Ultrastructure of phagocytes and oocysts of Nematopsis sp. (Apicomplexa, Porosporidae) infecting Crassostrea rhizophorae in Northeastern Brazil

This work describes the detailed ultrastructural morphology of the phagocyte imprisoning an oyster of Nematopsis (Apicomplexa) found in Crassostrea rhizophorae, in the city of Maceió (AL), Brazil. The highly infected hosts had halfopen leaflets with weak, slow retraction of the adductor muscles. Variable number of ellipsoid oocytes, either isolated and or clustered, was found between myofibrils of the adductor muscle. Each oocyst was incarcerated in a parasitophorous vacuole of host uninucleated phagocyte. The oocysts were composed of a dense wall containing a uninucleate vermiform sporozoite. The wall of the fine oocysts was composed of homogeneous electron-lucent material formed by three layers of equal thickness, having a circular orifice-micropyle obstructed by the operculum. The oocysts presented ellipsoid morphology with their wall was surrounded by a complex network of numerous microfibrils. Important details of the taxonomic value were visualized such as the ultrastructural organization of the oocyst wall and the organization of the micropyle and operculum, beyond the microfibrils that protrude from the oocyst wall only observed by transmission electron microscopy (TEM) and that may aid in the identification of the species. However, in order to clarify the systematic position of the species reported of the genus Nematopsis, it is important to proceed with genetic analyses.


Introduction
The microorganisms that may cause diseases or pathological conditions in several commercial bivalve species have not been investigated in detail.Many occurrences of mass mortality among farmed and wild bivalves been recently reported, and the losses to mussel and oyster farms that occur annually have primarily been attributed to poor environmental conditions, even if diseases may have caused them (BOEHS et al., 2010;BOWER et al., 1994;BRADBURY, 1994;CHENG, 1967;LAUCKNER, 1983;MOSS et al., 2007;PERKINS, 1991;POWELL & KIM, 2015;SPRAGUE, 1970).
An intriguing situation exists with regard to molecular data (phylogeny).Although there is information on the 18S rDNA sequences of the Nematopsis sp. that have been described, it is surprising that it was not possible to obtain any information from the oocyst stage (sporogony) that would allow comparative analysis on this taxonomic group.Molecular analyses were only performed on the cephaline stages of gregarines, which were described as species of the genus Nematopsis (BELAFASTOVA, 1996;PRASADAN & JANARDANAN, 1996;SHANAVAS et al., 1989).
Despite various attempts to apply current standard molecular biology technologies that have been developed in our laboratory (ICBAS/UP) for phylogenetic analyses on the SSU rDNA gene sequences, we have not obtained any positive results.This is an enigmatic situation, given that it has not been possible to create a compatible sequential primer.It is curious that, although this is a cosmopolitan genus, no phylogenetic results developed from oocysts in the sporogonic phase among the species of this genus have yet appeared.
Among all the species of the genus Nematopsis that have been described (around 40 species), we found that a few species (e.g.N. mytella and N. gigas) have been described based only on a few comparative ultrastructural analyses on the oocyst phase (AZEVEDO & MATOS, 1999;AZEVEDO & PADOVAN, 2004;PADOVAN et al., 2003).
The aim of the present study was to contribute towards better knowledge of the ultrastructural details of the oocysts of Nematopsis mytella, which is an apicomplexan species that has been described in marine bivalves (Crassostrea rhizophorae) from the Atlantic coast of Brazil, near the city of Maceió, state of Alagoas (Brazil).Additionally, the ultrastructural disorganization and disintegration of the tissues of the infected host specimens containing oocysts incarcerated in the host's phagocytes were observed and discussed.

Methodology
Four groups of ten wild C. rhizophorae Guilding, 1828 were collected monthly between September and December 2017 from mangroves near the city of Maceió (State of Alagoas), in the Atlantic coast of Brazil (09° 29' S, 35° 34' W).They were maintained in an aquarium with aeration for 3-5 days at a temperature of 20-25 °C and salinity of 2.5-3.5 ppm, the condition similar to their natural environment.
Oocysts in infected specimens of oysters were morphologically identified as the the genus Nematopsis.Some of these hosts presented gaping valves and their adductor muscles appeared to have weak contractile power.The oocysts were collected only from the adductor muscles, gill and mantle, and were prepared for common light microscopy (LM) and transmission electron microscopy (TEM) analyses.

