Katherina Brokordt

Effect of diet and temperature upon muscle metabolic capacities and biochemical composition of gonad and muscle in Argopecten purpuratus Lamarck 1819

Recently spawned Argopecten purpuratus broodstock were conditioned at two temperatures and fed three different diets (microalgae, microalgae mixed with lipids and microalgae mixed with carbohydrates) to examine changes in the biochemical composition of gonad and muscle as well as muscle metabolic capacities. During one experiment, scallops were fed at 3% of their dry mass per day whereas during a second experiment, they were fed at 6% of their dry mass per day. During both experiments, total gonadal levels of lipids and protein increased markedly during conditioning with the two mixed diets at 16 degrees C. These increases were less pronounced at 20 degrees C. Carbohydrate gonadal levels only increased during the second experiment at both temperatures and with the three diets. Of the major biochemical components of the adductor muscle, carbohydrate levels changed most during conditioning. Whereas muscle protein levels increased slightly with gonadal maturation, carbohydrate levels dropped considerably. Despite the marked drop in the levels of glycolytic substrates, only the activities of octopine dehydrogenase in the adductor muscle of the scallops conditioned at 16 degrees C consistently decreased. Muscle levels of glycogen phosphorylase were higher in mature than in recently spawned (control) scallops, suggesting a role in the transfer of glucose equivalents from the adductor muscle to other tissues.

Sex differences in reproductive investment: maternal care reduces escape response capacity in the whelk Buccinum undatum

We evaluated the effect of reproductive state and biochemical status of male and female whelks Buccinum undatum on their escape responses (foot contortions) when touched by their major predator, the sea star Leptasterias polaris. Gonad growth in males was not associated with a decrease in their general energetic and metabolic capacity, nor in their capacity for escape responses, but did reduce the rate of foot contortions during recuperation from exhaustive exercise. Similarly, the energetic status of females was good when the ovary was mature, suggesting that oogenesis did not require somatic reserves. On the other hand, the energetic status and muscle metabolic capacities of females dropped with egg laying. This decrease was associated with a reduced capacity for escape responses. Thus, egg laying resulted in (1) major drops in the carbohydrate and protein contents of the foot, (2) decreased activities of several glycolytic enzymes (LDH, ADH and APK) in the foot muscle, (3) a sharp drop in the digestive gland index, (4) a decrease in the number of foot contortions and (5) a decreased ability to recover from exhaustive escape exercise. Reproductive costs are much greater for females than males (as females must produce protective egg capsules, search for egg-laying sites and lay the capsules). Females have greater escape capacities than males, except directly after egg laying when their energetic reserves are virtually depleted. The greater capacity of females for escape responses may attenuate the risks associated with their greater boldness in stealing food from their predator L. polaris, particularly prior to egg laying.

Physiological performance of juvenile Haliotis rufescens and Haliotis discus hannai abalone exposed to the withering syndrome agent

Withering syndrome (WS) is a serious chronic disease caused by infection with the bacterium Candidatus Xenohaliotis californiensis, a Rickettsiales-like organism (WS-RLO) that affects multiple abalone species in both natural and farmed populations. However, there is no available information regarding the effects of this disease on the physiological performance of infected abalone. We studied the effect of different levels of infection on components of energy balance and physiological indices (rates of absorption and assimilation, O/N ratio, and scope for growth) in the abalone species Haliotis rufescens and Haliotis discus hannai. Juveniles were exposed to C. X. californiensis transmission for 130 days, during which time the presence/absence of WS-RLOs was evaluated by PCR (following DNA sequencing-based confirmation of 100% identity with the sequence of C. X. californiensis from California), and the prevalence and intensity of infection were evaluated via histological analysis. Among H. rufescens juveniles exposed to the bacterium, 92% became infected (positive by histology), and the intensity of infection ranged from low (degree 1) to moderate (degree 2). In contrast, no H. discus hannai juveniles were positive for WS-RLO by histology, although 23% were positive by PCR, possibly indicating incipient WS-RLO infection that did not develop during the experimental period or to mere presence of WS-RLO DNA in the sample. Infection of H. rufescens juveniles by WS-RLOs negatively affected all components of the energy balance and physiological indices, such as scope for growth and the O/N ratio, in direct relation to the degree of infection. The most strongly affected functions were the rate of ingestion, standard metabolism, and production of feces, which were reduced by 60-80% in the most highly infected individuals. The reduced energy intake in the organisms produced a strong energy imbalance such that the energy available for growth was reduced by 49% in infected organisms. In contrast, juveniles of H. discus hannai carrying the bacterium developed no infection and showed no alterations of physiological function. Our results indicate that the level of early infection by WS-RLOs may exert a negative effect on physiological activity in H. rufescens, even when the disease is not evident.

Molecular characterization of an inhibitor of NF-κB in the scallop Argopecten purpuratus: First insights into its role on antimicrobial peptide regulation in a mollusk.

