Journal of Invertebrate Pathology 110 (2012) 118–125

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Journal of Invertebrate Pathology

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Reaction of the mussel Mytilus galloprovincialis (Bivalvia) to Eugymnantheainquilina (Cnidaria) and Urastoma cyprinae (Turbellaria) concurrent infestation

Ivona Mladineo a,⇑, Mirela Petric b, Jerko Hrabar b, Ivana Bocina c, Melita Peharda a

a Institute of Oceanography and Fisheries, Šetalište I. Meštrovica 63, P.O. Box 500, 21 000 Split, Croatiab University of Split, Center of Marine Studies, Livanjska 5/III, 21 000 Split, Croatiac Faculty of Natural Sciences, Department of Biology, University of Split, Teslina 12, 21 000 Split, Croatia

a r t i c l e i n f o

Article history:Received 22 August 2011Accepted 5 March 2012Available online 15 March 2012

Keywords:Mytilus galloprovincialisEugymnanthea inquilinaUrastoma cyprinaeTEMApoptosis

0022-2011/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.jip.2012.03.001

⇑ Corresponding author. Fax: +385 21 358 650.E-mail address: mladineo@izor.hr (I. Mladineo).

a b s t r a c t

In total 480 individuals of Mytilus galloprovincialis were sampled monthly from October 2009 to Septem-ber 2010, at the shellfish farm in the Mali Ston Bay, south Adriatic Sea (Croatia) in order to assess theextent of pathology imposed by two parasites, Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae(Turbellaria). Although a deteriorating impact on host reproduction or condition index was lacking, weevidenced ultrastructural and functional alteration in host cells at the attachment site. Ultrastructuralchanges included hemocytic encapsulation of the turbellarian and cell desquamation in medusoid infes-tation. Caspase positive reaction inferred by immunohistochemistry (IHC) was triggered in cases of tur-bellarian infestation, in contrast with hydroids, suggesting that the former exhibits more complex host–parasite interaction, reflected in the persistent attempts of the parasite to survive bivalve reaction. Wehave evidenced that both organisms trigger specific host reaction that although not costly in terms ofhost reproductive cycle or growth, results in mild tissue destruction and hemocyte activation. A lowerdegree of tissue reaction was observed in cases of hydroid infestation, compared to turbellarian.

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1. Introduction

Turbellarians in general are accepted as hermaphroditic free-living Platyhelminthes, and include species that form particularassociations, from simple facultative shelter associations to obli-gate endoparasitism (Jennings, 1997). These associations are rela-tively of undefined and unresolved character, mostly just termedendosymbiotic (Jennings, 1997), although for some, evidence ofobligate parasitism (Ball and Khan, 1976) or predatory behavior(Bytinski-Salz, 1935; Galleni et al., 1980) has been found. Blurreddemarcation between putative types of turbellarians’ associationswith other organisms is evidenced in case of some Paravortexspp. that inhabit intestine and stomach of inter-tidal bivalves. Dur-ing intertidal periods representing ‘‘harsh’’ environmental condi-tions, turbellarians migrate into digestive glands, feeding onpartially digested food while employing the hosts own digestiveenzymes in the process (Jennings, 1997). Such shifts from endo-commensalism to endoparasitism, induced by sudden ambientalchanges may be interrelated with nutritional basis offered in thehost environment, where turbellarians choose particular nutri-tional strategy depending on the availability (Jennings, 1997). Sim-ilar behavior is not uncommon also for other groups of organisms,

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like Trichodina spp. protozoans (Lafferty, 1997). Even though para-sitic turbellarians are morphologically heterogeneous, it seemsthat partial loss of their epidermal ciliation and gut, developmentof a muscular sectorial pharynx, as well as epidermal specializa-tions are main features of adaptation to parasitism (Jondelius,1991; Cannon, 2005). Furthermore, many turbellarians show sig-nificant increase in metabolic dependence on their hosts, mani-fested by changes in intestinal structure and decreasingproduction of endogenous digestive enzymes. Parasitic turbellari-ans affect their hosts in different ways, usually inhibiting andretarding host gametogenesis, growth and overall condition, or di-rectly inducing tissue damage (Robledo et al., 1994). Rarely, sud-den mortalities of the hosts are observed (Reisinger, 1930) andmost authors agree that their infestations result in negligiblepathology (see Cannon, 2005). In contrast with gut-associatedParavortex spp., Urastoma cyprinae occurs on the gills of various bi-valve species (Samler, 2001) and gill disruption caused by this tur-bellarian has been evidenced so far only in the mussel Mytilusgalloprovincialis (Robledo et al., 1994), while in some cases a con-spicuous mucus coat (Winstead et al., 2004; Crespo-Gonzálezet al., 2010) or hemocytes cluster (Cáceres-Martínez et al., 1998)has been observed on the parasitation site.

