Protist (Coccidia) and Related Diseases

Coccidia of the genus Aggregata is the most widely distributed coccidian in cephalopods. Damages caused to the hosts include mechanical (tissue injury), biochemical (malfunction of digestive enzymes), and molecular (affects cellular immune response) effects. However, coccidiosis is not a fatal disease to the cephalopod host; it severely weakens its innate immunity making it vulnerable to secondary infections. Therefore, coccidia of the genus Aggregata are considered the most dangerous parasite for cephalopods affecting wild species of notable economic importance for fishery and aquaculture activity. The pathology caused by coccidiosis to the most important European cephalopod species is the subject of the present chapter.

is highly prevalent in most of the studied cephalopod hosts and reaches severe levels of infection that compromise the host well-being at functional, biochemical, and molecular levels. Traditional techniques of detection are still used until advanced protocols are being designed. Detection of coccidiosis is particularly important in species of aquaculture relevance such as Octopus vulgaris and Sepia officinalis, both caught from the wild for rearing and naturally infected by Aggregata spp. Hence, the study of coccidiosis in cephalopods including characterization, detection, and eradication of the parasite is under research. The mentioned protistan that infects cephalopods hosts is far from being well known. However, the up-to-date knowledge regarding biological aspects of the parasites and histopathological damage caused to the cephalopod host is presented in this chapter.

Etiology and Epidemiology
Coccidiosis is caused by an obligate, intracellular protozoa classified in the phylum Apicomplexa, family Aggregatidae. To date, 10 species have been described worldwide in octopus, squid, and cuttlefish, being Aggregata the single genus recognized (Table 9.1), and all the species known are parasites causative of coccidiosis (Hochberg 1983). The intensity of infection can be as high as 82 Â 10 6 sporocyst/digestive tissue of the cephalopod host (Pascual et al. 1996;Gestal 2000). The prevalence of infection varies among the cephalopod species. The highest prevalence (98-100%) is recorded in O. vulgaris and S. officinalis (Pascual et al. 1996). In contrast, the lowest prevalence of infection (3%) has been recorded in the flying squid, Todarodes sagittatus and all of them in the NE Atlantic . In the wild O. vulgaris from Adriatic and Ionian seas, 100% of prevalence were observed, whereas 98% were recorded in octopuses from Tyrrhenian Sea with no mortalities associated with coccidiosis reported from both localities (Tedesco et al. 2017).
Identification of Aggregata spp. relies on the morphological characterization of sporogonial stages (size, shape, number of sporozoites per sporocyst and ornamentation sporocyst) and parasite-host specificity. To date, the best known species are A. octopiana, parasite of O. vulgaris, and A. eberthi, which infects S. officinalis. A. octopiana characterizes because of the spiny sporocyst and eight sporozoites inside each. In contrast, smooth sporocysts harboring three sporozoites characterize A. eberthi ( Fig. 9.1) (Dobell 1925;Gestal et al. 1999). The Aggregata species from the  Narasimhamurti (1979) Adriatic differs morphologically from those in the NE Atlantic, Tyrrhenian, and Ionian seas in the appearance of the cyst wall (smooth in the former, spiny in the latter) and some minor variations in the size of oocysts and sporocysts (Tedesco et al. 2017). All the species show the same number of sporozoites (being eight), and the characterization of the SSU rRNA gene that complements morphological and morphometric descriptions provides molecular support that distinguish among Aggregata species from NE Atlantic, Adriatic, Ionian, and Tyrrhenian seas (Kopečná et al. 2006;Tedesco et al. 2017). Phylogeny indicates three lineages of which Adriatic isolates significantly separated from those of the Ionian and Tyrrhenian seas although being more related to Ionian isolates than the rest. These observations all together evidence the existence of different Aggregata species or subspecies complex in octopuses from different seas (Tedesco et al. 2017).

