In vivo and in vitro assessment of host-parasite interaction between the parasitic copepod Brachiella thynni (Copepoda; Lernaeopodidae) and the farmed Atlantic bluefin tuna (Thunnus thynnus).

The Atlantic bluefin tuna (ABFT; Thunnus thynnus) today represents one of the economically most important species for Croatian fisheries industry. Although the most diverse and abundant parasitofauna is usually found in the largest specimens of wild ABFT, the opposite was observed in captivity where parasite populations significantly decline by the end of the farming cycle. Copepod Brachiella thynni, is a skin parasite frequently parasitizing tuna, whose population also decreases in number throughout the rearing process. In order to better understand the immunity mechanisms underlying ABFT reaction to B. thynni infection, we studied expression profiles of immunity related genes; interleukin 1ß (il1ß), tumour necrosis factors (tnfα1, tnfα2), complement component 4 (c4) and caspase 3 (casp3), in peripheral blood leukocytes (PBLs) during in vitro stimulation by B. thynni protein extracts (i.e. antigens) and in infected tissues at B. thynni parasitation site. Finally, a histopathological analysis of semi-thin and ultra-thin sections of tissues surrounding B. thynii attachment site was performed to evaluate the severity of parasite-induced lesions and identify involved cell lineages. In vitro stimulation of ABFT PBLs with B. thynii antigens caused a dose-depended upregulation of selected genes, among which tnfα1 showed the highest induction by both concentrations of B. thynni protein extract. However, targeted genes were not significantly upregulated in the infected tissue. Also, no significant alterations in ultrastructure of epithelial layers surrounding B. thynii attachment site were noticed, except local tissue erosion, necrosis of squamous epithelium and proliferation of rodlet and goblet cells. Our results suggest that B. thynii has evolved strategies to successfully bypass both innate immune response and the connective-tissue proliferation processes. Therefore, the observed disappearance of this copepod by the end of the rearing process is more likely related to its limited lifespan on the host and its inability to complete the life cycle in the rearing cages, rather than host's reaction.

Expression analysis of the Atlantic bluefin tuna (Thunnus thynnus) pro-inflammatory cytokines, IL-1ß, TNFα1 and TNFα2 in response to parasites Pseudocycnus appendiculatus (Copepoda) and Didymosulcus katsuwonicola (Digenea).

Pro-inflammatory cytokines play an important role in teleost defence against numerous types of pathogens, therefore are often used as biomarkers during various infections. In order to evaluate Atlantic bluefin tuna IL-1ß, TNFα1 and TNFα2 induction by PAMPs, we quantified their expression during in vitro stimulation of peripheral blood leukocytes by LPS and Poly I:C. Furthermore, their role in acute and chronic parasitic infection was examined during natural infection of Pseudocycnus appendiculatus (Copepoda) and Didymosulcus katsuwonicola (Digenea), as well as during leukocyte exposure to total protein extracts isolated from two parasite species. Induction of ABT IL-1ß and TNFα2 by PAMPs and protein extracts from D. katsuwonicola and P. appendiculatus, as well as during natural infection with two parasites, suggests these cytokines play an important role in inflammation, being engaged in controlling parasite infections, in contrast to ABT TNFα1. Cellular innate response to the digenean D. katsuwonicola showed rather chronic character, resulting with parasite encapsulation in connective tissue. Mast cells, eosinophils, goblet cells, and occasional rodlet cells found at the site of infection, along with the induction of TNFα2, suggest the presence of a moderate inflammatory reaction that fails to seriously endanger digenean existence. In contrast, copepod P. appendiculatus, attached to the gill epithelium by clamping, caused direct tissue disruption with undergoing necrotic or apoptotic processes, and extensive proliferation of rodlet and goblet cells. Differential expression patterns of target cytokines in tissue surrounding two parasites and in vitro PBL model suggest that quality and quantity of tuna immune response is conditioned by parasite adaptive mechanisms and pathogenicity.

A plant-based diet supplemented with Hermetia illucens alone or in combination with poultry by-product meal: one step closer to sustainable aquafeeds for European seabass.

BACKGROUND: Increasing demand for high-value fish species and pressure on forage fish is challenging aquaculture to ensure sustainable growth by replacing protein sources in aquafeeds with plant and terrestrial animal proteins, without compromising the economic value and quality of the final fish product. In the present study, the effects of a plant protein-based diet (CV), two plant-based diets in which graded amounts of plan protein mixtures were replaced with Hermetia illucens meal alone (VH10) or in combination with poultry by-product meal (PBM) (VH10P30), a fishmeal (FM) diet (CF) and an FM diet supplemented with H. illucens (FH10) on growth performance, gut health and homeostasis of farmed subadult European seabass were tested and compared. RESULTS: Fish fed the VH10 and VH10P30 diets showed the highest specific growth rates and lowest feed conversion ratios among the tested groups. Expectedly, the best preservation of PI morphology was observed in fish fed the CF or FH10 diets, while fish fed the CV diet exhibited significant degenerative changes in the proximal and distal intestines. However, PBM supplementation mitigated these effects and significantly improved all gut morphometric parameters in the VH10P30 group. Partial substitution of the plant mixture with insect meal alone or PBM also induced most BBM genes and activated BBM enzymes, suggesting a beneficial effect on intestinal digestive/absorption functions. Regarding intestinal microbiota, fish fed diets containing H. illucens meal (FH10, VH10, VH10P30) had the highest richness of bacterial communities and abundance of beneficial genera such as Lactobacillus and Bacillus. On the other hand, fish fed CV had the highest microbial diversity but lost a significant component of fish intestinal microbiota, the phylum Bacteroidetes. Finally, skin pigmentation most similar to that of farmed or even wild seabass was also observed in the fish groups fed CF, FH10 or VH10P30. CONCLUSION: Plant-based diets supplemented with PBM and H. illucens pupae meal have great potential as alternative diets for European seabass, without affecting growth performance, gut homeostasis, or overall fitness. This also highlights the importance of animal proteins in diets of European seabass, as the addition of a small amount of these alternative animal protein sources significantly improved all measured parameters.

