Unveiling the effect of dietary essential oils supplementation in Sparus aurata gills and its efficiency against the infestation by Sparicotyle chrysophrii

Introduction

Nutritional therapies provide an important strategy for preventing and/or treating diseases¹. Among different options, such as the use of probiotics, prebiotics, immunostimulants and organic acids, phytogenics have gained interest as feed additives within aquafeeds². Phytogenics are plant-based natural substances derived from herbs, spices or extracts similar to essential oils (EOs), which are reputed for their beneficial properties and efficacy on performance and health in animal production³. In aquafeeds, EOs as dietary additives have been reported to stimulate appetite, improve feed utilization and growth, and boost the innate immunity⁴.

Thymol, carvacrol, cinnamaldehyde and EOs from clove, coriander, star anise, ginger, garlic, rosemary, mint among others, have been used either individually or as blends in animal nutrition^5,6. Among phytogenics, oregano (Origanum vulgare) is the most common because of its richness in carvacrol and thymol^7,8. These compounds have a wide range of properties such as antimicrobial⁹, immunostimulant and anti-oxidative activities^10,11, and the ability to enhance intestinal absorption¹², to improve growth¹³ and even to reduce cumulative mortality¹¹. The effectiveness of garlic (Allium sativum) extract as an immunostimulant, antimicrobial and antiparasitic agent has been demonstrated in several fish species^14–18; the inclusion of garlic extract in fish diets is effective against monogenean parasites^19,20.

Combinations of different EOs are promising strategies for functional feeds; however, evaluating their effectiveness in front of a biological challenge such as an ectoparasite infestation, as well as deciphering their mode of action is necessary prior to their recommendation as feed additives²¹. Under this context, the aim of this work was to evaluate the functional response of gilthead seabream, the most important farmed fish in the Mediterranean basin, to the dietary administration of a microencapsulated combination of garlic, carvacrol and thymol EOs. In teleosts, gills are one of the main mucosal barriers containing an associated-lymphoid tissue (GIALT) with innate and adaptive immune components that pathogens encounter upon first contact with the host^22,23. Thus, we ran a nutritional trial with the assessment of gill’s transcriptomic profiling in order to describe for the first time the main metabolic and immune pathways regulated by these EOs in this lymphoid tissue, as well as the histochemical properties of mucins produced by branchial mucous cells. Moreover, the efficiency in controlling the infestation by S. chrysophrii was also assessed through an in vivo cohabitation challenge trial.

Results

Discussion

The use of garlic, carvacrol and/or thymol EOs in functional aquafeeds has been tested and demonstrated both in vitro and in vivo for its effectiveness in fighting against bacterial and parasitic infections^2,25,26. The anthelmintic properties of EOs and gill’s response to their dietary administration are poorly understood and available information is scarce. Hence, most of the existing studies are focused in the effects of the above-mentioned EOs on intestinal health^10,12,27,28, as well as in their use in balneation treatments against parasitic organisms^{15,16,29–31}. As far as we know, this is the first study describing the gill’s response in gilthead seabream to the administration of the above-mentioned EOs as a feed additive, as well as the mechanisms underlying its antiparasitic properties.

The dietary inclusion of microencapsulated garlic, carvacrol and thymol EOs did not affect fish growth. Contrarily, previous studies suggested them as growth promoters in several fish species^{13,25,27,32–38}. Although the reasons explaining such differences are out of scope in this study, the utilization of different EO doses, administration length and diet formulation could be among the potential factors that may explain such results. Regardless of the fact that growth has been traditionally considered as one of the main end-points and/or key performance indicators for additive testing, some existing literature might not be trustworthy due to constrains in the reproducibility of the compounds studied³⁹. Under this context, the use of natural plant-derivates or extracts may lead to variability and discrepancy of results, as opposed to the utilization of similar synthetic compounds, which favours the reproducibility and robustness of the studies. Moreover, SGR values of gilthead sea bream fed both control (2.1 ± 0.07% BW/day) and the EOs supplemented diet (2.03 ± 0.01% BW/day) compared favourably to those reported by Mongile et al.⁴⁰ for this species also reared under summer conditions (1.5 ± 0.1% BW/day).

