Ontogenesis of opercular deformities in gilthead sea bream Sparus aurata: a histological description
Abstract
The aim of this study was to characterize histological changes during opercular osteogenesis in farmed gilthead sea bream Sparus aurata larvae from 7 to 69 days post hatching (dph) and compare normal osteogenesis with that of deformed opercles. Mild opercular deformities were first detected in 19 dph larvae by folding of the opercle's distal edge into the gill chamber. Here, the variation in the phenotype and the irregular bone structure at the curled part of the opercles is described and compared with the histology of normal opercles. Results indicated that deformed opercles still undergo bone growth with the addition of new matrix by osteoblasts at the opercular surface, especially at its edges. No significant difference was found in bone thickness between deformed and normal opercles. In addition to differences in bone architecture, differences in collagen fibre thickness between normal and deformed opercles were also found.
Introduction
Morphological abnormalities are a critical issue for Mediterranean finfish mariculture with many parts of the body being implicated, such as overall shape, pigmentation, scales, skeleton and swimbladder, observed both during intensive rearing and grow-out phases (Divanach et al., 1996). The gilthead sea bream Sparus aurata L. 1758 is a Mediterranean food-fish species that represented c. 158 × 103 t of the 73·8 × 106 t of global aquaculture production in 2014 (FAO, 2016). In this species skeletal deformities are a major component of abnormalities affecting the quality of hatchery-produced fish (Andrades et al., 1996; Divanach et al., 1996; Koumoundouros et al., 1997; Boglione et al., 2013). These deformities appear during both early larval and juvenile stages in hatchery farms and in adult fish from grow-out farms, leading to increased mortality rates, diminished growth (Andrades et al., 1996; Fernández et al., 2008) and decreased market value (Boglione et al., 2001; Fernández et al., 2008). Previous studies have described different types of skeletal malformations in this species, including lordosis and kyphosis in the vertebral column (Chatain, 1994; Andrades et al., 1996; Fernández et al., 2008), deformed upper and lower jaw (Boglione et al., 2001; Fernández et al., 2008), abnormal caudal-fin development (Koumoundouros et al., 1995; Boglione et al., 2001) and branchiostegal ray deformities in close association with gill cover (opercular) deformities (Koumoundouros et al., 1997).
Among these skeletal deformities, malformations of the opercular complex can show a high and unpredictable prevalence in farmed S. aurata, affecting up to 80% of the reared population (Paperna, 1978; Francescon et al., 1988; Andrades et al., 1996). Opercular deformities mainly begin during the larval period and continue occurring until juvenile or adult sizes (Boglione et al., 2001). As in most teleosts, the opercular complex of S. aurata is composed of a preopercle, interopercle, opercle and subopercle, with the latter two comprising the bony cover of the gills and being the parts most frequently affected. Opercular deformities can be unilateral or bilateral, with a folding of the opercle or subopercle inside or outside the gill cavity during the early larval stage (Koumoundouros et al., 1997; Galeotti et al., 2000; Beraldo et al., 2003; Verhaegen et al., 2007). As the function of the opercular complex is not only for protection of the gill but also for respiration, opercular deformities might indirectly cause gill diseases by a lowered resistance to environmental stress (Paperna et al., 1980). Exposed gills can decrease respiratory efficiency, as well as reduce market value (Divanach et al., 1996; Beraldo & Canavese, 2011).
