Osteological abnormalities in laboratory reared sea-bass (Dicentrarchus labrax) fingerlings

Aquaculture, 97 ( 199 1) I69- I80 Elsevier Science Publishers B.V., Amsterdam

169

Osteologicalabnormalitiesin laboratory reare sea-bass (Dicentrarchus labrax) fingerlings Ch. Daoulas, A.N. Economou and I. Bantavas National Centre for Marine Research,Aghios Kosmas, 16604 Helinikon. Athens, Greece (Accepted 14 January 1991)

ABSTRACT Daoulas. Ch., Economou, A.N. and Bantavas, I., 1991. Osteological abnormalities in laboratory reared sea-bass (Dicenttwchus labrax) fingerlings. Aquaculture, 97: 169- 180. Two groups of juvenile sea-bass, a random group and a group selected on the criteria of the appearance of macroscopically visible body deformations or anomalies in behaviour, were collected from the rearing tanks, treated for cartilage-bone staining with alizarin and alcian-blue, and examined for osteological abnormalities. The me&tic characters of the stained specimens were also examined. The percentage occurrence and the interrelationship of the various abnormality types are described. The most frequently encountered abnormalities were those involving spinal column and vertebral deformiiies and these were often associated with swimbladder deficiencies.

INTRODUCTION

A high frequency of occurrence of physical defects, often associated with growth depensation and increased mortality rates, seems to be a property of laboratory- or hatchery-produced fishes (Riley and Thacker, 1963; Riley, 1966; Houde, 1971; Aronovich, 1977; Barbaro et al., 1984; Shimizu, 1987). Available evidence suggests that abnormalities are induced during the embryonic and postembryonic periods of life, through a mechanism that is not yet well understood (Houde, 1973 ). The most significant causative agent seems to be unfavourable temperature, but factors such as oxygen depletion, light intensity, dietary deficits, certain metabolites, etc., are also known to produce aberrations from normal development (Brooke, 1975; Fends, 1975; MC-. Cormick, 1978; Coombs and Hiby, 1979; Oozzki and Hirano, 1985; Akiyama et al., 1986; Seikai et al., 1987; Korwin-Kossakowski, 1988; Wiegand et al., 1989; Caris and Rice, 1990). Reared populations of sea-bass have been reported to present an extremely high incidence of morphological abnormalities and swimbladder deficiencies, including spinal column and jaw deformations ( Barahona-Fernandes, 1982; 00448486/9

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CH.DAOULA!SETAL.

Johnson and Katavic, 1954). As sea-bass is a species of great importance to Mediterranean aquaculture, the high rate of abnormalities appearing in hatchery-produced fish has severely impaired the development of the industry. This is because abnormalities are often associated with reduced viability, and because of depression of prices and lower market demand for the deformed fish. So far, the presence and extent of skeletal abnormalities in sea-bass has been determined by microscopic examination in larvae and radiographs in adults. In this study, we present a record of osteological anomalies occurring in juvenile bass reared in the laboratory using bone staining techniques. MATERIALS AND METHODS

Sea-bass eggs were spawned and naturally fertilized on 6 February 1989, in a tank where an acclimatized broodstock was kept in ambient water temperature ( 12- 13OC ) . About 300 000 fertilized eggs were transferred to cylindroconical tanks of 250 ! capacity with a continuous supply of air and renewal of water, and incubated at a density of 1200 eggs 1-l and temperature maintained at 14°C. The larvae hatched out on 10 February (percentage survival during incubation 65%) and 2 days later 130 000 larvae were placed in a cylindrical tank of 1800 1 for further rearing. Temperature was allowed to increase gradually up to 17.5-l 8 “C on day 20 from hatchi.gngand thereafter it remained constant until day 50. During weaning (started on day 50 ) the temperature gradually increased again until it reachGd 20-2 1 a C. Live food (nauplii of Artemia safina) was offered from day 6 to day 50 at a density of 1 nauplius ml- ’ . Controlled illumination with fluorescent lamps gave a 12 : 12 h light-dark photoperiod with a light intensity of 1800 lux at the tank surface. Weaning took place from day 50 to day 65 by adding pelleted food to the diet and progressively reducing the proportion cf A. salina offered. A population of about 11 000 juveniles survived to day 100 (percentage survival from hatching 8.5%) with body standard lengths ranging from 13.9 to 22 mm (mean: 16.4 t- 1.19 mm). No grading concerning body size, nor removal of fish with uninflated swimbladders took place over the entire period of rearing. On 24 May ( 103 days from hatching) two lots of juvenile fish were collected for osteological examination: a group of 111 fish taken randomly from the tank, and a group of 73 fish selected individually on the criteria of the appearance of macroscopically visible body deformations or anomalies in swimming performance, abnormal buoyancy and lack of adequate response to stimulation. Both groups, referred to as the random and the selected group, were preserved in a 10% neutral formalin solution (sodium phosphate buffer ) . The purpose of including the selected group was twofold: first, to ensure that an adequate number of defective fish was available for analysis; and second,

