Aquaculture 198 Ž2001. 369–386 www.elsevier.nlrlocateraqua-online
High prevalence of skeletal deformity and reduced gill surface area in triploid Atlantic salmon žSalmo salar L./ J. Sadler a,) , P.M. Pankhurst a , H.R. King b a
School of Aquaculture, UniÕersity of Tasmania, Locked Bag 1-370, Launceston, Tasmania 7250, Australia b Salmon Enterprises of Tasmania Pty. Ltd., P.O. Box 1, Wayatinah, Tasmania 7140, Australia Received 29 August 2000; received in revised form 15 December 2000; accepted 18 December 2000
Abstract The prevalence of skeletal deformity throughout the development of different populations Žall-female triploid, mixed-sex triploid, all-female diploid and mixed-sex diploid. of Tasmanian Atlantic salmon Ž Salmo salar L.. was determined to elucidate the possible effects of ploidy or sex status on the incidence of deformity. Populations were produced and maintained under commercial conditions in freshwater until individuals had attained a wet weight of approximately 80 g, at which time each population was divided and either retained in freshwater ŽFW smolt. or transferred to seawater ŽSW smolt., where fish were held for a further 2 months. Whole fish were sampled throughout this period from hatching Ž4708 days post-fertilisation.. The prevalence of skeletal deformities was significantly higher in triploid populations. Jaw deformity, including lower jaw deformity syndrome ŽLJD., occurred in up to 2% of triploid fry, 7% of triploid FW smolt, 14% of triploid SW smolt and 1% of diploid FW smolt. The prevalence of LJD was highest in triploid FW smolt. Short opercula were observed in up to 22% of triploids and 16.6% of diploids. Up to 60% of triploids and 4% of diploids suffered from the absence of primary gill filaments Žgill filament deformity syndrome ŽGFD.. during FW development prior to SW transfer, then, up to 50% of triploid FW smolt and 60% of triploid SW smolt suffered from GFD. There was no significant difference in the sex ratio of each deformity type. An index of gill surface area ŽGSA. was significantly reduced in normal triploids and triploids afflicted with GFD, compared to diploid counterparts. It is likely that the reduction of GSA affects an individual’s capacity for metabolic gas exchange under vigorous exercise or suboptimal environmental conditions. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Skeletal deformity; Ploidy; Sex; Gills; Lower jaw deformity
)
Corresponding author. NORTAS Pty. Ltd., 100 Mornington Road, Mornington, Tasmania 7218, Australia. E-mail address:
[email protected] ŽJ. Sadler..
0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 5 0 8 - 7
370
J. Sadler et al.r Aquaculture 198 (2001) 369–386
1. Introduction Skeletal deformities are common in cultured fish populations due to the absence of natural selective pressures that may result in the mortality of afflicted fish. The incidence of skeletal deformities in cultured fish impairs the management of a reliable, high quality harvest and results in financial loss to producers. Lower jaw deformity syndrome ŽLJD., which is characterised by the downward curvature of the lower jaw ŽBruno, 1990; Jungawalla, 1991; Hughes, 1992; Lee and King, 1994; King and Lee, 1993; Quigley, 1995; McGeachy et al., 1996; Branson and Nieto, 1999; Goicoechea et al., 1999. and other skeletal deformities, including short opercula ŽSutterlin et al., 1987; Baeverfjord et al., 1997., scoliosis, lordosis ŽMcKay and Gjerde, 1986. and gill deformities ŽHughes, 1992; Baeverfjord et al., 1997. have been previously recorded in cultured Atlantic salmon Ž Salmo salar L... In Tasmania, lower jaw deformity has been reported in up to 30% of commercially produced all-female triploid Atlantic salmon during the seawater ŽSW. phase of the lifecycle ŽJungawalla, 1991; Hughes 1992., and subsequently represents considerable financial loss to sea farmers as all-female triploid populations are an important component of the annual harvest. The factors that contribute to the occurrence of skeletal deformity may be either genetic or environmental or both Žreviewed by Hickey, 1972; McKay and Gjerde, 1986.. Contributing factors within the culture environment may include the presence of pathogenic organisms, inappropriate physical parameters Žlight, temperature, salinity, dissolved oxygen, flow rate, pH. or the presence of heavy metals or other teratogenic substances Žreviewed by Hickey, 1972; Chatain, 1994; Andrades et al., 1996.. It has been suggested that therapeutants such as malachite green and formaldehyde may have teratogenic effects ŽAlderman, 1991; Alderman and Clifton-Hadley, 1993; Edgell and Lawseth, 1993.. In addition, failure to achieve swimbladder inflation, often a result of suboptimal culture conditions, has been associated with an increased rate of deformity ŽDaoulas et al., 1991; Chatain, 1994; Andrades et al., 1996.. Nutritional deficiencies, including phospholipids, vitamins A, B, C, or D and minerals such as manganese, phosphorous, magnesium and zinc Žreviewed by McKay and Gjerde, 1986. may also contribute to abnormalities under culture conditions. An environmental disturbance at any stage of fish development, particularly during organogenesis, can cause the cessation or dissociation of growth processes of different tissues, which can result in abnormal development ŽStockard, 1921.. Some studies suggest LJD is associated with the production of triploid Atlantic salmon ŽJungawalla, 1991; Hughes, 1992; Lee and King, 1994; King and Lee, 1993; McGeachy et al., 1996., whereas other studies do not specify the ploidy status of affected fish ŽBruno, 1990; Quigley, 1995; Branson and Nieto, 1999; Goicoechea et al., 1999., which may mean that diploid fish can also be affected by LJD. Therefore, it is unclear whether the incidence of LJD is associated directly with ploidy status and the potential differential physiology that may result from the altered cell morphology and genetic heterozygosity of triploid fish Žreviewed by Benfey, 1999., or whether the incidence of LJD is associated with environmental factors. In addition, the majority of reports show that LJD occurs in all-female triploids ŽHughes, 1993; King and Lee, 1993; Lee and King, 1994., and although one study showed LJD can occur in mixed-sex
