Characterization of Scale Abnormalities in Pinfish

Characterization of scale abnormalities in pinﬁsh, Lagodon rhomboides,from Biscayne Bay, Florida

Jone Corralesa, Laurie Beth Nyea, Sean Baribeaua, Nancy J. Gassmanb & Michael C. Schmalea,c aDivision of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149, U.S.A. bWater Resources Division, Broward County Department of Natural Resource Protection, Ft. Lauderdale, FL, U.S.A. cSenior author for correspondence (e-mail: [email protected])

Received 5 November 1998 Accepted 19 April 1999

Key words: gross pathology, estuarine ﬁsh, deformity

Synopsis

Correlations between marine habitat degradation and the prevalence of abnormalities and diseases in populations can provide a starting point for understanding the effects of changes in environmental conditions on marine organisms. The present study characterized the features of scale disorientation (SD), a common morphological anomaly encountered in pinfish, Lagodon rhomboides, in Biscayne Bay, Florida (U.S.A.). Scale disorientation consisted of discrete patches of scales rotated dorsally or ventrally away from the normal scale position without any projection of the scales outwards from the body surface. The direction of scale growth within the patches varied from normal to a minor misalignment to a complete reversal of direction. The severity of SD, defined as the percentage of body surface area affected, varied from 1 to 34% with a mean of 9.3%. Affected fish monitored in the laboratory demonstrated a proportional growth of SD areas such that the percentage of body surface affected did not change as the fish grew. Scale disorientation was more prevalent in the northern region of the bay, an area known to be more contaminated. Scales from SD areas exhibited significantly abnormal morphology with larger average focus diameter, smaller size, more elongate shape and fewer radii relative to normal scales. Experimental removal of scales demonstrated that normal scales regrew in normal orientation and morphology while those from SD areas regrew in abnormal orientations and morphologies. Experiments in which fish were exposed to acute and chronic injuries indicated that these physical traumas were insufficient to directly induce formation of scale disorientations typical of those seen in the wild. Observations of pinfish in the laboratory revealed that SD areas can appear spontaneously in normal juvenile and adult fish. These new SD areas developed relatively rapidly, did not require prior scale loss and remained stable in size after first appearance. Although the etiology of SD remains unknown, the significant difference in prevalence of this syndrome between regions of Biscayne Bay having different levels of sediment contaminants suggests that environmental factors may be important in development of SD.

Introduction 1996). The correlation of abnormal conditions with the presence of specific environmental or other factors can An important indicator of the effects of habitat degra- serve as a first step in understanding the etiology of dation on fishes and invertebrates in aquatic envi- such syndromes. A critical step in this process is the ronments is a change in the prevalence of diseases description of specific abnormalities in sufficient detail or deformities in exposed populations (Sindermann to allow design and testing of hypotheses concerning 206 the roles of environmental or other factors in develop- Materials and methods ment of such abnormalities. We have previously described distribution patterns Collection of fish of several types of abnormalities in fish and invertebrates in Biscayne Bay, Florida (U.S.A) and proposed Biscayne Bay is a shallow tropical estuary within a possible relationship between these abnormalities Miami-Dade county Florida, U.S.A. (Figure 1). This and habitat degradation (Gassman et al. 1994). The body of water is bounded on the east by a series of most prevalent abnormality observed in this study barrier islands. The bay receives urban runoff from was scale disorientation (SD) and the most commonly the city of Miami, particularly in the northern region. affected species was the pinfish, Lagodon rhomboides. Agricultural and suburban runoff impacts the southern The present study focuses on this scale abnormality bay from the southern region of the county. In addi- in pinfish in Biscayne Bay. This syndrome is char- tion to direct runoff, input to the bay is provided by acterized by a rotation of scales dorsally or ven- a large number of rivers, streams, creeks and canals trally away from the normal scale position. The draining the county. Four sites in the northern region direction of growth of the individual scales within of the bay (north of Rickenbacker Causeway) and four the patch varies from normal to a minor misalign- south bay sites were sampled monthly from August ment to a complete reversal of direction without 1993 to September 1994 (Figure 1). Sites were selected any projection of the scale outwards from the body based upon previous knowledge of the distribution of surface. pinfish. Sampling was conducted at night using paired The anatomy and developmental patterns of fish roller trawls (3m×4.6m×0.9m) with 1 cm2 mesh scales and the relationship of scale morphology to net. Nets were deployed for two, ten minute intervals genetic and environmental factors have been well at each site. The combination of mesh size and finger described (Blair 1942, Yamada 1961, Fouda 1979, bars on the trawl mouth typically limited the catch to Sire 1986, Bereiter-Hahn & Zylberberg 1993). How- individuals 30–250 mm in length. The prevalence of ever, abnormalities in scale morphology or arrange- pinfish with SD was estimated from surveys made over ment have rarely been reported. Taki (1938) described the total 14 month period of the study. To facilitate a a similar phenomenon in sole, Zebrias japonicus,in detailed analysis of SD, all pinfish with this abnormal- Japanese waters and suggested it might arise during ity encountered in surveys in July and September 1994 embryonic development. Gunter (1941, 1945, 1948) were collected and frozen for later study. reported three cases of disoriented scales in redfish, Sciaenops ocellata, in Nueces Bay and Mesquite Bay, Quantification of disorientation patterns of scales Texas. In pinfish, SD has also recently been observed in the Indian River Lagoon, Florida (Browder personal Scale disorientations were diagnosed by visual inspec- communication). tion and by touch. In order to document patterns of In Biscayne Bay, the occurrence of SD was docu- arrangement of scales within disoriented regions, a mented by Overstreet (1986), Skinner & Kandrashoff scale by scale examination was made of four such (1988) and Browder et al. (1993), sometimes referred patches on three fish. The angle of growth of each scale to as ‘Kandrashoff syndrome’. In 1992–1993, SD was was recorded relative to that of normal scales in adja- observed in several species of fish in this region, includ- cent areas outside of the affected patch. Directions were ing sea bream, Archosargus rhomboidalis, blue striped measured as degrees of deviation from normal, grouped grunt, Haemulon sciurus, bermuda chub, Kyphosus into 30 degree increments (i.e., 0◦ = normal, free edge sectatrix, and pinfish (Gassman et al. 1994). The high- toward the tail of the fish and 180◦ = reversed, free est prevalences were observed in pinfish with levels edge toward the head of the fish). reaching 15% at some locations. Thus, while the occur- Severity of SD was defined as the surface area of rence of scale disorientation has been documented each patch relative to the total surface area of the in the past, it has never been thoroughly character- individual fish. These areas were expressed as the per- ized in any species. The present study describes in centage of the body surface on a single side of a fish detail the characteristics of SD in pinfish and evalu- which was affected by SD. Scale disorientation areas ates several potential mechanisms for development of were measured on all pinfish collected which exhib- this syndrome. ited SD (n = 140). These areas were measured either 207

