4.1. Extracellular vesicle biogenesis
Most studies regarding EV biogenesis in platyhelminthic parasites have focused on the identification of the different proteins involved disregarding their biological functionality. Thus, platyhelminth EV biogenesis in general remains poorly understood, and has not been addressed in a monogenean fish parasite before.
Recently, the proteins required for EV biogenesis in helminths have been considered as well-conserved throughout different phyla. However, the degree of conservation of these proteins in Monogenea, with Protopolystoma xenopodis (Polyopisthocotylea, Polystomatidae) as the only representative of such class, raised questions due to the lack of orthologues and the low conservation of their interacting regions [23].
In regard to EV biogenesis by the ESCRT-dependent pathway, ESCRT-0, ESCRT-II and ESCRT auxiliary proteins are almost fully conserved in platyhelminths, whereas some proteins involved in ESCRT-I and ESCRT-III appear to be lacking in Trematoda (ESCRT-I: VPS28, VPS37 and MVB12; ESCRT-III: CHMP6 and IST1) and Monogenea (ESCRT-I: TSG101, VPS37 and MVB12; ESCRT-III: CHMP2, CHMP3, CHMP5 and IST1) [23]. The in silico analysis of S. chrysophrii revealed that the only missing protein required for EV biogenesis related to the ESCRT-dependent pathway was VPS37 (ESCRT-I), as no orthologue of any H. sapiens isoform (UniProt: VPS37a: Q8NEZ2; VPAS37b: Q9H9H4; VPS37c: A5D8V6; VPS37d: Q86XT2) nor Schistosoma bovis (Digenea, Schistosomatidae) VPS37 protein (GenBank: RTG90235.1) was confidently identified. It is to mention that, FYVE and VHS domains for HGS (ESCRT-0) were found and that a single isoform orthologue was found for STAM (ESCRT-0), MVB12 (ESCRT-I), CHMP4 (ESCRT-III) and VPS4 (ESCRT-related component) in S. chrysophrii, as opposed to the several isoforms of these proteins present in H. sapiens (STAM1/2, MVB12a/b, CHMP4a/b/c, and VPS4a/b). Moreover, from the analysed SNAREs, YKT6 was identified unlike in P. xenopodis, and VAMP7 remained the only unidentified protein in S. chrysophrii, in agreement with most Nematoda clades and parasitic platyhelminths [23]. In view of these findings, we assert that exosome production in S. chrysophrii could be primarily driven by the ESCRT-dependent pathway as described for adult Fasciola hepatica (Digenea, Fasciolidae) [41]. However, VPS37 is key for the modulation of the ternary complex formation of ESCRT-I and PDCD6IP/ALIX[42] and yet it is missing in S. chrysophrii and in trematodes, or highly divergent from their H. sapiens homologues. Specific species adaptations cannot be ruled out, and moreover, specific EV biogenesis pathways may acquire relevant roles depending on the parasite’s life stage as observed in Schistosoma japonicum (Digenea, Schistosomatidae; [43]. In the context of the ESCRT-dependent pathway, a non-canonical ESCRT-associated route revolving around the programmed cell death 6-interacting protein (PDCD6IP/ALIX) has been identified in EV biogenesis. In mammals, PDCD6IP/ALIX recruits ESCRT-III and facilitates the sorting and delivery of tetraspanins to exosomes. Additionally, PDCD6IP/ALIX can associate with transmembrane syndecan proteins via syntenin, thereby promoting the membrane budding steps of ILVs biogenesis [44–46]. In the current study, the non-canonical ESCRT-associated analysed protein representatives were identified, including syndecan, syntenin, and Bro1-domain containing protein, herein classified as PDCD6IP/ALIX. However, due to low homology (Table 1), further studies on S. chrysophrii PDCD6IP/ALIX are required. Moreover, tetraspanin CD63, and other membrane organiser proteins such as flotillin1/2 (FLOT1/2) appeared to be conserved in S. chrysophrii, in agreement with previous in silico studies [23].
