Adriana Muhlia-Almazán

Influence of molting and starvation on the synthesis of proteolytic enzymes in the midgut gland of the white shrimp Penaeus vannamei

We investigated the effect of starvation as a stimulant of the digestive system on digestive proteinase activities in the white shrimp Penaeus vannamei. The starved organisms were sampled periodically according to the molting stage and compared with a continuously fed group. Molting stage was included as an independent variable. Most analyzed variables, except for trypsin, were more affected by starvation than by molting, indicating that starvation is a stimulant that masks the effect of molting and showing that food or alimentary stress is more conspicuous than physiological ones. We found that starvation is a stimulant that surpasses the effect of molting, and because it affects the activity of digestive proteinases, studies of starving organisms in combination with tools of molecular biology, can be a helpful working model in the understanding of mechanisms of regulation of digestive enzyme activity. In the starved organisms, trypsin and chymotrypsin activities were similar, suggesting dependence of one to the other. Changes in proteolytic activities and the number of protein bands in electrophoresis showed evidence of synthesis regulation in the midgut gland of white shrimp.

Invertebrate trypsins: a review

Food protein hydrolysis, a crucial step in digestion, is catalyzed by trypsin enzymes from the digestive apparatus of invertebrates. Trypsin appeared early in evolution and occurs in all phyla and, in the digestive systems of invertebrates, it became the most abundant proteinase. As in vertebrates, invertebrate trypsin is also present in several forms (isoenzymes). Its physiological importance in food protein digestion in several invertebrate species has emerged with compelling evidence; and several other physiological functions, such as regulation of digestive functions, are now settled. Recent advances in the knowledge of invertebrate trypsin synthesis, regulation, genetics, catalytic characteristics; structure, evolution, as well as inhibition, especially in non-Drosophilidae insects and in some crustaceans are reviewed. Most of the existing information is largely based on the use of several tools, including molecular techniques, to answer many still open questions and solve medical, agricultural, and food quality problems.

Differential expression of trypsin mRNA in the white shrimp (Penaeus vannamei) midgut gland under starvation conditions

Regulation of the expression of trypsin encoding genes in juveniles white shrimp (Penaeus vannamei) was studied during starvation for up to 120 h. Since molting is now recognized as a physiologically important process in penaeids, specimens were selected according to molt stage. Starvation led to immediate weight loss and a sharp decrease in hepatosomatic index. Specific DNA probes for trypsin were synthesized and trypsin mRNA concentration was evaluated by dot blot hybridization. Trypsin-encoding RNA levels were strongly influenced by starvation, resulting in an increase after 24 h of starvation, followed by a steep decline to lower level compared to those obtained in fed shrimp. A possible mechanism for regulation of trypsin activity during transcription is proposed. The biological implications of our findings for transcriptional regulation of trypsin mRNA during starvation are discussed.

Shrimp thioredoxin is a potent antioxidant protein

Thioredoxin (TRX) is a main component of the redox homeostasis machinery in the cell and it is required for ribonucleotide reductase function among others. In invertebrates, the redox balance is compromised during disease and changes in the physiological state and it is one of the components of the innate immune response. In this work, the shrimp (Litopenaeus vannamei) LvTRX cDNA was sequenced, cloned and over-expressed in bacteria to further characterize the function of the recombinant protein. LvTRX was able to reduce insulin disulfides and it was a better antioxidant compared to reduced glutathione and ascorbic acid, by means of the Trolox Equivalent Antioxidant Capacity (TEAC) assay. Interestingly, LvTRX contains aside of the canonical active site CXXC disulfide motif, one Cys (C73) residue in the interface of a putative dimer previously reported for human TRX. Using qRT-PCR, we found that shrimp LvTRX is mainly expressed in gills and pleopods; the variation of LvTRX mRNA upon hypoxia and re-oxygenation is not statistically significant. LvTRX stands as an important antioxidant that must be considered in future physiological and immune challenges studies.

Effects of dietary protein on the activity and mRNA level of trypsin in the midgut gland of the white shrimp Penaeus vannamei

Protein food modulates the activity of proteases of the midgut gland of Penaeus vannamei. Shrimp fed with food containing 15, 30 and 50% protein exhibited differences in trypsin and chymotrypsin activity and trypsin mRNA levels. Shrimp fed with 30% protein showed higher trypsin and chymotrypsin activities than those fed 15 or 50% protein. An additional paralogue trypsin was observed with electrophoretic analysis in shrimp fed 30% protein. Shrimp fed 30% protein showed the highest trypsin to mRNA concentration, suggesting that trypsin genes expression is regulated transcriptionally.

Gene expression and protein levels of thioredoxin in the gills from the whiteleg shrimp (Litopenaeus vannamei) infected with two different viruses: the WSSV or IHHNV.