Figure 1. Sampling locations for pinfish, Lagodon rhomboides, in Biscayne Bay, Florida. Sample sites were subdivided into four northern region sites (light circles) and four southern region sites (dark circles). Full names for sites are provided in Table 1, corresponding to two letter codes shown on this map. from photographs or by tracing the outline of the fish over time in laboratory held fish were quantified on (exclusive of the head and fins) and each SD patch onto scanned images of photographs or traces using NIH a transparent plastic sheet, one side at a time. Areas Image software on a Power Macintosh computer. Body of SD regions were estimated using a grid measure- surface areas were also subdivided into regions above ment system. Measurements of changes in SD patches and below the lateral line. Prior to parametric analyses 208

(by ANOVA) of severity differences between groups, muscle tissue anterior to the caudal peduncle (con- these percentage measures were transformed using nat- stituting a chronic injury) with colored beads on the ural logarithms. monofilament used to identify individuals. This pro- tocol was based on Patzner’s (1984) tagging method. Fish were observed weekly for 6 months to detect any Scale morphology changes in the conformation of the scales or any other skin anomaly. A subset of 26 pinfish from 5 sample sites were selected for analysis of the morphology of disoriented scales. Ten disoriented scales were collected from the cen- Results ter of each patch with SD and ten normal scales from the opposite side at the equivalent location. The 520 Orientation of scales in disoriented patches scales (260 per scale type) were stored in 70% ethanol and examined using a stereo microscope at a mag- Disoriented scales were generally confined to a sin- nification of 16–40×. Four variables were measured gle discrete location on one side of each pinfish for each scale: anterior–posterior length (major axis), (Figure 2). The disorientations reflected light differ- dorso-ventral length (minor axis), diameter of the focus ently and appeared as a disruption of the normal color in the center of the scale and number of radial lines pattern of the fish. Patches of disoriented scales felt (radii) emanating from near the focus. In addition, a rough to the touch compared to the surrounding scales. fifth variable, the ratio of major to minor axes, was The direction of growth of disoriented scales typically calculated as a measure of scale shape. varied within a single patch, between patches on the same fish, and from fish to fish (Figure 3). Even though Experimental manipulations

Juvenile and adult pinfish were collected in the field by trawl or hook and line and transported to the laboratory for observations and manipulations of scales. Fish were held in 190 to 750 liter aquaria with flow-through or recirculating sea water and fed a diet of commercial trout chow and frozen shrimp. Fish were observed daily for major changes in appearance of skin and monthly for detailed examination of scales to detect changes in scale conformations indicative of early stages of SD. Fish collected with SD or which developed SD in the laboratory were photographed or traced periodically to document changes in surface area of the affected region relative to the size of the fish. Fish were anesthetized using MS-222 at 120 mg l−1 during any potentially trau- matic procedures. Scale removal experiments were conducted by care- fully pulling individual scales out of scale pockets in groups of 10–12 scales, leaving the scale pockets intact. In some experiments, these patches were also scraped with a scalpel blade to remove the epidermis and some Figure 2. Proportional growth of an area of scale disorientation dermal layers, ablating the scale pockets. in a pinfish, Lagodon rhomboides. An individual fish observed Contusion injury experiments were conducted in over a period of nine months exhibited an increase in surface which adult pinfish were injured by a physical impact area of abnormal scales which was proportional to the increase in −1 −1 somatic growth such that relative surface area affected remained (acute injury) consisting of a 0.022 kg m sec impact constant. Patch of disoriented scales indicated by arrow.a–16 induced by the free falling of a round, 120 g lead weight April 1996, fork length of fish = 75mm.b–17December 1996, from a height of 0.32 m. These fish were also tagged fork length = 111 mm. Scale bar corresponds to 1 cm in both with monofilament line inserted through the dorsal photographs. 209

