Abstract

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 half-open 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.

Keywords:
Oyster; microparasite; estuarine environment; mollusks; Ostreidae

Resumo

Este trabalho descreve a morfologia ultraestrutural detalhada do fagócito encarcerando um oocisto de Nematopsis (Apicomplexa) encontrado em Crassostrea rhizophorae, na cidade de Maceió (AL), Brasil. Os hospedeiros muito infectados apresentavam valvas entreabertas com retração fraca e lenta dos músculos abdutores. Número variável de oócitos de forma elipsoide, isolados e ou agrupados foi encontrado entre as miofibrilas do músculo abdutor. Cada oocisto estava encarcerado num vacúolo parasitóforo do fagócito uninucleado do hospedeiro. Os oocistos eram compostos por uma parede densa contendo um esporozoíto vermiforme uninucleado. A parede dos oocistos finos era composta de material electron-lucente homogêneo formado por três camadas de espessura igual, possuindo um orifício circular - micrópila, obstruída pelo opérculo. Os oocistos apresentavam morfologia elipsoide, sua parede era circundada por uma complexa rede de numerosas microfibrilas. Detalhes de valor taxonômico importantes foram visualizados tais como: a organização ultraestrutural da parede do oocisto e a organização da micrópila e do opérculo, além das microfibrilas que se projetam da parede do oocisto, estrutura apenas observada em microscopia eletrônica de transmissão (MET) e que pode auxiliar na identificação da espécie. Contudo, para esclarecer a posição sistemática da maioria das espécies relatadas do gênero Nematopsis é importante prosseguir com as análises genéticas.

Palavras-chave:
Ostra; microparasita; ambiente estuarino; moluscos; Ostreidae

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 Boehs G, Villalba A, Ceuta LO, Luz JR. Parasites of three commercially exploited bivalve mollusc species of the estuarine region of the Cachoeira River (Ilhéus, Bahia, Brazil). J Invertebr Pathol 2010; 103(1): 43-47. http://dx.doi.org/10.1016/j.jip.2009.10.008. PMid:19850046.
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Among the parasites infecting two types of hosts (mollusks and crustaceans) ( PRYTHERCH, 1938 Prytherch HF. Life-cycle of a sporozoan parasite of the oyster. Science 1938; 88(2289): 451-452. http://dx.doi.org/10.1126/science.88.2289.451. PMid:17736942.
http://dx.doi.org/10.1126/science.88.22... , 1940 Prytherch HF. The life cycle and morphology of Nematopsis ostrearum sp. nov. a gregarine parasite of the mud crab and oyster. J Morphol 1940; 66(1): 39-64. http://dx.doi.org/10.1002/jmor.1050660106.
http://dx.doi.org/10.1002/jmor.10506601... ), we highlight the genus Nematopsis Schneider, 1892 (Apicomplexa: Porosporidae). This is a cosmopolitan parasitic genus of apicomplexans with around 40 nominal species that have been reported in different geographic areas ( ABDEL-BAKI et al., 2012 Abdel-Baki AA, Al-Quraishy S, Dkhil MA, Al-Nasr I, Oliveira E, Casal G, et al. Ultrastructural characteristics of Nematopsis sp. oocysts (Apicomplexa: Porosporidae), a parasite of the clam Meretrix meretrix (Veneridae) from the Arabian Gulf, Saudi Arabia. Folia Parasitol (Praha) 2012; 59(2): 81-86. http://dx.doi.org/10.14411/fp.2012.012. PMid:22779107.
http://dx.doi.org/10.14411/fp.2012.012 ... ; AZEVEDO & MATOS, 1999 Azevedo C, Matos E. Description of Nematopsis mytella n. sp. (Apicomplexa), parasite of the mussel Mytella guyanensis (Mytelidae) from the Amazon estuary and description of its oocysts. Eur J Protistol 1999; 35(4): 427-433. http://dx.doi.org/10.1016/S0932-4739(99)80052-2.
http://dx.doi.org/10.1016/S0932-4739(99... ; AZEVEDO & PADOVAN, 2004 Azevedo C, Padovan I. Nematopsis gigas n. sp. (Apicomplexa), a parasite of Nerita ascencionis (Gastropoda, Neritidae) from Brazil. J Eukaryot Microbiol 2004; 51(2): 214-219. http://dx.doi.org/10.1111/j.1550-7408.2004.tb00548.x. PMid:15134258.
http://dx.doi.org/10.1111/j.1550-7408.2... ; BOWER et al., 1994 Bower SM, McGladdery SE, Price IM. Synopsis of infectious diseases and parasites of commercially exploited shellfish. Annu Rev Fish Dis 1994; 4: 1-199. http://dx.doi.org/10.1016/0959-8030(94)90028-0.
http://dx.doi.org/10.1016/0959-8030(94)... ; OZER & GUNEYDAG, 2015 Ozer A, Guneydag S. Seasonality and host-parasite interrelationship of Mytilus galloprovincialis parasites in Turkish Sea coast. J Mar Biol 2015; 95(8): 1591-1599. http://dx.doi.org/10.1017/S0025315415000740.
http://dx.doi.org/10.1017/S002531541500... ; SPRAGUE, 1970 Sprague V. Some protozoan parasites and hyperparasites in marine bivalve molluscs. In: Snieszko SF. A symposium on diseases of fishes and shellfishes. Washington: American Fisheries Society; 1970. p. 511-526. (Special Publ; no. 5). ; THÉODORIDÈS, 1962 Théodoridès J. Grégarines d’invertébrés marines de la région de Banyuls. I. Eugrégarines parasites de crustacés décapodes. Vie Milieu 1962; 13: 95-122. ; TUNTIWARANURUK et al., 2004 Tuntiwaranuruk C, Chalermwat K, Upatham ES, Kruatrachue M, Azevedo C. Investigation of Nematopsis spp. oocysts 7 species of bivalves from Chonburi Province, Gulf of Thailand. Dis Aquat Organ 2004; 58(1): 47-53. http://dx.doi.org/10.3354/dao058047. PMid:15038451.
http://dx.doi.org/10.3354/dao058047 ... ). Their vegetative and reproductive phase takes place in crustaceans (schizogonic phase), while the sporogonic phases take place in bivalves ( PRYTHERCH, 1938 Prytherch HF. Life-cycle of a sporozoan parasite of the oyster. Science 1938; 88(2289): 451-452. http://dx.doi.org/10.1126/science.88.2289.451. PMid:17736942.
http://dx.doi.org/10.1126/science.88.22... , 1940 Prytherch HF. The life cycle and morphology of Nematopsis ostrearum sp. nov. a gregarine parasite of the mud crab and oyster. J Morphol 1940; 66(1): 39-64. http://dx.doi.org/10.1002/jmor.1050660106.
http://dx.doi.org/10.1002/jmor.10506601... ). It has also been reported that the sporogonic phase can take place in gastropods ( AZEVEDO & PADOVAN, 2004 Azevedo C, Padovan I. Nematopsis gigas n. sp. (Apicomplexa), a parasite of Nerita ascencionis (Gastropoda, Neritidae) from Brazil. J Eukaryot Microbiol 2004; 51(2): 214-219. http://dx.doi.org/10.1111/j.1550-7408.2004.tb00548.x. PMid:15134258.
http://dx.doi.org/10.1111/j.1550-7408.2... ).

