Can turbid water and refuges with aquatic macrophytes increase the survival and growth of Brycon amazonicus larvae?

The productivity of aquatic organisms and higher concentration of suspended solids that occur in areas flooded by Whitewater Rivers and the presence of aquatic macrophytes are conducive to the initial development of matrinxã (Brycon amazonicus). The objective of this study was to evaluate whether turbid waters and refuges with aquatic macrophytes improve the survival and growth of the larvae of Brycon amazonicus, a species of great interest in Amazonian fish farming. For this, two experiments were conducted using a completely randomized design and with four replicas per treatment up to 120 hours after hatching (HAH), In experiment I: Brycon amazonicus larvae at 24 HAH were subjected to three treatments with different types of water: clear water (CW); clayey water (AW) and green water (GW). Experiment II: larvae with 24 HAH were maintained in clear water (CW); clear water with refuge (CWR); green water (GW); green water with refuge (GWR); clayey water (AW) and clayey water with refuge (AWR). Experiment I showed that clayey water, followed by green water, increased the survival of larvae (73.92 and 54.32%). Growth was best in larvae maintained in green water. In experiment II, the use of aquatic macrophytes did not influence the survival and growth of the larvae. Thus, we suggest the use of turbid (clayey and green) waters without refuge to increase the survival and growth of Brycon amazonicus larvae.


Introduction
Brycon amazonicus is noted for being a fish of economic importance and has potential in aquaculture in Brazil and other South American countries (Muller, Villacorta-Correa & Carvalho, 2019) due to it being well accepted in the consumer market, and for having good growth performance (Zaniboni-Filho, Reynalte-Tataje & Weingartner, 2006;Gomes & Urbinati, 2010). However, one of the main difficulties for large-scale production is the low survival rate due to its aggressive behavior and cannibalism , which can result in more than 90% mortality during larviculture (Bernardino et al., 1993;Romagosa et al., 2001).
As such, a number of studies have been developed in an attempt to mitigate the effect of cannibalism, increase survival and improve the zootechnical performance of Brycon amazonicus in breeding systems, which included darkening of incubators Senhorini & Soares, 1995); supply of planktonic organisms (Atencio-García et al., 2003); different levels of luminous intensity (Lopes, Villacorta-Correa & Carvalho, 2018), stocking densities and water temperature (Barros, Villacorta-Correa & Carvalho, 2019), as well as the use of common salt as a modulator of aggressiveness (Oliveira, Duncan & Carvalho, 2020). These studies are mostly based on the manipulation of environmental factors in order to reduce cannibalism and potentiate the production of species of commercial interest, as highlighted by Howell et al. (1998) and Franke et al. (2013).
Turbidity and the use of refuges are environmental conditions that are of fundamental importance for the survival of some species of fish in the larval phase. Improvement in the survival and growth of larvae reared in turbid environments, which can be induced by the addition of microalgae or clay (Shaw, Pankhurst & Battaglene, 2006), have been used as part of the protocols for some species such as Seriola lalandi (Stuart & Drawbridge, 2011), Esox lucius (Salonen & Engstrom-Ost, 2013) and Sander lucioperca (Ljubobratović et al., 2019).
Some studies have shown a positive relationship between the use of shelters and survival and growth in the larviculture of certain fish species, for example, Monopterus Cuchia and Clarias batrachus grow well with water hyacinths (Narejo, Rahmatullah & Rashid, 2003;Sahiduzzaman, Tauhiduzzaman & Rahman, 2018). Mitellbach (1984) and Gotceitas (1990) used macrophytes as shelters for Lepomis gibbosus and Lepomis macrochirus larvae, respectively, and demonstrated increased survival and reduced predation, both interspecific and intraspecific.
For the larvae of B. amazonicus, turbidity and the presence of aquatic macrophytes are of paramount importance in their natural habitat, since during this phase of development, their assemblages colonize areas flooded by whitewater rivers that have high loads of suspended clay and silt, which is of Andean origin (Junk, 1983), and results in muddy waters with high turbidity (33 to 94 NTUs) and the presence of macrophyte banks due to the contribution of these nutrients (Sioli, 1968, Araújo-availability, visual ability to detect predators and ease of escape, which increase the chances of survival and better growth of the larvae (Sanchez-Botero et al., 2003;Sanchez-Botero et al., 2007;Cajado et al., 2018).
The manipulation of these characteristics of the natural habitat of Brycon amazonicus larvae can potentiate their production in captivity; however, studies using turbid waters and natural refuges with Neotropical freshwater species are still incipient. Thus, this study evaluated whether the use of turbid waters and natural refuges improve the survival and growth of Brycon amazonicus larvae, a species with great potential in Amazonian fish farming and one which has been gaining prominence at the national level.