There are several studies that focus on opercular deformities in S. aurata (Koumoundouros et al., 1997; Galeotti et al., 2000; Boglione et al., 2001) and in other marine finfish belonging to the Perciformes, such as sharpsnout sea bream Diplodus puntazzo (Walbaum 1792) (Boglione et al., 2003), barramundi Lates calcarifer (Block 1790) (Fraser & de Nys, 2005), European perch Perca fluviatilis L 1758 (Lindesjöö et al., 1994) and European sea bass Dicentrarchus labrax (L. 1758) (Barahona-Fernandes, 1982). Additionally, the bone remodelling process in deformed opercular structures of juvenile S. aurata has been demonstrated by Ortiz-Delgado et al. (2014). It is still unknown, however, what aspects of osteogenesis are involved in generating this abnormal phenotype. Histological analyses of opercular deformities in S. aurata have so far identified the earliest onset and correlated body size of its appearance during larval development (Koumoundouros et al., 1997; Galeotti et al., 2000; Beraldo et al., 2003) and provided a comparative analysis of the tissue distribution between normal and deformed opercular bones at the juvenile stage (Ortiz-Delgado et al., 2014).
The opercular bones are cranial dermal bones that undergone intramembranous ossification. During this process, mesenchymal cells differentiate into osteoblasts and bone is formed without a cartilaginous precursor (Huysseune, 2000; Sire & Huysseune, 2003; Franz-Odendaal et al., 2006). Sparus aurata is a derived teleost species, having acellular bone with osteoblasts moving with the ossification surface as bone matrix is being deposited, instead of getting enclosed within the matrix (Moss, 1961; Witten & Huysseune, 2009). These osteoblasts then produce osteoid (non-mineralized bone matrix) that subsequently mineralizes, resulting in a bone matrix devoid of osteocytes (Ekanayake & Hall, 1988). During bone formation, the active and mature osteoblasts secrete collagen type I, as well as other matrix proteins toward the bone formation surface (Bart, 2008). Despite this general description of ossification, details regarding histological correlates of abnormal osteogenesis in early ontogeny are still missing.
Thus, the aim of this study was to characterize histological changes during opercular osteogenesis of farmed S. aurata and compare normal osteogenesis with that observed in deformed opercles.
Materials and methods
Source of material
Specimens were provided by the commercial hatchery Maricoltura Rosignano di Solvay, Rosignano di Solvay, Italy. Larvae were raised in indoor tanks, using a semi-closed circuit of filtered natural seawater. The rearing period spanned from 11 April till 22 June 2012 and comprised two phases: for the first 18 days post hatching (dph), larvae were kept in 6000 l larval rearing tanks (initial density of 100 larvae l−1) and at 19 dph, the larvae were transferred to 500 l pilot tanks at a density of 30 larvae l−1, until 69 dph. They were fed live food (rotifers, INVE Artemia AF strain and INVE Artemia EG strain) followed by INVE Start S, Start l, Wean S and Grow S artificial dry feeds (INVE Aquaculture Inc.; www.inveaquaculture.com), starting on day three until the end of the rearing period (69 dph). During the rearing period, the following environmental variables were controlled: water temperature 18·5° C; dissolved oxygen concentrations 6–12 mg l−1; light intensity 1–17 dph at 250 lx, 18–24 dph at 300 lx, 25–34 dph at 400 lx and 35–69 dph at 500 lx; and salinity 34–38. In this study, a total of 32 specimens between 3·1 and 27·7 mm standard length (LS) were analysed histologically.
Histology
All sampled specimens were killed using an overdose of MS-222 and were fixed and stored separately in a 10% neutral buffered formalin (for paraffin-wax sectioning) or 3% glutaraldehyde (for epon sectioning).
For the epon embedding, larvae were fixed in a glutaraldehyde solution at room temperature and post-fixed in OsO4 for 2 h. For decalcification, the specimens were immersed in 14% EDTA at room temperature for 7 days, washed in a phosphate buffered saline, PBS (pH 7·4), then dehydrated in graded ethanol and finally embedded in epon. The horizontal semithin sections (1–2 µm) were cut on a microtome with a diamond knife (MICROM HM360; www.hylandscientific.com) and stained with toluidine blue (Carson & Hladik, 2009).