Diversity and Phylogenetic Analyses of Bacterial Symbionts in Three Whitefly Species from Southeast Europe.

Bemisia tabaci (Gennadius), Trialeurodes vaporariorum (Westwood), and Siphoninus phillyreae (Haliday) are whitefly species that harm agricultural crops in many regions of the world. These insects live in close association with bacterial symbionts that affect host fitness and adaptation to the environment. In the current study, we surveyed the infection of whitefly populations in Southeast Europe by various bacterial symbionts and performed phylogenetic analyses on the different symbionts detected. Arsenophonus and Hamiltonella were the most prevalent symbionts in all three whitefly species. Rickettsia was found to infect mainly B. tabaci, while Wolbachia mainly infected both B. tabaci and S. phillyreae. Furthermore, Cardinium was rarely found in the investigated whitefly populations, while Fritschea was never found in any of the whitefly species tested. Phylogenetic analyses revealed a diversity of several symbionts (e.g., Hamiltonella, Arsenophonus, Rickettsia), which appeared in several clades. Reproductively isolated B. tabaci and T. vaporariorum shared the same (or highly similar) Hamiltonella and Arsenophonus, while these symbionts were distinctive in S. phillyreae. Interestingly, Arsenophonus from S. phillyreae did not cluster with any of the reported sequences, which could indicate the presence of Arsenophonus, not previously associated with whiteflies. In this study, symbionts (Wolbachia, Rickettsia, and Cardinium) known to infect a wide range of insects each clustered in the same clades independently of the whitefly species. These results indicate horizontal transmission of bacterial symbionts between reproductively isolated whitefly species, a mechanism that can establish new infections that did not previously exist in whiteflies.

Three Anisakis spp. isolated from toothed whales stranded along the eastern Adriatic Sea coast.

Knowledge concerning cetacean ecology in the Mediterranean is limited but important for sustainable planning and enforcement of appropriate conservation measures. Any information that might help to elucidate their ecology is essential. We explored the population and genetic structures of Anisakis spp. nematodes isolated from four toothed whale species - bottlenose dolphins (Tursiops truncatus), striped dolphins (Stenella coeruleoalba), Risso's dolphins (Grampus griseus) and Cuvier's beaked whales (Ziphius cavirostris) - stranded along the eastern Adriatic Sea coast (1990-2012) to reveal more information on host ecological patterns. Lower parasite prevalence was observed in resident dolphin species compared with occasionally occurring species, as well as in young compared with adult dolphins, indicating different feeding habits related to age. No unequivocal relationship between the biological traits of a host (age, body length, body mass and blubber depth) and Anisakis population parameters was observed. Phylogenetic analysis revealed a new geographical record of Anisakis simplex sensu stricto (1.96%) and Anisakis physeteris (1.31%) in the Adriatic Sea in addition to resident Anisakis pegreffii (96.73%). In an assessment of the Adriatic Sea and oceans worldwide, the genetic structure of Anisakis revealed that A. pegreffii populations do not differ among various final host species but do differ with respect to geographical location in contrast to previously accepted Anisakis panmixia.

Quantification and in situ localisation of abcb1 and abcc9genes in toxicant-exposed sea urchin embryos.

A multixenobiotic resistance (MXR) mechanism mediated by ABC binding cassette (ABC) transport proteins is an efficient chemical defence mechanism in sea urchin embryos. The aim of our work was to evidence whether exposure to sub-lethal doses of specific contaminants (oxybenzone (OXI), mercuric chloride (HgCl2) and trybutiltin (TBT)) would induce MXR transporter activity during embryonic development (from zygote to blastula stage) in purple sea urchin (Paracentrotus lividus) embryos. Further, we present data on molecular identification, transport function, expression levels and gene localisation of two ABC efflux transporters-P-glycoprotein (ABCB1/P-gp) and sulfonylurea-receptor-like protein (ABCC9/SUR-like). Partial cDNA sequences of abcb1 and abcc9 were identified and quantitative PCR (qPCR) evidenced an increase in mRNA transcript levels of both ABC transporters during the two-cell, as well as an overall decrease during the blastulae stage. Calcein-AM efflux activity assay indicated the activation of multidrug resistance-associated protein/ABCC-like transport in the presence of HgCl2 and TBT in exposed blastulae. The in situ hybridisation of the two-cell and blastula stages showed ubiquitous localisation of both transcripts within cells, supporting qPCR data. In conclusion, ABCB1 and ABCC9 are constitutive, as are HgCl2, TBT and OXI-inducible ABC membrane transporters, coexpressed in the zygote, two-cell and blastula stages of the P. lividus. Their ubiquitous cell localisation further fortifies their protective role in early embryonic development.