Investigations of skeletal anomalies in triploid Atlantic salmon (Salmo salar L. 1758) in freshwater : with particular focus on lower jaw deformity (LJD)

Triploid Atlantic salmon are a valuable part of production in Tasmania. In fact, not undergoing sexual maturation, triploids are used to ensure appropriately sized harvest fish all year round. Nevertheless, triploids tend to develop a higher prevalence of skeletal anomalies than diploids affecting mainly the lower jaw and the vertebral column and impacting fish performance, welfare and value deleteriously. The general intent of this PhD research project was to make a useful contribution to Atlantic salmon industry in order to understand and find a possible solution or mitigate the problem of skeletal anomalies of obvious relevance to economic efficiency as well as animal welfare in Tasmania and worldwide. This research explored the occurrence of several skeletal anomalies in farmed diploid and triploid Atlantic salmon in freshwater that represent a critical phase in development. The aims were to improve our knowledge on occurrence and causative factors of skeletal anomalies in triploids (and diploids) and in particular of lower jaw deformity (LJD), a skeletal anomaly mainly affecting triploid Atlantic salmon, and to understand the molecular mechanisms underlying it. Two experiments (the first containing two sub-experiments) and an extensive molecular investigation have been undertaken during this PhD research project in order to produce valuable information and fulfil the predetermined aims. In the first experiment, diploids and triploids were maintained in controlled conditions for nine months (from incubation to 60 g) and repeatedly screened (visually and by x-ray) for the occurrence of several categories of skeletal anomalies (i.e. lower jaw anomalies, opercular shortening, spinal anomalies) and the effect of standard and elevated temperature regimes (14 and 18 °C) on prevalence of skeletal anomalies was tested in the pre-smolt stage (8-60 g). This experiment showed that LJD started to be displayed at 8-10 g and the prevalence increased only in triploids up to 11% at 60 g implying that occurrence can be at any time in development. In diploids, LJD prevalence was approaching to zero throughout the experiment. A high prevalence of opercular shortening was observed (average prevalence in both ploidies 85.8%) and shortened lower jaw (SJ) was common in both ploidies (highest prevalence observed 11%). In triploids, the change in prevalence over time of LJD and SJ indicated a possible developmental link between the two jaw anomalies. No effect of temperature was observed on prevalence of lower jaw skeletal anomalies. A radiological assessment (n = 240 individuals) showed that at both temperature regimes tested triploids had a significantly (P < 0.05) lower number of vertebrae and higher prevalence of deformed individuals and that elevated temperature resulted in more regions of the vertebral column affected irrespective of the ploidy. Our findings confirmed that LJD occurs predominantly in triploids and at any time during freshwater phase and overall ploidy was more influential than temperature on development of skeletal anomalies. The second experiment, developed following the results of the first experiment, focused on LJD developmental patterns in triploids and LJD concurrence with another lower jaw anomaly affecting Atlantic salmon, shortened lower jaw (SJ). Triploid individuals (~12 g) only were collected and selected at a commercial hatchery, allocated into two separate groups according to their lower jaw condition (normal -NOR- or affected by SJ -SJ-), grown out in controlled conditions and assessed (visually and by image analysis) over three months for concurrence of SJ and LJD. The main aim of the experiment was to verify concurrence of lower jaw skeletal anomalies in the two groups and understand through their developmental patterns independency or dependency of the conditions. Furthermore, effect of lower jaw anomalies on growth and recovery from opercular shortening was tested. Prevalence of jaw anomalies increased in both groups over time (NOR group 0 to 24.5%; SJ group 17 to 31%). SJ and LJD occurred both independently and concurrently whereas another lower jaw anomaly, misaligned lower jaw (MA), concurred exclusively with them. All three anomalies could be concurrent. Severity of both LJD and SJ increased in the SJ group only. Opercular shortening recovery was observed in both groups but at a slower rate in the SJ group. The SJ group specific growth rate (SGR) was significantly (P < 0.05) lower than the NOR group. This study demonstrated the concurrence of SJ, LJD (and MA) in triploids and showed possible deleterious consequences deriving from the condition/s. The last research chapter, describes the first molecular analysis ever undertaken on fish affected by LJD. Two independent sample sets of triploid Atlantic salmon, experiment-sourced (60 g) (from the first experiment) and commercially produced (100 g) displaying LJD and with a normal lower jaw were used to delineate multigenicity of the condition. The main aim was to investigate, by using transcriptome analysis, real-time qPCR and in-situ hybridization, molecular mechanisms underlying the condition and to detect possible candidate genes providing clues on tissue/s responsible for the trait. The use of two independent sample sets at different developmental stages helped to strengthen the outcome of the analyses and verify differential gene expression consistency. The effect of temperature on gene regulation was also tested. A total of eleven genes, some detected through transcriptome analysis (fbn2, gal and gphb5) and others known to be related to skeletal physiology (alp, bmp4, col1a1, col2a1, fgf23, igf1, mmp13, ocn), were tested in the two independent sample sets. Gphb5, a recently discovered hormone, was significantly (P < 0.05) down-regulated in LJD affected fish in both sample sets suggesting a possible hormonal involvement. In-situ hybridization detected gphb5 expression in oral epithelium, teeth and skin of the lower jaw. Col2a1 showed the same consistent significant (P < 0.05) down-regulation in LJD suggesting a possible cartilaginous impairment as a distinctive feature of the condition. Significant (P < 0.05) differential expression of other genes found in either one or the other sample set highlighted the possible effect of stage of development or condition progression on transcription and showed that anomalous bone development, likely driven by cartilage impairment, is more evident at larger fish sizes. The present study improved our understanding of LJD suggesting that a cartilage impairment likely underlies the condition and col2a1 may be a marker. In addition, the involvement of gphb5 urges further investigation of a hormonal role in LJD and skeletal physiology in general.