OSTEOLOGICAL ABNORMALITIES IN SEA-BASS FINGERLINGS

!71

to ascertain whether macroscop!cally visible deficiencies are reflections of particular bone abnormalities. After a period of fixation of 1 month, all fishes were examined under a binocular microscope for the presence or absence of swimbladder. Subsequently they were treated for cartilage-bone staining according to Taylor and Van Dyke ( 1985 ) and were examined under a binocular microscope for meristic characters and occurrence of osteological abnormalities. The abnormalities were classified into 15 types as following: ( 1) fusions in trunk vertebrae; (2 ) fusions in caudal vertebrae; (3) fusion of the last caudal vertebra with the urostyle; (4) fusion of the head with the first trunk vertebra; ( 5 ) abnormal neural spines; (6 ) abnormal hemal spines; ( 7 ) abnormal pleural ribs; (8) deformed vertebral bodies; (9) abnormal soft rays in the 2nd dorsal fin; ( 10) abnormal soft rays in the anal fin; ( 11) abnormal rays in the caudal fin; ( 12) deformed upper jaw; ( 13) deformed lower jaw; ( 14) abnormal spinal column curvature; ( 15) absence of inflated swimbladder. Fish normal in all respects were grouped in type 0. Type 14 included all categories of spinal column deformity (scoliosis, lordosis, kurtosis etc. ) . The set of data on the presence or absence of each abnormality type in each fish was treated separately for the two groups in order to define possible interrelationships between abnormality types. The Bray-Curtis similarity measure (Bray and Curtis, 1957), based on extremely standardized values (binary data), was calculated: s= lOO( l-

C(Xi-Yi) C(Xi+j>’

where Xi and yi take values 0 or 1, representing the presence or absence of abnormality types x and y in the ith fish. The index takes values between 0 (no fish with abnormality types x and y simultaneously present ) and 100 (all fish with abnormality x also had abnormality y ). A similarity matrix was produced and classification was performed using the average linkage clustering technique (Sokal and Sneath, 1963 ) . RESULTS

Meristic characters

The mean numbers of vertebrae and fin rays ( 2 95% confidence limits) in the 184 fish examined are given in Table 1. With the exception of the pelvic fin rays, the number of the other fin rays showed an unexpectedly high variation. There was a difficulty in identifying the last vertebra to be included in the trunk vertebral counts because in a number of fish the hemal canals were still open. Due to this uncertainty, and also the frequent fusion of the last

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TABLE 1 Meristic counts of laboratory-reared sea-bass. Values for wild populations, included for comparison, are from: (a) Giinther ( 1859), (b) Whitehead et al. ( 1986) and (c) Moreau ( 1881) Character

w-t-sxt()os

Range

(a)

Trunk vertebrae Caudal vertebrae Total vertebrae I st dorsal fin rays (spines) 2nd dorsal fin rays (spines) 2nd dorsal tin rays (soft ) Pectoral fin rays Pelvic tin rays (spines) Pelvic tin rays (soft ) Anal tin rays (spines) Anal fin rays (soft ) Caudal fin rays Dorsal procurrent caudal rays Ventral procurrent caudal rays

9 54+0.11 14.33 + 0.11 23.98 + U.03 8.17kO.12 1.08+0.14 13.03+0.11 17.24kO.11

9-12 12-15 23-25 7-1rJ l-2 1 l-16 15-21

12 13 25 9 1 12-13

1

5 2.11 +o.os 11.67+0.10 17.01+0.03 14.i)3+0.21 12.57f0.21

l-3 10-14 16-18 12-18 9-16

(b)

8-9 1 12-13

ic)

13 12-13 25-26 8-9 1 12-13 15-16 1 5 r: 1;;~1-j 17

caudal vertebra with the urostyle, the couats of the trunk and caudal vertebrae do not necessarily reflect the true meristic situation. Osteolcgical abnormalities

