Growth and fatty acid composition of pikeperch (Sander lucioperca L., 1758) larvae under altered feeding protocol including the copepod Apocyclops panamensis (Marsh, 1913)

Alternative live feeds for small and sensitive fish early life stages such as pikeperch (Sander lucioperca L., 1758) can improve the larval quantity, quality and performance in aquaculture. Therefore, this study evaluated the cyclopoid copepod Apocyclops panamensis (Marsh, 1913) as live feed for pikeperch larviculture from day 11 post hatch (dph) in two independent experiments. In both experiments, pikeperch larvae had the highest specific growth rate (SGR) when they fed on Brachionus plicatilis until dph 11 and A. panamensis until dph 16–18. SGR was related to a decrease in total fatty acids (FAs), saturated FAs and monounsaturated FAs in pikeperch larvae, indicating their use as energy for growth. Within the polyunsaturated FAs, docosahexaenoic acid (DHA) increased in larvae fed with A. panamensis and coincided with the highest SGR suggesting that DHA is accumulated in larvae as structural FA. Our study demonstrated a suitable pikeperch larval fatty acid composition for growth after feeding A. panamensis compared with Artemia sp. from dph 11 until dph 16 and previously fed with B. plicatilis. Moreover, it highlighted the importance of the dietary PUFAs in pikeperch rearing, specifically of linoleic acid (LA) from dph 4 until dph 11 and of DHA from dph 11 onwards.

Freshwater copepods are part of the natural diet of pikeperch larvae and thus, might fulfil the nutritional requirements.They have a higher nutritional value than rotifers and Artemia spp.due to their high natural amounts of PUFAs, free amino acids and antioxidant pigments 16 .For these reasons, the use of copepods in aquaculture has increased.Copepods are popular for ornamental fishes 17 and have shown promising results for halibut larvae (Hippoglossus hippoglossus) 18 , winter flounder larvae (Pseudopleuronectes americanus) 19 , Atlantic cod (Gadus morhua) 20 , fat snook (Centropomus parallelus) 21 and ballan wrasse (Labrus bergylta) 15 .Despite some copepods having high-PUFA content with low-PUFA diets 22,23 for some copepods, it is essential to provide high-PUFA diets since enrichment techniques are not appropriate 10 .
Ballesteros-Redondo et al. 24 evaluated the potential of Apocyclops panamensis (Marsh, 1913) as live feed for larviculture.When A. panamensis was fed with Isochrysis galbana at 0.5-1 10 5 cells mL -1 per day, copepod culture seemed to be adequate in terms of their fatty acid composition (1.8-2.6% of DHA and DHA/EPA ratio of 2.5-2.9) to rear fish larvae 25 .However, A. panamensis had no advantage for pikeperch larvae between dph 4 -10 in comparison to B. plicatilis 4 .Peterka et al. 24 found nauplii of cyclopoid copepods in the stomach of pikeperch larvae, and El Kertaoui et al. 11 reported a need of 3.5% of EPA + DHA for pikeperch larvae, which coincides with the fatty acid composition of A. panamensis 24 .The authors hypothesized that A. panamensis is an adequate live feed organism as a second live feed organism following the application of rotifers and improving the fatty acid composition.The present study evaluates the effect of A. panamensis on pikeperch larval growth and fatty acids composition between dph 11-18 after fed with B. plicatilis or Artemia sp. from dph 4 to dph 10 and compares it with the use of Artemia sp. between dph 11-16.

Live feed
Zooplankton as well as microalgae were obtained from Aquacopa GmbH, Jabel, Germany, and were cultivated at the facilities of the University of Rostock.According to Ferreira et al. 27 , Brachionus plicatilis (Müller 1786) was fed with Nannochloropsis sp.and, according to Ballesteros-Redondo et al. 24 , Apocyclops panamensis was fed with Isochrysis galbana.Artemia eggs (ArtemioPur, JBL GmbH & Co. KG, Germany) were hatched and a maximum of 24 h old Artemia nauplii was fed to the larvae.The density of each zooplankton culture was measured daily to harvest the amount needed to feed the pikeperch larvae (see experimental diets below).Besides that, three samples of B. plicatilis (67,500 individuals per sample) and A. panamensis (210,000 individuals per sample) were taken 24 h after the last supply of microalgae.Furthermore, three samples of recently hatched Artemia sp. were collected (40,000 individual per sample).To collect the individuals of each zooplankton organism, each culture was filtered through a 50 µm net, the organisms were collected with a minimum of water content in glass vials for subsequent lyophilisation.Afterward, samples were weighed and an amount between 1.3 and 1.9 mg dry weight (DW) of each sample was taken for fatty acid analyses.One mg DW for B. plicatilis corresponded to 1048 ± 186 individuals (ind.), for Artemia sp.296 ± 36 ind.and for A. panamensis 4369 ± 533.

