Supplementary Information:

Transcriptome profiling of a multiuse model stagnalis () for ecoimmunological research

Otto Seppälä, Jean-Claude Walser, Teo Cereghetti, Katri Seppälä, Tiina Salo & Coen M. Adema

Contents Table S1. GenBank accession numbers of the sequences of proteins/genes relevant for immune function, stress responses, antioxidation and metabolism that were used as references in BLAST similarity searches to identify orthologs in the L. stagnalis reference transcriptome ...... 3 Table S2. Summary of the annotated transcripts linked to proteins with a role in immune defence, stress-responses, or metabolism. The total number of identified transcripts, the number of transcripts with unique open reading frames (ORFs), an example domain architecture using SMART and PFAM domainsa (when available in the annotation database), and transcripts representing unique ORFs for each examined factor are presented. Transcript ID the domain architecture refers to is in bold ...... 7 Fig. S1. Principal component analysis (PCA) plots showing the variation in transcriptome-wide expression profiles of the experimental snails using the first five principal components (PCs) after internal normalisation in sleuth. Additionally, the proportion of total variance each PC explained in the data is presented ...... 14 Fig. S2. Expression levels of individual transcripts found to represent annotated factors related to TLR signalling pathway in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity...... 15 Fig. S3. Expression levels of individual transcripts found to represent the annotated antibacterial defence factors in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using its dynamic range. Transcripts related to each factor are clustered according to their similarity...... 15 Fig. S4. Expression levels of individual transcripts found to represent annotated factors related to phenoloxidase/melanisation-type reaction in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity...... 16 Fig. S5. Expression levels of individual transcripts found to represent annotated factors related to non-self recognition in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity...... 16 Fig. S6. Expression levels of individual transcripts found to represent annotated cytokines in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity...... 17 Fig. S7. Expression levels of individual transcripts found to represent annotated factors related to the production of reactive oxygen species (ROS) in units of transcripts per million (TPM) for each

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experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity...... 18 Fig. S8. Expression levels of individual transcripts found to represent annotated factors related to apoptosis in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity...... 19 Fig. S9. Expression levels of individual transcripts found to represent the annotated stress-response factors in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity...... 19 Fig. S10. Expression levels of individual transcripts found to represent the annotated antioxidant enzymes in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity...... 20 Fig. 11. Expression levels of individual transcripts found to represent annotated factors related to metabolism in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity...... 20

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Table S1. GenBank accession numbers of the sequences of proteins/genes relevant for immune function, stress responses, antioxidation and metabolism that were used as references in BLAST similarity searches to identify orthologs in the L. stagnalis reference transcriptome.

Component Reference(s) Non-self recognition

PGRP AEH26026.1, AHB30456.1, XP_013064763.1, XP_013064406.1, XP_013062325.1, ABO40832.1, ABK76645.1, NP_001298227.1, XP_013063186.1, XP_013062465.1, XP_013062456.1, XP_005100262.1, XP_005093494.1, ABO40831.1, ABO40830.1, ABO40829.1

GNBP ABO40828.1

FREP Q8VCM7.1, ADA82222.1, CBM41040.1, AEO50745.1, O70165.1

Galectin ACO36044.1, NP_034837.2, ABQ09359.1, ACO53607.1, BAF75419.1

Chi-lectin O35744.2, P36222.2, Q1RQ16, Q1RQ22

C-type lectins AAX19697.1, ACS72237.1, AFA36633.1, ABO26594.1, P11226.2

F-box lectins Q96EF6.1, Q80UW2.1

F-type lectins Q7SIC1.1, ACF94293.1, ADJ40024.1, AFO64339.2, ADR63290.1

I-type lectins Q9Y286.1, AAH47244.1, AAA20148.1, P13597.1, Q91Y57.3

L-type lectin P49257.2, Q12907.1, EKC31192.1, EKC20002.1

M-type lectin Q925U4.1, Q91VV3, EKC26076.1, EKC23948.1

P-type lectins CAA45423.1, EKC18244.1, NP_000867.2, EKC35005.1

R-type lectins BAA36393.1, BAL61208.1, Q2HZ94

Intelectins ACC62155.1, CAJ32677.2, ACC62157.1, ACC62156.1, NP_001082570.1

Pentraxins AAZ68032.1, ABG37054.1, P02743.2, P02741.1

TLR pathway

TLR AGB93809.1

MyD88 XP_013086371.1, XP_013086405.1

IRAK XP_013088618.1

TRAF XP_013069680.1, XP_013069680.1

TBK XP_013083625.1

TAK XP_013073733.1, XP_013073731.1, XP_013073732.1, XP_013073736.1, XP_013073734.1, XP_013073735.1

TRF XP_013089600.1, XP_013073636.1, XP_005088853.1, XP_005104410.1, XP_012934632.1

TAB XP_013096269.1, XP_013096268.1, XP_005094474.1, XP_005094473.1, XP_014776667.1

SARM XP_013080872.1, XP_013080871.1 3

IKKα XP_005096610.2

IKKγ Nemo XP_013073873.1

LITAF XP_013089264.1, XP_013089261.1, XP_013089265.1, XP_013089270.1, XP_013089262.1, XP_013089269.1

IκB NP_001298198.1

NF-κBp65 NP_001298191.1, ACN73461.1

NF-κBp100 XP_022318641.1, XP_022318640.1

MPEG XP_013065342.1

Cytokines

MIF NP_001298224.1, XP_013075404.1, XP_005103629.2, XP_012939653.1, AQY19126.1

IL17 XP_013096467.1, XP_013090467.1, XP_013090466.1, XP_013085850.1, XP_013085675.1, XP_013080675.1, XP_013065647.1, XP_021365521.1, AGZ03660.1, XP_021365518.1, XP_009058928.1, XP_021365519.1, XP_022315060.1, XP_022310682.1, AKM49935.1, XP_009048356.1, XP_005097238.1, XP_011438620.1, NP_001292220.1, EKC33705.1, AKM49938.1, XP_022286467.1, XP_021365533.1, XP_022344404.1, XP_022344403.1, NP_001295831.1, OWF44912.1, OWF56800.1, XP_021364761.1, XP_011438445.1, AKQ98198.1, XP_009051888.1, NP_001295781.1, AKQ98197.1, XP_011414373.1, XP_022318598.1, XP_022315342.1

