Characterization of the digestive enzymes of juvenile Dormitator latifrons (Richardson, 1844).

The Pacific fat sleeper, Dormitator latifrons, is an omnivorous freshwater fish that primarily feeds on detritus. Our understanding of the digestive physiology of this species still needs to be completed, particularly concerning the characterization of its digestive enzymes. This information is crucial in guiding the design of diets that promote optimal digestion of this species, which has the potential for aquaculture. Thus, this study aimed to optimize enzymatic methods and characterize the digestive enzymes of the digestive tract regions: anterior region (AR), middle region (MR), posterior region (PR), and hepatopancreas (HP). Total acid protease, total alkaline protease, amylase, and lipase activities were measured. The enzymatic methods were optimized at an eco-physiological temperature of 25 °C based on extract volume, extract dilution, incubation time, pH, and CaCl2 concentration to determine specific activity (U/mg of protein). The optimal pH for acid protease (AR) was pH 2.0; while for alkaline protease, the optimal pH was between 7.5 and 11.0. For AR, chymotrypsin was pH 7.0; for the remaining digestive regions, it was pH 9.0-11.0. The optimal pH for amylase was 6.0 to 7.5 (all regions), and for lipase, it was between 7.0 and 11.0, with two apparent in vitro activity peaks (PR). HP experimental samples showed low or no chymotrypsin, amylase, and lipase activity. CaCl2 did not affect enzyme activity except for amylase and lipase (only in PR and HP, respectively). The acid proteolytic activity (pH 2.0) found in AR and the proteolytic inhibition by pepstatin suggest the presence of a stomach.

Transition from endogenous to exogenous feeding in longfin yellowtail Seriola rivoliana larvae under simultaneous effects of daily temperature fluctuation and rotifer Brachionus rotundiformis enrichment.

Temperature and nutrition are suggested as the primary factors affecting larval survival during the transition from endogenous to exogenous feeding in fish. However, little is known about its simultaneous impact during this period. In this study, Seriola rivoliana eggs were subjected to a constant 24 °C (CTE) and a daily temperature fluctuation (DTF) between 22.8 and 25.2 °C until oil droplet exhaustion (5.5 days after hatching). On the other hand, marine fish larvae mostly rely on live feed, with certain nutritional deficiencies such as poor long-chain fatty acids. Thus, rotifer Brachionus rotundiformis enrichment was simultaneously evaluated with temperature using three enrichment diets: Ori-green, S.presso, and a Domestic emulsion. For this purpose, the five experimental groups were established in triplicate using six 100-L tanks with three 10-L containers inside (18 experimental units in total). One hundred eggs were incubated, using a green water system, and 10 rotifers mL-1 were offered at mouth opening. After oil droplet exhaustion, survival was only affected by temperature (P < 0.01), being higher at DTF compared to CTE. At the same stage, Domestic emulsion resulted in bigger larvae than Ori-green. In a further assay at 3.7 DAH, the relative expression of the trypsin gene was higher at Domestic emulsion compared to S.presso and Ori-green. This study indicates that daily temperature fluctuation can improve larval performance and low levels of EPA and DHA in Domestic emulsion enriched rotifers were not critical for Seriola rivoliana at first feeding.

In vitro digestibility of specific dsRNA by enzymes of digestive tract of shrimp Litopenaeus vannamei / Digestibilidad in vitro del dsRNA específico por enzimas del tracto digestivo del camarón Litopenaeus vannamei

ABSTRACT Objective. The digestibility of specific dsRNA by action of the enzymes of digestive tract of the whiteleg shrimp Litopenaeus vannamei was determined in vitro. Materials and methods. Digestive enzyme activity (amylase, lipase, protease, DNase and RNase) was measured in the stomach, digestive gland, and anterior, middle, and posterior intestine of juvenile shrimp, and the digestibility of DNA, RNA and the dsRNA-ORF89, specific to WSSV, was determined by in vitro assays, as well as electrophoretic and densitometric analyses. Results. The highest enzymatic activity was found in the digestive gland: amylase (81.41%), lipase (92.60%), protease (78.20%), DNase (90.85%), and RNase (93.14%). The highest digestive capacity against DNA, RNA, and dsRNA was found in the digestive gland (5.11 ng of DNA ​​per minute, 8.55 ng of RNA per minute, and 1.48 ng dsRNA per minute). Conclusions. The highest digestibility of dsRNA-ORF89, specific to WSSV, was found in the digestive gland, whereas the lowest digestibility was observed in the posterior intestine. This is the first report regarding the digestibility of dsRNA-ORF89 by whiteleg shrimp digestive tract enzymes, with potential therapeutic importance in shrimp culture to prevent WSSV disease through balanced feed.