For the paraffin embedding, the formalin-fixed samples were washed with tap water for 8 h to remove formalin. Specimens were decalcified in 14% EDTA solution for 7 days, washed in PBS at pH 7·4, then dehydrated with ethanol and xylene, cleaned and embedded in paraffin wax (Histosec Merck; www.merck.com). Horizontal sections of 5 µm were deparaffinized in xylene, hydrated in graded ethanol, then washed in distilled water and stained with a picrosirius red (PSR) solution in order to visualize collagen thickness. The protocol was conducted according to the methods of Junqueira et al. (1979). Evaluation of collagen fibre patterns was purely qualitative, focusing on marked differences in the thickness of the fibres between normal and abnormal bone.
All sections were covered with a cover glass using DPX (Sigma Aldrich; www.sigmaaldrich.com), examined with light microscopes (Olympus BX41; www.olympus.com and Leica Polyvar; www.leica.com) and photographed with a digital camera (Olympus-Color View I and Olympus-Color View 8). Additionally, PSR sections were observed using ordinary and polarized light. Under polarized light, PSR staining shows collagen as brilliant red on a yellow background (Junqueira et al., 1979; Kiernan, 2008). A qualitative analysis of the relative thickness of the collagen fibres was based on the birefringence colour of the stained collagen, changing from bright red–orange (thicker collagen fibres) to green (thinner collagen fibres) (Junqueira et al., 1982; Dayan et al., 1989; Montes & Junqueira, 1991).
Bone thickness measurement and bone lining cell counts
Bone thickness measurement and bone lining cell counts were done on a series of histological images of five normal and five deoperculated specimens. Abnormal phenotypes were identified based on externally visible signs of opercular reduction or opercular shape distortions under a stereomicroscope. For this analysis, specimens were used from 40 to 69 dph. As it has been shown that opercular deformation is completely left–right decoupled (Verhaegen et al., 2007), left and right opercles are considered as separate specimens for this analysis. Seven horizontal sections from top to bottom of each opercle were sampled for bone thickness calculation [Fig. 1]. These sections symmetrically cut the left and right gill covers of each fish. In order to estimate the error that could be induced in the bone measurements, due to the fact that the orientation of the fish head in the blocks may not be perfectly vertical, modelling was undertaken of how and how much the cross section of the opercle (as it is cut in the histological sections) differs from if it would be cut in a plane that is perfectly perpendicular to the body symmetry plane (when the fish head would be embedded perfectly vertical). By this method, two surfaces were obtained: the first obtained from extruding the contour of the opercular section perpendicular to the actual histological section and the second obtained by extruding the contour of the opercles, but now in a plane that goes parallel with the body symmetry plane. To quantify this potential difference in measured bone thickness in the histological section (i.e. the actual cutting plane of the histological sections) v. the modelled section (i.e. horizontal cutting plane perpendicular to the midsagittal symmetry plane), 30 measurements of bone thickness were taken on a randomly taken position across the opercles (15 from the left opercle, 15 from the right opercle). The data from both sections were normally distributed based on the result of Shapiro - Wilk normality test-histology: W = 0·9513, P > 0·05 and modelled: W = 0·9518, P > 0·05). An ANOVA showed that there was no significant difference in the measurements taken on the histological versus modelled sections (F1,58 = 0·0013, P > 0·05). The thickness of the opercle was then measured at four positions in each section: (p1) at the anterior border of the opercle (close to the suspensorium), (p4) at the posterior border of the opercle (close to the gill slit), (p2) and (p3) at the middle of the opercle equally spread between p1 and p4 [Fig. 1(c), (d)]. All measurements were taken using analySIS docu 5·0 software (www.olympus-sis.com). The bone lining cells were counted on the surface of the bone matrix layer of the opercle in the same sections used for bone-thickness measurements.
). (b) Horizontal section stained with toluidine blue, showing a normal [right side and (c)] and deformed [left side and (d)] opercle. p1, p2, p3, p4, Positions where bone thickness was measured.Statistical analysis
A principal component analysis (PCA) was performed to examine overall patterns of variation across the 28 thickness measurements and seven osteoblast counts and was compared across normal v. deformed opercles. An analysis of covariance (ANCOVA) on the first principal component (PC1) score with log-transformed LS as the covariate was performed to test whether there is an overall size-independent difference in opercle thickness and osteoblast counts in deformed v. normal opercula. All these analyses were performed using PAST 2.17b (copyrighted by Hammer & Harper; http://nhm2.uio.no/norlex/past/download.html) (Hammer et al., 2001).