Lipid-modifying proteins, involved in the ESCRT-independent pathway and MV formation (Table 1), seem to be the least conserved in Monogenea and among other helminths, most probably due to the inability of helminth species to synthesise fatty acids de novo [47]. Thus, SMPD1/3, SMPDL3a/b, S1PR1/3, PLA2 remained unidentified in our analysis and were consistent with the finding in P. xenopodis. However, three further differences were noticed. Unlike in P. xenopodis, SMS2 and sphingosine kinase (SPHK) were identified. Interestingly, PLB-like 2 protein, apparently conserved throughout all helminth phyla including P. xenopodis[23] remained unidentified in S. chrysophrii. Furthermore, a single DGK isoform ortholog was identified in S. chrysophrii as opposed to the two isoforms present in H. sapiens.
The release of MVs are regulated by small Rab GTPases, including Rho-associated coiled coil-containing protein kinase and the GTP-binding protein ADP-ribosylation factor 6 (ARF6), which have been identified as positive regulators of vesicle budding in cancer cells [22, 48]. In the current study, Rho-associated protein kinase 1 and 2 (ROCK1/2) were identified together with ARF6 and ARF1; interestingly, a third ARF protein besides ARF6/1 was identified in the protein cargo from S. chrysophrii EVs, raising questions about the possible presence of other ARF isoforms and their role in monogenean EVs. Moreover, DIAPH3, previously reported in the platyhelminths, Echinococcus multilocularis (Eucestoda, Taeniidae) and Schistosoma spp. [23] was identified in S. chrysophrii, adding Monogenea as the third platyhelminth class where DIAPH3 has been identified. Other signalling proteins such ARRDC1, apparently absent in all platyhelminth classes remained also absent in S. chrysophrii, once again in agreement with previous findings [23].
Finally, regarding the proteins required for RNA sorting into exosomes, hnRNPQ was previously reported to be exclusive of Nematoda [23]. However, it was also identified in the trematode Opisthorchis viverrini (Digenea, Opisthorchiidae; GenBank: OON14851.1), from which a single orthologue was found in S. chrysophrii.
4.2. Extracellular vesicles isolation and transmission electron microscopy (TEM)
The present study showed that the second day of in vitro maintenance of S. chrysophrii was the best time frame for EV collection with a nanoparticle concentration of 2.33 × 1010 ± 1.12 × 109 particles · mL− 1 (mean ± SD) and a sample purity of 6.89 × 108 particles · µg protein− 1. Previous studies proposed that a purity of 1 × 108 particles · µg protein− 1 represent high-quality vesicular preparations from Schistosoma spp. and other helminth parasites [49]. Since the purity of the analysed samples is in the same order of magnitude, given the target organism, we consider the EV samples to have a high purity.
So far, two different EV subpopulations (exosomes and MVs) have been identified in adult trematode specimens in Fasciola spp., Schistosoma mansoni (Digenea, Schistosomatidae) and O. viverrini [41, 50–52]. Similarly, the current EV protein composition of S. chrysophrii indicated that S. chrysophrii has two distinctive EV subpopulations. Furthermore, we isolated nanoparticles in a 53.5–918.5 nm range, which correspond to exosomes (30–100 nm), and MVs (150–1000 nm) from the medium in which adult S. chrysophrii specimens were kept. Nevertheless, the nanoparticle population profile showed a greater abundance of those with a size of ≈ 200 nm, and EVs morphologically compatible with MVs, were identified by TEM in the opisthaptor region adjacent to S. chrysophrii’s clamps. Therefore, it is reasonable to argue that MVs could have a more relevant role as effector components in S. chrysophrii – S. aurata – associated microbiota interactions.
The clamp covering syncytial cytoplasm containing different vesicle types, multivesicular structures and large vacuoles has been described from several monogeneans, and vesicles in the process of exocytosis from the syncytium surface were observed in Gotocotyla bivaginalis (Polyopisthocotylea, Gotocotylidae) and Chimaericola leptogaster (Polyopisthocotylea, Chimaericolidae) [53–55]. For the latter, differences in the syncytial surface and composition were also described depending on its luminal or external orientation in the clamp, where the syncytial thickenings at the clamp distal tips were considered clamp lips. However, the excretory/secretory importance of the secreted vesicles was not further discussed. Yet, exocytosis of syncytial material, such as secretory granules, vesicles and vacuoles was also suggested as a mechanism of monogenean protection against immunological, ionic and osmotic damage by the gill mucus secretion or water forces [55]. In S. chrysophrii, the occurrence of MVs in the tegument syncytium-lamellar membrane interphase could suggest a protection mechanism but, considering the tight host-parasite-microbiota contact, a mechanism of modulation on the fish host or host-associated microbiota via parasite’s ESPs, should not be ruled out.