Experimental set-up
Two independent experiments took place in October 2020 and March 2022.
The first experiment (E1) was performed with fertilized pikeperch eggs from INAGRO, Belgium, transported cooled (< 10 °C) and brought to the experimental facilities of the University of Rostock, Germany.Upon arrival, the temperature was slowly raised and at a water temperature of 12 °C the eggs were transferred to an incubator.Within the next 48 h, the water temperature was continuously increased until 14 °C was reached.Three days after the transfer to the incubator, pikeperch larvae hatched.Larvae hatched within 24 h were stocked in 43 L tanks at 16 °C.Pikeperch larvae were maintained in a recirculating aquaculture system (RAS), including water treatment (mechanical and biological filtration as well as UV light treatment) under a light regime of 16L:8D, salinity of 0ppt, constant temperature and oxygen concentration.Two tanks with 43 L contained larvae at a density of 50 individuals per litre.While one tank was fed with B. plicatilis according to Ballesteros-Redondo et al. 4 , the other tank was fed with Artemia sp.(ArtemioPur, JBL GmbH & Co. KG, Germany).Both were fed from 4-10 dph three times per day (09:00, 12:00 and 15:00).Sixteen floating sub-units of 1 L were operated in parallel in two further 43 L tanks in the same recirculation system, arranged in four groups (each 4 replicates).On dph 10 after the last feeding time, larvae were stocked into the sub-units.Eight sub-units contained larvae fed previously with B. plicatilis and the other 8 sub-units larvae fed previously with Artemia sp.Four sub-units each were stocked with 25 larvae L -1 , which were fed with 600 A. panamensis ind.larva -1 day -1 , and the other sub-units with 50 larvae L -1 and fed with 300 ind.larva -1 day -1 from dph 11 until dph 18 (Fig. 1).
For the second experiment (E2) pikeperch larvae were obtained directly from the Pikeperch facility of the Mecklenburg-Vorpommern Research Centre for Agriculture and Fisheries in Hohen Wangelin (Mecklenburg-Western Pomerania, Germany).Larvae were transported at 15ºC to the experimental facilities of the University of Rostock at an age of 0dph and stocked into the experimental tanks at 16ºC.Pikeperch larvae were maintained in the same RAS as during the first experiment.Larvae were stocked at a density of 50 larvae L -1 and fed 340 B. plicatilis larva -1 day -1 from dph 4 until dph 10.After last feeding at dph10, again 50 larvae L -1 were stocked in 6 floating sub-units.As mortality increased during the first experiment from dph 16, in the second experiment the larvae were fed from dph 11 until dph 16 in two groups, one with A. panamensis and the other with Artemia sp. at 340 ind.larva -1 day -1 (Fig. 1).The period from dph 0-10 was analysed in E1 to monitor the effect of the first feeding period on the second period (dph 11-18) and in E2 to have the reference to compare with E1.
In addition, we performed daily siphoning of the bottom and removal of the dirt, dust and lipid layer at the water surface.Survival rate (E1 N = 4 and E2 N = 3) was calculated from dph 11, when the feeding with A. panamensis started, by counting recorded dead larvae from siphoning every day as follows: where N i is the initial number of larvae and T D the total number of dead larvae found, cumulated over the experimental days.
All methods were carried out in accordance with German guidelines and regulations.
The experiments with pikeperch larvae were conducted within the German Animal Welfare Act guidelines and were approved by the authorities, in our case the Mecklenburg-Western Pomerania State Office for Agriculture, Food Safety and Fisheries, based in Rostock.The authors complied with the ARRIVE guidelines.According to the animal experiment permit issued (permission number 7221.3-1.1-051/19), in E1, 30 larvae were taken at random at dph 0 and dph 11 in each treatment.At dph 18, 7 larvae from each replicate were taken.In E2, 30 larvae were taken at random at dph 0, 4 and 11.At dph 16, 7 larvae from each replicate were taken.In both experiments, larvae were first cooled down until 12 °C, and subsequently until 0 °C to anesthetize them.Pictures for measurements were taken and larvae were killed by cutting the spinal cord and frozen for fatty acid analyses.
