DOI: 10.5061/dryad.63xsj3v8v
title: Data from: First evidence of a genetic basis for thermal adaptation in a schistosome host snail

description: Freshwater snails play a key role in the transmission of schistosomiasis,
 a tropical parasitic disease affecting over 150 million people. Adaptation
 of these snails to local climatic conditions is a critical factor in
 determining how climate change and other environmental factors influence
 disease transmission dynamics, yet this potential adaptation has remained
 unexplored. Bulinus truncatus is the schistosome intermediate host snail
 with the widest geographic distribution and is therefore an important
 factor determining the maximum range of urogenital schistosomiasis. In
 this study, we assessed the local adaptation capacity of B. truncatus to
 temperature through an integrative approach encompassing phenotypic,
 ecophysiological, and genomic data. Ten snail populations from diverse
 thermal environments were collected in three countries, with eight
 populations reared in a common garden. The F2 generation (N= 2,304) was
 exposed to eight chronic temperature treatments (± 36
 snails/population/temperature treatment) and various life-history traits
 were recorded for over 14 weeks. Subsequently, ecophysiological analyses
 were conducted on the ten last surviving snails per population. Genotyping
 the parental generation collected in the field using a
 genotyping-by-sequencing (GBS) approach, revealed 12,875 single nucleotide
 polymorphisms (SNPs), of which 4.91 % were potentially under selection. We
 observed a significant association between outlier SNPs, temperature, and
 precipitation. Thermal adaptations in life-history traits were evident,
 with lower survival rates at high temperatures of warm-origin snails
 compensated for by higher reproduction rates. Cold-origin snails, on the
 other hand, exhibited higher growth rates adapted to a shorter growing
 season. Ecophysiological adaptations included elevated sugar and
 haemoglobin contents in cold-adapted snails. In contrast, warm-adapted
 snails displayed increased protein levels but also more oxidative damage.
 Furthermore, heightened phenoloxidase levels indicated a more robust
 immune response in snails from parasite-rich regions. These morphological
 and physiological differences provide convincing evidence for a genetic
 basis of local adaptation. This in turn holds profound implications for
 the snail’s response to climate change, future schistosomiasis risk, and
 the effectiveness of schistosomiasis control measures.
description: All data were obtained through an extensive common garden
 experiment in which second generation snails from different origins were
 subjected to different temperature treatments. Life-history parameters
 such as growth, fecundity and survival were measured during 14 weeks.
 After the experiment, a subset of snails were analysed physiologically.
 The DNA from the parental generation snails coming from the field was
 extracted and single nucleotide polymorphisms (SNPs) were identified using
 a genotyping by sequencing approach.
 <strong>Field Collection and experimental
 design</strong> <em>Bulinus truncatus
 </em>snails were collected in the second half of 2021 from ten
 localities in three countries (France, Senegal and Zimbabwe) spanning a
 latitudinal and temperature gradient. About 100 snails were collected per
 locality. Additionally, a French (Corsican) strain that has been
 maintained in the lab since 2014 was included. Each locality was screened
 for <em>B. truncatus</em> and snails were collected from every
 habitat where they were found. We chose French sampling sites on the
 border between the Corsican montane broadleaf and mixed forests and the
 Tyrrhenian-Adriatic Sclerophyllous and mixed forests ecoregions in the hot
 summer Mediterranean climate as this area represents the cold limit of the
 distribution range of <em>B. truncatus</em>. This area is
 characterised by a high seasonality with average air temperatures ranging
 between 7 °C in winter and 27 °C in summer. Senegalese snails were
 collected in the Sahelian Acacia savanna ecoregion characterised by a hot
 desert climate which represents the warm limit of the distribution range
 with less strong seasonality and average air temperatures ranging from 30
 to 39 °C. Finally, the snails from Zimbabwe originate from the Zambezian
 and Mopane woodlands (Triangle and Malilangwe populations, characterised
 by a hot, semi-arid steppe climate) and the Southern Miombo woodlands
 (Imire, characterised by a subtropical highland climate) ecoregions where
 temperatures show less seasonality and are more temperate with averages
 between 14 and 22.6 °C. The field-collected parental (P) snails were bred
 to the second (F2) generation in a common garden at 24 °C and a 12:12 h
 day-night regime, fed <em>ad libitum </em>with pesticide-free
 lettuce (dried at 55 °C for 12 h), and green macro-algae were added to
 enrich the water with oxygen. About 100 random egg masses from the P
 generation were transferred to start the F1 generation. As many eggs as
 possible were collected from the F1 generation to breed at least 300 F2
 snails per locality, needed for the experiments. DNA
 was extracted from the whole body (from the P generation collected in the
 field) using the E.Z.N.A.® mollusc DNA kit according to the manufacturer’s
 protocol (Omega Bio-tek). To verify the morphological identification from
 the field, a subsample of five snails from each population was genotyped
