Exploring the biological and socio-economic potential of new-emerging candidate fish species for the expansion of the European aquaculture industry – the DIVERSIFY project (EU FP7-GA603121)

by Rocio Robles, Dissemination Leader (CTAQUA, Spain), Constantinos C Mylonas, Project Coordinator (HCMR, Greece), Costas Tsigenopoulos, Reproduction & Genetics - pikeperch leader (HCMR, Greece), Ivar Lund, Nutrition - pikeperch leader (DTU, Denmark), Pascal Fontaine, Larval husbandry - pikeperch leader (UL, France), Patrick Kestemont, Grow out husbandry - pikeperch leader (FUNDP, Belgium)

DIVERSIFY project run between 2013 and 2018 and included six European finfish species (see April issue International Aquafeed). The combination of biological, technological and socioeconomic research activities developed in DIVERSIFY are expected to support the diversification of the EU aquaculture industry and help in expanding production, increasing aquaculture products and development of new markets.

Following the previous international Aquafeed issues on the project DIVERSIFY in which we presented the project achievements on halibut and meagre, this month we present the project results on pikeperch (Sander lucioperca).



Pikeperch, S. lucioperca, is a freshwater fish considered to have the highest potential in Europe for inland aquaculture diversification (Fig.1). Pikeperch flesh has a neutral taste lending to different forms of preparation. Moreover, the filets do not have bones --unlike carp, which competes on the same market segment. Year-round production of pikeperch requires production in RAS (Recirculation Aquaculture Systems). The RAS also allow to produce at high densities, 80-100 kg m-3. Recognized by a survey addressed to fish farmers, DIVERSIFY identified the major bottlenecks for further expansion of pikeperch culture today: (a) lack of knowledge of the genetic variability of the used broodstocks, (b) low larval survival (typically 5-10%); a high incidence of deformities, and (c) high sensitivity to stressors, handling and husbandry practices that result in high and sudden mortalities. All these bottlenecks have been addressed by the research carried out in DIVERSIFY.



Identification of genetic relationships among different broodstocks, inbreeding phenomena and loss of heterozygosity is important in aquaculture, since it may result in subsequent reproductive and productive failure (reduced progeny survival, growth, food conversion efficiency and increased frequency of deformities). It is also important to know how the domesticated stocks differ from their wild counterparts, which could potentially be a future source of fish to include in breeding programs. Overcoming the above bottlenecks is very important to reduce production costs and, therefore, expand the aquaculture production of pikeperch in the EU.

The first task of DIVERSIFY for pikeperch was to assess the genetic variability of captive broodstocks in commercial farms in Europe operating in RAS, and then compare this variability with that of wild populations. A total of 21 populations/broodstocks were sampled and analyzed, which included 13 captive broodstocks and eight wild origin populations. The results have indicated that some broodstocks have adequate genetic variation, but as some of them originate from few fish, attention should be paid in the future to establish breeding programmes. In general, there was agreement with the stock origin and our studies provided evidence that pikeperch populations in Europe are part of at least two genetically differentiated groups . The first group is found in northern Europe from the Netherlands/Denmark to the West, Poland (at least) to the East, and Finland to the North. The second group comprises all remaining populations in Central Europe to as south as Tunisia (and probably Spain, Italy and northern Greece). Based on this grouping, it can be stated that most analyzed populations seemed to contain fish of a single origin; nevertheless, in few domesticated populations this ratio varied from 5-19%, possibly due to the mixing of fish from multiple sources.



In the area of pikeperch nutrition, trials have shown that pikeperch larvae require both high dietary inclusion levels of phospholipids and essential Long Chain (LC) PUFAs to perform optimally. This requirement is unusual for freshwater fish larvae and is more commonly observed in marine species. Phospholipid level is generally low in dietary oils used in fish feeds, but some fish oils may have high concentrations. Phospholipids may be particularly important in fish larvae, as these lipids have an important function during larval development and are particularly present in larval brain and cellular membranes. Phospholipids may improve digestion and lipid feed utilization and have positive benefits in larval development. It was thus important to determine optimal phospholipid levels and levels of essential FA (EFA) in dry feeds for pike perch larvae on the performance and development.

Three dietary levels of phospholipids were tested in larval dry feed diets: Total phospholipid level ranged3.7 % ww (PL1), 8.2% ww (PL2) and 14.4 % ww (PL3) to evaluate their effect on larval growth and development. Additionally, supplementation of EFA in three other diets (PL1H1-PL3H3) was tested: 0.5 % ww PL1H1, 2 % ww PL2H2 and 3.4 % ww PL3H3. Larvae were fed the dry diets from 10 days until 30 days after hatching.