Some of the most frequently encountered osteological abnormalities are illustrated in the photographs presented in Fig. 1. Fig. 2 shows for both groups the percentage occurrence of each abnormality type (out of the total number of fish of each group), with type 0 indicating normal fish. It is remarkable that of the 73 !ish of the selected group, 11 were found with no abnormalities, while of the 111 fish of the random group, only four were found to be normal in all respects. The relatively high frequency of abnormal fish in the random group is the result of the unexpectedly high contribution of swimbladder anomalies in this group. The fact that 11 fish with no abnormalities were found in the selected group suggests that the macroscopical criteria used by us for selecting the fish which comprised this group did not involve only skeletal abnormalities. In both groups, the most prevalent abnormality types were absence of a functional swimbladder, neural and hemal spine defects, and spinal column deformity. Common, but less frequent were lower jaw defects and fusions or deformations of vertebrae. Spinal and vertebral abnormalities made a higher contribution in the selected grouo, indicating a gross relationship between morphological and skeletal anomalies. In Table 2, the fish of the two groups are ranked into four categories according to the extent of spinal column deformity. Because spinal deformities

OSTEOLOGICAL ABNORMALITIES IN SEA-BASS FINGERLINGS

Fig. 1. Common Deformed spinal and pleural ribs, vertebra with the

types of osteological abnormalities encountered in sea-bass fingerlings. a, d. column and vertebral bodies, vertebral fusions. b, c. Abnormal neural spines vertebral fusions. e. Fusion in caudal vertebrae. f. Fusion of the last caudal urostyle. g. Deformed lower jaw. h. Abnormal rays in the caudal fin.

CH. DAOULAS ET AL.

174

0

1

9

2345676

10

11

12

13

14

15

Type of abnormality Fig. 2. Percentage of fish with each of the encountered abnormality types. TABLE 2 Grouping of fish according to spinal column attitude and presence or absence of swimbladder Number

Spinal column attitude Normal spine

Small d&rmity

Medium deformity

Serious deformity

20 3 23

8 17 25

6 7 13

10 2 12

44 29 73

27 38 65

13 20 33

1

4 2 6

47 64 111

.

Selected group With uninflated swimbladder With inflated swimbladder Total Randofl*rgroup With unil:!iatcd 1 swimbladder With inflated swimbladder Total

: 7

have been reported to be associated with swimbladder malfunction, Table 2 also gives the number of fish under each category having or lacking a functional swimbladder. The results are confusing in some respects. As already mentioned, a higher proportion of fish in the random group (57.7%) than in the selected group (39.7%) were found without swimbladder. Also, most fish with non-inflated swimbladder in the selected group had an abnormally shaped column. And last, fish with inflated swimbladder in both groups presented a high percentage of spinal deformity. Chi-square analysis (2 x 5 contingency table) was performed to test if absence or presence of swimbladder is independent of spinal attitude. For the

OSTEOLOGICAL ABNORMALlTIES IN SEA-BASS FINGERLINGS

175

selected group x 2= 7.47 (d.f. = 4, P=O. 11) and for the random group x2= 33.47 (P= very small), suggesting that the relative numbers differ in the categories with and without swimbladder. Further tests (2 x 2 tables) were performed by combining columns to test the nature of departure from independence. In comparing fish with a normal spinal coltimn and fish with a minor or major spinal deformity, spinal deformi?y was found to be significantly associated with an uninflated swimbladdt:r in the selected group (x 2= 9.98, P= very small), but not in the random one (~‘-0.04, P=O.84). In comparing fish with an intense deformity and fish with either a normal spine or low and medium degrees of spinal deformity, intense deformity was associated with an inflated swimbladder in the selected group (x 2=3.19, P=O.O7), but not in the random one (x2=1.54, P= 0.2 1). It should be mentioned, however, that our observations during the examination of the stained individuals indicated that intense spinal deformity was often associated with what appeared as a very distended swimbladder. Unfortunately, the degree of distention of swimbladder could not be quantified, and therefore its possible association with spinal attitude crnnot be confirmed from our material.

Interrelation between abnormality types Numbers of abnormalities per fish are shown in Fig. 3. The affinities between abnormality types are shown in the dendrograms of Figs. 4 and 5. In both groups, abnormalities in caudal vertebrae, fin rays and upper jaw were either unrelated (types 2,9,10 and 12 ) or presented a very weak affinity to each other (types 3 and 11). Abnormalities involving trunk vertebrae and 3. _Percel;tage Selected group

0

1

2

3

4

5

m

6

Random group

7

Number of abnormalities per fish Fig. 3. Frequencydistributionof numberof abnormalitiesperfish.

0

CH. DAOULAS ET AL.

