Interaction between Dietary Vitamin D3 and Vitamin K3 in Gilthead Seabream Larvae (Sparus aurata) in Relation to Growth and Expression of Bone Development-Related Genes

Vitamins D and K are essential fat-soluble nutrients that intervene in bone development processes among other biological functions. The present study is aimed at investigating the potential combined effect of dietary supplementation with vitamin D3 (cholecalciferol) and vitamin K3 (menadione) in gilthead seabream (Sparus aurata) larvae. For that purpose, seabream diets were supplemented with different combinations of vitamin D3/vitamin K3 (mg/kg diet) as follows: 0.00/0, 0.06/70, 0.06/170, 0.13/70, 0.13/170, 0.40/70, and 0.40/170. Feeding gilthead seabream larvae (22 days post hatch) for 21 days with the diets supplemented with 0.06-0.13 mg/kg vitamin D3 and 70 mg/kg vitamin K3 (diets 0.06/70 and 0.13/70) led to the highest larval growth and survival and the highest expression of important biomarkers of both bone development and health, such as bmp2, osx, and mgp, and calcium homeostasis, such as pthrp and casr. However, the increased supplementation with both vitamins at 0.40 mg/kg vitamin D3 and 170 mg/kg vitamin K3 (diet 0.40/170) reduced larval growth and survival, downregulated bmp2 and pthrp expressions, and upregulated osx and mgp, causing an unbalance in the relative expression of these genes. The results of the present study have shown the interaction between vitamin D3 supplementation and vitamin K3 supplementation in larval performance and gene expression related to bone development and calcium homeostasis, denoting the significance of a correct balance between both vitamins in larval diets.


Introduction
Culturing marine fish larvae at commercial hatcheries is a demanding task, given their high sensitivity to biotic and abiotic factors [1]. Skeletal anomalies in larvae are more likely to develop during embryonic and postembryonic development, making it crucial to ensure proper nutrition for the larvae [2][3][4][5]. Micronutrients play a critical role in ensuring proper nutrition, but imbalances can lead to skeletal anomalies and affect larval performance during rearing [5][6][7][8]. Therefore, feed plays a significant role in larval performance, and the nutritional requirements for marine fish larvae differ from those of juveniles and broodstock [9,10].
The larval stage is marked by numerous morphological and physiological changes that may affect nutrient requirements [11], which also vary across species [10,12,13]. Hatcheries typically feed larvae live prey, such as rotifers and Artemia, which have varying nutritional compositions and may lack some essential vitamins and minerals [11,12,[14][15][16].
Among the different nutrients studied, fat-soluble vitamins D and K play crucial roles in the skeletal development of aquatic animals [17]. Vitamin D is vital for regulating plasma calcium and maintaining bone health, as well as for receptor-based biological functions in various tissues such as the gills, kidney, and intestine [18]. Vitamin K is essential for blood coagulation and the posttranslational modification of vitamin K-dependent proteins, which play critical roles in bone metabolism and growth control [19]. In juvenile fish studies, replacing fishmeal and fish oil with terrestrial ingredients in feed may decrease the dietary content of these vitamins, underscoring the need to establish their optimal dietary levels [20]. Several marine fish species have proposed individual dietary requirements for both vitamins, including Atlantic salmon (Salmo salar) postsmolt, Wuchang bream (Megalobrama amblycephala) fingerling, orange-spotted grouper (Epinephelus coioides) juveniles, and gilthead seabream (Sparus aurata) juveniles [18,[21][22][23][24][25][26][27][28][29][30]. However, quantitative requirements for larvae are still scarce, and only a few species, such as European seabass (Dicentrarchus labrax) and gilthead seabream for vitamin D and Senegalese sole (Solea senegalensis) for vitamin K, have had their optimal levels determined [31][32][33]. Additionally, live prey used in commercial hatcheries exhibit wide variations in their vitamin D and vitamin K contents [15], which could contribute to the high incidence of skeletal anomalies. Moreover, deficiencies or imbalances in either vitamin can lead to skeletal anomalies and affect the overall performance of the larvae during rearing.
Studies on humans or terrestrial animals demonstrate that vitamin D and vitamin K interact with each other to improve bone health and other biological functions [34][35][36][37][38][39][40][41][42][43][44]. However, this type of study is very scarce in fish [17], and none of them has been conducted during larval stages. The bone-forming cells (osteocytes) and boneresorbing cells (osteoclasts) play significant roles in bone formation and remodeling in fish [19,45,46]. Proliferation and differentiation of these bone-forming cells are regulated by bone biomarkers [46][47][48][49][50]. The bone biomarkers analyzed in the present study and their role in regulating bone and calcium metabolism are listed in Table 1.
The bone-forming molecules work as a cascade during the bone development process and help in regulating other physiological pathways in fish [51]. The up-and downregulation of these biomarkers can be modulated by dietary nutrients [19,20]. An abnormal expression of bone biomarkers causes severe problems in skeletal development and might lead to skeletal anomalies in cultured fish [52]. In marine fish species, the effect of dietary vitamin D and vitamin K on skeletal development has been studied individually by very few authors [20,22,[31][32][33][53][54][55]. Although the vitamin D and K interaction studies in fish species are scarce, a series of trials on Atlantic salmon studied the effects of dietary vitamin D and vitamin K on growth, bone minerals, and health performance [17,56]. However, the combined effect of dietary vitamins D and K has not yet been studied in gilthead seabream larvae, despite its importance to understand the versatile functions of these vitamins in growth and skeletal development. Thus, considering that our previous studies determining the dietary vitamin D 3 [32] and vitamin K 3 [57] requirements in gilthead seabream larvae pave the way to study the interaction between both vitamins, the present study is aimed at understanding the potential interaction between vitamin D 3 and vitamin K 3 in gilthead seabream larvae concerning larval performance and expression of selected genes related to bone development and calcium regulation.

