Steinhausia mytilovum in Mytilus galloprovincialis (The Case of Atlantic Northwest Africa-Morocco)

Steinhausia mytilovum (Field, 1924) is a microsporidian parasite that infects female individuals of Mediterranean mussel, Mytilus galloprovincialis . The aim of this work was to evaluate the incidence and histopathological effect of the microsporidian in the host from the Atlantic coast of northwest Africa. Samples were collected monthly during two years (March 2009 - March 2011) at three sites; north, middle and south, along the Moroccan coast. The prevalence showed to be the highest in the north (0% - 26.66%) and south (0% - 75%), respectively, while mussels from the middle sampling site showed no infection of S. mytilovum . The seasonality of S. mytilovum was investigated as well, although no significant variation of its incidence was observed in respect to season. The most usual histopathological characteristic of the S. mytilovum infection was hemocytic infiltration in gonads, showing a statistically significant relationship to the S. mytilovum infection ( P <.001). Further investigation is recommended to study influences of the biological and physical parameters on the infections of S. mytilovum in the natural populations of the Mediterranean mussel along the Moroccan coast.


INTRODUCTION
Steinhausia mytilovum (Chitridiopsidae, Microsporidia) is an intracellular parasite of bivalve oocytes that develops on either the surface or interior of its nucleus [1]. It can damage the mussel female gametes (Mussel Egg Disease) but unlikely is lethal to the host [2]. The microsporidian could be observed in mussels from early stage of gametogenesis to spawning [3].
Firstly described in the Mediterranean mussel Mytilus galloprovincialis Lamarck, 1819 along the Italian coast [4], it has a larger geographical distribution recognized today [5]; over the European Atlantic coasts from Norway [6] to Greece [7].
Previously, S. mytilovum has been observed in oocytes of Eurhomalea lenticularis from Chile, showing no evidence of cell damage in oocyte of the host [8]. It was also in the mussel Mytella guyanensis from the Amazon river estuary [9], as well as in ova of mussels from Atlantic and west coasts of USA [10,11]. It affected the condition index of infected Mytilus galloprovincialis from Greece and induced a strong hemocytic infiltration within affected gonadal follicles [7].
With the exception of approximately dozen tonnes of harvested Cassostrea gigas (oyster) and Mytilus galloprovincialis (mussels), there are almost no products of marine aquaculture in Morocco [12]. To bridge this gap, the fishery sector in Morocco developed in 2009 a strategy plan called "Halieutis" [13,14], integrated by coupling in the institutional frame of cooperation with the European Union [13].
Therefore, research of pathologies occurring in natural wild populations is necessary to collect fundamental information to secure future sustainable aquaculture.
The aim of this study was to evaluate incidence and effect of S. mytilovum in oocytes of the Mediterranean mussel along the Moroccan Atlantic coast, focusing on three natural biotopes of the host; Moulay Bousselham in the north, Cap Beddouza in the middle and Imessouane in the south.
Monthly in two years (March 2009 -March 2011), 30 bivalves with shell length equal or over 40 mm measured from the umbo, were sampled and shipped on ice to the laboratory, where they were processed immediately. The sex of mussel was determined with histological inspection. Gonads were cut at the central portion of the mantle, fixed in Davidson's solution for 24 hours, dehydrated in ascending ethanol concentrations and embedded in paraffin blocks. Histological sections (2 µm) were stained in hematoxylineosin Y and studied under light microscope (Leica DM2500).
In total, 720 bivalves were studied by histology at Cap Beddouza. Samples from Imessouane site of July 2009 and August 2009 were discarded because of unsatisfying fixation procedure (N=660), while samples from Moulay Bousselham in January 2010, January 2011 and March 2011 were not taken due to environmental conditions (N=630). The overall sample including the three sites was 2010 individuals.
Prevalence was evaluated according to Bush et al. [15]. Further, due to its location in the oocytes, Steinhausia mytilovum infects only females or hermaphrodites (Table 1). Thus, its prevalence was calculated only from female and hermaphrodite data [2,5]   In Imessouane site, S. mytilovum was more frequently observed during the two years of study (Fig. 4)  In order to determine the side effect of Steinhausia mytilovum on infected gonads, histological sections were inspected for the presence of hemocytic infiltration. Females from Imessouane site revealed heavy hemocytic infiltrations in the gonad, in the connective tissue and epithelia of gonadal follicles of several infected mussels. This condition was observed in some individuals and could not be linked to any detectable infected oocyte. (Table 2 the result of logistic regression method in the

Steinhausia mytilovum in oocytes of Mytilus galloprovincialis Imessouane sampling site, between March 2009 and March 2011
In order to determine the side effect of on infected gonads, histological sections were inspected for the presence of hemocytic infiltration. Females from Imessouane site revealed heavy hemocytic infiltrations in the gonad, in the connective tissue and epithelia of gonadal follicles of several nfected mussels. This condition was observed in some individuals and could not be linked to any Table 2), revealed the result of logistic regression method in the case of the dependant variable (presence or absence of hemocytic infiltration) according to the descriptive variable (presence or absence of Steinhausia). Logistic regression showed that the existence of microsporidian infection had a relationship with the presence of hemocytic infiltration; the infection by Steinhausi that there was hemocytic infiltration ( ( Table 2).