Pathogenesis
Coccidiosis is a disease that affects the digestive tract of cephalopods. The Aggregata spp. life cycle is heteroxenous; asexual merogonial stages develop inside the gut of the intermediate host, which is a crustacean ( Fig. 9.2). The infective stage called merozoite invades the intestinal mucosa of the crustaceans and develops intracellularly into meronts that remains awaiting until the intermediate host is eaten by the cephalopod. Merogonial stages ingested by the cephalopod are able to infect the whole digestive tissue tract of the mollusk and epithelial cells in additional tissues.
Unusual organs of infection such as the connective tissue of gills, mantle, arms, visceral mass and mesentery of the host have been recorded when it harbor an intense infection. It is named extraintestinal coccidiosis, and most of the time, it is macroscopically identified because the presence of white oocysts on the tissue (Figs. 9.3 and 9.4) simply opening gently the mantle cavity of the cephalopod (Sykes and Gestal 2014). Extraintestinal infection has been recorded in cephalopods reared in floating cages from the NE Atlantic.
Either the site of extraintestinal infection, the parasite causes hypertrophy of invaded cells, hemocytic infiltration and activates phagocytosis by hemocytes ( Fig. 9.5) (Gestal et al. 2002a;Pascual et al. 2006). In the Adriatic Sea, Aggregata spp. has been also identified in experimentally reared O. vulgaris (250-500 g) fished from wild (Island of Brač, Adriatic Sea) and held individually in 4 m 2 concrete tanks. After 8 months of rearing without expected weight gain, animal ceased the feeding, became excitated and aggressive, lacked the capacity of camouflage, and developed epidermal lesions on the mantle and arms ( Fig. 9.5), with visible miliary protozoan oocysts (Mladineo and Jozić 2005). Extraintestinal gamogonial and sporogonial Aggregata stages infecting arms and gills triggered notable infiltrated hemocytes and replacement of the host tissue (Mladineo and Bočina 2007). After merogonial stages are ingested by the cephalopod, the establishment of the infection inside the mollusk takes place. The infection initiates with gamogony, the formation of gametes, which occurs by unequal fission of merozoites inside the cephalopod digestive tract. Macrogametes (♀) and microgametes (♂) are formed. The macrogamete is the most conspicuous one due to their round to ovoid in shape, central nucleus, and large nucleolus that stains darker than the rest of the cell when H&E is used (Fig. 9.6). Although the infection can be recorded in the whole digestive tract of the mollusk, no infection is recorded in the stomach because the lumen is covered by a wide cuticle that reduces the nutrient intake (Garri and Lauria de Cidre 2013). A similar but thick cuticle covers the crop and esophagus (Garri and Lauria de Cidre 2013) that make nutrients and space available for infection, although in low frequency (Gestal et al. 2002a). Therefore, the target organs of infection are the non-cuticularized cecum, a tubular, spiraled organ and the gut of the cephalopod (Gestal et al. 2002a). In these tissues, the macrogamete once fertilized by a microgamete produces a zygote that in turn suffers from multiple fission, and gives rise to the sporogonial stage where oocysts, sporocysts and the infective sporozoites are formed (Gestal et al. 2002a) ( Fig. 9.7). The ultrastructural analysis by transmission electron microscopy (TEM) of the sporoblast formation shows the presence of multiple nuclei inside each oocyst ( Fig. 9.8), while each sporoblast has a single nucleus and a thin sporoblast cover ( Fig. 9.9). The development of each sporoblast into sporocyst results in the formation of the sporozoites inside (the specific number is species specific) and a thickening of the sporocyst wall ( Fig. 9.10). The infection by macrogametes and initial sporogonial stages induces a strong hemocytic infiltration, distention and rupture of the tissue infected ( Fig. 9.11). The cecum wall is covered by longitudinal folds lined with cylindrical, simple, and ciliated epithelium (Garri and Lauria de Cidre 2013) that can be totally replaced by the parasite (Fig. 9.12). The infection by coccidia triggers hemocytic defensive activities such as phagocytosis of sporogonial stages, encapsulation of oocysts and gametes by flattened hemocytes and then connective tissue (Fig. 9.12) in order to isolate the parasite (Castellanos-Martínez and Gestal 2013). Mature sporocysts  are released to the lumen of the cephalopod digestive tract in order to discharge the parasite to the aquatic medium in the feces of the mollusk and make them available for infecting a crustacean intermediate host (Hochberg 1983). The release of the parasite causes detachment of the epithelial cells and rupture of the connective tissue. As a result, the mucosal folds atrophy and ulcers in the digestive tissue tract of the host appear ( Fig. 9.13). Coccidiosis originates acidification of the mucosal lumen that affects the stability of the digestive enzymes and nutrient absorption. It is exacerbated by the destruction of the mucosal intestinal epithelium after parasite release that also impedes the nutrient uptake of the cephalopod host (Gestal et al. 2002b). Signs of coccidiosis in cephalopods include malabsorption syndrome, decrease of cephalopod condition, the number of hemocytes, plasmatic protein, and even the concentration of copper, the respiratory pigment of hemocyanine in the hemolymph, is affected (Gestal et al. 2002b(Gestal et al. , 2007. Intense coccidiosis also alters the cephalopod immune response. Immune genes involved in pathogen recognition and regulation are up-regulated in the cecum of highly infected hosts indicating active detection of parasite-derived ligands (Castellanos-Martínez et al. 2014a). The antioxidant gene peroxiredoxin, responsible to protect the host against oxidative stress, is up-regulated in the cecum of cephalopod with high parasite load. However, peroxiredoxin is weakly expressed in hemocytes of such hosts, suggesting that coccidia might suppress the respiratory burst and, thus, affect the octopus cellular immune response (Castellanos-Martínez et al. 2014a, b).