Another frequent symbiont isolated from M. galloprovincialiswith intriguing life cycle is hydroidomedusan polyp Eugymnantheainquilina (Cnidaria, Hydrozoa). Beside simple epibionts, hydroidshave evolved strict symbiotic associations with many marine and

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some fresh-water organisms. However bivalve-inhabiting hydroidsare typically regarded as commensal organisms, in spite of lack ofexperimental evidence for such assumption (Piraino et al., 1994).Their life cycle involves a solitary hydroid stage and a short-livedeumedusoid released free (Cerruti, 1941; Kubota, 1983). Interest-ingly, observations on hydroid-bivalve interaction span from theciliar loss in the M. galloprovincialis mantle epithelium (Cerruti,1941), significant loss of condition (Galinou-Mitsoudi et al., 2002;Rayyan et al., 2004) through mutualistic relationship where hydro-ids ingest digenean sporocysts parasitizing mytilid tissue (Pirainoet al., 1994), to no evident effect imposed by hydroid pedal diskattachment to the M. galloprovincialis mantle (Tiscar, 1992).According to Boero and Bouillon (2005) who considered as ‘‘para-sitic’’ the association in which at last part of guest body is embed-ded in the host tissue or the guest lives inside the host, there aresome 80 hydroids acting as parasites.

To our knowledge there are limited number of studies evaluat-ing the simultaneous impact of turbellarian and hydroid on theirbivalve host (Piraino et al., 1994; Brun et al., 2000). From theabove-mentioned data, it is evident that parasitic or commensalstatus of these two taxa is still controversial and differs consider-ably among published literature. Since from ecological and evolu-tionary point of view their intriguing associations giveopportunities to explore the mechanisms of the development of aparasitic life style and the conditions that affect such relations,the purpose of this paper is to report correlations of subcellular,cytological and histological pathology caused by turbellarian U.cyprinae and cnidarian E. inquilina with mussel growth and repro-duction. This was accomplished by following (1) prevalence, inten-sity and abundance of U. cyprinae and E. inqulina in the AdriaticSea’s commercially important bivalve M. galloprovincialis; (2)reproductive biology and gonadal condition index of infested ver-sus uninfested bivalve; (3) changes imposed by inhabiting organ-isms on bivalve tissue inferred by TEM and detection ofapoptosis-specific enzyme caspase, through 1-yr period.

2. Materials and methods

2.1. Sampling

In total, 480 individuals of M. galloprovincialis were sampledmonthly from October 2009 to September 2010, at the shellfishfarm in the Mali Ston Bay, south Adriatic Sea, Croatia(42�5104900E, 17�4005900N). Forty mussels, grouped into two sizecategories, i.e., <40 mm TL (small) and >60 mm TL (large), were col-lected at depths of 1–3 m from aquaculture ropes and transportedto the laboratory in plastic containers filled with seawater. Prior toall measurements, mussels were cleaned of their epibionts. Indi-vidual live mussel was opened, bysus dissected, and the internalorgans and intervalvar water were carefully examined for the pres-ence of organisms under a dissecting microscope. Afterwards, mus-sels gills were dissected and placed under the light microscope tocount the turbellarians, in the anterior, middle and posterior partof the gills. Ecological terms describing parasitological dynamic:prevalence (P), mean intensity (I) and mean abundance (A) ofinfestation were used according to Bush et al. (1997).

Shell length (TL) of each mussel was recorded as the maximummeasure along anterior-posterior axis to the nearest 0.1 mm usingcallipers. Total weight (Wt) and shell weight (Ws) were recorded toan accuracy of 0.01 g, while mantle weight (Wm) to 0.1 mg usingan analytical balance. The gonadal condition index (GCI) was calcu-lated using formula GCI = (Wm/(Wt �Ws)) � 100 (Suárez et al.,2005).

Temperature and salinity were measured using YSI – Pro probeat 4 m depth near the aquaculture facility.

2.2. Histological analysis

For histological analysis of the reproductive tissue, sections ofthe mussel mantle (N = 480) were fixed in Davidson’s solutionand processed for routine histological preparation. The tissue wasdehydrated in a graded ethanol series (from 70% to 100%), clearedin xylene and embedded in paraffin. Histological sections were cutat 5 lm, stained with hematoxylin-eosin and then mounted formicroscopic analysis. Each section was examined, sexed and as-signed to five stages of gonadal development: resting, developing,ripe, spawning and spent, according to Seed (1976). Each gameto-genic stage was given a numerical score from 0 to 5, based on thematurity of the follicles and gametes; i.e, (0) inactive, (3) earlydeveloping, (4) late developing, (5) ripe, (2) spawning and (1)spent. From these histological preparations, a mean gonad index(MGI) was determined by multiplying the number of mussels ateach stage by the numerical score of the stage and dividing thesum by the total number of individuals in the sample (Seed, 1976).