RESUMEN Objetivo. La digestibilidad del dsRNA específico para el virus de la mancha blanca (WSSV) por acción de las enzimas del tracto digestivo del camarón Litopenaeus vannamei fue analizada in vitro. Materiales y métodos. Se midió la actividad de enzimas digestivas (proteasa, amilasa, lipasa, ADNasa y ARNasa) en el estómago, la glándula digestiva, el intestino anterior, medio y posterior en juveniles de camarón patiblanco y se evaluó la digestibilidad de ácidos nucleicos ADN, ARN y dsRNA-ORF89 especifico contra el virus WSSV, por análisis electroforéticos y densitometría. Resultados. La actividad enzimática más alta se encontró en la glándula digestiva del camarón: amilasa (81.41%), lipasa (92.60%), proteasa (78.20%), ADNasa (90.85%) y ARNasa (93.14%). Se evidenció la capacidad digestiva del camarón patiblanco contra el ADN, ARN y dsRNA-ORF89 encontrando en la glándula digestiva la mayor digestión (5.11 ng de ADN por minuto, 8.55 ng de ARN por minuto y 1.48 ng de dsRNA por minuto). Conclusiones. La mayor digestibilidad del dsRNA-ORF89, específico contra el virus WSSV, se encontró en la glándula digestiva y la menor en el intestino posterior. Este es el primer informe relacionado con la digestibilidad del dsRNA-ORF89 por las enzimas del camarón patiblanco con potencial importancia terapéutica en el cultivo de camarón para prevenir la enfermedad del WSSV a través del alimento balanceado.

[Predatory capacity of Macrobrachium tenellum on Aedes aegypti larvae in lab conditions]. / Capacidad depredadora del langostino Macrobrachium tenellum sobre larvas de Aedes aegypti en condiciones de laboratorio.

INTRODUCTION: in the last few years, a lot of importance has been given to natural predators against Aedes aegypti. Several organisms have been studied both in lab and in the field so as to find out their capacity to devour mosquito larvae. High densities of Macrobrachium tenellum are found in natural conditions, it is not aggressive and may stand wide ranges of temperature, rates of salinity and oxygen concentrations. OBJECTIVE: to evaluate the predatory capacity of Macrobrachium tenellum on Aedes aegypti larvae in lab conditions. METHODS: very young Macrobrachium tenellum prawns measuring A(3.0-3.5cm) and B (4.5-5 cm) were used. The mosquito larvae were obtained after hatching of egss from adult females kept in entomological cages. Five, ten, fifteen and twenty Aedes aegypti larvae were placed per treatment per rank, whereas the second bioassays adjusted the number of larvae to 30, 40, 50 and 80 larvae per treatment per rank. RESULTS: Macrobrachium tenellum showed high rate of larval consumption for the two ranks and treatments. In the highest density (80 larvae), the consumption was 95% of larvae at 24 hours for rank A and 100% for rank B. CONCLUSIONS: Macrobrachium tenellum may be considered as a potential biological control agent, due to its abundant presence in natural conditions, its resistance to different environmental conditions and to its voraciousness seen in this study.

Efecto del nivel proteico de la dieta sobre el desarrollo de juveniles de Macrobrachium tenellum (Smith, 1871) / Effect of dietary protein level on the development of juveniles of Macrobrachium tenellum (Smith, 1871)