Breeding conditions
The research was carried out in the Aquaculture Laboratory at the Experimental Farm of the Federal University of Amazonas (UFAM), Manaus, Brazil. The larvae were produced using the pituitary technique with raw pituitary extract of carp, according to the adjusted protocol of Romagosa, Narahara & Fenerich-Verani (2018). After extrusion, the oocytes were fertilized and stored in 60 L incubators with constant water renewal at a temperature of 26.76 ± 0.9 °C and at a density of 1 g of eggs per liter of water.

Experimental design and type of research
The study was divided into two experiments. In experiment I, the effect of three types of water on the survival and zootechnical performance of B. amazonicus larvae from 24 to 120 HAH was evaluated. In experiment II, we tested the effect of the use of natural refuges on the survival and zootechnical performance of B. amazonicus larvae from 24 to 120 HAH This research is of a quantitative nature that, according to Botelho and Cruz (2013), presents as its reference the analysis of statistical data, which transforms information into numbers in order to analyze and interpret events that occurred or that may occur through statistical analysis, i.e., the data are quantified and measured (Fachin, 2006).

Experiment I
To evaluate the effect of the different types of water, three experimental treatments were compared: CW (clear water), AW (clayey water) and GW (green water), with four replicates each and in an entirely randomized experimental design. The clear and clayey waters came from nurseries with few suspended solids and clay, respectively, while the green water was obtained via culture of the microalgae Chlorella sp., which was produced in the laboratory.
The larvae were previously acclimated from 20 HAH in 4 L trays for 4 hours and then stored at a standardized density of 45 larvae/L, which is a value close to that used by Barros et al. (2019). Larvae with a length of 5.247 ± 0.363 mm and weight of 1.211 ± 0.225 mg were transferred to plastic trays (40 x 30 x 8 cm) and a useful volume of 4 L with a constant aeration system. Cleaning and renewal of 10% of the water volume of the experimental units were carried out on a daily basis.
The feeding of the larvae consisted of cladocera (Moina micrura and Diaphanosoma sp.) from controlled production, in the proportions proposed by Jomori et al. (2013) of 1250 organisms/larva, divided into eight daily meals. Water quality variables, such as temperature (°C), dissolved oxygen (mg L -1 ) and pH, were measured using a digital system (Minipa, MV-363), a digital oximeter (HI 9146) and a digital pH meter (pH-221) respectively (Table 1).

Experiment II
In order to evaluate the effect of the use of the aquatic macrophyte Cabomba furcata as a refuge, the larvae of B.
amazonicus were subjected to six treatments: CW (clear water); CWR (clear water with refuge); GW (green water); GWR (green water with refuge); AW (clayey water); AWR (clayey water with refuge), each with four replicates, in an entirely randomized experimental design. A total of 25 Cabomba furcata leaves with an average leaf area of 6.90 cm 2 were inserted in each tray in the treatments with clear water with refuge, green water with refuge and clayey water with refuge. The inserted leaves occupied 14.94 % of the total tray area, which is equivalent to 179.33 cm 2 of 1200 cm 2 . The waters used in this experiment follow the same pattern as experiment I, except for the clayey water, which was collected from the Solimões River, near where it meets the Negro River, which is a spawning area of this species.
At 20 hours after hatching, the larvae were acclimated for four hours and stored at a length of 5.583 ± 0.275 mm and a weight of 1.535 ± 0.288 mg under the same conditions as experiment I. Cleaning, water volume renewal, larvae feeding were performed, as well as monitoring of survival rate and zootechnical indices as described in experiment I. The variables of water quality, temperature (°C), dissolved oxygen (mg L -1 ) and pH were measured using a digital system (Minipa, MV-363), a digital oximeter (HI 9146), and a digital pH meter (pH-221) respectively. The mean values of the physicochemical variables of the water from experiment II are shown in Table 2.