2.3. Immunohistochemistry

Paraffin-embedded section of infested and uninfested musselgills and mantle were deparaffinised in xylene, rehydrated in eth-anol and water and incubated for 30 min in 0.1% H2O2, avoidingendogenous peroxidase activity. After washing with phosphate-buffered solution (PBS), sections were incubated in sodium citrateor ethylenediaminetetraacetic acid (EDTA) buffer for 10 min at95 �C and cooled to room temperature. To avoid background activ-ity, sections were incubated in 10% normal goat serum for 20 min.Afterwards, sections were incubated with primary antibodies (a-human caspase 3 active, aa 163-175 of human caspase 3, RandDSystems, cat. number AF835) according to their own protocolsand later with mouse secondary antibody for 30 min at room tem-perature, washed in PBS, stained with diaminobenzidine tetrahy-drochloride solution (DAB), and counter-stained withhematoxylin (Pavlovsky and Vagunda, 2003). Additional tissue sec-tions were cut from equivalent sampled area and were incubatedwith PBS, instead of primary antibodies, in order to obtain negativecaspase controls. The sections were analyzed using Olympus BX 40light microscope.

2.4. Transmission electron microscopy (TEM)

Small fragments of infested and uninfested gills and mantlewere collected and fixed in 3.5% paraformaldehyde and 3% glutar-aldehyde in 0.1 M PBS (phosphate buffer solution). Tissue waspostfixed in 1% osmiumtetroxide for 1 h, then dehydrated in anascending series of acetone and embedded in Durcopan resin. Sem-ithin sections were stained with 1% toluidine and examined underan Olympus BX 40 light microscope. Ultrathin sections (0.05 lm)were made from the chosen area of interest and stained with ura-nyl acetate and lead citrate. Material was examined under FEI Mor-gagni 268D (FEI, Eindhoven, The Netherlands) electron microscope.

2.5. Statistical analysis

Prior to statistical analysis, obtained data were tested for nor-mality (Anderson–Darling test) and when necessary log-trans-formed. Sterne’s exact 95% confidence limits were calculated forprevalences, bootstrap 95% confidence limits (number of bootstrapreplications = 2.000) for mean abundances, variance to mean ration(var/mean ratio) as measure of over dispersion and exponent of thenegative binomial (k) for the parasite skewness, using QuantitativeParasitology 3.0 software (Reiczigel and Rózsa, 2005). Since para-sites typically exhibit an aggregated (right-skewed) distributionwithin a host population, the negative binomial model represents

120 I. Mladineo et al. / Journal of Invertebrate Pathology 110 (2012) 118–125

the observed data following the maximum-likelihood method(Bliss and Fisher, 1953). For statistical analysis of differences ofprevalence, mean abundance, and mean intensity between thesexes, size categories and sampling months, exact unconditionaltest and bootstrap t-tests were used according to Rózsa et al.(2000). Exact unconditional test for the comparison of two preva-lences is more sensitive in detecting differences in case of smallsamples (n1, n2 < 100) (Reiczigel et al., 2008).

A non-parametric Mann-Whitney test was used to compare theGCI of infested and uninfested mussels, with hydroid infested mus-sels discarded from the turbellarian infested mussel and vice versa.Two digenea-infested mussels were excluded from the analysis.Contingency tables and v2 test were used to compare distributionof mussels into classes of gonad stages between uninfested and in-fested individuals. For this analysis only small mussels were used,because the ratio of uninfested versus infested individuals in smallmussels was 49:191 and in the case of large mussels 4:236. Pear-son’s correlation coefficient r was applied to determine the degreeof association between parasite parameters, hosts gonadal condi-tion index and environmental parameters. A p value of less than0.05 was considered statistically significant. Statistical analysiswas performed using MINITAB 15 Software.

3. Results

3.1. Bivalve population structure and life cycle

A total of 480 M. galloprovincialis individuals were collected, ofwhich 32% were males, 30% females and for 38% was not possibleto determine sex. The sex-ratio did not differ statistically from theexpected 1:1 (v2: p = 0.486). Shell length ranged from 26.1 to40.0 mm (mean ± SD; 34.35 ± 3.01 mm) for small and from 60.2to 86.7 mm (71.05 ± 5.58 mm) for large mussels. Total fresh weightranged from 0.91 to 4.75 g (2.31 ± 0.67 g) and from 9.56 to 36.15 g(18.16 ± 4.95 g) for small and large mussels, respectively. In totalsample, no statistically significant differences in shell length(Mann–Whitney test: p = 0.324) and in total fresh weight (Mann–Whitney test: p = 0.146) were noted between males and females.

