Incidence of systemic granulomatosis is modulated by the feeding sequence and type of enrichment in meagre (
 Argyrosomus regius
 ) larvae

Systemic granulomatosis is the most frequent disease in juvenile and adult meagre, but studies regarding the first appearance of granulomas in larvae do not exist. In order to evaluate this, meagre larvae were fed four different feeding regimes as follows: RS and RO (rotifer enriched with Easy DHA Selco or Ori‐Green from 3 to 30 dph respectively), RAS and RAO (rotifer enriched with Easy DHA Selco or Ori‐Green from 3 to 21 dph and Artemia enriched with Easy DHA Selco or Ori‐Green from 12 to 30 dph respectively). All treatments were also fed with commercial microdiet from 20 to 30 dph. At 30 dph weight, length, specific growth rate and survival were significantly higher in Artemia‐fed larvae, regardless of the enrichment. Microscopic first appearance of granulomas was observed in 20 dph larvae fed RS and RO. At 30 dph granulomas and thiobarbituric acid reactive substances (TBARS), values were significantly higher in RS and RO‐fed larvae than in RAS and RAO‐fed larvae. The results showed that granulomas first appeared in meagre larvae at 20 dph when fed rotifers only. Conversely, a reduced appearance of granulomas and lipid peroxidation occurs when Artemia is included in the feeding sequence reinforcing the hypothesis of a nutritional origin of the systemic granulomatosis.


Introduction
Meagre (Argyrosomus regius) is one of the fast-growing species proposed as a candidate for marine fish diversification on commercial aquaculture in Mediterranean and Eastern Atlantic coasts, mainly due to attractive attributes for the market such as large size, good processing yield (Jiménez, Pastor, Grau, Alconchel & Cárdenas, 2005;Duncan et al., 2013), low fat content, excellent taste (Poli et al., 2003), firm texture and great capacity to adapt to captivity (El-Shebly, El-Kady, Hussin & Yeamin Hossain, 2007).
Nevertheless, one of the most critical points in meagre production is the fish health considering the systemic granulomatosis as the more frequent disease in this species (Ghittino et al., 2004). The pathology is characterised by the presence of multiple granulomas in several tissues being liver, heart and kidney the most affected organs (Ruiz et al., 2018). Although systemic granulomatosis is a disease of unknown aetiology, there is evidence that it could be triggered by a nutritional imbalance (Ruiz et al., 2018;Cotou et al., 2016).
In the last years the studies on first life stages of meagre have been focused on larval density (Estevez, Treviño & Gisbert, 2007;Roo, Hernandez-Cruz, Borrero, Schuchardt & Fernandez-Palacios, 2010), live prey feeding sequence (Roo et al. 2010) and light conditions (Vallés & Estévez 2013) in order to standardize the culture protocols.
Additionally, El Kertaoui et al. (2017) studied the effect of vitamin E and C on growth parameters and in the fatty acids protection against oxidation in meagre larvae, while the omega 3 (n-3) polyunsaturated fatty acids (PUFA) requirement has been recently studied by Carvalho et al. (2018). However, there is not any study evaluating the relationship between nutrients and the appearance of systemic granulomatosis in meagre larvae.
The first live feed traditionally used in intensive aquaculture are rotifers (Brachionus spp.) due to their small body size (Theilacker & McMaster 1971;Støttrup, 2000). Rotifer are fed by filtration making it easy to enrich, furthermore they have high population growth rate at high density. In a later stage of development, when the mouth of the larvae is big enough, Artemia nauplii are used as a live prey. However, rotifer and Artemia do not cover all of the larvae nutritional requirements and for that reason it is necessary to enrich the live food in order to improve nutritional value for the marine fish larvae (Boglino et al., 2012). Presently, commercial production of meagre involves the use of both rotifers and Artemia, although the effect that these feeding sequences might have on the development and appearance of systemic granulomatosis is unknown.
On the other hand, typical visceral granulomatosis has been experimentally induced in turbot (Scophthatmus maximus) fed diets deficient in vitamin C (Messager, Ansquer, Metailler & Person-Le-Ruvet, 1986). Also in turbot, Coustans, Guillaume, Metailler, Dugornay & Messager, (1990) confirmed the role of ascorbic acid deficiency in the development of visceral granulomatosis and showed that the pathological condition was exacerbated by hypovitaminosis of B -complex. Vitamin C deficiency has also been responsible for the appearance of granulomatosis in the kidney of sea bream (Sparus aurata) (Alexis, Karanikolas & Richards, 1997). Ruiz et al. (2018) observed that high dietary levels of vitamin C, E and K reduced the severity and incidence of systemic granulomatosis in meagre.
The objective of the present study was to evaluate the first appearance and incidence of systemic granulomatosis in meagre larvae. In order to do so, meagre larvae were fed rotifers enriched with two different commercial products with divergent levels of vitamins and with or without Artemia in the feeding sequence. This study will help to understand when the systemic granulomatosis first appears in the life cycle on meagre.

