Evaluation of the intra and interspecific variability in the genus Perkinsus. Proteomic analysis of the parasite and its interaction with the host

Clam culture industry is rising every year worldwide. The most produced species is the Manila clam Ruditapes philippinarum. China is the main producer country followed by Italy, South Korea, USA and Spain. The introduction of the Manila clam in Europe was between 1970 and 1980. R. philippinarum is well adapted and grows faster than native species R. decussatus, for this, is the most produced species in Europe. The production of venerids in Galicia is a very important socio-economic resource. The production of Manila clam in Galicia rose in the last years and now is the most produced species too. The infection by Perkinsus olseni is one of the most serious diseases affecting clams. The parasite is highly distributed worldwide and affect to a large list of molluscs. Mortality of R. decussatus and R. philippinarum was associated with P. olseni infection in south Europe as well as with R. philippinarum mortality in several Asian countries. Two species of the genus Perkinsus, P. olseni and P. marinus, are included in the list of notifiable diseases of the World Organization for Animal Health, which indicates the interest to stop the expansion of the disease. The knowledge of P. olseni is very scarce in comparison with P. marinus which provokes massive mortalities in oysters Crassostrea virginica in USA. The economic transcendence of the perkinsosis justifies the research for a better knowledge of the disease and minimizes their effects. With this purpose, this study was developed in order to know (1) the variability of P. olseni along the Spanish coast, with the emphasis in virulence variability among populations, and (2) the modulation of the Manila clam protein expression due to P. olseni infection, with emphasis in the search of protein markers of resistance to the disease. The study of the variability was done analyzing the genetic population structure as well as the proteome of P. olseni clons derived from several regions of the Spanish coast. Furthermore, the proteome of P. olseni was compared with the proteome of two species of the genus, P. marinus and P. chesapeaki, in order to amplify the perspective of the variability of P. olseni. The analysis of the modulation of the Manila clam protein expression due to P. olseni infection was focused in haemocytes and haemolymph. The effect of the exposition to P. olseni in clams was measured at short time and a long time with the aim in the modulation of the immune response of the clam to the parasite

Genetic Variability in Ruditapes Decussatus Clam Combined With Perkinsus Infection Level to Support Founder Population Selection for a Breeding Program

[Abstract] Clam farmers worldwide face several challenges, including irregular seed supply and high mortalities due to pathogenic organisms such as "Perkinsus olseni". In Europe, there is a high unmet consumer demand for native clam species such as "Ruditapes decussatus". The high market value of "R. decussatus" makes the culture of this species potentially more attractive than that culture of the alien species "Ruditapes philippinarum". Thus, there is a market opportunity in breeding and producing "R. decussatus" at an industrial scale. A selective breeding program to improve "R. decussatus" performance will be carried out in Portugal; and the first critical step to develop such a breeding program is the establishment of a founder population. In this study, intra- and interpopulation genetic diversity was assessed using 13 microsatellite markers in eight natural beds located in Portugal, Spain and Italy. Also, allele and genotypic frequencies of each microsatellite locus were assessed discriminating between clams infected and non-infected by "P. olseni". All locations showed similar values for several genetic diversity parameters. Analyses of population differentiation (FST, Bayesian clustering and AMOVAs) revealed five genetically differentiated regions: Rías Altas and Rías Baixas (NW Spain), North/Central Coast of Portugal, Gulf of Cadiz and Adriatic Sea. Significant differences in the allelic and genotypic frequency distribution between infected clams and non-infected ones at four microsatellite loci are reported suggesting that resistance to the disease could have a genetic basis. Moreover, a positive or negative relationship between the frequency of certain alleles and the parasite infection was inferred. Further studies should confirm the potential use of those alleles as genetic markers for "P. olseni" infection. Integrating results of genetic diversity within and between populations and Perkinsus infection levels, a founder population for a "R. decussatu" breeding program is proposed, composed by individuals from Barallobre (Rías Altas), Pontevedra or Cangas (Rías Baixas), Óbidos (North/Central Coast of Portugal), Algarve (Gulf of Cadiz) and Venice (Adriatic Sea).Governo da República Portuguesa; 22390 (16/SI/2016) (CENTRO-01-0247-FEDER-022390)Ministério do Mar (Portugal); FA_01_2017_00