Additionally, it was tested to ascertain whether opercular deformation involved localized differences in thickness, by considering the section position and the measurement position as additional dependent variables instead of as random repeated measures. Differences in bone thickness between opercular phenotypes (normal and deformed) were tested by means of generalized linear mixed models. Controls for potential colinearity among the different measures were made by adding the individual specimen (being a single opercle from one side of a specimen) intercept and their interactions (slopes) with the section and measurement position variables as random effects into the model. A similar analysis was performed on the osteoblast counts, although now only the section position was added as fixed effects and random slope. All analyses were done in SAS 9.2 (SAS company; www.sas.com) with the GLIMMIX procedure, using respectively Gaussian and Poisson error structure. This procedure fits generalized linear mixed models by likelihood-based techniques conditional on normally distributed random effects. All statements of statistical significance refer to the 0·05 level.
Results
Histological ontogeny of normal and abnormal opercula
Stage 7 dph
No opercular bones have formed yet in the opercular skin fold at 7 dph. In this stage, mesenchymal cells aggregated within the opercular skin fold. This is the region where the bone will form in the later stage. A few elongated fibroblasts can be found around the mesenchymal condensation [Fig. 2(a)].
) are visible within the opercular skin fold; bone is still absent; (b) 14 dph (3·9 mm LS), opercle is visible between the thin opercular epithelium, showing more concentrated osteoblasts (
) at the edge; (c) 19 dph (4·7 mm LS), a straight opercular bone, showing osteoblasts (
) concentrated at the edge and interspersed at the inner bone surface; (d) 33 dph (4·8 mm LS), opercle tapers towards the posterior edge, with osteoblasts (
) at high density distributed at the posterior edge. A few bone-lining cells (
) lie at the outer bone surface. (e)–(h) 40–61 dph, rounded and cuboidal osteoblast cells (
) located mainly at the posterior edge of both opercle and subopercle, flattened osteoblasts interspersed at the inner mineralized bone surface and flat elongate bone-lining cells (
) distributed at the outer mineralized bone surface. Sizes of 40 dph, 47 dph, 54 dph and 61 dph specimens were 8·3 mm, 11 mm, 13·4 mm, 13·9 mm LS, respectively. GA, gill arch; HC, hyosympletic cartilage; MC, mucous gland; Op, opercle; OS, opercular skin fold; sOp, subopercle.Stage 14–19 dph
The opercle is visible as a flattened dermal bone, attached to the opercular process of the hyosympletic cartilage. A mineralized bone layer lies within the connective tissue of the opercular skin fold. At this stage, mucous glands could be seen below the outer opercular epithelium. It is difficult to distinguish a normal from an abnormal opercle c. 14 dph. In most cases, histological sections showed rounded osteoblasts that were stained dark blue, which are more concentrated at the posterior border of the bone [Fig. 2(b)].
The normal opercle of 19 dph larvae shows a straight cross-sectional profile and is covered by thin outer and inner skin layers. The outer epithelial layer is very thin and interspersed with mucous glands lying within the epidermal layer. The bone forming cells (osteoblasts) were concentrated at the opercle's posterior edge and have a rounded shape. This is clearly distinct from the fattened osteoblasts that are distributed at the inner opercle surface. At this stage, the deformed opercle is posteriorly curved slightly inwards into the gill chamber [Fig. 3(a)]. Osteoblasts appear to be concentrated at high densities on both the inner and outer sides of the curved-part [Fig. 2(c)].