In Brazil, most studies have aimed to investigate the Nematopsis oocyst stages and have only reported on the morphology of this genus, while some have been based on histopathology, but exclusively, in light microscopy ( BRANDÃO et al., 2013 Brandão RP, Boehs G, Silva PM. Health assessment of the oyster Crassostrea rhizophorae on the southern coast of Bahia, northeastern Brazil. Rev Bras Parasitol Vet 2013; 22(1): 84-91. http://dx.doi.org/10.1590/S1984-29612013005000007. PMid:23538501.
http://dx.doi.org/10.1590/S1984-2961201... ; BRITO et al., 2010 Brito LO, Barros JCN, Gálvez AO, Barros FN. Presence of Nematopsis sp. (Protozoa, Apicomplexa) in the oyster, Crassostrea rhizophorae (Guilding, 1828), cultivated in the state of Pernambuco, Brazil. World Aquac 2010; 41(1): 60-62. ; CEUTA & BOEHS, 2012 Ceuta LO, Boehs G. Parasites in the mangrove mussel Mytella guyanensis (Bivalvia: Mytilidae) in Camamu Bay, Bahia, Brazil. Braz J Biol 2012; 72(3): 421-427. http://dx.doi.org/10.1590/S1519-69842012000300002. PMid:22990810.
http://dx.doi.org/10.1590/S1519-6984201... ; PINTO & BOEHS, 2008 Pinto TR, Boehs G. Nematopsis sp. (Apicomplexa: Eugregarinida) em Mytella guyanensis (Lamarck, 1819) (Bivalvia: Mytillidae) da região estuarina do rio Cachoeira, Ilhéus, Bahia, Brasil. Braz J Vet Res Anim Sci 2008; 45(2): 95-100. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2008.26705.
http://dx.doi.org/10.11606/issn.1678-44... ; QUEIROGA et al., 2015 Queiroga FR, Vianna RT, Vieira CB, Farias ND, Silva PM. Parasites infecting the cultured oyster Crassostrea gasar (Adanson, 1757) in Northeast Brazil. Parasitology 2015; 142(6): 756-766. http://dx.doi.org/10.1017/S0031182014001863. PMid:25553815.
http://dx.doi.org/10.1017/S003118201400... ; SABRY et al., 2007 Sabry RC, Gesteira TCV, Boehs G. First record of parasitism in the mangrove oyster Crassostrea rhizophorae (Bivalvia: Ostreidae) at Jaguaribe River estuary - Ceará, Brazil. Braz J Biol 2007; 67(4): 755-758. http://dx.doi.org/10.1590/S1519-69842007000400024. PMid:18278331.
http://dx.doi.org/10.1590/S1519-6984200... ). Few species have been described on the base of concomitant observations through both light and transmission electron microscopy ( AZEVEDO & CACHOLA, 1992 Azevedo C, Cachola R. Fine structure of the Apicomplexa oocyst of Nematopsis sp. of two marine bivalve molluscs. Dis Aquat Organ 1992; 14(1): 69-72. http://dx.doi.org/10.3354/dao014069.
http://dx.doi.org/10.3354/dao014069 ... ; AZEVEDO & MATOS, 1999 Azevedo C, Matos E. Description of Nematopsis mytella n. sp. (Apicomplexa), parasite of the mussel Mytella guyanensis (Mytelidae) from the Amazon estuary and description of its oocysts. Eur J Protistol 1999; 35(4): 427-433. http://dx.doi.org/10.1016/S0932-4739(99)80052-2.
http://dx.doi.org/10.1016/S0932-4739(99... ; AZEVEDO & PADOVAN, 2004 Azevedo C, Padovan I. Nematopsis gigas n. sp. (Apicomplexa), a parasite of Nerita ascencionis (Gastropoda, Neritidae) from Brazil. J Eukaryot Microbiol 2004; 51(2): 214-219. http://dx.doi.org/10.1111/j.1550-7408.2004.tb00548.x. PMid:15134258.
http://dx.doi.org/10.1111/j.1550-7408.2... ; MAGALHÃES et al., 2006 Magalhães A, Silva W, Matos E, Azevedo C. Ultrastructural morphology of Nematopsis mytella parasite Apicomplexa of bivalve molluscs of south of Brazil. Rev Cienc Agrar 2006; 46: 251-259. ; PADOVAN et al., 2003 Padovan IP, Corral L, Tavares LA, Padovan PA, Azevedo C. Fine structure of the oocyst of Nematopsis mytella (Apicomplexa, Porosporidae), a parasite of the mussel Mytella falcata and of the oyster Crassostrea rhizophorae (Mollusca, Bivalvia) from the northeastern Atlantic coast of Brazil. Braz J Morphol Sci 2003; 20: 121-124. ).

