Embryonic development of Anodontites trapesialis (Lamarck, 1819) (Bivalvia: Mycetopodidae)

Desenvolvimento embrionário de Anodontites trapesialis (Lamarck, 1819) (Bivalvia: Mycetopodidae)

BIOLOGY

Embryonic development of Anodontites trapesialis (Lamarck, 1819) (Bivalvia: Mycetopodidae)

Desenvolvimento embrionário de Anodontites trapesialis (Lamarck, 1819) (Bivalvia: Mycetopodidae)

Silva-Souza, AT.I, ^*; Guardia-Felipi, P.^II; Arrebola, NR.^III

^ILaboratório de Ecologia de Parasitos de Organismos Aquáticos - LEPOA, Departamento de Biologia Animal e Vegetal, Centro de Ciências Biológicas, Universidade Estadual de Londrina - UEL, CEP 86051-990, Londrina, PR, Brazil

^IIPrograma de Pós-graduação em Ciências Biológicas, Departamento de Biologia Animal e Vegetal, Universidade Estadual de Londrina - UEL, CEP 86051-990, Londrina, PR, Brazil

^IIIDepartamento de Histologia, Universidade Estadual de Londrina - UEL, CEP 86051-990, Londrina, PR, Brazil

ABSTRACT

The phases of embryonic development of Anodontites trapesialis lasidia are described for the first time. Adult specimens were obtained from two fish farms located in Londrina, Paraná, Brazil. The internal demibranchs of 120 individuals were studied using a routine histological technique; 70 of these carried eggs and/or larvae in the marsupium and were utilized for the description of the phases of embryonic development. The demibranchs of five specimens were evaluated by scanning electron microscopy to detail the morphology of the larvae. Five phases of development were established: phase I, corresponding to the initial stage of cleavage with the formation of apical cells; phase II, including the stages of the morula and blastula; phase III, where the gastrula forms; phase IV, where the larva formed is still inside the egg envelope; and phase V, where the lasidium can still be identified immediately after eclosion.

Keywords: fish farming, Anodontites trapesialis, morphology, embryogenesis, lasidium.

RESUMO

As fases do desenvolvimento embrionário das lasídias de Anodontites trapesialis são descritas pela primeira vez. Espécimes adultos foram obtidos de duas pisciculturas localizadas no município de Londrina, Paraná, Brasil. As demibrânquias internas de 120 indivíduos foram estudadas por técnicas histológicas rotineiras; 70 apresentavam ovos e/ou larvas no marsúpio e foram utilizadas para a descrição das fases do desenvolvimento embrionário. As demibrânquias de cinco espécimes foram avaliadas por microscopia eletrônica de varredura para detalhar a morfologia da larva. Cinco fases do desenvolvimento foram estabelecidas: fase I, correspondente ao estágio inicial da clivagem com formação das células apicais; fase II, incluindo os estágios de mórula e blástula; fase III, na qual se forma a gástrula; fase IV, na qual a larva formada ainda encontra-se dentro do ovo; e fase V, na qual a lasidium pode ser identificada imediatamente após a eclosão.

Palavras-chave: piscicultura, Anodontites trapesialis, morfologia, embriogenia, lasidium.

1. Introduction

The bivalves of the family Mycetopodidae incubate their eggs in internal demibranchs, in a so-called marsupium (Castellanos and Landoni, 1990). The egg gives rise to the lasidium, a temporary ectoparasite of fish and present only in the families Mutelidae in Africa and Mycetopodidae in South America (Parodiz and Bonetto, 1963; Wächtler et al., 2001). When mature, the larvae are passed from the marsupium to the branchial canal and eliminated to the exterior through the exhalant opening (Castellanos and Landoni, 1990). In the environment, it needs to find a host fish to complete its development, to which it adheres by means of an adhesive organ (Bonetto and Ezcurra, 1962; Silva-Souza and Eiras, 2002).

Descriptions of the general characteristics of the family Mycetopodidae, for the majority, are based on studies with species of the genus Anodontites Bruguière, 1792 (Castellanos and Landoni, 1990).

According to Simone (1994), the first studies on Anodontites trapesialis were carried out by Simpson (1900). Later, its anatomy was detailed by Veiteinheimer (1973) and Hebling (1976). The species has been utilised in environmental monitoring studies (Callil and Junk, 1999; Jacomini, 2002; Tomazelli et al., 2003) and, more recently, in aquaculture works (Tello-Panduro et al., 2003, 2004; Vivanco et al., 2003).

Studies on the reproduction of A. trapesialis are rare. The only one on the gametogenesis and dynamics of reproduction was conducted by Callil and Mansur (2007), but nothing was recorded on the embryonic development of the lasidia.

Thus, due to the scarcity of information, the aim of the present work was to describe the development of the eggs of A. trapesialis in the marsupium up to the eclosion of the lasidia.

2. Material and Methods

Monthly collections of specimens of Anodontites trapesialis were performed in the period of May 2006 to April 2007, in fish culture tanks on the properties of "Toca do Jacaré" (23° 24' 46.8" S, 51° 08' 33.5" W) and "Paraíso da Tilápia" (23º 25' 16.7" S, 51° 09' 13.6" W), located in the Distrito Espírito Santo, Londrina, PR, Brazil.