J. Sadler et al.r Aquaculture 198 (2001) 369–386
371
triploid populations ŽMcGeachy et al., 1996., there was no indication as to whether the fish affected by LJD were male or female. Whether LJD is found only in female fish is unknown, but it is possible that the deformity is congenital and associated with the sex-determining locus. Furthermore, reports of the incidence of lower jaw deformity in Atlantic salmon stocks from Canada ŽMcGeachy et al., 1996., Scotland ŽBruno, 1990., Ireland ŽQuigley, 1995., Norway ŽDr. Tore Hastein ŽCentral Veterinary Laboratory, Oslo., personal communication, 1996. and Tasmania ŽJungawalla, 1991; Hughes, 1993; King and Lee, 1993; Lee and King, 1994., indicate that the deformity is present following SW transfer, although the time of onset is unknown. More recently, scientists working in Chile have reported that LJD can occur in Atlantic salmon smolt just prior to SW transfer ŽBranson and Nieto, 1999; Goicoechea et al., 1999.. It remains unclear whether LJD is associated with culture conditions or a specific stage of development of the fish. The aim of the current study was to describe the prevalence and temporal onset of various skeletal deformities, including LJD, in four different populations of Tasmanian Atlantic salmon Žall-female triploid, mixed-sex triploid, all-female diploid and mixed-sex diploid. to determine whether skeletal deformities are associated with either ploidy status or sex. In addition, we examined whether the prevalence of deformities varied between smolt either held in freshwater ŽFW. or transferred to SW. Examining the incidence of deformity throughout ontogeny of the different population types is fundamental to determining the possible mechanisms by which these deformities occur, and thereby has the potential to improve management procedures and harvest quality. This study records for the first time a gill filament deformity ŽGFD. and compares the relative total gill surface area ŽGSA. of fish afflicted with GFD to that of diploid and triploid fish exhibiting normal gill structure.
2. Methods and materials 2.1. Fish production and husbandry All-female diploid, all-female triploid, mixed-sex diploid and mixed-sex triploid Atlantic salmon populations were produced in May 1996 using standard commercial techniques at the Salmon Enterprises of Tasmania ŽSALTAS. hatchery at Wayatinah, Tasmania. Populations were not related to one another and were each produced by fertilising eggs pooled out of 15–18 females with sperm pooled out of 7–12 males. All-female populations were produced by fertilising the eggs of normal females with milt from sex-reversed genotypic females. Sex reversal of genotypic female broodstock was induced by oral administration of 17a-methyldihydrotestosterone at first feeding. Triploidy was induced by hydrostatic pressure treatment of fertilised eggs ŽJungawalla, 1991.. During the FW phase, the four populations were each maintained in one of four tanks under standardised husbandry conditions at ambient temperature Ž8–108C. and photoperiod. At 17 months post-fertilisation, the populations were split in two groups, one was maintained at the SALTAS hatchery in FW ŽFW smolt., whereas the other was
J. Sadler et al.r Aquaculture 198 (2001) 369–386
372
transferred to the School of Aquaculture, University of Tasmania, Launceston, where each population was maintained in one of four tanks within a closed recirculating seawater system ŽSW smolt., at a temperature of 12.5 " 0.58C, salinity of 33 " 1 ppt and stocking density of 15 kg my3 . FW smolt were freeze branded for identification purposes and maintained communally in a 44 m3 tank at ambient temperature and a stocking density of 2 kg my3 . FW and SW smolt were fed to satiation daily with GIBSONS Salmon Growers Diet Ž45% protein, 22% lipid; Gibsons, Cambridge, Tasmania.. 2.2. PreÕalence of deformity during deÕelopment Fish were randomly sampled from each population at various stages throughout development ŽTable 1.. Fish were killed by terminal anaesthesia in 25 ppm Benzocaine and total wet weight ŽTWWt.. and fork length ŽFL. were recorded. Specific growth rate at each stage of development was determined using the formula: Specific Growth Rate ŽSGR. s 100 = lnŽŽWfrW0 .rt., where Wf s final total wet weight Žg., W0 s initial total wet weight Žg. and t s time Ždays. between initial and final weight check. Gross skeletal deformities were determined by examination of the external morphology of freshly euthanased fish and were characterised as follows: A jaw in which the
Table 1 Developmental stages and total number of fish sampled Ž n. for determination of deformity prevalence throughout the development of four Atlantic salmon populations; all-female triploids ŽFT., mixed-sex triploids ŽMT., mixed-sex diploids ŽMD. and all-female diploids ŽFD.. Accumulated temperature units ŽATU. in 8 days represents development post-fertilisation Development stage Fertilisation Hatching ALEVINS
Swim up and First Feeding FRY
PARR
SMOLT Sea transfer SW SMOLT FW SMOLT
Weeks Žpost-fertilisation.