Common light microscopy and morphological analysis on oocysts
The oocysts were examined and photographed using a Leitz microscope equipped with a Nomarski differential interference contrast (DIC) system.Morphometric analysis on the oocysts was conducted using fresh material and all measurements included the mean ± SD, range of variation and number of spores measured (range, n).

Transmission Electron Microscopy (TEM)
Samples of small infected fragments from the adductor muscle, gills, mantle and digestive gland (hepatopancreas) of C. rhizophorae were fixed in 4-5% glutaraldehyde that was buffered in 0.2 M sodium cacodylate (pH 7.4) for 20 to 24 h.They were then washed in the same buffer and postfixed in 2% osmium tetroxide, which was also buffered in the same solution for 3 to 4 h.All of these steps were performed at 4 °C.The samples were then dehydrated in an ascending graded series of ethanol and propylene oxide.The dehydrated samples were embedded in a series of propylene oxide and EPON mixtures, ending in EPON.Semi-thin sections were cut and stained with methylene blue-Azure II.Ultra-thin sections were cut using a diamond knife and were double-contrasted with uranyl acetate and lead citrate.The semi-thin sections were observed in LM, and the ultra-thin sections were examined and photographed using a transmission electron microscope (JEOL 100CXII; JEOL Optical), operating at 60 kV.

Common light microscopy observations
Observations under the light microscope enabled identification of the presence of oocysts in several organs (adductor muscle, gills, mantle and digestive gland) of C. rhizophorae.The infecting oocysts were easily observed among different host cells and were identified in freshly squashed preparations as Nematopsis sp.(Figures 1A-B), previously described in other bivalves species.This identification was matched using semi-thin sections (Figure 1D and Figure 2A) and through ultrastructural analysis (Figures 2B-E).The phagocyte cytoplasm contained variable numbers of parasitophorous vacuoles (PVs), each containing a single oocyst (Figures 1D and 1E).These oocysts seemed to be morphologically similar with similar dimensions and similar internal organization.No measured or morphological differences between the oocysts collected from the different organs were found.Thirty-one out of the 40 specimens (77.5%) of C. rhizophorae examined were infected by oocysts.The prevalence of oocysts varied according to the organ.It was observed that the prevalence of oocysts in the adductor muscles and in the gills were higher than the prevalence in the digestive gland and in the mantle, although it was not possible to quantify.
Our analyses based on the LM were oriented towards observing and describing exclusively the oocysts infecting the adductor muscles and, simultaneously, the host reaction due the presence of the parasite.The oocysts occurred singly or in groups, and the groups contained variable numbers of oocysts (up to 13) that were randomly dispersed throughout the adductor muscle tissue.Each oocyst was observed to be incarcerated in a phagocyte (Figures 1A-E).Single oocyst was more frequently incarcerated in individualized PVs of the host cells and these cells were identified as phagocytes (Figures 1A-E).On rare occasions, two oocysts were incarcerated in the same PV, as a result of contact between two neighboring PVs.The oocysts generally occupied a central position in the PV (Figures 1B-E).The oocysts were unicellular structures (15.6 ± 0.6 µm long and 11.1 ± 0.7 µm wide; n = 50) composed of an oocyst wall with an apical operculum surrounding a vermiform uninucleate cell, which was designated a sporozoite (Figures 1B-E).