Material and methods 2.1. Fish
Meagre eggs were obtained from an induced spawning from broodstock from the ECOAQUA facilities at University of Las Palmas de Gran Canaria (ULPGC; Telde, Canary Islands, Spain) where the experiment was carried out. Eggs were volumetrically counted and set into 12 light grey colour cylindrical fibreglass tanks (4 triplicate treatments) of 170 l capacity at a density of 50 egg l -1 . All tanks were equipped with continuous aeration and supplied with filtered UV-sterilized seawater at an increasing rate from 5% h -1 to a 100% h -1 , to guarantee good water quality during the trial. Water entered the tanks at the bottom and exited at the surface. Oxygen (4.5-6.5 g l -1 ), salinity (0.034 mg l -1 ) and temperature ( Figure 1) .
Larvae were fed with enriched rotifers twice daily (9:00 and 14:00) from 3 to 21 (RAS and RAO treatments) or 3 to 30 (RS and RO treatments) days post hatching (dph). Larvae fed diets RAS and RAO were fed enriched Artemia twice daily (11:00 and 15:00) from 12 to 30 dph. Additionally, larvae from all the dietary treatments were co-fed with inert microdiets from 20 to 30 dph at a feeding rate of 10-15 % of the biomass.  Figure 1 Meagre (Argyrosomus regius) larvae feed sequence from 3 dph to 30 dph.

Live prey culture and enrichment protocol
Rotifers and Artemia were cultured at the ECOAQUA facilities at the University of Las Palmas de Gran Canaria (ULPGC; Telde, Canary Islands, Spain). Rotifers were cultured at a density of 400 rotifers ml -1 in 500 L enrichment troncoconical-tanks.
Enrichment tanks were well aerated (5 mg l -1 O2) with filtered and UV-treated seawater (0.037 mg l -1 ). Rotifer were enriched with Ori-Green (0.15 -0.25 g million -1 rotifers, RO dietary treatment) for two hours before being fed to the meagre larvae or with Easy DHA Selco (0.6 g l -1 , RS dietary treatment) for 24 hours.
Artemia cysts were hatched at 27° C and 0.030 mg l -1 salinity until 100 % hatch was achieved. Then they were rinsed with sea water and transferred to a culture tank at 24° C. Artemia was enriched with Ori-Green (0.8 g million -1 Artemia, RAO dietary treatment) for 12 hours or with Easy DHA Selco (0.6 g million -1 Artemia, RAS dietary treatment) for 24 hours before being fed to the larvae.

Sample collection
Samplings were performed at day 10, 20 and 30 dph. On each sampling 50 larvae were sacrified in ice and measured for total length. After measurement, that the 50 larvae were fixed in 4 % buffered formalin for histological analysis. Another 30 larvae were collected to determine dry weight at each sampling point. Additional 30 larvae were collected for biochemical and TBARS analysis and stored at -80 °C until analysis.

Growth and survival
Every 10 days, 50 larvae from each tank were sampled and measured for dry weight (100 °C for 24 hours) and total length (TL) using a profile projector (Mitutoyo PJ-3000A, Kanagawa, Japan). Final survival was determined at 30 dph by counting remaining alive larvae in experimental tanks.

