Understanding the Digestive Peptidases from Crustaceans: from Their Biochemical Basis and Classical Perspective to the Biotechnological Approach.

Scientific studies about decapod crustaceans' digestive physiology have increased, being an important topic with novel results in the last years. This revision aims to show how the study of crustacean peptidases has evolved, from the classical biochemical characterization studies to the assessment of their usefulness in biotechnological and industrial processes, with emphasis on commercial species of interest to world aquaculture and fisheries. First studies determined the proteolytic activity of the midgut gland crude extracts and evaluated the optimum biochemical properties of specific enzymes. Peptidase's identity was determined using inhibitors and specific protein substrates on tube tests and electrophoresis gels. Later, various studies focused on the characterization of purified peptidases and their gene expression. Recently, the integrated mechanisms of enzyme participation during the digestive process of food protein have been established using novel techniques. Scientific research has revealed some of the potential biotechnological applications of crustacean peptidases in the food industry and other processes. However, the knowledge field is enormous, and there is much to explore and study in the coming years.

Energy reserves mobilization: Strategies of three decapod species.

In food deprivation assays, several different responses have been observed in crustaceans. However, studying energy reserves utilization among more than one species during the same starvation period has not yet been performed, particularly to discern whether the responses are due to intrinsic and/or environmental factors. We hypothesize that decapod species with similar feeding habits have the same strategies in the use of energetic reserves during starvation, even though they inhabit different environments. The aim of this study was to compare the energy reserves mobilization of three decapods species (Cherax quadricarinatus, Palaemon argentinus and Munida gregaria) with similar feeding habits, exposed to similar food deprivation conditions. The crayfish, shrimp and squat-lobster were experimentally kept at continuous feeding or continuous starvation throughout 15 days. Every 3rd day, the midgut gland index (MGI), and the glycogen, lipid and protein contents were measured in the midgut gland (MG) and pleon muscle. Palaemon argentinus mobilized more reserves during starvation, followed by C. quadricarinatus, and the last M. gregaria. The starved shrimps presented low MGI, whereas MG showed a reduction in glycogen (from day 6 to 15), lipid (from day 3 to 15), and protein levels (at day 9 and 15) while in their muscle, lipid reserves decreased at days 3 and 6. In C. quadricarinatus, the most affected parameters in the MG were MGI, glycogen (from day 6 to 15), and lipids (at day 12 and 15). In the MG of M. gregaria only the glycogen was reduced during fasting from 3 to 15 days. Even though the three studied species have similar feeding habitats, we found that their energetic profile utilization is different and it could be explained by the habitat, life span, temperature, organ/tissue, and metabolism of the species. Our results may be useful to understand the several different responses of crustaceans during starvation.

Liposoluble vitamins in Crustacean feed: Metabolic and Histological responses.

Vitamins are vital for normal growth and survival of living organisms and they are distributed in feedstuffs in small quantities. This review is focused on the liposoluble vitamins (A, D, E and K) in the diets and metabolic responses of the Argentine penaeoid shrimps Pleoticus muelleri and Artemesia longinaris, distributed along the South American coast line. Growth, survival and histological analyses serve as indicators of the nutritional value derived from vitamin deficiency. Liposoluble vitamins are also related to stress, antioxidant defense and immune response of shrimps. Effective diet for shrimp culture that provide not only macronutrients including protein and lipid but also micronutrients such as vitamins for optimal growth is an ever improving subject. This review may help formulating suitable feeds for shrimps.

Long-Term Starvation and Posterior Feeding Effects on Biochemical and Physiological Responses of Midgut Gland of Cherax quadricarinatus Juveniles (Parastacidae).

We investigated the effect of long-term starvation and posterior feeding on energetic reserves, oxidative stress, digestive enzymes, and histology of C. quadricarinatus midgut gland. The crayfish (6.27 g) were randomly assigned to one of three feeding protocols: continuous feeding throughout 80 day, continuous starvation until 80 day, and continuous starvation throughout 50 day and then feeding for the following 30 days. Juveniles from each protocol were weighed, and sacrificed at day 15, 30, 50 or 80. The lipids, glycogen, reduced glutathione (GSH), soluble protein, lipid peroxidation (TBARS), protein oxidation (PO), catalase (CAT), lipase and proteinase activities, and histology were measured on midgut gland. Starved crayfish had a lower hepatosomatic index, number of molts, specific growth rate, lipids, glycogen, and GSH levels than fed animals at all assay times. The starvation did not affect the soluble protein, TBARS, PO levels and CAT. In starved juveniles the lipase activity decreased as starvation time increased, whereas proteinase activity decreased only at day 80. The histological analysis of the starved animals showed several signs of structural alterations. After 30 days of feeding, the starved-feeding animals exhibited a striking recovery of hepatosomatic index, number of molts, lipids and glycogen, GSH, lipase activity and midgut gland structure.