TNF XP_013076951.1, XP_013061250.1, XP_013061249.1, XP_013061248.1, XP_013061247.1, XP_013096914.1, XP_013096721.1, XP_013096720.1, XP_013096719.1, XP_013096712.1, XP_013096711.1, XP_013096710.1, XP_013094785.1, XP_013094784.1, XP_013094783.1, XP_013094495.1, XP_013094494.1, XP_013086591.1, XP_013082590.1, XP_013082589.1, XP_013082588.1, XP_013082587.1, XP_013076957.1, XP_013073494., XP_013073185.1, XP_013072204.1, XP_013072202.1, XP_013070598.1, XP_013067540.1, XP_013067539.1, XP_013067535.1, XP_013070598.1, XP_013077259.1, XP_022338094.1, XP_022338095.1, XP_011450914.1, EKC35160.1, XP_021346927.1, XP_021364181.1, XP_013073494.1, XP_012936811.1, XP_021346926.1, OWF56727.1, XP_021340535.1

Antimicrobial defence

Macins AFR36920.1, CK989857.1

LBP/BPI AGG82435.1, AKM45823.1, AKM45822.1, AKM45821.1, AKM45820.1

LAAO NP_001191524.1, AAN78211.1, Q17043.1, P35903.1, AAR14185.1, AAR14187.1, AAR14186.1

Lysozymes AGQ50333.1, AGQ50332.1, AGQ50331.1, ADR70996.1, ADR70995.1, AGQ50337.1, AGQ50335.1, AGQ50330.1, ADV36303.1, AGQ50336.1, AGQ50334.1, AOX15710.1, AOX15709.1, AOX15708.1, AOX15707.1, XM_013226064.1

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Biomphalysin AGG38744.1, NP_001298219.1

Glabralysin A0A2C9L301, A0A2C9M7U6, A0A2C9M7H5, A0A2C9L4Y1, A0A2C9LNR7, A0A2C9KPA0

Production of reactive oxygen species

NOX XP_005090645.1, XP_005096802.1, XP_012938230.1

DUOX XP_012942535.1, XP_012943483.1

DUOXA XP_021362528.1, XP_022343917.1

NOS NP_001191470.1, AGI44587.1

Peroxidasin XP_013088968.1, XP_005110224.1

Glutaredoxin XP_013084576.1, XP_013061972.1, XP_005104531.1, XP_005110052.1

GST XP_013075526.1, XP_012944480.1, XP_013071551.1, XP_013060671.1, XP_012945427.1, RUS76363.1, XP_013097111.1, AEI27296.1, XP_005109181.1, XP_013070482.1, AXN72681.1, XP_013095077.1, XP_009049471.1, RUS84494.1, XP_005094586.1, XP_005106935.1, XP_012941915.1

Nucleoredoxin EKC27452.1, XP_013073587.1, XP_005098394.1, XP_013074893.1

Peroxiredoxin XP_013086205.1, XP_013087397.1, XP_013091924.1, ACI42883.1

Phenoloxidase/melanisation-type reaction proPO CCQ18551.1, NP_476812.1, AGC54940.1, BAF98646.1

Laccase XP_013079632.1, XP_013079631.1, XP_013086277.1, XP_013074984.1, XP_013074983.1, XP_013074982.1, XP_013068526.1, XP_013085808.1, XP_013062555.1, XP_013064545.1, XP_013079036.1, XP_013085832.1, XP_013085833.1, XP_013085834.1, XP_013088628.1, XP_013090126.1, XP_013067361.1, XP_013067388.1, XP_013074978.1, XP_013074979.1, XP_013074980.1, XP_013074981.1, ESO98873.1, ESO97324.1, ESO96136.1, ESO89911.1, XP_009059385.1, XP_009053251.1, XP_009051926.1, XP_009050501.1, ESO89918.1, XP_009059392.1, ESO90420.1, ESO89919.1, ESO84603.1, XP_009064708.1, XP_009059393.1, XP_009058892.1, XP_012937904.1, XP_012944262.1, XP_012942999.1, XP_012942636.1, XP_012940646.1, XP_012945653.1, XP_012934797.1

Tyrosinase XP_013080599.1, XP_013065857.1, APC92582.1, EKC35331.1, EKC38463.1, OWF48254.1, APC92581.1, XP_021374137.1, AMB26746.1, ASR73340.1, ALG64484.1

Apoptosis

FAIM1 XP_013071009.1

HTRA2 XP_013072351.1

AIF XP_013095497.1, XP_013095498.1

IAP XP_013075317.1, XP_013064429.1, XP_013064681.1, XP_013075313.1, XP_013060630.1, XP_013090049.1, XP_013064690.1, XP_013075320.1, XP_013082134.1, XP_013084114.1, XP_013084111.1 5

Bcl-Bax XP_013075909.1, XP_013089117.1

PARP XP_013090488.1, XP_013062683.1, XP_013062670.1, XP_013065538.1, XP_013068782.1, XP_013062696.1, XP_013067682.1, XP_013092390.1, XP_013096616.1

Caspase XP_013067788.1, XP_013065656.1, XP_013073999.1, XP_013074008.1, XP_013088924.1, XP_013094549.1, XP_013095352.1