, gill chamber). (a) 19 Days post hatching
(dph) and (b) 26 dph showing mildly folded opercles with osteoblasts at a high density and mucous glands at the curved part (
). (c) Specimen at 40 dph, showing the opercle curled outwards at the posterior edge, covered with osteoblasts (
) and bone lining cells (
) on the outer bone matrix surface. (d) Specimen at 40 dph, showing opercle curled outwards with the non-mineralized osteoid surrounding at the curved point (
). Osteoblasts can also be observed at this point (
) with a high amount of osteoblasts at the posterior edge (
). (e) Specimen at 47 dph and (f) 61 dph showing inward folded opercles with thick inner skin layer (*), containing adipose-like cells (
). Osteoblasts (
) present at the inner bone surface and posterior edge. Bone lining cells (
) distributed at the outer bone surface. (g) Intensive inward folding into the gill cavity of the opercle in a 61 dph specimen, showing a branchiostegal ray lying within
the folded area. Br, branchiostegal ray; GA, gill arch; MC, mucous gland; Op, opercle; sOp, subopercle. Scale bars 50 µm.Stage 26–33 dph
Compared with previous stages, the normal opercle has become elongated posteriorly and now almost completely covers the gill cavity. The cross section of the opercle now also tapers towards the posterior border. The connective tissue of the dermis layers that cover the opercle was much thicker compared with that of younger larvae. The presence of rounded osteoblasts being concentrated at the posterior edge of the opercle (versus a few flat bone lining cells at the outer surface of opercle) indicates that most of the matrix deposition occurs here [Fig. 2(d)]. In an abnormal opercle, the number of rounded osteoblast that concentrate at the posterior edge was larger compared with that of a normal opercle [Fig. 3(b)].
Stage 40–47 dph
The bone matrix in both normal and abnormal opercles stained darker at this stage compared with previous stages, suggesting that the matrix was more mineralized from these stages on. In a normal opercular phenotype, the rounded and cuboidal osteoblasts were located mainly at the posterior edge of both the opercle and subopercle. Flat and elongated bone lining cells were present at the outer bone surface [Fig. 2(e), (f)].
At this stage, most of the observed deformities were mild with the opercle folding inward into the gill cavity [Fig. 3(e)]. In some rare cases, the opercle curled outwards at the posterior or intermediate region [Fig. 3(c), (d)]. A thin layer of mineralized bone matrix with surrounding osteoid (paler line between the dark bone matrix and the layer of bone cells) and several osteoblasts could be found at the curved point of an outward folded opercle [Fig. 3(d)]. Although normal and deformed opercles showed some differences in their histological phenotype, there seems to be no obvious difference in the distribution of osteoblasts around the edges of the opercular elements. In several deformed ones, the connective tissue layer contained cells that have the phenotype of adipose cells [Fig. 3(e)].
In one 47 dph specimen, an irregularity in the opercular shape was found. It displayed a zigzag pattern at its posterior edge and was only observed in a few sections throughout the whole opercle. Numerous osteoblasts were also observed at this position [Fig. 4(a)].
) around. (b) Deformed opercular complex in a 47 dph larva, showing the opercle folding inside and the subopercle folding outside of the gill cavity. (c) Spiky protuberance with osteoblasts (
) distributed at the tip, suggesting new bone is formed on the inner side of the deformed opercle in 47 dph specimen. (d) Larva 69 dph and (e) 61 dph showing irregular surface in a showing deformed opercles, showing several bony spikes budded bones (
) and a chamber formed by a bony strut (*) in the folded area. The loose connective tissue was found inside the chamber. cOp, Curled opercle; GA, gill arch; Op, opercle; sOp, subopercle. Scale bars 50 µm. (a)–(d) epon embedding with toluidine staining; (e) paraffin embedding with PSR staining.In another deformed phenotype, the gill cover showed the double folding in a 47 dph specimen, with the opercle folded inside and the subopercle folded outside of the gill cavity [Fig. 4(b)].
