Fish gills are responsible for several vital physiological functions other than respiration, such as osmoregulation, excretion of nitrogenous waste, pH regulation, hormone production  and homeostatic regulation of copper . Compromised gill function would inevitably alter these physiological functions, hence the importance of assuring gill health. Despite the impact on animal welfare and the economic repercussions, little is known about the effect of S. chrysophrii in GSB. The plasma proteomic profile from healthy and S. chrysophrii-infected GSB was assessed via a SWATH-MS analysis to better understand the host-parasite interactions and broaden the knowledge of the pathogenesis of sparicotylosis.
The discriminant analysis of the abundance of all detected proteins formed three different groups (C, M/L and H). The dispersal among the healthy GSB (C) group was clearly greater than among diseased fish (M/L and H groups) (Fig. 1A). However, differentially abundant proteins in the M/L group showed a greater disparity than in the H group, which in turn seemed closer to the C group phenotype in both the PLS-DA model and heatmap (Fig. 1). These observations suggest that the hosts suffer a profound imbalance when suffering mild (M/L) S. chrysophrii-infection intensities, as observed by Piazzon et al. 2019 . In GSB surviving to high parasitic burdens (H), the detected proteomic profile seems to indicate an onset of compensation mechanisms to restore homeostasis, getting closer to the profile of C fish. In sparicotylosis, such homeostasis recovery might be the result of the monogeneans’ intimate coevolution with their hosts [41, 42]. Under farming conditions, with high stocking densities, exposure to environmental stressors, high infection pressures and recurrent sparicotylosis infections once the disease is established, high mortality is reported, even after receiving treatment. Apparently, under these harsh farming conditions, fish would be unable to recover homeostasis, resulting the pathogenic effect of the parasite more devastating. Hence, future proteomic plasma studies of moribund GSB from sea cages suffering sparicotylosis would shed some light on this issue. However, the current results were obtained in a time-limited exposure (10 weeks) and under a relatively bounded parasite multiplication context compared to an enzootic farm. Under these conditions, the observed attempt of homeostatic restitution in highly infected fish raises the question of whether an earlier stimulation of these mechanisms by some dietary or health interventions would be feasible, in order to mitigate the effects of the disease.
Sparicotyle chrysophrii modulated proteins involved in several biological processes in GSB. Among them, the levels of various proteins increased and decreased in a complex network of interactions. The main pathways severely modulated by S. chrysophrii were those related to haemostasis, lipid metabolism and transport, and the immune system (Fig. 3).
Polyopisthocotylean monogeneans have been described as hematophagous parasites [43, 44], but it has not been until recently that the haematophagous nature of S. chrysophrii has been demonstrated experimentally . From the current study, we can discern a clear negative impact on the GSB’s haemostasis. Blood haemoglobin significantly dropped as the parasite burden increased (Fig. 4). These low Hb values mirrored the plasma proteomic results, where the main Hb constituents, alpha-2 and beta globins, negatively and significantly correlated with the infection intensity (Table 1, Fig. 4). In addition, alpha-1-microglobulin, a radical scavenger dealing with heme toxicity and erythroprotective anti-haemolytic effects in humans , presented a positive and significant correlation (Table 1). This suggests that S. chrysophrii-infected GSB suffer from haemolytic anaemia, as occurs in hosts facing a haemolytic insult, which leads to the release of Hb and free heme groups from erythrocytes, increasing the oxidative stress . Overall, these results would imply anaemia and oxygen transport impairment, explaining hypoxia and lethargy signs observed in parasitised fish.
The coagulation cascade also seems to be triggered by sparicotylosis. In mammalian [47, 48] and fish  blood, the extrinsic pathway is initiated following tissue damage and subsequent exposure of subendothelial tissue factor (TF) to Factor VII, whereas the intrinsic pathway is triggered by the exposure of a foreign negatively charged surface to Factor XII. Both pathways converge in Factor X, after which the common pathway of the coagulation cascade follows, resulting in the production of thrombin and leading to clot formation and final restoration of haemostasis [47, 48]. In the current study, most proteins involved in the coagulation cascade were represented in cluster B of the K-means analysis (Fig. 2). In GSB suffering sparicotylosis, both intrinsic (Factor IX) and extrinsic (Factor VII) pathways of the coagulation cascade as well as common pathway of the coagulation cascade (Factor X and Factor V) were modulated (Additional file 2). It is noteworthy that, unlike in the M/L group, all coagulation factors in cluster B were upregulated in the H group (Fig. 2), in agreement with the significantly positive correlation of Factor X with the infection intensity (Table 1). Thus, the coagulation capacity of GSB apparently increased when high parasitic burdens were reached. Similarly, in several tick species, different proteins with anticoagulant properties affecting the intrinsic, extrinsic and common coagulation pathways [50–52] have been described and characterised, suggesting that these haematophagous parasites can modulate their host’s haemostasis at different levels.