Biochemical analysis
Larvae and feeds biochemical composition analysis were conducted following standard procedures (AOAC, 2010). Lipids of larvae and feeds were extracted with a choloroform:methanol (2:1 v/v) mixture as described by Folch, Lees, & Stanley Sloane, (1957).
Fatty acids from total lipids were prepared by transmethylation as described by Christie (1982). Fatty acid methyl esters (FAMES) were separated and quantified by gas-liquid chromatography following the conditions described by Izquierdo, Arakawa, Takeuchi, Haroun, & Watanabe, (1992).
TBARS were measured in triplicate from extracted total fatty acids (10 mg ml -1 ) according to Burk, Trumble, & Lawrence (1980). The concentration of vitamin E was determined in the dietary treatments (Table   3). The α-TOH was injected (50 µl

Histopathology
In order to estimate the first appearance of granulomas, every 10 days (10, 20, 30 dph) 50 larvae per tank (150 larvae per treatment) were sacrificed in ice and fixed in 4 % buffered formalin for histological analysis. The samples were dehydrated in a series of different concentrations of ethanol and embedded in a paraffin block. The samples were cut at 4 μm, fixed to the microscope slide, heated and finally stained with haematoxylin and eosin (H&E), Ziehl-Neelsen (ZN) (Martoja & Martoja-Pearson, 1970), Fite-Faraco method (Fite, Cambre, & Turner, 1947) and Gram stain (Gregersen, 1978). Then, the samples were used for histopathological evaluation.

Statistical analysis
All statistical analyses were done with Statgraphics (Statgraphics Centurion XVI Growth, survival, fatty acid composition and percentage of granulomas were analysed by two-way analysis of variance (ANOVA). A significance level of 0.05 was used.

Growth and survival
Significant differences in larvae dry weight and total length were found among the dietary treatments at 20 dph, being higher in larvae fed with Artemia regardless of the enrichment (Figure 1). This superior growth in Artemia-fed larvae was maintained at 30 dph. Specific growth rate (SGR) was significantly higher in larvae fed dietary treatments RAS and RAO (15.7 and 16.6 %, respectively) compared with those fed RS and RO (11.6 and 8.3 %, respectively) ( Table 4). Survival was significantly higher in larvae fed with Artemia (Table 4). All these differences could be explained by the factor "live prey" as indicated by the two-way ANOVA, with "enrichment media" also affecting dry weight at 20 dph. No interactive effect between "live prey" and "enrichment media" was observed for any of the performance parameters. LP*EN, interaction between live prey and enrichment. * p < 0.05 ** p < 0.01. n.s. n.s. n.s.
Data expressed as means of three technical replicates per batch of larvae. SGR, specific growth rate; LP, live prey; EN, enrichment; LP*EN, interaction between live prey and enrichment. Different superscript letters denote differences among treatments identified by one-way ANOVA. ** p < 0.01

Histopathology
First observation of granulomas under the microscope was at 20 dph in liver of fish fed rotifer enriched with Selco (RS; 1.3 %) and with Ori-Green (RO; 2.0 %), without significant differences (Figure 2).

Figure 2
Granuloma in the liver of meagre larvae fed with experimental diets at 20 dph.
The incidence of granulomas in meagre larvae increased after 30 dph, mainly in larvae fed rotifer-only together with microdiet. The percentage of affected larvae with granulomas was significantly higher in RS and RO-fed larvae (10.7 and 15.3 %, respectively) compared with larvae fed RAS (0 %) and RAO (0.7%) (Figure 3). "Live prey" was the only factor driving the differences in the appearance of granulomas at 30 dph. Regarding the morphology of the granulomas, all were at initial stages of development and could be observed as isolated and irregular aggregated of macrophages with scattered lymphocytes ( Figure 4A) that later were forming concentric layers with a necrotic centre ( Figure 4B). The main affected organ with granulomas was liver, followed by kidney. The specific stainings (Ziehl-Neelsen, Fite-Faraco and Gram stain) were all negative, discarding a possible infectious origin ( Figure 5A, 5B and 5C).