Figure 3. Orientation of growth of disoriented scales from representative pinfish, Lagodon rhomboides, collected from Biscayne Bay. a – Diagram showing direction of growth of individual scales within an area of scale disorientation (each arrow represents one scale). Major axes of orientation of the fish are indicated by large arrows, including the direction of normal scale growth (open arrowhead, posterior direction). Scales with normal orientation outside of the disoriented area are not shown. Dashed line denotes exclusion of head region for calculation of surface area involved in scale disorientations. b–e – Summaries of directions of scale growth for four patches of scale disorientation indicating, by vector length, the percentage of total scales within a patch growing in a specific orientation (summarized in 30◦ increments). Calibration scale indicates relationship between length of vectors and percentage of scales in each group(b–fish1, left side, large-sized SD; 252 scales, 28.3% coverage of body surface. Note that this is the same fish as shown in a, c – fish 2, left side, intermediate sized SD; 134 scales, 9.4% coverage of body surface, d – fish 3, right side, intermediate sized SD; 34 scales, 4.5% coverage of body surface, e – fish 3, left side, small sized SD; 13 scales, 1.4% coverage of body surface). disoriented scales grew in multiple directions within a 10 scales. This two-dimensional measuring system was patch, subpatches which consisted of disoriented scales adequate for these studies as all scales were essentially growing in the same direction were observed within the flat relative to the body surface and were never project- primary patch. The direction of growth of contiguous ing up from the surface of the fish. In addition, these subpatches deviated from 30◦ to 180◦ (complete rota- areas of SD were never observed to involve any ulcer- tion of the scale). Although a predominant direction of ation or other overt damage to the epidermal layers. growth was sometimes observed within a patch, this was not always the case and no trends or patterns of Prevalence and severity of SD scale orientations were detected among the 4 patches analyzed. A comparison between numbers of scales in Prevalence of pinfish with SD varied between sites from each patch and surface area affected indicated that 1% 5–24% (Table 1). The prevalence varied significantly of the body surface was equivalent to approximately between sites overall and the northern sites exhibited 210

Table 1. Prevalence and severity of scale disorientations (SD) in pinfish, Lagodon rhomboides. Prevalence levels of pinfish with SD were calculated based on all fish collected from August 1993 to September 1994. Analysis of severity of SD was based on affected fish collected in July and September 1994. Some fish had SD on both sides so that the total sample size is based on the number of fish sides analyzed, rather than the number of fish. Sites are arranged from north to south as divided by the Rickenbacker Causeway (see Figure 1). Average severity was measured as the mean percent surface area with SD above and/or below the lateral line. Site (letter code) Prevalence of SD Severity of SD (mean percent surface area affected)

% ﬁsh affected Fish (sides % above % below % total (Affected ﬁsh/total sample size) analyzed) lateral line lateral line body area Biscayne Canal (BC) 19.3 (34/176) 7 (9) 8.3 0.1 8.4 Little River (LR) 23.8 (24/101) 0 (0) Sunset Harbor (SH) 11.6 (17/147) 3 (3) 14.2 2.8 17 Miami River (MR) 8.0 (8/100) 0 (0) Sum for northern region 15.81 (83/524) 10 (12) 9.8 0.8 10.6 Rickenbacker Caus. (RC) 5.0 (7/139) 0 (0) Matheson Hammock (MH) 10.1 (159/1569) 14 (16) 7.2 0.6 7.7 Black Point (BP) 13.5 (164/1213) 34 (40) 9.2 0.5 9.7 Turkey Point (TP) 12.1 (247/2043) 82 (88) 8.9 0.3 9.2 Sum for southern region 11.6 (577/4964) 130 (144) 8.8 0.4 9.2 Total 12.0 (660/5488) 140 (156) 8.92,3 0.4 9.3

1Northern sites exhibited significantly higher prevalences of SD than southern sites (G-test, p < 0.05). 2Severity of SD above the lateral line varied significantly between sites overall (ANOVA, p < 0.05) [while percentages affected below the lateral line and of the total surface area were not significantly different by site (ANOVA, p > 0.05).] 3The percentage area involved above the lateral line was significantly greater than that below the lateral line (t-test, p ≤ 0.001).

a slightly but significantly higher prevalence than the The mean percent surface area affected per pin- southern sites (p < 0.05, G-test). Severity of SD var- fish was 9.3% (Table 1). Of 156 affected fish sides ied between sites with a small but significant differ- measured, 85.2% had less than 15% of the surface ence overall among sites in percentage of body surface area covered by SD (Figure 4). The smallest SD patch area above the lateral line affected with SD (p < 0.05, observed was 1.3% of the surface area, corresponding ANOVA).However, there was no significant difference to roughly 13 scales. The largest SD regions covered in the mean severity between fish at northern compared 34% of the body surface, an area equivalent to the entire to southern sites (p > 0.05, ANOVA). fraction of the body surface above the lateral line. Cor- The distribution of SD patches on the bodies of relation between size of fish and relative area of scale affected pinfish was highly skewed such that the vast disorientation was not significant overall or when cal- majority of disoriented scales were found on the dorsal culated for southern region fish only (Figure 5, Pearson region of the body. This was reflected in both the pro- correlation, p > 0.05). This correlation was significant portion of fish exhibiting SD only above the lateral line for fish from the northern region only (Pearson corre- (88.5% of fish) and the average surface area affected lation, r = 0.48, p < 0.05). However, the significance above the lateral line (8.9% above vs 0.4% below, of this correlation was not robust and relied heavily on Table 1). Only a small number of affected fish (11.5%) one fish, the largest individual in this group. exhibited any SD below the lateral line and these areas were contiguous with SD regions above the lateral line. Scale morphology No pinfish was observed with SD only below the lateral line despite the fact that most of the body surface Scales collected from areas of SD were significantly of the fish (66%) was located below the lateral line. different in morphology from normal scales based on The majority of affected pinfish (88.6%) exhibited SD all five variables measured (Figure 6, Table 2). Disori- on only one side of the body with no preference shown ented scales were significantly smaller in both major for either the left or right side (Chi-square, p > 0.05). and minor axes than normal scales (t-test, p < 0.05). 211