The total body length as well as yolk sac and oil droplet sizes were measured in both experiments under a stereo light microscope (SZX10 Olympus, Hamburg, Germany) connected to a UC30 digital camera (Olympus, Hamburg, Germany) and the software package cellSens Dimension 1.6 (Olympus Soft Imaging Solutions, Hamburg, Germany).Yolk sac volume and oil droplet volume were calculated according to Bischoff et al. (2018).Finally, we calculated the specific growth rate (SGR) [% d -1 ] (E1 N = 4, E2 N = 3) as follows assuming linear growth 4, 28 : where L t and Lo represent the average length of the larvae at time t and time t = 0.
Fatty acid analyses of zooplankton as well as pikeperch larvae were performed at Greifswald University, in the Laboratory of Animal Ecology.The freeze-dried samples were transferred to extraction tubes, and dichloromethane:methanol (2:1, v:v) and nonadecanoic acid methyl ester as an internal standard was added to the samples.After ultrasonic treatment for > 5 s samples were kept under a nitrogen atmosphere at -25 °C until further analysis, which was done according to Wacker et al. 29 .Fatty acids were transesterified into fatty acid methyl esters (FAMEs) with methanolic HCl (Sigma-Aldrich Chemie, Taufkirchen, Germany) 3031 and FAMEs were analysed by gas chromatography (6890N, Agilent Technologies, Böblingen, Germany) with helium as carrier gas 32 .For verification, mass spectra were recorded using a gas chromatograph-mass spectrometer (Pegasus 4D GC-TOFMS, LECO Instruments, Mönchengladbach, Germany).
Statistical analyses were performed by using the software IBM SPSS Statistics, Version 27.Normal distribution was tested using the Shapiro-Wilk Test.To analyse differences between means, Analysis of Variance (oneway ANOVA) or t-Test for independent samples was applied when normality was proven.Without normality, (1) www.nature.com/scientificreports/ the Kruskal-Wallis or Mann-Whitney U test was applied.All significance levels α were set to 0.05.Data was reported as mean ± s.d.

Discussion
Our study showed that A. panamensis is suitable as live feed for the rearing pikeperch larvae from dph 11 until dph 16.According to Ballesteros-Redondo et al. 4 , B. plicatilis was a suitable live feed for pikeperch larvae from dph 4 until dph 10.In the present study (E1), pikeperch larvae fed initially with B. plicatilis and followed by A. Vol:.( 1234567890) ).These SGRs of both treatments were higher than in Imentai et al. 33 (1.37% d -1 ) and in experiment 1 of Ballesteros-Redondo et al. 4 .Our SGR of the larvae first fed with B. plicatilis and subsequently with A. panamensis exceeded the so far highest SGR of 3.0% d -1 when feeding solely 340 B. plicatilis per larva per day 4 .Consequently, the pikeperch larvae in the present study performed best in comparison to earlier studies and life feed combinations during this early life cycle stage.With the increasing growth, energy and nutrient requirements of the pikeperch larvae from dph 11 onwards, larvae fed with the copepod A. panamensis had the highest survival rates of 72% and 82% until dph 16 (E1) for protocols B + Apo300 and B + Apo600, respectively.However, the mortality drastically increased from dph 16.This might indicate that A. panamensis should be best used until dph 16 (for a five-days feeding period).However, the mortality increase might be caused by the start of cannibalism 34 .On dph 18, after feeding A. panamensis, the longest size was achieved in protocol B + Apo300 (7.13 mm).Nevertheless, larval length is higher in other studies 2,33 .Difference in breeders, genetics or initial larval quality makes the larval length on dph 18 difficult to compare.Thus, future studies should include more parameters such as initial fatty acid composition and larval weight 28 .Despite this, the SGR from dph 11 until dph 18 was lower for B + Apo300 and B + Apo600 compared with Art + Apo300 and Art + Apo600, which suggests a better larval development despite the initial supply with Artemia sp.This result indicated that larvae previously fed with Artemia sp.during the first 10 days were able to compensate a slower growth from dph 0 to dph 11 by feeding with A. panamensis afterwards.Nevertheless, B + Apo300 had a high SGR of 4.33% d -1 (dph 11-18), even higher than SGR data by Ballesteros-Redondo et al. 4 and Imentai et al. 35 .Therefore, A. panamensis supplied an adequate level of energy and nutrient supply for the pikeperch larvae and was consequently well suitable to obtain an adequate larval development, independent of the feeding protocol B + Apo300, B + Apo600, Art + Apo300, and Art + Apo600.Nevertheless, our data showed the importance of including the growth data for the different live feeds since similar results with different initial live feeds might make the growth process of the larvae up.