Stress responses

HSP70 XP_013081556.1, XP_013082114.1, ABB45831.1, XP_013091813.1

HSP90 XP_013072750.1

HSF XP_013085217.1

Antioxidant enzymes

SOD AAP93637.2, XP_013070344.1

MnSOD NP_001298192.1

CAT ACO59957.1

Glutathione ACO59958.1 peroxidase

Glutathione ACO59956.1 reductase

Metabolism

PP1 XP_013085822.1

Ubiquitin NP_001298238.1

Ferritin XP_013080834.1

ADH XP_013073559.1

ERR XP_013094385.1, XP_013080351.1

RXR NP_001298239.1

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Table S2. Summary of the annotated transcripts linked to proteins with a role in immune defence, stress-responses, or metabolism. The total number of identified transcripts, the number of transcripts with unique open reading frames (ORFs), an example domain architecture using SMART and PFAM domainsa (when available in the annotation database), and transcripts representing unique ORFs for each examined factor are presented. Transcript ID the domain architecture refers to is in bold.

Component # transcripts # unique Example of domain Transcript IDs ORFs structure with unique ORFs Non-self recognition

PGRPs 8 7 14145_c0_g1_i1 32865_c0_g1_i1 34108_c0_g1_i1 36537_c0_g1_i1 36963_c0_g1_i1 42094_c0_g6_i1 42956_c0_g1_i1

GNBPs 8 4 41774_c0_g1_i2 47227_c0_g2_i1 47391_c1_g1_i1 48085_c1_g8_i11

FREPs 2 2 36764_c0_g1_i2 45250_c0_g1_i1

Galectins 20 6 42521_c0_g1_i2 44043_c0_g3_i1 44175_c0_g1_i1 47859_c0_g2_i1 47859_c0_g2_i5 48059_c1_g2_i1

Chi-lectins 11 8 33218_c0_g1_i1 44081_c0_g1_i1 44227_c0_g2_i2 45634_c0_g3_i1 47306_c0_g5_i1 48292_c1_g1_i3 26259_c0_g1_i1 45255_c1_g3_i3

L-type lectins 3 2 44959_c0_g1_i1 48268_c2_g1_i1

M-type lectins 9 3 37655_c0_g1_i1 39342_c0_g1_i1 47242_c4_g2_i1

TLR pathway

TLR 1 1 28651_c0_g1_i1

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MyD88 7 1 47065_c0_g1_i1

TRAF 1 1 22931_c0_g1_i1

TRF 1 1 34891_c0_g1_i1

TAB 1 1 24669_c0_g1_i1

SARM 1 1 46010_c7_g2_i1

IKKα 2 1 35196_c0_g1_i1

IKKγ Nemo 6 1 47446_c1_g1_i1

LITAF 6 2 45095_c0_g1_i1 46794_c1_g1_i1

IκB 2 2 39554_c0_g1_i1 46270_c2_g2_i1

NF-κBp65 4 2 38589_c1_g1_i1 43263_c0_g2_i1

MPEG 3 2 46200_c0_g1_i1 47480_c0_g1_i1

Cytokines

MIF 2 1 36453_c0_g1_i1

IL-17 3 3 110902_c0_g1_i1 4951_c0_g1_i1 17215_c1_g1_i1

TNF 63 23 15848_c0_g1_i1 20877_c0_g2_i1 34263_c0_g1_i2 37129_c0_g1_i1 37407_c0_g1_i1 37593_c0_g1_i1 39166_c0_g1_i1 41927_c0_g1_i1 42168_c1_g1_i2 42302_c0_g4_i1 42612_c1_g1_i1 42736_c0_g1_i1 43123_c0_g2_i2 8

43870_c0_g1_i2 44512_c0_g1_i2 45558_c0_g3_i1 45927_c0_g1_i1 46059_c2_g1_i2 46501_c1_g2_i1 47286_c3_g3_i2 47468_c1_g1_i1 47690_c7_g2_i10 48316_c0_g1_i1

Antimicrobial defence

Macins 7 6 18198_c0_g1_i1 18890_c0_g1_i1 34989_c0_g2_i1 35624_c0_g1_i1 41887_c0_g3_i1 43018_c0_g6_i3

LBP/BPI 4 3 25950_c0_g1_i1 45368_c0_g6_i1 46649_c0_g1_i1

LAAO 6 3 47821_c4_g3_i1 47893_c0_g9_i1 48351_c1_g3_i1

Lysozymes 11 4 28373_c0_g1_i1 35313_c1_g1_i1 36143_c0_g3_i1 44233_c1_g1_i1

Lymnaea-lysins 4 3 38389_c0_g1_i1 47349_c1_g38_i2 5994_c0_g2_i1

Stagnalysins 3 3 34104_c0_g1_i1 42332_c1_g1_i1 48069_c5_g1_i1

Production of reactive oxygen species

NOX 9 3 40220_c1_g2_i1 36496_c0_g1_i1 36278_c0_g1_i1

DUOX 6 2 42865_c0_g1_i3 46890_c0_g1_i1

DUOXA 1 1 38205_c0_g1_i1

NOS 3 1 45570_c1_g1_i1

Peroxidasin 16 6 40499_c0_g1_i1 45939_c0_g1_i1 9

46236_c0_g1_i2 46303_c1_g1_i1 47256_c0_g1_i1 48181_c2_g1_i1

Glutaredoxin 7 4 34293_c0_g2_i1 34343_c1_g2_i1 4572_c0_g2_i1 45783_c0_g1_i1

GST 32 17 1429_c0_g2_i1 31273_c0_g1_i1 36750_c0_g1 38055_c0_g1_i2 39155_c0_g1_i1 40581_c0_g1_i1 40585_c0_g1_i1 41000_c0_g2_i1 43131_c0_g1_i1 44207_c5_g1_i1 44723_c0_g5_i1 46105_c0_g60_i1 46670_c0_g1_i1 46742_c1_g1_i1 46851_c0_g1_i2 46851_c0_g1_i6 46929_c1_g35_i1