 through DNA barcoding using the protocol described in Maes et al. (2022)
 and identification of the specimens relied on a BLAST search against the
 NCBI database GenBank. After DNA barcoding, eight populations from three
 countries identified as <em>B. truncatus </em>(see results
 section)<em> </em>were selected for the life history
 experiment while all populations identified as <em>B. truncatus
 </em>were included in the genetic analyses. The Imire population
 from Zimbabwe was identified as <em>Bulinus tropicus
 </em>(Krauss, 1848) and excluded from the experiment and genetic
 analyses. The F2 snails were individually placed in 100
 ml plastic cups filled with aged tap water at four to eight weeks old. The
 experiment was started in 12 different batches, each batch containing
 three snails per locality for each temperature treatment (3 snails x 8
 localities x 8 temperatures x 12 batches = 2304 snails in total, 36
 snails/population/temperature). The temperature was increased/decreased by
 2 °C/day starting from 24 °C until the snails reached their experimental
 temperature (4, 8, 12, 18, 24, 28, 32 or 36 °C) at the start of the
 experiment (time t=0). A constant temperature was permanently monitored
 using Hobo onset data loggers (tidbit v2 Temp logger). The snails were fed
 <em>ad libitum</em> with dried lettuce and green algae, and
 the water was refreshed weekly or when the oxygen level dropped below 5
 mg/ml. The lettuce was changed daily in the high-temperature treatments
 (24, 28, 32 and 36 °C) to prevent its decomposition and associated oxygen
 depletion. The snails were kept at the experimental temperatures for 14
 weeks. <strong>Response
 variables</strong> Each week the number of egg
 masses per snail was counted, dead snails were counted and removed, and
 shell height (apex to bottom of aperture) was measured under a
 stereomicroscope (Ceti Steddy-B) with a built-in ruler. The ten last
 surviving snails per treatment and locality combination were taken out of
 their shell, the body was weighed with an accuracy of 0.01 mg (Mettler
 Toledo AB135-S, Columbus, Ohio, USA) and stored at -80 °C for
 ecophysiological analyses. Egg masses were collected in weeks one to seven
 and in week 12 and stored in 98 % ethanol to later quantify the number of
 eggs per clutch under a stereomicroscope. The critical
 thermal maximum and minimum (CT<sub>max</sub> and
 CT<sub>min</sub>,<sub> </sub>respectively;
 Bartnicki et al., 2021; Morgan et al., 2018) were measured on a separate
 subset of F2 snails that were not included in the chronic temperature
 experiment. For measuring the CT<sub>max</sub>, the snails
 were individually put in 12 ml plastic tubes filled with aged tap water
 and placed in a heating block (Thermo Fisher Scientific). The water
 temperature increased by 0.1 °C/min (following Johansson &
 Laurila, 2017). For measuring the CT<sub>min</sub>, the snails
 were also individually put in 12 ml plastic tubes filled with aged tap
 water and placed in a water bath. The water temperature was decreased by
 0.1 °C/min using a cooler. Before both runs, the size of each snail was
 measured under a stereomicroscope with a built-in ruler. Snails were
 considered fainted when they showed no response to tactile stimuli (i.e.
 the snails did neither retreat in their shell nor retreat their
 tentacles). After the test, snails were allowed to recover and only snails
 that fully recovered within 10 min were considered for the analysis (5 out
 of 120 snails died in the CT<sub>max</sub> experiment, none in
 the CT<sub>min</sub> experiment). For the
 preferred temperature experiment, ten aluminium U-profiles (lanes) of 2 m
 in length were attached to create ten replicas and filled with aged tap
 water to establish a temperature gradient to assess the preferred
 temperature (T<sub>p</sub>) (cf. Johansson & Laurila,
 2017). One side of the profiles was placed on a cooling plate, and the
 other side on a heating plate. The temperatures of both plates were
 adjusted to generate a stable temperature gradient of 24 °C in the middle
 of each lane and an increase/decrease of 1 °C/10 cm in the direction of
 the heater/cooler (min: 14 °C max: 34 °C). Ten snails that were not
 included in the chronic temperature experiment were randomly selected from
 each locality and randomly assigned to one of the ten lanes. The snails
 were placed in the middle at 24 °C and the temperature at the position of
 each snail was measured every 15 min for 4 h. The first 2 h were
 considered as acclimation time while the preferred temperature was defined
 as the average temperature during the last two hours. We assumed that
 there were no differences in movement speed between the populations
 (Dillon et al., 2012). <strong>Ecophysiological
 analyses</strong> The last surviving individuals
 per population and temperature treatment were weighed and stored at -80 °C
 until further analyses. Whole-body snail samples were homogenised, 15 X
 diluted in phosphate-buffered saline (PBS) and centrifuged at 4 °C for 10
 min at 13,000 g. A minimum of 8 mg snail wet weight was required to run
 all ecophysiological analyses. If this weight was not reached, we pooled
 two snails from the same locality and temperature treatment to obtain the
 minimum required weight. Various ecophysiological
 parameters were examined to gain a comprehensive understanding of the
 snail’s overall well-being and to identify nuanced signs of local
 adaptation that might not be evident through life history traits alone.