Despite a suitable B. plicatilis supply and adequate water quality in E2, the larval growth was low during the first few days.On dph 11, the total body length (5.47 mm) was still similar to the initial length (5.17 mm).The total FA contents until dph 11 almost did not decrease as in normally developing larvae or in starving larvae 1 .The larvae did not consume their FA reserves (fed on Brachionus) while the growth and survival rates were still low.However, from dph 11 onwards, the use of A. panamensis significantly improved larval performance compared to the use of Artemia sp.On dph 16, the survival rate for B + Art was slightly higher (94.0%) than in B + Apo (87.9%), the latter similar to B + Apo600 in E1 (82%).These survival rates were higher than in Imentai et al. 5 , who reported survival rates of 35 -68% on dph 16, and Yanes-Roca et al. 2 , who reported survival rates of 35-75% on dph 21.However, our calculated survival rates only considered dph 11 onwards to study the effect of A. panamensis as a live feed.Therefore, larval survival and growth rates should be reported at the change of live feed organism.On dph 16, the larvae fed with B + Art were smaller compared with B + Apo and thus, the SGR was significantly higher in B + Apo (2.95% d -1 ) compared with B + Art (1.32% d -1 ).The SGR for B + Apo was also higher compared with Imentai et al. 5 , who fed pikeperch larvae with different combinations of B. plicatilis and/or Artemia sp.They reported the maximum SGR of 2.41% d -1 between dph 11-17 (according to our own calculations).Consequently, our larval growth data demonstrate that A. panamensis had a distinctly positive effect on the growth of pikeperch larvae between dph 11 and dph 16 in comparison to Artemia sp. as live feed.
The applied B. plicatilis had lower total FAs and PUFAs contents than Artemia sp. per dry weight (Table 1).Consequently, the pikeperch larvae fed with B. plicatilis showed a lower amount in total FAs and PUFA contents than the larvae fed with Artemia sp.However, the highest SGR was found for the larvae fed with B. plicatilis, which especially contained a higher amount of LA than Artemia sp.(Table 1).LA seems to be a highly relevant FA in the diet for pikeperch, as suggested by Ballesteros-Redondo et al. 4 , Bischoff and Kubitz et al. 3 and Yanes-Roca et al. 2 .Yanes-Roca et al. 36 stated that pikeperch larvae might have the capacity to desaturate and elongate fatty acids with 18 carbons like LA to obtain DHA during the first 12 days of life.However, Reis et al. 37 demonstrated that pikeperch larvae cannot biosynthesize DHA at dph 20.Recently, Perez and Reis et al. 38 have shown that B. plicatilis esterifies C18 PUFAs into phospholipids.An increase in dietary polar lipids increased the growth rate of pikeperch and showed earlier digestive structure development 12 .Phospholipids in the diet might contribute to a better absorption and transport of long-chain fatty acids through enhanced lipoprotein synthesis 40 .This is supported by the fact that total FAs, MUFAs, PUFAs, EPA and DHA contents are lower in larvae fed with B. plicatilis which, at the same time, had the highest SGR, demonstrating that all these groups of FAs might have been used for growth and that the LA possibly as polar lipid plays a crucial role during these first days of larval development.Our results showed the importance of including the study of the lipids in form of neutral and polar lipids.Thereby growing larvae use up their larval storages from the yolk sac.With all their PUFA lipid storages, and by growing and increasing their body weight and by producing and accumulating non-lipid biomass, the relative content of PUFA decreases.In contrast, the slower-growing larvae (after feeding Artemia sp.) might just use up energy (carbohydrates and SFA) without growing due to a less appropriate diet thus, increasing their proportion of MUFAs and PUFAs.This suggests that B. plicatilis might have a suitable fatty acid composition in the adequate form of polar lipids for pikeperch larvae.