Since S. chrysophrii is an ectoparasite and not an intravascular parasite, we suspect that the intrinsic pathway could be, in part, triggered by RBCs fragment remnants  resulting from haemolysis. In contrast, the activation of the extrinsic pathway, may be due to tissue disruption induced by the parasite’s feeding mechanisms and its haptor.
Lipid metabolism and transport
Different parasitic species, ranging from Protozoa to Metazoa, have been described to alter the lipidic profile of their host species in fish  as well as in higher vertebrates [55–64]. In particular, Platyhelminthes are unable to synthesise fatty acids de novo ; thus, they rely on the host’s lipid reservoir to ensure their survival. Several fatty acid binding proteins (FABPs) have been identified in trematode species such as Schistosoma spp., Fasciola spp, and most recently in the diplozoid monogenean Eudiplozoon nipponicum [66, 67]. Even though FABPs have been described to play a role in fatty acid uptake in F. hepatica from host blood and in immunomodulation, their function in monogeneans remains unknown . Our results show that apolipoprotein B-100 (ApoB-100) and apolipoprotein A-II (ApoA-II) were negatively and significantly correlated with the infection intensity (Table 1; Fig. 5). In addition, cholesterolaemia values in plasma samples of S. chrysophrii - infected GSB were significantly lower than in the C group, supporting our proteomic results (Fig. 5). Reduction of plasma cholesterol in GSB was also triggered by environmental stressors [68, 69]) and dietary intervention involving the replacement of fish meal and oil by vegetable ingredients [32, 70]. The latter provoked a simultaneous drop in plasma cholesterol and blood Hb, which was reversed by a butyrate additive in the diet. This finding could open a path for the use of butyrate as a mitigation strategy for the effects of sparicotylosis.
ApoB-100 is a crucial structural component in very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL), predominantly composed of triglycerides and cholesteryl esters, respectively. ApoA-II, on the other hand, is associated with high-density lipoproteins (HDL2 and HDL3), predominantly composed of cholesteryl esters [71, 72]. Our results suggest a dependency of S. chrysophrii on its host’s lipid reservoir. However, the mechanisms by which S. chrysophrii would rely on the host’s lipids remain unknown.
Plasma lipoproteins (LDLs and HDLs) play an essential role in host defence as a component of the immune system [73–75] and against bacterial, viral and parasitological infections  in mammals. Hence, in our fish-parasite model, an alteration in lipoprotein levels could render the host more susceptible to secondary infections. Moreover, other roles in haemostasis have been granted to LDLs and HDLs, thus somewhat contributing to the control of haematological parameters, such as RBC membrane stability .
Differences regarding the host’s immune response have been observed between monopistholocotylean and polyopisthocotylean monogenean parasites . These differences have been suggested to lie in the different feeding strategies , since polyopisthocotylean monogeneans are generally considered haematophagous. Hence, they need to evade the host’s immune response to ensure their feeding and survival. Our study shows how S. chrysophrii infection changes the abundance of several complement proteins (factor H, factor B, factor I, C1q, C3, C4, C5, C6, C7, C8; Additional file 2), inducing an inhibition of the alternative pathway as the infection intensity increases (Fig. 6). In the same line, other studies with the same host and parasite species revealed that this parasite downregulated c3 splenic expression  and lowered complement levels in serum . A local downregulation of c3 expression has also been described in other monogenean infections [78, 79]. The depletion of complement effectors in GSB plasma worsened during the infection, compromising the fish immunocompetence. Further in-depth studies regarding the host’s immune response during monogenean parasite infections and, in particular, polyopisthocotylean monogeneans are required to fully understand the exact mechanisms involved. Our results point to a complex network regulating the innate immune response, including SERPINs and ceruloplasmin, which may indirectly modulate the complement system, resulting in neutrophil activation and inflammation.