Focus diameter was used as an index to classify scales into two categories: ontogenic scales which probably were in place since the larva period and regenerated scales which developed as replacements following loss of ontogenic scales during the post- larva life of the fish. Due to the more rapid growth required for replacement scales to reach appropriate size, these scales would be expected to have a larger focus diameter. Based on a size frequency distribution of diameters, there was a clear demarcation at 0.2 mm suggesting that scales with larger focus sizes were regenerated scales while smaller diameters indicated ontogenic scales (Figure 7). Based on this crite- rion, 79% of scales from normal areas were ontogenic compared with 62% of scales from SD areas. Figure 4. Frequency distribution of size of scale disorientation Normal and SD scales were subdivided for further relative to the body surface area of affected pinfish. Frequency expressed as number of affected fish sides (each side measured analysis into ontogenic and regenerated scales based separately, N = 156 affected sides from 140 fish). The majority on focus diameter to control for the effects of the dif- (85.2%) of scale disorientations covered 15% or less of the body ferent percentages of regenerated scales within these surface area. The largest disorientations covered nearly 35% of groups on scale morphology averages. A comparison the body surface, equivalent to the entire area above the lateral of ontogenic normal with ontogenic SD scales showed line (34% of the body surface). the same variations in morphology seen for the groups as a whole (Table 2). With the exception of a slightly larger average focus diameter in regenerated normal scales, these relationships were also observed in com- parisons of regenerated scales although the differences were generally not significant due the large amount of variation in morphology seen in regenerated scales. Thus, these data indicate that SD scales were smaller, more elongate and had fewer radii than normal scales regardless of whether the scales were classified as ontogenic or regenerated. In addition, regenerated scales from either normal or SD regions were slightly larger, more variable in morphology and exhibited more radii than ontogenic scales.

Experimental manipulations Figure 5. Relationship of size of scale disorientation to fish size. Correlation between fork length and relative size of scale disorientation was not significant overall or when calculated for southern Preliminary studies were conducted in the laboratory region fish (•) only (Pearson correlation, p > 0.05). Correlation to address several questions raised by data from field- was significant for fish from the northern region () only (Pearson collected fish. Several groups of normal juvenile and correlation, r = 0.48, p < 0.05). However, significance of this adult pinfish as well as a group of fish collected with correlation was dependent on the largest fish in this group. SD were observed in the laboratory with and without manipulations. Both juvenile and adult pinfish were In contrast to normal scales which were essentially observed to develop SD spontaneously when held in round (major/minor axis = 1.0), SD scales also exhib- the laboratory with no other manipulations. These areas ited a relatively smaller minor axis, making them more of SD were typical of those observed in field-collected elongate (t-test, p < 0.05). The number of radii animals. Three of 15 juveniles (20%) developed SD observed in SD scales was significantly fewer than in lesions spontaneously over a 5.5 month period (exper- normal scales (t-test, p < 0.05). iment 1, Table 3). In experiments involving removal of 212

Figure 6. Morphology of a normal scale (a) and three scales (b, c, d) from a region of scale disorientation. The normal scale was collected from an equivalent area on the opposite side of the ﬁsh from the disorientation. Orientation of scales relative to body of ﬁsh is indicated (except for SD scale d for which orientation could not be determined). Scale bar corresponds to 2 mm.

Table 2. Morphology of normal and disoriented scales from pinﬁsh collected from Biscayne Bay. Scales from each area subdivided based on focus diameter into ontogenetic or regenerated categories. All values as mean values in mm ± standard deviation (N = 26 ﬁsh).

All scales Ontogenetic scales Regenerated scales Normal Disoriented Normal Disoriented Normal Disoriented scales scales scales scales scales scales Number of scales 260 260 204 160 56 100 Focus diameter (mm) 0.21 0.29 0.39 0.52∗ 0.10 0.02 0.11 0.03∗ 0.72 0.41∗ 0.71 0.47 Scale width (mm) 2.25 0.73∗ 2.03 0.72 2.17 0.63∗ 1.92 0.56 2.52 0.90 2.24 0.85 Scale length (mm) 2.25 0.63∗ 2.12 0.65 2.18 0.51∗ 2.01 0.56 2.52 0.82 2.30 0.75 Length/width ratio 1.00 0.08 1.10 0.16∗ 1.02 0.08 1.07 0.15∗ 1.01 0.09 1.05 0.17 Number of radii 9.80 1.70∗ 8.20 2.30 9.76 1.69∗ 8.27 2.29 10.48 1.69∗ 8.30 2.31

∗Significantly larger value within a normal-disoriented pair (t-test, p < 0.05). 213 scales from adult fish, unmanipulated (control areas) time required to completely form an area of SD was were also seen to develop spontaneous SD in a total less than one month. of 2 of 14 fish (14%) after 3 months of observation Observations of SD regions in the laboratory indi- (experiments 3 and 5). The exact timing of develop- cated that these patches generally increased in size only ment of these spontaneous SD events could not be in proportion to the growth of the fish (Figures 2, 8). determined due to the difficulty of constantly monitor- Surface areas of SD measured as a percentage of total ing scale alignment in numerous fish (the vast majority body area remained within 1% of initial observations, of which remained totally normal). However, based on even over periods as long as 8 months. This strictly pro- detailed monthly monitoring of all fish, the estimated portional growth was observed even from the earliest detectable appearances of the spontaneous SD events, suggesting that these lesions appeared essentially ‘fully formed’ (Figure 8). Scale disorientations were never observed to decrease in relative or absolute surface area. Six of 8 SD areas manipulated by removal of 10–12 scales from the center of each patch showed very slight increases in patch size over time (experiment 5, Table 3). These increases were in the form of small added patches on an edge of an existing patch and thus may have been unrelated to the scale removal manipulations. Experiments designed to determine if physical injuries to scales, scale pockets and/or underlining dermal layers were capable of provoking development of SD indicated that such injuries do not appear to induce SD in this species. No new areas of SD appeared Figure 7. Frequency distribution of focus diameter of scales from within 3–6 months of injury in regions subjected to normal and disoriented regions. Open bars correspond to scales scale removal, scale removal with epidermal scraping from normal areas and solid bars represent scales from disoriented to remove scale pockets, contusion injuries or chronic areas. Ten normal and ten disoriented scales were measured from injuries due to monofilament tags (experiments 2, 3 and 26 pinfish yielding a total of 260 scales per scale type. Scales 4, Table 3). In addition, fish were sometimes involved in having a focus diameter of less than 1 mm were grouped in 0.1 mm categories. Larger scales were grouped in 1 mm classes. Scales fights in laboratory tanks during which varying num- with a focus diameter≤0.2 mm were considered to be ontogenic bers of scales were lost (a pattern of injury which is while larger scales were considered to be regenerated. likely to be common in the wild). Scale regeneration in

