bioRxiv preprint doi: https://doi.org/10.1101/2020.02.03.932509; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 The parasite Schistocephalus solidus secretes 2 proteins with putative host manipulation functions 3 Chloé Suzanne Berger1,2,3, Jérôme Laroche2, Halim Maaroufi2, Hélène Martin1,2,4, 4 Kyung-Mee Moon5, Christian R. Landry1,2,4,6,7, Leonard J. Foster5 and Nadia Aubin- 5 Horth1,2,3 6 1Département de Biologie, Université Laval, Québec, QC, Canada 7 2Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec, QC, 8 Canada 9 3Ressources Aquatiques Québec (RAQ), Institut des sciences de la mer de Rimouski, 10 Québec, Canada 11 4Département de Biochimie, Microbiologie et Bioinformatique, Université Laval, Québec, 12 QC, Canada 13 5Department of Biochemistry & Molecular Biology, Michael Smith Laboratories, 14 University of British Columbia, Vancouver, Canada V6T 1Z4 15 6PROTEO, Le réseau québécois de recherche sur la fonction, la structure et l’ingénierie 16 des protéines, Université Laval, Québec, Canada 17 7Centre de Recherche en Données Massives (CRDM), Université Laval, Québec, 18 Canada 19 Corresponding author: Nadia Aubin-Horth ([email protected]) 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.03.932509; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 20 ABSTRACT 21 Background: Manipulative parasites are thought to liberate molecules in their 22 external environment acting as manipulation factors with biological functions implicated 23 in their host’s physiological and behavioural alterations. These manipulation factors are 24 part of a complex mixture called the secretome. While the secretomes of various 25 parasites have been described, there is very little data for a putative manipulative 26 parasite. It is necessary to study the molecular interaction between a manipulative 27 parasite and its host to better understand how such alterations evolve. 28 Methods: Here, we used proteomics to characterize the secretome of a model 29 cestode with a complex life cycle based on trophic transmission. We studied 30 Schistocephalus solidus during the life stage in which behavioural changes take place in 31 its obligatory intermediate fish host, the threespine stickleback (Gasterosteus 32 aculeatus). We produced a novel genome sequence and assembly of S. solidus to 33 improve protein coding gene prediction and annotation for this parasite. We then 34 described the whole worm’s proteome and its secretome during fish host infection using 35 LC-MS/MS. 36 Results: A total of 2 290 proteins were detected in the proteome of S. solidus, and 30 37 additional proteins were detected specifically in the secretome. We found that the 38 secretome contains proteases, proteins with neural and immune functions, as well as 39 proteins involved in cell communication. We detected Receptor-type tyrosine-protein 40 phosphatases, which were reported in other parasitic systems to be manipulation 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.03.932509; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 41 factors. We also detected 12 S. solidus-specific proteins in the secretome that may play 42 important roles in host-parasite interactions. 43 Conclusions: Our results suggest that S. solidus liberates molecules with putative 44 host manipulation functions in the host and that many of them are species specific. 45 46 Keywords: Schistocephalus solidus, secretome, proteomics, manipulation factor, 47 parasite, behaviour 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.03.932509; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 48 BACKGROUND 49 Parasites have major impacts on their hosts, including on their morphology (1), 50 physiology (2) and behaviour (3, 4). To induce these complex changes in their hosts, it 51 has been proposed that parasites produce, store and release manipulation factors that 52 interfere with the host physiological and central nervous systems (5–7). These 53 manipulation factors are thought to be part of a complex mixture of molecules called the 54 secretome, which is a key element of parasite-host interactions (6). The secretome of a 55 parasite includes lipids (8), nucleic acids (9) and proteins (10), which are sometimes 56 protected inside extracellular vesicles (11). Using molecular and bioinformatics 57 approaches, the proteomic fraction of secretomes of parasites infecting humans (12) 58 and livestock (13) have been described, both in terms of protein composition and 59 function (14,15) (see Table 1 for a review). 60 61 The secretomes that have been examined so far are enriched in peptidases and 62 proteases (12,15), which are known to weaken the host immunity barriers. Other 63 secreted proteins, such as paramyosin in the blood fluke Schistosoma mansoni, have 64 been shown to help the parasite to escape the host immune response, while secreted 65 proteins involved in calcium functions have impacts on the host neural activity (12). In 66 the context of behavioural manipulation, the secretome is a logical potential source of 67 manipulation factors. However, the secretome content of a behaviour-manipulating 68 parasite has rarely been investigated, to the point that secretomes are referred to as 69 “the missing link in parasite manipulation” (7). The literature contains several reports 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.03.932509; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 70 from which it is possible to infer a list of putative manipulation factors, which would 71 target the neural and the immune systems of the hosts and induce behavioural changes 72 (Table 1). Our knowledge regarding if and how many proteins with neural and immune 73 functions can be found in the secretomes of manipulative parasites is very limited, and 74 is based in many cases on inferred proteins rather than actual detection. 75 76 One particularly powerful model to study behavioural manipulation is the cestode 77 Schistocephalus solidus (16). This parasite exhibits a complex life cycle based on 78 trophic transmission that includes three hosts: a copepod, the threespine stickleback 79 (Gasterosteus aculeatus, obligatory intermediate fish host) and a fish-eating bird, in 80 which S. solidus reproduces (16,17). S. solidus infects the threespine stickleback’s 81 abdominal cavity through the ingestion of a parasitized copepod (18). The 82 consequences of the infection by S. solidus on the threespine stickleback’s morphology 83 (19), physiology (20), immune system (21), and behaviour (22) are well-documented. 84 For example, sticklebacks infected by S. solidus show drastic behavioural changes that 85 result in a loss of the anti-predator response (16): infected fish are more exploratory 86 (23), less anxious (24) and bolder in the presence of a predator (25) than non-infected 87 fish. 88 89 Most of these behavioural alterations seen in infected fish appear after several weeks, 90 when the worm has grown to reach the infective stage within its intermediate host (i.e. 91 larger than 50 mg) (26). The infective stage coincides with the time at which S. solidus 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.03.932509; this version posted July 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 92 is ready to reproduce in the bird (16,27), which also generally corresponds to the 93 activation of the immune response in the host. In the first phase of infection, the 94 adaptive immune response is generally not activated in the fish. It is only when the 95 worm reaches the infective stage that an ineffective up-regulation of the respiratory 96 burst activity occurs (21). Nevertheless, activation of the immune response through the 97 production of reactive oxygen species (ROS) by granulocytes has been shown to occur 98 early during infection, depending on the genotype of the stickleback population (126). 99 Several studies have suggested that the manipulation of the stickleback’s behaviour 100 increases the worm’s transmission rate to its final avian host (16). Yet, the adaptive 101 value for S. solidus of such behavioural changes has never been demonstrated (28), 102 and it is possible that these behavioural modifications in the fish may solely result from 103 a side effect of infection (7), such as the effect of the parasite mass burden or of the 104 activation of the host immune response (24, 109). To demonstrate that behavioural 105 changes in the host are the result of direct parasitic manipulation, the first step requires 106 to determine if the parasite can liberate molecules in its external environment, and if 107 yes, to study their functions in relation with the host’s phenotype perturbations.