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 Belafastova IP. Gregarines of the genus Nematopsis (Eugregarinida, Porosporidae) - parasites of the Black Sea invertebrates. Parazitologiya 1996; 30: 159-173. ; PRASADAN & JANARDANAN, 1996 Prasadan PK, Janardanan KP. Nematopsis idella sp. n. two cephaline gregarines from freshwater prawns in Kerala. Acta Protozool 1996; 35: 239-243. ; SHANAVAS et al., 1989 Shanavas KR, Prasadan PK, Janardanan KP. Nematopsis rosenbergii n. sp. (Apicomplexa: Cephalina) from the brackishwater prawn, Macrobrachium rosenbergii (De Man). Arch Protistenkd 1989; 137(2): 161-164. http://dx.doi.org/10.1016/S0003-9365(89)80007-7.
http://dx.doi.org/10.1016/S0003-9365(89... ).

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 C, Matos E. Description of Nematopsis mytella n. sp. (Apicomplexa), parasite of the mussel Mytella guyanensis (Mytelidae) from the Amazon estuary and description of its oocysts. Eur J Protistol 1999; 35(4): 427-433. http://dx.doi.org/10.1016/S0932-4739(99)80052-2.
http://dx.doi.org/10.1016/S0932-4739(99... ; AZEVEDO & PADOVAN, 2004 Azevedo C, Padovan I. Nematopsis gigas n. sp. (Apicomplexa), a parasite of Nerita ascencionis (Gastropoda, Neritidae) from Brazil. J Eukaryot Microbiol 2004; 51(2): 214-219. http://dx.doi.org/10.1111/j.1550-7408.2004.tb00548.x. PMid:15134258.
http://dx.doi.org/10.1111/j.1550-7408.2... ; PADOVAN et al., 2003 Padovan IP, Corral L, Tavares LA, Padovan PA, Azevedo C. Fine structure of the oocyst of Nematopsis mytella (Apicomplexa, Porosporidae), a parasite of the mussel Mytella falcata and of the oyster Crassostrea rhizophorae (Mollusca, Bivalvia) from the northeastern Atlantic coast of Brazil. Braz J Morphol Sci 2003; 20: 121-124. ).