Nucleoredoxin 7 4 33046_c0_g1_i1 39371_c0_g1_i1 44859_c0_g2_i1 45653_c0_g1_i2

Peroxiredoxin 9 5 27522_c0_g1_i1 34959_c0_g2_i1 41634_c2_g1_i1 46977_c0_g1_i1 47425_c0_g2_i6

Phenoloxidase/melanisation

Laccase 19 8 35966_c0_g1_i1 38462_c0_g1_i1 45077_c0_g1_i1 47321_c2_g2_i1 47844_c2_g5_i1 48217_c1_g1_i10 38987_c2_g2_i1 45762_c4_g1_i3

Tyrosinase 2 2 26246_c2_g1_i1 46270_c3_g2_i1

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Apoptosis

FAIM1 1 1 44771_c0_g1_i1

HTRA2 3 2 36021_c0_g1_i1 37936_c0_g1_i1

AIF 1 1 40505_c0_g1_i1

IAP 25 10 30243_c0_g1_i1 41110_c0_g1_i1 42472_c1_g1_i1 46127_c2_g1_i1 47200_c0_g1_i1 47310_c1_g1_i4 47642_c0_g1_i11 48019_c3_g3_i1 48046_c0_g1_i3 48048_c3_g1_i2

Bcl-Bax 1 1 44934_c1_g1_i3

PARP 21 b

Caspase 20 8 33457_c0_g1_i1 37005_c0_g1_i1 37479_c0_g2_i1 42025_c0_g1_i2 43539_c0_g1_i1 45052_c4_g2_i1 48203_c0_g1_i2 48216_c2_g3_i10

Stress responses

HSP70 13 5 40510_c1_g2_i1 41463_c0_g1_i1 44096_c1_g1_i1 46830_c1_g3_i1 47977_c0_g5_i1

HSP90 1 1 47161_c0_g4_i1

HSF 1 1 29117_c0_g1_i1

Antioxidant enzymes

SOD 6 2 41920_c0_g1_i2 45899_c0_g2_i1

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MnSOD 1 1 28257_c0_g1_i1

CAT 2 2 40830_c0_g1_i1 44469_c0_g1_i1 glutathione 5 3 36171_c0_g2_i1 peroxidase 37235_c0_g1_i1 47906_c1_g7_i1 glutathione 1 1 44983_c1_g1_i1 reductase

Metabolism

PP1 2 1 36580_c0_g1_i1

Ubiquitin 13 8 109816_c1_g1_i1 33535_c0_g1_i1 40537_c0_g1_i1 44027_c1_g1_i2 44915_c1_g1_i1 45261_c0_g1_i1 46234_c4_g1_i1 46544_c1_g3_i2

Ferritin 3 3 125081_c0_g1_i1 33209_c0_g1_i1 43780_c0_g5_i1

ADH 3 1 47996_c0_g14_i4

ERR 2 2 36130_c1_g1_i4 42943_c0_g1_i1

RXR 5 3 33793_c0_g1_i1 41983_c1_g1_i1 42786_c1_g1_i1 aSMART/PFAM domains: ADH_N – alcohol dehydrogenase GroES-like domain; ADH_zinc_N_2 – zinc-binding dehydrogenase; Aerolysin – aerolysin toxin; AIF_C – apoptosis-inducing factor, mitochondrion-associated, C-term; Amino_oxidase – flavin-containing amine oxidoreductase; An_peroxidase – heme-dependent peroxidase; An_peroxidase – animal heme-dependent peroxidase; ANK – ankyrin repeats; Bax1-I – inhibitor of apoptosis-promoting Bax1; BIR – baculoviral inhibition of apoptosis protein repeat; BPI1 – bactericidal permeability-increasing protein / lipopolysaccharide-binding protein / cholesteryl ester transfer protein N-terminal domain; BPI2 – bactericidal permeability-increasing protein / lipopolysaccharide-binding protein / cholesteryl ester transfer protein C-terminal domain; CASc – caspase, interleukin-1 beta converting enzyme homologues; Catalase – catalase for enzymatic detoxification of hydrogen peroxide; CC2- LZ – leucine zipper of domain CC2 of NEMO, NF-kappa-B essential modulator; Cu-oxidase – multicopper oxidase; DEATH – DEATH domain, bind each other to form oligomers of factors 12

