Jorge Hernández-López

Activation of the prophenoloxidase system of the brown shrimp Penaeus californiensis Holmes)

Abstract The prophenoloxidase (proPO) system of the brown shrimp Penaeus californiensis was activated using different preparations and inductors. In shrimp, this system is confined inside the hemocytes and can be obtained by lysing or centrifuging the cells. In both cases, the shrimp proPO system showed a high spontaneous activation because the proPO activating enzyme was also released. In addition, the shrimp proPO system could be activated by laminarin, but not by bacterial LPS. Although SDS and other chemicals are also capable of activating shrimp proPO, this activation proceeds in different way since it can not be inhibited by proteinase inhibitors.

cDNA cloning of the lysozyme of the white shrimp Penaeus vannamei.

Lysozyme, an antibacterial protein, has been implicated in innate immunity in invertebrates, but its activity in shrimp remained to be determined. We cloned the white shrimp lysozyme cDNA using a PCR strategy and detected its activity in haemocytes using a lytic-zone assay against Micrococcus luteus. The cloning was based on a reported EST (dbEST BE18831). The deduced amino acid sequence resulted in 150 amino with 46% identity to hen egg white lysozyme. RT-PCR was used to detect lysozyme mRNA in haemocytes. Analysis of the amino acid sequence of the shrimp lysozyme showed that it belongs to the C-type family of lysozymes. Furthermore, the lysozyme amino acid sequence contained extra residues at its C-terminus, which are characteristic of marine invertebrates. This information will be useful in future studies on the molecular mechanisms of immunity in marine invertebrates.

Prophenoloxidase from brown shrimp (Penaeus californiensis) hemocytes

Prophenoloxidase (proPO) was purified from blood cells of the brown shrimp Penaeus californiensis by ultracentrifugation and dye affinity chromatography. The isolated proPO is a 114-kDa monomeric protein as determined by SDS-PAGE. This protein can be hydrolyzed by proteinases, producing a 107-kDa active phenoloxidase (PO). The isoelectric point for both protein forms was 7.35. The PO reaction using L-DOPA as substrate, has an optimum pH of 8, and was poorly inhibited by sodium azide, thiourea and EDTA, but strongly inhibited by diethyl thiocarbamate. According to the substrate affinity and inhibition characteristics, this phenoloxidase was classified as a tyrosinase-like phenoloxidase. Purified proPO was not activated by bacterial lipopolysaccharides or beta-glucans.

EFFECT OF CALCIUM ON THE PROPHENOLOXIDASE SYSTEM ACTIVATION OF THE BROWN SHRIMP (PENAEUS CALIFORNIENSIS, HOLMES)

Abstract The soluble prophenoloxidase (proPO) system of the brown shrimp (Panaeus californiensis) was obtained by centrifuging hemocytes (15,000 g) in low salt buffers. In these samples, proPO spontaneous activation was observed when calcium (>5 mM) was present in the buffers. Stable samples can be obtained in divalent cation-free buffer, and the sole addition of Ca2+ resulted in the proPO activation. In contrast, Ca2+ was not able to induce spontaneous activation in samples depleted of proPO activating enzyme (PPAE) obtained by passing the sample through a Blue Sepharose column. In addition, protease inhibitors like melittin and soybean trypsin inhibitor blocked the Ca2+-induced spontaneous activation, indicating this cation is required for the proPO proteolytic activation. Although Ca2+-induced spontaneous activation was not observed with intact hemocytes, this cation was necessary for the activation of proPO by β-glucans. Plasma Ca2+ concentration of the brown shrimp is 8 mM, as determined by absorption spectroscopy. Thus, these results suggest Ca2+ activates PPAE and then PPAE transforms proPO to an active form when both proteins are released from the cells after the stimulus.

Detection of white spot syndrome virus in filtered shrimp-farm water fractions and experimental evaluation of its infectivity in Penaeus (Litopenaeus) vannamei.

Detection of white spot syndrome virus in filtered shrimp-farm water fractions and experimental evaluation of its infectivity in Penaeus (Litopenaeus) vannamei Héctor M. Esparza-Leal , César M. Escobedo-Bonilla , Ramón Casillas-Hernández , Píndaro Álvarez-Ruíz , Guillermo Portillo-Clark , Roberto C. Valerio-García , Jorge Hernández-López , Jesús Méndez-Lozano , Norberto Vibanco-Pérez , Francisco J. Magallón-Barajas d,⁎

Experimental evidence of metabolic disturbance in the white shrimp Penaeus vannamei induced by the Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV).

The Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) is a single-stranded DNA virus that infects several penaeid shrimp species, provoking economic losses in farmed shrimp populations estimated at several million of dollars. Furthermore, IHHNV has historically been considered an important threat for wild shrimp populations, but its real measurable impact remains unknown. Currently no treatments are available against IHHNV, and research to develop potential antiviral strategies depends on a detailed understanding of the viral life cycle. However, the exact pathophysiological events underlying the development of metabolic changes in IHHNV-infected shrimp are still unknown. Thus, the biochemical changes caused by the IHHNV infection in plasma and hepatopancreas of the economically important shrimp species Penaeus vannamei were evaluated. Glucose, lactate, total protein, glycogen, triacylglycerides, cholesterol, and total lipids were measured in healthy and IHHNV-infected shrimp. Significant changes were observed in energy substrates (glucose, lactate, triacylglycerides and cholesterol), in plasma and hepatopancreas. These changes may indicate a temporal sequestration of the host-cell metabolic pathways by the virus to maximize its replication and propagation.

Quantification of pathogenic marine vibrio using membrane filter technique

Abstract A rapid and simple methodology based on a membrane filter technique was designed to detect and quantify marine vibrios involved in fish and shellfish diseases, as well as human gastrointestinal disorders. By placing the membrane in TCBS medium and incubating it at 40–42°C, it was possible to differentiate between pathogenic and non-pathogenic vibrios. A presumptive identification was also done by growing cultures at different salinities and by using a few biochemical tests.

Specific diversity of the entomopathogenic fungi Beauveria and Metarhizium in Mexican agricultural soils.

Prior knowledge of the local population structure of entomopathogenic fungi is considered an important requisite when developing microbial control strategies against major pests of crops such as white grubs. An extensive survey in the estate of Guanajuato, one of the main agricultural regions of Mexico, was carried out to determine the abundance and diversity of entomopathogenic fungi in soil. Soil collected from 11 locations was baited for entomopathogenic fungi using Galleria mellonella. In addition, all isolates were morphologically identified and selected isolates of Beauveria and Metarhizium isolates identified using Bloc and ITS or Elongation Factor 1-α and ITS sequence information respectively. Genotypic diversity was then studied using microsatellite genotyping. The proportion of isolates belonging to each genus varied amongst all locations. The species Beauveria bassiana, B. pseudobassiana and Metarhizium robertsii were found, with B. bassiana being the most abundant and widely distributed. Microsatellite genotyping showed that the 36 B. bassiana isolates were grouped in 29 unique haplotypes, but with no separation according to geographical origin.

Efecto de la adición del rotífero Brachionus rotundiformis (Tschugunoff, 1921) sobre la calidad del agua y la producción, en cultivos super-intensivos de camarón blanco del Pacífico Litopenaeus vannamei (Boone, 1931)

Se evaluo la efectividad del rotifero Brachionus rotundiformis como alimento natural exogeno combinado con alimento balanceado sobre los parametros de produccion de Litopenaeus vannamei y la calidad del agua. Se probaron diferentes concentraciones de rotiferos: 0, 5, 10, 15 y 20 rotiferos mL-1 durante 45 dias. No se encontraron diferencias en los niveles de fosfatos entre los tratamientos. El pH fue estadisticamente menor en los tratamientos con 15 y 20 rotiferos mL-1. Los nitratos, nitritos y amonio fueron significativamente menores en los tratamientos con baja concentracion de rotiferos (0 y 5 mL-1), mientras que los valores mas elevados se presentaron en las concentraciones mas altas (15 y 20 mL-1) sin que estas llegaran a ser toxicas. El peso ganado, biomasa, supervivencia y tasa de crecimiento especifico de L. vannamei fueron significativamente mayores en los tratamientos con mayor numero de rotiferos (15 y 20 mL-1), mientras que los valores mas bajos se encontraron en el tratamiento control (0 mL-1). El factor de conversion alimenticia (FCA) fue estadisticamente menor en el tratamiento con mayor concentracion de rotiferos, mientras que el factor de conversion alimenticia (FCA) mas alto se encontro en el tratamiento control. Se concluye que el uso del rotifero B. rotundiformis en altas concentraciones mejora los parametros de produccion del camaron blanco del Pacifico sin llegar a deteriorar de manera importante la calidad del agua

Role of fumonisin B1 on the immune system, histopathology, and muscle proteins of white shrimp (Litopenaeus vannamei)

White shrimps, Litopenaeus vannamei, were tested in two indoor trials to determine the effect of fumonisin B1 on (i) immune response, (ii) histopathology, and, (iii) muscle proteins. Trial 1: (0, 0.5, 0.75 and 1.0μg/g of FB1 levels, 18-day duration; shrimp 5-6g) to evaluate the FB1 effect on the immune system and histopathology response. Trial 2: (0.0, 0.5, 0.75 and 1.0μg/g of FB1 levels, 16-day duration; shrimp 5-6g) to detect FB1 effect on muscle proteins. Prophenoloxidase activity was affected by all FB1 concentrations tested. Both, total haemocyte count and phenoloxidase activity decreased by the 18th day in shrimp exposed to FB1. Marked histological changes in the hepatopancreas of shrimp fed on diet containing FB1, at the all FB1 levels tested, as well as a necrotic tissue were observed. Changes in both, electrophoretic patterns and thermodynamic properties of myosin extracted from shrimp exposed to FB1 were also observed.