Experimental data supporting adaptive locomotor responses to salt stress in the mud-tidal gastropod populations (Batillaria)

This article describes the experimental locomotor data used to study the general and adaptive responses to salt stress of the northern Pacific intertidal gastropod Batillaria attramentaria. The data were obtained from a series of 30-day experiments on snails acclimated to different salinity regimes. Snails were collected from coastal areas on the eastern and western sides of the North Pacific Ocean. The data consist of three parts: 1) raw videos recording the locomotion of the snails when exposed to novel artificial salinity regimes in laboratory settings, 2) Spectral Time-Lapse results of movement distance of the snails extracted from the recorded videos, and 3) CO1-gene sequences isolated from individuals collected from four sampling sites. A Linear Mixed-effect Model inference procedure was applied in an attempt to assess the impacts of geographic distribution and genetic composition on the locomotor response to salt stress in the snail B. attramentaria. The locomotor dataset we present are the first reports of locomotor response to salt stress of the snail B. attramentaria, that is valuable for further exploration and understanding of the impacts of environmental changes on the physiology and adaptive capacity of living marine molluscs.


a b s t r a c t
This article describes the experimental locomotor data used to study the general and adaptive responses to salt stress of the northern Pacific intertidal gastropod Batillaria attramentaria . The data were obtained from a series of 30-day experiments on snails acclimated to different salinity regimes. Snails were collected from coastal areas on the eastern and western sides of the North Pacific Ocean. The data consist of three parts: 1) raw videos recording the locomotion of the snails when exposed to novel artificial salinity regimes in laboratory settings, 2) Spectral Time-Lapse results of movement distance of the snails extracted from the recorded videos, and 3) CO1 -gene sequences isolated from individuals collected from four sampling sites. A Linear Mixedeffect Model inf erence procedure was applied in an attempt to assess the impacts of geographic distribution and genetic composition on the locomotor response to salt stress in the snail B. attramentaria . The locomotor dataset we present are the first reports of locomotor response to salt stress of the snail B. attramentaria , that is valuable for further exploration and understanding of the impacts of environmental changes on the physiology and adaptive capacity of living marine molluscs.
© 2021 The Author(s

Value of the Data
• These locomotor data present significant insights into the response to artificial salt stress of the intertidal gastropod Batillaria attramentaria in laboratory settings. • These data could be utilized for further statistical analyses including a Linear Mixed-effect Model to study adaptive response to salt stress of introduced snails, which were accidentally introduced to a novel osmotic environment (Elkhorn Slough, CA, USA) approximately 80 years ago. • These data are also valuable for further research on forecasting the impacts of environmental changes on the physiology and adaptive capacity of living marine molluscs.

Data Description
The raw locomotor videos recorded snail movement during a 30-day period in which snails were exposed to different artificial salinity regimes. The intertidal snails Batillaria attramentaria (G. B. Sowerby I, 1855) were collected from coasts of the northeast and northwest Pacific Ocean. Sample collection details are provided in Table 1 . The locomotor performance of the snail B. attramentaria in response to salt stress is presented as movement distance (. txt format file) and movement trails (. tiff format file) ( Fig. 1 ); the former was to assess the impacts of geographic distribution and genetic composition on the locomotor responses of the snails.
The data comprise three geographic variables (origin o , location lo, and population p ), one genetic variable ( CO1 genetic lineage li), and one temporal variable time t (video recording date). Ho et al. [1] found that B. attramentaria acclimated to an extremely low salinity of 13 PSU exhibited significantly shorter movement distance than individuals acclimated to normal or moderately altered salinities of 23, 33, and 43 PSU [Supplemental Table A2 and Fig. 3 A in [2] . In addition, the introduced snails, which were accidentally introduced to north America [Monterey Bay, Elkhorn Slough, CA , USA , [3 , 4] , exhibited shorter movement distance than those from native habitats [Supplemental Table A2 and Fig. 3 B, C, D in [2] . These findings suggest that the introduced snails may have adapted or acclimated to the novel osmotic conditions of their new habitat, where salinity fluctuations are wider than their native habitats. Here, we present how snails responded over the courses of 30-day experiments by applying a Linear Mixed-effect Model (LMM) to movement distance. We discovered that snails significantly changed their locomotor performance after 30 days with F Time (1,14) = 12.42 and P -value < 0.0 0 01 ( Table 2 A and Fig. 2 ). Means of movement distance of the snails recorded every two days over a 30-day period are presented in Table 2 B and Fig. 2 . Subsequent Tukey post-hoc tests revealed that snails significantly increased their movement distance over the experiment period with d Day 30-Day 2 = 0.12 m ± 0.031 and P -value < 0.005 ( Table 3 ). On the other hand, we also observed that significant increases in locomotion occurred between two pairs of consecutive recording dates, Day 4 vs. Day 6 and Day 18 vs. Day 20 ( Table 3 and Fig. 2 ). This result is graphically presented in Fig. 3 , which shows the trend of movement distance change between groups of snails experimentally exposed to different salinities.

