kou mound outhe effect of rearing conditions on development of saddleback syndrome and caudal fin...

Ž .Aquaculture 200 2001 285–304www.elsevier.comrlocateraqua-online

The effect of rearing conditions on development ofsaddleback syndrome and caudal fin deformities in

ž /Dentex dentex L.

G. Koumoundouros a,), P. Divanach a, M. Kentouri a,b

a Institute of Marine Biology of Crete, P.O. Box 2214, 71003 Iraklio, Crete, Greeceb Biology Department, UniÕersity of Crete, P.O. Box 1470, 71110 Iraklio, Crete, Greece

Received 23 October 2000; received in revised form 28 January 2001; accepted 1 February 2001

Abstract

The development of saddleback syndrome and of caudal fin deformities in Dentex dentexŽ . Ž . Ž .Linnaeus, 1758 was compared under two rearing methods, extensive E and semi-extensive S .The osteological appearance and the meristic characters of the reared fish were compared to thatof D. dentex juveniles, collected from the natural environment. All the wild juveniles werenormal in respect to their osteological appearance, while the reared specimens presented skeletal

Ž .deformities resulting from different rearing methods applied. Saddleback syndrome 4.0–4.4%Ž .and severe external deformities of the caudal fin 14.3–15.0% characterised exclusively the

semi-extensive populations, while the extensive populations presented severe abnormalities of theŽ . Ž .pre-ural centra 25.0–25.6% with significantly higher frequency than the S reared fish 5.8% .

Saddleback syndrome was expressed as a lack of one to all the hard spines of the dorsal fin,accompanied by shape, number and position abnormalities of the related pterygiophores. Caudalfin deformities were mainly characterised by the lack of the upper lepidotrichia orrand derma-totrichia, accompanied by severe deformities of the supporting elements of the upper lobe. Thesaddleback syndrome and severe caudal fin deformities were anatomically and ontogeneticallyrelated to each other, originating at the early larval stage as a result of abnormalities of theprimordial marginal finfold and of the posterior tip of the notochord. In respect to the dominantphenotypes, the meristic characters of the normal reared fish were not differentiated from those ofthe wild, but they presented higher variability.

) Corresponding author. Tel.: q30-81-242022; fax: q30-81-241882.Ž .E-mail address: [email protected] G. Koumoundouros .

0044-8486r01r$ - see front matter q2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0044-8486 01 00552-X

( )G. Koumoundouros et al.rAquaculture 200 2001 285–304286

The results are discussed in view of the aetiology, applications and research targets for theerasure of skeletal malformations. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Dentex dentex; Saddleback; Caudal fin; Deformities; Meristic characters; Rearing

1. Introduction

The presence of morpho-anatomical abnormalities in fish is a frequent and importantŽproblem for aquaculture Komada, 1980; Matsuoka, 1987; Boglione et al., 1993; Marino

et al., 1993; Chatain, 1994a; Divanach et al., 1997; Koumoundouros et al., 1997a,b;.Takeuchi et al., 1998 , due to their negative effect on the biological performance, the

marketing image, the commercial value and the production benefits of the reared fishŽBarahona-Fernandes, 1982; Chatain, 1987, 1994b; Bolla and Holmefjord, 1988;

.Boglione et al., 1994 . Abnormalities have been reported in the skeleton, body pigmenta-tion, scales or the swimbladder of a variety of fish species, without exhibiting any

Žspecies-specific expression in respect to their anatomical form reviewed by Divanach et.al., 1996 . In the natural environment, although their frequency is relatively low, their

strong correlation with pollution and other environmental disturbances further underlinesŽthe importance of their study Sloof, 1982; Bengtsson et al., 1988a,b; Pohl, 1990;

.Lindesjoo et al., 1994; von Westernhagen and Dethlefsen, 1997 .¨¨Rearing and general environmental conditions seriously affect the appearance of

Žmorpho-anatomical abnormalities in respect to both their incidence and type Francescon.et al., 1988; Boglione et al., 1994; Koumoundouros et al., 1997a . In most of the cases

however, the identification of the causative factors is difficult due to large numbers,commonly expressed symptoms and frequently cooperative effect, as well as due to the

Ž .wide developmental phases during which they act reviewed by Divanach et al., 1996 .Of the most severe morpho-anatomical deformities is the partial or complete absence of

Ž .the dorsal fin saddleback syndrome, after Tave et al., 1983 and those of the caudalskeleton, both of which have been reported in a big variety of species under naturalŽ . ŽBrowder et al., 1993; Lemly, 1993; Honma, 1994 or aquaculture conditions Komada,1980; Matsuoka, 1987; Valente, 1988; Boglione et al., 1993; Marino et al., 1993;

.Chatain, 1994b; Koumoundouros et al., 1997a , due to both heritable and environmentalreasons.