Transmission Electron Microscopy (TEM) observations
Only the oocysts collected from the adductor muscles were ultrastructurally analyzed and described, as was reported.Observations on the fine structure confirmed that each PV contained a single oocyst (Figures 2B and 2C).The PV was formed by a parasitophorous membrane of irregular outline that was in close contact with the cytoplasm of the phagocyte (Figures 2B and 2C).The matrix of the PV was mainly occupied by a complicated network of numerous irregular and anastomosed microfibrils around the oocyst, projecting from the oocyst wall towards the PV membrane (Figures 2B-E).These double microfibril layers formed a complex anastomosed network projecting from the oocyst wall, in which the layer adhering to the oocyst wall had microfibrils that were thicker than those in more distant layers with more anastomosed contact with the PV membrane (Figures 2B-E).
Through observations on serial ultrathin sections, it was confirmed that each oocyst contained a single vermiform sporozoite and that the oocyst measurements (length and width) matched the LM observations (Figures 1B and 1C).The oocyst wall thickness was 0.8 ± 0.3 µm (n = 25) and this wall was formed only by homogeneous electron-dense material (Figures 2B-E).The apical region of the oocyst contained a circular micropyle (sometimes designated by a micropore) of diameter 0.9 ± 0.3 µm (n = 15).This was covered by an operculum formed by material of electron density similar to that of the wall material (Figure 2D).In favorable serial ultrathin sections, the operculum presented arcuate morphology (∩-shaped).Operculum occupied the cylindrical space of the micropyle (Figures 2D and 3) The first signs of lysis occurred in the phagocytes when the nucleus became pyknotic (Figure 2C) and the PV membranes disappeared.The cytoplasm became lighter and numerous vesicles appeared in this region (Figures 2C and 2E).All oocysts infecting the adductor muscle, gills and mantle showed similar measurements (15.3 ± 0.6) (n=10) and ultrastructural morphology, thus suggesting that all the oocysts found in different organs of the same specimens belonged to the same species.A schematic drawing of the oocyst morphology (Figure 3) was made on observations from light and serial ultrathin sections.
In Brazil, the genus Nematopsis has been intensively studied along the Brazilian Atlantic coast based mainly on light microscopy observations (BRANDÃO et al., 2013;BRITO et al., 2010;Figure 3. Schematic drawing of a longitudinal section of the operculum (Op) system of a Nematopsis sp.oocyst showing the operculum (Op) located in the apical region of the oocyst wall (Wa), which blocks the micropyle (Mcp).The internal part of the oocyst is occupied by the sporozoite (Sz) and the external complex system of microfibrils (Mf ) projects from the oocyst wall.PADOVAN et al., 2003;PINTO & BOEHS 2008;QUEIROGA et al., 2015;SABRY et al., 2007).The data from these studies do not allow comparative ultrastructural descriptions in relation to species that had previously been described (AZEVEDO & MATOS, 1999;AZEVEDO & PADOVAN, 2004;MAGALHÃES et al., 2006).These descriptions of undetermined Nematopsis species (BRITO et al., 2010;COVA et al., 2015;LUZ & BOEHS, 2015;PINTO & BOEHS, 2008;SABRY & MAGALHÃES, 2005) seem to correspond to the morphological characteristics of oocysts of the genus Nematopsis infecting Brazilian fauna that had previously been described.
Comparison of our results with the morphological and ultrastructural organizations of species of this genus that had previously been described showed that the oocyst organizations were highly similar.Thus, these data based on oocyst morphology confirm that the parasite described here belongs to the genus Nematopsis (AZEVEDO & MATOS, 1999;AZEVEDO & PADOVAN, 2004;MAGALHÃES et al., 2006), despite some morphological and ultrastructural differences.Detailed ultrastructural comparisons of the oocysts described in the present manuscript seem to confirm that they are similar to those of the species Nematopsis mytella that had previously been described (AZEVEDO & MATOS, 1999;MAGALHÃES et al., 2006;PADOVAN et al., 2003).
Today around 40 named species that belong to the genus Nematopsis have been described.In addition, several unnamed species have been attributed to this genus.Given the need for detailed morphological data and the lack of molecular data, these unnamed species may correspond to nominal species that have previously been described.Most of these unnamed Nematopsis were described based on LM observations and some of them were based on doubtful host specificity (ABDEL-BAKI et al., 2012;AZEVEDO & CACHOLA, 1992;BRITO et al., 2010;KUA et al., 2013;PINTO & BOEHS 2008;QUEIROGA et al., 2015;SABRY et al., 2007;SOTO et al., 1996;SUJA et al., 2016;TUNTIWARANURUK et al., 2004).Only a few of these species were identified using ultrastructural observations (AZEVEDO & MATOS, 1999;AZEVEDO & PADOVAN, 2004).
One important methodology providing an important means of diagnosing ultrastructural organizations allows details and rigor in comparing the different structures of these parasites, such as structural organization of the operculum and micropyle.Most species of the genus Nematopsis that have previously been described were only based on LM observations and on the parasite specificity of the hosts (BELAFASTOVA, 1996;CHAKRABORTI & BANDYOPADHYAY, 2010;JIMÉNEZ et al., 2002;KUA et al., 2013;THÉODORIDÈS, 1962;TUNTIWARANURUK et al., 2004).These observations enabled visualization of details of taxonomic value, such as the ultrastructural organization of the oocyst wall, and organization of the micropyle and operculum.The ultrastructural morphology of the micropyle and the microfibrils that adhere to Nematopsis oocysts seem to be important taxonomic features.However, these were not exploited to identify differences in the specificity of this genus.On the other hand, the surrounding layers of microfibrils that project from the oocyst wall towards the PV membrane, a structure only observed via TEM, may form a further characteristic that has the capacity to influence species identification.This merits exploitation in future studies.
Unfortunately, to date, no phylogenetic studies have been conducted to correlate the genomic DNA of the oocysts of some of the 40 named species based on the morphology of the oocysts.Only a few species (N.idella Prasadan & Janardanan, 1996;N. annulipes Prasadan & Janardanan, 2001;N. messor Prasadan & Janardanan, 2001;and N. quadratum Prasadan & Janardanan, 2001) have been described based on studies on the gregarine stage of the life cycle in crustacean hosts.Studies on the genomic DNA of the oocysts (sporogonic phase) during which the oocyst phase develops are of fundamental importance in relation to naming the species that are described in the schizogonic phase.This phase develops with morphological aspects that are not compatible with the morphology of the oocyst stages.No other species of the genus Nematopsis have been described using phylogenetic data based on genomic DNA on the oocysts stages infecting mollusks, or at least no such results have ever been published.
To clarify the systematic position of most of the reported species of the genus Nematopsis, detailed morphological studies on oocysts and molecular investigations on the SSU rDNA sequences based on genomic DNA isolated from oocyst stages need to be conducted.