8 9 10 11 12
13 14 15 19 31 45
ATU Ž8 days. 0 f 400 470 528 583 642 706 f 720 772 843 913 1510 2350 3118 4026 4202 4220 4810 4928
FT Ž n.
MT Ž n.
MD Ž n.
FD Ž n.
26 123 94 125 254
55 29 95 15 108
82 58 115 99 127
24 86 94 113 104
209 20 200 200 200 200 30 100
34 30 200 200 200 200 30 100
32 110 200 200 200 200 30 100
200 20 200 200 200 200 30 100
97 100
85 100
47 100
56 100
J. Sadler et al.r Aquaculture 198 (2001) 369–386
373
symphyseal joint was not aligned with the median longitudinal plane of bilateral symmetry was defined as a laterally curved lower jaw. In contrast, LJD was characterised by the downward curvature of the lower jaw and was subjectively defined as either mild ŽMLJD. or severe ŽLJD.. A long lower jaw extended anteriorly beyond the rostral cartilage and upper jaw, whereas a short lower jaw had a length less than or equal to 2r3 the length of the upper jaw. Fish diagnosed with short opercula deformity were characterised by shortening of the operculum such that the distal tips of the gill filaments were exposed. In some cases the opercula bone was reduced in anterio–posterior length and in other cases there was an inward folding of the posterior edge of the operculum so that the folded tissues, including dermal bone, projected into the branchial chamber. GFD was diagnosed by the complete absence of a number of primary gill filaments on the branchial arches of a fish ŽFig. 1.. Other types of deformity, collectively referred to as Anon-cranialB deformities, included kyphosis ŽBruno and Poppe, 1996., scoliosis ŽBruno and Poppe, 1996., lordosis ŽBruno and Poppe, 1996., spiralled tails, reduced number of muscle myomeres and Siamese Ždouble-headed. fish. The number of fish afflicted with each deformity type was recorded for each population, at each stage of development, and expressed as a percentage of the total number of fish sampled at each stage. Total percentage prevalence of deformity was defined as the total number of fish affected by one, or a number of different deformity types expressed as a percentage of the total number of fish observed within each population, at each stage of development.
Fig. 1. Gross morphology of a branchial arch from an Atlantic salmon SW smolt with Ža. a complete complement of primary gill filaments and Žb. primary gill filaments absent, this being a characteristic of gill filament deformity syndrome ŽGFD.. Abbreviations: Ebsepibranchial, Cbs ceratobranchial, pgf s primary gill filaments, mgf s zone of missing gill filaments. Scale bar s6.0 mm.
374
J. Sadler et al.r Aquaculture 198 (2001) 369–386
All fish sampled for assessment of gross deformity were then fixed in 10% neutral buffered formalin. 2.3. Gill surface area A random subsample of fixed diploid and triploid SW smolt Ž48108 days old. was examined to determine the relative gill surface area of normal diploid Ž n s 26. and normal triploid Ž n s 18. fish and that of fish afflicted with GFD Ž n s 48.. The whole branchial apparatus Žeight branchial arches. from each fish was carefully dissected and laid flat under a glass plate, to expose the maximum two-dimensional area represented by each branchial arch. Photographic contact prints were then made and an image analysis program ŽAnalytical Imaging station 3. was used to determine the total two-dimensional GSA Žmm2 . for each fish. 2.4. Sex and ploidy status of fish populations At the late FW parr Ž42028 days. and SW smolt Ž48108 days. stages, in addition to the assessment of gross deformity, a section of gonad tissue was dissected from each of the freshly euthanased fish and was fixed in Bouin’s fixative for 24 h, dehydrated in an ascending ethanol series, embedded in paraffin, sectioned at 5 mm and stained with haematoxylin and eosin, prior to examination at the light microscope level to determine sex status. All-female populations were 100% female and mixed-sex populations had an equal sex ratio Ž1 male:1 female.. The sex ratio of fish afflicted with each deformity type was recorded. Population triploidy rates were determined using fish sampled for sex determination at 42028 days. Blood was collected from 100 fish from each triploid population and 50 fish from the mixed-sex diploid population and smeared onto glass slides. Blood smears were air dried, fixed in 70% ethanol and stained with haematoxylin. Erythrocyte nucleus length ŽENL. was measured in 10 randomly selected red blood cells from each fish using an image analysis program ŽCue-2 Image Analysis.. Mean ENL values were determined for each fish and frequency histograms of mean ENL values from each triploid population were compared to those of the diploid population. Any triploid fish with a mean ENL value equal to, or less than the maximum diploid mean ENL value, was considered to be a possible diploid ŽThomas and Morrison, 1995.. Within each triploid population, 96% of the all-female and 100% of the mixed-sex populations were considered to be triploid fish. The ploidy status of deformed fish was confirmed using the latter technique. 2.5. Statistical analysis A paired t-test ŽMicrosoft Excel version 5 software. was used to compare the prevalence of deformity at each stage of development between: Ži. all-female diploids and all-female triploids, Žii. mixed-sex diploids and mixed-sex triploids, and Žiii. diploids and triploids Žmixed-sex and all-female populations pooled.. In addition, for
J. Sadler et al.r Aquaculture 198 (2001) 369–386
375
each population, comparisons of the incidence of deformity were made between FW smolt Ž49288 days. and SW smolt Ž48108 days.. Analysis of covariance ŽANCOVA. was used to compare GSA between: Ži. normal triploid fish and triploid fish afflicted with GFD and Žii. normal diploid and normal triploid fish, using total wet weight as a covariable. In each case, the relationship between gill surface area and total wet weight was similar between each of the groups compared, such that the respective regressions had the same slope. This fulfilled one of the assumptions required for ANCOVA analysis.