Objetivo.Evaluar el efecto de cinco niveles de proteína cruda (PC) en alimentos balanceados sobre el crecimiento, sobrevivencia y tasa de conversión alimenticia (FCA) en juveniles de Macrobrachium tenellum. Materiales y métodos. Se alimentó por 60 días a juveniles de M. tenellum (0.31±0.01 g y 32.62±1.10 mm) con niveles de 20, 25, 30, 35 y 40% de PC en el alimento. Los organismos fueron distribuidos al azar en 15 tinas experimentales de 64 L (15 org./tina) bajo condiciones controladas (5.95±0.41 ppm de oxígeno, 29.89±0.72°C, y pH 8.44±0.15) y alimentados con el 10% de su peso vivo. Resultados. El porcentaje de sobrevivencia fue del 98.22±3.96% sin diferencias significativas entre los tratamientos (p>0.05). Los organismos alimentados con un 40% de PC tuvieron un peso significativamente mayor (p<0.05) respecto a los demás tratamientos (cambio de peso de 0.54±0.02g; incremento de peso de 173.60±12.99%; y tasa de crecimiento específico de 1.68±0.08). El FCA fue significativamente mejor (p<0.05) en los organismos alimentados con 35 y 40% de PC (2.85±0.18 y 2.40±0.05, respectivamente) que los demás tratamientos. Conclusiones. Los organismos juveniles de M. tenellum alimentados con niveles altos de proteína (40%), se desarrollaron más rápido que organismos que recibieron una menor concentración de proteína bajo las condiciones experimentales establecidas en este estudio.

Objective. To evaluate the effect of five levels of crude protein (CP) in balanced feed on the survival, growth and feed conversion ratio (FCA) in juveniles of Macrobrachium tenellum. Materials and methods. Juveniles of M. tenellum (0.31±0.01g and 32.62±1.10mm) were fed for 60 days with 20, 25, 30, 35 and 40% of CP in feed. The organisms were randomly distributed in 15 experimental tanks (15 org /tank) under controlled conditions (5.95±0.41 ppm of oxygen, 29.89 ± 0.72 °C, and pH 8.44±0.15) and fed with 10% of its live weight. Results. The survival percentage was 98.22±3.96% with no statistical difference between treatments (p>0.05). The organisms fed with 40% CP in their diet had a significantly higher weight (p<0.05) compared to the other treatments (weight change of 0.54±0.02g; weight increase 173.60±12.99%, and specific growth rate of 1.68±0.08). The FCA was significantly better (p<0.05) in organisms fed with 35 and 40% CP (2.85±0.18 and 2.40±0.05, respectively) than other treatments. Conclusions. Juveniles of M. tenellum fed with high protein levels (40%) developed faster than organisms which received a lower concentration of the protein under the experimental conditions established for this study.

Extraction systems for isolating esterases having interfacial adsorption / Sistemas de extracción para el aislamiento de esterasas con adsorción interfacial

En el presente trabajo se optimizaron las condiciones de extracción de esterasas con actividad en interfaces, a partir de la anémona marina Stichodactyla helianthus y del camarón peneido Litopenaeus vannamei Las esterasas interfaciales, cuya presencia en estas especies había sido informada previamente, presentan características funcionales que las hacen muy atractivas para su empleo industrial. Los homogenados de los animales se trataron con los detergentes Tritón X-100, Tween 20 y Tween 80 en dos concentraciones cada uno: la Concentración Micelar Crítica (CMC) y la mitad de ésta. Además se empleó NaCl 0,5 mol/L y n-butanol a las proporciones 5, 10 y 20%. Cada variante fue comparada con el método tradicional de extracción con agua destilada, que fue tomado como control. Los mejores resultados se obtuvieron empleando n-butanol al 20%, para recuperar las actividades esterasa y fosfolipasa, y al 10%, en el aislamiento de la actividad lipasa. La efectividad de este solvente en el aislamiento de estas enzimas con afinidad por las interfaces lípido/agua, pudiera estar dada por su capacidad para romper los agregados entre estas moléculas y causar la desorción de las mismas a los restos de membrana y tejidos presentes en la preparación.

Interfacial esterases present great functional versatility, making them very attractive molecules for industrial applications. The conditions for extracting interfacial esterases previously detected in the sea anemone Stichodactyla helianthus and the shrimp Litopenaeus vannamei were optimised in this work. Animal homogenates were treated with Triton X-100, Tween 20 and Tween 80 detergents at two different concentrations: critical micellar concentration (CMC) and half of that concentration; 0.5 mol/L NaCl and n-butanol at 5%, 10% and 20% v/v ratios were also tested. Each procedure was compared to the control extraction method using distilled water. The best results were obtained with 20% n-butanol for recovering esterase and phospholipase activity whilst 10% n-butanol extraction was the most effective for lipase activity isolation. This solvent’s suitability for isolating interface-activated enzymes could be explained by its ability to dissociate biomolecule aggregates and cause enzyme desorption from the membranes and tissues remaining in the preparation.