involved in signaling pathways, frequently associated with immunity or apopotosis; Deltameth_res – deltamethrin resistance; DuoxA – dual oxidase maturation factor; EHf – EF-hand, calcium binding motif; FAD_binding_1 – flavoprotein pyridine nucleotide cytochrome reductase; FAD_binding_8 – FAD binding domain associated with ferric reductase NAD binding proteins and the heavy chain of Cytochrome b-245; FAD_binding_8 – FAD-binding domain; FAIM1 – Fas apoptotic inhibitory molecule; FBG – fibrinogen-like domain; Ferric_reduct – ferric reductase like transmembrane component; Ferritin – ferritin-like domain; Flavodoxin_1 – flavodoxin-like domain; GLECT – galectin; Glutaredoxin – glutaredoxin, oxidation repair enzyme; Glyco_18 – O-Glycosyl hydrolases; Glyco_hydro_16 – glycosyl hydrolases family 16; Glyco_hydro_47 – glycoside hydrolase family 47; GSHPx – glutathione peroxidase; GST_C – glutathione S-transferase, C- terminal domain; GST_N_3 – glutathione S-transferase; HATPase_c – histidine kinase-like ATPases; HOLI – ligand binding domain of hormone receptors; HSF – heat shock factor; HSP70 – heat shock protein 70; HSP90 – heat shock protein 90; IG – immunoglobulin; IL17 – interleukin- 17; IPT – ig-like, plexins, transcription factors; IRF – interferon-regulatory factor; IRF-3 – interferon-regulatory factor 3; Lectin_leg-like – L-type lectin domain; LITAF – LPS-induced TNF- activating factor; LRR – leucine-rich repeats; Macin – macin family; MACPF – membrane-attack complex / perforin; MATH – meprin and TRAF homology; MIF – macrophage migration inhibitory factor; NAD_binding_1 – oxidoreductase NAD-binding domain; NAD_binding_6 – ferric reductase NAD binding domain; NO_synthase – nitric oxide synthase; PBRP – animal peptidoglycan recognition protein; PDZ – domain present in PSD-95, Dlg, and ZO-1/2, proteins that anchor receptor proteins to the cytoskeleton, to help form signal transduction complexes; PP2Cc – serine/threonine phosphatases family 2C, catalytic domain; Pyr_redox_2 – pyridine nucleotide- disulphide oxidoreductase; Pyr_redox_3 – pyridine nucleotide-disulphide oxidoreductase; Pyr_redox_dim – pyridine nucleotide-disulphide oxidoreductase, dimerisation domain; Redoxin – redoxin domain with oxidoreductase activity; RHD_DNA_bind – Rel homology DNA-binding domain; RING – ring finger; S_TKc – serine/Threonine protein kinases, catalytic domain; SAM – sterile alpha motif; Sod_Cu & Sod_Fe_N & Sod_Fe_C – catergories of superoxide dismutase; Thioredoxin – thioredoxin, small redox proteins involved in responses to reactive oxygen species and cell-cell communication; TIR – Toll/interleukin-1 receptor; TNF – tumour necrosis factor family; Trypsin_2 – trypsin-like peptidase domain; Tyrosinase – tyrosinase, enzymatic domain of copper-type 3 enzymes involved in melanization contributing to pigmentation or immune defence; UBQ – ubiquitin homologues; zf-TRAF – TRAF-type zinc finger; ZnF_C4 – c4 zinc finger in nuclear hormone receptors. bDetermining which sequences cover a complete ORF not possible.

13

PC2 PC3 PC4 PC5 −2e+05 0e+00 2e+05 −2e+05 −1e+05 0e+00 1e+05 −50000 0 50000 −50000 0 50000 −5e+05 0e+00 PC1 5e+05

Treatment −2e+05 Untreated Anaesthesia Wounding

E. coli 0e+00 M. lysodeikticus PC2 Healthy gonad Trematode-infected gonad Elevated temperature Starvation 2e+05 −2e+05 −1e+05 PC3 0e+00

10 1e+05

5 −50000 0 PC4

Proportion of explained variance (%) 0

1 2 3 4 5 PC 50000 Fig. S1. Principal component analysis (PCA) plots showing the variation in transcriptome-wide expression profiles of the experimental snails using the first five principal components (PCs) after internal normalization in Sleuth. Additionally, the proportion of total variance each PC explained in the data is presented.

14 TLR 28651_c0_g1_i1 0.1 0.2 0.3 0.4 0.5 0.6 47065_c0_g1_i2 47065_c0_g1_i1 47065_c0_g1_i4

MyD88 47065_c0_g1_i8 0 0.5 1 1.5 47065_c0_g1_i3 47065_c0_g1_i5 0.2 0.3 0.4 0.5 0.6 0.7 47065_c0_g1_i7 TRAF 22931_c0_g1_i1 1 1.1 1.2 1.3 1.4 1.5 1.6 TRF 34891_c0_g1_i1 TAB 24669_c0_g1_i1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 SARM 46010_c7_g2_i1 35196_c0_g1_i1 IKKα 0.5 0.6 0.7 0.8 0.9 35196_c0_g1_i2 47446_c1_g1_i5 0 0.2 0.4 0.6 47446_c1_g1_i3 IKKγ 47446_c1_g1_i4 0 0.2 0.4 0.6 0.8 1 1.2 Nemo 47446_c1_g1_i6 47446_c1_g1_i1 47446_c1_g1_i2 46794_c1_g1_i3 46794_c1_g1_i5 45095_c0_g1_i2

LITAF 0 0.5 1 1.5 2 46794_c1_g1_i1 45095_c0_g1_i1

Component of TLR signalling pathway 46794_c1_g1_i2 39554_c0_g1_i1 IκB 2 2.5 3 3.5 4 46270_c2_g2_i1 38589_c1_g1_i1 43263_c0_g2_i2 NF-κB 0 0.5 1 1.5 43263_c0_g1_i1 43263_c0_g2_i1 46200_c0_g1_i1

MPEG 46200_c0_g1_i2 1 2 3 47480_c0_g1_i1 Untreated Anaesthesia Wounding E. coli M. lysodeikticus Healthy gonad Trematode-infected Elevated Food Transcript ID Expression level (TPM) gonad temperature deprivation Treatment Fig. S2. Expression levels of individual transcripts found to represent annotated factors related to TLR signalling pathway in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity.

