The pattern of digestive enzyme activity and growth of marine fish larvae mangrove red snapper Lutjanus argentimaculatus (Forsskål, 1775)

. Mangrove red snapper Lutjanus argentimaculatus is a highly valuable marine fishery and should be cultivated to ensure sustainability. The growth is crucial for larvae rearing, and the digestive enzyme influences the larval growth. This study aimed to determine the pattern of digestive enzyme activity and its relationship to larval growth. Digestive enzymes consisting of protease and amylase were quantified using a spectrophotometer, while lipase was tested using a titration method. Total length and body weight used to measure larval growth. Samples of larvae were collected at 5, 10, 15, 20, and 25 Days After Hatching (DAH). According to the results, digestive enzyme activity was detected at 5 DAH larvae, even though the levels were deficient. Further, the digestive enzyme activity increased and exponentially correlated (r = 0.96) to larval age. The total length and body weight indicated larvae growth, which performed polynomial (r = 1) and exponential (r = 0.99) correlation, respectively, to larval age. Based on this study's finding, the digestive enzyme activity of mangrove red snapper L. argentimaculatus larvae increased in line with the increasing larval age. In addition, there was also a very close correlation between digestive enzyme activity and larval growth.


Introduction
Mangrove red snapper (Lutjanus argentimaculatus) is a marine fishery commodity that has high economic value [1].This fish is dominantly caught in Eastern Indonesia [2].It is worried that the high market demand for this fish will have an impact on decreasing its population in nature.Therefore, culturing this fish in a hatchery is essential to be done in order to fulfill the market demand for this species as well as maintain their sustainability in nature.
The larval stage largely determines the success of marine fish seed production.Several factors influence the life of the larvae.Feed is one of the keys to success surviving in the larval stage [3].Feed is also a source of energy for larval growth and maintenance for larval growth and larval survival.
Larval growth is a crucial consideration for hatchery activities since it is correlated with the success of the hatchery itself.The good growth of the larvae most strongly influences the success of seed production.The good larval growth and successful larvae metamorphosis into seeds are indicators of success in hatchery activities.
Several factors influence larval growth, and one of them is digestive enzyme activity.The digestive enzyme plays an important role in the hydrolyzing process of the feed consumed by larvae so that the feed becomes a simple form that can be absorbed by the body's cells [4].Some digestive enzymes are protease, amylase, and lipase, each of which has a role in the digestion of protein [5], carbohydrate [6] and fat [7].
Digestive enzyme activity is a good indicator of digestion capacity, when the enzyme activity is high, it can be inferred that physically, the larvae have been able to process the feed [8].Digestive enzyme activity can also be an indicator of the utilization of the feed provided [9].Thus, the activity of digestive enzymes is also one factor that influences the growth of fish larvae [10].
The activity of digestive enzymes is influenced by several factors, such as larval age [11], type of feed [12], and development of digestive system structure [13].The structure of the digestive system that is still simple will correlate with the low production of digestive enzymes [14].
The increase in the activity of the digestive enzyme indicates an increase in the ability of larvae to hydrolyze feed, which is then used for their growth.Thus, it is crucial to be aware of the activity of the larval digestive enzymes in hatchery activities since it can reveal the larvae capacity to utilize the feed provided.
The pattern of digestive enzyme activity in several types of marine fish larvae in Indonesian water was known, such as groupers Epinephelus coioides [15], E. fuscogutattus [16], and Cromileptes altivelis [17], the silver pompano Trachinotus blochii [18] and rabbitfish Siganus guttatus [19].However, the activity of digestive enzymes in mangrove red snapper larvae L. argentimaculatus, which is also a marine fish species, is still unknown.Therefore, it is necessary to conduct research related to this topic.
This study aimed to determine the pattern of digestive enzyme activity of mangrove red snapper larvae and their relation to larval growth.The results of this study can become primary biological data as a reference for feeding management for larvae rearing of mangrove red snapper in the hatchery.