Konstanzová et al. [54] found in Paradiplozoon homoion (Polyopisthocotylea, Diplozoidae) glandular cells with vesicles at the sclerite base, apparently responsible for the secreted vesicles in the syncytium cytoplasm. These authors described three clearly differentiated vesicle types by their electron density and diameter, and suggested that vesicles may have a storage function. In our case, a more heterogenous vesicle population from 50 nm to 400 nm diameter was found in the cytoplasm of the tegument syncytium, but the question of whether these have a storage or secretory fate remains unsolved. In regard to ultrastructure, Konstanzová et al. [54] and Mergo [56] also described that clamps were covered by a very thin syncytial layer, compared to the overall body surface in P. homoion and Diplostamenides spinicirrus (formerly Microcotyle spinicirrus; Polyopisthocotylea, Microcotylidae), respectively. The reduction of the syncytial thickness in clamps is a trait shared among other monogeneans and was attributed to an adaptation to increase the grasping ability to the host tissue in the limited space between gill lamellae [53–56]. In S. chrysophrii, no tegumental MVB containing ILVs nor exosomes were identified on the outer body surface, perhaps due to the way these parasites interact with their host, as opposed to endoparasitic helminths. However, the lack of secretion of exosomes or MVs through tegument in other regions of the parasite’s body attributed to the sample preparation, should not be ruled out.
In any case, the intimate contact between S. chrysophrii clamps and their hosts’ secondary lamellae pinpoints the opisthaptor as an optimal area for host recognition and continuous parasite-host-microbiota cross-talk. Furthermore, haemorrhagic mechanical microlessions inflicted by the haptoral clamps in the gill epithelium [57], might facilitate host exposure to EVs. Future research might focus on the ability of S. chrysophrii EVs to internalise into host cells, especially since a cell-polarity regulator protein (LLGL scibble cell polarity complex component 2) has been identified in the current EV proteome analysis [58].
4.3. Extracellular vesicles proteome analysis
Proteases are considered a mechanism for the uptake of haemoglobin, a nutrient-rich source from which iron and amino acids resulting from the digestion of globins can be obtained. Hematophagous parasites, such as S. chrysophrii, have a broad repertoire of peptidases to conduct the digestion of their host’s haemoglobin. This process is achieved by a multienzymatic network cascade in which clan CA (cathepsins B, C and L) and AA (cathepsin D) cysteine peptidases account for most of the proteolytic activity together with aminopeptidases [59, 60]. So far, they have been identified in Nematoda, Trematoda, Cestoda [61–63], and Monogenea [7, 9, 13, 14]. The liver fluke, F. hepatica presents an extracellular digestion phase in its gut lumen [64], and presumably so does Eudiplozoon nipponicum (Polyopisthocotylea, Diplozoidae) [8]. However, Riera-Ferrer et al. [65] went a step beyond by suggesting that S. chrysophrii induces an intravascular haemolysis in its host, prior to ingesting the resulting blood-meal. Thus, ESPs and EV-originating peptidases and peptidase inhibitors might have a crucial role in such parasite’s feeding strategies.