18198_c0_g1_i1 34989_c0_g2_i1 18890_c0_g1_i1 35624_c0_g1_i1 Macin 0 1 2 3 4 41887_c0_g3_i3 41887_c0_g3_i1 43018_c0_g6_i3 25950_c0_g1_i1 46649_c0_g1_i1

LBP/BPI 0 1 2 3 4 45368_c0_g6_i1 45368_c0_g6_i2 47893_c0_g9_i1 48351_c1_g3_i1 47821_c4_g3_i1 LAAO 47821_c4_g3_i5 1 2 3 4 5 47821_c4_g3_i4 47821_c4_g3_i6 44233_c0_g1_i1 44233_c0_g1_i2 44233_c0_g1_i4 28373_c0_g1_i1 44233_c0_g1_i5

Lyzosyme 36143_c0_g3_i1 0 1 2 3 4

Antibacterial defence factor 44233_c0_g1_i3 44233_c1_g1_i1 35313_c1_g1_i1 35313_c0_g1_i1 35313_c1_g1_i2 47349_c1_g38_i2 38389_c0_g1_i2 Lymnaea lysin 0 0.5 1 1.5 2 2.5 3 3.5 38389_c0_g1_i1 5994_c0_g2_i1 34104_c0_g1_i1

Stagnalysin 42332_c1_g1_i1 0.8 1 1.2 1.4 1.6 48069_c5_g1_i1 Untreated Anaesthesia Wounding E. coli M. lysodeikticus Healthy gonad Trematode-infected Elevated Food Transcript ID Expression level (TPM) gonad temperature deprivation Treatment Fig. S3. Expression levels of individual transcripts found to represent the annotated antibacterial defence factors in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity.

15 5 1.5 2 5 1.5 4 2 4 4 2 4 3 1.5 1.5 3 3 1 1 3 1.5 1 1 2 2 2 2 0.5 0.5 1 0.5 1 0.5 1 1 1 Expression level (TPM) Expression level (TPM) 0 0 0 0 0 0 Transcript ID Transcript ID 47859_c0_g2_i7 44175_c0_g1_i5 44175_c0_g1_i4 47859_c0_g2_i5 47859_c0_g2_i2 44175_c0_g1_i2 44175_c0_g1_i1 42521_c0_g1_i5 44175_c0_g1_i3 44175_c0_g1_i6 42521_c0_g1_i6 47859_c0_g2_i4 47859_c0_g2_i6 48059_c1_g2_i3 48059_c1_g2_i1 47859_c0_g2_i1 48059_c1_g2_i2 47859_c0_g2_i3 42521_c0_g1_i2 44043_c0_g3_i1 45762_c4_g1_i3 47321_c2_g2_i1 47321_c2_g2_i3 48217_c1_g1_i3 47321_c2_g2_i2 47321_c2_g2_i4 38462_c0_g1_i1 47321_c2_g1_i3 45077_c0_g1_i1 47844_c2_g5_i1 47321_c2_g1_i2 38987_c2_g2_i1 45077_c0_g1_i2 48217_c1_g1_i10 48217_c1_g1_i12 20307_c0_g1_i1 35966_c0_g1_i1 48217_c1_g1_i2 48217_c1_g1_i6 47391_c1_g1_i3 47391_c1_g1_i2 47391_c1_g1_i4 41774_c0_g1_i3 47391_c1_g1_i1 41774_c0_g1_i2 47227_c0_g2_i1 48085_c1_g8_i11 44959_c0_g1_i1 48268_c2_g1_i1 48268_c2_g1_i5 39342_c0_g1_i1 47242_c4_g2_i6 47242_c4_g2_i1 47242_c4_g2_i8 47242_c4_g2_i2 47242_c4_g2_i3 47242_c4_g2_i7 37655_c0_g1_i1 47242_c4_g2_i5 42094_c0_g6_i1 42956_c0_g1_i1 36537_c0_g1_i1 34108_c0_g1_i1 14145_c0_g1_i1 32865_c0_g1_i1 34108_c0_g1_i2 36963_c0_g1_i1 36764_c0_g1_i2 45250_c0_g1_i1 26259_c0_g1_i1 44227_c0_g2_i2 48292_c1_g1_i5 33218_c0_g1_i1 44227_c0_g2_i3 45255_c1_g3_i1 48292_c1_g1_i3 45634_c0_g3_i1 47306_c0_g5_i1 44081_c0_g1_i1 45255_c1_g3_i3 26246_c2_g1_i1 46270_c3_g2_i1 Food Food deprivation deprivation Elevated Elevated temperature temperature gonad gonad Trematode-infected Trematode-infected Transcripts related to each factor are clustered according to their related to each factor Transcripts Healthy gonad Healthy gonad Transcripts related to each factor are clustered according to their similarity. Transcripts 16 M. lysodeikticus M. lysodeikticus Treatment Treatment E. coli E. coli Wounding Wounding Anaesthesia Anaesthesia Untreated Untreated

similarity. Fig. S4. Expression levels of individual transcripts found to represent annotated factors of individual transcripts found to represent annotated Fig. S4. Expression levels in units of transcripts per million to phenoloxidase/melanisation-type reaction related using snail. Heatmap shows the variation for each factor (TPM) for each experimental its dynamic range. Tyrosinase Laccase

Factors with phenoloxidase activity phenoloxidase with Factors

Fig. S5. Expression levels of individual transcripts found to represent annotated factors related to non-self recognition in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. L-type lectin Chi-type lectin Galectin FREP GNBP PGRP M-type lectin Non-self recognition factors recognition Non-self 2.5 2 4