Gonadal condition index (GCI) of small mussels varied from6.17 to 24.9 (12.25 ± 3.01), while for the large ones it varied from8.38 to 32.41 (17.67 ± 4.2) (Fig. S.1). No statistically significant dif-ferences in GCI were noted between the sexes of small (Mann–Whitney test: p = 0.203) and large mussels (Mann–Whitney test:p = 0.465). Furthermore, differences in GCI between the hydroid in-fested and uninfested mussels and turbellarian infested and unin-fested mussels, did not differ statistically (for hydroid infested:Mann–Whitney test: p = 0.396; for turbellarian infested: Mann–Whitney test: p = 0.119). Also, no statistically significant differencein the distributions of gonad stages between uninfested and in-fested small mussels was noted (v2 = 9.81; df = 5; p = 0.081)(Fig.S.2).

No statistically significant correlations were found between theGCI and the number of turbellarians for each mussel size (smallmussels: Pearson’s r = �0.062, p = 0.337; large mussels: Pearson’sr = �0.041, p = 0.527) (Fig. S.3).

High values of the mean gonad index (MGI) of mussels were re-corded in November and December, corresponding to the latedeveloping and maturing period. Afterwards, in January MGI de-creased corresponding to the winter spawning event. In March,an increase of MGI indicated a new reproductive cycle (Fig. S.1).An inverse fluctuation of gonadal condition index (GCI) and meangonad index (MGI) was observed from April to September; MGI de-creased reaching its minimal values, while GCI increased.

Histological analysis revealed that gametogenesis of M. gallo-provincialis has already started in early autumn (October)

(Figs. S.4 and S.5) when the first mussel sample was taken. Firstmature individuals were noted in November and maturity periodextended to March. Another maturity period, however less pro-nounced, was reached in May and June. Spawning period was alsoextended, and individuals in spawned stage were noted fromNovember to July with main spawning intensity occurring duringthe winter months (January–February). In summer, most individu-als were in their resting period, with no development of gametes.

3.2. Turbellarian and hydroid population dynamic

The overall prevalences of turbellarian U. cyprinae and hydroidEugymnythea inquilina were 88.8% and 35.0%, respectively. A verylow prevalence (0.42%) of sporocysts of an unidentified digenetictrematode in two mussels, female (TL = 63.8 mm) and male(TL = 68.3 mm) was observed in January and March in the digestiveand gonadal tissue (Fig. S.6).

Overall abundance of turbellarians was 16.17. The smallest andlargest infested mussel measured 26.1 mm and 86.7 mm TL,respectively. Data on the prevalence, mean intensity, and meanabundance of U. cyprinae parasitizing M. galloprovincialis are pre-sented in Table 1. Turbellarians were aggregated across host pop-ulations, and most mussels harbored low numbers of parasites(<20 parasites per bivalve) (Fig. S.7). The greatest number of para-sites (N = 268) harbored an individual of 78.9 mm TL in July. Thebootstrap t-test showed significant difference in mean intensity(p < 0.001) and in mean abundance (p < 0.001) between smalland large mussels. However, no significant differences betweenthe sexes in mean intensity (bootstrap t-test: p = 0.647) and meanabundance (bootstrap t-test: p = 0.525) were noted. Furthermore,exact unconditional test for the comparison of two prevalencesshowed no significant differences of U. cyprinae prevalences be-tween males and females of both sizes (small mussels: p = 0.687;large mussels: p = 0.345).

Turbellarians were most frequent in the anterior part of the gills(Fig. 1). Comparison of intensity showed significant differencesamong anterior, medial and posterior gill site (bootstrap t-test:p < 0.001). Temporal variation of the turbellarian presence wasnoted in both small and large mussels. In small ones intensity lev-els were lowest in autumn (October–November) and highest inApril for small, or in July for large bivalves (Fig. 2B).

Throughout the year, E. inquilina was present each month andM. galloprovincialis mantle was covered with numerous hydroids,of which some were noticed with the formation of medusa bud(in summer months). The smallest mussel inhabited by E. inquilinahad size of 26.2 mm. Temporal variation of hydroid occurrence isshown in Fig. 2A.

Seawater temperature ranged from 11.6 to 23.3 �C, in Februaryand July, respectively, and salinity varied from 33.94 to 39.15 ppt,in February and May, respectively. There was no correlation be-tween these environmental parameters and both turbellarian andhydroid infestation.

3.3. Ultrastructure of parasitation site

Turbellarians (Fig. 3a–c) were observed as whitish spots dis-seminated throughout gill filaments. Semithin sections revealedtranslucent cellular capsule, relatively thick, but loosely surround-ing immobile organism, consisting mainly of hemocyte fractionand vacuolated lacy-like matrix. Hemocytes were small cells show-ing a large, usually decentered nucleus, typically with dispersedheterochromatin and delamination of nuclear membrane (Fig. 3dand g). Crescent-shaped condensed chromatin was observed inhemocyte nuclei. Cytoplasm was filled abundantly with free ribo-somes, enlarged mitochondria, while rough- and smooth-surfacedendoplasmic reticulum was rarely observed, as well as Golgi

Table 1Total mean prevalence (%) with Stern’s exact 95% confidence limits (CI), mean intensity and mean abundances with bootstrap 95% confidence limits (CI) and variance to meanratio of turbellarian Urastoma cyprinae isolated from Mytilus galloprovincialis through the year (N of hosts = 20 for each sampling month; N total = 240 per size category). Exponentof the negative binomial (k) showed statistical difference between observed and expected frequencies at p = 0.05⁄.

