Changes in digestive enzymes activities during the initial ontogeny of wolf cichlid , Parachromis dovii ( Perciformes : Cichlidae )

1División Académica Multidisciplinaria de Jalpa de Méndez, Universidad Juárez Autónoma de Tabasco, Carretera Nacajuca-Jalpa de Méndez R/a Rivera Alta, 86200, Jalpa de Méndez, TB, Mexico. cafq22@hotmail.com, https://orcid.org/0000-0002-5407-4991 2Laboratorio de Acuacultura (DACBIOL), Universidad Juárez Autónoma de Tabasco, Carretera Villahermosa-Cárdenas Km 0.5, 86139, Villahermosa, TB, Mexico. (CAA-G) alvarez_alfonso@hotmail.com, https://orcid.org/0000-0001-9240-0041 (corresponding author), (RG-Z) rocio7224@hotmail.com, https://orcid.org/0000-0002-0346-0841 3Escuela de Ciencias Biológicas, Universidad Nacional, Campus Omar Dengo, Heredia l86-3000, Costa Rica. (SV-C) silvia.valverde. chavarria@una.cr, https://orcid.org/0000-0003-2823-459X, (JBU-R) juan.ulloa.rojas@una.cr, https://orcid.org/0000-0003-4464-1136 Introduction


Introduction
The Cichlidae family is composed of about 1,000 species and is widely distributed around the tropics.It has showed a remarkable commercial importance for fisheries, sport fishing and aquaculture (Luna-Figueroa, Figueroa-Torres, 2000).In Costa Rica and in most Central America countries, one of the most important species is the Wolf cichlid, guapote lagunero (Parachromis dovii Günther, 1864), which has a tasty flesh, good performance in earthen pond culture systems and in addition, it is used as biological control for undesired reproduction in tilapia culture (Nonell, Rojas, 1995;Hernández, 1992;Barrera, Paz, 2006).However, this species still has some production problems (e.g.low growth rate) related to malnutrition especially at early stages; which had affected negatively its production at commercial scale.

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For this reason, the knowledge of its digestive physiology is essential for maximizing its growth by knowing the fish capability to metabolize protein sources needed for tissue synthesis (Kolkovski, 2001).Further, studies on the digestive system development and enzymatic capacity should bring information to understand the nutritional status, the digestive enzyme activity and related processes to generate better strategies for control and improvement of survival at the earlier stages (larvae) (Moyano, 2006;Zambonino-Infante, Cahu, 2007).
These studies have allowed the understanding of digestive enzymes variability, which reflects changes in fish morpho--physiology and feed use during larvae period.Specifically, for the P. dovii, in Costa Rica only one study has been done related to the effect of feed on growth and proteolytic digestive activity in larvae (Quirós-Orlich et al., 2014), evaluating the live feed substitution (Artemia Leach nauplii) by an inert diet.Further, studies related to the digestive physiology of wolf cichlid are needed to improve its growth by using specific diets, before to recommend its commercial scale culture.For this reason, this research focused on the digestive proteases, lipases and amylases of P. dovii during early ontogeny.

Material and methods
Larvae and sampling.The larvae were obtained from broodstock of P. dovii (1 male: 1 female) (adult specimens are included in Ichthyological collection ROM 84151), at the hatchery unit of the Escuela de Ciencias Biológicas of Universidad Nacional de Costa Rica, maintained in fiberglass tanks with a water recirculation system by air flow.Larvae were obtained from spontaneous spawns and after hatching; all were transferred and randomly distributed into aquaria (11 L each, 23 larvae L -1 ) of a recirculation system.The water temperature was kept at 25-29 o C, dissolved oxygen at 6.0 g L -1 minimal and NH 3 lower than 0.06 g L -1 .
Larvae were fed to satiety with Artemia nauplii (Great Salt Lake, Artemia International®) four times daily (9:00, 11:30, 14:30 and 17:00 h) from the beginning of exogenous feeding (6 day after hatching, DAH) until the end of the larval period (30 DAH).The larvae were fed with a pelletized formulated feed SilverCup® (45% protein and 10% lipids) at the same larvae feeding frequency.After the programmed feedings, the surplus food was cleaned by suction with a hose to avoid excess nutrients and the subsequent contamination of the water.

Growth and survival.
The specific growth rate (SGR) for weight and total length was calculated at the end of the trial by the formula: SGR = (lnW f -lnW i / t 2 -t 1 )*100 (% body length day -1 or % body weight day -1 ), where: ln: natural logarithm, W f : final length or weight (mm or mg), W i : initial length or weight (mm or mg), t 2 : final day, t 1 : initial day.Survival rates were determined by counting remaining larvae at the end of the experiment and it was corrected for number of sampled larvae.