Considering that some undetermined species of Nematopsis occurring in several hosts belonging to different species ( ABDEL-BAKI et al. 2012 Abdel-Baki AA, Al-Quraishy S, Dkhil MA, Al-Nasr I, Oliveira E, Casal G, et al. Ultrastructural characteristics of Nematopsis sp. oocysts (Apicomplexa: Porosporidae), a parasite of the clam Meretrix meretrix (Veneridae) from the Arabian Gulf, Saudi Arabia. Folia Parasitol (Praha) 2012; 59(2): 81-86. http://dx.doi.org/10.14411/fp.2012.012. PMid:22779107.
http://dx.doi.org/10.14411/fp.2012.012 ... ; AZEVEDO & CACHOLA, 1992 Azevedo C, Cachola R. Fine structure of the Apicomplexa oocyst of Nematopsis sp. of two marine bivalve molluscs. Dis Aquat Organ 1992; 14(1): 69-72. http://dx.doi.org/10.3354/dao014069.
http://dx.doi.org/10.3354/dao014069 ... ; BOEHS et al., 2010 Boehs G, Villalba A, Ceuta LO, Luz JR. Parasites of three commercially exploited bivalve mollusc species of the estuarine region of the Cachoeira River (Ilhéus, Bahia, Brazil). J Invertebr Pathol 2010; 103(1): 43-47. http://dx.doi.org/10.1016/j.jip.2009.10.008. PMid:19850046.
http://dx.doi.org/10.1016/j.jip.2009.10... ; BRANDÃO et al., 2013 Brandão RP, Boehs G, Silva PM. Health assessment of the oyster Crassostrea rhizophorae on the southern coast of Bahia, northeastern Brazil. Rev Bras Parasitol Vet 2013; 22(1): 84-91. http://dx.doi.org/10.1590/S1984-29612013005000007. PMid:23538501.
http://dx.doi.org/10.1590/S1984-2961201... ; BRITO et al., 2010 Brito LO, Barros JCN, Gálvez AO, Barros FN. Presence of Nematopsis sp. (Protozoa, Apicomplexa) in the oyster, Crassostrea rhizophorae (Guilding, 1828), cultivated in the state of Pernambuco, Brazil. World Aquac 2010; 41(1): 60-62. ; CANESTRI-TROTTI et al., 2000 Canestri-Trotti G, Baccarani EM, Paesanti F, Turolla E. Monitoring of infections by Protozoa by the genera Nematopsis, Perkinsus and Porospora in the smooth Venus clam Callista chione from the North-Western Adriatic Sea (Italy). Dis Aquat Organ 2000; 42(2): 157-161. http://dx.doi.org/10.3354/dao042157. PMid:11023256.
http://dx.doi.org/10.3354/dao042157 ... ; COVA et al., 2015 Cova AW, Serafim JM Jr, Boehs G, Sousa JM. Parasites in the mangrove oyster Crassostrea rhizophorae cultivated in the estuary of the Graciosa River in Taperoá, Bahia. Rev Bras Parasitol Vet 2015; 24(1): 21-27. http://dx.doi.org/10.1590/S1984-29612015012. PMid:25909249.
http://dx.doi.org/10.1590/S1984-2961201... ; KUA et al., 2013 Kua BC, Salleh MT, Noraziah MR. A case study of protozoan parasite gregarine Nematopsis spp. (Apicomplexa: Sporozoa) infection in mangrove oyster Crassostrea belcheri imported from Thailand. Trop Agricult Sci 2013; 36(3): 217-224. ; PINTO & BOEHS, 2008 Pinto TR, Boehs G. Nematopsis sp. (Apicomplexa: Eugregarinida) em Mytella guyanensis (Lamarck, 1819) (Bivalvia: Mytillidae) da região estuarina do rio Cachoeira, Ilhéus, Bahia, Brasil. Braz J Vet Res Anim Sci 2008; 45(2): 95-100. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2008.26705.
http://dx.doi.org/10.11606/issn.1678-44... ; QUEIROGA et al., 2015 Queiroga FR, Vianna RT, Vieira CB, Farias ND, Silva PM. Parasites infecting the cultured oyster Crassostrea gasar (Adanson, 1757) in Northeast Brazil. Parasitology 2015; 142(6): 756-766. http://dx.doi.org/10.1017/S0031182014001863. PMid:25553815.
http://dx.doi.org/10.1017/S003118201400... ; SABRY & MAGALHÃES, 2005 Sabry RC, Magalhães ARM. Parasitas em ostra de cultivo (Crassostrea rhizophorae e Crassostrea gigas) da ponta do Sambaqui, Florianopolis, SC. Arq Bras Med Vet Zootec 2005; 57(Supl. 2): 194-203. http://dx.doi.org/10.1590/S0102-09352005000800010.
http://dx.doi.org/10.1590/S0102-0935200... ; SUJA et al., 2016 Suja G, Kripa V, Mohamed KS, Shamal P, Sanil NK. Nematopsis sp. (Apicomplexa: Porosporidae) infection in Crassostrea madrasensis and its associated histopathology. J Mar Biol Assoc India 2016; 58(1): 29-33. http://dx.doi.org/10.6024/jmbai.2016.58.1.1890-04.
http://dx.doi.org/10.6024/jmbai.2016.58... ; TUNTIWARANURUK et al., 2004 Tuntiwaranuruk C, Chalermwat K, Upatham ES, Kruatrachue M, Azevedo C. Investigation of Nematopsis spp. oocysts 7 species of bivalves from Chonburi Province, Gulf of Thailand. Dis Aquat Organ 2004; 58(1): 47-53. http://dx.doi.org/10.3354/dao058047. PMid:15038451.
http://dx.doi.org/10.3354/dao058047 ... , 2015 Tuntiwaranuruk C, Boonmameepool J, Noppharatarphakul N, Upatham ES. Ultrastructure of gametocyst of parasitic protozoan, Nematopsis sp. in Black Tiger Shrimp Penaeus monodon from the Gulf of Thailand. Acta Protozool 2015; 54(2): 137-142. ), it is acceptable to think that many of the species thus described may correspond to previously described species. On the other hand, several Nematopsis species have been described from the gregarine stage of the schizogonic phase ( CHAMBOUVET et al., 2016 Chambouvet A, Valigurová A, Pinheiro LM, Richards TA, Jirků M. Nematopsis temporariae (Gregarinasina, Apicomplexa, Alveolata) is an intracellular infectious agent of tadpole livers. Environ Microbiol Rep 2016; 8(5): 675-679. http://dx.doi.org/10.1111/1758-2229.12421. PMid:27119160.
http://dx.doi.org/10.1111/1758-2229.124... ; CHAKRABORTI & BANDYOPADHYAY, 2010 Chakraborti J, Bandyopadhyay PK. First record of a parasitic septate gregarines (Apicomplexa: Sporozoea) in the shrimp Peneaus monodon in Sundarbans of West Bengal. J Parasit Dis 2010; 34(1): 40-43. http://dx.doi.org/10.1007/s12639-010-0002-7. PMid:21526032.
http://dx.doi.org/10.1007/s12639-010-00... ; JIMÉNEZ et al., 2002 Jiménez R, Barniol L, Machuca M. Nematopsis marinus n. sp., a new septate gregarine from cultured penaeoid shrimp Litopenaeus vannamei (Boone), in Ecuador. Aquacult Res 2002; 33(4): 231-240. http://dx.doi.org/10.1046/j.1355-557x.2002.00647.x.
http://dx.doi.org/10.1046/j.1355-557x.2... ; PRASADAN & JANARDANAN, 1996 Prasadan PK, Janardanan KP. Nematopsis idella sp. n. two cephaline gregarines from freshwater prawns in Kerala. Acta Protozool 1996; 35: 239-243. , 2001 Prasadan PK, Janardanan KP. Three new species of gregarines (Apicomplexa: Sporozoea: Porosporidae) in the estuarine crabs from Kerala, India. Acta Protozool 2001; 40: 303-309. ; SHANAVAS et al., 1989 Shanavas KR, Prasadan PK, Janardanan KP. Nematopsis rosenbergii n. sp. (Apicomplexa: Cephalina) from the brackishwater prawn, Macrobrachium rosenbergii (De Man). Arch Protistenkd 1989; 137(2): 161-164. http://dx.doi.org/10.1016/S0003-9365(89)80007-7.
http://dx.doi.org/10.1016/S0003-9365(89... ). These descriptions have not included the corresponding stage of the oocysts (schizogonic phase). This is a confusing situation and there is no morphological comparison with other species that have previously been described.