Larval
Rearing. Natural spawns of gilthead seabream larvae were obtained from selected broodstock (PROGENSA (Spanish National Breeding Program) project [2] from GIA (Grupo de Investigación en Acuicultura, ECOAQUA Institute, Las Palmas de Gran Canaria University (ULPGC), Spain). Larvae (initial total length 6:27 ± 0:46 mm, dry body weight 0:22 ± 0:03 mg, mean ± SD) previously fed rotifers (Brachionus plicatilis) enriched with ORIGREEN (Skretting, Norway) until 22 days post hatch (dph) [58] were randomly distributed in 12 experimental tanks at a density of 1200 larvae in each tank. After the distribution, larvae were fed with experimental diets for 21 days (43 dph). All tanks (200 L, light grey color cylinder fiberglass tanks) were supplied with filtered seawater (36 g L -1 salinity) at an increasing rate of 0.3-1.0 L min -1 as a flow-through system along the feeding trials. Water entered the tank through a bottom mesh and was let out from the top to ensure water quality. Water was continuously aerated (125 mL min -1 ), attaining 5-8 g L -1 dissolved oxygen and saturation ranging between 60 and 80%. Water temperature (20 ± 2°C), photoperiod (12 h light : 12 h dark), and light intensity (1700 lux) were maintained constant throughout the experimental period (21 days).