3. Results 3.1. Total deformity throughout deÕelopment During the period between hatching and swim-up Ž470–7068 days post-fertilisation. the total prevalence of skeletal deformity, including jaw deformities, short opercula, GFD and non-cranial deformities, was up to 20% in each population, irrespective of ploidy status Ž P ) 0.05, Fig. 2.. Following swim-up and during first feeding ŽF 9138 days., total deformity rates decreased to zero in all populations ŽFig. 2.. Following first feeding Ž) 10008 days., the incidence of skeletal deformity increased and was significantly higher in triploid populations compared to diploid populations Ž P - 0.05, Fig. 2.. Up to 65% of triploid individuals and 20% of diploid individuals suffered from gross skeletal deformities towards the end of smoltification Ž42028 days, Fig. 2.. These levels of deformity were subsequently maintained in populations of smolt regardless of whether the fish were maintained under FW conditions Ž49288 days., or were transferred to SW Ž48108 days, Fig. 2.. 3.2. Deformity of freshwater and seawater smolt The diversity of deformities did not differ between FW and SW smolt ŽFig. 2.. The prevalence of short opercula and lower jaw deformity was higher in smolt grown in FW compared to smolt from the same populations grown in SW Ž P s 0.069.. The specific growth rate of FW smolt from each population ŽSGR s 0.143–0.176%rday. was significantly higher than that for SW smolt in each population ŽSGRs 0.041– 0.063%rday, P - 0.05.. For stages of development other than the smolt stage, no correlation was found between specific growth rate and the percentage prevalence of lower jaw deformity or short opercula at each stage. 3.3. Jaw deformity The prevalence of jaw deformities, including short lower jaw, long lower jaw, LJD and laterally curved lower jaw, increased during ontogeny and remained below 7% in all populations at the termination of the experiment with the exception that jaw deformities occurred in 13.6% of all-female triploid SW smolt at 48108 days ŽFig. 3.. The prevalence of MLJD was significantly higher in triploid fish compared to diploid fish
376
J. Sadler et al.r Aquaculture 198 (2001) 369–386
Fig. 2. Prevalence Ž%. of gill filament deformity ŽGFD., short opercula, jaw deformity and non-cranial deformities in Ža. all-female triploid, Žb. mixed-sex triploid, Žc. all-female diploid and Žd. mixed-sex diploid populations of Atlantic salmon throughout development. Accumulated temperature units ŽATUs8 days. represent development post-fertilisation, FWs freshwater phase, SWssea water phase. Sample sizes as per Table 1.
J. Sadler et al.r Aquaculture 198 (2001) 369–386
377
Fig. 3. Prevalence Ž%. of mild lower jaw deformity, severe lower jaw deformity and other types of jaw deformity Žshort lower jaw, long lower jaw, laterally displaced lower jaw. in Ža. all-female triploid, Žb. mixed-sex triploid, Žc. all-female diploid and Žd. mixed-sex diploid populations of Atlantic salmon throughout development. Accumulated temperature units ŽATUs8 days. represent development post-fertilisation, FWs freshwater phase, SWssea water phase. Sample sizes as per Table 1.
378
J. Sadler et al.r Aquaculture 198 (2001) 369–386
Ž P - 0.05, Fig. 3.. LJD was observed exclusively in triploid fish under FW conditions only ŽFig. 3.. Jaw deformities occurred in both male and female fish. The relative contribution of jaw deformity to the total prevalence of deformity in each population remained below 20% throughout development in FW and was significantly lower than the contribution of short opercula and gill filament deformity ŽFig. 2.. At the SW smolt stage Ž48108 days., jaw deformity contributed to 25.4% of deformity in all-female triploids, 20% in mixed-sex triploids, 10% in all-female diploids and 37.5% in mixed-sex diploids ŽFig. 2.. 3.4. Short opercula Short opercula were initially observed after first feeding ŽG 15108 days., in both triploid and diploid fish; the prevalence of which fluctuated throughout development ŽFig. 2.. The prevalence of short opercula was significantly higher in triploid populations compared to diploid populations Ž P - 0.05., but did not differ with gender Ž P ) 0.05.. Short opercula were observed in up to 22% of fish from both the all-female and mixed-sex triploid populations and up to 18% of fish from each of the diploid populations ŽFig. 2.. At 40268 days, short opercula were present in up to 6.25% of mixed-sex triploid fish, but were not detected in other populations at this stage. Short opercula was the predominant type of deformity observed in diploid populations, but not in triploid populations ŽFig. 2.. For example, at 23508 days short opercula contributed to 90% and 100% of all deformities observed in the all-female diploid and mixed-sex diploid populations, respectively ŽFig. 2.. At the same stage of development, short opercula represented 48% and 26% of all deformities in the all-female triploid and mixed-sex triploid populations, respectively ŽFig. 2.. 3.5. Gill filament deformity syndrome Primary gill filaments were absent from between one and five branchial arches from either side of the body in fish with GFD. The proportion of primary gill filaments missing from any one branchial arch varied between arches and between fish. The prevalence of GFD in triploid fish was significantly higher than that in diploid fish throughout development Ž P - 0.05.. In triploid populations, GFD was initially observed at levels up to 8% at 15108 days ŽFig. 2., increasing with development and reaching a maximum of 60% in all-female triploids at 42028 days, and 50% in mixed-sex triploids at 40268 days. The prevalence of GFD in diploid fish ranged from 0% to 4% in mixed-sex diploids and 0–2% in all-female diploids throughout development ŽFig. 2.. There was no significant difference in the prevalence of GFD with population sex status, nor was the deformity associated with only female fish. Some fish were afflicted with both GFD and short opercula. GFD was the predominant deformity type observed in triploid population as it contributed between 51% and 100% of total deformity rates in all-female triploids and 68%–92% of that in mixed-sex triploids. In contrast, GFD contributed to between 0% and 13% of deformities for the all-female diploid population and between 0% and 38% of deformities for the mixed-sex diploid population ŽFig. 2..