Month Prevalence (%) (CI) Intensity (CI) Abundance (CI) Var/mean ratio k

Small musselsOctober 35 (16.7–57.6) 1.00 (/) 0.35 (0.15–0.50) 0.684 /November 50 (29.3–70.7) 3.50 (/) 1.75 (/) 10.278 0.343December 75 (52.6–89.6) 2.60 (2.07–3.07) 1.95 (1.30–2.50) 1.105 /January 85 (62.8–95.8) 3.59 (2.47–4.82) 3.05 (2.00–4.25) 2.294 2.195February 85 (62.8–95.8) 6.06 (4.12–8.94) 5.15 (3.30–7.75) 5.381 1.202March 95 (75.6–99.7) 5.84 (4.42–8.37) 5.55 (4.15–8.00) 3.461 2.988April 100 (83.3–100) 9.00 (6.70–11.80) 9.00 (6.70–11.80) 4.211 2.825May 85 (62.8–95.8) 8.82 (5.94–11.94) 7.50 (4.85–10.55) 6.182 1.039June 90 (68.0–98.2) 3.61 (2.56–4.78) 3.25 (2.20–4.45) 2.069 3.040July 90 (68.0–98.2) 5.17 (3.22–7.83) 4.65 (2.85–7.15) 5.462 1.164August 100 (83.3–100) 6.10 (4.40–8.20) 6.10 (4.40–8.20) 3.294 2.914September 60 (37.2–79.1) 2.25 (1.42–3.08) 1.35 (0.75–2.10) 1.893 1.297TOTAL 79.2 (73.6–84.0) 5.23 (4.59–5.94) 4.14 (3.56–4.76) 5.649 0.886

Large musselsOctober 95 (75.6–99.7) 6.42 (4.53–9.58) 6.10 (4.20–8.95) 4.796 1.766November 90 (68.3–98.8) 6.00 (4.39–8.11) 5.40 (3.70–7.40) 3.595 1.809December 100 (83.3–100) 35.50 (23.70–50.85) 35.50 (23.70–50.85) 28.778 1.240⁄

January 100 (83.3–100) 12.35 (8.95–17.15) 12.35 (8.95–17.15) 7.460 2.276February 100 (83.3–100) 21.60 (13.45–37.00) 21.60 (13.45–37.00) 29.763 0.838March 100 (83.3–100) 46.2 (32.00–71.70) 46.20 (32.00–71.70) 44.203 1.480⁄

April 95 (75.6–99.7) 23.58 (13.89–38.26) 22.40 (13.20–37.70) 32.497 0.736May 100 (83.3–100) 26.40 (14.80–47.10) 26.40 (14.8–47.10) 49.571 0.748June 100 (83.3–100) 31.15 (21.95–42.05) 31.15 (21.95–42.05) 17.830 1.845July 100 (83.3–100) 84.75 (55.65–118.65) 84.75 (55.65–118.65) 66.260 1.101August 100 (83.3–100) 25.50 (18.75–34.15) 25.50 (18.75–34.15) 12.906 2.498September 100 (83.3–100) 19.80 (14.15–31.50) 19.80 (14.15–31.50) 16.755 2.104Total 98.3 (95.8–99.6) 28.57 (24.12–33.32) 28.10 (23.75–32.99) 50.877 0.827⁄

Fig. 1. Gill site of turbellarian Urastoma cyprinae parasitation, A – anterior, M –middle, P – posterior on small (A) and large (B) Mytilus galloprovincialis.

Fig. 2. Environmental parameters, temperature (�C) and salinity (ppm), withprevalence of hydroid Eugymnanthea inquilina (A) and mean intensity of turbellar-ian Urastoma cyprinae (B) isolated from small (white) and large (black) Mytilusgalloprovincialis throughout the 1-yr period in the south Adriatic Sea.

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apparatus. Many empty vacuole and clear-looking endocytic vesi-cles of different sizes were scattered on cell periphery (Fig. 3f).

Cellular elements of the capsule were missing in the layer inclose contact with turbellarian ciliation, and bordering membranewas disrupted and highly exvaginated (Fig. 3e). Gill epitheliumshowed ciliar integrity in contact with enclosed turbellarians,retaining characteristic cell composition and architecture.