 Assessing the snails’ general condition involved determining fat, total
 sugar, and protein contents (for the full protocols, see Appendix S1).
 From these variables, the cellular energy allocation (CEA) was calculated
 as outlined in Gomes et al. (2015). The CEA integrates the energy
 available (Ea) and energy consumption (Ec) of an organism. Additionally,
 the haemoglobin content was measured to gauge the oxygen-binding capacity.
 Haemoglobin is absent in most freshwater snails, except for pulmonate
 species like <em>B. truncatus </em>(Lieb et al., 2006).
 Furthermore, the malondialdehyde (MDA) levels were quantified to assess
 oxidative damage to lipids (Miyamoto et al., 2012). Phenoloxidase (PO),
 instrumental in the immune response to various parasite species in snails
 (Le Clec’h et al., 2016), was also measured to evaluate the strength of
 the snails’ immune system. Lastly, the snails’ metabolic rate was
 quantified at the cellular level by assessing the activity of the electron
 transport system (ETS) (De Coen & Janssen, 2003).
 <strong>SNP genotyping</strong>
 DNA from the parental (P) generation snails was used to build a
 paired-end GBS reduced representation library (RRL) according to Elshire
 et al. (2011) and sequenced on an Illumina NovaSeq 6000 platform (Genomics
 Core, KU Leuven). High molecular weight DNA was digested with restriction
 enzyme <em>NsiI</em>; Illumina sequencing primers P1 and P2
 and adapters containing the barcodes were ligated to the resulting
 fragments. The NovaSeq run produced 2.01 x
 10<sup>9</sup> paired-end raw 101 and 92 bp reads (forward and
 reverse reads, resp.). Reads were demultiplexed using process_radtags from
 Stacks 2.5 (Catchen et al., 2013) and the sequence quality was checked
 using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). FastX trimmer (http://hannonlab.cshl.edu/fastx_toolkit/) was used to trim the first 7 bp of the forward reads and the first 6 bp of the reverse reads. Trimmed forward and reverse reads were mapped against the <em>B. truncatus </em>reference genome (Young et al., 2022) using Bowtie2 (Langmead et al., 2019) and SNPs were identified using GATK UnifiedGenotyper 3.7. The resulting vcf file containing all variants was further filtered to only include biallelic SNPs, a read depth between 20 and 100, a minimum genotyping quality of 20, a minimum allelic depth of 6, a maximum of 40 % missing data per locus, a maximum of 70 % missing data per individual and a minor allele frequency of 0.02. Since <em>B. truncatus </em>is presumed to be autotetraploid (Young et al., 2022), a ploidy test was carried out using nQuire (Weib et al., 2018). NQuire uses next-generation sequencing data to distinguish between different ploidy levels based on the frequency distributions at variant sites where only two bases are segregating. SNP identification was carried out with both diploid (n=2) and tetraploid (n=4) settings in UnifiedGenotyper and a PCA was carried out on both filtered vcf files to check if the two datasets gave similar outcomes. Since both datasets gave the same output, all downstream analyses were carried out using the diploid dataset since more analysis tools are available for diploid data.