J. Sadler et al.r Aquaculture 198 (2001) 369–386
379
The relative measure for GSA of triploid SW smolt with GFD Ž n s 48. ranged between 1132.37 and 1845.38 mm2 , whereas GSA for triploid Ž n s 18. and diploid SW smolt Ž n s 26. with normal gills, sampled at the same stage of development ranged between 1248.73–1840.76 and 1464.95–2040.81 mm2 , respectively ŽFig. 4.. Generally, fish with a low GSA also had a low TWWt. ŽFig. 5.. The relationship between GSA and TWWt. was similar in triploid fish with deformed gills and triploid fish with normal
Fig. 4. Frequency distribution Ž%. of total gill surface area ŽGSA. values Žsize classes are 100 mm2 . for Ža. triploid SW smolt with GFD, Žb. normal triploid and Žc. normal diploid SW smolt. Sample sizess 48, 18 and 26 for each group, respectively.
380
J. Sadler et al.r Aquaculture 198 (2001) 369–386
Fig. 5. Relationship between total gill surface area ŽGSA. with total wet weight ŽTWWt.s g. of Ža. triploids with GFD and normal triploids and Žb. normal triploid and normal diploid Atlantic salmon SW smolt Ž48108 days.. Black filled circles denote normal diploid fish, black filled triangles denote normal triploid fish, open triangles denote triploid fish with gill filament deformity ŽGFD.. Dashed lines indicate 95% confidence limits.
J. Sadler et al.r Aquaculture 198 (2001) 369–386
381
gills such that the respective regressions had similar slope ŽFig. 5.. As a result, it was possible to use ANCOVA to compare GSA values of each group using means that were adjusted to account for the covariance of GSA with weight. When comparing fish of a similar weight, there was no significant difference in mean GSA values between triploid SW smolt with GFD and triploid SW smolt with normal gills Ž P ) 0.05, Fig. 6a.. In the same fashion, ANCOVA comparing the mean GSA of normal triploid and normal diploid fish, with total wet weight the covariate, determined that the mean GSA of triploid SW smolt was significantly lower than that of diploid SW smolt, when comparing fish of a similar weight Ž P - 0.05, Fig. 6b..
Fig. 6. Comparison of relative mean total gill surface area ŽGSA"S.E.. between Ža. triploid Atlantic salmon SW smolt Ž48108 days. with gill filament deformity ŽGFD. and those with normal gills Žnormal triploids., and Žb. triploid and diploid SW smolt Ž48108 days. with normal gills. In each case, means are adjusted to account for covariance with total wet weight of the fish.
382
J. Sadler et al.r Aquaculture 198 (2001) 369–386
3.6. ‘Non-cranial’ deformities All deformities observed prior to first feeding consisted entirely of non-cranial deformities ŽFig. 2.. The contribution of non-cranial deformities to total deformity prevalence was negligible in all populations after first feeding ŽFig. 2..