Whole larvae composition, TBARS content and fatty acid profiles
There were not differences in the tissue lipid, protein or ash content among larvae fed the different experimental diets (Table 4). The level of lipid peroxides, as indicated by TBARS content (μmol g -1 larval tissues), was significantly higher in those larvae fed rotifer only together with microdiet (diets RO and RS) ( Table 4). The factor "enrichment media" did not drive the differences observed according to the two-way ANOVA, indeed only live prey factor influenced TBARS content.

Discussion
Significant differences in terms of dry weight, SGR and total length were observed at 30 dph being higher in meagre larvae fed with the combination of rotifers, Artemia and microdiets (diets RS and RO) regardless of the enrichment media used. Survival was also significantly higher in those larvae fed with Artemia (19.5 % RAS and 17.8 % RAO) compared with larvae fed rotifers only (14.8 % RS and 12.8 % RO). Similar results were obtained by Fernández-Palacios, Hernández-Cruz, Schuchardt, Izquierdo & Roo (2009) who showed that meagre larvae were significantly larger, and the survival was higher when co-fed with Artemia instead of rotifers only. The energy that is required to breakdown, absorb, transport and assimilate nutrients from a meal adds up to 25 % of the daily energy expenditures in fish (Secor, 2011). As larvae grow, the range of accessible particle size increase, however, the ingested prey also needs to supply mass and energy.
The growing larvae try to ingest the maximum quantity of nutrients required for growth, while maintaining a favourable ratio between the energy gained by the ingestion of the prey and the energy spent in the capture (Herbing, Gallager & Halteman, 2001;Puvanendran, Salies, Laurel & Brown, 2004). The lack of Artemia in the feeding sequence lead to poor survival and growth probably because of a high energy cost/benefit related to the small size of the prey (rotifers), indicating that it is necessary an intermediate feeding between rotifers and microdiet. The present study suggests that it is possible to wean meagre larvae directly from rotifer to an inert feed from 20 dph (survival 12.8-14.8 %), but a co-feeding period with Artemia implies higher survival and growth of the larvae. Survival and growth results were in the same range as those reported by Estevez et al. (2007) and Rodríguez-Rúa et al. (2007) for the same teleost species.
Apart from larvae performance, one of the aims of the present study was to assess the first appearance of granulomas in meagre larvae (10 to 30 dph). To the authors knowledge this is the first report of systemic granulomatosis in meagre larvae.
Specifically, the first observation of microscopic granulomas was at 20 dph on fish fed with rotifer only either enriched with Selco (RS; 1.3 %) or with Ori-Green (RO; 2.0 %).
On the contrary, fish fed with Artemia did not show any granuloma until 30 dph (only 0.7 % incidence; RAO-fed larvae). However, after 30 dph significant differences were observed in the percentage of granulomas among the dietary treatments, being higher in fish fed diet RO and RS (15.7 and 10.7 %, respectively) compared with fish fed RAO and RAS (0.7 and 0.0 %, respectively). In the present study, the concentration of vitamin E in rotifers (310.1-179.9 mg kg -1 ) was lower than in Artemia (410.7 and 368.0 mg kg -1 ) and was also influenced by the enrichment media, being higher the concentration in the diets RAS and RS compared with RAO and RO. The vitamin content of rotifer and Artemia has been previously studied by Meeren, Olsen, Hamre & Fyhn, (2008) being the level of vitamin E slightly lower in rotifer but those of vitamin C clearly lower (220.1 µg g -1 ) compared with Artemia (530.6 µg g -1 ). Vitamin C is a powerful antioxidant and its deficiency has been related with the appearance of granulomas in turbot (Messager et al., 1986;Coustans et al., 1990), sea bream (Alexis et al., 1997) and meagre (Ruiz et al., 2018). The lack of Artemia in the feeding sequence increased the appearance of granulomas in meagre larvae after 30 dph (diets RO and RS) related likely with the differences in antioxidant vitamins of the live prey. Nevertheless, feeding with enriched rotifer, followed by enriched Artemia and microdiet, could prevent the occurrence of granulomas in meagre larvae after 30 dph (diet RAS), being the period before feeding with microdiet, a critical point to the development of systemic granulomatosis in meagre.