Degree-days calculation.For the determination of the degrees-days: DD = [Tmax-Tmin/2]-To, where Tmax and Tmin are the maximum and minimum daily ambient temperature, respectively, and To is the temperature below which growth or development does not occur (often referred to as the base or threshold temperature, Hazel, Prosser, 1974;Sharpe, DeMichele, 1977) multiplying for the number of days after hatching (DAH).
Preparation of multi-enzymatic extracts.The viscera bulk was removed from each larva in cold conditions (kept on plates maintained in ice), by cutting off the tail, head and dorsal part of the body.Samples from the smallest larvae were taken by removing their head and tail.Viscera were split in two sections, to analyze stomach and intestine en-e180161[3] zymatic activity separately.Samples for alkaline enzymes determinations were homogenized with buffer 50 mmol L -1 Tris-HCl, CaCl 2 20 mmol L -1 , pH 7.5 (35 mg mL -1 ) and for acid enzymes determinations with buffer 100 mmol L -1 glycine-HCl, pH 2.0.Next, samples were centrifuged at 16 000 g, 4 °C for 30 min (Hettich Mikro 200).The supernatant was kept at -20 °C f or further enzymatic analysis.
Biochemical analysis.The soluble protein concentration was determined by Bradford (1976), using serum bovine albumin as standard.Total alkaline proteolytic activity was measured according to Walter (1984), using casein (0.5%) as substrate in a Tris-HCl 50 mmol L -1 , pH 9.0 buffer solution.Total acid proteolytic activity was determined following Anson (1938), using hemoglobin (0.5%) in a glycine-HCl 100 mmol L -1 , pH 2.0 buffer solution.One unit of enzymatic activity was defined as 1 µg tyrosine released per minute, using the 0.005 molar extinction coefficient at 280 nm.
The acid and alkaline phosphatase activities were measured according to Bergmeyer (1974), incubating the ex-tracts for the acid and alkaline phosphatase with 4-nitrophenyl phosphate 2% in citric/citrate (1:1 W/W) pH 5.5 and glycine-NaOH 50 mmol L -1 pH 10.1 buffer solutions, respectively.A unit of enzymatic activity was defined as 1µg of naphthyl released per minute, using a 0.0185 molar extinction coefficient at 405 nm.
Zymogram analysis.The classification of proteases was obtained by SDS-PAGE electrophoresis with discontinuous gels for alkaline proteases (Laemmli, 1970;García-Carreño et al., 1993).The enzymatic extracts were mixed with the sample buffer (Tris-HCl 50 mmol L -1 , pH 6.8, glycerol, SDS, bromophenol blue) at a v/v ratio of 1:1, and 20 µL of this mixture was applied on the gel wells (8.3 cm 9 6.1 cm 9 0.75 cm).The discontinuous zymograms consisted of a storage gel at 4% and a separating gel at 10%.The gel was equilibrated at 80 V for 15 min, and the electrophoresis was done at 100 V and 120 mA for 100 min (Mini Protean III BIORAD Laboratories, CA, USA).The gels were submerged in a 2% casein solution in Tris-HCl 50 mmol L -1 , pH 9 at 4°C for 60 min to allow the gels to absorb the casein and then incubated at 37 °C during 18 h to allow the substrate hydrolysis.After development of enzyme activity, gels were stained by using the same Coomassie brilliant blue R-250 solution.
Reference front (Rf) and molecular weight calculations.A low molecular weight marker (LRMWM) from Pharmacia Biotech (Uppsala, Sweden) was applied to each SDS-PAGE adding 5 µl well.The LRMWM contained the following proteins: 1) phosphorylase b (97 kDa), 2) serum bovine albumin (66 kDa), 3) egg albumin (45 kDa), 4) carbonic anhydrase (29 kDa), 5) trypsinogen (24 kDa), and 6) soybean trypsin inhibitor (20 kDa).The relative electromobility (Rf) for all zymograms was calculated according to Igbokwe, Downe (1978), and the molecular weight (MW) of each band with alkaline protease activity was calculated as the linear fit between the Rf and the decimal logarithm of the molecular weights of the proteins used as markers, using the software Quality One V 4.6.5 (Hercules, CA, USA).
The SDS-PAGE zymogram for alkaline proteases showed two bands that appeared at 6 DAH (19.2, 24.8 kDa), and a third band that was observed at 14 DAH (68 kDa), three bands were present until the end of the larviculture; however, the intensity of these bands increased gradually until 41 DAH (Fig. 4).