176

0 ..-i

40-

I

Lo

a .t

60-

(3 I 0"

30-

!

il

’

,

2

Abnormality

99

3

?O

12

9

types

Fig. 4. Similarity indices between abnormality types in the selected group. O-

20-

J) ._ 5 0 I g

60-

30-

kl

15

13 14

5

8

6

Abnormality

4

1

7

3

11

10

2

9

12

types

Fig. 5. Similarity indices between abnormality types in the random

group.

OSTEOLOGICAL

ABNORMALITIES

IN SEA-BASS FINGERLINGS

177

spinal column deformities ( 1,4,8 and 14)) abnormal neural and hemal spines and pleural ribs ( 5, 6 and 7 ), uninflated swimbladder ( 15 ) and defects in lower jaw ( 13) appeared to co-occur relatively frequently. In the selected group, abnormality types 1, 5 and 14 and types 4 and 15 formed two distinct but interrelated groups, which were linked to the other types at a lower similarity level. In the random group, the strongest associations were between abnormalities 5,8 and 14 and between 1 and 4. DISCUSSION

The particularly high frequency of swimbladder deficiencies and skeletal anomalies observed in the random group suggests an extreme vulnerability of our reared population of sea-bass to the acquisition of abnormalities, But while swimbladder abnormalities are common among hatchery- or laboratory-produced fish of this or other fish species (Nash et al., 1977; Johnson and Katavic, 1984; Barrows et al., 1988; Chapman et al., 1988; Battaglene and Talbot, 1990), records of skeletal abnormalities in larval and juvenile fish are rare (though, see Paperna, 1978; Shimizu, 1987), probably because the observation of such abnormalities requires a suitable bone staining technique. However, high frequencies of fish with deformed bodies have been reported (Houde, 197 1; Barahona-Fernandes, 1982; Barbaro et al., 1984), and possibly these body deformations involve skelet,al anomalies. Swimbladder and skeletal anomalies in the trunk region predominated in both groups (Fig. 2) and showed relatively high affinity indices (Figs. 3 and 4). Our data are not adequate to identify the causative agent(s) of the observed abnormalities, but seem to indicate cause-and-effect situations, e.g. that an abnormality was the mechanical stimulus for producing other abnormalities. However, the actual mechanism remains obscure. For example, an abnormally bent spinal column could have acted to generate vertebral or neural spine malfunctions, but spinal deformity could also be the result of an existing vertebral abnormality. The affinity (albeit weak) between lower jaw deformation and the class of the other interrelated abnormalities is more difficult to explain. Possibly, it suggests an association of jaw deformation with only one class member. In the selected group, the overwhelming proportion of individuals with noninflated swim.bladders, also presented spinal deformity. Almost all of them belonged to the categories with slight to medium seriousness of deformity (Table 2 ) . These two categories may represent spinal column attitudes which compensate the previous failure to activate the swimbladder. But why did the category with more serious spinal deformity have a higher percentage of individuals with inflated swimbladders in both groups? Two explanations, not mutually exclusive, are possible. First, that the hypertrophy of the swimbladder, that was likely to be encountered in many fish of this category (see Ke-

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suits j, z’epresents a compensatory mechanism tending to offset an existing serious spinal deformity. And second, that a combination between non-activated swimbladder and serious spinal deformity was lethal at an earlier stage, hence the apparent abundance of fish with this combination of abnormalities is underrepresented at a later stage. The second explanation seems more probable to us and can also account for the reason why fish with uninflated swimbladders were more common in the random group. Note that the majority of fish of the selected group presented spinal deformity. Assuming that combination of this defect with absence of a functional swimbladder reduces survivorship, differential mortality could have lowered the apparent abundance of fish with a functional swimbladder in the selected group. In contrast to the laboratory, records of abnormal fish from the field are extremely rare (e.g. Gartner, 1986). However, relatively high incidences of abnormalities have been reported from polluted habitats (Baumann and Hamilton, 1984; Bengtsson et al., 1988; Loganathan et al., 1989). Reared and wild fish also differ in gross morphological features, growth patterns and physiological parameters (Balbontin et al., 1973; Blaxter, 1975; Suda et al., 1987). Some meristic measurements from our laboratory-reared fish fell outside the range reported for wild populations of the species. The mechanism of meristic control in fishes involves some effect of temperature or other environmental factors during early development (Lindsey and Almason, 198 1) . Hence, the deviation in meristics from the normal observed in our reared population of sea-bass possibly reflects an effect of the aquarium environment during the period of meristic character determination, and may be considered as a form of environmentally induced abnormality. ACKNOWLEDGEMENTS

We thank Miss K. Anastassopoulou for technical assistance during the clearing and staining procedure, Dr. E. Bapathanassiou for statistical advice and three anonymous reviewers for constructive criticism on an earlier version of this article.

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