Gills are one of the main mucosal immune barriers in fish^22,41,42, but they also represent an ideal site for the attachment of ectoparasites⁴³, potentially inducing a host hypometabolic response, as suggested for S. chrysophrii infections⁴⁴. In fact, gills are considered one of the most active tissues in the protein synthesis with a significantly high plasticity in protein metabolism⁴⁵. Our transcriptional study revealed several biological processes associated to biogenesis and metabolic processes, including peptide biosynthesis, and protein and lipid metabolism that were predominantly up-regulated in the gills of gilthead seabream fed dietary EOs. Therefore, EOs would contribute to restore the gill's metabolic rate by increasing protein synthesis. Although gill’s protein turnover contribution is not significant to the whole body⁴⁵, our transcriptional profile is in accordance with studies that have reported the significant influence of garlic^33,34, carvacrol⁴⁶ and thymol^13,35 dietary administration on body and blood protein content upon the protein synthesis and metabolism. Collectively, our results provide new evidence for the biological activity of garlic, carvacrol and thymol, indicating these compounds also promote the protein synthesis and metabolism at mucosal level, particularly on gills (see text below); thus, increasing the overall activity of this tissue.

The vesicular trafficking processes are intrinsic to the secretory protein biogenesis⁴⁷. Despite the previous evidence reporting an augment in the proteins synthesis^{13,32–35,46}, the transcriptional regulation of vesicle-mediated transport by the dietary administration of garlic, carvacrol and thymol have not been described in fish to date. Genes involved in protein vesicle-mediated transport, such as the ER to Golgi vesicle-mediated transport process, were also positively regulated in the gills of fish fed dietary EOs, as for example the Secretion Associated Ras-related GTPase 1A (sar1a), the Rho Guanine Nucleotide Exchange Factor 7 (arhgef7), the Vesicle Transport Through Interaction With T-SNAREs 1B (vti1b) and some of the Rab Family of GTPases (rab11b, rab2a). The above-mentioned genes are known for their role in cellular trafficking pathways, like the RAB11-coding protein, which is recognized for its localization in recycling endosomes and its role in exocytic trafficking⁴⁸. In vitro studies in mammal cells have associated garlic⁴⁹, carvacrol and thymol to vesicle fusion and exocytic processes⁵⁰. Therefore, we may infer that the machinery implied in the activation of biogenic processes observed by dietary EOs is inherent to the activation of processes of secretory protein translocation by vesicles.

As previously mentioned, vesicle trafficking and exocytosis are intimately related processes. In this way, genes like Rab GTPases participate in the regulation of the exocytosis membrane trafficking pathway⁵¹. In our gill’s transcriptional analysis, exocytosis was one of the most positively regulated processes by dietary EOs. This finding is especially relevant since exocytosis is recognized by its important role in the immune response participating in neutrophil function⁵², in the immunological synapses between cells⁵³ and in the cell-mediated cytotoxicity⁵⁴. Remarkably, the tested EOs also positively regulated immune-related biological processes by means of myeloid leukocyte immunity activation. Besides, this response appeared to be orchestrated by neutrophil population, since neutrophil mediated immunity and neutrophil degranulation processes were boosted by dietary EOs. In gilthead seabream, acidophilic granulocytes are functionally equivalent to higher vertebrates neutrophils^55,56 being described as one of main phagocytes of this species^57,58 occurring also in mucosal tissues^59–61. Neutrophils’ granules are reputed for their antimicrobial, proteolytic and potential cytotoxic capacities^52,62, which are synthetized during myeloid cell differentiation, comprising specific protein biosynthesis and the early formation of secretory vesicles⁵². Remarkably, we detected the presence of DEGs associated with protein biosynthesis, vesicular transport and exocytosis (as formerly discussed), which could be in their turn up-regulated due to the immunostimulatory effect of dietary EOs upon acidophilic granulocytes degranulation process. For instance, Mitogen-Activated Protein Kinase 3 gene (mapk3) resulted up-regulated in the gills of the EOs diet group, with representation both in protein and lipid metabolic and in the vesicle-mediated transport processes. Mitogen-activated protein kinases (MAPK) are known to be involved on signalling pathways of neutrophil functional response^63,64. There is evidence of the activation of MAPK by ajoene (an organosulfur compound found in garlic) in the process of apoptosis of human cancer cells⁶⁵. Despite of the evidences of an acidophilic granulocyte-mediated immune response stimulation induced by the bioactive compounds of the EOs tested, the exact bioactive compounds and accurate mechanisms involved in the alleged immunomodulatory and antiparasite effects of these EOs still needs to be deciphered when tested in separate.