The thioredoxin (TRX) system in crustaceans has demonstrated to act as a cell antioxidant being part of the immune response by dealing with the increased production of reactive oxygen species during bacterial or viral infection. Since the number of marine viruses has increased in the last years significantly affecting aquaculture practices of penaeids, and due to the adverse impact on wild and cultured shrimp populations, it is important to elucidate the dynamics of the shrimp response to viral infections. The role of Litopenaeus vannamei thioredoxin (LvTRX) was compared at both, mRNA and protein levels, in response to two viruses, the white spot syndrome virus (WSSV) and the infectious hypodermal and hematopoietic necrosis virus (IHHNV). The results confirmed changes in the TRX gene expression levels of WSSV-infected shrimp, but also demonstrated a more conspicuous response of TRX to WSSV than to IHHNV. While both the dimeric and monomeric forms of LvTRX were detected by Western blot analysis during the WSSV infection, the dimer on its reduced form was only detected through the IHHNV infectious process. These findings indicate that WSSV or IHHNV infected shrimp may induce a differential response of the LvTRX protein.

Cathepsin B from the white shrimp Litopenaeus vannamei: cDNA sequence analysis, tissues-specific expression and biological activity

Cathepsin B is a cystein proteinase scarcely studied in crustaceans. Its function has not been clearly described in shrimp species belonging to the sub-order Dendrobranchiata, which includes the white shrimp Litopenaeus vannamei and other species from the Penaeidae family. Studies on vertebrates suggest that these lysosomal enzymes intracellularly hydrolize protein, as other cystein proteinases. However, the expression of the gene encoding the shrimp cathepsin B in the midgut gland was affected by starvation in a similar way as other digestive proteinases which extracellularly hydrolyze food protein. In this study the white shrimp L. vannamei cathepsin B (LvCathB) cDNA was sequenced, and characterized. Its gene expression was evaluated in various shrimp tissues, and changes in the mRNA amounts were compared with those observed on other digestive proteinases from the midgut gland during starvation. By using qRT-PCR it was found that LvCathB is expressed in most shrimp tissues except in pleopods and eye stalk. Changes on LvCathB mRNA during starvation suggest that the enzyme participates during intracellular protein hydrolysis but also, after food ingestion, it participates in hydrolyzing food proteins extracellularly as confirmed by the high activity levels we found in the gastric juice and midgut gland of the white shrimp.

The complete mitochondrial genomes of the yellowleg shrimp Farfantepenaeus californiensis and the blue shrimp Litopenaeus stylirostris (Crustacea: Decapoda).

Mitochondria play key roles in many cellular processes. Description of penaeid shrimp genes, including mitochondrial genomes are fairly recent and some are focusing on commercially important shrimp as the Pacific shrimp Litopenaeus vannamei that is being used for aquaculture not only in America, but also in Asia. Much less is known about other Pacific shrimp such as the yellowleg shrimp Farfantepenaeus californiensis and the blue shrimp Litopenaeus stylirostris. We report the complete mitogenomes from these last two Pacific shrimp species. Long DNA fragments were obtained by PCR and then used to get internal fragments for sequencing. The complete F. californiensis and L. stylirostris mtDNAs are 15,975 and 15,988 bp long, containing the 37 common sequences and a control region of 990 and 999 bp, respectively. The gene order is identical to that of the tiger shrimp Penaeus monodon. Secondary structures for the 22 tRNAs are proposed and phylogenetic relationships for selected complete crustacean mitogenomes are included. Phylogenomic relationships among five shrimp show strong statistical support for the monophyly of the genus across the analysis. Litopenaeus species define a clade, with close relationship to Farfantepenaeus, and both clade with the sister group of Penaeus and Fenneropenaeus.

The function of mitochondrial FOF1 ATP-synthase from the whiteleg shrimp Litopenaeus vannamei muscle during hypoxia

The effect of hypoxia and re-oxygenation on the mitochondrial complex F(O)F(1)-ATP synthase was investigated in the whiteleg shrimp Litopenaeus vannamei. A 660 kDa protein complex isolated from mitochondria of the shrimp muscle was identified as the ATP synthase complex. After 10h at hypoxia (1.5-2.0 mg oxygen/L), the concentration of L-lactate in plasma increased significantly, but the ATP amount and the concentration of ATPβ protein remained unaffected. Nevertheless, an increase of 70% in the ATPase activity was detected, suggesting that the enzyme may be regulated at a post-translational level. Thus, during hypoxia shrimp are able to maintain ATP amounts probably by using some other energy sources as phosphoarginine when an acute lack of energy occurs. During re-oxygenation, the ATPase activity decreased significantly and the ATP production continued via the electron transport chain and oxidative phosphorylation. The results obtained showed that shrimp faces hypoxia partially by hydrolyzing the ATP through the reaction catalyzed by the mitochondrial ATPase which increases its activity.

Invertebrates Mitochondrial Function and Energetic Challenges

Oliviert Martinez-Cruz1, Arturo Sanchez-Paz2, Fernando Garcia-Carreno3, Laura Jimenez-Gutierrez1, Ma. de los Angeles Navarrete del Toro3 and Adriana Muhlia-Almazan1 1Molecular Biology Laboratory, Centro de Investigacion en Alimentacion y Desarrollo (CIAD), Hermosillo, Sonora, 2Laboratorio de Sanidad Acuicola, Centro de Investigaciones Biologicas del Noroeste (CIBNOR), Hermosillo, Sonora, 3Biochemistry Laboratory, Centro de Investigaciones Biologicas del Noroeste (CIBNOR), La Paz, Mexico