4. Discussion The prevalence of skeletal deformity in Tasmanian cultured Atlantic salmon was not sex-linked, suggesting that if the observed skeletal deformity is of genetic origin, it is likely to be an autosomal condition Žnon-sex-linked genotype.. Other studies on Atlantic salmon have shown that deformities such as LJD ŽSutterlin et al., 1987; Quigley, 1995. and short opercula ŽSutterlin et al., 1987. can occur in both male and female fish, although McKay and Gjerde Ž1986. suggested that the higher incidence of a congenital spinal defect in male compared to female Atlantic salmon may be a result of differential growth rates in male and female fish. Deformities observed prior to the commencement of feeding in Atlantic salmon were all non-cranial and were generally not detected following swim-up. This suggests that abnormalities observed in larval fish were lethal. In contrast, those observed in post-larval fish prevailed, and as such were not necessarily lethal. Barahona-Fernandes Ž1982. reached a similar conclusion after examination of abnormalities in hatchery reared sea bass Ž Dicentrarchus labrax L... Deformities occurring after the commencement of feeding may have been influenced by culture parameters rather than genetic factors because the same genes determine characteristics of paired organs ŽBarahona-Fernandes, 1982. and yet short opercula, GFD and some jaw deformities, displayed asymmetrical manifestation. Differential growth rate and coincident higher incidence of jaw deformity in triploid and diploid FW smolt compared to SW smolt also suggests influence of the culture environment on prevalence of skeletal abnormality. Higher growth rates in FW smolt may contribute to skeletal abnormality in the current study, since growth rate related alteration of osteogenic activity could cause dissociation of normal skeletal developmental processes ŽStockard, 1921.. Lee and King Ž1994. suggested that growth rate may affect the incidence of LJD in triploid Tasmanian Atlantic salmon, although the results of their study were inconclusive. Nutritional factors are likely to impact on deformity rates post-embryology, after the commencement of feeding, and as such may explain the increasing prevalence of deformity, particularly GFD, later in development, especially if it impacts on the gill arch growth zones. The branchial bones have cartilagenous joints and their growth is similar to mammalian appendages ŽNorris et al., 1963., in which the epiphyseal region of new bone development is adjacent to the cartilagenous joints at either end of each bone. New bone is formed at each epiphyseal region as the branchial bones increase in length during ontogeny ŽNorris et al., 1963. and presumably primary gill filaments, which extend along the lateral edge of these bones, are added and develop in conjunction with the new bone as the bones grow. The incidence of GFD at later stages of
J. Sadler et al.r Aquaculture 198 (2001) 369–386
383
development may be caused by the failure of new primary gill filaments to develop in the zone of new bone growth at the cartilagenous epiphyseal region, as the ceratobranchial and epibranchial bones increase in length. If the cause is not nutritional deficiency, some other environmental disturbance during post-embryological development may disrupt the development of new primary gill filaments, culminating in the cumulative prevalence of GFD. Alternatively, since GFD occurred almost exclusively in triploid fish, there is a possibility that triploid induction treatment contributed to GFD. However, in another study diploid Atlantic salmon that retained their ploidy status despite being subject to triploid induction treatment did not display deformities, whereas triploid fish displayed a protrusive jaw deformity ŽSutterlin et al., 1987., suggesting the deformity was associated with the triploid condition, rather than the triploid induction process. The prevalence of skeletal deformity after the commencement of feeding was higher in triploid Atlantic salmon than in diploid fish and may be caused by any of three possible mechanisms. Firstly, Tasmanian Atlantic salmon stocks may be genetically prone to deformities since the Phillip River stock from which the Tasmanian Atlantic salmon stock originates, has a history of high deformity rate ŽBrian Glebe ŽAtlantic Salmon Federation, Canada., personal communication, 1999.. That triploid fish displayed higher prevalence of skeletal deformity compared to diploid populations may be a result of increased heterozygosity of genes ŽAllendorf and Leary, 1984. that control skeletal morphology in triploid fish. Secondly, deformities may result from the altered expression of genes under suboptimal environmental conditions, with modulation generated by morphogenic agents such as retinoic acid and growth factors Žreviewed by Koumoundourus et al., 1997; Hall and Miyake, 1995.. If suboptimal environmental conditions are the trigger for skeletal deformity then the higher susceptibility of triploid fish to deformity under standardised culture conditions indicates that triploids may have different environmental requirements to diploids. Finally, altered metabolism may contribute to the higher incidence of skeletal deformity in triploid fish. The capacity to metabolise nutrients, including the ability to absorb and utilise dietary phospholipids, vitamins A, B, C, or D and minerals such as manganese, phosphorus, magnesium and zinc, may be compromised in triploid fish due to differences in cellular morphology Žreviewed by Benfey, 1999. and increased heterozygosity ŽAllendorf and Leary, 1984., thereby affecting skeletal development. Interestingly, a study to examine the effects of reduced vitamin C on the incidence of LJD in triploid Atlantic salmon proved inconclusive ŽKing and Lee, 1993.. The most significant outcome of the present study is that triploid Atlantic salmon had significantly reduced GSA compared to their diploid counterparts, regardless of whether they had apparently normal gills or gills affected by GFD. That the incidence of GFD did not significantly decrease the total gill surface area of afflicted triploid fish compared to that of triploid fish with apparently normal gills, may indicate that fish with missing primary gill filaments are able to compensate by increasing the length of existing gill filaments; however this was not examined in the present study. Nevertheless, it is possible that the significant decrease in relative gill surface area observed in triploid fish compared to diploid fish may contribute to decreased rate of calcium uptake via the chloride cells of the gill lamellae ŽPayan et al., 1984; Wendelaar Bonga and Flik,
384
J. Sadler et al.r Aquaculture 198 (2001) 369–386
1991. thereby altering ionic regulation and osteogenic activity during development. In addition, the reduced relative gill surface area may affect respiration in triploid fish since fish regulate blood circulation in the gills, and although they do not utilise their full gill surface area under optimal pre-stress conditions, fish may require the full surface area of their gills for efficient blood gas exchange under conditions of hypoxia or physical exertion ŽHughes, 1966; Booth, 1978, 1979.. The repercussions of reduced GSA on respiration may be detrimental under traumatic conditions and, in the absence of differences of blood parameters ŽSadler et al., 2000a. and stress physiology ŽSadler et al., 2000b. between diploid and triploid populations, may explain high mortality levels of triploid fish observed during transport and following seawater transfer in the present study Žunpublished data., and similarly under suboptimal culture conditions, such as high water temperatures, low dissolved oxygen levels and crowding in previous reports ŽQuillet et al., 1987; Quillet and Gaignon, 1990; Aliah et al., 1991; Johnstone et al., 1991; Ojolick et al., 1995 reviewed by Benfey, 1999.. The effect of reduced gill surface area on respiration and ionic regulation in triploid fish under suboptimal conditions requires further investigation. In summary, it has been shown that the triploid Atlantic salmon used in this study are prone to a higher prevalence of skeletal deformity, particularly GFD and jaw deformity, than diploid salmon. Lower jaw deformity was occasionally detected in fish during early FW development, but was more prevalent in fish during the post-smolt stage both under FW and SW conditions, and could possibly be exacerbated by higher growth rates at this stage. The aetiology of skeletal deformity remains unclear, but it appears that the differential cell morphology and increased genetic heterozygosity of triploid fish may affect the processes of cellular differentiation, development andror metabolism. Consequently, the environmental requirements, particularly the nutritional requirements of triploid fish, or the ability of triploid fish to uptake and utilise nutrients, may differ to that of diploid fish and contribute to the higher incidence of skeletal deformity in triploid fish under standardised conditions. The reduced gill surface area of triploid fish may impact on either ionoregulation or respiratory efficiency under conditions of high oxygen demand and may contribute to high mortality under suboptimal culture conditions.