The specimens were located by touching the bottom of the tanks with the feet, collecting them manually and transporting them to the Laboratory of Ecology of Parasites of Aquatic Organisms (LEPOA), Department of Animal and Vegetal Biology, State University of Londrina, Londrina, PR, in plastic containers with water from the same tank, without mechanical aeration. In the laboratory, the animals were kept alive in a dry container for the elimination of the water contained in the paleal cavity, weighed (total weight in grams) and measured (total length in millimeters). Next, the gills were examined under a stereomicroscope for the verification of the presence of eggs and for morphological characterisation.

Samples of 1.0 cm² of both internal demibranchs of 120 specimens of A. trapesialis were fixed in Bouin's solution for 24 hours, preserved in 70% alcohol, dehydrated in the an ethanol series, cleared in xylol and embedded in Paraplast for histological analyses. Serial sections of 0.7 μm thickness were prepared with a Leica RM 2145 rotary microtome, mounted on slides and stained with hematoxylin and eosin (HE). The sections were analysed with a Zeiss Axiophot photomicroscope, and the images were obtained with a Leica DM 4500B microscope equipped with a DFC 300FX camera and with the Leica IM50 4.0 program.

For scanning electron microscopy (SEM), samples of demibranchs of five individuals selected randomly were fixed in a solution of 2% glutaraldehyde, 2% paraformaldehyde and 0.1 M cacodylate buffer, washed in 0.1 M cacodylate buffer, post-fixed in 1% osmium tetroxide, washed again in 0.1 M cacodylate buffer, and dehydrated in an increasing ethanol series. They were then critical-point dried, coated with a film of gold with thickness of 25 nm and analysed with a FEI Quanta 200 scanning electron microscope in the Electron Microscopy and Microanalysis Laboratory from PROPPG/UEL.

3. Results

A total of 120 specimens of A. trapesialis with a mean total weight of 159.24 ± 70.85 g (range: 24.69 g to 322.71 g) and mean total length of 119.44 ± 18.58 mm (range: 62.59 mm to 153.17 mm) were examined. Of these, only 70 showed eggs and larvae inside the marsupium of the internal demibranchs. When examined with a stereomicroscope, the marsupia with eggs in the initial phase had a beige colour, and during development they showed intensification in colouration.

The marsupium is lined by a simple cuboidal epithelial tissue that rests on loose connective tissue where blood cells are identified. This connective tissue fills the spaces between the lacunae, which are lined by an epithelial tissue similar to that of the marsupium (Figure 1a).

Histologically, it was possible to establish five phases of egg development in A. trapesialis, as described below.

• Phase I. The eggs observed in the initial phase are characterised by intense acidophilia. They are identified by their spherical and regular structure, covered by an acellular envelope appearing slightly basophilic. Initially, the contents of the egg are intensely acidophilic, and a thin layer of cell clusters appear at one of the poles (Figure 1b). At the end, there is an increase in the number of cells, which remain aggregated at one of the poles, accompanied by a reduction in the quantity and acidophilia of the remaining contents (Figure 1c). This phase appears to be the initiation of cleavage, where there appear to be a few apical cells.

• Phase II. When compared to the preceding phase, the eggs appear less acidophilic, and their envelope has an irregular surface. They are practically filled by an aggregate of cells whose nuclei are well visible and show little variation in size and coloration, which appears to correspond to the morula (Figure 1d). At the end, the cells are grouped and displaced, where part of the egg appears to be empty, corresponding to the blastula (Figure 1e).

• Phase III. This can be characterised as a joining of various layers of cells, which are grouped at one of the poles as an open ring in the shape of the letter C, constituting the gastrula. There are small, rounded nuclei with dense chromatin or loose chromatin and slightly basophilic cytoplasm (Figure 1f).

• Phase IV. The envelope is very irregular. It is possible to identify four constituents of the larva that localize at one of the poles: the anterior region consisting of two acidophilic lobes, with a homogeneous appearance and surface covered by cilia; wider medial region, constituted by a group of cells that have small, spherical slightly basophilic nuclei and slightly acidophilic cytoplasm; and a shorter posterior region which is less stained than the others. The space around the larva is filled with a structure that is irregular, acellular and slightly basophilic, suggesting that it could be the adhesive organ (Figure 1g).

• Phase V. The larva is indentified just after eclosion, generally close to the empty envelope. The anterior region, predominantly acidophilic in relation to the others, comprises two types of cells, one containing large, spherical nuclei and the other with small, elliptical nuclei. This whole region is covered by a large amount of cilia. The medial and posterior regions are more basophilic. In the medial region, the nuclei are spherical and have loose chromatin. The posterior region is shorter, bilobed, formed by cells with elliptical nuclei and dense chromatin. In a small portion of the anterior region, there is a structure similar to that seen inside the egg in phase IV, which confirms that it is the adhesive organ. The envelope of the eggs is identified as a thin structure of irregular shape and generally surrounded by pigmented cells (Figure 1h).