description: # Data from: First evidence of a genetic basis for thermal adaptation in a
 schistosome host snail
 [https://doi.org/10.5061/dryad.63xsj3v8v](https://doi.org/10.5061/dryad.63xsj3v8v) Data from Maes, T.; Verheyen, J.; Senghor, B., Mudavanhu, A., Schols, R., Hellemans, B., Geslain, E., Volckaert, F.A.M., Gante, H.F., & Huyse, T. (2025). First evidence of a genetic basis for thermal adaptation in a schistosome host snail, *Ecological Monographs.* ## Description of the data and file structure All data were obtained through an extensive common garden experiment in which second generation snails from different origins were subjected to different temperature treatments. Life-history parameters such as growth, fecundity and survival were measured during 14 weeks. After the experiment, a subset of snails were analysed physiologically. The DNA from the parental generation snails coming from the field was extracted and single nucleotide polymorphisms (SNPs) were identified using a genotyping by sequencing approach. ### Files and variables #### File: Datasets.zip **Description:** Folder containing all datasets used for the analyses. This zip file contains the following folders with the following files: ## Life-history and ecophysiology  * 01_Life-history-traits.csv: contains all the raw data for the survival, growth and egg mass analyses. Column names are as follows: | Column ID | Content | | :---------------------------------- | :----------------------------------------------------------------------------------------------------------- | | Batch | the batch number that included the individual | | Temperature | the temperature treatment (°C) | | Individual | the snail identifier | | Country | the country of origin | | Locality | the locality where the snail stems from | | Generation | the generation of the snail (parental: P, first generation: F1, second generation: F2) | | start-up\_date | the start date of snail acclimation | | Start\_experiment | the start date of the experiment after snails have acclimatised | | dead\_date | the date on which the snail was found dead | | Weeks\_survived | the number of weeks the snail has survived | | Size\_t0 | the size of the snail at the Start\_experiment date | | Size\_t1 to Size\_t14 | the size of the snail at week 1 to 14 | | Egg\_masses\_t1 to Egg\_masses\_t14 | the number of egg masses laid at week 1 to 14 | | Weight\_fysio\_(mg) | the weight of the snail if it was selected for ecophysiological analyses at the moment it was frozen (in mg) | | remarks | any peculiarities about the snail or observations made during the experiment | NA values indicate no data is available for the specific trait/timepoint because the snail died before that timepoint. * 02_nr_of_eggs-in-clutches.csv: the number of eggs per clutch laid per snail in certain weeks. | Column ID | Content | | :---------------------------------------- | :--------------------------------------------------------------------------------------- | | Individual | the snail identifier (the same individuals were used as in 01\_Life-history-traits.xlsx) | | Time | the week in which the egg masses were collected | | Country, locality, temperature, and batch | the same as in 01\_Life-history-traits.xlsx | | startSize | the size of the snail at time t=0 (i.e. the start of the experiment) | | Size | the size of the snail when the egg masses were collected | | nr\_of\_clutches | the number of egg masses collected at Time | | average\_clutch\_size | the average number of eggs per egg mass | | clutch size 1 to clutch size 15 | the number of eggs per egg mass | NA values indicate no data is available for a certain clutch number because the snail laid fewer clutches than this number at the time point specified in column "time". * 03_Tp_experiment.csv: the data from the preferred temperature experiment: | Column ID | Content | | :------------------------------- | :----------------------------------------------------------------------------------------------- | | Snail\_ID | the snail identifier (different from 01\_Life-history-traits.xlsx as different snails were used) | | Locality, Country and Generation | the same as in 01\_Life-history-traits.xlsx | | Snail\_size | the size of the snail when the experiment was carried out | | min\_15 to min\_240 | the temperature at the position of the snail (°C) after 15 to 240 minutes | * 04_ctmax_experiment.csv: the results of the CTmax experiment | Column ID | Content | | :------------------------------- | :-------------------------------------------------------------------------------------------------------------------------- | | Snail\_ID | the snail identifier (different from 01\_Life-history-traits.xlsx and 03\_Tp\_experiment.csv as different snails were used) | | Locality, Country and Generation | the same as in 01\_Life-history-traits.xlsx | | Snail\_size | the size of the snail when the experiment was executed | | CTmax | the temperature at which the snail fainted (°C) | | remarks | here it is mentioned if the snail died during the experiment | NA values indicate that the snail recovered after the experiment and no specific remarks were made. * 05_CTmin_experiment.csv: the results of the CTmin experiment | Column ID | Content | | :------------------------------- | :------------------------------------------------------------------------------------------------------------------------------------------------------ | | Snail\_ID | the snail identifier (different from 01\_Life-history-traits.xlsx, 03\_Tp\_experiment.csv, and 04\_CTmax\_experiment.csv as different snails were used) | | Locality, Country and