DISCUSSION
The increase of aquaculture industrialization provoked the frequent apparition of epizooties in marine bivalves [16,17] resulting in serious propagation, as witnessed by the disappearance of certain bivalve populations. The parasites are major disease-causing agents of marines bivalves [16], because bivalves facing permanent parasite infection have compromised immunity system [18,19,20,21].
In this study, the presence of Steinhausia mytilovum in female of mussels sampled during two years of monitoring was assessed for the first time along the Atlantic coasts of Morocco. Using histopathological approach systematic sampling of mussels from north (Moulay Bousselham), middle (Cap Beddouza) and south (Imessouane) of Atlantic Moroccan coast with systematic sampling allowed us to investigate geographic and seasonal dynamics of the Mussel egg disease.
The microsporidian S. mytilovum, intra-oocytic parasite of the mussel was identified in Morocco, which widens the area of its distribution along the Northwest Atlantic coast extending from Norway [6] to the northwest of Africa (Morocco).
The parasite was more prevalent in south (Imessouane) than in north (Moulay Bousselham), while mussels from the middle sampling site (Cap Beddouza) showed no infection of S. mytilovum. The difference of prevalence between the sampling sites could be explained by the wide variability of environmental conditions for the three studied populations.
Various developmental forms of S. mytilovum observed during this study were identical to those described in other populations of parasitized mussels [22,23,24,10] However, all three studied site in Morocco, showed a number of oocysts, cylindrical sporocysts without any track of spores. This shape could be considered as a premature stage or state of Steinhausia lysis. To avoid any doubt, these cases were not considered in our statistical analysis, moreover because they were not observed mussels studied along the Mediterranean coasts [22].
The highest prevalence of this parasite in the North of Morocco (26.66%) was lower than the one found in the females of Mytilus galloprovincialis in California [11], in Galicia (28.3%) [2] and in M'diq (76.5%) [22]. This maximum is also lower than that of the Moroccan south (75%). Previously, significant relationship between microsporidian prevalence and temperature or salinity was not found [22], possibly explaining the lack of seasonality of prevalence in Moulay Bousselham and Imessouane, although this coastal part of the Atlantic Moroccan coast is very dynamic between the seasons [29]. The temporal variation were studied in the Mediterranean coasts [22] and in the Atlantic [3]. [3] and [22] found a significant relationship between the infection of S. mytilovum and reproductive cycle of mussels, particularly strengthening during the spawning period.
The environmental conditions of water in a particular site would indirectly influence to the infections of S. mytilovum by determining the timing and intensity of reproduction. Indeed, the highest prevalence of S. mytilovum in mussels from Imessouane might be explained by the abundance of mature oocytes through entire seasons which was indicated by [30]. With regard to the environmental context in Imessouane, the temperature has a very dynamic rate of variations, with recesses in summer [30]. This region is subject to permanent upwelling entire seasons, which have the greatest impact on nutrient-enriched waters [29].
Furthermore, the histological effect of this parasite on infected tissue was investigated from samples collected in Imessouane, showing that hemocytic infiltrations in gonads was significantly related to the presence of the parasite (P<.001), suggesting that the existence of infected oocytes in mussel did multiply the risk of hemocyte infiltration at the gonads. This is accordance to most of previous reports [2,3,19], in contrast to [26]. In [23], intensive development of hemocytic infiltration limited the healthy growth of the other organs, which considering the potential risk upon the aquaculture in Imessouane, implicates the need for a continuous monitoring of the infection and impact of the S. mytilovum.
Finally, the absence of S. mytilovum at Cap Beddouza located in the middle of Moroccan coast, could have different yet unidentified reasons, related to host's immune status. Also, it should be noted that conversely to the mussels in north and in south, the spawning was periodic in this site (unpublished data). This point deserves further consideration by the study of the reproductive characteristics of each treated female and its conditions of life, in order to find an answer to this question.
In summary, this is a part of preliminary study aiming to a more detailed research of the extent of S. mytilovum along Moroccan coast: M'diq, Moulay Bousselham, Cap Beddouza and Imessouane.

CONCLUSION
During a 25-month period, we sought the presence of the parasite Steinausia mytilovum in Mytilus galloprovincialis in three sites along the Moroccan Atlantic coast: Moulay Bousselham, Cap Bedouzza, Imessouane, was assessed. There was a clear differentiation of the presence at these three sites. At Moulay Bousselham, we found the presence of S. mytilovum throughout the study period was evenly distributed over the seasons. At Cap Bedouzza, no cases of S. mytilovum were recorded. At Imessouane, the parasite was intensely present, seasonally uniform throughout the studied period. Moreover, its presence is strongly correlated with the gonad hemocytic infiltration.