Besides, in the present study the appearance of systemic granulomatosis was highly correlated with TBARS content in meagre larvae (R 2 =0.948, y=0.084x-4.3924), corroborating a possible relationship between lipid peroxidation and the appearance of granulomas. In this sense, TBARS levels were significantly higher in meagre larvae fed with rotifers only and microdiet (282.9-288.0 μmol g tissue -1 ) compared with larvae fed with rotifer, Artemia and microdiet (56.8-67.4 μmol g tissue -1 ). Moreover, a higher incidence of granulomas was observed at 30 dph in meagre larvae fed diets RS (10.7 %) and RO (15.7 %) with less analysed vitamin E contents in the diet (310.1 and 179.9 mg kg -1 , respectively) than in diets RAS (0 %) and RAO (0.7 %) with higher concentration of vitamin E (410.7 and 368.0 mg kg -1 , respectively). Betancor et al. (2011) observed that in sea bass (Dicentrarchus labrax) larvae the supplementation of vitamin E up to 3,000 mg kg -1 diet had an antioxidant effect reducing TBARS values, decreasing lipid peroxidation and reducing the incidence of muscular lesions. In juveniles of meagre, the dietary increase of vitamin E and C lead to a reduction in the percentage and severity of granulomas in liver and heart together with a decrease in TBARS content (Ruiz et al., 2018), suggesting that these vitamins have an important role in the development of systemic granulomatosis in this species.
El Kertaoui et al. (2017) determined that the requirements of n-3 PUFA for meagre larvae were a 3 % (1.5-1.7 % DHA) in order to improve lipid absorption, fatty acid profile and growth. The levels of n-3 PUFA in all diets in the present study probably fulfilled the requirements However, these fatty acids, specially DHA, are very prone to oxidation (Izquierdo et al., 2013) being necessary an adequate level of antioxidant nutrients to avoid lipid peroxidation. The combination of a high concentration of DHA together with low vitamin E and C levels can lead to an imbalance between prooxidant and antioxidant nutrients, as observed in a higher TBARS values in meagre larvae fed diets RO and RS.
When an imbalance between the generation and removal of ROS by cellular defences occurs a status of oxidative stress takes place. The oxidative stress has been related with some diseases such as haemolysis (Kawatsu, 1969), anaemia, liver degeneration (Cowey, Degener, Tacon, Youngson & Bell, 1984), jaundice (Sakai et al., 1989), skeletal alterations (Watanabe, Izquierdo, Takeuchi, Satoh & Kitajima, 1989;Lewis-McCrea & Lall, 2007) or muscular dystrophy (Betancor et al., 2012). In agreement, the results of this study suggest that the systemic granulomatosis could be related to this imbalance.
Vitamin E also seems to be related with the development of granulomas, as larvae fed with RAO and RO (enrichment media with the lowest vitamin E content) had a higher incidence of granulomas compared with larvae fed RAS and RS. On the other hand, the systemic granulomatosis in meagre is similar to the pathology produced by infectious agents such as Mycobacterium spp. (Gauthier & Martha, 2009) and Nocardia spp. (Labrie et al., 2008;Elkesh et al., 2012). The inability to determinate an infectious origin and the relation observed between the appearance of the systemic granulomatosis and the feeding sequence in meagre larvae reinforce the hypothesis of a nutritional origin of the disease.
Concluding, the inclusion of Artemia in the feeding sequence of meagre larvae significantly increased dry weight, total length, SGR and survival and reduced the incidence of systemic granulomatosis and TBARS content after 30 dph. Furthermore, larvae fed with diet RO and RS, with a higher concentration of DHA and lower vitamin E, had high TBARS values what suggests an imbalance between prooxidants and antioxidants. Feeding larvae with a combination of enriched rotifers and Artemia greatly decreased the incidence of granulomas, not finding any at 20 dph and just a 0.7 % at 30 dph in larvae fed RAO. This is the first study to report the appearance of granulomas in meagre larvae and to determine the implication of antioxidant nutrients in their development. Further investigation is required to understand the pathogenesis of the granulomas as consequence of an imbalance nutritional supply.