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NOTE

Report from the Mediterranean coast of Spain (Southern Catalonia) of parasitism

of Octopus vulgaris by Aggregata octopiana and Dicyema acuticephalum

K. B. Andree1*, N. Carrasco1, D. Furones1, N. Duncan1, A. Estevez1, P. Querol2 and I. Gairin1

1IRTA-Aquaculture Center, San Carlos de la Rapita, Tarragona, Spain; 2Research Group of Aquaculture and Biodiversity, Institute of Animal Science

and Technology, Universitat Politecnica de Valencia, Valencia, Spain

Abstract Presented herein from a study of an inert diet trial for octopus aquaculture are observations of Aggregata octopiana, with fully documented sporogonic development, and a report of Dicyema acuticephalum in the renal tubules of Octopus vulgaris from the northeastern Mediterranean Sea (southern Catalonia). Prevalence for the A. octopiana infection of the intestinal epithelium was 100% (8/8), while prevalence of D. acuticephalum was ~12.5 % (1/8).

* Corresponding author’s e-mail: karl.andree@irta.es

The common octopus, Octopus vulgaris (Cuvier, 1797) has been under investigation as a new species for developing a more diversified aq-uaculture market due to its high nutritional value, rapid growth rate [around 13% of body weight per day with natural food] (Vaz-Pires et al., 2004), and its high market price (Prato et al., 2010). However, its culture is limited by larval survival, difficulty to obtain an afford-able feed source (Quintana et al., 2008), and possibly also very high densities of intestinal parasites (Gestal et al., 2002a, 2002b). In recent years, artificial diets for O. vulgaris have shown improvement and different pellet diets have

been tested (Quintana et al., 2008; Estefanell et al., 2011; García-Garrido et al., 2011). The observations of parasites presented herein were part of a study to determine growth and feed efficiency of formulations of dry extruded diets (Querol et al., 2013), hence selected animals from the study population were examined beforehand and at the end to document any parasitism. As octopus has become a species of interest for aquaculture, parasites that may adversely affect its production in culture also become important.

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A variety of protozoan and metazoan parasites of cephalopods have been catalogued previ-ously in Spain (Pascual et al., 2006). However, cephalopods and their parasites are generally not as well-studied as marine vertebrates and their parasitic taxa. Parasites identified in O. vulgaris (Pascual et al., 2006) encompass nema-todes, cestodes, and members of the Dicyemid Mesozoans (Dicyema spp.) and Apicomplexa (Aggregata spp.). Among the first parasites to be described in octopus in the Mediterranean (Dobell, 1914; Goodrich, 1950), Aggregata sp. completes its life cycle through ingestion of an infected crustacean host (Goodrich, 1950; Gestal, 2002c). It has received some consider-able attention in studies of octopus culture as it is commonly found in the intestinal tissues (Gestal et al., 2007; Mladineo and Jozic, 2005). In contrast, descriptions of Dicyema spp. infections in O. vulgaris are fewer (Nouvel, 1947; Pascual et al., 2006), and its life cycle is still unknown. Therefore, we present a report of two parasites observed during a dietary trial in the common octopus, O. vulgaris from the southern coast of Catalonia, describing stage-by stage the sporo-gonic development of A. octopiana in the host’s intestinal epithelium.

Specimens of octopus (O. vulgaris) (n = 22) were caught near Alfacs Bay, Ebro Delta, 40º37′12′′ N; 0º35′ 34′′E; Spain (W. Mediterranean) during April 2011. Animals were kept in open flow sea-water systems in the Instituto de Recerca y Tec-nologia Agroalimentaries (IRTA). Eighteen octo-pods with a similar weight (955.8 ±85.5 g) were selected for the dietary assay, and divided into control and experimental diet groups. Animals were maintained in flow-through water condi-tions with temperature of 18.4 ± 1.3°C. In order to acclimate the octopi to laboratory conditions,

they were fed golden grey mullet (Liza aurata) and crab (Liocarcinus depurator) for 10 days prior to the growth tests on formulated pellet feed (Querol et al., 2013). None of the fresh feed used for acclimation was examined for presence of parasite infective stages.

The four remaining animals that were initially collected were humanely sacrificed by immer-sion in ice water until immobile and then bi-secting the central nervous system with a large knife. Afterwards, they were dissected to obtain samples of all major organs for histological ex-aminations and documentation of parasitism, or other health changes prior to the dietary trial. Two animals from the control and two from the experimental diet were also examined at the end of the study. The tissues were fixed in Davidson´s fixative, and stained by standard hematoxylin/eosin method.

Aggregata sp. was observed in various devel-opmental stages in the intestinal epithelium of all animals examined (Figure 1), while Dicyema sp. was present at very low abundance in the kidney tubules of a single animal, sacrificed prior to the beginning of the dietary trial (Figure 3). An inflammatory response to atypical Ag-gregata infections was noticed in the branchia (Figure 2), as reported previously (Pascual et al., 2006). The intestine in particular had a very intense parasite load along the entire length, enabling the observation of a variety of sporo-zoites and other developmental stages of Ag-gregata sp. (Figure 1).