Results showed either a specific effect of the EFA, Ω-3 fatty acids or a combined effect of phospholipids and fatty acids. Combined supplementation of up to 14.5% phospholipids with EFA, Ω-3 fatty acids lead to the highest growth (Fig. 2) and lowest anomalies. Survival was much lower for larval groups reared on the lowest phospholipid level PL1 and PLH1. The highest phospholipid EFA level improved enzymatic activity in the larval digestive tract, which was likely due to a higher maturation of the gut followed by growth improvement. Several of the proteins expressed in the liver (which is the main metabolic organ in the body) such as FAS (fatty acid synthase) showed a marked increase, when larvae were fed low levels of EFA in the diets, suggesting a higher energy demand of these smallest larvae. An increase in dietary phospholipids from 3.7 up to 8.2% did not lower the incidence of skeletal deformities, but inclusion of 14.5 % phospholipids significantly reduced the incidence of severe skeletal anomalies, and was lowest in larvae fed 14.5 % phospholipids + EFA.

Combinations of nutritional requirements and husbandry rearing conditions during early ontogeny are poorly studied in pikeperch. The substitution of marine oils with vegetable oils has reduced stress tolerance and caused neurophysiological changes in pike perch larvae, but effects of environmental cues are limited. Saline water influences on a range of physiological functions during early fish larval ontogeny and may affect FA metabolism, so that larvae are better able to convert non-essential fatty acids to essential fatty acids and thus have less requirement for essential fatty acids provided by the food (Fig. 3).

Results of an experiment with pikeperch larvae fed different sources of non-essential fatty acids and reared at three different salinities (0, 5 and 10 ppt) showed that salinity had no effect on the growth performance of the larvae. Larvae possessed a marked specificity to incorporate and esterify essential Ω-3 fatty acids especially ARA (arachidonic acid), EPA (eicosapentanoic acid) and DHA (docosahexanoic acid) into lipids. Salinity had no effect on the ability of larvae to esterify and incorporate unsaturated PUFA precursors and thus to biosynthesize lipid classes containing essential fatty acids. A confinement stress test caused high acute mortality in all groups (50-70%), however significantly lowest for a control group given high levels of essential Ω-3 fatty acids. The prevalence of severe skeletal anomalies was generally high, affecting over 75% of the larval population with negative effects by increase in salinity.

It is recommended that essential Ω-3 fatty (EPA + DHA) must be supplied in diets of pikeperch larvae for normal development and to reduce stress sensitivity. The results pointed out a high occurrence of deformities and increased incidence at higher salinities.


Larval husbandry

Until now several bottlenecks have influenced the reduced the success of pikeperch larval rearing. Three major bottlenecks have been identified: (1) high mortality due mainly to cannibalism, (2) high rate of deformities and (3) a large size heterogeneity between larvae cohorts at various ontogenic development stages. Using a pilot scale larval rearing system (RAS, ten 700 L tanks, Fig. 4) and based on existing protocols used by the SMEs, successive experiments were conducted using factorial designs (4 factors tested with 8 experimental units) which are efficient methods to successfully optimize larval protocols. Such methodology allows (i) to integrate the effects of each simple factor tested and interactions between them, (ii) to rank and evaluate the effects induced by factors or interactions, (iii) to identify rapidly an optimal combination of factors that increase larval survival, and (iv) to establish a first modeling of the complex multifactorial determinism of output variables. This method has been already applied successfully in fish larviculture. Our objective was to study successively the effects of environmental, nutritional and population variables. For each experiment, the choice of these factors was a trade-off between data available in the literature and the constraints of our system (i.e. the impossibility for varying the temperature in each tank). From each experiment, according to results obtained, the most influential factors and modalities were conserved and integrated in the following experiment in order to optimize the protocol.

Effects of environmental factors: The effects of light intensity (5 or 50 lx), water renewal rate (50 or 100% per hour), water current direction (at the bottom or the surface of the tank) and time of tank cleaning (morning or afternoon) were studied. The multifactorial experimental design was based on the application of 8 combinations of factors. From the spawn of a domesticated broodstock 500,000 newly hatched larvae (<1 dph) were obtained from the SME Asialor (Pierrevillers, France). Then larvae were distributed into 8 tanks (62,500 per tank, 90 larvae L-1), where water temperature was initially kept at 15-16°C. Photoperiod was fixed at 12 h of light and 12 h of darkness with a progressive increase of light intensity (from 0 to 5 or 50 lx) from 07:30 to 08:00 and a decrease of light intensity (from 50 or 5 to 0 lx) from 19:30 to 20:00. Temperature was incrementally increasing by 1°C per day to 20°C. The frequency of feeding was a meal every 1.5 hours during the light period. Dissolved oxygen was maintained above 6 mg L-1.

