Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns.

BACKGROUND: Anisakis simplex sensu stricto and Anisakis pegreffii are sibling species of nematodes parasitic on marine mammals. Zoonotic human infection with third stage infective larvae causes anisakiasis, a debilitating and potentially fatal disease. These 2 species show evidence of hybridisation in geographical areas where they are sympatric. How the species and their hybrids differ is still poorly understood. RESULTS: Third stage larvae of Anisakis simplex s.s., Anisakis pegreffii and hybrids were sampled from Merluccius merluccius (Teleosti) hosts captured in waters of the FAO 27 geographical area. Specimens of each species and hybrids were distinguished with a diagnostic genetic marker (ITS). RNA was extracted from pools of 10 individuals of each taxon. Transcriptomes were generated using Illumina RNA-Seq, and assembled de novo. A joint assembly (here called merged transcriptome) of all 3 samples was also generated. The inferred transcript sets were functionally annotated and compared globally and also on subsets of secreted proteins and putative allergen families. While intermediary metabolism appeared to be typical for nematodes in the 3 evaluated taxa, their transcriptomes present strong levels of differential expression and enrichment, mainly of transcripts related to metabolic pathways and gene ontologies associated to energy metabolism and other pathways, with significant presence of excreted/secreted proteins, most of them allergens. The allergome of the 2 species and their hybrids has also been thoroughly studied; at least 74 different allergen families were identified in the transcriptomes. CONCLUSIONS: A. simplex s.s., A. pegreffi and their hybrids differ in gene expression patterns in the L3 stage. Strong parent-of-origin effects were observed: A. pegreffi alleles dominate in the expression patterns of hybrids albeit the latter, and A. pegreffii also display significant differences indicating that hybrids are intermediate biological entities among their parental species, and thus of outstanding interest in the study of speciation in nematodes. Analyses of differential expression based on genes coding for secreted proteins suggests that co-infections presents different repertoires of released protein to the host environment. Both species and their hybrids, share more allergen genes than previously thought and are likely to induce overlapping disease responses.

Ultrastructural changes and structure and mobility of myowater in frozen-stored hake (Merluccius merluccius L.) muscle: relationship with functionality and texture.

The ultrastructural changes and the main Raman spectral features of water (3100-3500 and 50-600 cm(-)(1) ranges) in frozen-stored hake were studied with the aim of connecting these changes with loss of some functional properties such as water holding capacity, and with modifications of muscle texture. The following results were obtained: (a) The changes in the spaces between myofibrils can be related to modifications of shear resistance. (b) The behavior of the strong 160 cm(-)(1) band can be related to conformational transitions of muscle proteins, to changes in the structure of muscle water, and/or to alterations in protein-water interactions. (c) There were intensity changes in the nu(s)(OH) band that may be attributable to transfer of water to larger spatial domains during frozen storage.

Structural changes of hake (Merluccius merluccius L.) fillets: effects of freezing and frozen storage.

Structural changes in hake (Merluccius merluccius L.) fillets as affected by freezing method and frozen storage temperature have been studied through Raman spectroscopy and related to changes in texture and functionality. Changes in protein secondary structure were observed due to storage temperature, accompanied by changes in apparent viscosity and shear resistance. Samples at -10 degrees C showed greater structural alteration than at -30 degrees C in terms of increase of beta-sheets at the expense of alpha-helices. An increase of unordered protein structure was found only in samples stored at -10 degrees C. Exposure of buried tryptophan residues was observed at both storage temperatures. The decrease of the deltaCH(2) band upon storage suggested an increase of hydrophobic interactions of aliphatic residues. Except for liquid air frozen fillets, all samples showed a decrease of the nuO-H/nuC-H band ratio compared to the fresh ones, this decrease being higher the harsher the conditions.

Characteristics of the salt-soluble fraction of hake (Merluccius merluccius) fillets stored at -20 and -30 degrees C.

