Introduction
Challenges related to sea lice prevention and treatment remain major cost drivers in salmon farming (Abolofia et al., 2017). The deployment of cleaner fish, such as ballan wrasse (Labrus bergylta ), has been used as one of several strategies to prevent and control sea lice infestations. S keletal anomalies are amongst the most common challenges in farmed ballan wrasse (Fjelldal et al., 2021) compromising fish welfare by altering feeding and swimming behavior and increasing stress susceptibility (Boglione et al., 2013). These anomalies have multifactorial causes and are often linked to inadequate early nutrition ( Lall and Lewis-McCrea, 2007; Fang et al., 2024). During the weaning phase, it is therefore crucial to use appropriate diets tailored to the species’ nutritional needs. This study aimed to better understand the impact of early nutrition on growth performance, osteogenesis, and skeletal anomaly incidence in ballan wrasse larvae, using bone staining and comparative transcriptomic analyses.
Material and methods
Ballan wrasse larvae (Mowi Stord, Norway) at two days post-hatching (dph) were stocked at a density of 115 larvae.L-1 in 200 L tanks at SINTEF/ NTNU SeaLab (Trondheim, Norway). Larvae were reared with a 24L:0D photoperiod, oxygen saturation was maintained above 90 %, salinity at 34psu and temperature was gradually increased from 11.8 ± 0.2 to 15.7 ±0.1 °C. The experiment was conducted in triplicates with four experimental feeding regimes differing in the weaning onset . The live feed protocol included Acartia tonsa copepodites (NII-III, 160–210 μm) from 4 to 12 dph, rotifers (Brachionus ibericus ), enriched for 24 hours with Larviva Multigain (Biomar, Denmark), from 10 to 22 dph, and large barnacle nauplii (Cryo-L; Semibalanus balanoides NI, 320µm) from 18 to 44 dph. A formulated diet (WINWrasse, SPAROS) was introduced at 5, 17, 24 and 32 dph for Protocols 1, 2, 3 and 4 , respectively. From 45 dph onwards, larvae were fed exclusively with the formulated diet. Larvae were sampled from each tank at 4, 16, 23, 31, 45 and 56 dph to determine standard length (SL), dry weight (DW), carbon and nitrogen content (CN), specific growth rate (SGR) and daily weight increase (%DWI). For gene expression analysis, 5-10 larvae per tank were collected at 16, 23, 31, 45, and 56 dph for sequencing. For the analyses the R package glmmSeq (Rivellese et al., 2022) was used to fit negative binomial mixed-effects models at the individual gene level. Additionally, 6–10 larvae per tank were sampled at 31, 45, and 56 dph for assessment of osteogenesis and skeletal anomalies, using a modified double-staining method (Gavaia et al., 2000) and an adapted scoring system (Sæle et al., 2004; Sørøy, 2012; Sæle et al., 2017).
All procedures complied with the Norwegian Animal Welfare Act of 20 December 1974, No. 73 (Sections 20–22, amended 19 June 2009). The experiment was approved by the Norwegian Food Safety Authority (FOTS ID 30573, reference number 23/243983).
Results and Discussion
At 56 dph, significant differences were observed in DW between Protocols 3 and 4 (p<0.05; One-way ANOVA) . A notable peak in mortality occurred across all groups between 21– 26 dph, with Protocol 1 showing significantly higher cumulative mortality than Protocol 2 at 24 dph (p<0.05; One-way ANOVA). Growth patterns up to 30 dph were consistent with previous studies (Sørøy 2012; Høyland 2015) and both growth and mortality followed the tendency described by Malzahn et al. (2022) for a group fed copepod and Cirripedia nauplii. Possible contributing factors to the mortality peak may include appetite fluctuations, environmental conditions, and pathogen presence.
Urostyle flexion was observed from 6.3 ± 0.10 mm SL and fully ossified vertebrae in larvae measuring 7 mm SL, which was consistent with previous studies in ballan wrasse larvae (Sørøy 2012; Høyland 2015; Malzahn et al., 2022). Protocol 4 had significantly more fully ossified vertebrae (p < 0.05; One-way ANOVA ) compared to P rotocols 1 and 2. At 45 dph, the most common anomaly in P rotocols 2 and 3 was abnormal arches and/or spines (A/S) and lordosis was most prevalent in P rotocol 1 (41.1%). In Protocol 4, twisted spines was the most frequent anomaly (30.3%) at 45 dph and increased across all Protocols by 56 dph. This increasing trend aligns with previous studies (Sørøy 2012; Høyland 2015), suggesting that twisted spines develop in later stages of ossification. The higher prevalence of this anomaly in Protocol 4 at 45 dph may be linked to its more advanced vertebral ossification at that time point. While anomalies in arches and spines are often not considered severe due to their minimal impact on external morphology, severe cases of spine anomalies can negatively impact fish performance.
Transcriptomic analysis revealed a significant upregulation of genes involved in the oxidative stress response, particularly in P rotocol 1. Similar trends were observed for genes involved in the activation and response of the immune system against microbial infections, innate and adaptive immune response and stress resistance. We hypothesize that the introduction of formulated diets altered the bacterial load and community composition, thereby activating antibacterial immune responses.
Conclusion
In conclusion, the results of this trial emphasize the potential for refining rearing protocols for ballan wrasse larvae by incorporating advances in nutrition, environmental management, and developmental biology to enhance survival, growth, and skeletal development. Furthermore, we highlight the importance of better understanding ossification types and skeletal anomaly prevalence and integrating this knowledge with improvements in early-life-stage nutrition and longitudinal studies.
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