Figure 1 .
Figure 1.Different aspects of oocysts of Nematopsis sp. that were obtained from the host adductor muscle of Crassostrea rhizophorae.These oocysts were incarcerated in the host's phagocytes.Observations via light microscopy, differential interference contrast and transmission electron microscopy.A-Clusters containing some oocysts (Oc) each incarcerated in a phagocyte (Pgc), observed using DIC; B-Oocysts (Oc) observed using DIC, showing the internal sporozoite (Sz) surrounded by the oocyst wall (Wa); these oocysts were included in parasitophorous vacuoles (PVs) of the phagocytes (Pgc); C-Composition of an isolated oocyst (Oc): oocyst wall (Wa), operculum (Op) and sporozoite (Sz) observed using DIC; D-Semi-thin section through an oocyst (Oc) showing the sporozoite (Sz) and the parasitophorous vacuole of the host's phagocyte (Pgc) surrounded by other host cells (*); E-Semi-thin section showing oocysts (Oc) incarcerated by a phagocyte (Pgc): the oocysts are located among the host cells (*).Each oocyst shows an internal sporozoite (Sz), surrounded by the oocyst wall (Wa) and containing numerous microfibrils (Mf ) projecting into the parasitophorous vacuole membrane.All scale bars in µm.

Figure 2 .
Figure 2. Some morphological aspects of the different organelles and structures of the oocytes and the surrounding host cells.A-Semi-thin cross-section through an oocyte (Oc) showing the internal sporozoite (Sz) and numerous microfibrils (Mf ) projecting from the oocyst wall; B-Transverse section through an oocyst (Oc) incarcerated in a parasitophorous vacuole (PV), showing the internal sporozoite (Sz) surrounded by the oocyst wall (Wa) from which numerous microfibrils (Mf ) radiate, projecting towards the phagocyte (Pgc); C-Ultrastructural detail of the peripheral zone of the oocyst wall (Wa) showing irradiation of different types of microfibrils (Mf ) projecting from the oocyst wall towards the parasitophorous vacuole membrane of the phagocyte (Pgc).The phagocyte shows a pyknotic nucleus (Nu) and the parasitophorous vacuole membrane is partially destroyed (arrows); D-Ultrastructural detail of the apical region of the oocyst, showing the oocyst wall (Wa), the sporozoite (Sz) and the operculum (Op) plugging the micropyle (Mcp).Nearby, several sections of the microfibrils (Mf ) are located in the parasitophorous vacuole (PV); E-Ultrastructural detail of the peripheral zone of the oocyst showing some aspects of degradation of the microfibrils (Mf ) and the phagocyte periphery (Pgc) in which the membrane is in contact with microfibrils that appear to be disrupted.All scale bars in µm.