From dph 11 to dph 18 (E1), there was a higher decrease in PUFAs in larvae fed with more PUFAs Art + Apo (10.7%) than fed with B + Apo (2.7%).The use of B. plicatilis might have improved the absorption of the LC-PUFAs by the larvae.We therefore suggest that the first live feed might affect the future success of the larvae development although this effect was not shown by an improved SGR based on the larval length.This result highlighted the importance of measuring the survival and larval growth when changing live feed organisms.Moreover, including larval weight in future studies might allow us to detect the effect of the first live feed on the larval growth.There was an increase in DHA for all treatments in E1 after feeding A. panamensis. A. panamensis is characterised by its higher content of DHA in comparison with B. plicatilis and Artemia sp.(Table 1).This higher DHA content has already been reported in copepods 41 and in particular for Apocyclops species (for A. royi 23,42,43 and for A. panamensis 24 ).Therefore, our data demonstrate that pikeperch larvae could ingest and digest A. panamensis, and consequently could utilize the supplied nutrients.This underlined the possibility of rearing pikeperch larvae from dph 11 until dph 18 with this marine copepod species.However, since saltwater copepods do not survive long in freshwater, freshwater copepod species should be studied since they survive longer and might supply suitable nutrient composition to the freshwater fish species 3 .
A. panamensis also seemed to fulfill the nutritional requirements of the pikeperch larvae after first feeding on B. plicatilis better than feeding Artemia sp.In E2, the total FA concentrations, SFAs and MUFAs decreased more in B + Apo between dph 11 and dph 16, coinciding with a higher growth.When fish larvae grow, they require more energy.Both groups of fatty acids are used through the ß-oxidation to obtain adenosine triphosphate (ATP).This suggests that the pikeperch larvae used these groups of FAs as energy for growth.However, PUFAs decreased more in larvae fed with Artemia sp., which had a higher content of PUFAs than A. panamensis in our study (Table 1).This allows the conclusion that the PUFAs profile of Artemia sp.lacks important single fatty acids and that the FAs provided through A. panamensis were used.Although the total PUFAs decreased for both protocols, DHA only decreased in larvae fed with Artemia sp. as also shown by Yanes-Roca et al. 36 .The pikeperch fed with A. panamensis instead increased their DHA content.While some FAs might have been used as energy for growth, in larvae fed with A. panamensis DHA accumulated, which is an important component used in fish retina 39 .Consequently, we demonstrate a better pikeperch larval fatty acid composition after feeding with A. panamensis compared with Artemia sp.Although Artemia sp. has more EPA, the larvae fed with B + Art increased their EPA content less than the larvae fed with B + Apo.This shows that the incorporation of these nutrients is more efficient when feeding A. panamensis.As mentioned before, phospholipids may enhance lipoprotein synthesis that improves absorption and transport of long-chain fatty acids 40 .Higher content of phospholipids in copepods compared to Artemia spp. 41might explain a better incorporation of EPA by the pikeperch larvae in our study.Consequently, our data demonstrate an improvement in pikeperch larviculture by the use of A. panamensis compared to Artemia sp.
Our pikeperch larvae kept during the experiments a minimum amount of 120 µg total FAs, 20 µg SFAs, 30 -40 µg MUFAs, 60 µg PUFAs, 4 µg EPA and 20 µg DHA per mg DW, and is definitively higher than those reported in starving larvae 1 .Furthermore, the content of PUFAs in our experiments was higher than those reported by Ballesteros-Redondo et al. 4 and by Bischoff and Kubitz et al. 3 , which also reported lower SGR although the initial PUFAs contents were higher.Consequently, our study highlights the importance of the dietary PUFAs in pikeperch rearing, specifically of LA, from dph 4 until dph 11 and of DHA from dph 11.
It must be considered that the better performance of pikeperch larvae with A. panamensis occurred during the 5 consecutive days after an initial 10 days B. plicatilis feeding.This suggests an adequate timing and availability of both live feed organisms, making larviculture of pikeperch more complex.The high cost of copepod production is another constrain to be considered by the pikeperch hatcheries.Therefore, the economic viability and production efficiency of the combined Brachionus sp. and A. panamensis use must be further assessed.
Nevertheless, a more favourable dietary fatty acid composition will allow fish larvae to reach higher growth rates and thus allow the larvae to feed earlier and with less effort on bigger prey such as small fish.These other fish as prey items will then perfectly fit the nutritional requirements of the fish.

Figure 2 .
Figure 2. Total body length ± s.d.(mm) at the different days post hatch (dph) in experiment 1 (E1) and in experiment 2 (E2) with the different feeding protocols.Lines until dph 11 represent initial diet and different colour are different live feeds.Bifurcation at dph 11 shows change in diet.From dph 11, different colours are different live feeds and, in E1, continues or discontinuous lines are different quantities of feed.

Figure 3 .
Figure 3. Fatty acids main group's dynamics during both experiments (E1 and E2) under different feeding protocols (mean ± s.d.).For E2, black line until dph 11 is B. plicatilis diet.Bifurcation at dph 11 shows the change in diet.