Proteins linking haemostasis and immune system
Serine-protease inhibitors (SERPINs)
Serine proteases are conserved enzymes throughout evolution that have a crucial role in several physiological processes, including blood coagulation, fibrinolysis, inflammation and immune response. SERPINs obtain their name from serine protease inhibitors. Still, they are a superfamily of proteins that primarily regulate the proteolytic pathways of serine and cysteine proteases [80–83].
It has been described that protease inhibitors may have a leading role in host-parasite interactions and, more specifically, in evasion mechanisms and survival on the parasite’s behalf [84–86]. Interestingly, two protease inhibitors similar to SERPING1 in terms of function, EnSerp1 and EnKT1, have been characterised in the monogenean Eudiplozoon nipponicum [87, 88].
Some authors suggested that some digenean trematode parasites could have the ability to modulate the host’s SERPINs . In several parasitic species, including ticks [50, 51, 89], copepods , digenean [91–94], and monogenean [66, 87, 88, 95–98] trematodes, endogenous proteases and protease inhibitors potentially acting as virulence factors have been described and characterised.
In the current study, three SERPINs were differentially expressed upon infection; SERPINA1 (α1–antitrypsin), SERPIND1 (heparin coagulation factor II) and SERPING1 (C1-inhibitor; C1INH) (Additional file 2). SERPINA1 was grouped in cluster C (more abundant in M/L group), whereas both SERPIND1 and SERPING1 were grouped in cluster B (more abundant in H) (Fig. 2).
SERPINA1 inhibits neutrophil elastase, a serine protease with microbiocidal effects involved in the acute phase of the inflammation process and tissue remodelling . We relate this observation to an early impairment of an inflammatory response elicited by neutrophils. This event could be driven by a deliberate modulation of SERPINA1 by S. chrysophrii as an evasion mechanism, in order to enable attachment to gill filaments. Similar effects on SERPINA1 have been observed in murine Toxoplasma gondii infections , but no records are available on SERPINA1 being modulated by fish parasites.
Both SERPIND1 and SERPING1 have a key role in coagulation; however, SERPING1 also affects the immune system. SERPIND1 is known to, directly and indirectly, inhibit thrombin in the common pathway of the coagulation cascade [80, 81, 100], thus preventing the fibrinogen and platelet activation and ultimately preventing the clot formation and haemostasis restoration. At a haemostatic level, SERPING1 inhibits several components within the intrinsic coagulation pathway (plasma kallikrein, activated Factor XII (FXIIa) and XI (FXIa)) as well as fibrinolytic proteases (plasmin, tPA and uPA). Further SERPING1 inhibitory abilities extend to both C1s and C1r, proteases responsible for the activation and proteolytic activity of the C1 complex of the classic complement pathway [47, 48, 80, 81, 83, 101–103].
Thus, the significantly higher abundance of SERNPIND1 and SERPING1 in GSB with high parasitic burdens could imply an anticoagulant and innate immunosuppressor effect in these hosts (Fig. 2). However, within cluster B, SERPING1 shows a significant negative correlation with the infection intensity (Table 1). SERPINs operate within a complex physiological modulation network, and further SERPIN-targeted studies are needed to unravel this paradox, as well as the opposing coagulant and anticoagulant actions of serpins and the coagulation cascade. Counter-regulation evidenced by our results might be the effect of the host response aiming for homeostatic balance or a host vs parasite modulation.
Ceruloplasmin is an acute-phase protein that has been associated with inflammation, severe infection and tissue damage in mammals and fish. Ceruloplasmin has also been described as a copper-carrying protein, ultimately having a role in hypoxic vasodilation and ischemia-reperfusion cytoprotection [104–106], and having the ability to oxidise toxic plasma ferrous iron into its ferric form to be transported by transferrin . Moreover, under hyperammonaemia conditions, the intrinsic pathway of the coagulation cascade is triggered, and the functional activity of platelets decreases. However, ceruloplasmin can prevent haemostatic disorders by restoring platelet functionality and preventing hypercoagulation .
Henry et al. 2015 previously described no significant differences in ceruloplasmin activity in GSB after a 10 week-long S. chrysophrii infection. Our results suggest an initial increase in plasma ceruloplasmin levels in M/L followed by a later decrease during the course of infection in H group. Thus, we hypothesize that fish with lower infection intensities were in an acute phase of the disease, while H GSB restored their ceruloplasmin to control levels in agreement with the observations of Henry et al. 2015.