Dentex dentex is a Sparidae fish of high commercial value, which is newlyintroduced in aquaculture and has attracted wide interest of many scientists in respect to

Ž .its reproduction and physiology Alarcon et al., 1998; Pavlidis et al., 1999, 2000 , larval´Ž .rearing Glamuzina et al., 1989; Sweetman, 1992; Pastor et al., 1995 , nutrition

ŽEfthimiou et al., 1994; Tibaldi et al., 1996; Company et al., 1999; Mourente et al.,. Ž .1999a,b , immunology Efthimiou, 1996a , morphological and osteological ontogeny

ŽHolt, 1899; Lo Bianco, 1909; Ranzi, 1933; De Gaetani, 1938; Koumoundouros et al.,.1998, 1999a,b, 2000a . Although morpho-anatomical abnormalities have been reported

Ž .at the juvenile stage on the skull, fins and vertebrae of D. dentex Efthimiou, 1996b noone study exists about their ontogeny and the inducing factors.

( )G. Koumoundouros et al.rAquaculture 200 2001 285–304 287

The present study examines the ontogeny of saddleback syndrome and caudal findeformities in D. dentex and the effect of rearing conditions on the differentialappearance of this deformity.

2. Materials and methods

The study was based on two extensively and two semi-extensively reared larvalŽ .populations of common dentex. The extensive method of larval rearing E was chosen

Žbecause the biotic and abiotic conditions are very close to natural ones Divanach, 1985;.Kentouri, 1985 and because the produced fish populations develop morpho-anatomical

Žabnormalities of very low intensity and those only rarely Koumoundouros et al.,. Ž .1997a . The semi-extensive method S was chosen due to its high approach to the

methodologies followed for the mass production of fish juveniles.The entire experimental period was divided into three rearing phases, the autotrophic

Ž . Žegg incubation and yolk-sac larval stage , the larval 3.6–15.5 mm total length, TL,. Ž1–17 days post-feeding and that of weaning and pre-growing 15.5–46.0 mm TL,.18–44 days post-feeding . The methodology, which was followed for each phase, is

Ž .analytically presented by Koumoundouros et al. 1999a . As the water temperature in theŽ .tanks of the larval rearing phase 24.58C was higher than in the tanks of the autotrophic

Ž .phase 18.58C by 68C, an acclimation process of 2 h was followed prior to the transferof the larvae to the new conditions. Fish survival during the larval rearing phase was4.0–6.9% for the two extensive populations and 4.9–5.5% for the two semi-extensive.Fish survival during weaning and pre-growing was 75.7% and 81.8% for the extensiveand semi-extensive population, respectively.

The frequency of the osteological deformities was estimated on random samples ofŽ .60–90 specimens taken from each reared population at the end of the a larval rearing

Ž .phase, and b weaning and pre-growing phase. The individuals with a TL less than 25.0Ž .mm were stained for bone and cartilage Dingerkus and Uhler, 1977 , while the larger

Ž . Žspecimens )25.0 mm TL were exposed to X-rays Senograph 500 t exposer, Kodak.MinR, 24–26 KV, 400–600 mA, 300–600 s; Koumoundouros et al., 2000b .

To determine the developmental and morphological prodroms of the osteologicaldeformities, random samples of 30 specimens were taken every 2 days from each rearingtreatment throughout the larval rearing phase and every 4 days thereafter. All the

Žmorphologically abnormal specimens, as well as 160 extensively ranging between 3.9. Ž .and 28.5 mm TL and 172 semi-extensively ranging between 3.8 and 29.4 mm TL

Ž .reared specimens were stained for bone and cartilage Dingerkus and Uhler, 1977 .In all the samples, observations and photographs were made on anaesthetized

Ž y1 .ethylenglycol-monophenylether, Merck, 0.2–0.5 ml l specimens using a stereo-scopic microscope. Measurements of the total length were carried out on these pho-tographs to the nearest 0.1 mm. Specimens were then fixed individually in phosphate

Ž .buffered 5% formalin pHs6.8, Markle, 1984 and stored in the dark at roomtemperature. Larvae larger than 14.0 mm TL were fixed in phosphate buffered 10%formalin.


A meristic analysis of the different populations was carried out by counting thenumber of pterygiophores, spines, lepidotrichia, and dermatotrichia of all the fins, aswell as the number of vertebrae. Pectoral and pelvic fins elements were examined onboth the sides of the body. All the meristic characters were counted in the stained

Žspecimens, after they had attained their final expression Koumoundouros et al., 1999a,. Ž .2000c . The Fluctuant Asymmetry Index FAI of pectoral and pelvic fins was estimated

for each population separately, by using the variance of the arithmetic differenceŽ .between the two sides of the body Palmer and Strobeck, 1986 .

For the determination of the osteological and meristic wild standard of D. dentex, 41Ž .juvenile fish 93–185 mm standard length were collected from the natural environment

Ž .during bottom trawl service carried out in the Gulf of Iraklion Crete, Greece duringspring. A trawl with a cod-end bag liner of 22 mm stretched mesh size was used. More

Ž .details about the fishing operation are provided in Machias et al. 1998 . The fin meristiccharacters were counted by examining the individuals under a stereomicroscope. Radio-graphies of the individuals were used for the counting of the vertebrae and theexamination of any possible osteological deformity.

Ž .Drawings were made using computer software MacDraw Pro 1.0v. , from pho-tographs of the different skeleton anatomical regions of the specimens. The developmen-

Ž .tal stages were defined in accordance with Kendall et al. 1984 . Osteological elementsŽ . Ž .were described according to the terminology of Harder 1975 and Matsuoka 1987 .