Natural actomyosin (NAM) extracted in 0.6 M NaCl from hake fillets stored at -20 and -30 degrees C for up to 49 weeks was studied. The extracted protein decreased as storage progressed and became poorer in myosin while the proportion of actin remained constant. Two major peaks composed of myosin plus actin and actin plus tropomyosin plus troponins were obtained by size exclusion chromatography. SDS-PAGE analysis of the protein retained in the precolumn filter showed that there was protein aggregated by covalent bonding. Surface hydrophobicity increased while Ca(2+)-ATPase activity, apparent viscosity, and SH groups decreased as storage progressed. The loss of Ca(2+)-ATPase activity was due mainly to denaturation of the extracted myosin, whereas the minimum viscosity values occurred earlier and were not directly due to the lower proportion of myosin in the extracts. Thus, the extracted NAM exhibited changes during frozen storage. The temperature-dependent difference was mainly quantitative due to a smaller amount of protein extracted at -20 degrees C.

Thermal gelation of chicken, pork and hake (Merluccius merluccius, L) actomyosin.

Response surface methodology compared the effects of protein concentration (PC, 5-15 mg mL(-1)), ionic strength (NaCl concentration, IS, 0.1-0.7 M) and pH (5.2-7.5) on the gelation properties (penetration test) of natural actomyosin (NAM) from chicken, pork and hake muscle. Results indicated that, for Ln work of penetration (Ln WP), models had r(2) of 0.705 (p < 0.01) for NAM from pork, 0.813 (< 0.001) for NAM from chicken and 0.264 from hake (p > 0.05). The maximum work of penetration did not differ widely among the three samples, regardless of the fact that pH, NaCl level and protein concentration were different in each case. It was found that hake NAM only formed gels within a very narrow range of PC, pH and IS compared with chicken and pork NAM. In the latter two, maximum WP levels were found in gels formed within a pH range of 5.5-6.0 and ionic strength of 0.1-0.2 (NaCl, M). This suggests that hake protein is more sensitive to changes in environmental factors than that from pork or chicken.

Effect of added lipids on the texture of minced hake (Merluccius merluccius L.), megrim (Lepidorhombus whiffiagonis W.) and sardine (Sardina pilchardus W.) during frozen storage.

The effect of added cod liver oil and oxidized cod liver oil on the measurement of texture in minced hake (Merluccius merluccius L.), megrim (Lepidorhombus whiffiagonis W.) and sardine (Sardina pilchardus W.) has been measured during frozen storage (-18 degrees C). The results show that added neutral and oxidized lipids, even at high rancidity levels, do not affect shear resistances measured by the Kramer shear-compression cell in non-formaldehyde forming species such as megrim and sardine, over the frozen storage period. However, in a formaldehyde-forming species such as hake, in the presence of neutral and oxidized lipids at the end of the storage period, the values of shear resistances may be lower than in the absence of these lipids, probably owing to formation of less formaldehyde in these cases. Although it is very difficult to estimate the importance of a single compound or group of compounds on texture, these results seem to indicate that formaldehyde is a much more important factor than oxidized lipids in changes of texture in fish.

Effect of prolactin and glucocorticoids on P-enolpyruvate carboxykinase activity in liver and mammary gland from diabetic and lactating rats.

The administration of 2 bromo-alpha-ergocryptine, to reduce serum prolactin decreased the activity of cytosolic P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) about 50% in both liver and mammary gland of lactating animals. Adrenalectomy had similar effects to those of bromo-alpha-ergocryptine. In contrast, there was a 50% increase in enzyme activity in the mammary gland of diabetic, lactating rats and a 10-fold increase in liver as compared with normal rats. P-enolpyruvate carboxykinase activity in mammary gland as liver is coordinately regulated by prolactin, glucocorticoids and insulin.

Influence of starvation/refeeding transition on lipogenesis and NADPH producing systems in adipose tissue, mammary gland and liver at mid-lactation.

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and malic enzyme are enzymes involved in NADPH synthesis. Their specific activities and glucose utilization by isolated cell systems have been measured in adipose tissue and mammary gland from mid-lactating rats during starvation/refeeding transition. Starvation for 24 h produced a 75-90% decrease in the specific activities of these NADPH producing systems in mammary gland. Acinis isolated from the gland of starved rats had a lower production of CO2, fatty acids and triacylglycerols from (1-14C)glucose and (6-14C)-glucose than did gland from control rats. The activities of these enzymes in adipose tissue were very low and did not undergo any measurable alteration with starvation. The ability of adipocytes from well fed lactating rats to synthesize fatty acids from (1-14C)glucose was completely blocked. However, starvation is accompanied by a marked decrease in glucose incorporation into triacylglycerols. All the variations observed "in vivo" and "in vitro" in mammary gland returned almost to normal values by refeeding the starved lactating rats.