Data analysis
The data were analyzed for discrepant values and then tested for normality using the Shapiro-Wilk test. In experiment I, the survival rate (S %) was compared between the experimental treatments using one-way analysis of variance (ANOVA), followed by the Tukey post-hoc test. Zootechnical parameters (with the exception of the specific growth rate) were compared using the Kruskal-Wallis test, followed by the Dunn's test for multiple comparisons. The specific growth rate (SGR) was compared using one-way ANOVA. In experiment II, the survival rate (S %) and the specific growth rate (SGR) were compared between treatments using one-way ANOVA, which was followed by the Fisher LSD post-hoc test. The relative condition factor (Kn) and final weight (MFW) were compared between the treatments using the Kruskal-Wallis test, with the MFW being followed by the Tukey test, for multiple comparisons. The MFL was compared using the Holm-Sidak test followed by the Tukey test, for multiple comparisons.

Experiment I
The survival rate of the B. amazonicus larvae was significantly influenced by the different types of water ( Figure 1).
Clayey and green waters increased the survival rate during the experimental period, while larvae kept in clear water presented the lowest survival rates (one-way ANOVA; F=56.91; P=< 0.001).
There was no significant difference in specific growth rate (SGR) and relative condition factor (Kn) between treatments. The final weight and length were not significantly different in clear or in clayey water, though these indices were significantly higher in larvae kept in green water when compared to those kept in clayey water (Kruskal-Wallis, P=0.004; P=0.036), as can be seen in Table 3.

Experiment II
The survival rates of the larvae in the treatments with clear water, in the presence and absence of a refuge, were significantly lower than the treatments with green and clayey water with and without a refuge. There was greater survival of the larvae kept in green water without shelter when compared to the larvae kept in clayey water. The presence of a refuge did not influence survival in either green or clayey water (one-way ANOVA; F=20.71; P=<0.001). The differences between experimental treatments are shown in Figure 2. No difference was observed between treatments for the values of the condition factor (Kn) during the experimental period (Kruskal-Wallis; P=0.987). TGE was lower in larvae kept in clear water, with or without a refuge. Those kept in green water had a higher TGE than those kept in clayey water without a refuge. The TGE of those who were maintained in the green Research, Society andDevelopment, v. 10, n. 15, e227101522737, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i15.22737 water with a refuge and those in clayey water without a refuge did not differ significantly (one-way ANOVA; F=20.20; P=<0.001).
The mean final weight values (Table 4) were higher for larvae raised in green water with or without a refuge, and there was a significant difference in relation to those raised in clear water with or without a refuge and clayey water without a refuge (Tukey; P=0.001). The mean final length had lower values for larvae kept in clear water with or without a refuge and the greatest lengths were achieved in green water with or without a refuge and in clayey water without a refuge, as shown in Table 4 (one-way ANOVA; F=157,343; P=<0.001).