Table 3. Laboratory observations of development of scale disorientations (SD) spontaneously and in response to experimental manipulations.

Experiment Fish Number Observation Number of SD occurrences (percentage) in response to speciﬁc number type of ﬁsh time, months manipulations Spontaneous, no Scale Scale removal + Contusion Tagging manipulation removal skin abrasion injury injury 1 Normal juveniles 15 5.5 3 (20%) n/a n/a n/a n/a 2 Normal juveniles 12 3 0 0∗ 0∗ n/a n/a 3 Normal adults 8 3 1 (13%) 0 0 n/a n/a 4 Normal adults 8 6 0 n/a n/a 0 0 5 Adults with SD 6 (8)• 3 1 (17%) 6 (75%)# n/a n/a n/a

∗Two areas of scale removal and 3 areas of scale removal with skin abrasion exhibited very slight abnormalities in scale orientation at 10 months post manipulation (at which time there were 4 ﬁsh remaining in this group). #Six areas of SD from which scales were removed developed adjacent, contiguous small patches of SD. •Sample size in this experiment was 8 SD patches on 6 ﬁsh (denominator for manipulations was 8, for spontaneous cases was 6). n/a – not applicable (manipulation not used in that experiment). 214

changes were noted (13 and 26 months post injury) and did not show any further development of these patches in size or degree of disorientation. Abnormal scales removed from SD areas were replaced by scales in the same abnormal orientation as the original scales (experiment 5, Table 3). Morphology of scales removed in these experiments was compared to that of replacement scales which grew in their place. Although these data indicated high levels of variation between scales, several significant trends were evident. Replacement, normal scales exhibited signiﬁcantly larger focus diameter, more radii and somewhat larger size as compared to original, normal scales (Table 4). These trends were also evident in scales regenerating into SD areas where the replacement scales had signiﬁcantly larger focii and more radii. Although the disoriented replacement scales still tended to have fewer radii than the normal replacement scales.

Discussion

Numerous types of skin lesions, particularly ulcers, affecting scales as well as the epidermis and underly- ing layers have been documented in fishes in response Figure 8. Proportional growth of areas of scale disorientation to pollutants, infectious diseases and unknown factors (, •) observed in the laboratory. Two fish collected in the field (Sindermann 1996). However, aside from the SD phe- with scale disorientation and two fish which developed this syndrome spontaneously in the laboratory (N, ) were observed for nomenon reported here, the occurrence of areas of dis- 4–8 months in the laboratory. a – Relative surface area of patches oriented scales appears to be an extreme rarity in most of disoriented scales did not change over these time periods. species of fish. Scale disorientation was reported in b – Surface area is also shown in comparison to changes in length three individual redfish, Sciaenops ocellata, as a bilat- of these fish. The fish indicated by the ‘•’ symbol is the same erally symmetrical condition on the posterior half of the individual shown in Figure 2. body (Gunter 1941, 1945, 1948). Disoriented scales were observed in groups, and no single disoriented scale was found isolated among the normal scales. injured areas was always complete (regenerated scales Gunter (1948) suggested that the causative agent of had reached approximately the same size as adjacent, this condition must have been present during the for- undisturbed scales) within 4 weeks of the injury. There mation of the dermis in the embryo. These studies con- was no evidence of abnormal growth patterns in any cluded that a hereditary cause was more probable than of these areas within this time frame. However, several an environmental one due to the symmetry of the con- fish observed for 10 months post-injury exhibited very dition observed. However, no evidence was provided slight scale alterations at sites of injury (experiment 2, to support this hypothesis. The only other report of Table 3). These changes were much less pronounced scale disorientation in marine fish involves two indi- (smaller deviations from normal) than typical in SD viduals of a sole, Zebrias japonicus (Taki 1938). These regions and would NOT have been scored as SD in wild lesions were similar in arrangement to those described caught fish. These altered areas were not observed at in the present studies with abnormalities observed both 3 months post injury and thus, must have developed in growth directions of scales and in scale morphol- after the affected scales had already regenerated in a ogy. However, affected areas also exhibited abnormal normal pattern. Two of these fish with slight disorien- color patterns, a feature not seen in pinfish. The author tations were monitored beyond the point at which these suggested that these areas might have resulted from 215

Table 4. Morphology of original and replacement scales from experimentally manipulated normal adult ﬁsh and ﬁsh with scale disorientation. Mean values in mm standard deviation.