Diagnosis
Signs of infection are not easily visible in the infected cephalopod hosts. Gross detection of coccidia can be realized by direct observation of macroscopic white cysts when the host is highly infected (Mayo-Hernández et al. 2013), and small pieces of digestive epithelia can be observed in feces as a result of severe infection (Dobell 1925;Hochberg 1990). Fecal oocyst count is a common method for detecting coccidia, although it is not yet standardized in cephalopods. Oocyst and sporocyst detection in feces is being under standardization as a diagnostic approach in O. vulgaris. Presently, the challenge is focused on the design of molecular probes for detecting coccidia in the cephalopod feces that avoid the need to sacrifice the host, as is currently done, and provide certainty on experiments that uses    (Gestal et al. 2002a;Sykes and Gestal 2014;Sykes et al. 2017). However, while this approach is reached, the positive detection of developmental stages of Aggregata spp. is still performed by standard histological procedure of the digestive tissue tract of dead hosts. Further stain with hematoxylin-eosin is enough to clarify the presence of parasitic stages and assess tissue damage.

Concluding Remarks
Coccidiosis is a common, chronic disease in cephalopods that destroy the cecum and intestinal mucosa of host, although extraintestinal infection can also affect mantle, arms, and gills connective tissue. From the 10 Aggregata species described, all of them are pathogenic and causative of coccidiosis in cephalopods that can be hard to detect. However, malabsorption syndrome, decrease in the number of hemocytes, plasmatic protein, and iron in hemolymph, as well as up-regulation of immune genes, are among the notable signs of disease and may be accompanied by discharge of small pieces of tissue lumen. The age of cephalopods when they are infected by Aggregata spp. for the first time has not been determined; however, specimens as small as 5 mm mantle length have been found infected in the Ria of Vigo (NW Atlantic). Any risk group based on age or length has been determined and studied for differences in severity effects of the infection. However, clear differential effects have been noted regarding the number of sporocyst infecting the digestive tract of hosts. The cephalopod rearing is still depending on wild specimens; therefore, detection of coccidian species has become an important task to prevent fattening of severely infected hosts. Preventive strategies of coccidiosis are difficult to apply in cephalopods, and no coccidiostats are available to date. Currently, avoiding the intake of infected food is the best strategy to prevent coccidia infection or reinfection, particularly, to those specimens reared in the wild.