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.

Results

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 1 AB), previously described in other bivalves species. This identification was matched using semi-thin sections ( Figure 1 D and Figure 2 A) and through ultrastructural analysis ( Figures 2 BE). The phagocyte cytoplasm contained variable numbers of parasitophorous vacuoles (PVs), each containing a single oocyst ( Figures 1 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.

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
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.

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 1 AE).Single oocyst was more frequently incarcerated in individualized PVs of the host cells and these cells were identified as phagocytes ( Figures 1 AE). 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 1 BE).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 1 BE).

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 2 2C). The PV was formed by a parasitophorous membrane of irregular outline that was in close contact with the cytoplasm of the phagocyte ( Figures 2 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 2 BE). 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 2 BE).

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 1 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 2 BE). 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 2 D). In favorable serial ultrathin sections, the operculum presented arcuate morphology (∩-shaped). Operculum occupied the cylindrical space of the micropyle ( Figures 2 D and 3 )

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.

The first signs of lysis occurred in the phagocytes when the nucleus became pyknotic ( Figure 2 C) and the PV membranes disappeared. The cytoplasm became lighter and numerous vesicles appeared in this region ( Figures 2 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.

Discussion

The morphology and ultrastructural analyses on the parasite oocysts and phagocytes in the infected adductor muscle, gills and mantle of the C. rhizophorae showed that this parasite belonging to the genus Nematopsis had characteristics similar to those observed in hosts from different regions worldwide ( ABDEL-BAKI et al., 2012 Abdel-Baki AA, Al-Quraishy S, Dkhil MA, Al-Nasr I, Oliveira E, Casal G, et al. Ultrastructural characteristics of Nematopsis sp. oocysts (Apicomplexa: Porosporidae), a parasite of the clam Meretrix meretrix (Veneridae) from the Arabian Gulf, Saudi Arabia. Folia Parasitol (Praha) 2012; 59(2): 81-86. http://dx.doi.org/10.14411/fp.2012.012. PMid:22779107.
http://dx.doi.org/10.14411/fp.2012.012 ... ; AZEVEDO & CACHOLA, 1992 Azevedo C, Cachola R. Fine structure of the Apicomplexa oocyst of Nematopsis sp. of two marine bivalve molluscs. Dis Aquat Organ 1992; 14(1): 69-72. http://dx.doi.org/10.3354/dao014069.
http://dx.doi.org/10.3354/dao014069 ... ; AZEVEDO & MATOS, 1999 Azevedo C, Matos E. Description of Nematopsis mytella n. sp. (Apicomplexa), parasite of the mussel Mytella guyanensis (Mytelidae) from the Amazon estuary and description of its oocysts. Eur J Protistol 1999; 35(4): 427-433. http://dx.doi.org/10.1016/S0932-4739(99)80052-2.
http://dx.doi.org/10.1016/S0932-4739(99... ; AZEVEDO & PADOVAN, 2004 Azevedo C, Padovan I. Nematopsis gigas n. sp. (Apicomplexa), a parasite of Nerita ascencionis (Gastropoda, Neritidae) from Brazil. J Eukaryot Microbiol 2004; 51(2): 214-219. http://dx.doi.org/10.1111/j.1550-7408.2004.tb00548.x. PMid:15134258.
http://dx.doi.org/10.1111/j.1550-7408.2... ; BRANDÃO et al., 2013 Brandão RP, Boehs G, Silva PM. Health assessment of the oyster Crassostrea rhizophorae on the southern coast of Bahia, northeastern Brazil. Rev Bras Parasitol Vet 2013; 22(1): 84-91. http://dx.doi.org/10.1590/S1984-29612013005000007. PMid:23538501.
http://dx.doi.org/10.1590/S1984-2961201... ; KUA et al., 2013 Kua BC, Salleh MT, Noraziah MR. A case study of protozoan parasite gregarine Nematopsis spp. (Apicomplexa: Sporozoa) infection in mangrove oyster Crassostrea belcheri imported from Thailand. Trop Agricult Sci 2013; 36(3): 217-224. ; PADOVAN et al., 2003 Padovan IP, Corral L, Tavares LA, Padovan PA, Azevedo C. Fine structure of the oocyst of Nematopsis mytella (Apicomplexa, Porosporidae), a parasite of the mussel Mytella falcata and of the oyster Crassostrea rhizophorae (Mollusca, Bivalvia) from the northeastern Atlantic coast of Brazil. Braz J Morphol Sci 2003; 20: 121-124. ; SHANAVAS et al., 1989 Shanavas KR, Prasadan PK, Janardanan KP. Nematopsis rosenbergii n. sp. (Apicomplexa: Cephalina) from the brackishwater prawn, Macrobrachium rosenbergii (De Man). Arch Protistenkd 1989; 137(2): 161-164. http://dx.doi.org/10.1016/S0003-9365(89)80007-7.
http://dx.doi.org/10.1016/S0003-9365(89... ; TUNTIWARANURUK et al., 2004 Tuntiwaranuruk C, Chalermwat K, Upatham ES, Kruatrachue M, Azevedo C. Investigation of Nematopsis spp. oocysts 7 species of bivalves from Chonburi Province, Gulf of Thailand. Dis Aquat Organ 2004; 58(1): 47-53. http://dx.doi.org/10.3354/dao058047. PMid:15038451.
http://dx.doi.org/10.3354/dao058047 ... ).