Eggs source
This study used eggs that came from the natural spawning of mangrove red snapper broodstocks, which have been domesticated in rearing tanks at the Institute of Mariculture Research and Fisheries Extension (IMRAFE) that was located in Bali.Firstly, all of the eggs were selected, and then only the fertilized eggs were used.

Rearing of larvae
The rearing of larvae was conducted in the hatchery at IMRAFE, using two concrete rectangular tanks with a maximum capacity of 6,000 L each.Every tank was equipped with an aeration system as a source of dissolved oxygen for the larvae.
Phytoplankton Nannochloropsis oculata in volume 40 L tank was added to the larvae rearing tanks starting at 2 Days After Hatching (DAH).The phytoplankton was produced in mass culture of phytoplankton tanks at IMRAFE.Phytoplankton was given every morning to larvae rearing tanks.The phytoplankton is the source of feed for zooplankton that was given as live feed for the larvae.In addition, the phytoplankton in the medium for larval rearing was also a shade or dimmer for the sunlight that entered the larval tank.
Larvae were fed by live feed zooplankton rotifers Brachionus rotundiformis.Zooplankton were started given at 2 DAH in the afternoon.The density was 5 rotifers/ml in the beginning.The number of given zooplankton increased in line with the larval age.Other feeds given to the larvae were artificial feed in the form of micro pellets and zooplankton nauplii Artemia, which were started at 11 and 20 DAH.

Larval samples
This study used larvae at 5, 10, 15, 20, and 25 DAH as samples.The number of samples used for enzyme activity analysis was 80-500 larvae, depending on the age of the larvae.While the number of samples used for measuring total length was five larvae for each age.

Digestive enzyme activity
Protease and amylase activities were analyzed spectrophotometrically according to the method of [20], while lipase activity was analyzed by titration method according to [21].Protease activity was measured using casein as substrate, tyrosine as standard, and the absorbance was read at a wavelength of 578 nm.Amylase activity was measured with starch as substrate, maltose as standard, and absorbance at a wavelength of 540 nm.Lipase activity was measured using soybean oil as substrate and titrated by 0.01 M KOH.

Larval growth
Larval growth was observed from the total length and body weight.Larval total length was measured microscopically using SZH stereoscopic microscope equipped with a micrometer.While larvae were weighed with a digital balance with an accuracy of 10 -4 g.

Parameters and analysis
Parameters observed were larval digestive enzyme activity, which consisted of protease, amylase, and lipase, and larval growth, which included total length and body weight.Data were mean ± standard deviation values.The data were displayed in graphical form between ages and each parameter variable.Each of these graphs also analyzed the correlation of the parameters.Furthermore, the data were analyzed descriptively.