Salinity experiments
Snails were kept at 25 °C and on a 12h Light:12h Dark photocycle for two days to reduce any effects of transportation. After this acclimation period, we conducted 30-day experiments on the four collected sets of samples separately. Snails were randomly equally divided into five groups    with 20 individuals per group for the native-population samples and 10 individuals per group for the introduced-population sample. Five groups of each population were cultured separately in plastic aquaria [40 × 23 × 21 cm 3 , with an inclined layer of sea sand on the bottom and fresh aerated artificial sea, Supplementary Figure S1 in [5] at different salinities of 13, 23, 33, and 43 PSU for 30 days. We marked each of the snails in each group with different colors of nail polish (Innisfree, ROK). All individuals were fed with excised fresh seaweed (Ottogi, ROK) every two days throughout the experiments.

Locomotion recording and analyzing
We recorded snails and tracked their movement for one hour every two days from 9:00 to 15:00 throughout the laboratory experiments. To monitor the snails' performance and track their movement distance, we placed each snail in the center of a single disposable Petri dish which was filled with artificial seawater and recorded them using a Sony Nxcam (AVCHD Progressive MPEG2 SD). Snails from the same group were recorded at the same time. The camera was mounted on a tripod, and the camera was situated above the twenty Petri dishes to record all the dishes at once. Saline water was freshly prepared with overnight-aerated distilled water and Instant Ocean Sea Salt (United Pet Group Inc., Cincinnati, OH, USA). After being recorded, all snails were conveyed back to their corresponding aquaria. All recorded videos were uploaded to a computer for computational analyses. Video analyses were then conducted following the protocol described in our earlier study [5] . Briefly, we used a series of software packages such as AVS Video Editor v.7.1.2.262 to increase the play-back rate of the recorded videos, Avidemux v.2.6.12 to crop videos, and Spectral Time-Lapse (STL) toolbox [6] implemented in Matlab release R2014a (MathWorks Inc., Natick, Massachusetts, USA) to estimate the movement distance of snails. Movement distance of the snails was measured in meter.

Statistical analyses
A Linear Mixed-effect Model (LMM) was employed to (1) assess the impacts of salinity exposure ( es), geographic distribution ( g : population p , location lo, and origin o), and CO1 genetic lineage ( li ), as well as their interactions ( es × o, es × o + li, es × lo, es × lo + li, es × p, es × p + li, es × li, es × li + o, es × li + lo , and es × li + p ) on the snails' locomotion, and (2) model the snails' movement distance over time due to salinity stress. In the analysis, es refers to the salinity level used in the laboratory experiments including four levels: 13, 23, 33, and 43 PSU; g indicates the geographic location of each population p (Hacheon in Korea, Nemuro city and Matsushima bay in Japan, and Elkhorn Slough in the USA); location lo ( Korea, Japan, or the USA); origin o (native or introduced); and CO1 lineage li is defined as either Tsushima (comprising the Hacheon and Nemuro populations) or Kuroshio (comprising the Matsushima and Elkhorn Slough populations) based on the individual's position in a CO1 phylogenetic tree. Subsequently, we conducted multimodel inference and model averaging [7] to select the best-fit model that best described snail locomotor response using the corrected Hurvich and Tsai's Criterion (AICC). The models were compared based on AICc values using the MuMIn package MuMIn 1.9.13 [8] for R 3.0.2. The model with the lowest AICc value and those satisfying a AICc ≤ 6 cut-off rule [9] were considered the best-fit models. Following the multimodel inference procedure, we conducted post-hoc multiple comparison tests of the models to examine the effects of each explanatory factor. Additionally, we used MuMIn to perform model averaging and estimate the importance of predictor variables by summing the weights of models where the variables appeared. The significance level was set at α = 0.05 for all statistical tests.
For the present study, we additionally conducted LMM to the movement distance data in order to assess the impact of temporal variable time t on the response to salt stress of the snails. Subsequently, we conducted Tukey post-hoc tests to examine the difference in movement distance of the groups of snails between dates over the course of acclimation experiments.

Ethics Statement
We confirm that all experiments comply with the ARRIVE guidelines and were be carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments , or the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).

CRediT Author Statement
Phuong-Thao Ho: Conceptualization, Data curation, Formal analysis, Methodology, Writing -original draft, Writing -review and editing; Hoa Quynh Nguyen: Formal analysis; Writingreview and editing; Elizabeth MA Kern: Writing -original draft, Writing -review and editing; Yong-Jin Won: Funding acquisition, Writing -review and editing.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.