In aquaculture relevant species, some studies reported an increase of blood neutrophil number after therapeutic balneation with EOs, which was also effective against monogeneans^29,66,67. Neutrophils function involves the interplay of many different receptors, ion channels and signalling pathways, such as changes in intracellular Ca²⁺ levels, for instance⁶⁸. Accordingly, garlic EO organosulfur compounds were recently demonstrated to activate human neutrophil functional activity through the activation of Ca²⁺ flux⁶⁴, whereas ajoene and allicin were described as potent inhibitors of neutrophil ROS production⁶⁴. Similar results were also attributed to thymol redox properties⁶⁹, whereas in fish, dietary carvacrol (0.05%) significantly reduced leukocyte ROS release in seabass¹¹. Although neutrophils and other circulating leukocytes have a critical role in the innate immune defence against pathogens, such helminth parasites⁷⁰ including monogeneans⁷¹; and that acidophilic granulocytes have been particularly identified in gilthead seabream gill's response to ectoparasite infections⁵⁹; its activity comprises a significant tissue damage associated to the ROS released during the inflammatory process. For instance, it was proposed that gilthead seabream may control S. chrysophrii infection through ROS action produced by immune cells. Although parasite evasion mechanisms were also suggested, this response may potentiate secondary infections if it is not properly controlled⁷². Although it might seem contradictory, the sustenance of self-protective antioxidant mechanisms is vital for the correct functioning of the immune system, preventing oxidative damage by ROS that escort leukocyte activity; particularly in neutrophil-mediated immune response⁷³. Outstandingly, these antioxidant properties were also highlighted in the transcriptomic profiling of the gills in fish fed dietary EOs, where several genes involved in oxidation–reduction processes were positively regulated. Aquafeeds containing natural garlic (4%), garlic powder (3.2%) and garlic oil (0.25%) promoted glutathione peroxidase, superoxide dismutase and catalase activities in tilapia serum and liver³³. In seabass, after cadmium-induced toxicity, the up-regulation of genes coding for these enzymes in the liver improved the antioxidant capacity of individuals fed a diet containing garlic powder (2%)⁷⁴. Similarly, an increased antioxidant activity in rainbow trout fillet was associated to dietary carvacrol (1.2%) or thymol (0.6%)¹⁰, and in channel catfish, a commercial product containing O. heracleoticum EO (0.05%) enhanced plasma antioxidant activity³². In accordance with the above-mentioned studies, our transcriptomic data showed that dietary EOs promoted the up-regulation of Glutathione Peroxidases (gpx1, gpx7) and Glutathione S-transferases (gstm1, gstk1, mgst2). Additionally, Mitochondrial Thioredoxin (txn2), a key antioxidant protein that participates in the removal of ROS and cytotoxicity^75,76, was also up-regulated by dietary EOs. In our study Peroxiredoxins 1 and 3 (prdx1, prdx3) were also up-regulated. Additionally, the up-regulation of the Epoxide Hydrolase 1 (ephx1) was promoted by dietary EOs; this enzyme has a detoxifying function, playing an important role in cellular and organ defence against exogenous toxicity compounds⁷⁷. Thus, it appears that tested EOs may exert an important antioxidant action to counteract the impact of the high amounts of ROS released by the previously referred stimulation of acidophilic granulocyte’s activity, evidencing the importance of their joint supplementation.

After a proinflammatory phase starred by neutrophils and their ROS production, the induction of a resolution phase is mandatory to prevent persistent harmful inflammation and oxidative stress in the host cells⁷⁶; thus, the activation of the anti-inflammatory response is needed to minimize such side-effects⁷⁸. Therefore, the leukocyte activation together with the up-regulation of a repertoire of anti-inflammatory cytokines (i.e. il7, il6r, il20ra and il21r) is representative of this intimate coordination between both processes, suggesting also an added anti-inflammatory response triggered by the dietary EOs. The modulation of cytokine expression and immune cell stimulation are the mechanisms attributed to the biological activity of garlic compounds⁷⁹. In addition, there are also processes associated to the anti-inflammatory bioactivity of carvacrol and thymol^80,81. For instance, carvacrol was described as an inhibitor of human neutrophil elastase⁸². In our study, the Serpin Family B Member 1 (serpinb1), a protein that inhibits neutrophil elastases protecting tissue from damage at inflammatory sites, was also up-regulated by dietary EOs, corroborating its anti-inflammatory properties. In fish, the up-regulation of inflammatory cytokine genes was observed in the gut of tilapia fed dietary garlic powder (1.0%)¹⁷. In juvenile gilthead seabream, the dietary inclusion of a commercial encapsulated combination of carvacrol and thymol EOs (0.01%) resulted in an enhancement of the intestine absorptive capacity that was attributed to the induction of anti-inflammatory and anti-proliferative gene markers¹². The same commercial additive also demonstrated an immunostimulatory effect in juvenile hybrid tilapia²⁸.