In another deformed phenotype of a 47 dph specimen, a spiky protuberance was found on the inner surface of the opercle, suggesting new bone is formed on the inner side. The osteoblasts were also observed at the tip of the spike, indicating bone deposition is occurring at this position, which could be the onset of a bony trabecula being formed [Fig. 4(c)].
Stage 54–69 dph
The normal opercle in 54–69 dph larvae showed a similar structure to that in the previous stage (40–47 dph). Two differences were observed in the malformed opercles, however: a severe deformity with the substantial inward folding of the opercle [Figs 2 (g), (h), 3(f), (g)] and the presence of a bony trabecula spanning the curved part of the opercle, creating a chamber filled with loose connective tissues [Fig. 4(d), (e)].
In cases of intensive inward folding, the inner skin layer was thicker at the folded area and contained several adipose-like cells inside [Fig. 3(f)]. In addition, osteoid deposition could be observed at the inner surface of the inward folded opercle [Fig. 3(f)]. A rare observation was a branchiostegal ray enclosed in the inward curved part of a strongly curled opercle at this stage [Fig. 3(g)].
Bone thickness calculation and bone lining cell counts
The ANCOVA on the PC1 scores (with PC1 describing 68·2% of the total variation in bone thickness) showed that deformed opercles did not differ from normal ones in their overall thickness (test for homogeneity of slopes: F1,17 = 0·495, P > 0·05; test for intercepts: F1,18 = 0·016, P > 0·05) (Fig. 5).
; y = 61·798x − 64·811, r2 = 0·553 and deformed (
, y = 86·263x − 91·448, r2 = 0·570) larvae.The complex test on interaction effects between the different opercular phenotype, section and positional measurements also showed there was no significant difference in overall bone thickness between normal and abnormal opercles (P > 0·05). There was, however, a significant difference in bone thickness for the separate sections (P < 0·05) and for the positional measurement (P < 0·001). This was not the case for the interaction effect between phenotype and section (P > 0·05), indicating that thickness of both normal and deformed opercles is similar from upper to lower of sections of the opercle. As phenotype*measurement-position gave a significant result (P < 0·05), it suggests that there was a significant difference between phenotypes taking into consideration the measurement-position. Based on the details of the analyses and after correcting for standard length, this significant difference was located at the first measurement-position (p1) where deformed fish had an average opercle thickness of 8·37 (± 0·59) µm whereas for normal ones, this was 6·76 (± 0·57) µm.
For bone-lining cell counts, both the one-way ANCOVA (test for homogeneity of slopes: F1,15 = 0·61, P > 0·05; test for intercepts: F1,16 = 0·00005, P > 0·05) showed that there was no significant difference in the number of bone lining cells between normal and deformed opercles (Fig. 6). According to the complex test, the bone lining cell amount between the phenotypes, between the sections, as well as for their interaction effect between both were not significant (P > 0·05).
; y = 71·99x − 74·66, r2 = 0·659 and deformed (
, y = 102·64x − 106·82, r2 = 0·618) larvae.Collagen fibre thickness
Differences between normal and deformed opercles in collagen fibre thickness were demonstrated by PSR staining and visualizing under polarized light. While the normal opercles showed a homogeneous red colour under polarized light [Fig. 7(a), (b)], deformed opercles showed brighter stained orange regions with a lack of colour at some sites [Fig. 7(c), (d)]. The intensively deformed opercle presented a more heterogeneous colour staining where the brighter orange, yellow and greenish regions were narrow strips, aligned in a parallel direction [Fig. 7(e), (f))].
Discussion
Earlier histological studies of opercular malformations in S. aurata provide a general description of opercular anomalies, reporting on the folding or curvature of the opercular bones and tissue proliferation around the folded area (Paperna, 1978; Galeotti et al., 2000; Beraldo et al., 2003; Ortiz-Delgado et al., 2014). Still, a clear understanding of how the skeletal formation and deformation in the opercle becomes established during ontogeny and how it relates to changes in associated tissues is lacking. Using regular histological techniques, particular traits were characterized that reflect changes in both the osteogenesis and the differentiation of tissues surrounding the bone.