The shrimp mitochondrial FoF1-ATPase inhibitory factor 1 (IF1).

The whiteleg shrimp species Litopenaeus vannamei is exposed to cyclic changes of the dissolved oxygen concentration of seawater and must neutralize the adverse effects of hypoxia by using ATP as energy source. In crustaceans, the mitochondrial FOF1-ATP synthase is pivotal to the homeostasis of ATP and function prevalently as a FOF1-ATPase. Hitherto, it is unknown whether these marine invertebrates are equipped with molecules able to control the FOF1-ATPase inhibiting the ATP consumption. In this study, we report two variants of the mitochondrial FOF1-ATPase Inhibitory Factor 1 (IF1) ubiquitously expressed across tissues of the Litopenaeus vannamei transcriptome: the IF1_Lv1 and the IF1_Lv2. The IF1_Lv1, with a full-length sequence of 550 bp, encodes a 104 aa long protein and its mRNA amounts are significantly affected by hypoxia and re-oxygenation. The IF1_Lv2, with a sequence of 654 bp, encodes instead for a protein of 85 aa. Both proteins share a 69 % homology and contain a conserved minimal inhibitory sequence (IATP domain) along with a G-rich region on their N-terminus typical of the invertebrate. In light of this characterization IF1 is here discussed as an adaptive mechanism evolved by this marine species to inhibit the FOF1-ATPase activity and avoid ATP dissipation to thrive in spite of the changes in oxygen tension.

In vivo and in vitro protein digestibility of formulated feeds for Artemesia longinaris (Crustacea, Penaeidae)

This study was undertaken to determine the in vivo crude protein apparent digestibility in the prawn Artemesia longinaris, using feeds with 0.25 percent of chromic oxide and animal (fish meal, meat and bone meal and squid protein concentrate) and plant (soybean meal) ingredients. Three replicate groups of prawn were fed and the feces were collected. The rate of protein hydrolysis was measured in vitro using midgut gland enzyme extract from the prawns fed the respective feeds and was compared with those found with enzyme extract of wild prawn. The in vivo apparent digestibility coefficients showed significant differences among the feeds (P<0.05). Fish meal feed presented the highest digestibility (92 percent); intermediate digestibility (83 percent) was found for meat and bone meal feed, and the less digestible feed (63 percent) was that containing soybean meal and squid proteins concentrate. No significant differences in the in vitro protein digestibility were found among the experimental feeds. The results indicated the limitation of in vitro enzyme assays and that it should be complemented by in vivo studies.

O objetivo do presente trabalho foi determinar a digestibilidade aparente in vivo da proteína bruta de ingredientes de origem animal (farinhas de peixe, osso e carne e concentrado de proteína de lula) e ingredientes vegetais (farinha de soja) em camarões Artemesia longinaris utilizando rações contendo 0,25 por cento de óxido de cromo. Três grupos de camarões, utilizados como replicatas, foram alimentados e as fezes coletadas. A velocidade de hidrólise da proteína de cada ração foi medida in vitro utilizando extrato enzimático da glândula do intestino médio dos camarões alimentados com a ração correspondente e foi comparado com aqueles obtidos com o extrato enzimático de camarões selvagens. Os coeficientes de digestibilidade aparente in vivo mostraram diferenças significativas entre as rações testadas (P<0,05). A farinha de peixe apresentou a maior digestibilidade (92 por cento), enquanto valores intermediários de digestibilidade (83 por cento) foram encontrados para a farinha de carne e ossos. A ração contendo farinha de soja e concentrado de proteína de lula resultou em menor digestibilidade (63 por cento). Não houve diferença significativa entre os valores de digestibilidade in vitro para as rações testadas. Estes resultados indicam a limitação inerente dos ensaios enzimáticos in vitro, os quais poderiam ser complementados com estudos in vivo.