Carlos Azevedo

The Kingdom “Protista” or “Protoctista” encompasses a diverse group of microparasites, which includes the phylum Haplosporidia Caullery & Mesnil, 1899, where around 50 species, most of them reported as pathogenic, are described. This phylum divided into four genera: Minchinia, Haplosporidium, Urosporidium and Bonamia. Most species of haplosporidia infect bivalves and gastropods, and may infect other taxonomic groups. The taxonomic nomenclature was very controversial at the time the name of the phylum was created, with a nomenclatural variation, with the unanimously accepted designation of Haplosporidia prevailing, defended by Caullery & Mesnil in 1899. The first haplosporidian species described were the genera Minchinia Labbé, 1896 and Haplosporidium Caullery & Mesnil, 1899. However, the taxonomic characteristics described for each of the genera were not based on the same morphological aspects of the same organelles. Most haplosporidian species in this taxonomic group were initially described based on comparative morphology by LM observations; subsequently, ultrastructural morphology and, more recently, molecular technology of SSU rDNA sequences have allowed us to clarify the taxonomic position of some species. In few species the life cycle has been reported, although there are isolated states without continuity of development and cellular differentiation.

Ultrastructural and histopathological ocasional parasite survey of some samples of the teleostean, Trachurus trecae (Carangidae), collected on the Atlantic coast of Angola were found infected by prokaryotic organisms identified as belonging to two morphotypes of Gram-negative bacteria located inside on extracellular space of the connective tissue of the endomysium among the skeletal muscle fibers. By serial ultrathin sections was observed that the majority of bacteria were cylindrical morphology, some appeared in division measuring 2.8 ± 0.7 µm long x 0.9 ± 0.3 µm in diameter, other having spherical form measuring 2.8 ± 0.4 µm in diameter. The ultrastructural organization of the myofibres contacting with of region where the bacteria were located, present the myofibrils ultrastructurally disorganized, without the typical muscular striations. The pathogenic effects of the two bacterial association seemed to give rise to the myofibrils lysis and concomitant disintegration and rupture of the wall of the muscle fibres, forming a recess where the bacteria live in close proximity. Muscle fiber wall in contact with the local where the bacteria were localized appeared lysed, disorganized and without the typical striations where, among them, some bacteria were observed. Some aspects of apparent penetration of bacteria through the degraded wall of the myofibers into the interior of the fibers, were observed and discussed. This is the first description of the pathogenic activity of bacteria in muscle tissue of a teleost from the aquatic fauna of Angola.

This summary aims to provide a detailed review of the ultrastructural morphology of the microspore prototype of different species of microsporidia (Phylum Microsporidia Balbiani, 1882), found and described in the Brazilian fauna, showing with illustrations of schemes and ultrastructural aspects of images of the particularities of the structures that are part of the morphology of microspores. The subject of this microparasitological study were species of economic interest of fish, molluscs and crustaceans, but microsporidioses can occur in immunocompromised patients, predominantly those infected with HIV. On the other hand, emphasis was placed on particularities cell division processes that occur in certain life cycles of some microsporidian species. This review was exclusively based on the work published by our group of fellow researchers, with special emphasis on brazilian colleagues.

During a survey Myxozoa, four specimens of the sheepshead (18 ± 1.5 cm and 59 ± 2.5 g) (Archosargus probatocephalus) were collected in the Ipioquinha river (Maceió/AL). Transmission electron microscopy observations revealed erythrocyte agglutinations in gill capillaries located near spherical cysts containing myxospores of the genus Henneguya. This hemagglutination partially or totally obstructed the gill capillaries. Erythrocytes occurred in close adherence to each other, with a closed intercellular space. A few lysed erythrocytes were observed among agglutinated cells. The reduced lumen of the capillaries was partially filled with amorphous dense homogenous material adhering to the erythrocytes. In addition, heterogeneous masses of irregular lower electron density were observed in the reduced channel of the capillary. The agglutinated erythrocytes appeared dense and homogenous, lacking cytoplasmic organelles. The nuclei had the appearance of normal condensed chromatin masses, generally without visible nucleoli. This occurrence of hemagglutination only in the capillaries located in close proximity to the developing myxozoan cysts suggests that parasite development may be a factor triggering erythrocyte agglutination. This is supported by previous experimental studies that showed a probable correlation between parasitic infections and hemagglutination. Nonetheless, further studies are necessary in order to better understand the physicochemical processes involved in this phenomenon.

A histopathological survey was conducted to investigate the presence of microparasites in fish Archosargus probatocephalus in a river near Maceió, Brazil. Light microscope observations of fragments of gill showed the presence of small cysts containing numerous myxospores that were morphologically identified as Henneguya. Transmission electron microscopy observations further revealed several gill cells containing groups of prokaryotic cells within large cytoplasmic vacuoles. Each infected host cell displayed a single vacuole containing a variable number of Rickettsia-like cells (up to 11), some of which presented the dumbbell shape characteristic of binary fission. The Rickettsia-like cells were pleomorphic, without a nucleus and with chromatin dispersed in the cytoplasm. They had a thin electron-dense wall of Gram-negative type. The morphology of these prokaryotic was similar to those of the order Rickettsiales and was described as a Rickettsia-like organism. Histopathological evaluation showed that several vacuole membranes had a lysed appearance. Some had ruptured, thus allowing direct contact between the Rickettsia-like organism and the cytoplasm of the host cell. The rupturing of the branchial epithelium may have contributed towards reduction of the surface area of the gills, but it is not possible to say that this was the cause of the host’s death.