The numbering of the dorsal and anal fin elements followed a caudal direction.Statistical comparisons of the mean meristic characters between the different popula-

tions were made by means of Mann–Whitney U-test. The distribution of the meristiccharacters in the different populations was compared by means of Kolmogorov–Smirnoftest. G statistic was applied to test any differences of deformities occurrence between thedifferent populations. For the comparison of FAI between the different populations,

Ž .normality was tested and then F-test was applied Sokal and Rohlf, 1981 .

3. Results

The specimens, which were collected from the natural environment, did not presentany morpho-anatomical abnormality, while the reared specimens exhibited osteologicaldeformities, which were differentiated both quantitatively and qualitatively as a result ofthe different rearing methodologies applied. Both the S populations presented the

Ž .saddleback syndrome SBS at a frequency of 4.0–4.4% at the end of the larval rearingphase to 3.2–3.4% at the end of the pre-growing phase. In addition, S populations were

Ž .characterized by the presence of severe external deformities of the caudal fin CfD-sbs ,ranging between 14.3% and 15.0% at the end of the larval rearing phase to 7.5–8.2% at

Ž .the end of the pre-growing phase Table 1 .

3.1. Saddleback syndrome

Saddleback syndrome was externally expressed as a lack of the anterior one to evenall the hard spines of the dorsal fin. As it was shown by the osteological analysis of the


Table 1Ž . Ž .Frequency % evolution of the saddleback syndrome SBS and of the correlated caudal fin deformities

Ž . Ž . Ž .CfD-sbs in the extensively E and semi-extensively S reared populations

Deformity Population First appearance End of larval End ofŽ .age in days post-feeding rearing phase pre-growing phase

SBS E1 0 0 0E2 0 0 0

Ž .S1 3.1 14 4.4 3.2Ž .S2 2.8 14 4.0 3.4

CfD-sbs E1 0 0 0E2 0 0 0

Ž .S1 13.0 10 15.0 8.2Ž .S2 12.0 11 14.3 7.5

deformed specimens, the main internal characteristic of the saddleback was the abnormalshape, number and position of the anterior proximal pterygiophores, combined with the

Žlack of all the distal pterygiophores which were associated with the missing rays Fig..1a . Additionally, in the 39.1% of the specimens with SBS, 1–2 hard spines of the

dorsal fin were atrophied.The osteological analysis of the semi-extensively reared D. dentex, with normal

external morphology in respect to the SBS, revealed that the 43.1% of these fish boreŽ .anatomical abnormalities at the dorsal fin of low intensity SBS-l . These consisted of

Ž . Ž .Fig. 1. a Saddleback syndrome in a specimen of 11.0 mm TL. b Fusions of the anterior-most pterygio-phores in a specimen of 17.5 mm TL. Open area, cartilage; stippled area, ossification. The ossification state ofthe rays is not drawn. Prd, predorsal; Prx, proximal pterygiophore; R, lepidotrichium; Rd, distal radial. S, hardspine; Scale bars are equal to 0.5 mm.


fusions and deformations of the anterior supporting elements of the dorsal fin, with thefusion of the proximal pterygiophore 1, distal pterygiophore 1 and proximal pterygio-

Ž .phore 2 to be the most frequent 40.0%, Fig. 1b . The atrophy of the dorsal hard spineswas presented by only the 6.4% of the fish with SBS-l.

The E populations did not present any type of skeletal deformities of the dorsal fin.

3.2. Caudal fin deformities associated to the saddleback syndrome

The main external characteristic of the CfD-sbs was the lack of the upper lepi-dotrichia orrand dermatotrichia, as well as the atrophy, curvature or fusion or the

Ž .remaining of them Table 2 . They were associated with severe abnormalities of theŽ . Ž .supporting elements of the upper lobe of the caudal fin Fig. 2 , expressed as a fusion

Ž .orrand deformation of the hypural elements 3–5, b fusion, atrophy or deformation ofŽ . Ž .the two pre-ural vertebrae and of the urostyle, c lack of the hypurals 3–5, and d the

Ž .development of one extra epural element Table 2 .The parallel analysis of the CfD-sbs with the SBS clearly showed that the two types

of abnormalities were closely associated to each other. The CfD-sbs deformitiespresented a frequency gradient from the semi-extensively reared fish with a normal

Ž .dorsal fin, to those with deformations of only the dorsal supporting elements SBS-l andŽ . Ž .then to those bearing the saddleback syndrome SBS Table 2 .