Discussion
The growth (in weight and total length average) and survival rate of P. dovii larvae was higher compared to that found by Quirós-Orlich et al. (2014), who reported a SGR of 12.2±0.1% and 3.4±0.1 % for weight and length, respectively, and a survival rate of 98.99% during a period of 20 days (from 8 to 28 DAH).In that study, P. dovii larvae were feeding with Artemia nauplii (at satiation) until 15 DAH, followed by Artemia nauplii substitution by formulated feed.The activity of digestive enzymes is used as an indicator of the larvae capability to hydrolyze nutrients and, to some extent, as a guide to elucidate the enzyme affinity to specific substrates according to feed types (Zambonino-Infante, Cahu, 2001).
Accordingly, our results show that P. dovii larvae had acid proteolytic activity (pepsin-like) since 6 DAH (9.49 mm, 168 DD, 0.056 mm.ºC.days), in agreement with the observation of a stomach with gastric glands at the time of first feeding (Valverde-Chavarría et al., 2013).This early appearance of a stomach with gastric glands has also been reported for Petenia splendida (Treviño et al., 2011).In addition, it is also possible that some of this early acid activity (6 DAH) corresponds to other acid enzymes, such as cathepsin, since the enzymatic extracts were made using the whole larvae (Moyano et al., 1996).Further, the pepsin-like activity is also a good indicator of the transition from larvae to juvenile as it has been reported for perch, Sander lucioperca (Linnaeus), common pandora, Pagellus erythrinus (Linnaeus) (Suzer et al., 2006;Hamza et al., 2007) and freshwater species as M. urouphthalmus (López-Ramírez et al., 2011), P. splendida (Treviño et al., 2011;Uscanga-Martínez et al., 2011) and O. niloticus (Uscanga-Martínez et al., 2010).The difference in the time of detection of pepsin activity and gastric glands may be due to the species variation.Recent study has demonstrated daily rhythms of digestive enzyme activity in fish larvae (Mata-Sotres et al., 2016).To improve larval rearing and feeding practices, the functional development of digestive system in several species from the genus of Lutjanus has been evaluated in Pacific red snapper, Lutjanus peru (Nichols, Murphy), the appearance of the gastric glands and pyloric caeca was around 24 DAH (Peña et al., 2017), and pepsin secretion was detected around 25 DAH (Moguel-Hernandez et al., 2014).
The result from the current study together with previous published data suggests that digestive enzymes of fish larvae are presented at a low level before the onset exogenous feeding and their activities increase to a high level during exogenous feeding.In contrast to a taxonomically similar species onset points of trypsin as M. urophthalmus (López--Ramirez et al., 2011) andA. trimaculatus (Toledo-Solís et al., 2015), amylase and lipase in pompano, Trachinotus ova-tus (Linnaeus), are triggered by internal mechanisms, rather than dietary stimulation; for this reason, the fluctuations in specific enzyme activities covered the period of morphological differentiation in the digestive tract and the development of digestive glands (Ma et al., 2015).According to this study, gastric glands were first observed on 14 DPH, but the pepsin activity of crimson snapper was not detected until 17 DPH.Such development may cause by the feeds shifting from rotifers to Artemia nauplii.
The reduction of the specific activity levels for most of the digestive enzymes tested at the transition from larvae to juvenile (between 22 and 30 DAH, 20.83 mm, 550 DD, 0.057 mm.ºC.days and 23.42 mm, 810 DD, 0.028 mm.ºC.days, respectively) has been reported for other species and it seems to be related to physiological changes that occur during larval development, such as the increment of other soluble proteins in the extracts, the appearance of other hormones or enzymes, or may be genetically programmed (Zambonino-Infante, Cahu, 2001;Lazo et al., 2007).It is well known that digestive enzymes play great roles in the catabolism of yolk, the processes of energy metabolism and growth regulation during embryonic developmental stages in fishes (Zhao et al., 2016).Accordingly, enzymes represent important links between the nutrients and the basal metabolism and growth (Santos et al., 2016).Furthermore, some specific enzymes correlate closely with gamete quality (Jia et al., 2013).
Larvae of P. dovii have a high capability to digest protein from different sources, because of the presence of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A. These enzymes are capable to hydrolyze peptide bonds (functioning as endopeptidases or exopeptidases), releasing peptides and amino acids, which, in turn, are easily absorbed by the larvae to fulfill its requirements and complete the metamorphosis to juvenile (e.g.scale formation, spine growth).The latter is favored by the early appearance of a functional stomach (Valverde-Chavarría et al., 2013) with pepsin-like activity since 6 DAH (9.49 mm, 168 DD, 0.056 mm.ºC.days).