Functional and molecular mmune response of rainbow trout (Oncorhynchus mykiss) following challenge with Yersinia ruckeri

Currently, aquaculture production of rainbow trout (Oncorhynchus mykiss) is a multibillion dollar industry; nevertheless, the development of this sector has not been exempt from pitfalls related to the recurrent presence of pathogens of bacterial origin. This is the case of Yersinia ruckeri, the etiologic agent of the infectious pathology known as Enteric Red Mouth Disease (ERM), causing serious economic losses that can be as high as 30–70% of production. Although several studies have been performed regarding pathogen features and virulence factors, more information is needed about the host defense mechanism activation after infection. Given this perspective, this study aimed to evaluate rainbow trout’s short-term innate immune response against infection with Y. ruckeri. A series of factors linked to the innate immune response were evaluated, including determination of hematological parameters, oxidative stress biomarkers, and analysis of the expression of immunerelated genes. Results showed a significant decrease in several hematological parameters (white blood cell count, hematocrit, neutrophils, monocytes, lymphocytes, and thrombocytes) and oxidative stress indicators (SOD) between the control and infected groups. In addition, there were significant differences in the level of gene expression between infected individuals and the control group. Most of these genes (il-1b, il-8, il-10, tnf-a1, tnf-a2, socs3, mmp-9, cath, hsp-70, saa, fer, pcb) were upregulated within the first 24 h following infection. Results from this study showed more insights into the short-term immune response of rainbow trout to infection with Y. ruckeri, which may be useful for the establishment of biomarkers that may be used for the early detection of ERM.info:eu-repo/semantics/publishedVersio

Effect of the alveolate parasite Perkinsus olseni infection on sexual maturation and spawning efficiency of the clam Ruditapes decussatus

The effect of Perkinsus olseni infection on the reproduction ability of clams has been underestimated so far. Although some studies found evidence of reduction of egg production and delay in gonad maturation after infection, the total effect of the infection is still unclear. In this study, Ruditapes decussatus clams from a naïve population were injected with two different doses of P. olseni parasites, a low dose leading to a light infection and a high dose leading to a heavy infection. Clams were maintained during 2 months for maturation, and at the end of the experiment, the spawning was induced, the number of larvae release and mortality were evaluated. During the maturation period, infection level, gonadal stage, condition index, gross biochemical composition and oxidative status of progenitors were evaluated at days 0, 30 and 60 post-injection. The effects of P. olseni infection on clams showed alterations on biochemical parameters, namely lipid peroxidation, a significant mortality and a delayed gonad maturation, with a greater effect in the highly infected individuals. The reproductive capacity of the clams was impaired in both infected groups showing a lower production and a higher mortality rate of larvae. Finally, this study indicates that the production of natural beds with a high prevalence of P. olseni could be compromised by a deregulation of the natural reproduction cycle and a decrease in larvae production by infected animals, probably due to a combination of lower egg production and lower lipid reserves in larvae from infected clams

Health status in gilthead seabream (Sparus aurata) juveniles fed diets devoid of fishmeal and supplemented with Phaeodactylum tricornutum