All the individuals with more than four caudal lepidotrichia missing bore severeŽ .kypho-lordosis at the caudal peduncle. The SBS was highly associated 68.1% with

Table 2Ž .Frequency % of the severe caudal fin deformities in the different groups of reared fish

Ž . Ž . Ž .Abnormalities S-n % SBS-l % SBS %

PU2 lack 1.4 2.1 5.3PU2–PU3 deformations 1.4 6.4 5.3Hy lack 4.1 6.4 0ANaB-Ep1 fusion 2.7 4.3 0Hy’s fusions 11.0 12.8 52.6Extra-numeral Ep 4.1 6.4 0Miscellaneous 17.8 25.5 21.1

1,2 1,3 2,3Subtotal 42.5 63.9 84.3PCR lack 21.0 38.3 63.2PCR atrophy 0 2.1 15.8PCR curvature 3.2 6.4 0PCR fusion 3.2 0 0Extra-numeral PCR 0 0 5.3

4,5 4,6 5,6Subtotal 27.4 46.8 84.3n 73 47 19

SBS, fish with saddleback syndrome; SBS-l, fish with deformations of only the dorsal supporting elements;S-n, normal, in respect to the SBS, semi-extensively reared fish; n, number of examined individuals; Ep,epural; Hy, hypural; ANaB, specialised neural arch; PCR, caudal lepidotrichia; PU, pre-ural centra. Subtotal

Ž . Ž .frequencies with the same superscript are significantly different at p-0.05 3 , p-0.01 1, 4 or atŽ .p-0.001 2, 5, 6 .


Ž . Ž .Fig. 2. Characteristic types of caudal deformities, correlated to the saddleback syndrome; a 16.0 mm TL, b19.5 mm TL. Open area, cartilage; stippled area, ossification. The ossification state of the rays is not drawn.Ep, epural; HP, haemal process; Hy, hypural; ANaB, specialised neural arch; NP, neural process; PCR, upperŽ . Ž . Ž .-up and lower -lo caudal lepidotrichia; PrH, parhypural; PU, pre-ural centra; SCR, upper -up and lowerŽ .-lo caudal dermatotrichia; Ur, urostyle; UrN, uroneural. Scale bars are equal to 0.5 mm.

deformations of the neural spines and centra of the pre-hemal vertebrae. This type ofvertebral deformity was less frequent in the specimens with deformations of only the

Ž .dorsal supporting elements 27.6% and completely absent in the fish with normal dorsalfin.

3.3. Meristic characters

All the wild juveniles of D. dentex had 24 vertebrae, which were divided into 10Ž .pre-hemal and 14 hemal including urostyle . In the majority of the examined fish

Ž .92.7% , the dorsal fin was composed by 20 proximal pterygiophores which bore XIŽ .spines and 11 lepidotrichia, while in the rest specimens 7.3% dorsal fin presented 19

proximal pterygiophores with XI spines and 10 lepidotrichia. Nine proximal pterygio-phores and III spines and eight lepidotrichia articulating on them composed the anal fin.

Ž .The dominant wild type of the caudal fin presented nine upper in the 97.6% of fish andŽ .eight lower lepidotrichia in the 100% of fish , as well as by 9–11 upper and 9–10


Table 3Ž . Ž .Frequency % , mean and standard deviation SD of the meristic characters in the different fish populations

W E S-n SBS-l SBS

PCRup 3 2.1 5.34 5.35 2.1 5.36 5.37 5.1 2.1 10.58 2.4 18.6 31.9 26.39 97.6 100.0 76.3 61.7 31.610 10.5Mean"SD 9.0"0.2 9.0"0.0 8.7"0.6 8.4"1.1 7.7"2.0

1, 2 3, 4, 5 3, 6 1, 4 2, 5, 6n 41 88 59 47 19

PCRlo 7 1.78 100.0 100.0 98.3 100.0 100.0Mean"SD 8.0"0.0 8.0"0.0 8.0"0.1 8.0"0.0 8.0"0.0n 41 92 59 47 20

SCRup 2 7.73 15.44 7.77 7.78 3.4 12.0 23.19 24.3 17.2 28.0 38.510 67.6 79.3 56.011 8.1 4.0Mean"SD 9.8"0.6 9.8"0.5 9.5"0.8 6.8"2.7

7 8 9 7, 8, 9n 37 29 25 13

SCRlo 9 27.0 3.4 24.0 7.710 59.5 89.7 68.0 38.511 8.1 6.9 8.0 53.812 5.4Mean"SD 9.9"0.8 10.00"0.3 9.8"0.6 10.5"0.7

10 11 12 10, 11, 12n 37 29 25 13

A II,8 2.3 4.5III,7 1.7III,8 100.0 95.0 97.1 83.7 50.0III,9 3.3 2.9 14.0 45.5

A Hard Mean"SD 3.0"0.0 3.0"0.0 3.0"0.0 3.0"0.2 3.0"0.2A Soft Mean"SD 8.0"0.0 8.0"0.2 8.0"0.2 8.1"0.4 8.5"0.5

13 14 15 16 13, 14, 15, 16n 41 60 34 43 22

D 10 4.8I,10 4.8I,11 4.8III,11 9.5IX,11 4.8V,10 4.8V,11 14.3VI,12 4.8


Ž .Table 3 continued

W E S-n SBS-l SBS

D VII,10 4.8VII,11 9.5VIII,11 9.5VIII,9 4.8X,10 9.5X,11 4.8 4.8X,12 3.8 5.7 4.8XI,10 7.3 24.5 28.6 42.9XI,11 92.7 69.8 60.0 42.9XI,12 1.9 2.4XI,9 2.9XII,10 2.4XII,11 2.9XIV,9 4.8

D Hard Mean"SD 11.0"0.0 11.0"0.2 11.0"0.3 10.9"0.3 6.3"3.517 18 19 20 17, 18, 19, 20