The samples of internal demibranchs analysed by scanning electron microscopy showed details of the eggs on the wall of the marsupium (Figure 2a) and confirmed the modifications of the envelope during development (Figure 2b-d).

Figure 3 shows the details of the lasidium: ciliated anterior lobes (a and b), middle region with larval shell (c) and posterior lobes with hooks curved downward and inward (d). It is noted that the adhesive arises from the anterior region in the dorsal portion, close to an opening that appears to be the mouth (Figure 3a, b). It is noted that the hooks of the right posterior lobe are not present.

4. Discussion

The examination of the specimens of Anodontites trapesialis in the present study revealed the presence of eggs only in the internal demibranchs, as found by Bonetto and Ezcurra (1962) for Anodontites trapesialis forbesianus (Lea, 1860), Mansur (1974) for Monocondylaea minuana d'Orbigny, 1835 and Veiteinheimer-Mendes and Mansur (1978) for Mycetopoda legumen (Martens, 1888).

In A. trapesialis, as reported by Mansur (1974) for M. minuana, the marsupium occupies almost all of the internal demibranch, leaving only a narrow ventral strip and some aquifer tubes in the anterior and posterior regions. On the other hand, in Mycetopoda legumen, the marsupium occupies only the anterior half of the demibranch (Veiteinheimer-Mendes and Mansur, 1978).

Contrary to that described by Maldonado et al. (1990), who observed only small whitish masses corresponding to mature larvae, in the present study, the presence of the lasidia in the marsupium conferred a brown coloration.

Larvae with a body divided into three regions, observed for Anodontites trapesialis studied here, constituted a peculiar characteristic of the lasidium cited by Bonetto and Ezcurra (1962, 1965), Parodiz and Bonetto (1963), Veiteinheimer-Mendes and Mansur (1978), Castellanos and Landoni (1990), Maldonado et al. (1990) and Wächtler et al. (2001).

Bonetto and Ezcurra (1962) and Castellanos and Landoni (1990) reported that the adhesive organ arises from the medial region in the ventral position of the larva. However, scanning electron microscopy showed that in the larvae of Anodontites trapesialis, it originates from the dorsal portion of the anterior region, close to an opening that resembles a mouth. In histological sections, an accumulation of cells in a circular arrangement can be seen in the region corresponding to the mouth. However, no digestive tract appears to be contiguous with it, and no reference to the presence of a mouth in lasidia was found in the available literature. Therefore, further studies are necessary for such speculations to be clarified.

The medial and posterior regions concur with those described in the literature, also with respect to the larval shell (Bonetto and Ezcurra, 1962; Castellanos and Landoni, 1990).

With respect to the hooks in the posterior region, a group of six to seven was found present in each lobe, similar to that described by Bonetto and Ezcurra (1962) in A. trapesialis forbesianus. On the other hand, Anodontites trapezeus (Spix, 1827) shows approximately 11 short hooks in each of the lobes, and Mycetopoda siliquosa (Spix, 1827) has seven or eight hooks (Bonetto and Ezcurra, 1965). In Mycetopoda legumen, the number of hooks varies from four to seven (Veiteinheimer-Mendes and Mansur, 1978). The hooks are responsible for establishing the first firm contact with the body of the host, in which the larva will complete its metamorphosis and reach the juvenile stage (Wächtler et al., 2001).

It is known that mollusks show holoblastic spiral cleavage. However, characteristics of the egg during development generally are not described for the majority of species. Most studies of this nature refer to marine species of economic importance (Mouëza et al., 1999).

With respect to freshwater species, Maldonado et al. (1990) reported the occurrence of three phases of egg development in the marsupium of Anodontites (Anodontites) soleniformes Orbigny, 1835 of Bolivia, without providing any description. In the present study, it was possible to describe, for the first time, five distinct phases of egg development in the marsupium of Anodontites trapesialis, where the last corresponded to the lasidia immediately after eclosion.

Acknowledgements - Thanks go to Mr. Helio, owner of Pesque-Pague "Toca do Jacaré", and to Mr. Nelson, owner of the fish farm "Paraíso da Tilápia," for permitting the collections in the tanks, and to Aparecido de Souza, Edson Santana da Silva and Wanner Galves for their help with the collections. Dr. Celia G. T. de Jesus Andrade, Juca A. Barrera San Martin and Osvaldo Capello from EMML / PROPPG / UEL assisted with the SEM analyses. Renata Baldo Rodrigues Carvalho from the Histology Laboratory, UEL, helped with the histological procedures. Dr. Claudia Tasso Callil from Universidade Federal do Mato Grosso confirmed the identification of the species. Dr. A. Leyva helped with the translation of the manuscript. CAPES provided financial support.

Received April 1, 2010

Accepted June 7, 2010

Distributed February 28, 2011

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Publication Dates

• Publication in this collection
  11 Mar 2011
• Date of issue
  Feb 2011

History

• Received
  01 Apr 2010
• Accepted
  07 June 2010