In fact, the gene ontology analyses from the purified EVs inferred biological processes related to blood (Negative regulation of coagulation, Iron ion transport, Haemocyte migration and Intracellular iron ion homeostasis; Fig. 4A), consistent with the negative haemostatic impact [65, 66] and catalytic process (Regulation of catalytic activity and Proteolysis) observed in infected S. aurata. Moreover, molecular functions revealed Ferric and Metal ion binding, Ferroxidase activity, Glutathione transferase and enzymatic activities related to Oxidoreductase, Transferase, Hydrolase-Peptidase, Lyase, Isomerase, Ligase and Translocase enzymes (Fig. 4B). A total of eight peptidases were identified in S. chrysphrii EVs (Table 3) among which a single cysteine peptidase, belonging to the clan CA, family C2 (calpain), involved in signal transduction, cellular differentiation, cytoskeletal remodelling, vesicular trafficking and in MV biogenesis, was identified [67, 68]. Interestingly, two metallo- catalytic-type aminopeptidases were identified, namely, a leucyl aminopeptidase (clan MF, family M17) and an alanyl aminopeptidase (clan MA, family M1), both with overlapping, but distinct substrate specificity profiles [69]. These aminopeptidases have been described as essential for the survival of Plasmodium falciparum (Apicomplexa, Plasmodiidae), the causative agent of malaria in humans, and are involved in the final steps of haemoglobin catabolism [69, 70]. In addition, the aminopeptidase leukotriene A4 hydrolase (clan MA family M1), classified as an ether hydrolase (EC 3.3.2.6), was identified in the EVs of S. chrysophrii. This protein is related to the ESCRT-independent EV biogenesis pathway [71]; however, Haeggström et al. [72] suggested that the peptidase activity of leukotriene A4 hydrolase could have an additional extracellular role. While it is tempting to suggest an intravascular haemolytic event taking place given the current findings and previous studies, further mechanistic, functionality, protein-characterisation and EV-internalisation studies are required. It is noteworthy that, from the identified peptidases, the most represented were threonine proteasome-related peptidases (clan PB, family T1; Table 3) in agreement with E. nipponicum secretome profile observations [14] and F. hepatica EV results [41], implying that isolated EVs present a substantial protein turnover role.
Free haem groups resulting from haemoglobin digestion elicit high toxicity and oxidative stress. Thus, hematophagous parasites require a haem-detoxification route to ensure their viability. Several haem-detoxification mechanisms have been suggested for different hematophagous parasites [73], in which high-affinity haem-binding enzymes belonging to the glutathione S-transferase family, which engage haem catabolic processing, have been proposed [74, 75]. Glutathione S-transferase has been identified in ESPs in platyhelminths including Trematoda [76] and Monogenea, where Vorel et al. [14] described an abundant transcription of this group of enzymes in E. nipponicum. Furthermore, glutathione S-transferase enzymes have been identified in EVs of several trematodes [41, 49, 77, 78]. In the present study, a glutathione S-transferase class-mu 28 kDa isozyme, homologous to that of S. japonicum (accession no: P26624.1; Additional file 4: Dataset S3) was identified in S. chrysophrii EVs, and its glutathione transferase activity molecular function was inferred from the GO analysis (Fig. 4B). Such results are in line with Vorel et al. [14] observations and suggest that monogeneans belonging to the Polyopisthocotylea subclass are able to detoxify haem groups through this catalytic process.
In the same line, iron is essential for multiple biological processes [79], and as such, haemoglobin remains the main source of iron acquisition in parasitic blood-feeders. However, iron homeostasis must be precisely regulated as iron deficiency results in survival impairment, and an excess of this trace metal results in high toxicity similar to that of haem [80, 81]. To counteract the toxic effects of free iron ions, ferritins, known as iron storage proteins, bind to iron [82]. In the current study, we identified a single ferritin (EC 1.16.3.1) in isolated S. chrysophrii EV samples (Additional file 4: Dataset S3). Iron ion transport and intracellular iron ion homeostasis biological processes (Fig. 4A) and ferric ion binding, metal ion binding and ferroxidase activities related to molecular function (Fig. 4B) were inferred from the GO analyses. The origin of free iron is unknown, and while one could suggest that these ions are a result of haem catabolic detoxification by glutathione S-transferase, there is no solid evidence on other organisms. Interestingly, ferritins are among the most transcribed genes in E. nipponicum [14] and given their presence in EVs of several trematode species [49, 77, 83], it has been suggested that these play a significant role in the feeding pathways of Trematoda.
Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) plays a significant role as an inflammatory mediator within the mammalian immune system. In addition to promoting platelet aggregation, it triggers various immune responses, such as chemotaxis, aggregation and degranulation of neutrophils and eosinophils. Additionally, PAF induces oxidative burst, leading to the production of reactive oxygen species, and stimulates the synthesis of two important cytokines, interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha) [84, 85]. In this study, we identified the ester hydrolase, platelet-activating factor acetylhydrolase (PAF-AH; EC 3.1.1.47), which by removing the sn-2 acetyl group from PAF silences its biological activity [86]. Thus, PAF-AH might ultimately inhibit the host’s coagulation cascade and extravasation of granulocytes. From the resulting GO analysis, a Negative Regulation of the Coagulation biological process was inferred (Fig. 4A), in agreement with the impairment of the host’s haemostasis and immune system (depletion of complement effectors and Ig light chain variable domains), which was observed from the plasma proteome of experimentally S. chrysophrii-infected S. aurata [65]. Nevertheless, a significant increase in eosinophilic granular cells, the functional equivalent of mammalian neutrophils in S. aurata [87], was determined from 28 to 50 days post parasite exposure [28]. In this context, the previously mentioned identification of a leukotriene A4 hydrolase suggests that S. chrysophrii might also secrete eicosanoids as proinflammatory mediators, as described for S. japonicum [88, 89]. Furthermore, human PAF-AH has been described to bind to very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) [90], in rapport with the detrimental impact on the lipid transport and metabolism described in S. chrysophrii-infected S. aurata, which presented plasma apolipoprotein impairment and hipocholesterolemia [65]. However, such PAF-AH role in Nippostrongylus brasilensis (Nematoda, Heligmonellidae) was unsubstantiated [91], as opposed to Leishmania major (Euglenozoa, Trypanosomatidae) whose PAF-AH seems to possess the lipid-binding abilities described in human PAF-AH, and has been categorised as a virulence factor given its relevant role in the parasite’s survival [92]. Altogether, these observations raise questions on the possible pathogenic roles of the identified protein in S. chrysophrii and further studies are required.
The knowledge of the energy metabolism in the class Monogenea still remains somewhat elusive. Previous studies on S. chrysophrii, E. nipponicum and Diclidophora merlangi (Polyopisthocotylea, Diclidophoridae) have suggested a need for oxygen in this parasite class and hypothesised they have an aerobic metabolism [14, 28, 93]. In the current study, three oxidoreductases, five transferases and three lyases (Additional file 4: Dataset S3) related to energy metabolism were identified, gathering more evidence on the nature of S. chrysophrii metabolism. Moreover, according to the gene ontology analyses, biological processes related to glycolysis, gluconeogenesis and the pentose-phosphate pathways (Fig. 4A) were identified, all indicative of an aerobic metabolism. Similar results were observed in other platyhelminths, including Monogenea, at a transcriptomic level [14], ESPs of Trematoda [76] and in EVs from Trematoda and Cestoda [41, 94].
Besides the previously discussed proteins, proposed EV-marker proteins such as 14-3-3 and heat shock protein 70 (HSP70), both commonly found in eukaryotic EVs were found in S. chrysophrii vesicles (Additional file 4: Dataset S3), in agreement with other helminth species [51, 78, 83, 95]. Heat shock protein 90 (HSP90), previously identified in S. japonicum EVs [96], and four out of eight subunits belonging to the T-complex protein 1 (TCP1) ring complex, a group II chaperonin related to heat shock protein 60 (HSP60) exhibiting ATP-dependent protein folding (Additional file 4: Dataset S3) [97–99], previously identified in F. hepatica [41], were also identified in S. chrysophrii EVs. Altogether, the presence of HSP70/ 90 and TCP1 is reflected in the Protein folding and refolding and Cellular response to unfolded protein biological processes (Fig. 4A) and Unfolded protein binding, ATP-dependent protein folding chaperon and HSP70 protein binding molecular functions (Fig. 4B) inferred from the GO analyses.
Other proposed EV-marker proteins correspond to those presenting an EF-hand domain (PF13499), which have been identified in several platyhelminth species and have been proposed to be characteristic of Trematoda and Cestoda EVs protein cargo [95]. In the S. chrysophrii-derived EVs from the present study, three proteins presenting an EF-hand domain (PF13499.9; sorcin, calmodulin and calcineurin B homologous protein 1; Additional file 4: Dataset S3) were identified. Moreover, the presence of MVs was indicated by seven proteins of the Ras family, as well as two Rho-associated proteins, and an ADP-ribosylation factor [21, 22, 48, 100, 101] consistent with the NTA and TEM results.
