Cloning and molecular characterization of two ferritins from red abalone Haliotis rufescens and their expressions in response to bacterial challenge at juvenile and adult life stages.

Ferritins are ubiquitous proteins with a pivotal role in iron storage and homeostasis, and in host defense responses during infection by pathogens in several organisms, including mollusks. In this study, we characterized two ferritin homologues in the red abalone Haliotis rufescens, a species of economic importance for Chile, USA and Mexico. Two ferritin subunits (Hrfer1 and Hrfer2) were cloned. Hrfer1 cDNA is an 807 bp clone containing a 516 bp open reading frame (ORF) that corresponds to a novel ferritin subunit in H. rufescens. Hrfer2 cDNA is an 868 bp clone containing a 516 bp ORF that corresponds to a previously reported ferritin subunit, but in this study 5'- and 3'-UTR sequences were additionally found. We detected a putative Iron Responsive Element (IRE) in the 5'-UTR sequence, suggesting a posttranscriptional regulation of Hrfer2 translation by iron. The deduced protein sequences of both cDNAs possessed the motifs and domains required in functional ferritin subunits. Expression patterns of both ferritins in different tissues, during different developmental stages, and in response to bacterial (Vibrio splendidus) exposure were examined. Both Hrfer1 and Hrfer2 are most expressed in digestive gland and gonad. Hrfer1 mRNA levels increased about 34-fold along with larval developmental process, attaining the highest level in the creeping post-larvae. Exogenous feeding is initiated at the creeping larva stage; thus, the increase of Hrfer1 may suggest and immunity-related role upon exposure to bacteria. Highest Hrfer2 expression levels were detected at trochophore stage; which may be related with early shell formation. Upon challenge with, the bacteria an early mild induction of Hrfer2 (2 h post-challenge), followed by a stronger induction of Hrfer1 at 15 h post-challenge, was observed in haemocytes from adult abalones. While maximal upregulation of both genes in the whole individual occurred at 24 h post-challenge, in juveniles. A significant increase in ferritin protein levels from 6 h to 24 h post-challenge was also detected. Our results suggest an involvement of Hrfer1 and Hrfer2, and of ferritin proteins in the immune response of H. rufescens to bacterial infection.

Molecular characterization of two ferritins of the scallop Argopecten purpuratus and gene expressions in association with early development, immune response and growth rate.

Ferritin is involved in several iron homoeostasis processes in molluscs. We characterized two ferritin homologues and their expression patterns in association with early development, growth rate and immune response in the scallop Argopecten purpuratus, a species of economic importance for Chile and Peru. Two ferritin subunits (Apfer1 and Apfer2) were cloned. Apfer1 cDNA is a 792bp clone containing a 516bp open reading frame (ORF) that corresponds to a novel ferritin subunit in A. purpuratus. Apfer2 cDNA is a 681bp clone containing a 522bp ORF that corresponds to a previously sequenced EST. A putative iron responsive element (IRE) was identified in the 5'-untranslated region of both genes. The deduced protein sequences of both cDNAs possessed the motifs and domains characteristic of functional ferritin subunits. Both genes showed differential expression patterns at tissue-specific and early development stage levels. Apfer1 expression level increased 40-fold along larval developmental stages, decreasing markedly after larval settlement. Apfer1 expression in mantle tissue was 2.8-fold higher in fast-growing than in slow-growing scallops. Apfer1 increased 8-fold in haemocytes 24h post-challenge with the bacterium Vibrio splendidus. Apfer2 expression did not differ between fast- and slow-growing scallops or in response to bacterial challenge. These results suggest that Apfer1 and Apfer2 may be involved in iron storage, larval development and shell formation. Apfer1 expression may additionally be involved in immune response against bacterial infections and also in growth; and thus would be a potential marker for immune capacity and for fast growth in A. purpuratus.

Potential Response to Selection of HSP70 as a Component of Innate Immunity in the Abalone Haliotis rufescens.

Assessing components of the immune system may reflect disease resistance. In some invertebrates, heat shock proteins (HSPs) are immune effectors and have been described as potent activators of the innate immune response. Several diseases have become a threat to abalone farming worldwide; therefore, increasing disease resistance is considered to be a long-term goal for breeding programs. A trait will respond to selection only if it is determined partially by additive genetic variation. The aim of this study was to estimate the heritability (h2) and the additive genetic coefficient of variation (CVA) of HSP70 as a component of innate immunity of the abalone Haliotis rufescens, in order to assess its potential response to selection. These genetic components were estimated for the variations in the intracellular (in haemocytes) and extracellular (serum) protein levels of HSP70 in response to an immunostimulant agent in 60 full-sib families of H. rufescens. Levels of HSP70 were measured twice in the same individuals, first when they were young and again when they were pre-harvest adults, to estimate the repeatability (R), the h2 and the potential response to selection of these traits at these life stages. High HSP70 levels were observed in abalones subjected to immunostimulation in both the intracellular and extracellular haemolymph fractions. This is the first time that changes in serum levels of HSP70 have been reported in response to an immune challenge in molluscs. HSP70 levels in both fractions and at both ages showed low h2 and R, with values that were not significantly different from zero. However, HSP70 induced levels had a CVA of 13.3-16.2% in young adults and of 2.7-8.1% in pre-harvest adults. Thus, despite its low h2, HSP70 synthesis in response to an immune challenge in red abalone has the potential to evolve through selection because of its large phenotypic variation and the presence of additive genetic variance, especially in young animals.