Acknowledgements This research was funded by an APAI awarded to P.M. Pankhurst and was supported by Salmon Enterprises of Tasmania ŽSALTAS., Wayatinah, Tasmania. We thank Polly Hilder and Mark Hilder for technical assistance in fish husbandry. Thanks to the staff of SALTAS for their assistance and provision of the smolt.
References Alderman, D.J., 1991. Malachite green and alternatives as therapeutic agents. In: De Pauw, N., Joyce, J. ŽEds.., Aquaculture and the Environment. European Aquaculture Society, Ghent, Belgium, pp. 234–244.
J. Sadler et al.r Aquaculture 198 (2001) 369–386
385
Alderman, D.J., Clifton-Hadley, R.S., 1993. Malachite green: a pharmacokinetic study in rainbow trout Oncorhynchus mykiss ŽWalbaum.. J. Fish Dis. 16, 297–311. Aliah, R.S., Inada, Y., Yamaoka, K., Taniguchi, N., 1991. Effects of triploidy on haematological characteristics and oxygen consumption of ayu. Nippon Suissan Gakkaishi 57, 833–836. Allendorf, F.W., Leary, R.F., 1984. Heterozygosity in gynogenetic diploids and triploids estimated by gene-centromere recombination rates. Aquaculture 43, 413–420. Andrades, J.A., Becerra, J., Fernandez-Llebrez, P., 1996. Skeletal deformities in larval juvenile and adult stages of cultured gilthead sea bream Sparus aurata L. Aquaculture 141, 1–11. Baeverfjord, G., Lein, I., Asgard, T., Rye, M., 1997. Shortened operculae in Atlantic salmon Salmo salar L. fry reared at high temperatures. Proceedings of the 8th International European Association of Fish Pathology Conference, Edinburgh, Scotland. p. 15ABSTRACT. Barahona-Fernandes, M.H., 1982. Body deformation in hatchery reared European sea bass Dicentrarchus labrax ŽL..: types, prevalence and effect on fish survival. J. Fish Biol. 21, 239–249. Benfey, T.J., 1999. The physiology and behaviour of triploid fishes. Rev. Fish. Sci. 7, 39–67. Booth, J.H., 1978. The distribution of blood flow in the gills of fish: application of a new technique to rainbow trout. J. Exp. Biol. 73, 119–129. Booth, J.H., 1979. Circulation in trout gills: the relationship between branchial perfusion and the width of the lamellar blood space. Can. J. Zool. 57, 2183–2185. Branson, E.J., Nieto, D., 1999. Jaw deformities in Atlantic salmon Ž Salmo salar . in Chile. Proceedings of the 9th International European Association of Fish Pathology Conference, Rhodes, Greece. p. 31ABSTRACT. Bruno, D.W., 1990. Jaw deformity associated with farmed Atlantic salmon Ž Salmo salar .. Vet. Rec. 126, 402–403. Bruno, D.W., Poppe, T.T., 1996. The Colour Atlas of Salmonid Diseases. Academic Press, London. Chatain, B., 1994. Abnormal swimbladder development and lordosis in sea bass Ž Dicentrarchus labrax . and sea bream Ž Sparus auratus .. Aquaculture 119, 371–379. Daoulas, Ch., Economou, A.N., Bantavas, I., 1991. Osteological abnormalities in laboratory reared sea-bass Ž Dicentrarchus labrax . fingerlings. Aquaculture 97, 169–180. Edgell, P., Lawseth, D., 1993. Use of salt solutions to control fungus Ž Saprolegnia sp.. infestations on salmon eggs. Prog. Fish Cult. 55, 48–52. Goicoechea, O., Enr’quez, R., Paredes, E., Molinari, E., 1999. Jaw and other skeletal deformities in Chilean farmed Atlantic salmon Ž Salmo salar .. Proceedings of the 9th International European Association of Fish Pathology Conference, Rhodes, Greece. p. 31cABSTRACT. Hall, B.K., Miyake, T., 1995. Divide, accumulate, differentiate: cell condensation in skeletal development. Int. J. Dev. Biol. 39, 81–893. Hickey, C.R., 1972. Common Abnormalities in Fishes: Their Causes and Effects. NY Ocean Science Laboratory, New York, 21 pp. Hughes, G.M., 1966. The dimensions of fish gills in relation to their function. J. Exp. Biol. 45, 177–195. Hughes, D., 1992. Lower jaw deformity in farmed Tasmanian Atlantic salmon Salmo salar ŽSalmoniformes, Teleostei.. Final report. Barriers and Breakthroughs. Papers from 1992 SALTAS Research and Development Seminar. SALTAS, Hobart, Tasmania, pp. 17–64. Hughes, D., 1993. Lower jaw deformity in farmed Tasmanian Atlantic salmon—when does the problem start? Paper presented at the Biennial Symposium on Applied SEM Imaging and Microanalysis February. 20 pp. Johnstone, R., McLay, H.A., Walsingham, M.V., 1991. Production and performance of triploid Atlantic salmon in Scotland. In: Pepper, V.A. ŽEd.., Proceedings of Atlantic Canada Workshop on Methods for the Production of Non-maturing Salmonids, Feb. 19–21, Dartmouth, Nova Scotia. Dept. Fisheries and Oceans, St. Johns, Newfoundland, Canada, pp. 15–33. Jungawalla, P., 1991. Production of non-maturing Atlantic salmon in Tasmania. In: Pepper, V.A. ŽEd.., Proceedings of the Atlantic Canada Workshop on Methods for the Production of Non-maturing Salmonids, Feb. 19–21, Dartmouth, Nova Scotia. Dept. Fisheries and Oceans, St. Johns, Newfoundland, Canada, pp. 47–71. King, H., Lee, P., 1993. Progress report: jaw deformity and respiratory physiology of triploids. Seeking and Solving: Papers from the SALTAS Research and Development Review Seminar. SALTAS, Wayatinah, Tasmania, Australia, pp. 37–44.
