Tandem Mass Tag-based proteomic analysis of protein changes in superchilled crayfish (Procambarus clarkii) presoaked with carrageenan oligosaccharides.

To assess the effectiveness of carrageenan oligosaccharides (COs) in enhancing superchilling storage of crayfish, the physicochemical features of muscle and protein abundance in the refrigerated sample (RS), superchilled sample (SS) and COs soaked superchilled sample (CS) were evaluated. Microstructural and SDS-PAGE analyses suggested that CS exhibited fewer pores, with a microstructure and protein subunits distribution more similar to RS. Tandem Mass Tags quantitative proteomic analysis revealed 66 up-regulated differentially abundant proteins (DAPs) in the CS vs. SS batch, including myosin light chain 2, neural cadherin, integrin beta, lectin-like protein, toll-1, reticulon-1, and moesin/ezrin/radixin homolog 1, which facilitate cells adhesion and maintain membrane/cytoskeleton integrity. Eukaryotic Clusters of Orthologous Groups results confirmed that COs treatment increased the stability of crayfish myofibrillar proteins by up-regulating DAPs, which were concentrated in functional categories such as "posttranslation modification, protein turnover, chaperones", "signal transduction mechanisms", "energy production and conversion", and "cytoskeleton".

A transporter that allows phosphate ions to control the polymorph of exoskeletal calcium carbonate biomineralization.

The SLC20A2 transporter supplies phosphate ions (Pi) for diverse biological functions in vertebrates, yet has not been studied in crustaceans. Unlike vertebrates, whose skeletons are mineralized mainly by calcium phosphate, only minute amounts of Pi are found in the CaCO3-mineralized exoskeletons of invertebrates. In this study, a crustacean SLC20A2 transporter was discovered and Pi transport to exoskeletal elements was studied with respect to the role of Pi in invertebrate exoskeleton biomineralization, revealing an evolutionarily conserved mechanism for Pi transport in both vertebrates and invertebrates. Freshwater crayfish, including the study animal Cherax quadricarinatus, require repeated molt cycles for their growth. During the molt cycle, crayfish form transient exoskeletal mineral storage organs named gastroliths, which mostly contain amorphous calcium carbonate (ACC), an unstable polymorph long-thought to be stabilized by Pi. RNA interference experiments via CqSLC20A2 dsRNA injections reduced Pi content in C. quadricarinatus gastroliths, resulting in increased calcium carbonate (CaCO3) crystallinity and grain size. The discovery of a SLC20A2 transporter in crustaceans and the demonstration that knocking down its mRNA reduced Pi content in exoskeletal elements offers the first direct proof of a long-hypothesized mechanism by which Pi affects CaCO3 biomineralization in the crustacean exoskeleton. This research thus demonstrated the distinct role of Pi as an amorphous mineral polymorph stabilizer in vivo, suggesting further avenues for amorphous biomaterial studies. STATEMENT OF SIGNIFICANCE: • Crustaceans exoskeletons are hardened mainly by CaCO3, with Pi in minute amounts • Pi was hypothesized to stabilize exoskeletal amorphous mineral forms in vivo • For the first time, transport protein for Pi was discovered in crayfish • Transport knock-down resulted in exoskeletal CaCO3 crystallization and reduced Pi.

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.

La langosta de agua dulce Cherax quadricarinatus es una especie tropical de gran interés para la acuicultura. Se midió la concentración de vitelogenina (Vg) en hemolinfa, ovario y hepatopáncreas de hembras adultas de esta especie, por medio de ELISA. Las mediciones fueron hechas en los cuatro períodos del ciclo reproductivo: pre-reproductivo, reproductivo medio, reproductivo tardío y post-reproductivo. Se detectaron variaciones significativas en los niveles de Vg tanto en hemolinfa como en hepatopáncreas, se observó el mayor valor durante el período reproductivo medio. Además, tales variaciones se correlacionaron positivamente con el índice hepatosomático. Se observó además una correlación positiva de los niveles de Vg entre hepatopáncreas y ovario. Estos resultados apoyan evidencias previas sobre el papel central del hepatopáncreas como sitio de síntesis de Vg, en esta especie, y también enfatizan la importancia de la hemolinfa para el transporte de la Vg del hepatopáncreas al ovario. Para propósitos de acuicultura, la medición de Vg en hemolinfa podría ser utilizada como un método no lesivo, con el fin de constatar la actividad reproductiva de hembras de C. quadricarinatus.

Óxidos de colesterol en langostinos frescos y congelados, crudos y a la plancha / Cholesterol oxidation products in fresh and frozen shrimps, raw and grilled

Los óxidos de colesterol (COPs) se relacionan con diferentes efectos tóxicos entre los que destacan su implicación en los procesos de aterosclerosis. Se estudió la presencia de óxidos de colesterol en langostinos comercializados en fresco y en congelación, tanto en crudo, como sometidos a una tecnología culinaria habitual (plancha). La determinación se realizó por cromatografía de gases acoplada a espectrometría de masas (CG-EM). En los langostinos frescos se detectaron todos los COPs analizados con excepción del 7 α-hidroxicolesterol, presentando una cantidad total de 33,15 µg COPs/g grasa. Por el contrario, en los langostinos comercializados congelados sólo se detectaron el 7-ketocolesterol y el 7ß-hidroxicolesterol, dando lugar a una cantidad total de 2,38 µg COPs/g grasa. Estos resultados indican la gran efectividad de la comercialización bajo condiciones de congelación de este tipo de alimentos en cuanto a ralentizar la formación de COPs. El tratamiento culinario incrementó el contenido de COPs en ambos tipos de langostinos, alcanzando 55,43 µg COPs/g grasa en los frescos y sólo 13,06 µg COPs/g grasa en los congelados (AU)

Cholesterol oxidation products (COPs) have been related to different toxic effects, being the atherosclerotic process one of the best known. The presence of cholesterol oxides in freshly and frozenly commercialised shrimps, both raw and grilled, was studied. The determination was made by gas chromatography-mass spectrometry (GCMS). Fresh shrimps showed significant amounts of all analysed COPs, except for 7α-hydroxycholesterol, accounting in total for 33.15 µg COPs/g fat. In contrast, in frozen commercialised shrimps only 7-ketocholesterol and 7ß-hydroxycholesterol were detected. These results point out the great effectiveness of the commercialisation of this type of products under freezing, in terms of to the minimisation of the COPs formation. The cooking method (grilling) increased the COPs content in both types of shrimps, reaching 55.43 µg COPs/g fat in fresh shrimps and only 13.06 µg COPs/g fat in frozen ones (AU)