Experimental Diets.
Seven experimental microdiets of isoproteic (64:8 ± 0:56%) and isolipidic (20:48 ± 0:52%) contents were prepared containing four different levels of vitamin D 3 (VD-cholecalciferol) (Sigma-Aldrich, CAS-67970) combined with two different levels of vitamin K 3 (VK-menadione) (Sigma-Aldrich, CAS-58275) and one deficient diet (nonsupplemented) ( Table 2). The chosen dietary vitamin levels were based on our previous study in gilthead seabream larvae fed different level of dietary vitamin D 3 [32] and vitamin K 3 [57]. The microdiets were prepared by grinding and sieving the ingredients below 125 microns. The ingredients were mixed in the following order: squid powder, water-soluble components, lipids, and fat-soluble vitamins, and finally, gelatin was mixed to obtain a homogenized mix. Then, the mix was pressed through a compress pelletizer (Severin, Suderm, Germany) and dried in the oven at 38°C for 24 h (Ako, Barcelona, Spain). After drying, pellets were grounded using a grinder (Braun, Kronberg, Germany) and sieved (Filtra, Barcelona, Spain) to achieve a pellet size of 250 and 500 μm. Then, the microdiets were stored at refrigerated condition (2-8°C) throughout the experiment to avoid vitamin deterioration. Proximate analysis of the feed was conducted at GIA laboratories (Table 3). Each diet was tested in triplicates, and the diets were fed manually from 8.00 to 20.00 with 45-minute intervals for 21 days. Daily feed supply was increased from 3 g to 5 g along with the pellet size of 250-500 μm, gradually during the experimental period. Larvae were periodically observed under stereoscope (Leica, M125, using Leica Application Suite software, Wetzlar, Germany) to determine the feed acceptance.

Larval Performance.
Growth performance was determined at each sampling points at 7 th , 14 th , and 21 st days of feeding, by measuring total length and dry body weight. 2 Aquaculture Nutrition Total length of 20 larvae (anesthetized with clove oil) from each tank was measured by microscope (Leica microsystem, Leica Application Suite software, Germany). Whole-body weight was determined by triplicates of 10 starved larvae washed with distilled water and dried in a glass slide at an oven at 110°C for 24 h, followed by 1 h periods until constant weight was achieved. To evaluate the survival rate of larvae, the total live larvae from each treatment were collected and counted at the end of the experimental period.       Different letters in a row denote significant differences between groups fed different diets (mean ± SD, n = 3, p < 0:05).

Aquaculture Nutrition
72°C for 30 s, and 95°C for 1 min, and a final denaturation step from 58 to 95°C for 10 s. The nucleotides of the housekeeping and target gene primers are listed in Table 4.

Skeletal Anomalies.
To determine the occurrence of skeletal anomalies, 150 larvae per treatment (i.e., 50 larvae per tank) were sampled at 43 dph and fixed in 4% formalin in phosphate buffer, pH 7.2, 0.1 M. Fixed larvae were stained for whole mount staining and examined under stereomicroscope (Leica M125, Wetzlar, Germany), and larval image (photographed using Leica DFC295 digital camera, Leica, Wetzlar, Germany) was processed using the Leica application suite (LAS 32167, Leica, Wetzlar, Germany). The stained larvae were examined for severe skeletal anomalies [5].
2.6. Data Analysis. All data were statistically analyzed using IBM SPSS Statistics v26.0. (IBM Corp., Chicago, IL, USA) and are expressed as means ± S:D. Data were treated for normality and homogeneity of variances using Levene's statistic, and means were compared to understand the statistical difference among the groups using Tukey's post hoc test (p < 0:05). To determine the effect of individual diet, oneway analysis of variance (ANOVA) was used, and for the interaction, two-way ANOVA was performed. SigmaPlot 12.3 (Systat Software, Inc., California, USA) was used to plot data in a three-dimensional mesh surface response plot to illustrate the interaction effect.

Larval Performance.
Microscopic observation showed that all the experimental diets were well accepted by gilthead seabream larvae. After a period of 21-day feeding, the best survival was found in larvae fed 0.06/70 VD/VK diet and the lowest in those fed the 0.40/170 VD/VK diet (p = 0:08) ( Table 5). The two-way ANOVA denoted a significant negative effect of increased dietary vitamin K 3 levels on survival rates. Regarding the growth of larvae, there was no significant difference in total length on day 7 (29 dph) and day 14 (36 dph) (Figure 1). At the end of the trial, due to high mortality, no samples were recorded for length and weight in the group of larvae fed with high 0.40/170 VD/VK diet; hence, the highest growth in terms of total length was found in fish fed the 0.13/70 diet and the lowest in those fed the 0.40/70 diet (Table 5) 6 Aquaculture Nutrition   Aquaculture Nutrition supplementation level (70 mg/kg), but it did not at the higher supplementation level of vitamin K 3 (170 mg/kg), denoting the interaction between both vitamins. Besides, further increase in dietary vitamin D 3 from 0.13 to 0.40 mg/kg significantly reduced larval total length. Accordingly, the two-way ANOVA denoted that there was no significant effect on day 7 and day 14. But on day 21, there was a significant effect of dietary VD on total length, with an interaction between both vitamins. No significant effect was found on dry body weight, which is a less sensitive parameter at larval stages.