D Soft Mean"SD 10.9"0.3 10.8"0.5 10.7"0.6 10.6"0.6 10.6"0.821 21

n 41 53 35 42 21D Prx 18 2.6 10.0

19 7.3 25.4 34.2 47.7 20.020 92.7 74.6 63.2 52.3 45.021 10.022 5.023 5.024 5.0Mean"SD 19.9"0.3 19.8"0.4 19.6"0.6 19.5"0.5 20.2"1.5

22, 23 24 22 23, 24n 41 67 38 44 20

A Prx 8 1.5 4.39 100.0 95.5 97.4 86.4 47.810 3.0 2.6 13.6 47.8Mean"SD 9.0"0.0 9.0"0.2 9.0"0.2 9.1"0.4 9.4"0.6

25 26 27 28 25, 26, 27, 28n 41 66 38 44 23

Ž .Pectorals rightrleft 14,14 4.9 1.8 3.014,15 4.9 1.8 2.9 6.1 5.915,14 1.8 11.815,15 70.7 71.9 70.6 63.6 70.615,16 2.4 8.8 8.8 9.1 5.916,15 9.8 5.3 5.9 9.1 5.916,16 7.3 8.8 11.8 9.1n 41 57 34 33 17

Ž .Pelvics rightrleft I,5–I,5 97.6 100.0 100.0 100.0 100.0I,4–I,5 2.4n 41 59 35 38 19

lower dermatotrichia, which however were hardly discriminated on the radiographiesŽ . Ž .Table 3 . The majority of the wild fish 82.9% was bilaterally symmetrical with


Table 4Ž .Fluctuant asymmetry index FAI of the pectoral fins in the different groups of wild and reared fish

W E S-n SBS-l SBSns41 ns57 ns34 ns33 ns17

FAI 0.174 0.177 0.178 0.246 0.309FAI 100 101.7 102.3 141.4 177.6r

W, wild fish; E, extensively reared fish; SBS, fish with saddleback syndrome; SBS-l, fish with deformations ofonly the dorsal supporting elements; S-n, normal, in respect to the SBS, semi-extensively reared fish; n,

Ž . Ž .number of examined individuals. The relative FAI FAI was estimated as a percentage % of the FAI of ther

wild fish.

Ž .respect to the number of the pectoral lepidotrichia, with the dominant type 70.7%bearing 15 lepidotrichia on each side of the body and the FAI to be equal to 0.174Ž . Ž .Tables 3 and 4 . The dominant pelvic type 95.1% was bilaterally symmetric and bore

Ž .I spine and five lepidotrichia, while the rest specimens 4.9% bore one less pelvicŽ .lepidotrichion on the one side of the body Table 3 .

Generally, wild, normal E and normal S specimens were not differentiated from eachŽ .other in respect to the dominant types, the mean value Table 3 , as well as the

Ž .distribution of the meristic characters Table 5 . Only the S specimens presented lessŽdorsal Prx from the wild juveniles, as well as less PCRup from the E specimens Table

.3 . The Fluctuant Asymmetry Index was 0.177 and 0.178 for the E and S population,Ž .and equal to that of the wild juveniles p)0.05, Table 4 .

The SBS deformity significantly affected not only the meristics of the dorsal fin, butalso the other meristic characters, presenting either new types, altered frequencies of the

Ž .dominant types or different mean values of the meristic characters Tables 3 and 5 . Themeristic deviation from the normal types increased as the severity of the SBS deformityrose, with the SBS-l deviating from the frequency distribution of the normal specimens

Ž .less than the SBS did Tables 3 and 5 . No one SBS individual presented the dominanttype of dorsal fin spines and lepidotrichia, while the SBS-l individuals differentiated

Žfrom the normal S fish only in respect to the relative frequency of XI,10 to XI,11 1:1. Ž .vs. 1:2 in the normal fish Table 3 . Similarly, the 35.0% of the SBS individuals

Ž .deviated from the normal number of the dorsal proximal pterygiophores 20 by 1–2 lessto 1–4 more, while the SBS-l individuals were differentiated from the normal S fish

Žonly in respect to the relative frequency of 19–20 pterygiophores 1:1 vs. 1:2 in the

Notes to Table 3:E, extensively reared fish; SBS, fish with saddleback syndrome; SBS-l, fish with deformations of only thedorsal supporting elements; S-n, normal, in respect to the SBS, semi-extensively reared fish; W, wild fish. n,number of examined individuals. PCRup, upper caudal lepidotrichia. PCRlo, lower caudal lepidotrichia.SCRup, upper caudal dermatotrichia. SCRlo, lower caudal dermatotrichia. A, anal fin. D, dorsal fin. Hard,spines. Soft, lepidotrichia. Prx, proximal pterygiophores. Means marked with the same underlined number are

Ž . Žsignificantly different at p-0.05 3, 6, 10, 11, 12, 16, 21, 22, 24, 28 , p-0.01 1, 2, 5, 13, 14, 15, 23, 25,. Ž .26, 27 or p-0.001 4, 7, 8, 9, 17, 18, 19, 20 .