Generation | the same as in 01\_Life-history-traits.xlsx | | Snail\_size | the size of the snail when the experiment was executed | | CTmin | the temperature at which the snail fainted (°C) | | remarks | here it is mentioned if the snail died during the experiment | NA values indicate that the snail recovered after the experiment and no specific remarks were made. * 06.1_ecophysiology_pooled_samples.csv: spreadsheet outlining which samples were pooled for physiological analyses because the separate snails did not have enough mass. | Column ID | Content | | :-------- | :-------------------------------------------------------------------------------------------------------- | | poolnr. | the number of the pool as used in 06\_ecophysiology.csv | | sample | the sample numbers (individual) as in 01\_Life-history-traits.xlsx that are included in the pooled sample | * 06_ecophysiology.csv: spreadsheet containing all the ecophysiological data collected | Column ID | Content | | :-------------------------------- | :------------------------------------------------------------------------------------------------------------------------------ | | Pool | the number of the pool as specified in 06.1\_ecophysiology\_pooled\_samples.xlsx | | Locality, Country and Temperature | the same as in 01\_Life-history-traits.xlsx | | Weeks\_survived | the number of weeks the snail survived before being frozen. | | Weight | the total weight of the pooled sample (mg) | | migfat\_mgsample | the total fat content in the sample (µg/mg sample) | | migprotein\_mgsample | the total protein content in the sample (µg/mg sample) | | migsugar\_mgsample | the total sugar content in the sample (mµ/mg sample) | | O2\_mgsnail | the metabolic rate of the snails expressed as the molar amount of O2 used per minute per mg protein (nmol O2/minute/mg protein) | | nmolMDA\_mgfat | malondialdehyde levels expressed as nmol MDA per mg fat (nmol MDA/mg fat) | | hemoglobine\_mgprotein | hemoglobin content expressed per mg protein | | nmolPO\_min\_mgprotein | the phenoloxidase activity expressed as nmol dopachrome formed per minute per mg protein (nnmol PO/min/mg protein) | | CEA | the cellular energy allocation =Ea/Ec (unitless) | | Ea | the total energy available (unitless) | | Ec | the total energy consumption (unitless) | NA values indicate that data for this trait is not available for the specified individual because of measurement errors of the spectrofotometer. If this occurred while measuring the protein content, other parameters such as the ETS activity, hemoglobin content, PO activity, CEA, Ea and Ec could not be calculated since these depend on the protein content. # Genetic analyses * 01_population_map.csv: file linking each individual to its respective population. | Column ID | Content | | :------------ | :------------------------------------------------------------------------------------------------------------------------------------- | | Sample | the sample ID for the sequenced individual (different from the Life-history and physiological data as different individuals were used) | | Locality | the locality where the snail was caught | | ID\_structure | identifier used in the structure analysis | * 02_sample_list.csv: file linking each individual to its respective population and spatial coordinates (used in the landscape genomics analyses) | Column ID | Content | | :-------- | :------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | sample | the sample ID for the sequenced individual (the same as in 01\_population\_map.csv) | | Locality | the locality where the snail was caught | | X | x-coordinates of the locality where the snail was caught (NA values introduced for the laboratory population as the exact location of origin can not be determined) | | Y | y-coordinates of the locality where the snail was caught (NA values introduced for the laboratory population as the exact location of origin can not be determined) | * variants_BA_DP20100_GQ20_QUAL20_AD6_miss0.40_missind0.70_maf0.02.vcf: all filtered SNPs used for the downstream analyses in the variant call format. * neutral_BA_DP20100_GQ20_QUAL20_AD6_miss0.40_missind0.70_maf0.02.vcf: only the neutral SNPs as identified by both Bayescan and PCadapt in the variant call format. Outlier loci have been removed from this dataset. * outliers_BA_DP20100_GQ20_QUAL20_AD6_miss0.40_missind0.70_maf0.02.vcf: only the outlier SNPs as identified by both Bayescan and PCadapt in the variant call format. ## Code and software **Data viewing** All data can be viewed using spreadsheet viewing software such as Microsoft office Excel or google sheets. The vcf files can be viewed with any text editor but it is advised to use one that does not read in the whole file at once. **Scripts to analyse the data** All scripts to process the files and reproduce the results have been included in this repository and are provided with the necessary comments that should allow anyone to run the code in R. The scripts are organised in the following way: * ecophysiology: folder containing all the scripts for the ecophysiological analyses. Each script is named after the parameter that is analysed in that script. * life-history analyses: folder containing all the scripts for the life-history analyses. Each script is named after the parameter that is analysed in that script. * genetic analyses: folder containing the scripts used for the genetic analyses. These include the basic population statistics, pca analyses, outlier detection and landscape genomics. The naming of the script folders corresponds to the naming of the dataset folders from which they analyse the data.