Normal area scales Disoriented area scales Original scales Replacement scales Original scales Replacement scales Nofﬁsh 4 4 6 6 N of scales 80 80 60 60 Focus diameter (mm) 0.54 0.42 1.53 0.40∗ 0.54 0.53 1.20 0.50∗ Scale width (mm) 2.56 0.26 2.75 0.28∗ 2.86 0.52 2.95 0.59 Scale length (mm) 2.46 0.19∗ 2.39 0.24 2.82 0.45 2.73 0.46 Length/width ratio 0.97 0.10∗ 0.88 0.09 1.00 0.19 0.94 0.16 Number of radii 11.24 1.84 14.87 3.03∗ 10.47 3.32 12.30 4.54∗

∗Signiﬁcantly larger value within an original-replacement scale pair (t-test, p < 0.05).

physical–chemical influences during the formation of in large SD areas in older (larger) sized fish. However, scales in the fish embryo. such a negative correlation was not observed, suggest- Scale disorientation in pinfish from Biscayne Bay ing that SD patch size is most likely determined by the has been noted by several authors (Skinner & SD process itself. In addition, a study of prevalence of Kandrashoff 1988, Gassman et al. 1994) but a thorough SD in pinfish in Biscayne by Gassman et al. (unpub- description of this phenomenon has been lacking. The lished data) demonstrated that larger fish were more present study determined that scale disorientation in frequently affected by SD than were smaller fish, sug- pinfish is highly variable in the extent of coverage and gesting a lack of significant negative selection pressure orientation of affected scales. However, many aspects on affected fish. The similarity in the severity in SD of the SD syndrome are highly stereotyped. Disori- observed in northern and southern regions of the Bay ented scales were always oriented flat relative to the also suggests that a similar process of SD was operat- body surface, rather than protruding from the surface ing in these areas, in spite of the significant difference of the fish. SD scales were always observed in dis- in prevalence of SD observed in these areas. crete patches rather than scattered across the body sur- The process of SD development may involve face. All SD patches were found to involve the dorsal changes occurring at the time at which scales initially region of the body and to be limited primarily to this develop during growth of larvae. Such a process would region. These lesions appeared to be self-limiting with be expected to produce fish exhibiting SD throughout no evidence that SD progresses to a more extensive or their lives, from the time of earliest recruitment. How- invasive syndrome such as ulceration of the dermis. In ever, laboratory observations have indicated that at least addition, SD were found to remain constant in area rel- some pinfish develop SD later in life, well past the early ative to the size of the fish over time, suggesting that juvenile period. These observations suggest that SD can the number of scales involved remained constant as the occur in response to events encountered by the fish at fish grew (the only exception to this observation was essentially any time in life. The relative rapidity of SD the addition of small, contiguous SD areas to several development in the laboratory, from a normal morphol- existing SD patches following manipulation of these ogy to a completely formed SD, also suggests that this patches by scale removal). is not a gradual process but more likely the result of a The frequency distribution of SD areas showed that sudden change in growth characteristics of cells in the smaller SD (<10% body coverage) were much more affected area. The hypothesis that SD development is common than those affecting larger areas and that there a rapid, all-or-none, event is further supported by the was an apparent upper limit in severity (≈34% of body apparent lack of relative growth of SD areas observed area). Such a distribution could be produced either by over long periods of time in the laboratory. a negative selection pressure on larger SD areas or by Discrimination between ontogenic and disoriented some inherent property of the SD process. A selec- scales based on focus diameter revealed that while tion effect would be expected to result in a decrease SD areas contain a slightly higher proportion of 216 regenerated scales, that the majority of scales in these to determine if, as expected, SD scale morphology patches are ontogenic. This indicates that formation of began to diverge from normal only after scales began to SD does not require loss of scales but suggests that SD change orientation, or if these scales were morpholog- areas may be more subject to scale loss and replace- ically abnormal prior to the development of a visible ment than normal areas of skin. disorientation. The significant differences in morphology of scales These observations raise the question of what mech- in SD areas relative to those from normal skin clearly anism could produce a rotation of intact scales and demonstrate that the SD process alters the growth of their associated scale pockets as well as the abnor- individual scales in addition to turning them in place. mal scale morphology. Presumably, rotation could be A small portion of the differences between average accomplished by an increase or decrease in prolifera- SD and normal scale morphology can be attributed to tion (or mortality) of cells at one side of a scale pocket the slightly higher percentage of replacement scales in resulting in an uneven, asymmetrical growth pattern. these regions. However, even when this difference was Similarly, the abnormalities in morphology of affected factored in, SD scales were significantly smaller, more scales suggests that alterations have occurred in the ori- elongate and had fewer radii. In addition, an increase entation or relative secretion rates of the scleroblasts in number of radii in replacement scales relative to or epidermal cells in the scale pockets responsible for ontogenic scales was conspicuous in the present study production and mineralization of layers of scale mate- and has been reported by others (Yamada 1961). Thus, rial. Such changes might be capable of producing many the reduced number of radii seen in SD scales is oppo- types of abnormalities in scale shape and appearance. site to what would be expected if the process of scale However, the nature of these mechanisms can not be growth in SD areas were similar to that involved in scale determined from the data presented here. A preliminary replacement. Radii are thought to provide flexibility to histological study of areas of the skin affected by SD the scale (Bereiter-Hahn & Zylberberg 1993, Yamada has indicated that such regions are often characterized 1961) suggesting that a scale with fewer radii would by hyperplasia and dysplasia within the epidermis and be less flexible and therefore might be more likely to a thickening of the scales themselves (Schmale unpub- be lost from the fish during physical trauma (although lished data). Hyperplasia of the dermis and inflamma- the observed higher rate of scale loss in SD areas could tory