2 1.5 3 1.5 1 2 1

0.5 1 0.5 Expression level (TPM) 0 0 0 Transcript ID 48316_c0_g1_i1 48316_c0_g1_i6 43870_c0_g1_i3 47468_c1_g1_i2 42612_c1_g1_i1 46501_c1_g2_i1 43870_c0_g1_i1 47690_c7_g2_i10 48366_c1_g1_i2 42168_c1_g1_i5 42168_c1_g1_i7 37593_c0_g1_i2 44512_c0_g1_i5 34263_c0_g1_i2 37407_c0_g1_i1 41927_c0_g1_i2 81680_c0_g1_i1 42302_c0_g4_i4 20877_c0_g2_i1 42302_c0_g4_i2 47690_c7_g2_i3 37129_c0_g1_i2 44512_c0_g1_i2 48149_c3_g6_i11 47286_c3_g3_i5 42302_c0_g4_i1 47286_c3_g3_i4 37593_c0_g1_i1 47690_c7_g2_i16 42168_c1_g1_i2 47690_c7_g2_i14 20877_c0_g1_i1 34263_c0_g1_i1 34263_c0_g1_i4 47690_c7_g2_i15 47690_c7_g2_i8 47468_c1_g1_i1 45558_c0_g3_i1 45558_c0_g3_i5 41598_c0_g1_i2 45558_c0_g2_i2 47427_c2_g2_i8 15848_c0_g1_i1 48366_c1_g1_i1 47286_c3_g3_i2 48366_c1_g1_i3 46501_c1_g2_i3 39166_c0_g1_i1 43123_c0_g2_i2 37129_c0_g1_i1 42736_c0_g1_i1 41927_c0_g1_i1 46059_c2_g1_i4 48316_c0_g1_i3 43870_c0_g1_i2 46059_c2_g1_i2 45927_c0_g1_i1 47468_c1_g1_i3 46059_c2_g1_i1 46059_c2_g1_i3 45927_c0_g1_i2 42736_c0_g1_i2 47286_c3_g3_i3 4951_c0_g1_i1 110902_c0_g1_i1 17215_c1_g1_i1 36453_c0_g1_i1 36453_c0_g1_i2 Food deprivation Elevated temperature gonad Trematode-infected Healthy gonad 17 M. lysodeikticus Treatment Transcripts related to each factor are clustered according to their similarity. Transcripts E. coli Wounding Anaesthesia Untreated

Fig. S6. Expression levels of individual transcripts found to represent annotated cytokines in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. TNF IL-17 MIF Category of cytokines of Category 3 1.2 4 4 1.2 5 2.5 2.5 1.5 1 1 3 4 3 2 3 2 0.8 0.8 1 3 1.5 2 1.5 0.6 2 2 2 1 0.6 1 0.4 0.5 1 1 1 0.2 0.5 0.5 1 0.4 Expression level (TPM) 0 0 0 0 0 0 0 0 Transcript ID 46851_c0_g1_i2 46851_c0_g1_i3 46851_c0_g1_i1 35109_c0_g1_i1 37634_c0_g1_i2 31273_c0_g1_i1 43131_c0_g1_i4 38055_c0_g1_i2 43131_c0_g1_i2 36750_c0_g1_i1 39155_c0_g1_i1 37634_c0_g1_i1 43131_c0_g1_i1 38965_c1_g1_i2 46851_c0_g1_i6 38055_c0_g1_i1 46105_c0_g60_i1 46670_c0_g1_i1 38965_c1_g1_i1 44723_c0_g5_i1 46929_c1_g35_i1 40585_c0_g1_i1 44207_c5_g1_i1 46851_c0_g1_i4 37296_c0_g2_i1 46742_c1_g1_i1 17067_c0_g1_i1 40581_c0_g1_i1 37296_c0_g2_i2 43131_c0_g1_i3 1429_c0_g2_i1 41000_c0_g2_i1 4572_c0_g2_i1 34343_c1_g2_i1 45783_c0_g1_i2 4572_c0_g1_i1 34293_c0_g2_i1 34293_c0_g2_i2 45783_c0_g1_i1 47774_c2_g1_i1 28492_c0_g1_i1 45570_c1_g1_i1 47256_c0_g1_i1 46236_c0_g1_i2 46236_c0_g1_i5 47256_c0_g1_i2 48181_c2_g1_i2 48181_c2_g1_i4 46303_c1_g1_i3 47256_c0_g1_i3 47256_c0_g1_i4 46303_c1_g1_i2 46236_c0_g1_i4 46303_c1_g1_i1 48181_c2_g1_i1 48181_c2_g1_i3 40499_c0_g1_i1 45939_c0_g1_i1 42865_c0_g1_i4 42865_c0_g1_i3 42865_c0_g1_i1 42865_c0_g1_i6 46890_c0_g1_i1 46890_c0_g1_i2 33046_c0_g1_i1 44859_c0_g2_i1 44859_c0_g2_i4 39371_c0_g1_i1 44859_c0_g2_i3 45653_c0_g1_i2 45653_c0_g1_i3 36496_c0_g1_i1 40220_c1_g2_i1 36278_c0_g1_i4 36278_c0_g1_i5 36278_c0_g1_i3 40220_c1_g2_i2 36278_c0_g1_i1 36278_c0_g1_i2 36278_c0_g1_i6 46977_c0_g1_i1 27522_c0_g1_i1 41634_c2_g1_i1 46977_c0_g1_i4 46977_c0_g1_i3 34959_c0_g2_i1 47425_c0_g2_i6 41634_c2_g1_i2 46977_c0_g1_i2 38205_c0_g1_i1 Food deprivation Elevated temperature Transcripts related to each factor are clustered according Transcripts gonad Trematode-infected Healthy gonad 18 M. lysodeikticus Treatment E. coli Wounding Anaesthesia Untreated

to their similarity. Fig. S7. Expression levels of individual transcripts found to represent annotated factors related to the production of reactive oxygen species (ROS) in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Peroxiredoxin Nucleoredoxin GST Glutaredoxin Peroxidasin NOS DUOX DUOXA NOX Components of the production of ROS of production the of Components FAIM1 44771_c0_g1_i1 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 37936_c0_g1_i1

HTRA2 36021_c0_g1_i1 0.5 1 1.5 2 37936_c0_g1_i2

AIF 40505_c0_g1_i1 1.5 1.6 1.7 1.8 1.9 2 2.1 48019_c3_g3_i1 47200_c0_g1_i1 47310_c1_g1_i4 48046_c0_g1_i3 48048_c3_g1_i2 48048_c3_g1_i3 42472_c1_g1_i1 30243_c0_g1_i1 47310_c1_g1_i8 46127_c2_g1_i2 47642_c0_g1_i3 47962_c0_g2_i3