Hydoids were observed attached by pedal disk on mantle epi-thelium of the host, forming a medusa bud in summer months

(Fig. 4a and c). Semithin sections revealed thickening and disrup-tion at the attachment site, with loss of host ciliature and occur-rence of few large hemocytes among tissue debris (Fig. 4c and d).Hydroid pedal disk cells had basally located nuclei and in the apicalpart in contact with the host, many small, dark-stained granulewere observed (Fig. 4e and f). Host cells at the contact surface

Fig. 3. Turbellarian Urastoma cyprinae (Urastomidae, Platyhelminthes) infesting gills of cultured mussel Mytilus galloprovincialis: (a) Wet mount of adult U. cyprinae; (b)Histological section of paraffin-embedded gills (g) infested by single turbellarian (t). Arrow shows remains of hemocyte encapsulation enveloping the parasite. H&E staining;(c) Histological section of isolated paraffin-embedded turbellarian, partially enveloped by hemocyte encapsulation (arrow); (d) Higher magnification of semithin section ofthe turbellarian (t) enveloped by translucent hemocyte encapsulation (e). Note chromatine condensation in hemocytes nuclei (arrow). Toluidine staining; (e) TEM section ofthe turbellarian epithelium with cilia (t) in close contact with translucent hemocyte-derived encapsulation (e). Note fragile and lace-like appearance of the capsule (arrow).Scale bar = 10 lm; (f) TEM section of hemocytes forming encapsulation around turbellarian. Note abundant empty vacuole in cytoplasm. Scale bar = 5 lm; (g) TEM section ofhemocyte nucleus (n) showing delamination of nuclear envelope (arrow). Note empty vacuole in cytoplasm (⁄). Scale bar = 1 lm; (h) Caspase positive staining in the musselgill (g) epithelium has been observed in few cells (arrows) in direct contact with the turbellarian (t). IHC staining; (i) Caspase positive staining in uninfested mussel gills hasbeen noticed in a few migratory hemocytes among epithelial cells. IHC staining; (j) Caspase positive staining has been noticed in hemocyte encapsulation (e) surroundingturbellarian (t). Note hemocyte with large nucleus demonstrating morphological characteristics of apoptosis with peripheral crescent-shaped condensed chromatin alongnuclear membrane and caspase activity in cytoplasm (arrow). IHC staining; (k) Negative control reaction without caspase positive staining in hemocyte encapsulation (e)surrounding turbellarian (t). IHC staining; (l) Higher magnification of caspase positive staining in a single cell (arrow) of the mussel gill (g) epithelium, in direct contact withthe turbellarian (t). IHC staining.

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showed vacuolated cells, with centrally located nuclei, scarce inchromatin (Fig. 4g).

3.4. IHC staining of apoptosis

Apoptotic activity evidenced through caspase IHC at parasita-tion site was observed in cytoplasm of epithelial gill cells whenturbellarian capsule was disrupted (Fig. 3h). When turbellarianswere enclosed, capsule matrix and occasionally enlarged hyalino-cytes showed caspase activity (Fig. 3j and k). Uninfested gill tissueis shown in Fig. 3i.

Caspase activity in the hydroid-parasitized mantle, was ob-served only subepithelially in the mantle connective tissue, butnot in cells in direct contact with the parasite (Fig. 4i–l).

4. Discussion

Provoked by high prevalence of turbellarian U. cyprinae and cni-darian E. inquilina in the cultivated mussel M. galloprovincialis, weattempted to determine if infested versus uninfested bivalve sufferretardation in gametogenesis, one of the main hallmarks of bivalveparasitation. In contrast with Rayyan et al. (2004), we have evi-denced that despite their heavy infestation rate, both turbellarianand hydroid cause only mild effects on their host M. galloprovincial-is and evidence of significant condition loss, or effects on reproduc-tive effort, like disruption of reproduction, were not observed.However, a solid comparison could have been biased by a remark-ably small number of uninfested mussels, mainly classified in smallcategory.

The annual reproductive cycle of M. galloprovincialis in the east-ern Adriatic Sea reported in this study is congruent with previousstudy (Hrs-Brenko, 1971) and we have not evidenced any impactof two parasites on the host reproduction and condition. However,we have observed ultrastructural and functional deviation in hostcells at the parasite attachment site. The turbellarian U. cyprinaewas previously detected in many bivalves throughout the world,including oysters Crassostrea virginica and Ostrea edulis and mus-sels Mytilus edulis and M. galloprovincialis (Cáceres-Martínezet al., 1998; Winstead et al., 2004; Francisco et al., 2010 and refer-ences therein). Although U. cyprinae was sometimes consideredeither as parasite or commensal, Lauckner (1983) regarded it asharmless. According to Jennings (1971), remarkably few turbellar-ian species are parasitic in the generally accepted sense of theterm. This was further fortified by a report of free-living U. cyprinaeamong algae or on marine mud along the eastern Adriatic coast(Westblad, 1955), as well as demonstration of fulfillment of U. cyp-rinae reproduction cycle outside its host (Crespo-González et al.,2005). Same authors proposed a life cycle for this species involvinga sexual maturation period in the bivalve gills and a reproductionperiod including cocoon secretion, egg laying, and hatching en-tirely completed in the external environment. The presence ofwell-developed eyespots supports the hypothesis that a free periodoccurs, during which the eyespots enable the appropriate photo-tactic responses to happen, perhaps to take the animal to the sed-iments to find shelter and a mate (Cannon and Lester, 1988). Incontrast, several authors have evidenced U. cyprinae parasitic nat-ure describing damages of gill filaments, with disorganization andlamellar hyperplasia (Robledo et al., 1994; Villalba et al., 1997;Cáceres-Martínez et al., 1998; Francisco et al., 2010). Burt and