changes are also observed in some cases and bac- also be explained by the increased likelihood of dis- teria are occasionally observed in these regions. The orientated scales being dislodged by water flow and/or stratum compactum of the dermis is intact and the lat- rubbing against other scales in the normal swimming eral body musculature below the dermis does not show motion of the fish). However, an understanding of why abnormalities in SD areas, suggesting that this phe- formation of radii is so much reduced in affected scales nomenon is restricted to the skin (the epidermis, der- is hindered by a lack of data on mechanisms control- mis and scales). Thus, the process of physical rotation ling formation of these structures in either ontogenic of scales might actually be driven by a proliferation or replacement scales in normal skin (Geraudie et al. of epithelial cells surrounding the scale pockets. How- 1998). ever, demonstration of such a mechanism would require Data on scale regeneration in SD areas show that a time series analysis of the developing abnormality in where abnormal scales are removed, replacement order to determine if the hyperplasia precedes, follows scales are similarly abnormal, albeit with the additional or develops concurrently with changes in scale ori- features typical of replacement scales (such as larger entation. If epidermal and/or dermal hyperplasia was focal diameter and increase in number of radii). These the dominant mechanism for scale rotation, then an data also demonstrate that regardless of any damage understanding of the factors producing the hyperplas- to scale pockets associated with SD, the scale forming tic response in the epithelial cells should reveal the tissues in these areas are still capable of rapid regen- necessary and sufficient conditions for development of eration of scales. This observation also reinforces the these lesions. hypothesis that the alterations which led to formation Unfortunately, investigation of the mechanisms of the SD are relatively fixed after the disorientation responsible for rotation and abnormal morphology of has appeared, such that replacement scales in an SD disoriented scales may be greatly complicated by the patch achieve the same, abnormal, orientation as the apparent stability of SD areas after initial development. original scales. Additional studies would be required This stability could result from a resolution of some or 217 all of the pathogenic processes leading to the SD. In possibility of the maintenance of genetically isolated such a scenario, the time available for productive obser- populations within Biscayne Bay. This rules out the vations would be restricted to the brief interval (perhaps hypothesis that different distributions of genotypes are several weeks) during which the rotation was actually responsible for prevalence differences in SD in pinfish taking place. This would require that observations be between the northern and southern regions of the Bay. restricted to the small percentage of fish developing Genetic factors might contribute indirectly to such a SD spontaneously in the laboratory unless the agent(s) distribution pattern if differences in selection pressures responsible for development of SD could be identified for or against the SD syndrome existed in the north and and utilized to induce SD experimentally. south Bay. For example, given a uniform genetically Perhaps the most important question raised by this based incidence of SD throughout the Bay, differences study is that of the nature of the etiologic agent or in selection pressure for this trait in different regions agents which trigger the development of an SD. As in of the Bay might yield variations in prevalence such the investigation of any pathological process, four gen- as those observed in the present study. In an area with eral categories of agents must be considered: genetic, strong negative selection pressure the presence of SD infectious, physical and chemical. Studies of skin ulcer- might be reduced by differential mortality favoring sur- ations in a variety of fish species have demonstrated vival of unaffected fish (a similar but less likely sce- that these can be caused by injuries from fishing gear nario could be envisioned based on positive selection (Mellergaard & Bagge 1998), bacterial and fungal pressure favoring the survival of SD fish). However, as infections (Hilger et al. 1991, Noga et al. 1988), a com- discussed above, data from the present study suggest bination of infections and injury (Ludemann 1993) and a lack of selection pressure for or against SD in these chemical agents (Minchew & Yarbrough 1977, Fournie fish. et al. 1996). Agents in any of these categories might Bacterial, protozoan, viral and fungal infections of play direct or indirect roles in SD formation. Several fishes have been shown to produce necrosis, granu- potential etiologic factors can be discounted based on lomas, hyperplasia and hypertrophy in the epidermis data from the present study, Gassman et al. (1994) and and dermis (Noga 1996). Any of these phenomena information on the life history of this species. could conceivably produce abnormal growth of scales. Inherited mutations have been shown to result in sev- While bacterial colonization of scale pockets has been eral types of diseases and gross abnormalities in fishes observed in some pinfish, our preliminary histologi- including a variety of vertebral deformities (Schultz cal analyses were too limited in sample size to allow 1963, Tave et al. 1982, 1983), albinism (Rothbard & any correlation between presence or extent of infection Wohlfarth 1993) and melanomas (Schartl et al. 1982). and the occurrence of SD (Schmale unpublished data). However, we have previously proposed that the distri- No other potential pathogens were observed in these bution pattern of the SD syndrome in Biscayne Bay, regions. However, such observations may not be infor- with significantly higher prevalences in the northern mative if they are made after the SD formation process bay relative to the southern bay over several years, is completed. is not consistent with a genetic syndrome (Gassman Our experiments on the effects of physical injuries on et al. 1984). This conclusion is based on the life his- the growth of scales in pinfish, while preliminary, have tory of similar species (family Sparidae) which, typical demonstrated that several types of acute and chronic of most marine fishes, spawn pelagic eggs that hatch injuries are not sufficient to result in development of into pelagic embryos with a prolonged developmental SD. Although several fish treated by scale removal period (25–60 days, Hussain et al. 1981, Battaglene & with or without skin abrasion developed very slight Talbot 1992). These pelagic stages result in dispersal disorientations of scales approximately 10 months fol- of offspring over great distances with mixing among lowing injury, these changes