To enhance fish general health, feeds can be supplemented with health-promoting additives, reducing the need to use chemotherapeutics. Incorporation of marine algae biomasses in aquafeeds has been shown to improve fish immune status by enhancing innate immune response. This study evaluated the effects of Phaeodactylum tricornutum incorporation in feed by two different processes, either as freeze-dried biomass or broken cell wall biomass, on fish health status and performance. Triplicate groups of gilthead seabream juveniles (13.3 ± 0.3 g) were either fed a control diet (CTRL) with an extreme (i.e., 0% fishmeal), nutritionally balanced, formulation, or two experimental diets formulated as the CTRL with 1% inclusion of the microalga P. tricornutum at the expense of wheat meal: BC diet contains P. tricornutum broken cells and WC diet microalgae whole cells. After 2 and 12 weeks of feeding, blood was collected for hematological procedures, whereas plasma and mucus were sampled for immune parameters. Head-kidney, liver, and white skeletal muscle were also collected for gene expression measurements. No major differences were observed in hematological nor plasma humoral parameters after 12 weeks irrespective of dietary treatment. Arrays of 29–31 genes were analyzed in the different tissues, revealing an early dietary effect (2 weeks) in a tissue-specific pattern. In the liver, the major effect was found in the GH/IGF axis and in muscle there was a late downregulation of myostatin (mstn) gene, mainly due to WC diet, even though all fish had similar growth performance. Regarding the head-kidney, BC diet led to alpha-2-macroglobulin (a2m) gene upregulation. Also, the same treatment showed increased mucus alternative complement pathway and bactericidal activity at 2 and 12 weeks, respectively. Hence, it seems that BC diet has a potential stimulatory effect that might be relevant as a prophylactic measure before a predictable stressful event.This work has been funded under the EU FP7 by the MIRACLES project No. 613588: Multi-product Integrated bioRefinery of Algae: from Carbon dioxide and Light Energy to high-value Specialties and by national funds through FCT - Foundation for Science and Technology within the scope of UIDB/04423/2020 and UIDP/04423/2020. B. Reis, L. Ramos-Pinto, B. Costas, and S. Engrola were supported by the Portuguese Foundation for Science and Technology (PD/BDE/129262/2017, PD/BDE/114436/2016, IF/00197/ 2015, and IF/00482/2014, respectively)

Comparative analysis between synthetic vitamin E and natural antioxidant sources from tomato, carrot and coriander in diets for market-sized dicentrarchus labrax

Synthetic vitamin E is commonly used in aquafeeds to prevent oxidative stress in fish and delay feed and flesh oxidation during storage, but consumers’ preferences tend towards natural antioxidant sources. The potential of vegetable antioxidants-rich coproducts, dried tomato (TO), carrot (CA) and coriander (CO) was compared to that of synthetic vitamin E included in diets at either a regular (CTRL; 100 mg kg−1) or reinforced dose (VITE; 500 mg kg−1). Natural antioxidants were added at 2% to the CTRL. Mixes were then extruded and dried, generating five experimental diets that were fed to European sea bass juveniles (114 g) over 12 weeks. Vitamin E and carotenoid content of extruded diets showed signs of degradation. The experimental diets had very limited effects on fish growth or body composition, immunomodulatory response, muscle and liver antioxidant potential, organoleptic properties or consumer acceptance. Altogether, experimental findings suggest that neither a heightened inclusion dose of 500 mg kg−1 of vitamin E, nor a 2% inclusion of natural antioxidants provided additional antioxidant protection, compared to fish fed diets including the regular dose of 100 mg kg−1 of vitamin E.info:eu-repo/semantics/publishedVersio

Dietary Methionine Improves the European Seabass (Dicentrarchus labrax) Immune Status, Inflammatory Response, and Disease Resistance

Methionine presents a pivotal role in the regulation of many cellular events with crucial impact on the immune system, such as in processes involved in the control of inflammation and polyamines synthesis. Accordingly, the present study aimed to assess the modulatory effects of dietary methionine on the European seabass (Dicentrarchus labrax) immune status, inflammatory response and disease resistance to Photobacterium damselae subsp. piscicida (Phdp). For this purpose, fish were randomly distributed in three independent groups (three replicates per group) and each was fed the corresponding diet: a control diet (CTRL) formulated to meet the established amino acid requirements for the species; a diet supplemented with methionine at 0.5% of feed weight relative to the CTRL diet (8.2% of methionine concentration above CTRL); and one supplemented with methionine at 1% of feed weight to the CTRL diet (11.8% of methionine concentration above CTRL). To evaluate the immune status of fish fed with each of the diets before being submitted to bacterial infection fish were sampled from each group at 2 and 4 weeks after the beginning of feeding. Non-sampled fish were injected intraperitoneally with Phdp (5 × 103 cfu/fish) at 4 weeks after initiation of feeding and the inflammatory response (at 4, 24, and 48 h post-infection) and survival (lasting 21 days post-infection) evaluated. Fish hematological profile, peripheral cell dynamics, plasma humoral immune parameters, leucocyte migration to the inflammatory focus and head-kidney gene expression were evaluated. Results show that methionine dietary supplementation improves seabass cellular immune status without evidence of activation of pro-inflammatory mechanisms. Additionally, the observed enhanced immune status provided by methionine supplementation translated into an improved immune response to infection, as higher cellular differentiation/proliferation and recruitment to the inflammatory focus, improved plasma humoral immune parameters and modulation of key immune-related genes was observed. Lastly, after a bacterial challenge, higher survival was observed in fish fed supplemented diets, ultimately corroborating the positive effect of methionine administration for 4 weeks in the cellular immune status