In Brazil, the genus Nematopsis has been intensively studied along the Brazilian Atlantic coast based mainly on light microscopy observations ( BRANDÃO et al., 2013 Brandão RP, Boehs G, Silva PM. Health assessment of the oyster Crassostrea rhizophorae on the southern coast of Bahia, northeastern Brazil. Rev Bras Parasitol Vet 2013; 22(1): 84-91. http://dx.doi.org/10.1590/S1984-29612013005000007. PMid:23538501.
http://dx.doi.org/10.1590/S1984-2961201... ; BRITO et al., 2010 Brito LO, Barros JCN, Gálvez AO, Barros FN. Presence of Nematopsis sp. (Protozoa, Apicomplexa) in the oyster, Crassostrea rhizophorae (Guilding, 1828), cultivated in the state of Pernambuco, Brazil. World Aquac 2010; 41(1): 60-62. ; PADOVAN et al., 2003 Padovan IP, Corral L, Tavares LA, Padovan PA, Azevedo C. Fine structure of the oocyst of Nematopsis mytella (Apicomplexa, Porosporidae), a parasite of the mussel Mytella falcata and of the oyster Crassostrea rhizophorae (Mollusca, Bivalvia) from the northeastern Atlantic coast of Brazil. Braz J Morphol Sci 2003; 20: 121-124. ; PINTO & BOEHS 2008 Pinto TR, Boehs G. Nematopsis sp. (Apicomplexa: Eugregarinida) em Mytella guyanensis (Lamarck, 1819) (Bivalvia: Mytillidae) da região estuarina do rio Cachoeira, Ilhéus, Bahia, Brasil. Braz J Vet Res Anim Sci 2008; 45(2): 95-100. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2008.26705.
http://dx.doi.org/10.11606/issn.1678-44... ; QUEIROGA et al., 2015 Queiroga FR, Vianna RT, Vieira CB, Farias ND, Silva PM. Parasites infecting the cultured oyster Crassostrea gasar (Adanson, 1757) in Northeast Brazil. Parasitology 2015; 142(6): 756-766. http://dx.doi.org/10.1017/S0031182014001863. PMid:25553815.
http://dx.doi.org/10.1017/S003118201400... ; SABRY et al., 2007 Sabry RC, Gesteira TCV, Boehs G. First record of parasitism in the mangrove oyster Crassostrea rhizophorae (Bivalvia: Ostreidae) at Jaguaribe River estuary - Ceará, Brazil. Braz J Biol 2007; 67(4): 755-758. http://dx.doi.org/10.1590/S1519-69842007000400024. PMid:18278331.
http://dx.doi.org/10.1590/S1519-6984200... ). The data from these studies do not allow comparative ultrastructural descriptions in relation to species that had previously been described ( AZEVEDO & MATOS, 1999 Azevedo C, Matos E. Description of Nematopsis mytella n. sp. (Apicomplexa), parasite of the mussel Mytella guyanensis (Mytelidae) from the Amazon estuary and description of its oocysts. Eur J Protistol 1999; 35(4): 427-433. http://dx.doi.org/10.1016/S0932-4739(99)80052-2.
http://dx.doi.org/10.1016/S0932-4739(99... ; AZEVEDO & PADOVAN, 2004 Azevedo C, Padovan I. Nematopsis gigas n. sp. (Apicomplexa), a parasite of Nerita ascencionis (Gastropoda, Neritidae) from Brazil. J Eukaryot Microbiol 2004; 51(2): 214-219. http://dx.doi.org/10.1111/j.1550-7408.2004.tb00548.x. PMid:15134258.
http://dx.doi.org/10.1111/j.1550-7408.2... ; MAGALHÃES et al., 2006 Magalhães A, Silva W, Matos E, Azevedo C. Ultrastructural morphology of Nematopsis mytella parasite Apicomplexa of bivalve molluscs of south of Brazil. Rev Cienc Agrar 2006; 46: 251-259. ). These descriptions of undetermined Nematopsis species ( BRITO et al., 2010 Brito LO, Barros JCN, Gálvez AO, Barros FN. Presence of Nematopsis sp. (Protozoa, Apicomplexa) in the oyster, Crassostrea rhizophorae (Guilding, 1828), cultivated in the state of Pernambuco, Brazil. World Aquac 2010; 41(1): 60-62. ; COVA et al., 2015 Cova AW, Serafim JM Jr, Boehs G, Sousa JM. Parasites in the mangrove oyster Crassostrea rhizophorae cultivated in the estuary of the Graciosa River in Taperoá, Bahia. Rev Bras Parasitol Vet 2015; 24(1): 21-27. http://dx.doi.org/10.1590/S1984-29612015012. PMid:25909249.
http://dx.doi.org/10.1590/S1984-2961201... ; LUZ & BOEHS, 2015 Luz MSA, Boehs G. Parasites in the oyster Crassostrea rhizophorae from farmed and natural stocks in the Bay of Camamu, Bahia, northeastern Brazil. J Parasitol Vector Biol 2015; 76: 120-128. ; PINTO & BOEHS, 2008 Pinto TR, Boehs G. Nematopsis sp. (Apicomplexa: Eugregarinida) em Mytella guyanensis (Lamarck, 1819) (Bivalvia: Mytillidae) da região estuarina do rio Cachoeira, Ilhéus, Bahia, Brasil. Braz J Vet Res Anim Sci 2008; 45(2): 95-100. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2008.26705.
http://dx.doi.org/10.11606/issn.1678-44... ; SABRY & MAGALHÃES, 2005 Sabry RC, Magalhães ARM. Parasitas em ostra de cultivo (Crassostrea rhizophorae e Crassostrea gigas) da ponta do Sambaqui, Florianopolis, SC. Arq Bras Med Vet Zootec 2005; 57(Supl. 2): 194-203. http://dx.doi.org/10.1590/S0102-09352005000800010.
http://dx.doi.org/10.1590/S0102-0935200... ) 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 C, Matos E. Description of Nematopsis mytella n. sp. (Apicomplexa), parasite of the mussel Mytella guyanensis (Mytelidae) from the Amazon estuary and description of its oocysts. Eur J Protistol 1999; 35(4): 427-433. http://dx.doi.org/10.1016/S0932-4739(99)80052-2.
http://dx.doi.org/10.1016/S0932-4739(99... ; AZEVEDO & PADOVAN, 2004 Azevedo C, Padovan I. Nematopsis gigas n. sp. (Apicomplexa), a parasite of Nerita ascencionis (Gastropoda, Neritidae) from Brazil. J Eukaryot Microbiol 2004; 51(2): 214-219. http://dx.doi.org/10.1111/j.1550-7408.2004.tb00548.x. PMid:15134258.
http://dx.doi.org/10.1111/j.1550-7408.2... ; MAGALHÃES et al., 2006 Magalhães A, Silva W, Matos E, Azevedo C. Ultrastructural morphology of Nematopsis mytella parasite Apicomplexa of bivalve molluscs of south of Brazil. Rev Cienc Agrar 2006; 46: 251-259. ), 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 Azevedo C, Matos E. Description of Nematopsis mytella n. sp. (Apicomplexa), parasite of the mussel Mytella guyanensis (Mytelidae) from the Amazon estuary and description of its oocysts. Eur J Protistol 1999; 35(4): 427-433. http://dx.doi.org/10.1016/S0932-4739(99)80052-2.
http://dx.doi.org/10.1016/S0932-4739(99... ; MAGALHÃES et al., 2006 Magalhães A, Silva W, Matos E, Azevedo C. Ultrastructural morphology of Nematopsis mytella parasite Apicomplexa of bivalve molluscs of south of Brazil. Rev Cienc Agrar 2006; 46: 251-259. ; PADOVAN et al., 2003 Padovan IP, Corral L, Tavares LA, Padovan PA, Azevedo C. Fine structure of the oocyst of Nematopsis mytella (Apicomplexa, Porosporidae), a parasite of the mussel Mytella falcata and of the oyster Crassostrea rhizophorae (Mollusca, Bivalvia) from the northeastern Atlantic coast of Brazil. Braz J Morphol Sci 2003; 20: 121-124. ).