Gills of gilthead seabream infested with S. chrysophrii showed an over-expression of apoptosis, cell proliferation and inflammation processes⁴⁴. Thus, under an infective process, the combination of the pro- and anti-inflammatory mechanisms is also required for a successful pathogen eradication⁷⁸. Although our transcriptomic profiling of gills was conducted at the end of the nutritional trial before fish being exposed to the ectoparasite, the build-up of a local former anti-inflammatory response induced by dietary EOs might delay the effect of the inflammatory outcome associated to S. chrysophrii; thus, potentially reducing tissue damage. This regulation observed in our study could enhance tissue protection and regeneration mechanisms involved in gill's responses against ectoparasites, not only by the stimulation of an inflammatory response, but also by means of antioxidant and anti-inflammatory processes.

One of the common characteristics of mucosal-associated lymphoid tissues, including the GIALT, is the presence of mucus-secreting cells⁴². The main components of mucus are mucins, high molecular weight glycoproteins (GPs) with numerous carbohydrate chains O-glycosidically linked to a protein core. Both commensal and pathogenic microorganisms^83–85, and likewise monogenean parasites^86,87, use this mucosal GPs as receptors for their attachment. A high variety of mucin oligosaccharides forms an extensive repertoire of attachment sites with different carbohydrate specificities⁸⁸. Peculiarly, sialic acids and related saccharide residues can serve as receptor sites for binding exogenous macromolecules such as those of bacterial, viral or parasitical aetiology, playing an important role as “decoy” for pathogens, in such a strategy where the sialylated mucins are shed with the anchored sialic-binding pathogen^89,90. Under parasite infections, qualitative changes of fish mucus occur, mainly in the mucin glycosylation pattern^91–93. Thus, increases in sialic acid and N-acetylglucosamine terminal in mucins are described in as a host defence against helminths⁹⁴. Nevertheless, many pathogens and parasites have evolved to disrupt the mucin barrier; for instance, several digestive mucins were down-regulated and a significant reduction of MC positive for sialic acid was observed in gut-parasitized gilthead seabream^95–97.

In our study, it is relevant to highlight an increase of carboxylated and/or sulphated GPs containing sialic acid and of N-acetylglucosamine/β-d-GlcNAc residues in gills of fish fed dietary EOs. Neutral GPs lubricate, facilitate gas exchange and regulate the acidity of mucous secretions, whereas acid carboxylated and sulphated GPs are more viscous, a characteristic associated to their antibacterial and antiparasitic properties^85,98–100. The higher presence of acid GPs coupled with the increase in sialic acid and N-acetylglucosamine in mucins may be associated to an enhanced protection against S. chrysophrii attachment. Furthermore, MCs hypertrophy observed in gills of fish fed dietary EOs indicated a potentiation of the mucosal secretion and renewal, boosting its protective function. These results may be a consequence of the synergy between garlic, carvacrol and thymol EOs, since such histochemical differences and antiparasitic effects were not observed when garlic was tested by separate (Supplementary Information 1). Transcriptomics revealed that ALG9 Alpha-1,2-mannosyltraferase (dibd1), which is involved in N-glycan biosynthesis¹⁰¹, was positively regulated by dietary EOs, which is one of the most common post-translational modifications of proteins¹⁰². Additionally, we found an up-regulation of O-Sialoglycoprotein Endopeptidase like 1 (osgepl1), whose enzyme is for long commercially used and recognized for its mucin-degrading activity, and which increase might indicate an enhance of the proteolytic mucin degradation¹⁰³, which is characteristic of the host GPs “shedding” defence mechanism. In this way, although not evidenced among the main representative processes in our gill's transcripteractome outcome, the regulation of some of pathways such as translational elongation, peptide biosynthesis, cellular protein metabolism, intracellular protein transport, and exocytosis-related processes, might be correlated with those of the mucosal surface.