386
J. Sadler et al.r Aquaculture 198 (2001) 369–386
Koumoundourus, G., Gagliardi, F., Divanach, P., Boglione, C., Cataudella, S., Kentouri, M., 1997. Normal and abnormal osteological development of caudal fin in Sparus aurata L. fry. Aquaculture 149, 215–226. Lee, P., King, H., 1994. Effects of reduced dietary energy on the incidence of jaw deformity in Tasmanian Atlantic salmon. Reports from SALTAS Research and Development Programme. Salmon Enterprises of Tasmania, Wayatinah, Tasmania, pp. 61–69. McGeachy, S.A., O’Flynn, F.M., Benfey, T.J., Friars, G.W., 1996. Seawater performance of triploid Atlantic salmon in New Brunswick aquaculture. Bull. Aquat. Assoc. Can. 96, 1–5. McKay, L.R., Gjerde, B., 1986. Genetic variation for spinal deformity in Atlantic salmon Salmo salar. Aquaculture 52, 263–272. Norris, W.P., Chavia, W., Lambard, L.S., 1963. Studies on calcium metabolism in a marine teleost. Ann. N. Y. Acad. Sci. 109, 312–336. Ojolick, E.J., Cusak, R., Benfey, T.J., Kerr, S.R., 1995. Survival and growth of all-female diploid and triploid rainbow trout Ž Oncorhynchus mykiss . reared in chronic high temperature. Aquaculture 131, 177–187. Payan, P., Girard, J.P., MayerGostan, N., 1984. Branchial ion movements in teleosts: the roles of respiratory and chloride cells. In: Hoar, W.S., Randall, D.J. ŽEds.., Fish Physiology, vol. XB, Academic Press, Florida, pp. 39–60. Quigley, D.T.G., 1995. A lower jaw deformity in juvenile and adult Atlantic salmon Ž Salmo salar L... Bull. Eur. Assoc. Fish Pathol. 15, 206–209. Quillet, E., Gaignon, J.L., 1990. Thermal induction of gynogenesis and triploidy in Atlantic salmon Ž Salmo salar . and there potential interest for aquaculture. Aquaculture 89, 351–364. Quillet, E., Chervassus, B., Krieg, F., 1987. Characterisation of auto- and allo-triploid salmonids for rearing in seawater cages. In: Tiews, K. ŽEd.., Selection Hybridisation and Genetic Engineering in Aquaculture. Heenemann Verlag, Berlin, pp. 239–252. Sadler, J., Wells, R.M.G., Pankhurst, P.M., Pankhurst, N.W., 2000a. Blood oxygen transport, rheology and haematological responses to confinement stress in diploid and triploid Atlantic salmon Salmo salar. Aquaculture 184, 349–361. Sadler, J., Pankhurst, N.W., Pankhurst, P.M., King, H., 2000b. Physiological stress responses to confinement in diploid and triploid Atlantic salmon. J. Fish Biol. 56, 506–518. Stockard, C.R., 1921. Developmental rate and structural expression: an experimental study of twins, double monsters and single deformities, and the interaction among embryonic organs during their origin and development. Am. J. Anat. 28, 115–266. Sutterlin, A.M., Holder, J., Benfey, T.J., 1987. Early survival rates and subsequent morphological abnormalities in landlocked, anadramous and hybrid Žlandlocked=anadramous. diploid and triploid Atlantic salmon. Aquaculture 64, 157–164. Thomas, P., Morrison, R., 1995. A method to assess triploidy in swimup rainbow trout. Australas. Aquacult. 9, 61–63. Wendelaar Bonga, S.E., Flik, G., 1991. Calcium regulation in fish. In: Lahlou, B., Vitiello, P. ŽEds.., Aquaculture: Fundamental and Applied Research. American Geophysical Union, Washington DC, pp. 47–59.