Increasing fish demand worldwide makes it necessary to impel the development of the aquaculture industry to compensate for the stagnation of fisheries extraction (FAO 2020). The expansion of aquaculture of new fish species and geographical areas brings new health challenges that must be faced with research in fish diseases and wellness. Among pathogens, parasites go often unnoticed as natural factors affecting fish populations, however natural mortalities and decreases in fish captures related to parasite outbreaks have sometimes been reported (Lloret et al. 2012). In contrast, parasitoses are known as main threats to several fish cultures worldwide, as several parasites proliferate in culture conditions provoking abnormally severe pathologies (Dyková 2006, Ogawa 2015).
Mediterranean aquaculture has been focused in two fish species, the European seabass Dicentrarchus labrax and the gilthead seabream Sparus aurata, both cultures affected by several parasites; e.g. the microsporidian Enterospora nucleophila and the monogenean Diplectanum aequans (in D. labrax) or the myxozoan Enteromyxum leei and the monogenean Sparicotyle chrysophrii (in S. aurata) (Dezfuli et al. 2007, Fleurance et al. 2008, Antonelli et al. 2010, Palenzuela et al. 2014). With the aim of diversifying Mediterranean aquaculture, new fish cultures are being developed, including highly valuable species of fast growth such as the ABT, (which life cycle has been closed in culture conditions) (De la Gándara et al. 2017). The culture of the bluefin tuna species (Thunnus spp.) has experienced a fast expansion worldwide, partly caused by the overexploited stocks. In both wild and cultured tunas, parasites are known as the main pathological agents, including external (as copepods related to mild to severe damage; see Hayward et al. 2009) and internal parasitoses (aporocotylid trematodes causing significative mortalities in tuna juveniles (Ogawa et al. 2011, Shirakashi et al. 2012). The only microsporidian reported to date in the ABT is M. milevae, although it has only been found in isolated episodes (Mladineo et al. 2011, Mladineo and Lovy 2011). The disease herein described is provoked by a different microsporidian species (G. thunni sp. nov.) related to conspicuous and severe pathologies in fish and may become a relevant problem for the culture of the bluefin tunas, in particular for the Mediterranean ABT.
Currently, 35 species of Glugea have been described (Azevedo et al. 2016, Mansour et al. 2020). Glugeathunni sp. nov. possess the morphological traits of the genus Glugeasensu Lom (Lom 2002): unpaired nuclei throughout development, thin membrane-like wall of parasitophorous vesicle, monomorphic mature spores and isofilar polar tube coiled in single row. This diagnosis would include the new described species in the subclade G1 described by Mansour et al. (Mansour et al. 2016) including mostly Mediterranean parasites. However, Lom’s (Lom 2002) generic description would not include the six congeneric species more recently described, mostly from the Red Sea and Arabian Gulf, included in the subclade G2 (Mansour et al. 2016), in which polar tubes are arranged in several rows (this trait not described in G. epinephelusis) (Zhang et al. 2004, Wu et al. 2005, Mansour et al. 2016). Within the subclade G1, other similar species to the new Glugea species are G. anomala, G. gasterostei, G. hertwigi, G. plecoglossi, G. sardinellensis and G. stephani, based on the range of number of coils and the spore width range; however, the spore of G. thunni sp. nov. is shorter in mean (Canning et al 1982, Takvorian and Cali 1983, Takahashi and Egusa 1977a, Lovy et al. 2009, Tokarev et al. 2015, Mansour et al. 2016). Within this group, the most similar species is G. sardinellensis with a similar spore shape and the same range of number of coils (13–14): however, the new species is different from G. sardinellensis by the above-mentioned shorter spore and the much larger maximum size of the xenomas (probably related with the host size: T. thunnus vs. Sardinella aurita).
Regarding the molecular results from both phylogenetic trees with long and short sequences, the distribution of the Glugea species in the present study were identical to the ones observed in (Mansour et al. 2016). Glugeathunni sp. nov. is included in the G1 group cited above, which is congruent with the morphological similarity. However, the relationships between species within this G1 group are not well resolved due to the short sequences available and the low genetic divergences obtained in the SSU-LSU genes (Figure 5 and Table 2). The two closest species genetically to G. thunni sp. nov. are G. hertwigi, from the intestine of Osmerus epperlanus and G. anomala, from the muscle of Gasterosteus aculeatus. Low but significant differences among these species are observed only by using longer sequences of G. anomala, G. hertwigi and G. thunni sp. nov. (used in the second alignment of present work): p-distances range from 0.4% of differences between G. thunni sp. nov. vs.G. hertwigi to 1.1% from G. hertwigi vs. G. anomala (Figure 6 and Table 3). The phylogenetic tree resulting from the long sequences revealed G. anomala as the closest species to G. thunni sp. nov., instead of G. hertwigi. Surprisingly, contrary to the morphological information, G. sardinellensis was the most clearly distant species to G. thunni sp. nov. among those of the G1 group with at least 2.4–2.7% of differences in respect to their other relatives (Figure 5 and Table 2). An additional sequence labelled as “G. plecoglossi” (KY882286, unpublished) exists in GenBank. This microsporidian could have been inaccurately identificated, as its sequence is different to those of G. plecoglossi from other studies but almost identical to G. thunni sp. nov. In absence of morphological confirmation, molecular results indicate that “G. plecoglossi” (KY882286) and G. thunni n. sp could be the same species. This information could be useful to determine the transmission path in ABT cultures, as “G. plecoglossi” (KY882286) was found in a clupeid (Sardina pilchardus, Clupeidae), a fish that is commonly used as bait to feed tuna in the Spanish farms (e.g., Sardinella aurita, Clupeidae). It is worth mentioning that the other microsporidia species in bluefin tunas, Microsporidium sp. and M. milevae (Zhang et al. 2004, Mladineo and Lovy 2011), are not included in these comparisons as they are not genetically or morphologically close.