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 Abdel-Baki AA, Al-Quraishy S, Dkhil MA, Al-Nasr I, Oliveira E, Casal G, et al. Ultrastructural characteristics of Nematopsis sp. oocysts (Apicomplexa: Porosporidae), a parasite of the clam Meretrix meretrix (Veneridae) from the Arabian Gulf, Saudi Arabia. Folia Parasitol (Praha) 2012; 59(2): 81-86. http://dx.doi.org/10.14411/fp.2012.012. PMid:22779107.
http://dx.doi.org/10.14411/fp.2012.012 ... ; AZEVEDO & CACHOLA, 1992 Azevedo C, Cachola R. Fine structure of the Apicomplexa oocyst of Nematopsis sp. of two marine bivalve molluscs. Dis Aquat Organ 1992; 14(1): 69-72. http://dx.doi.org/10.3354/dao014069.
http://dx.doi.org/10.3354/dao014069 ... ; BRITO et al., 2010 Brito LO, Barros JCN, Gálvez AO, Barros FN. Presence of Nematopsis sp. (Protozoa, Apicomplexa) in the oyster, Crassostrea rhizophorae (Guilding, 1828), cultivated in the state of Pernambuco, Brazil. World Aquac 2010; 41(1): 60-62. ; KUA et al., 2013 Kua BC, Salleh MT, Noraziah MR. A case study of protozoan parasite gregarine Nematopsis spp. (Apicomplexa: Sporozoa) infection in mangrove oyster Crassostrea belcheri imported from Thailand. Trop Agricult Sci 2013; 36(3): 217-224. ; PINTO & BOEHS 2008 Pinto TR, Boehs G. Nematopsis sp. (Apicomplexa: Eugregarinida) em Mytella guyanensis (Lamarck, 1819) (Bivalvia: Mytillidae) da região estuarina do rio Cachoeira, Ilhéus, Bahia, Brasil. Braz J Vet Res Anim Sci 2008; 45(2): 95-100. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2008.26705.
http://dx.doi.org/10.11606/issn.1678-44... ; QUEIROGA et al., 2015 Queiroga FR, Vianna RT, Vieira CB, Farias ND, Silva PM. Parasites infecting the cultured oyster Crassostrea gasar (Adanson, 1757) in Northeast Brazil. Parasitology 2015; 142(6): 756-766. http://dx.doi.org/10.1017/S0031182014001863. PMid:25553815.
http://dx.doi.org/10.1017/S003118201400... ; SABRY et al., 2007 Sabry RC, Gesteira TCV, Boehs G. First record of parasitism in the mangrove oyster Crassostrea rhizophorae (Bivalvia: Ostreidae) at Jaguaribe River estuary - Ceará, Brazil. Braz J Biol 2007; 67(4): 755-758. http://dx.doi.org/10.1590/S1519-69842007000400024. PMid:18278331.
http://dx.doi.org/10.1590/S1519-6984200... ; SOTO et al., 1996 Soto M, Pascual S, Rodrigues H, Gestal C, Abollo E, Arias C, et al. Nematopsis spp. Schneider, 1892 (Apicomplexa: Gregarinida) in bivalve molluscs off Ria de Vigo (Galicia, NW Spain). Bull Eur Assoc Fish Pathol 1996; 16(5): 157-160. ; SUJA et al., 2016 Suja G, Kripa V, Mohamed KS, Shamal P, Sanil NK. Nematopsis sp. (Apicomplexa: Porosporidae) infection in Crassostrea madrasensis and its associated histopathology. J Mar Biol Assoc India 2016; 58(1): 29-33. http://dx.doi.org/10.6024/jmbai.2016.58.1.1890-04.
http://dx.doi.org/10.6024/jmbai.2016.58... ; TUNTIWARANURUK et al., 2004 Tuntiwaranuruk C, Chalermwat K, Upatham ES, Kruatrachue M, Azevedo C. Investigation of Nematopsis spp. oocysts 7 species of bivalves from Chonburi Province, Gulf of Thailand. Dis Aquat Organ 2004; 58(1): 47-53. http://dx.doi.org/10.3354/dao058047. PMid:15038451.
http://dx.doi.org/10.3354/dao058047 ... ). Only a few of these species were identified using ultrastructural observations ( AZEVEDO & MATOS, 1999 Azevedo C, Matos E. Description of Nematopsis mytella n. sp. (Apicomplexa), parasite of the mussel Mytella guyanensis (Mytelidae) from the Amazon estuary and description of its oocysts. Eur J Protistol 1999; 35(4): 427-433. http://dx.doi.org/10.1016/S0932-4739(99)80052-2.
http://dx.doi.org/10.1016/S0932-4739(99... ; AZEVEDO & PADOVAN, 2004 Azevedo C, Padovan I. Nematopsis gigas n. sp. (Apicomplexa), a parasite of Nerita ascencionis (Gastropoda, Neritidae) from Brazil. J Eukaryot Microbiol 2004; 51(2): 214-219. http://dx.doi.org/10.1111/j.1550-7408.2004.tb00548.x. PMid:15134258.
http://dx.doi.org/10.1111/j.1550-7408.2... ).