In this experiment (39 days), it was demonstrated that weaned juveniles of 0.50±0.06 g mean body weight can be produced in 5 weeks, but survival rates (0.3-2.6%) were very low. Finally, it appears that a water inlet at the bottom of the tank is better to reduce size heterogeneity. Considering the growth results, we recommend to apply a light intensity of 50 lx, a water renewal rate of 100%, a cleaning of the tank during the afternoon and an inlet of the water at the bottom level. According to behavior, this first experiment allowed us to know that it is possible to determine the 'personality' on pikeperch juveniles and maybe highlight in a future experiment the link between personality and cannibalism.

Effects of nutritional factors: a second experiment (53 days) was done in order to evaluate the effects of four feeding factors: the timing of the beginning of weaning (at 10 or 16 dph), the method of food distribution (continuous or discontinuous during the lighting period), the implementing or not of a co-feeding approach (6 day before the weaning period) and the weaning duration (3 or 9 days). Larvae (240,000, 30,000 larvae per tank ca. 43 larvae L-1) were obtained from the SME Asialor (Pierrevillers, France). The results suggest, that a later onset and longer duration of weaning followed by discontinuous feeding will improve larval survival, growth and reduce deformities in pikeperch populations.

Effects of population factors: A third experiment (52 days) tested, the effects of the initial larvae density (50 or 100 larvae L-1), sorting out fish jumpers (yes or not), stocking sibling or not sibling larval group (larvae from one or two females) and female weight (< 2.8 kg or > 3.3 kg). Larvae (420,000) were obtained from the SARL Asialor (Pierrevillers, France) and transferred to the UL experimental platform (UR AFPA, Vandœuvre-lès-Nancy, France). Results obtained in the platform larval facilities suggest that the higher final biomass could be correlated to a higher initial larvae density (100 larvae L-1) and to the use of larvae supplied by bigger females, but independent of jumper sorting and the use of sibling population.


Identification of optimal combinations of factors

According to the best results obtained in the previous experiments, an optimal combination of factors (Table 1) was proposed to improve pikeperch larval rearing and tested in the same rearing system using 7 replicates (52 days).


Environmental conditions grow out

the area of grow out, the studies identified the optimal conditions for improving growth and welfare of pikeperch in aquaculture and characterized the effects of major husbandry and environmental factors on growth and physiological status of this species. a screening experiment, eight factors considered as relevant for the welfare of pikeperch were compared in two modalities using a fractional multifactorial design (28-4). Each experimental unit represented a combination of eight factors in two modalities which included grading, stocking density (15 vs 30 kg/m3), feed type (floating vs sinking), light intensity (10 vs 100 lux), light spectrum (red vs white), photoperiod (long vs short), dissolved oxygen (60 vs 90 %) and temperature (21 vs 26°C). Fish sampling occurred on days 36 and 63. Stress markers – glucose, cortisol and brain serotonergic activity – and changes in humoral immune activities and immune gene expression in kidney were assessed. Light intensity and the type of feed clearly appeared as directive factors for pikeperch culture (Fig. 5). The use of a sinking feed led to the best results in terms of final individual weight, the specific growth rate and the weight heterogeneity. High light intensity affected survival. The main influence on physiological and immune status was imposed by light characteristics, including intensity, spectrum and photoperiod, as well as temperature.

Pikeperch is sensitive to its light environment. Its preference for dark environments are explained by specific adaptations of its retina, including a tapetum lucidum that is a specific anatomo‐histological tissue which greatly amplifies the eye sensitivity to light. It was shown that light intensity and light colors can both affect vision of various fish species, affecting food intake, reproduction, growth and even survival. It is thus essential to maintain fish in optimal light environment. However, the effects of the light environment, including the light intensity and the light spectrum, on the physiology and immunity of pikeperch, and more generally of teleost, are poorly documented. And considering results from the multifactorial experiment, an in vivo experiment was performed in order to further validate and deepen the effects of the light intensity and light spectra on stress status, humoral innate immune response and expression profiles of immune-relevant genes in pikeperch.

A stock of 1000 pikeperch juveniles were distributed in 24 indoor 100‐L tanks of a recirculating aquaculture system. After an acclimation of 30 days in constant conditions (spectrum: white; light intensity at water surface: 10 lx; photoperiod: 12L(8:00‐20:00)/12D) new light conditions were applied, with six tanks per experimental condition: 10‐lx white; 10‐lx red; 100‐lx white; and 100‐lx red. Light intensity was measured at water surface and spectra included a white (industrial white—Osram, cool white 840 Lumilux) and a red color (red filter, 610 nm; Loomis). Samplings occurred during scotophase at 04:00 and photophase at 16:00, at both days 1 and 30. To avoid repetitive stressful events on fish and potential artifacts on results, 12 tanks (three per condition) were assigned at each time of sampling.

The results defined that the use of a high light intensity was followed by long-term stress and an immune suppression. Light spectrum has only little influences. In addition, results demonstrated that high stress status may have impacted melatonin production and secretion by the pineal organ. The drop in circulating melatonin and the increase in stress status may both be involved in the immune suppression.

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