Early development of the S. aurata opercular complex, with the formation of a flattened opercle at 7–14 dph is consistent with previous observations by Koumoundouros et al. (1997), Galeotti et al. (2000) and Beraldo et al. (2003). Faustino & Power (2001) indicated that the opercle is the first dermal bone of the opercular series to appear and ossifies at 3·7 mm notochord length (LN) (approximated 3·7 mm Ls). The current study confirms this, as the opercle is still absent in 7 dph larvae with 3·1 mm Ls but was first observed in 14 dph larvae with 3·9 mm Ls.
The ontogeny of a normal opercle from 7 to 69 dph mainly involves appositional bone deposition of acellular bone. A similar period of change in shape and size of the opercle without bone resorption and remodelling was reported in zebrafish Danio rerio (Hamilton 1822) from three to 38 dph (Kimmel et al., 2010). During early ontogeny, osteoblasts were concentrated around the posterior edge of the opercle, indicating that most matrix addition occurs at the edges, as has also been observed during early development in D. rerio (Kimmel et al., 2010).
With respect to the onset of opercular malformations, this study also corresponds to previous studies. The first histological indication of opercular deformation in the current study was at 19 dph (4·5 mm LS). Galeotti et al. (2000) detected it at 17 dph using histology, while it could only be observed at 23 dph (6·1 mm LT) using the alizarin red S staining method (Koumoundouros et al., 1997). This difference in the initial detection of opercular deformities can be explained by the limitations of in toto clearing and staining with alizarin staining for early ossification when bone matrix has not calcified yet. Although the histological aberration leading to a phenotypic deformation may start at the very beginning of opercle ossification, it still remains difficult to identify the actual deformed phenotype at this point. The opercle skin fold was seen at 7 dph when there is no bone present, but it ossifies as a very thin opercle at 14 dph. It can be expected that at this stage, an unsupported opercular skin fold is more susceptible to altered external influences, especially mechanical ones such as an excessive water flow in rearing tanks. This can cause mechanical damage to a developing opercular bone, influencing bone morphogenesis (Hall, 2015). Furthermore, minor deviations of a proper positioning of the opercular bone within this skin fold at this stage of early development may become consolidated during later development (Galeotti et al., 2000; Beraldo et al., 2003).
The presence of irregular bone matrix, observed at the curved point of deformed opercles (40 dph), could indicate abnormal mineralization during early skeletogenesis. This result is in agreement with Galeotti et al. (2000), who showed irregular mineralization within the bone matrix in deformed opercles of 30–50 days-old larvae. Accordingly, Koumoundouros et al. (1997) pointed out that any mechanical stress or abnormal position of the opercular complex at the moment it begins to ossify (after 6·0 mm LT or about 20 days old in S. aurata larvae), could lead to abnormal calcium deposition, thus causing deformities to appear. Some studies have proposed that nutritional factors affect bone mineralization, as a predisposition to opercular deformities during early ontogeny. Gapasin et al. (1998) concluded that reared milkfish Chanos chanos (Forsskål 1775) fed highly unsaturated fatty acid + vitamin C enriched live food had a lower incidence of opercular deformities. Also diets with low DHA levels have been shown to postpone early mineralization and increase the risk of opercular deformities (Izquierdo et al., 2013). A first exploratory comparison of mineralization patterns, using micro-computed tomography (CT)-data, showed an increased mineralization in the folded opercle of a 65 dph larvae (Morel et al., 2010). Although both aberrant mineralization and deformed opercles were recognized during S. aurata larval stadia, causal relationships are still unknown. Further insights in this could be obtained from a more comprehensive comparison of CT-data during normal and abnormal development (work in progress).