We observed sporogony initiating as nuclear organising centers developing along the surface of the invaginated pansporoblast membrane (Figure 1d and e), and these dense areas

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Figure 1. Intestine of the octopus showing progressive stages of sporogony of Aggregata octopiana: a) oocysts; b) fertilized oocyst; c) initial condensation of chromatin-like material along the margin of the oocyst internal membrane (triangles); d) residual body visible (RB). Notice invaginations beginning to form along the inner membrane and the sporocyst bodies beginning to develop; e) sporocyst bodies and what appears to be chromatin-like material (triangles) becoming more condensed; f) bodies of the individual sporocysts beginning to develop; g) walls between individual sporocysts are formed and immature sporocysts are visible displaying an oval shape; h) more mature and more rounded sporocysts; i) sporocysts becoming more compact and rounded; j) mature sporocysts with mature coiled sporozoites visible within (higher magnification - arrow). Scale bar images a-i = 100 µm; j = 20 µm, inset = 10 µm.

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become foci for individual sporocyst develop-ment (Figure 1f – j). Completion of sporocyst development resulted in sporocysts of approxi-mately 11 µm diameter encasing approximately 8 sporozooites. Although we did not perform other morphometric characterisation, observed meristic values indicate that the apicomplexan belongs to Aggregata octopiana. Prevalence of in-fection was 100% among animals examined and no discernible difference in evaluated infection

intensity was evident among infected animals. In kidney tubules of a single animal examined prior to the dietary trial, both vermiform and infusoriform embryos of Dicyema spp. were ob-served (Figure 3). No morphological traits typi-cally used for species identification (eg. calotte morphology) were visible by histology for species identification to be confirmed. However, a presumptive identification of D. acuticephalum could be assigned based only upon the host

Figure 2. Branchial tissue: a) granuloma (arrowhead); b) parasite stages of Aggregata octopiana (arrows). Scale Bar = 100 µm.

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specificity for O. vulgaris (Fuyura and Tsuneki, 2003) and size of the infusoriform embryo (see inset, Figure 3). The intensity of infection in the kidney by this parasite appeared to be low.

During this study we did not observe any clini-cal signs in the parasitised animals. No other parasite taxa were observed during the course of the study.

While A. octopiana has been previously seen elsewhere around the Iberian Peninsula (Gestal et al., 2002c; Mayo-Hernandez et al., 2013), this is a new geographical record from the Ebro Delta, in Southern Catalonia. This apicomplexan is common in reared animals with prevalences approaching 100% (Estévez et al., 1996), al-though lower prevalence values may be seen in wild animals (Gestal et al., 2002c; Mladineo and

Figure 3. Dicyema acuticephalum in renal tissue: a) infusoriform embryos (arrows). Inset (Scale Bar = 10 µm): margins of ciliary fringe of an infusiform embryo (arrowheads); b) vermiform embryo (arrowhead). Scale Bar = 60 µm.

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Jozic, 2005). In cultured octopus, some infections with a high parasite abundance in the intestinal epithelium have been suggested the cause of a “malabsorption syndrome”, caused by dis-equilibrium of the digestive enzymes (Gestal et al., 2002a, 2002b). Therefore, the elimination of intestinal Aggregata might in fact augment the food conversion ratio and could benefit aqua-culture. Alternate hosts for these heteroxenous parasites are crustaceans (Goodrich, 1950), sug-gesting that the prevalence of this parasite in octopus may be altered under captive rearing by providing thermally-processed feed (Mladineo and Jozic, 2005); frozen crab and fish parts. It has been considered possible that a sterile inert dietary regimen might aid in reducing the total parasite load by eliminating the continued intro-duction of new viable infective parasite stages, but the dietary regimen undertaken in this study was too short (6 weeks) to offer some answers. In this study, we identified the parasite as A. octopiana by characteristics of the final spo-rocyst development, sporocyst diameter and number of sporozoites per sporocyst, previ-ously published for this species (Estévez et al., 1996). The number of sporozoites per sporocyst were not obvious by the methodology used, but the estimate of 8 sporozoites per sporocyst fits within the range of 6-15 described for A. octopiana (Estévez et al., 1996).

To sustain the lifecycle of Aggregata sp. in nature requires both cephalopods and crustaceans, suggesting that the life histories of both parasite and hosts have evolved together, consequently leading to a tolerance of A. octopiana in intestinal tissues of O. vulgaris, with few adverse effects to its health, aside from the afore-mentioned “malabsorption syndrome”. In contrast, atypi-cal A. octopiana infections of branchial tissues

have been reported previously (Pascual et al., 2006) and in this study (Figure 2). The observed granuloma formations at this site suggests there may be an active immune reaction to the para-site, speaking in favour of a reduced host’s tolerance for colonisation of atypical tissue sites (Halliday, 1976).

We provide here some data acquired during the dietary study for future reference of bio-geographic distribution of parasites and some useful imagery for assisting others studying parasites of octopus. In future work, for the objective of commercial aquaculture develop-ment, longer term trials using inert pelleted feed and culture in closed recirculation systems should be attempted as a means to reduce para-site burdens and possible negative pressures on health from these infections. The ability to culture parasite-free animals may require completion of the octopus life cycle in captivity, grown on an inert diet.

We wish to acknowledge funding obtained from JACUMAR (Secretary of Sea, Ministry of Agriculture, Food and Environment) and Conselleria d′Agricultura, Pesca, Alimentacio i Aigua (Generalitat Valenciana) with specific funding from the project National Plan for the Optimization of the Grow-out of the Octopus, O. vulgaris. We also are indebted to Sr. José Ramon Tomas Martí for his help in the capture of wild animals for this study.

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