Bone Biomarker Gene Expression.
Among the genes related to bone metabolism, the relative gene expression of the bone morphogenic protein 2 (bmp2) was not affected by the diet after 7 days of feeding the experimental diets ( Table 6) (p > 0:05), whereas at day 21, it was significantly upregulated in fish fed 0.06/70 diet in comparison to those fed the 0.40/170 diet. The runx2 expression was not affected by the different dietary vitamin D 3 and vitamin K 3 levels tested neither at day 7 nor at day 21 ( Table 6). The relative expression of osterix (osx) showed a similar expression pattern as bmp2, with no differences at day 7 (p < 0:05), but an upregulation at day 21 in fish fed 0.06/70 diet (Table 6). Moreover, in osx gene expression, a significant (p < 0:05) interaction between both vitamins was found at day 21 by the two-way ANOVA, denoting that although at the lowest vitamin D 3 levels the increase in vitamin K 3 Table 6). The osteocalcin (oc) expression was significantly downregulated in fish fed diet 0.13/170 at day 7 (p < 0:05), the two-way ANOVA suggesting an effect of vitamin D 3 levels (p = 0:055, Table 6). At day 21, besides the mild vitamin D 3 effect (p = 0:06), the two-way ANOVA also denoted the significant (p < 0:05) downregulatory effect of vitamin K 3 increase (Table 6 and Figures 3(a)  and 4(a)). After 7 days of feeding, the matrix-GLA protein (mgp) was significantly (p < 0:05) lowest in fish fed diet 0.13/ 170 and highest in fish fed 0.40/170 followed by those fed 0.40/70 (Table 6), and the two-way ANOVA analysis suggested the downregulatory effect of vitamin D 3 increase (p = 0:056) and the interaction of both vitamins leading to an upregulation at the highest dietary levels of both vitamins (p = 0:067). At day 21, mgp expression followed a similar pattern at day 7, with the downregulation of this gene in fish fed the 0.40/70 diet followed by those fed intermediate levels of these vitamins (p < 0:05) and the highest in fish fed 0.06/70 followed by those fed 0.40/170 diet. The two-way ANOVA suggested the downregulatory effect of vitamin D 3 (p = 0:054) and vitamin K 3 (p = 0:059) and showed a significant interaction between both vitamins (p < 0:05, Figures 3(b) and 4(b)). Moreover, there was a strong lineal regression (R 2 = 0:92, p < 0:05; y = 1:1285x − 0:2077) between mgp and oc relative expressions at day 7 ( Figure 5). At day 21, a lineal regression (R 2 = 0:5242, y = 1:1635x + 0:0946) was also observed between mgp and oc relative expressions (Figure 6), except for the value at highest level of both vitamins (0.40/170 diet) that showed a lower expression in oc in relation to the mgp expression.
Regarding the expression of other genes related to vitamin D and K metabolism, the calcium-sensing receptor (casr) expression at day 7 was significantly (p < 0:05) highest in fish fed 0.40/170 diet in comparison to those fed 0.13/170, whereas at day 21, no significant differences were found among the different dietary groups (Table 6 and Figures 3(c) and 4(c)). The expression of the parathyroid hormone receptor 1 (pthr1) and the parathyroid hormonerelated protein (pthrp) at day 7 and increase in dietary vitamin K 3 tend to downregulate these genes leading to the significantly (p < 0:05) lowest expression in fish fed the 0.13/170 diet, except in those fish fed the highest vitamin D 3 levels, which showed the highest expression (Table 6 and Figures 3(d) and 3(e) and 4(d) and 4(e)). Moreover, there were strong logarithmic regressions between casr expression and pthr1 (R 2 = 0:8325, y = 0:4795lnðxÞ + 0:8173) or pthrp (R 2 = 0:8975, y = 0:6917lnðxÞ + 1:0387) relative expressions at day 7 ( Figure 7). After 21 days of feeding, increase in vitamin K 3 downregulated both parathyroid hormonerelated genes, which showed the lowest expression in fish fed 0.06/170 and 0.13/170 diets. In the case of pthrp, the twoway ANOVA also suggested (p = 0:053) the interaction between both vitamins, and, hence the increase in vitamin K 3 in fish fed the highest levels of dietary vitamin D 3 did not upregulate pthrp expression. There was also an exponential regression (R 2 = 0:9162, y = 0:2552e1:4825x) between casr and pthrp expressions except for the value at highest level of both vitamins (0.40/170 diet) that showed a lower expression in pthrp in relation to the casr expression ( Figure 8). However,   (Table 7).