were relatively minor larvae spawned over a wide geographic area. Numer- compared with what is encountered in typical SD. ous studies of other fish species in the tropical west- Additionally, if physical injury were sufficient to pro- ern Atlantic Ocean with such life histories has clearly duce SD, this would imply that differences in injury demonstrated a complete lack of significant popula- rates were encountered by pinfish in northern and tion isolation or any impediment to gene flow across southern region of the Bay. Although no data are avail- the entire Caribbean Sea and Gulf of Mexico region able on this variable, anecdotal information indicates (Victor 1984, Lacson 1992). Thus, there is no practical that the number and frequency of commercial roller 218 trawl shrimp fishing operations is much higher in the chemically contaminated sediments. In addition, they bay south of Rickenbacker Causeway. Thus, the likeli- found that fish exhibiting a demersal life style were sig- hood of physical injury by this means would probably nificantly more likely to be affected than pelagic fish. be lower in the north bay, where SD is more preva- Although pinfish are not demersal, they typically feed lent, contradicting this as a simple explanation. Thus, on benthic plants and invertebrates and thus are likely while the likelihood of physical injuries resulting in SD to ingest sediments (Caldwell 1957). appears small, this potential agent can not be ruled out Several studies have examined the levels of chemi- by available data. Physical injury must also be consid- cal contaminants in sediments in Biscayne Bay1,2. Most ered as a potential co-factor that might play a contribut- recently, Long et al.3 examined concentration levels of ing role in development of SD. organic compounds and metals in sediments as well The localization of SD primarily to the body region as toxicity of sediments in Biscayne Bay and its trib- dorsal of the lateral line suggests that either the scales utaries. This study demonstrated that average levels of in this region of the body were inherently more suscep- chemical contaminants and toxicity of sediments were tible to development of SD or that this region was more clearly higher in the region of the basin north of the likely to be exposed to the etiologic agent of SD. This Rickenbacker Causeway than in the region to the south. raises the possibility that UV light, being one of the few The limited number of study sites in the present agents encountered with predominantly a dorsal expo- study combined with a lack of precise correspon- sure profile, might play a role in this syndrome. High dence of study site locations with those sampled by levels of UV exposure have been shown to cause skin Long et al. (1998) have prevented us from determining damage on the dorsal surface in several species of fish correlations between severity of SD and locations of creating a syndrome resembling ‘sun burn’ in mam- potentially toxic sediments. The generally higher con- mals (Rodger 1991). However, no data are available taminant levels observed in the northern bay do corre- to support this hypothesis or to determine if the dorsal spond to the trend which would be predicted if chemical skin is inherently more susceptible to SD. In addition, agents were an important component in the develop- there is no reason to believe that exposure to UV radi- ment of the SD syndrome. However, direct proof of ation would differ between the northern and southern such an etiology, including identification of the spe- regions of the Bay. cific agents responsible, would require experimental While differences exist in salinity and temperature exposure of pinfish of various ages to a variety of con- regimes of these regions of the bay, these differences taminant and/or sediment types found in Biscayne Bay. are typically small in comparison with the seasonal fluctuations which occur within any one area. However, either of these variables, as well as the range and rate Acknowledgments of variation of each, can not be disregarded as potential co-factors in the development of SD. These studies were supported by contracts from the The final group of potential etiologic agents are the South Florida Water Management District (C-8930 & organic and inorganic chemicals present in the water column and sediments of Biscayne Bay. Any chemi- 1 Corcoran, E.F., M.S. Brown, F.R. Baddour, S.A. Chasens & cal agent has the potential to be taken up via contact A.D. Freay. 1983. Biscayne Bay Hydrocarbon Study Final Report. with the skin or gills and/or by ingestion. Exposure to Bureau of Marine Science and Technology, Department of Natu- tributyl tin was shown to cause a hyperplasia of the ral Resources, State of Florida. 327 pp. 2 Corcoran, E.F., M.S. Brown & A.D. Freay. 1984. The study dermal layers of the skin in Atlantic salmon, resulting of trace metals, chlorinated pesticides, polychlorinated biphenyls in protruding scales (Bruno & Ellis 1988). Similarly, and phthalic acid esters in sediment of Biscayne Bay. MetroDate numerous vertebral abnormalities have been associated County, Florida, Department of Environmental Resource Man- with heavy metal and organic pollution of water and agement. 58 pp. 3 sediments (Bengtsson 1974, Benoit & Holcombe 1978, Long, E.R., G.M. Sloane, G.I. Scott, B. Thompson, R.S. Carr, Bengtsson et al. 1988, Middaugh et al. 1990). Fournie J. Biedenbach, T.L. Wade, R.J. Presley, K.J. Scott, C. Mueller, G. Brecken-Fols, B. Albrecht, J.W. Anderson & G.T. Chandler. et al. (1996) reported from a large-scale survey of estu- 1988. Magnitude and extent of chemical contamination and tox- arine fishes from the Gulf of Mexico and Mid-Atlantic icity in sediments of Biscayne Bay and vicinity (review draft), states that skin lesions (primarily fin erosion, ulcera- U.S. Department of Commerce, National Oceanic and Atmo- tions and papillomas) were more prevalent in areas with spheric Administration, Silver Spring. 219

C-6797) and by the National Institute of Health (PHS Gunter, G. 1941. A rare abnormality, a fish with reversed scales. P30 ES 05705). We also thank Walter and Michael Copeia 1941: 176. Kandrashoff and Joan Browder for introducing us to Hilger, I., S. Ullrich & K. Anders. 1991. A new ulcerative the concept of scale disorientation in these fish. All flexibacteriosis-like disease (‘yellow pest’) affecting young Atlantic cod Gadus morhua from the German Wadden Sea. collections were conducted under permit from the Dis. Aquat. Org. 11: 19–29. Department of Environmental Protection of the State Hussain, N., S. Akatsu & C. El-Zahr. 1981. Spawning, egg and of Florida and the Biscayne National Park. All animal early larval development, and growth of Acanthopagrus cuvieri experiments were conducted with the approval of and (Sparidae). Aquaculture 22: 125–136. in accordance with the regulations of the University of Lacson, J.M. 1992. 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