Table 5Comparison of distribution of the meristic characters between the different fish populations

WrSBS-l WrSBS ErS-n ErSBS-l ErSBS S-nrSBS SBSrSBS-l)) ))) ) ))) )))PCRup

PCRlo))) ))) ))SCRup

)SCRlo))) ))) ))) )))D hard

))D softA hard

)) )) )A soft))D Prx

)) )) ))A Prx

E, extensively reared fish; SBS, fish with saddleback syndrome; SBS-l, fish with deformations of only thedorsal supporting elements; S-n, normal, in respect to the SBS, semi-extensively reared fish; W, wild fish.PCRup and PCRlo, upper and lower caudal lepidotrichia; SCRup and SCRlo, upper and lower caudaldermatotrichia; D hard, dorsal hard spines; D soft, dorsal lepidotrichia; A hard, anal hard spines; A soft, anallepidotrichia; D Prx, dorsal proximal pterygiophores; A Prx, anal proximal pterygiophores. ) p-0.05;)) p-0.01; ))) p-0.001. WrE, WrS-n and S-nrSBS-l comparisons did not present any statisticaldifferences in respect to the distribution of any character.

. Ž .normal fish Table 3 . The SBS-l and SBS fish presented 1 anal pterygiophore moreŽ .than the normal dominant type 9 , with a frequency of 5 for SBS-l and 18 times for

SBS individuals higher than the normal dominant type. The 4.3% of the SBS fishpresented one anal pterygiophore less than the normal dominant type. Lack of one analspine was observed in the 2.3% of the SBS-l and in the 4.5% of the SBS fish. Soft analrays presented significant differences between the normal and the SBS specimens inrespect to both the distribution and the mean value, due to the presence of 1 more soft

Ž .anal ray in the 45.5% of the SBS individuals Tables 3 and 5 . Although abnormal fishhad the same dominant pectoral type with the normal fish, they presented an increased

Ž . Ž .Fluctuant Asymmetry Index of 0.246 SBS-l and 0.309 SBS , which however was notŽ .statistically different from the FAI of the normal fish p)0.05, Table 4 .

3.4. DeÕelopmental prodroms of the SBS and CdF-sbs deformities

During the embryonic stage no morphological abnormalities were present, whileduring the yolk-sac larval stage they were present at a frequency of 3%. The majority ofthem consisted of the enlargement of the perivitellin space at hatching, while a small

Ž .percentage -1% of the yolk-sac larvae bore severe lateral curvatures of the noto-chord. Both these types of abnormalities were lethal and up to the onset of feeding theyhad disappeared.

Ž .Two days post-feeding, a malformation of the primordial marginal finfold PMF waspresented by a proportion of the larvae of all the populations, in the form of necrosis orlack of its posterior region. The semi-extensive populations presented twice the inci-


Ž .dence of PMF malformation than the extensive populations Fig. 3 . Additionally, 79.0%Ž .34 out of 43 of the semi-extensive malformed larvae bore severe deformities of the

Ž .posterior tip of the notochord Fig. 3 . In the extensive populations, this coincidence wasŽ . Ž .significantly p-0.001 smaller than in S population, and only the 23.5% 4 out of 17

of the PMF malformed larvae bore notochord deformities. The PMF deformity waspresent in all the reared populations up to the 11 post-feeding day, while later itdisappeared.

Although the SBS fish could be identified only after the full formation of the dorsalŽ .spines 9.2 mm TL; Koumoundouros et al., 2000c , the internal anatomic characteristics

of these specimens showed that their earlier identification is possible during theŽdevelopment of the proximal pterygiophores 5.8–7.6 mm TL; Koumoundouros et al.,

.2000c . According to these criteria, on 10th post-feeding day, two individuals of 6.5 mmTL presented characteristically deformed proximal pterygiophores which were accompa-

Ž . Ž .nied by a atrophy of the anterior dorsal part of the primordial marginal finfold and bŽ .severe deformities of the caudal fin Fig. 4a . These two associated deformities formed

two new criteria used for the identification of the SBS fish prior to the development ofthe proximal pterygiophores. Following this methodology, the two abnormal specimensŽ .4.4% of the sample of 4.8 and 5.2 mm TL were identified on seventh post-feeding day

Ž .as the earlier stage of SBS Fig. 3 , which additionally presented the PMF deformity.Finally, one specimen of 7.5 mm TL presented an alternative type of SBS, with only

10 central proximal pterygiophores and five lepidotrichia to be present. The caudal finwas also abnormal, presenting lack of lepidotrichia, fusions of hypural and epural

Ž .elements, as well as abnormal curvature of the notochord Fig. 4b .

3.5. Miscellaneous caudal fin deformities

Although the E populations did not have any SBS-l, SBS and CfD-sbs deformities,they presented some deformities of the caudal elements with significantly higherfrequency than the S populations. The E fish were characterized by the lack of the

Ž . Ž .pre-ural centrum 2 PU2 15.0–18.6% and by fusions or twists of the two posteriorŽ . Ž .pre-ural centra 7.0–10.0% Table 6 . In the E specimens, the PU2 lack was accompa-

Ž . Ž .nied by a lack of 13th hemal process HP13 in 10 out of 14 abnormal specimens , the

Fig. 3. Abnormalities of the primordial marginal finfold that are accompanied by deformation of the posteriorŽ . Ž .part of the notochord; a 4.8 mm TL, b 5.2 mm TL. Stippled area indicates the necrosis of primordial

marginal finfold. Nc, notochord; PmF, primordial marginal finfold. Scale bars are equal to 1.0 mm.