Larval digestive enzymes activity
The results of digestive enzyme activity showed that protease activity at the beginning of the observation, which was in 5-days old larvae, was still very low, but later, this activity increased in line with the increasing larval age (Figure . 1).The results also showed that protease activity had a positive correlation with larval age (r = 0.96) which was expressed in exponential form.Protease activity was significantly increased at 25 DAH larvae.Amylase activity was also very low in 5-days old larvae, then increased slightly until 20-days larvae, and had a substantial increase at 25 DAH larvae (Figure .2).Amylase activity was also correlated very closely with larval age (r = 0.96) in exponential correlation.Lipase activity was also still low in 5 and 10 days old larvae, but it began to increase starting from 15 DAH larvae and then increased very rapidly at 25 DAH larvae (Figure .3).Similarly to protease and amylase, lipase activity was also exponentially correlated very closely with the age of the larvae (r = 0.96).The findings of this study showed that the activity of digestive enzymes, including protease, amylase, and lipase, was still deficient in the early stages of larvae.This is due to the lack of feeding activity of larvae.Larvae at 5 DAH began to consume zooplankton rotifers B. rotundiformis as their exogenous feed.The number of rotifers consumed by the larvae at this early stage was only a few number, thus, the activity of the digestive enzymes of the larvae was also still very low and not readily apparent.In addition, the structure of the digestive system of the larvae itself was still straightforward at this early stage, which resulted in the limited secretion of digestive enzymes [13].In this period, the feed hydrolysis process was carried out by the zooplankton consumed by larvae because the live feeds had the ability to autolyze [22].
Further, the activity of digestive enzymes in 10 to 20 DAH larvae showed an incensement as the larval age increased.This is related to the increasing of feeding activity of larvae so that the digestive enzymes become more active in hydrolyzing the feed.Besides, the larval digestive system that was developed more complex also increases the secretion of larval digestive enzymes.This result is similar to the development of digestive enzyme activity in tiger grouper larvae E. fuscogutattus, which showed an increase with increasing larval feeding activity and a more complete structure of the larval digestive system [16].However, in this period, the live feed consumed by the larvae still plays a role as an exoenzyme due to the incomplete development of the larval digestive system, which restricts the secretion of digestive enzymes.The artificial feed that was given to larvae in this period was also believed to have a role in increasing the activity of digestive enzymes.
The significant increase in digestive enzyme activity was at 25 DAH larvae.Three factors were assumed to have caused this increase.First, 25 DAH larvae were in the metamorphosis stage, thus the larvae required a lot of energy to metamorphose.This increase in energy demand stimulated an increase in the feeding activity of the larvae, which then results in an increase in the activity of their digestive enzymes.The metamorphosis period of mangrove red snapper L. argentimaculatus larvae can begin in the range of 18 DAH [23] to 30 DAH [24].Second, it is related to the more complete formation of organs in the digestive system in fish, so that the secretion of digestive enzymes by these organs also increases.Third, it is related to the type of feed given to the larvae.Larvae were also given zooplankton Artemia started at 25 DAH larvae in addition to the live feed given previously.The addition of this live feed is believed to play a significant role in increasing the activity of larvae digestive enzymes.Feeding Artemia and artificial feed positively affected the absorption of feed nutrients in the larvae [12].This is linked to the increased activity of digestive enzymes during the hydrolysis of feed.
Several previous studies on different fishes indicated that the increased activity of digestive enzymes was closely related to the increasing age of larvae [25], the development of organ structure in the digestive system of fishes larvae [26,27,28], and the transitional period of the larvae become juveniles [29].The findings from this study indicate that the digestive enzyme activity is highly correlated with an increase in larval age, the development of the formation of the larval digestive system, and also the metamorphosis of the larvae, as described in the results of previous studies.
The high increase in enzyme activity also shows that larvae that have undergone metamorphosis can entirely consume artificial feed [8].This fact is also supported by the complete development structure of the digestive system in metamorphosing larvae [13], thus the larvae are able to secret their own digestive enzymes to hydrolyze the artificial feed and eliminate the requirement for exoenzymes derived from live feeds [22].

Larval growth
The larvae used in this study had total length of 2.17   The study results also showed that the larvae had growth in the form of an increase in total length and body weight gain.The total length of the larva increases polynomial, while the body weight increases exponentially with increasing age.The significant rise in body weight observed in 25 DAH larvae is in line with the metamorphosis period of the larvae, where in that period, the larvae have begun to change their body shape to be young fish.The growth of the larvae is also related to the activity of digestive enzymes, which are increasingly active in hydrolyzing the feed that is consumed by the larvae, which is then used in the process of larval growth.As a result, the activity of digestive enzymes can also be an indicator of the utilization of the feed that has been provided to them [9] as well as of their growth [10].

Conclusion
Based on the findings, the larval digestive enzyme activity of mangrove red snapper L. argentimaculatus increased in line with the increasing larval age.In addition, there was also a very close correlation between digestive enzyme activity and larval growth.
± 0.09 mm at 5 DAH and then increased to 8.18 ± 0.55 mm at 25 DAH (Figure.4).Larval total length correlated very closely, even perfectly (r = 1), with the larval age.Larval total length increased in polynomial correlation with the increase of larval age.Body weight of 5 to 15 DAH larvae was still very light and just showed enhancement at 20 DAH, then sharply increased at 25 DAH l (Figure.5).Larval body weight ranged from 3x10 -4 g up to 467x10 -4 g at 5 to 25 DAH As with larval total length, the increase in larval body weight was also very closely and exponentially correlated with the age of the larvae (r = 0.98).