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 Belafastova IP. Gregarines of the genus Nematopsis (Eugregarinida, Porosporidae) - parasites of the Black Sea invertebrates. Parazitologiya 1996; 30: 159-173. ; CHAKRABORTI & BANDYOPADHYAY, 2010 Chakraborti J, Bandyopadhyay PK. First record of a parasitic septate gregarines (Apicomplexa: Sporozoea) in the shrimp Peneaus monodon in Sundarbans of West Bengal. J Parasit Dis 2010; 34(1): 40-43. http://dx.doi.org/10.1007/s12639-010-0002-7. PMid:21526032.
http://dx.doi.org/10.1007/s12639-010-00... ; JIMÉNEZ et al., 2002 Jiménez R, Barniol L, Machuca M. Nematopsis marinus n. sp., a new septate gregarine from cultured penaeoid shrimp Litopenaeus vannamei (Boone), in Ecuador. Aquacult Res 2002; 33(4): 231-240. http://dx.doi.org/10.1046/j.1355-557x.2002.00647.x.
http://dx.doi.org/10.1046/j.1355-557x.2... ; KUA et al., 2013 Kua BC, Salleh MT, Noraziah MR. A case study of protozoan parasite gregarine Nematopsis spp. (Apicomplexa: Sporozoa) infection in mangrove oyster Crassostrea belcheri imported from Thailand. Trop Agricult Sci 2013; 36(3): 217-224. ; THÉODORIDÈS, 1962 Théodoridès J. Grégarines d’invertébrés marines de la région de Banyuls. I. Eugrégarines parasites de crustacés décapodes. Vie Milieu 1962; 13: 95-122. ; TUNTIWARANURUK et al., 2004 Tuntiwaranuruk C, Chalermwat K, Upatham ES, Kruatrachue M, Azevedo C. Investigation of Nematopsis spp. oocysts 7 species of bivalves from Chonburi Province, Gulf of Thailand. Dis Aquat Organ 2004; 58(1): 47-53. http://dx.doi.org/10.3354/dao058047. PMid:15038451.
http://dx.doi.org/10.3354/dao058047 ... ). 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. idellaPrasadan & Janardanan, 1996 Prasadan PK, Janardanan KP. Nematopsis idella sp. n. two cephaline gregarines from freshwater prawns in Kerala. Acta Protozool 1996; 35: 239-243. ; N. annulipesPrasadan & Janardanan, 2001 Prasadan PK, Janardanan KP. Three new species of gregarines (Apicomplexa: Sporozoea: Porosporidae) in the estuarine crabs from Kerala, India. Acta Protozool 2001; 40: 303-309. ; N. messorPrasadan & Janardanan, 2001 Prasadan PK, Janardanan KP. Three new species of gregarines (Apicomplexa: Sporozoea: Porosporidae) in the estuarine crabs from Kerala, India. Acta Protozool 2001; 40: 303-309. ; and N. quadratumPrasadan & Janardanan, 2001 Prasadan PK, Janardanan KP. Three new species of gregarines (Apicomplexa: Sporozoea: Porosporidae) in the estuarine crabs from Kerala, India. Acta Protozool 2001; 40: 303-309. ) 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.

Acknowledgements

This work was partially supported by the Engenheiro António de Almeida Foundation, Porto, Portugal; the Federal University of Alagoas, Maceió, Alagoas, Brazil; and the Research Foundation of the State of Alagoas (FAPEAL), Maceió, Alagoas, Brazil. Licence number 56475-1 of 15/November/2016, renewed in 09/May/2018 - MMA of Brazil. We would like to thank the helpful comments and suggestions of the anonymous reviewers, which contributed to the improvement of the manuscript. This manuscript is original and complies with the current laws of the countries in which were performed.

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