Fig. 4. Hydroid Eugymnanthea inquilina (Cnidaria) attached to the mantle of cultured mussel Mytilus galloprovincialis: (a) Wet mount of the hydrozoan showing medusa bud(⁄); (b) Hydrozoans (h) attached to the mantle inner fold (m): ⁄ – attachment site of the pedal disk, H&E staining; (c) Higher magnification of semithin section of thehydrozoan (h) attached to the bivalve mantle (m). Note fragmented clumps of host tissue (arrows) and granulocytic hemocytes (arrow). Toluidine staining; (d) Highermagnification of semithin section of the hydrozoan (h) attached to the bivalve mantle (m). Note abundant granule present in the pedal disk cells of the hydroid (arrows),fragmented clumps of bivalve cell cilia (c) and migrating granulocytic hemocytes (arrowhead). Toluidine staining; (e) TEM section of the attachment area of the hydroid (h)and host mantle (m). Scale bar = 5 lm; (f) Higher magnification of the contact between hydroid cells (h) and host mantle cells (m). Note amorphous material in the apical partof hydroid cells and basal electron dense granule (arrow), nucleus (n). Scale bar = 2.5 lm; (g) TEM section of host mantle cells (m) in close contact with hydroid. Notefragmentation of cilia (c), loss of basal bodies (arrow) and cell debris (⁄) at the attachment site. Scale bar = 5 lm; (h) Caspase positive staining in the bivalve mantle (m)connective tissue (brownish staining) infected with three hydrozoans (h). IHC staining; (i) Higher magnification of hydroids (h) attachment site showing subepithelial caspaseactivity (brownish staining) in the mantle (m) connective tissue. IHC staining; (j) False positive caspase activity in the apical mantle (m) epithelium (arrow) infected byhydroids (h). IHC staining; (k) Higher magnification of false positive caspase activity in the apical mantle (m) epithelium (arrow) infected by hydroids (h). IHC staining; (l)Negative control reaction of the hydroids (h) infection of mussel mantle (m), showing brownish staining in the apical part of mantle epithelium (arrow). IHC staining.

I. Mladineo et al. / Journal of Invertebrate Pathology 110 (2012) 118–125 123

Bance (1981) claimed U. cyprinae exhibits a marked negative pho-totaxis when removed from its bivalve host, while strong protrusi-ble pharynx and the presence in the gut of cells and melaningranules strongly suggest the worms feed on host cells lining thechamber. Our results speak in favor of harmful interaction of theturbellarian with the host gills, the later in turn succeeding, evenwhen parasite numbers are very high, to sequestrate turbellarianinside a cyst originating from mussel hemocytes, disabling itsmovements and consequent gill damage. Since we were not ableto evidence any absorption processes through turbellarian epider-mis as described by Bataller et al. (2003), we suggest that food con-sumption within hemocyte capsule is limited. Such capsule wascomposed of hemocytes, whose role resides in internal defense,such as recognition and phagocytosis or encapsulation of nonselfmaterials (Foley and Cheng, 1974), and coagulation (Moore andLowe, 1977; Nakayama et al., 1997) that could be engaged in theencapsulation process of U. cyprinae. Encapsulation of the turbel-larian within ‘‘small mucus-looking covering’’ or ‘‘mucus coat’’has been previously described (Winstead et al., 2004; Crespo-Gon-zález et al., 2010), but authors have suggested that the envelopederived from the secretion of turbellarian mucus glands (Batalleret al., 2003). Although the later have evidenced mucus secretionby TEM, turbellarians were not fixed in situ with bivalve gills in or-der to prove that the secreted mucus formed turbellarian envelope,whilst other authors (Winstead et al., 2004; Crespo-González et al.,2010) did not use TEM to define cyst origin.

Interestingly, Cannon and Lester (1988) reported two unidenti-fied Paravortex sp. that created skin and gill lesions in fish with noapparent pathology, except that specimens parasitizing gills whereencircled by a translucent ‘‘tube’’ that authors attributed to layersof epithelial cells, finding no evidence of necrosis or advanced

inflammatory reaction. Leucocytic infiltration and hyperplasiawere observed, not accompanied by cell necrosis, congruent toadequate defense mechanisms we have observed in the mussel.Successful protective answer to turbellarian infection in bivalvehas been evidenced also in the eastern oyster (C. virginica) wheregill mucus contained three proteases involved in the reaction toturbellarians (Brun et al., 2000).