Skeletal Studies.
Supplementation with different combinations of dietary VD and VK showed no significant difference in frequency of skeletal anomalies ( Figure 9). The occurrence of abdominal kyphosis and haemal lordosis ( Table 8) did not differ among the different dietary treatments. Similarly, two-way ANOVA also showed no effect on skeletal anomalies.

Discussion
Optimum dietary levels of either vitamin D or vitamin K are necessary for larval performance and skeletal development in marine fish larvae [31,32]. Despite the clear interaction between these vitamins in mammals [42], studies in fish are very scarce and none were conducted during larval stages [56,59]. The present study showed the significant interaction between both vitamins in larval performance and gene expression related to bone development, denoting the importance of a correct balance between both vitamins in larval diets. Regarding larval performance, neither growth nor survival was significantly reduced in fish fed diets without supplementation with vitamin D 3 or vitamin K 3 . However, an increase in both vitamins led to the best larval performance, suggesting that the basal dietary levels were only marginally deficient. Thus, the increase in dietary vitamin D 3 supplementation from 0.06 to 0.13 mg/kg improved the total length of larvae fed the lower dietary vitamin K 3 supplementation level (70 mg/kg). This agrees well with the growth-promoting effect of vitamin D observed in mammals [60] and fish [25,27,61], particularly during early developmental stages [31]. In contrast with the present results, in gilthead seabream juveniles, increased supplementation of vitamin D 3 had only a mild effect on growth improvement [20], confirming the stronger growth-promoting effect and importance of this vitamin in early stages. However, increase in vitamin D 3 from 0.06 to 0.13 mg/kg did not improve growth at the higher vitamin K 3 supplementation level (170 mg/kg), denoting a clear interaction between both vitamins confirmed by the 2-way ANOVA statistical analysis. The present study is the first one that shows the interaction effect on the larval growth. On the contrary, in the present study, the increase in vitamin K 3 supplementation level from 70 or 170 mg/kg did not affect seabream growth, in agreement with the lack of effect of vitamin K supplementation on growth of salmon fry [62].
In the present study, the further increase in the dietary vitamin D 3 up to 0.4 mg/kg significantly reduced the larval growth, denoting the negative effects of excessive dietary 13 Aquaculture Nutrition vitamin D 3 , and this conclusion is in agreement with the previous studies on seabream [32] and other species [18]. In turbot (Psetta maxima), the overdose of vitamin D 3 produced an intestinal inflammation and reduced the gut microbiota diversity [63] that could be related with an impairment of growth. Despite the increase in vitamin K 3 supplementation from 70 to 170 mg/kg did not affect seab-ream growth, at the highest vitamin D 3 and K 3 supplementation levels, the survival rate was significantly reduced. Poor survival rates may be attributed to the toxic effect of vitamin overdose, where the larvae fed 0.4 mg/kg vitamin D 3 with 70 mg/kg showed higher survival than larvae fed 170 mg/kg vitamin K 3 . This denotes that the highest dietary  vitamin D 3 content accompanied with the lower vitamin K 3 level had better effect on survival than in larvae fed on the higher vitamin K 3 content. This in agreement with the authors' previous study, where the larvae were fed on diets supplemented with vitamin D 3 at 0.50 mg/kg and vitamin K 3 at 173 mg/kg supplementation levels that reduced the larval survival rate [32]. Further studies in mammals and other species showed that the excess menadione supplementation would impair the growth and survival rate [23] and increase the binding capacity of vitamin D transporter to vitamin D, which results in increased vitamin D receptor activity. The vitamin D