Fig. 4. Different types of the early developmental stages of the saddleback syndrome and its correlated caudalŽ . Ž .fin deformities; a 6.5 mm TL, b 7.5 mm TL. Ep, epural; HP, haemal process; Hy, hypural; ANaB,

Ž . Ž .specialised neural arch; Nc, notochord; NP, neural process; PCR, upper -up and lower -lo caudallepidotrichia; Prd, predorsal; PrH, parhypural; Prx, proximal pterygiophore; R, lepidotrichium; Rd, distalradial. S, hard spine; SCR-lo, lower caudal dermatotrichia; Ur, urostyle. Scale bars are equal to 1.0 mm.

Ž . Ž .posterior-most modified neural arch ANaB in 3 out of 14 abnormal specimens or bothŽ .the HP13 and the ANaB in 1 out of 14 abnormal specimens . The only S specimen with

PU2 lack also presented a lack of the HP13.The differently reared populations were not differentiated in respect to the other

Ž .deformities of the caudal fin Table 6 . All of them presented extra-numerous epuralelements and hemal spines, detachment of the hemal spines from the arches, as well assmall fusions or and shape deformations small fusions of the various caudal elements.

Table 6Percentage of the internal caudal fin deformities, not correlated to the saddleback syndrome, in the extensivelyŽ . Ž .E and semi-extensively S reared populations

Ž . Ž . Ž . Ž .Abnormalities E1 % E2 % S1 % S2 %ns40 ns43 ns35 ns38

PU2 lack 18.6 15.0 2.9 0PU2–PU3 deformations 7.0 10.0 2.9 0Ep deformations 4.7 7.5 2.9 5.3Miscellaneous 14.0 12.5 14.3 21.1

1,2 3,4 1,3 2,4Total 44.3 45.0 23.0 26.4

n, number of individuals examined; PU, pre-ural centra; Ep, epural. Total frequencies with the samesuperscript are significantly different at p-0.01.


4. Discussion and conclusions

Environmental conditions and genotype can significantly affect the morphology ofŽ .fish in respect to the shape Divanach, 1985; Blaxter, 1988; Wimberger, 1992 ,

Ž .pigmentation Mansfield and Mansfield, 1982; Marliave, 1988 , allometric growth of theŽ .body Koumoundouros et al., 1995; Koumoundouros et al., 1999b , and meristic

Ž .characters reviewed by Lindsey, 1988 , as well as development of morpho-anatomicalŽ .abnormalities reviewed by Divanach et al., 1996 .

In the present study, we examined the effect of two different rearing methods,extensive and semi-extensive, on the development of morpho-anatomical abnormalitiesand on the meristic characters of D. dentex. In agreement to the general trend observed

Ž .in other studies Fowler, 1970; Lau and Shafland, 1982; Matsuoka, 1987 , the reared D.dentex presented larger meristic variability than the wild. Among the environmentalfactors that affect the meristic characters of fish, temperature plays the most important

Ž .role reviewed by Lindsey, 1988 . All the eggs used in the present study, came from thesame batch and the larval rearing was performed under the same abiotic conditions,leading to the same dominant meristic type and mean value of each character in thedifferently reared populations.

Ž .D. dentex spawns under an increasing photoperiod and temperature 16.5–21.48CŽ .Pavlidis et al., 2000 . In eastern Mediterranean, its larvae are found in June, inhabitingthe upper surface layer of well-stratified waters, where surface temperature ranges

Žbetween 228C and 268C Somarakis, Institute of Marine Biology of Crete, personal. Ž .communication . Lo Bianco 1909 also reports that larvae and juveniles of D. dentex

are neustonic and associated with drifting algae. Our experimental temperature condi-Ž .tions Koumoundouros et al., 1999a were in the ranges reported for spawning and larval

ontogeny, therefore explaining the similarity of the meristic characters between rearedand wild individuals.

4.1. Saddleback syndrome and caudal fin deformities

The two rearing methodologies that were followed in the present study, stronglyaffected the presence of morpho-anatomical abnormalities in D. dentex, with thedevelopment of saddleback syndrome and of caudal fin deformities favoured by thesemi-extensive method. The osteological deformity called saddleback syndrome was

Ž .first described in Oreochromis aureus Tave et al., 1983 and should not been confusedwith the pathogen-associated saddleback disease, which involves skin discoloration and

Ž .lesions Morrison et al., 1981 . This skeletal deformity is characterized by a bigvariability, presenting a continuous distribution from the lack of just the first dorsalspine to the complete absence of the dorsal fin and a simultaneous lack of the respective

Ž .pterygiophores. In many other fish species Browder et al., 1993 , including D. dentexŽ .Efthimiou, 1996b , the SBS is accompanied by abnormal orientation of the scales. Inthe present study, SBS was present in D. dentex with a high variability of expression,which however never extended to the complete absence of the dorsal fin. No scaleabnormalities were detected to be associated with the SBS, probably due to theirabsence, or to their small frequency or even due to the small intensity of expression at


the studied developmental stages. In contrast, the SBS was correlated in D. dentex withthe abnormal development of the upper caudal lepidotrichia and dermatotrichia, present-ing a gradient of abnormality incidence as the intensity of the SBS raised. Similarly to