Altogether, transcriptomic results suggest that dietary EOs may be promoting the synthesis and release of GPs detected at histological level, which might have a beneficial functionality on the gills and potentially reducing the S. chrysophrii attachment to gill’s surface. Accordingly, gilthead seabream fed dietary EOs showed a reduction of 78% of total parasite load when compared with the control group, with a decrease in the prevalence of most of the parasitic developmental stages as well. Garlic is known for its wide-spectrum of antimicrobial activity that is attributed to allicin and ajoene, which exert multiple inhibitory effects on thiol-dependent enzymatic systems¹⁰⁴. Similarly, garlic-based treatments demonstrated to be particularly effective in the fight against monogeneans and other parasites^18,105,106. Farmed barramundi fed diets containing a garlic extract (50 and 150 mL kg⁻¹) for 30 days showed a reduction of Neobenedenia sp. oncomiracidia stage¹⁹. Similarly, garlic extract administered by balneation (0.76 and 15.2 µL L⁻¹ allicin concentration) had also antiparasitic properties towards Neobenedenia sp.²². In guppy, both diet (10 and 20% garlic powder) and bath (7.5 and 12.5 mL L⁻¹ garlic extract; 1 g L⁻¹ fresh crushed garlic) garlic-based treatments were successful against Gyrodactylus turnbulli²⁰. The application of garlic bath treatments (3 ppt garlic oil; 300 mg L⁻¹ crushed garlic cloves) resulted effective against Trichodina sp. and Gyrodactylus sp. in Nile tilapia¹⁶. However, an in vitro treatment with a water–ethanol extract of garlic tested at different dilutions (1:10, 1:50 and 1:100) showed no overall antiparasitic effect on Neobenedenia sp.¹⁰⁷. The instability of free organosulfurs may lead to contradictory results in terms of the efficiency of garlic extracts and doses against monogenean parasites. These results highlight the benefits of encapsulating this type of compounds for dietary administration, since this process ensures their dietary stability, preventing inopportune interactions with the host and environment, and allowing their proper delivery in the gastrointestinal tract⁴.

Concerning carvacrol and thymol, several bath treatments with different EOs proved to be effective anthelmintics^29–31. The antiparasitic action of carvacrol against protozoans in chum salmon was associated to its presence in the skin of fish fed a diet supplemented with oregano EO at 0.02%¹⁰⁹. Thymol was also demonstrated to have antiparasitic effects against the protozoan Leishmania sp.¹⁰⁸ and sheep gastrointestinal nematode helminths¹⁰⁹. Nonetheless, there are few studies that accurately describe the antiparasitic effect of dietary phytochemicals against monogeneans in fish species; thus, the pathways and mechanisms of their action are not clear yet¹¹⁰. Regardless of this fact, present transcriptomic data from gills at the end of the nutritional trial provided the base line knowledge for deciphering the antiparasitic role of the tested EOs. While fish immunity against monogenean parasites is certainly multifactorial and innate factors seem to dominate the first response against this parasites^111–113, the participation of the mucosal adaptive factors, such as B-lymphocytes^42,114, immunoglobulins^115–117 or even specific antibodies^118–120, could be critical for longstanding parasite suppression^71,86. Responses against helminth parasites also include the expression of classical effector type 2 cytokines that will signal the recruitment of inflammatory cells and induce goblet cell hyperplasia leading to mucus production¹²¹. Nevertheless, the specific mechanisms modulated during the parasitic challenge were not evaluated in this work, thus further studies are needed to determine if a type 2 immune response is also implicated in the success of the diet-induced antiparasitic response. Studies focused on identifying the bioactive compounds responsible for the gill's response observed in the present work are currently in progress.

Concluding, we showed that the administration of a microencapsulated feed additive containing garlic, carvacrol and thymol EOs in gilthead seabream promoted the activation of protein biosynthetic processes in gills. These biogenic processes are highly related to the translation of mRNA into proteins, which in turn are actively mobilized by vesicular transport and exocytosis. This mechanism activates effector leukocytes like acidophilic granulocytes. The immune response promoted by dietary EOs is also supported by the active control of oxidation–reduction processes, the building of an anti-inflammatory local response and the changes in the histochemical properties of mucins produced by branchial MCs. The overall results of our study highlighted the main biological processes induced by this dietary EOs that might be responsible for the later antiparasitic response observed in gills against S. chrysophrii. The notorious effect of the tested dietary EOs suggests its application as preventive and active treatment for this particular ectoparasite and a promising alternative treatment for other infections, although further evaluation is needed in order to validate this hypothesis.

Methods

Supplementary information

Author contributions

A.E. and E.G. designed and carried out the experiments. Biological samplings were performed by E.G., A.E. and J.F. The transcriptomic analysis was performed by F.E.R.-L., E.V.V. and J.F.; J.B. and J.F. contributed to the transcriptomic results graphic design. C.S. and J.O.D. carried out the histochemical analyses. J.F. wrote the original draft. All the authors provided critical feedback and approved the final manuscript. The study was supervised by E.G., F.E.R.-L and L.T.

Competing interests

Joana P. Firmino is a current TECNOVIT-FARMFAES S.L employer conducting an Industrial PhD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary information

is available for this paper at 10.1038/s41598-020-74625-5.