receptor participates in the pathophysiological process by enhancing the metabolite effect in larvae [64,65]. The highest larval survival rate was recorded in larvae fed the diet supplemented with 0.06 and 70 mg/kg vitamin D 3 and vitamin K 3 , respectively. These larvae also showed, at the end of the trial, the highest expression of bmp2, osx, and mgp, which are important biomarkers of bone development and health. The bmp2 is an osteochondrogenic factor which initiates bone formation and bone healing, inducing the expression of other bmps, whereas osx regulates late osteogenesis and bone matrix mineralization [66,67]. In the present study, osx expression followed a linear relation with bmp2 expression, in larvae fed the diet supplemented with 0.06 and 70 mg/kg vitamin D 3 and vitamin K 3 , respectively, showing the highest values for both genes. The mgp promotes osteoblast proliferation and bone formation through the Wnt/β-Catenin signaling pathway [68], affecting mineralization and osteoclast differentiation [69]. The expression of mgp occurs during early development of gilthead seabream [70]. In the present study, mgp expression was associated with increased oc expression, with the highest values for both genes found in the larvae fed the diet supplemented with 0.06 and 70 mg/kg vitamin D 3 and vitamin K 3 , respectively. Osteocalcin is produced by osteoblasts and incorporated into the bone matrix, but it is also involved in energy metabolism and other metabolic functions [71]. Vitamin K plays an important role in the carboxylation of osteocalcin and its posterior binding to hydroxyapatite [17]. Besides, both vitamins regulate oc expression [72,73]. The highest expression of mgp and its correlation with oc in larvae fed the diet supplemented with 0.06 mg/kg vitamin D 3 and 70 mg/kg vitamin K 3 agree well with the combined effect of vitamin D and vitamin K on osteoblast activity and osteoclast formation, upregulation of osteoblastic genes, and bone health improvement found in other species [43,74]. Besides, vitamin D enhances the vitamin K-dependent proteins to induce bone formation along with vitamin K [38]. Thus, in mammals, the combination of both vitamins is more effective in the prevention of bone loss and fracture risk [42,75].
However, further elevation of vitamin K 3 supplementation from 70 to 170 mg/kg at 0.06 mg/kg vitamin D 3 supplementation downregulated bmp2, osx, and mgp, denoting the negative effect of increased vitamin K 3 . These results agree well with the indirect effect of vitamin K on bmp2 production translating into MK-7 [76]. Besides, vitamin K 3 intervenes in the activation by carboxylation of the matrix Gla protein (mgp), which is a main inhibitor of mgp expression. Moreover, the combined higher concentrations of both dietary vitamin D 3 and K 3 (0.40 and 170 mg/kg, respectively) further downregulated bmp2 expression and upregulated osx and mgp, causing an imbalance in the expression of osx in relation to bmp2 and in the expression of oc in relation to mgp. These results reflected the interaction of both vitamins denoted by the two-way ANOVA and agree well with the downregulation of bmp2 found in fish larvae fed high doses of vitamin D 3 [31,32]. Indeed, vitamin D is actively involved in the regulation of bmp2 and osx expression [77]. Whereas bmp2 is an osteochondrogenic factor, osx inhibits chondrogenesis, and the imbalance in the expression of both genes could be responsible for an impairment in bone formation, particularly in endochondral bones. This agrees well with the high incidence of cranial anomalies found in gilthead seabream larvae fed excessive levels of vitamin D 3 [32].