IAP 41110_c0_g1_i1 0 0.5 1 1.5 2 2.5 41110_c0_g1_i2 41110_c0_g1_i3 47962_c0_g2_i1 47962_c0_g2_i6 48019_c2_g1_i1 47642_c0_g1_i11 47962_c0_g2_i2 46127_c2_g1_i3 46127_c2_g1_i5 46127_c2_g1_i1 46127_c2_g1_i4 46127_c2_g1_i6

Bcl-Bax 44934_c1_g1_i3 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 48116_c1_g1_i2 45625_c0_g1_i2 48398_c1_g4_i2 42464_c1_g2_i1 48116_c1_g1_i9 45339_c0_g1_i2 45339_c0_g1_i5 47461_c0_g4_i5 47461_c0_g4_i7 47461_c0_g4_i8

PARP 29056_c0_g2_i1 0 0.5 1 1.5 2 34140_c0_g1_i1 Components of apoptosis 33385_c0_g2_i1 37433_c3_g2_i1 5046_c0_g1_i1 45339_c0_g1_i3 45339_c0_g1_i1 45339_c0_g1_i4 5046_c0_g2_i1 47461_c0_g4_i2 47461_c0_g4_i4 48216_c2_g3_i10 45052_c4_g2_i2 48203_c0_g1_i7 48203_c0_g1_i6 48216_c2_g3_i18 48216_c2_g3_i17 48216_c2_g3_i6 37005_c0_g1_i2 Caspase 48216_c2_g3_i8 48203_c0_g1_i2 48216_c2_g3_i3 0 0.5 1 1.5 2 2.5 42025_c0_g1_i2 45052_c4_g2_i1 33457_c0_g1_i1 42025_c0_g1_i3 48203_c0_g1_i5 48203_c0_g1_i8 37005_c0_g1_i1 37479_c0_g2_i1 43539_c0_g1_i1 Untreated Anaesthesia Wounding E. coli M. lysodeikticus Healthy gonad Trematode-infected Elevated Food Transcript ID Expression level (TPM) gonad temperature deprivation Treatment Fig. S8. Expression levels of individual transcripts found to represent annotated factors related to apoptosis in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity.

46830_c1_g2_i1 41463_c0_g1_i1 46830_c1_g2_i2 44096_c1_g1_i10 44096_c1_g1_i9 44096_c1_g1_i1 44096_c1_g1_i8 HSP 70 0 1 2 3 4 44096_c0_g1_i1 44096_c1_g1_i7 47977_c0_g5_i1 47977_c0_g5_i2 3.5 4 4.5 40510_c1_g2_i1

Stress-response factors 46830_c1_g3_i1 HSP 90 47161_c0_g4_i1 0.4 0.5 0.6 0.7 0.8 HSF 29117_c0_g1_i1 Untreated Anaesthesia Wounding E. coli M. lysodeikticus Healthy gonad Trematode-infected Elevated Food Transcript ID Expression level (TPM) gonad temperature deprivation Treatment Fig. S9. Expression levels of individual transcripts found to represent the annotated stress-response factors in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity. 19 45899_c0_g2_i2 45899_c0_g2_i3 41920_c0_g1_i2

SOD 0 0.5 1 1.5 2 2.5 45899_c0_g2_i5 41920_c0_g1_i4

45899_c0_g2_i1 2.9 3 3.1 3.2 3.3 3.4 3.5 MnSOD 28257_c0_g1_i1 40830_c0_g1_i1

CAT 1.8 2 2.2 2.4 2.6 2.8 3 44469_c0_g1_i1 47906_c1_g7_i2 36171_c0_g2_i1 47906_c1_g7_i1 1 2 3 4 Antioxidant enzyme glutathione peroxidase 37235_c0_g1_i1 47906_c1_g7_i3 1.6 1.8 2 2.2 glutathione reductase 44983_c1_g1_i1 Untreated Anaesthesia Wounding E. coli M. lysodeikticus Healthy gonad Trematode-infected Elevated Food Transcript ID Expression level (TPM) gonad temperature deprivation Treatment Fig. S10. Expression levels of individual transcripts found to represent the annotated antioxidant enzymes in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity.

36580_c0_g1_i1

PP1 0 0.2 0.4 0.6 0.8 1 1.2 36580_c0_g1_i2 109816_c1_g1_i1 44915_c1_g1_i2 46544_c1_g6_i1 40537_c0_g1_i1 44027_c1_g1_i2 44915_c1_g1_i1 Ubiquitin 44027_c1_g1_i1 0 1 2 3 4 5 6 45261_c0_g1_i1 46544_c1_g3_i2 40537_c0_g1_i3 46544_c1_g3_i4 33535_c0_g1_i1 46234_c4_g1_i1 33209_c0_g1_i1

Ferritin 125081_c0_g1_i1 3 4 5 6 7 43780_c0_g5_i1

Metabolism-related factor 47996_c0_g14_i1

ADH 47996_c0_g14_i4 2 2.5 3 3.5 4 4.5 47996_c0_g14_i7 36130_c1_g1_i4

ERR 0.2 0.4 0.6 0.8 1 1.2 42943_c0_g1_i1 33793_c0_g1_i1 41983_c1_g1_i2

RXR 42786_c1_g1_i2 0 0.5 1 1.5 41983_c1_g1_i1 42786_c1_g1_i1 Untreated Anaesthesia Wounding E. coli M. lysodeikticus Healthy gonad Trematode-infected Elevated Food Transcript ID Expression level (TPM) gonad temperature deprivation Treatment Fig. S11. Expression levels of individual transcripts found to represent annotated factors related to metabolism in units of transcripts per million (TPM) for each experimental snail. Heatmap shows the variation for each factor using the dynamic range. Transcripts related to each factor are clustered according to their similarity.

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