In recent years, molecular data has become an essential tool for taxonomical analyses of the microsporidia, however most of the species are only characterized by their ultrastructure, xenoma traits, host specificity or life cycle (Corradi and Keeling 2009, Azevedo et al 2016). The fact that only 14 sequences of Glugea spp. (including G. thunni sp. nov.) are available in Genbank makes it necessary to combine molecular analyses and other biological traits to elucidate the phylogenetic relationships of the microsporidians. In this context, the morphological and molecular classifications must be congruent. Several Glugea species recently described, which have been genetically included in G2 according to Mansour 2016, do not accomplish one of the diagnostic traits of the genus, which is the arrangement of polar tubes in a single row (Lom 2002) (several rows in G2). Based on the different morphology and the separation of G1 and G2 in phylogenetic trees, the inclusion of G2 species within genus Glugea seems doubtful. We also strongly recommend obtaining longer sequences, with similar coverage, in order to obtain more solid results to clarify the phylogenetic relationships among this diverse parasite group.
According to (Azevedo et al 2016), the species of Glugea either have preference for smooth musculature or connective tissues of visceral organs. Glugea thunni sp. nov. shares this habitat preference with G. hertwigi, one of the phylogenetically closest species (Lovy et al. 2009). The infection of visceral mesenteries in this investigation allowed a wide parasite dispersion, not only in the caecal mass, but also in other intestinal regions and the liver; moreover, this extensive infection had to have been achieved in a relatively short time, due to the young age of the specimen (five months). The impact of this parasite seems different to that of the other microsporidians in bluefin tunas; Microspora sp. was reported in the muscle of T. orientalis (Zhang et al 2010), which could affect product value, while M. milevae infects the muscularis mucosa of T. thynnus (Mladineo and Lovy 2011), which could affect the intestinal function. However, these Microspora spp. infections seem more localized than that which is associated to G. thunni sp. nov., and therefore their consequences appear to be milder. Moreover, the massive alterations of viscera associated with G. thunni sp. nov. is likely to cause rejection by the consumer.
The new species shows a high capability to spread within the host, reaching a high intensity, however the parasite was found in only one fish of the sea cage. Transmission of fish microsporidians is described as trophic and direct, although some crustaceans could also take part in the life cycle (Lom and Nilsen 2003, Lom and Dyková 2005). In culture conditions, a small number of crustaceans of the zooplankton can reach sea cages, but the most probable infection path of the parasite is through bait or by cannibalism. The transmission capability of these parasites among different tunas has been quite limited, however, in view of the severe consequences of the parasite, prevention measures are needful. The removal of dead fish is highly recommended, as well as, when possible, ill and moribund fish. Nonetheless, infected food appears to be the main issue to deal with this disease, as it is the most probable pathway for this parasite to have entered in the cultures, as tunas of this study were not captured from the wild for fattening. These ABTs were born in captivity and fed with thawed bait, mostly clupeids. Glugea thunni sp. nov. could also infect clupeids as although the type host is T. thynnus, clupeids are frequent hosts of Glugea spp. (Mansour et al 2016) and, more importantly, the new species sequence is the same as KY882286 in Genbank, an unpublished sequence apparently wrongly identified as “G. plecoglossi” from Sardina pilchardus (Clupeidae). Therefore, an adequate management of the bait is highly recommendable. Bait is routinely frozen (approximately, -18ºC) to avoid horizontal transmission of anisakid nematodes, an important concern for consumer health. This process would also affect G. thunni sp. nov. infectivity. The development of G. plecoglossi is known to be slowed at -16ºC (Takahashi and Egusa 1977b) and G. stephani experimental infection failed at -15ºC (Olson 1976). However, it is known that some microsporidians show a high resistance to low temperatures (up to -80ºC) (Maddox and Solter 1996). The harshness of this parasite makes it necessary to study its viability at low temperature.
In summary, it is very important to highlight the potential degree of damage of this microsporidian in cultures of ABT, one of the most expensive and appreciated fish worldwide. Nowadays, there are no effective treatments against microsporidian in fish, except for some sporadic and inconclusive reports (toltrazuril for G. anomala and fumagilin for G. plecoglossi, see Fleurance et al. 2008). Other fungicides or new therapeutic strategies to control microsporidian diseases are needed. Thus, prevention appears to be the most recommendable way to cope with disease, which requires knowledge of the transmission paths. Future investigations should therefore focus on: i) searching for the parasites in clupeids of bait to determine their role as possible disease entry; ii) studying the effect of low temperatures in the microsporidian infectivity; and iii) finding alternative ways to treat the food to inactivate the parasite. Despite the lack of this information, avoiding dangerous practices as the use of fresh and never frozen bait is highly recommendable to prevent this disease, especially when clupeid fishes are used as food.