Inhibitors of nuclear factor kappa B (IκBs) are major control components of the Rel/NF-κB signaling pathway, a key regulator in the modulation of the expression of immune-related genes in vertebrates and invertebrates. The activation of the Rel/NF-κB signaling pathway depends largely in the degradation of IκB proteins and thus, IκBs are a main target for the identification of genes whose expression is controlled by Rel/NF-κB pathway. In order to identify such regulation in bivalve mollusks, the cDNA sequence encoding an IκB protein was characterized in the scallop Argopecten purpuratus, ApIκB. The cDNA sequence of ApIκB is comprised of 1480 nucleotides with a 1086 bp open reading frame encoding for 362 amino acids. Bioinformatics analysis showed that ApIκB displays the conserved features of IκB proteins. The deduced amino acid sequence consists of a 39.7 kDa protein, which has an N-terminal degradation motif, six ankyrin repeats and a C-terminal phosphorylation site motif. Phylogenetic analysis revealed a high degree of identity between ApIκB and other IκBs from mollusks, but also to arthropod cactus proteins and vertebrate IκBs. Tissue expression analysis indicated that ApIκB is expressed in all examined tissues and it is upregulated in circulating hemocytes from scallops challenged with the pathogenic Gram-negative bacterium Vibrio splendidus. After inhibiting ApIκB gene expression using the RNA interference technology, the gene expression of the antimicrobial peptide big defensin was upregulated in hemocytes from non-challenged scallops. Results suggest that ApIκB may control the expression of antimicrobial effectors such as big defensin via a putative Rel/NF-κB signaling pathway. This first evidence will help to deepen the knowledge of the Rel/NF-κB conserved pathway in scallops.

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 (h 2) and the additive genetic coefficient of variation (CV A) 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 h 2 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 h 2 and R, with values that were not significantly different from zero. However, HSP70 induced levels had a CV A of 13.3–16.2% in young adults and of 2.7–8.1% in pre-harvest adults. Thus, despite its low h 2, 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.

Molecular characterization and protein localization of the antimicrobial peptide big defensin from the scallop Argopecten purpuratus after Vibrio splendidus challenge

Abstract Big defensins are antimicrobial peptides (AMPs) that are proposed as important effectors of the immune response in mollusks, chelicerates and chordates. At present, only two members of the big defensin family have been identified in scallop. In the present work, a cDNA sequence encoding a new big defensin homologue was characterized from the scallop Argopecten purpuratus, namely ApBD1. ApBD1 cDNA sequence comprised 585 nucleotides, with an open reading frame of 375 bp and 5’‐ and 3′‐UTRs of 41 and 167 bp, respectively. The deduced protein sequence contains 124 amino acids with a molecular weight of 13.5 kDa, showing characteristic motifs of the big defensin family and presenting 76% identity with the big defensin from the scallop A. irradians. Phylogenetic analysis revealed that ApBD1 is included into the cluster of big defensins from mollusks. Tissue‐specific transcript expression analysis by RT‐qPCR showed that ApBD1 was present in all tissues tested from non‐immune challenged scallops but it was most strongly expressed in the mantle. The transcript levels of ApBD1 were significantly up‐regulated in gills at 24 and 48 h post‐injection with the heat‐attenuated bacteria Vibrio splendidus. Additionally, immunofluorescence analysis using a polyclonal anti‐ApBD1 antibody showed that this protein was abundantly located in epithelial linings of gills and mantle; and also in digestive gland showing ApBD1‐infiltrating hemocytes from immune challenged scallops. This is the first time that a big defensin is detected and located at the protein level in a mollusk. These results suggest an important role of ApBD1 in the mucosal immune response of A. purpuratus. HighlightsA new big defensin homologue, ApBD1, was characterized in Argopecten purpuratus.ApBD1 mRNA is constitutively present in all tissues and strongly expressed in mantle.ApBD1 mRNA is upregulated in gills of scallops challenged with Vibrio splendidus.ApBD1 protein is located in gills and mantle epithelia, digestive gland and infiltrating hemocytes.Results suggest an important role of ApBD1 in the mucosal immune response of A. purpuratus.

Assessing Potential Predation Risk by Introduced Predators on Unattended Eggs in the Red-Tailed Tropicbird, Phaethon rubricauda, on Rapa Nui (Easter Island)

Anthropogenic impact has been heavy in remote oceanic islands, including the introduction of alien species, having negative effects on native seabirds. The isolated and subtropical Rapa Nui (Easter Island) is one of the few known breeding sites of the red-tailed tropicbird, Phaethon rubricauda in Chile (southeastern Pacific Ocean) where is listed as vulnerable. A relatively new breeding colony is found in the Rano Raraku volcano, where human-introduced species are present. We used hen eggs as a proxy for red-tailed tropicbird eggs to assess potential predation risk on unattended eggs. Each experimental egg was monitored by camera traps during 6 days. Three predatory species were identified on the records: the Brown rat Rattus norvegicus, the Polynesian rat Rattus exulans, and the raptor Chimango Caracara Phalcoboenus chimango. The most frequent species were the Rattus spp. A total of 45 predatory visits were recorded with a total time of 1.7 h, accounting for the 0.3% of the experimental time. Within this time of visits, all the potential predators spent time in both interacting activities (trying to prey on) and no-interacting activities with the experimental eggs. Only a Brown rat was able to prey on one of the eggs. Our results suggest that these invasive species are a low threat for unattended red-tailed tropicbird eggs at Rano Raraku, Rapa Nui. However, future research is needed to determine the potential negative effects over unattended red-tailed tropicbird nestlings that are easier for these predators to handle compared with an egg.

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

Abstract 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. HighlightsTwo ferritin sequences were characterized in red abalone, Hrfer1 and Hrfer2.Both ferritins are present in all tissues and strongly expressed in digestive gland and gonad.Hrfer1 transcripts increased along with larval development being highest in creeping post‐larvae.Both ferritins are upregulated in adults and juveniles challenged with Vibrio splendidus.Ferritin is upregulated at protein level in the juveniles after bacterial challenge.