Trypsin specific activity reached its maximum during first 14 DAH (15.10 mm, 364 DD, 0.041 mm.ºC.days), decreasing days after, while the chymotrypsin specific activity was low at 6 DAH (9.49 mm, 168 DD, 0.056 mm.ºC.days) and gradually increased to reach its maximum at 41 DAH (27.66 mm, 1189 DD, 0.023 mm.ºC.days).Fluctuations in the specific activity of trypsin and chymotrypsin have also been reported in larvae of other species such as S. ocellatus (Lazo et al., 2007), S. hasta (Nazemroaya et al., 2015), E. coioides (Guo et al., 2016), L. erythopterus (Cui et al., 2017) and A. sapidissima (Xiao-Qiang et al., 2017), which has been attributed to genetically programmed changes and variations in the natural food organisms (Zambonino-Infante, Cahu, 2001).Furthermore, the different interactions between alkaline enzymes such as trypsin and chymotrypsin may be considered good indicators of nutritional quality of larvae stages (Cara et al., 2003).The lack of trypsin in the first few days may be compensated by pinocytosis until the digestive tract becomes fully functional to digest protein in lumen itself (Sharma et al., 2016).
The increment of alkaline phosphatases activity reflects the enterocytes brush border development, which in turn, it is associated with the diminishing of cytosolic enzymes, agreeing with S. senegalensis larvae (Ribeiro et al., 2002).The latter is an indication of the digestive system maturation (which it is faster than skeletal system development) and, from the digestive physiology viewpoint, of the transition from larvae to juvenile stage (Balon, 1984;Kendall et al., 1984).The acid phosphatase was also present from 6 DAH (9.49 mm, 168 DD, 0.056 mm.ºC.days), with a maximal activity at 14 DAH (15.10 mm, 364 DD, 0.041 mm.ºC.days), which may indicate that not only the enterocytes are mature but also that nutrient absorption processes are more efficient.Similar trends for activity increments and microvilli development were found with D. labrax, S. aurata, R. kutum and A. sapidissima (Zambonino-Infante, Cahu, 1994;Moyano et al., 1996;KhosraviBakhtiarvandi, Abedian-Kenari,2015;Xiao-Qiang et al., 2017).The increment in activity of these enzymes is not only correlated to enterocyte maturation but also to genetic modulation; however, it may also vary due to composition of feed used (Zambonino-Infante, Cahu, 1994;2001;Gawlicka et al., 1995;Hakim et al., 2007).
The number of digestive enzyme isoforms expressed on the different species it is related with feeding habits of the species, so when counting with six bands of proteases activities, P. dovii may be considered a species of omnivore habits similar to M. urophthalmus (López-Ramirez et al., 2011); besides, this type and number of isoforms have been detected in other fish species such as Senegal sole, Solea senegalensis (Kaup) with seven types of bands (Sáenz-de Rodrigáñez et al., 2005), while in carnivore species, the number of bands is low as Atlantic bluefin tuna, Thunnus thynnus (Linnaeus) with three isoforms (Essed et al., 2002), spotted sand bass, Paralabrax maculatofasciatus (Steindachner) with two isoforms (Alvarez-González et al., 2010), common snook, Centropomus undecimalis (Bloch) with two isoforms (Jimenez-Martínez et al., 2012) and C. trimaculatus with six bands of proteases activities (Toledo-Solís et al., 2015).
According to the results of this study, P. dovii larvae have a complete digestive enzyme activity from 6 DAH (first feeding, 9.49 mm, 168 DD, 0.056 mm.ºC.days), that maximize after 14 DAH (15.10 mm, 364 DD, 0.041 mm.ºC.days) when larvae were fed with live food, which indicates that the exogenous feeding with a formulated diet can be started by this time.From the nutritional point of view, it is needed to search for the nutritional requirements of the larval stage and to evaluate the in vitro digestibility of diets and ingredients for this specie.
In conclusion the digestive enzymes, alkaline phosphatase, amylase, lipase, chymotrypsin, and trypsin in our study were present before the onset of exogenous feeding in P. dovii larvae.The relatively high specific activity of chymotrypsin and trypsin facilitate digestion of protein at an early stage.The pattern of primary digestive enzyme activity indicates early functional development of the digestive system.This study on digestive enzyme patterns will provide valuable information on the nutritional requirements of larvae and help establish feeding protocols for optimizing larval mass-rearing production.