Ž .what was reported for Sparus aurata Koumoundouros et al., 1997a , the caudaldeformities of D. dentex originated from developmentally earlier abnormalities of theposterior part of the notochord, thus indicating that the latter are also correlated to theSBS. The abnormalities of the notochord, caudal and dorsal fin, as well as of theprimordial marginal finfold were also associated in the case of a mutation in Oryzias

Ž .latipes, which induces the Adouble anal finB deformity Ishikawa, 1990 .Although the SBS has been reported in a variety of fish species under both natural

Ž . ŽBrowder et al., 1993; Lemly, 1993; Honma, 1994 and rearing conditions Komada,.1980; Tave et al., 1983; Valente, 1988; Efthimiou, 1996b , the causative factor was only

Židentified in a few cases. SBS had a heritable basis in reared O. aureus, Tave, 1986;.Tave et al., 1983 , while under natural conditions the development of SBS was attributed

Ž .to selenium pollution Lemly, 1993 . Environmental pollution could induce morpho-anatomical abnormalities directly, or by inducing mutations at some labile genetic locusshared by all fish groups, like regulatory genes which direct the differential development

Ž .of body segments Gehring, 1987 . Finally, the lack of genetic variability, as in the caseof small isolated natural populations or broodstocks, could induce developmental

Žinstability and lead to the formation of morpho-anatomical abnormalities Browder et.al., 1993 .

The direct involvement of the genetic factors in the development of SBS can beexcluded in the present study, as the differently reared populations originated from onecommon egg batch, but not their indirect through the interaction with the rearingenvironment. Although not statistically significant, the increase of the asymmetry of thepectoral fins with the intensity of SBS, indicates an increase of the developmental

Žinstability as a result of the Adevelopmental noiseB minor environmentally induceddepartures of the gene regulation and expression from some ideal developmental

. Ž .program Palmer and Strobeck, 1986; Lindsey, 1988 . The environmental factors whichwere different between the two rearing methods, and which could be critical for thedifferential development of SBS, were the quality and quantity of the larval food, as

Žwell as the water characteristics that are correlated to the feeding conditions spatial.distribution of the plankton, microbes, transparency .

The water chemistry should also be considered as a possible environmental differ-ence, as the date of tank filling with water was different for the two rearing methods.Using anatomical and developmental criteria, the present work demonstrates that theSBS originated from abnormalities of the primordial marginal finfold. Although theseexhibited different frequency of appearance and of correlation with notochord deformi-ties in the two rearing methods, they were common in both the extensively andsemi-extensively reared populations. Additionally, although the E populations did notpresent any one of the SBS related deformities of the caudal fin, they were significantlydifferentiated by the S populations in respect to the development of PU2 deformities.These results strongly support the hypothesis of a common SBS-CfD causative factor forall the reared populations, but with different expression because of the different rearingmethods. The large water temperature difference between the autotrophic and the


exotrophic phase could be the initial signal for the development of morphologicalŽ .abnormalities Divanach et al., 1996 , through a developmental pathway with environ-

ment-dependent expression. The hypothesis of differential development of morphologi-cal abnormalities due to the selective mortality of the individuals under different rearing

Ž .conditions Mair, 1992; Koumoundouros et al., 1997a , does not fit into the results ofthe present study. The continuous sampling and the detailed examination of theindividuals showed that no morphological abnormality was present during the au-totrophic phase, and that the SBS was completely absent from the extensively rearedpopulations during the entire ontogeny. The hypothesis of differential mortality how-ever, might be valid for the wild juveniles of the present study, which showed nodeformities, possibly due to severe selection pressure against deformed individuals in thenatural environment.

4.2. General considerations

Although the development of morpho-anatomical abnormalities has been an impor-tant problem for fish farming industry, a solution has not been found. The determinationof the causative factors of the morpho-anatomical abnormalities requires intensiveresearch effort in wide developmental periods and for a large variability of potentialimportant factors which either act cooperatively, or they have identical symptomsŽ .reviewed by Divanach et al., 1996 . The multivariate approach of the problem ofmorpho-anatomical abnormalities both under mass rearing conditions initially, and underexperimental conditions later, is the only way for the achievement of the solution.Following this approach, the trials can be focussed by detecting the developmental phaseat which morpho-anatomical abnormalities originate and can therefore reduce thenumber of factors to be considered. Additionally, it can serve for the early detection andelimination of reared fish populations with a high incidence of abnormalities.

Acknowledgements

This research was financed by the Greek Ministry of Development, AGeneralŽ .Secretariat of Research and TechnologyB IPER 94 YP: 17 . We would like to express

our thanks to Prof. E. Zouros for the kind supply of the wild specimens. We also wish toexpress our thanks to Dr. S. Efthimiou, Dr. J. Carrillo and Mr. S. Stefanaki for theirparticipation in the maintenance of fish populations and to M. de Wilde for her adviceconcerning the English language.

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kou mound outhe effect of rearing conditions on development of saddleback syndrome and caudal fin...

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