Mullets are ecologic and commercially important fish species. Their ubiquitous nature allows them to play critical roles in freshwater and marine ecosystems but makes them more vulnerable to diseases and parasitic infection. In this study, a myxozoan survey was performed on three species of mullet captured from a northern Portuguese river. The results disclose a high biodiversity, specifically due to the hyperdiversification of Myxobolus. Thirteen new species of this genus are described based on microscopic and molecular procedures: 7 from the thinlip grey mullet Chelon ramada, 2 from the thicklip grey mullet Chelon labrosus, and 4 from the flathead grey mullet Mugil cephalus. Myxobolus exiguus and Ellipsomyxa mugilis are further registered from their type host C. ramada, as well as six more myxospore morphotypes that possibly represent distinct Myxobolus species. Overall, the results obtained clearly show that the number of host-, site- and tissue-specific Myxobolus spp. is much higher than what would be expected in accordance to available literature. This higher biodiversity is therefore discussed as either being the result of the usage of poor discriminative criteria in previous studies, or as being a direct consequence of the biological and ecological traits of the parasite and of its vertebrate and invertebrate host communities. Bayesian inference, maximum likelihood and maximum parsimony analyses position the new species within a clade comprising all other Myxobolus spp. that infect mugiliform hosts, thus suggesting that this parasitic group has a monophyletic origin. Clustering of species in relation to the host genus is also revealed and strengthens the contention that the evolutionary history of mugiliform-infecting Myxobolus reflects that of its vertebrate hosts. In this view, the hyperdiversification of Myxobolus in mullet hosts is hypothesized to correlate with the processes of speciation that led to the ecological plasticity of mullets.

We describe, morphologically and molecularly, a new actinosporean from the intestinal epithelium of the freshwater oligochaete Ilyodrilus templetoni in the upper estuary of the River Minho, Northern Portugal. Mature actinospores resembled both echinactinomyxon and raabeia types, emphasizing the previously known lack of a clear boundary between these 2 collective groups. Historically, raabeia and echinactinomyxon types have been differentiated solely based on the shape of the valvular processes being curved or straight, respectively. Our observations, however, show that this morphological character is too variable for distinguishing between these 2 collective groups, since the actinospores of the raabeia described here displayed valvular processes that could either be straight, downward or upward curved. Several similar cases can be found in the available literature. Considering this overlap in actinospore morphology, we propose that echinactinomyxon be deemed invalid and its types be included in raabeia, as the latter constitutes the older of the 2 groups. Known echinactinomyxon types, however, should not be renamed as raabeia, as this would create unnecessary confusion. Accordingly, a more comprehensive definition of the raabeia collective group is provided. Phylogenetic analyses revealed polyphyletic clustering of raabeia/echinactinomyxon types among members of the myxosporean suborders Variisporina and Platysporina, reiterating the lack of agreement between actinosporean morphotypes and myxosporean genera. The new type described here specifically clusters within the Paramyxidium clade, alongside other SSU rDNA sequences of raabeia, echinactinomyxon, aurantiactinomyxon and synactinomyxon. Considering that most Paramyxidium spp. parasitize Anguilla anguilla (Linnaeus, 1758), future myxozoan surveys in the River Minho should include this species.

The Iberian Peninsula provides a unique freshwater ecosystem for native and endemic cypriniforms to thrive. Despite cypriniforms being hosts to multiple myxobolids worldwide, little research has been performed in this geographic location. In this study, the examination of three Iberian endemic cypriniforms showed that myxosporean richness in the Iberian Peninsula is underestimated, with three new and one known myxobolid species being reported based on morphological and molecular data (SSU).Myxobolus arcasiin. sp. is described from the kidney and gonads of the “bermejuela”Achondrostoma arcasii,M. duriensisn. sp. from the gills of the Northern straight-mouth nasePseudochondrostoma duriense, andThelohanellus paludicusn. sp. from the intestine of the Southern Iberian spined-loachCobitis paludica.Myxobolus pseudodisparGorbunova, 1936 is further reported from several organs ofP. duriense, and from the spleen ofA. arcasii. The occurrence ofM. pseudodisparin endemic Iberian species reveals that host-shift followed its co-introduction with central European leuciscids into this geographic location. Several other myxobolids originally described from barbels in central Europe have also been reported from the Iberian endemic cypriniformLuciobarbus bocagei. Nonetheless, except forM. musculi, the identification of these myxobolids inL. bocageiis here shown to be dubious and require molecular confirmation. Phylogenetic analyses revealM. arcasiin. sp. andM. duriensisn. sp. clustering within different lineages of leuciscid-infecting species, showing that myxobolids entered Leuciscidae as hosts multiple times during their evolution. Constituting the first myxobolid reported from the subfamily Cobitinae,Thelohanellus paludicusn. sp. stands alone in the tree topology.

An aurantiactinomyxon type is described from the marine naidid Tubificoides pseudogaster (Dahl, 1960), collected from the lower estuary of a Northern Portuguese River. This type constitutes the first of its collective group to be reported from Portugal, and only the fourth described from a marine oligochaete worldwide. Extensive morphological comparisons of new aurantiactinomyxon isolates to all known types without available molecular data are proposed to be unnecessary, given the artificiality of the usage of morphological criteria for actinosporean differentiation and the apparent strict host specificity of the group. Recognition of naidid oligochaetes as the hosts of choice for marine types of aurantiactinomyxon and other collective groups, suggests that the family Naididae played a preponderant role in the myxosporean colonization of estuarine communities. Molecular analyses of the type in study further infer its involvement in the life cycle of Paramyxidium giardi (Cépède, 1906) Freeman and Kristmundsson, 2018, a species that infects the kidney of European eel Anguilla anguilla (Linnaeus, 1758) and that has been reported globally, including from Portuguese waters. The low intraspecific difference registered in relation to Icelandic isolates of P. giardi (0.6%) is hypothesized to result from the emergence of genotypically different subspecies due to geographic isolation.