There is much evidence that, particularly during early developmental stages, both vitamin D and K are necessary   for the formation of strong bones and the prevention of skeletal anomalies [38,53,78]. Besides, feeding gilthead seabream with high levels of vitamin D 3 leads to cranial malformations [32], whereas excessive levels of vitamin K 3 cause a high incidence of kyphosis [57]. In the present study, despite the imbalance found in the relative expression of bone health related genes and their significant differences among larvae fed different vitamin supplementation levels, no significant differences were found in the incidence of skeletal anomalies. This lack of evidence on the skeletal anomalies could be attributed to the sufficient contents of vitamins D 3 and K 3 in the diet without supplementation of these vitamins, preventing the marked deficiency symptoms such as poor growth or high incidence of bone anomalies. On the other hand, the highest supplementation levels of vitamins D 3 and K 3 (0.40/170 mg/kg) caused an imbalance in the expression of some bone biomarkers, significantly reduced larval survival rate (7.92% vs. 28.39%), and induced the weakest and deformed larvae. Moreover, the supplementation levels of vitamins D 3 and K 3 tested in the present study were lower than those inducing skeletal anomalies in the previous studies [57]. Nevertheless, further studies should be conducted to find out the potential interaction between these two vitamins in relation to the occurrence of skeletal anomalies.
After 21 days of feeding the experimental diets, the expression of pthrp followed an exponential regression with casr expression, with the highest values found in larvae fed the diets 0.06/70 and 0.13/70. These results agree well with the highest survival and growth found in these larvae and with the upregulation of pthrp found in juveniles of seabream when dietary vitamin D is increased over the deficient levels [79]. Besides, the results also agree with the strong correlation between pthrp and casr found in mammals [80]. The parathyroid hormonerelated protein (pthrp) is related to the function of PTH and among other roles regulates endochondral bone development. The calcium-sensing receptor (CaSR) is a class C G-protein coupled receptor, sensitive to extracellular levels of calcium. It is expressed in the parathyroid gland, among other organs, and calcium inhibits parathyroid hormone (PTH) release. Both factors are the main regulators of calcium homeostasis [81], and their ratios are determinant for the correct balance of intra-and extracellular calcium.
However, the increase in the dietary vitamin K 3 supplementation level from 70 to 170 mg/kg at the lowest dietary vitamin D 3 supplementary level downregulated pthrp. Moreover, the highest supplementation level of both vitamins up to 0.40 vitamin D 3 mg/kg and 170 mg/kg vitamin K 3 markedly reduced the expression of pthrp in relation to cars expression. This unbalance in the expression of these two

Conclusions
The present study showed the interaction between vitamin D 3 supplementation and vitamin K 3 supplementation in larval performance and gene expression related to bone development and calcium homeostasis, denoting the significance of a correct balance between both vitamins in larval diets. Supplementation with 0.06-0.13 mg/kg vitamin D 3 and 70 mg/ kg vitamin K 3 led to the highest larval growth and survival and the highest expression of important biomarkers of both bone development and health, such as bmp2, osx, and mgp, and calcium homeostasis, such as pthrp and casr. However, the combined increase in the supplementation of both vitamins up to 0.40 mg/kg vitamin D 3 and 170 mg/kg vitamin K 3 reduced the larval growth and survival, downregulated bmp2 and pthrp expression, and upregulated osx and mgp, causing an unbalance in the relative expression of these genes.

Data Availability
The data used to support the findings of this study are included in the article. However, more data could be available from the corresponding author upon request.

Disclosure
This output reflects the views of the authors only, and the European Union cannot be held responsible for any use which may be made of the information contained therein.

Conflicts of Interest
The authors declare no conflicts of interest.