Trematode genetic patterns at host individual and population scales provide insights about infection mechanisms

Multiple parasites can infect a single host, creating a dynamic environment where each parasite must compete over host resources. Such interactions can cause greater harm to the host than single infections and can also have negative consequences for the parasites themselves. In their first intermediate hosts, trematodes multiply asexually and can eventually reach up to 20% of the host’s biomass. In most species, it is unclear whether this biomass results from a single infection or co-infection by two or more infective stages (miracidia), the latter being more likely a priori in areas where prevalence of infection is high. Using as model system the trematode Bucephalus minimus and its first intermediate host cockles, we examined the genetic diversity of the cytochrome c oxidase subunit I (COI) region in B. minimus from three distinct geographical areas and performed a phylogeographic study of B. minimus populations along the Northeast Atlantic coast. Within localities, the high genetic variability found across trematodes infecting different individual cockles, compared to the absence of variability within the same host, suggests that infections could be generally originating from a single miracidium. On a large spatial scale, we uncovered significant population structure of B. minimus, specifically between the north and south of Bay of Biscay. Although other explanations are possible, we suggest this pattern may be driven by the population structure of the final host

Shrimp immune response: A transcriptomic perspective

During the last 15 years, a significant amount of data related to the crustacean immune system has been generated, mainly led by the advances made in the field of transcriptomic analysis through massive mRNA sequencing technology. In order to shed new light on the genes and metabolic pathways associated with the immune system in shrimp species, we have carried out a meta-analysis using the current literature and the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI), which has 1195 published transcriptomic data sets until 31 December 2020, from different tissues and 14 Penaeus species. The transcriptomic analyses published so far have explored the effects of the challenge from viral and bacterial pathogens, as well as abiotic stressors, such as ammonia, temperature, salinity, oxygen deprivation and pH. They have also explored the effect of dietary factors and beneficial microbes (pre- and probiotics) in the immune response. In order to harness the vast amount of information available, multidisciplinary efforts have to generate better genomic tools to overcome the complexity of the genome and to implement the knowledge gained into commercial applications for the improvement of the culture conditions, adaptation of more sustainable feeds and producing strains showing more resilience/tolerance to diseases.Peer reviewe

Is pallial mucus involved in Ostrea edulis defenses against the parasite Bonamia ostreae?

Bonamia ostreae is an intrahemocytic parasite that has been responsible for severe mortalities in the flat oyster Ostrea edulis since the 1970́s. The Pacific oyster Crassostrea gigas is considered to be resistant to the disease and appears to have mechanisms to avoid infection. Most studies carried out on the invertebrate immune system focus on the role of hemolymph, although mucus, which covers the body surface of molluscs, could also act as a barrier against pathogens. In this study, the in vitro effect of mucus from the oyster species Ostrea edulis and C. gigas on B. ostreae was investigated using flow cytometry. Results showed an increase in esterase activities and mortality rate of parasites exposed to mucus from both oyster species. In order to better understand the potential role of mucus in the defense of the oyster against parasites such as B. ostreae, liquid chromatography and tandem mass spectrometry were used to describe and compare mucus protein composition from both species. In all oyster species, pallial mucus contains a high level of proteins; however, O. edulis mucus produced a variety of proteins that could be involved in the immune response against the parasite, including Cu/Zn extracellular superoxide dismutase, thioxiredoxin, peroxiredon VI, heat shock protein 90 as well as several hydrolases. Conversely, a different set of antioxidant proteins, hydrolases and stress related proteins were identified in mucus from C. gigas. Our results suggest an innate immunity adaptation of oysters to develop a specific response against their respective pathogens. The mucosal protein composition also provides new insights for further investigations into the immune response in oysters