Discussion
The results of experiment I demonstrated that the survival of B. amazonicus larvae was low when they were maintained in clear water and high when raised in turbid (clayey and green) waters. This response can be attributed to the decrease in the visibility of the larvae in the water column caused by the presence of clay and microalgae (Eiane et al., 1999), which reduces the chances of encounters between them and facilitates escape.
In their natural environment, Silva (2009) found greater abundance of Brycon amazonicus larvae and juveniles in floodplain areas of the Solimões River, which have typically white or "muddy" waters and lower water transparency. Lima & Araújo-Lima (2004) state that in rivers of clear and black waters the potential predation rates may be higher due to greater visibility of eggs and larvae by predators because of the few suspended solids in the water.
According to Hecht and Pienaar (1993), turbid waters simulate conditions of low light and greater availability of refuge, which ends up increasing the survival of the larvae. Studies that employed the manipulation of light intensity in the larviculture of B. amazonicus showed that at low intensity, larvae with 24, 48, 72, 120 and 240 HAH, showed an increase in their survival rate due to the reduction in the display of aggressive interactions in these periods (Lopes, Villacorta-Correa & Carvalho, 2018;Muller, Villacorta-Correa & Carvalho, 2019). In addition, the turbid waters provide an effective shelter due to the suspended solids, which facilitate hiding by the larvae (Maes et al., 1998;Snickars, Sandstrom & Mattila, 2004;Engström-Öst, Karjalainen & Viitasalo, 2006). This was observed during the cleaning of the experimental units in the treatment using clayey water (AW), which resulted in the highest survival rate, since the larvae did not swim to the surface and took shelter in the decanted clay at the bottom of the experimental units.
Another factor is that less swimming activity occurs in turbid waters due to a lower detection of threat by the larvae (Abrahams & Kattenfeld, 1997) and thus a reduced need to seek shelter (Gregory, 1993). Therefore, a reduced swimming activity decreases the likelihood of counterattacks between members of the group (Souza, Villacorta-Correa & Carvalho, 2014), as observed in Esox lucius (Lehtiniem, Engström-Öst & Viitasalo, 2005) and B. amazonicus (Lopes et al., 2019).
However, the effects of turbid water use are different for each species of aquatic animal (He et al., 2020). For aquatic animal species that rely on vision to catch prey, turbid water can affect the predator's vision and thus reduce aggressiveness and cannibalism. However, for those looking for prey without the aid of vision, water turbidity has little effect on cannibalistic behavior (Liao & Chang, 2010).
The larvae of B. amazonicus bred in green water had better growth when compared to clayey or clear water. This indicates that during B. amazonicus larviculture the microalgae acted as a food source for the cladocera (zooplankton) that remained in the experimental units after the feeding (Reitan, Rainuzzo, Oie & Olsen, 1997), which played an indirect role in improving the growth rate of the larvae (Reitan, Rainuzzo, Oie & Olsen, 1993;Tamaru, Murashinge & Lee, 1994). As such, there was an increase in the population density of cladocera, which caused greater prey availability, and was a preponderant factor for higher values of weight, length and specific growth rate. However, some studies have shown that the growth of larvae of some fish species slows down over time in green water because the increased turbidity induced by algae negatively Research, Society andDevelopment, v. 10, n. 15, e227101522737, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i15.22737 influences the ability of the larvae to capture prey (Salonen & Engstro¨mO¨st, 2010), as observed in the larvae of Rhombosolea tapirina (Shaw et al., 2006) and Esox lucius (Salonen & Engstro¨mO¨st, 2013). Some species show increased feeding when kept in green water, but decreased feeding in clayey water (Shaw et al., 2006). This corroborates our results in which the larvae of B. amazonicus bred in clayey water showed significantly lower growth rates compared to those bred in green water. The same occurred with Clupea harengus pallasi (Boehlert & Morgan, 1985), Lepomis macrochirus (Rafinesque) (Miner & Stein, 1993) and Gobiusculus flavescens (Fabricius) (Utne, 2005).
In experiment II, the use of a natural refuge did not significantly influence the survival rates of B. amazonicus larvae.
The best zootechnical performance values in green water, regardless of the presence of the aquatic macrophytes, can be attributed to the improvement in vision in moderately turbid waters (Naas, Naess & Harboe, 1992;Utne-Palm, 2002) and the fact that green water functions as a parallel production of live food (Reitan et al., 1993;Tamaru et al., 1994) during B. amazonicus larviculture, which increased the number of zooplankton in the experimental units. On the other hand, the clayey waters decreased the visibility of the larvae due to the higher level of turbidity and consequently less probability of finding the prey (Eiane et al., 1999).
The use of only one species of aquatic macrophyte and food (Cladocera) may have influenced the results, since, because they have a structural complexity of species-specific leaves and roots in lowland areas, the aquatic macrophytes harbor a great diversity of aquatic invertebrates and fish larvae, which are part of the diet of B. amazonicus, which in its early stages of development feeds on zooplankton (Leite & Araújo-Lima, 2002;Leite, 2004).

Final Considerations
It was observed that green and clayey waters, with and without refuges, were efficient for larviculture of Brycon amazonicus since the larvae presented higher survival rates and zootechnical index values, such as mean final weight, mean final length and specific growth rate, when compared to whitewaters. Turbid waters (green and clayey) can thus be employed to replace whitewaters in fish farms where they are commonly used during the larviculture of Brycon amazonicus.
Given the above, we suggest new studies on this topic that test different concentrations of clay and microalgae, i.e., determine different turbidity ranges (NTU) in the larviculture of Brycon amazonicus in order to verify the optimal level for survival and productive performance. In addition, the realization of larviculture in experimental units of larger volumes, such as incubators, would be interesting since it would reflect the reality of fish farms and indicate possible adaptations in this type of system. Another interesting future study would be to test the use of turbid waters during a longer period of larval development, and exceed the 120 HAE period of this study.
Farm (FAEXP) available for our research. To Mr. Paulo Sérgio de Oliveira (In memoriam) and André B. de Souza for their technical support.