Currently, treatment options against monogenean infections in aquaculture systems, including S. chrysophrii, remain scarce and hydrogen peroxide and formaldehyde baths in their licensed formulation are the only chemotherapeutants available. However, its use has already been banned in Italy, among other countries, due to safety concerns, and is not warranted worldwide in the coming years [3, 102, 103]. Together with the ongoing threat of emerging anthelmintic drug resistance, the current limitations for treating sparicotylosis constitute a compelling reason for further therapeutic or prophylactic purposes, aiming to broaden the range of action against this parasitosis and going a step ahead from potential emerging drug resistance. Most of the identified proteins in S. chrysophrii EVs have already been confirmed in other helminths including platyhelminths [95]. Several proteins including the aforementioned peptidases, non-enzymatic proteins and energy metabolism-related proteins have already been proposed as drug targets or vaccine candidates in Trematoda, Nematoda and some Apicomplexa parasitic species. Such protein target candidates identified in S. chrysophrii EVs are compiled in Table 4, overall, broadening the insight into novel strategies against this parasite.
Table 4
List of orthologs described as drug and vaccine target candidates from different hematophagous parasites including Trematoda, Nematoda and Apicomplexa found in the EVs’ proteomic study using Sparicotyle chrysophrii, Protopolystoma xenopodis, Gyrodactylus salaris and Microcotyle sebastis proteomes as reference.
Protein target candidates | References | Species and sequence hit |
| | Sparicotyle chrysophrii |
Proteases | | |
Leucine aminopeptidase (M17 family) | [49], [70], [104–107] | maker-contig_20098_pilon_pilon-augustus-gene-0.16-mRNA-1 |
Alanyl aminopeptidase (M1 family) | [70], [108] | maker-contig_6883_pilon_pilon-snap-gene-0.23-mRNA-1 |
Calpain | [49] | maker-contig_24278_pilon_pilon-snap-gene-0.33-mRNA-1 |
Carrier proteins | | |
Ferritin | [81] | maker-contig_34520_pilon_pilon-augustus-gene-0.7-mRNA-1 |
Cytoskeleton | | |
Dynein light chain | [49], [106] | maker-contig_41206_pilon_pilon-augustus-gene-0.3-mRNA-1 |
14-3-3 protein | [49], [106] | maker-contig_34517_pilon_pilon-snap-gene-0.19-mRNA-1 |
| | maker-contig_34517_pilon_pilon-snap-gene-0.19-mRNA-1 |
| | maker-contig_30173_pilon_pilon-snap-gene-0.10-mRNA-1 |
Heat shock protein 70 | [49], [106] | augustus_masked-contig_13257_pilon_pilon-processed-gene-0.1-mRNA-1 |
| | augustus_masked-contig_5207_pilon_pilon-processed-gene-0.2-mRNA-1 |
Annexin | [49] | maker-contig_38531_pilon_pilon-exonerate_est2genome-gene-0.0-mRNA-1 |
| | maker-contig_29652_pilon_pilon-augustus-gene-0.2-mRNA-1 |
| | maker-contig_12610_pilon_pilon-snap-gene-0.5-mRNA-1 |
Tubulin | [109] | maker-contig_8332_pilon_pilon-augustus-gene-0.19-mRNA-1 |
| | snap_masked-contig_2563_pilon_pilon-processed-gene-0.25-mRNA-1 |
Antioxidant/ Defence/ Detoxification | | |
Glutathione S-transferase | [49], [106] | maker-contig_7282_pilon_pilon-snap-gene-0.22-mRNA-1 |
Superoxide dismutase | [110] | maker-contig_45344_pilon_pilon-augustus-gene-0.2-mRNA-1 |
Metabolism | | |
Enolase | [49], [106] | snap_masked-contig_11270_pilon_pilon-processed-gene-0.1-mRNA-1 |
Fructose-bisphosphate aldolase | [106] [110] | maker-contig_31661_pilon_pilon-snap-gene-0.18-mRNA-1 |
| | Protopolystoma xenopodis |
Cytoskeleton | | |
Heat shock protein 70 | [49], [106] | A0A448WN47 |
Tubulin | [109] | A0A448X3E2 |
| | A0A3S5FFD8 |
| | A0A448WX98 |
| | A0A448XD95 |
| | Gyrodactylus salaris |
Cytoskeleton | | |
Tubulin | [109] | Q709M4 |
| | Microcotyle sebastis |
Cytoskeleton | | |
Annexin | [49] | B3GQS3 |