2-5.5 µm thickness (Figure 2F).SEM examination showed that the cirrus had no spines and was tapered.Next to the metraterm's opening, we could also observe some eggs of this trematode adhered to the cirrus (Figures 2G-H).

DISCUSSION
The male and female reproductive systems of O. fonsecai were described by Ruiz & Leão (12).Bothrops moojeni infection by this species was previously reported by Correa (3).However, there are no reports on the copulation of this trematode.
Patterns of sexual reproduction in platyhelminths were discussed by Nollen (8).
According to this author, digeneans have potential for both cross and selfinsemination, since they are almost exclusively hermaphrodite and, in most of them, the male and female systems share a common genital atrium or pore.In addition, he cited other papers in which insemination through Laurer's canal in the species

Diphterostomum brusinae and Haploporus benedeni; mating via metraterm in
Podoctyle fractum; reciprocal copulation in Fasciola hepatica; and self-insemination in Leucochloridiomorpha constatinae were observed.For many digeneans, crossfertilization is usual.On the other hand, at least in two trematodes (Liolope copulans and Collyriclum faba), Laurer's canal was used for the reception of the other individual's cirrus during copulation and for the conveyance of the spermatozoa received during this act to the seminal receptacle (4).

Figure 1 .
Figure 1.Specimen of Opisthogonimus fonsecai collected from the mouth of a Bothrops moojeni snake.