In this study, direct indicators (lacunae of Howship, reversal lines, active osteoclasts) of osteoclastic activity and bone remodelling could not unambiguously be observed because of the staining procedure applied. A previous study, however, showed that a bone remodelling process takes place in deformed opercular structures of S. aurata (Ortiz-Delgado et al., 2014). According to Witten & Huysseune (2009), bone remodelling can be linked to an increased mechanical stimulus, allowing the skeleton to adapt to changes in mechanical loading. The observation of bone remodelling during early ontogeny in deformed opercles might be a mechanotransduction effect from aberrant water flow against a folded opercle during respiration or suction feeding. Bone remodelling as a response to altered feeding conditions has been recorded in jaw bones of other teleosts (e.g. the cichlid Astatoreochromis alluaudi Pellegrin 1904; the orangespotted sunfish Lepomis humilis Girard 1858) (Huysseune et al., 1994; Hegrenes, 2001). In this study, the spiky protuberances and chamber formed by a bony strut in the folding area of extreme opercular deformities strongly suggests the appearance of excessive bone remodelling and a bone disorder. Increasing mineral density in deformed opercles (Morel et al., 2010) could be an indicator of a bone morphogenesis disorder at the early onset of the phenotypic deformity becoming apparent. To what degree it is the organic bone matrix deposition, rather than matrix mineralization that is involved still remains unclear.
Although qualitative histological differences between normal and deformed opercles could be observed from 7 to 69 dph, there were no significant overall differences in both bone thickness and number of bone lining cells on the bone surface. Histological observation in this study showed that most osteoblasts, which are involved in matrix outgrowth, are concentrated at the posterior edge of opercles. Although a thickened layer of connective tissues was found where the opercular bone is heavily folded [Fig. 3(f)], no increased bone thickness was observed. Clearly more detailed studies are required to resolve the causes of opercular deformations and effects of opercular osteogenesis on deformities during early ontogeny.
Collagen fibres of opercular bones, which were stained with PSR, were visualized under polarized light. Differences in coloration patterns of collagen fibres between normal and deformed opercular bones suggest that they differ in thickness. According to Junqueira et al. (1982), Dayan et al. (1989), Montes & Junqueira (1991), thickness of collagen fibres is displayed in PSR birefringence with orange or red stain representing thicker (thus stronger) fibres and greenish stains representing thinner fibres. In this study, normal opercles seemed to have thicker collagen fibres, with a homogeneous red staining, whereas deformed opercles displayed a much more heterogeneous staining with areas of red dye interspersed with areas of orange and even yellowish to greenish dye (some fibres did not stain at all). A similar description was given by Ortiz-Delgado et al. (2014) for opercular bone development in S. aurata at later stages (from 3 to 10 months age). A bone remodelling process that occurs within the deformed opercle can explain these differences in collagen thickness between normal and deformed opercles. Indeed, Fernández et al. (2012) mentioned that changes in bone composition and degradation of collagen fibres is always related to bone remodelling processes. In addition, collagen represents >90% of the organic matrix in bone (Tzaphlidou, 2008) and collagen fibrils are affected, with changes in compositional properties of collagen in abnormal bone (Bailey et al., 1992; Kafantari et al., 2000). As shown by Totland et al. (2011), matrix composition and structure of a bone can affect the mechanical properties of its bone. Therefore, thinner collagen fibres in deformed opercles might increase the risk of failing or instability when exposed to mechanical disturbance.
In conclusion, a qualitative and quantitative comparison of the early ontogenesis of normal versus deformed opercles in S. aurata is given using histological methods. Mild deformities were first detected in 19 dph larvae by folding of the opercle's edge into the gill chamber. No significant difference was found in bone thickness between deformed and normal opercles. Furthermore, differences in bone architecture and differences in collagen fibre thickness between normal and deformed opercles were found.
This work was financially supported by the scholarship program (no. 1081/QD-BGDDT) of Vietnam International Education Development (Ministry of Education and Training, Vietnam). The authors thank INVE and the staff of hatchery Maricoltura Rosignano di Solvay for their help in fish rearing and kind supply of specimens.