Another protective mechanism of cells, for example to intracel-lular parasite infections, is apoptosis. It ensures a death programtriggered by detection of an extra or intracellular unfavorable con-dition, i.e., deprivation of growth factors, DNA damage, or infection,leading to increased caspase and endonuclease activities, cleavageof target substrates, nuclear condensation, DNA fragmentation, andcell shrinkage (Guillermo et al., 2009), aiming at ‘‘altruistic suicide’’of the host cell, before the invading organism has a chance to mul-tiply and disperse throughout other host cells (James and Green,2004). Apoptotic activity in bivalves has been evidenced in Perkin-sus marinus-infected C. virginica hemocytes where protozoan mod-ulated and inhibited the process, in order to maintain itselfsecluded and propagate in the hemocytes (Hughes et al., 2010).Whilst protozoan parasites inhibit host cell apoptosis attemptingto perpetuate their life cycle within cells (Heussler et al., 2001;Lüder et al., 2001) it seems that extracellulary-living helminthsmodulate host apoptosis by its induction in target host immunecells. This has been proposed recently for intestinal nematodes,filariae, schistosomes and cestode Taenia crassiceps (Chen et al.,2002; Jenson et al., 2002; Kuroda et al., 2002; Lopez-Brioneset al., 2003) and our data show that similar mechanism developsin turbellaria-bivalve system. Caspase positive reaction inferredby IHC in M. galloprovincialis was triggered in cases of turbellarianinfestation, in contrast with hydroids, suggesting more complex

124 I. Mladineo et al. / Journal of Invertebrate Pathology 110 (2012) 118–125

host–parasite interaction and persistent attempts of the parasite tosurvive bivalve reaction. Even we have not evidenced a wide arrayof morphological features of apoptotic cells, including cell shrink-age, DNA fragmentation and membrane blebbing, disassembly ofthe nucleus and organelles and encapsulation of the products inmembrane-bound apoptotic bodies (Penninger and Kroemer,2003; Sokolova, 2009), in hemocyte encapsulation of the turbellar-ian and migrating gill hemocytes in parasite close contact, caspasepositive IHC staining and the crescent-shaped condensed chroma-tin, state in favor of undergoing apoptotic mechanism.

In case of hydroid E. inquilina infestation, mutualistic relation-ship between host and guest was experimentally demonstrated,where hydroid fed upon offered trematode sporocysts (Pirainoet al., 1994), although such behavior was not observed in vivo orin our study. As in case with turbellarian, dual observations havebeen given, spanning from mutualistic (Mattox and Crowell,1951; Kubota, 1983) to harmful effect on the host, manifested byciliar (Cerruti, 1941) or significant condition loss (Galinou-Mitsou-di et al., 2002; Rayyan et al., 2004). Our results state in favor of his-topathological alterations observed as disruption of mantleepithelium integrity in infested mussels. However, it needs to bepointed out that bivalve tissue reaction, inferred by TEM anddetection of apoptosis, is far less attenuated in comparison to tur-bellarians. Interestingly, a different fraction of hemocytes and inconsiderably fewer numbers than observed in turbellaria-infestedmussels, is engaged in hydroid infestation.

Population dynamic of hydroids followed patterns alreadyestablished for Mediterranean (Piraino et al., 1994), which wasnot fully the case with U. cyprinae (Crespo-González et al., 2004).In the present study, large mussels exhibited three pronouncedturbellarian-intensity peaks (December, March and July), suggest-ing another factor exempt higher temperature that seems to trig-ger parasite proliferation in cold season. Such observation iscontradictory to studies of other authors (Fleming et al., 1981;Fleming, 1986; Murina and Solonchenko, 1991; Robledo et al.,1994). In the Adriatic Sea mussels, abundances level and predilect-ed part of the gill were also in contrast with results reported earlier(Robledo et al., 1994; Crespo-González et al., 2010), and the diver-sity of results between reports strongly indicates that habitat con-ditions play an important role in shaping these variables.

In conclusion, it is evident that there is a very high turbellarianand hydroid infestation among cultured mussel population in theAdriatic, suggesting particular culturing and environmental condi-tions as shaping forces of parasites population dynamic. We haveevidenced that both organisms trigger specific host reaction thatalthough not costly in terms of reproductive cycle or growth, resultin tissue destruction and hemocyte activation, however to a lesserdegree in cases of hydroid infestation.

Acknowledgments

The authors are very grateful to Zeljka Trumbic and Daria Ezg-eta-Balic for their assistance with sample collections and prepara-tion, as well as anonymous referees whose comment significantlyimproved the manuscript.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jip.2012.03.001.

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