DOI: 10.5061/dryad.qbzkh18tc
title: Evaluating stress recovery and gill morphology during experimental supplementation of an endangered fish species

description: The efficacy of population supplementation (i.e., stocking) may be limited
 by physiological stress incurred upon transported and released fishes. In
 an experimental supplementation program for the critically endangered
 delta smelt (Hypomesus transpacificus), acclimation enclosures have been
 developed to facilitate stress recovery. However, the magnitude and
 duration of the stress response and the optimal time of release from
 enclosures remain unknown. To address these knowledge gaps, we quantified
 whole-body cortisol, glucose, lactate, and gill ionocyte morphology at 10
 time points across 72 hours following transport to enclosures at high and
 low densities. Cortisol and glucose peaked within 0.5 hours following
 transport into enclosures and recovered significantly by 27 hours. Lactate
 was moderately affected, and transport density did not affect cortisol,
 glucose, or lactate. Gill ionocyte surface area significantly increased in
 response to shifts in water chemistry throughout transport, and we
 observed three distinct types of microvilli on the surface of gill
 ionocytes, including two never previously reported. This work highlights
 the importance of ensuring stress recovery during the supplementation of
 delta smelt and other species.
description: # Evaluating stress recovery and gill morphology during experimental
 supplementation of an endangered fish species
 [https://doi.org/10.5061/dryad.qbzkh18tc](https://doi.org/10.5061/dryad.qbzkh18tc) ## Description of the data and file structure These data were collected using a variety of methods, as outlined in the manuscript text. Initial fish samples were collected in the field following a structured experimental design. Cortisol, glucose and lactate was collected in a lab environment using standard biochemistry techniques. Gill ionocyte morphology data was collected in a lab setting at a scanning electron microscopy facility at UC Davis. Exact methods of data collection and methods used are outlined in the text.  ### Files and variables #### File: Delta_Smelt_Transport_Stress_Carbonate_Chemistry.R **Description:** R script that performs the necessary data cleaning and statistical tests to determine shifts in water chemistry parameters across the experiment.  #### File: Delta_Smelt_Transport_Stress_Carbonate_Chemistry.csv **Description:** Excel file with data for transport water chemistry changes in carboys across time points.  ##### Variables * ID: An identifier for determining the location and replicate of the sample * Carboy: Identifier for the exact carboy (fish transport container) that the data was measured from * Density: Density of fish in each carboy * Treatment: The location that the carboy was transported to and the fish were released to * TimePoint: Time at which the water sample from the carboy water was taken  * Temp: Temperature in degrees Celsius  * DO: Dissolved oxygen of the carboy water measured by a YSI in mg/L * pH: pH of the carboy water measured by YSI * H_nM: Nano moles of hydrogen of the carboy water used in calculation of pCO2 * Drop: the amount of droplets used with a HATCH digital titration to initiate a color change in the water sample * Alk_mg: Alkalinity in mg/L calculated using HATCH digital titrator manual  * Alk_umol: Alkalinty transformed to micro moles * pCO2: Calculated pCO2 of the water sample from the corresponding carboy across transport #### File: Stress_Study_Stats.R **Description:** R script that performs the necessary data cleaning and statistical tests to determine changes in cortisol, glucose, and lactate concentrations throughout the experiment  #### File: Mort_Data.xlsx **Description:** Excel file including the data of fish mortalities from the field experiment  ##### Variables * Cage: Cage that mortalities were from * Density: Density of fish in cage * Hours Since Release: Discrete time points when fish were sampled, same as the hours since fish were released to cages  * Morts at sample: Amount of observed mortalities at each time point  #### File: Salinity_data.xlsx **Description:** Excel file including salinity data from the different study locations ##### Variables * Stage: Either the pre transport or post transport stage of moving fish from hatchery to field  * Salinity: Salinity at the pre and post transport stage * Destination: Final destination of transported fish #### File: Mort_Analysis.R **Description:** R script that performs the necessary data cleaning and statistical tests to analyze fish mortalities throughout the experiment #### File: Lactate_Data.xlsx **Description:** Excel file including the lactate concentrations of every fish sampled in the study  ##### Variables * Hours Since Release: Discrete time points when fish were sampled, same as hours since fish were released to cages  * Sample Point: Numeric identifier for the 10 sequential sampling points, 1:10 * Cage: Cage number that the specific fish belonged to * Fish number: Numeric identifier for the individual fish * Density: The total density of fish experienced by that specific fish during transport and within cages * Code: Unique identifier that incorporates sample point, cage, and fish number * Lactate: Concentration of lactate in ug of lactate per gram of fish mass * Location: Location where fish was released  #### File: SEM_Stats.R **Description:** R script that performs the necessary data cleaning and statistical tests to determine changes in gill monocyte morphology throughout the experiment  #### File: Cortisol_Data_corrected.xlsx **Description:** Excel file including the cortisol concentrations of every fish used in the study  ##### Variables * Hours Since Release: Discrete time points when fish were sampled, same as hours since fish were released to cages  * Sample Point: Numeric identifier for the 10 sequential sampling points, 1:10 * Cage: Cage number that the specific fish belonged to * Fish number: Numeric identifier for the individual fish * Density: The total density of fish experienced by that specific fish during transport and within cages * Code: Unique identifier that incorporates sample point, cage, and fish number * Cortisol: Concentration of cortisol in ng of cortisol per gram of fish mass * Correction factor: Multiplier used to correct cortisol values based on an updated cortisol calculation method  * Cortisol_corrected: Updated concentration of cortisol in ng of cortisol per gram of fish mass using the correction factor * Location: Location where fish was released  #### File: Glucose_Data.xlsx **Description:** Excel file including the glucose concentrations of every fish sampled in the study  ##### Variables * Hours Since Release: Discrete time points when fish were sampled, same as hours since fish were released to cages  * Sample Point: Numeric identifier for the 10 sequential sampling points, 1:10 * Cage: Cage number that the specific fish belonged to * Fish number: Numeric identifier for the individual fish * Density: The total density of fish experienced by that specific fish during transport and within cages * Code: Unique identifier that incorporates sample point, cage, and fish number * Glucose: Concentration of glucose in ug of glucose per gram of fish mass * Location: Location where fish was released  #### File: DS_SEM_Final5.csv **Description:** Excel file that includes data from analyzed SEM images of gill ionocytes necessary to calculate the surface area of the cells.  ##### Variables * Sample_ID: Individual ID applied to an image of an ionocyte cell used for analysis. All data from the image used in our analysis is provided in the proceeding columns. * Fish_ID: The ID of the fish that the cell belongs to * Cage: The cage that the fish that the image belongs to was collected from  * Density: Density of the cage that the fish that the image belongs to was in * Time_Point: Time point that the fish that the image was collected from was collected * Hours_After_Release: Discrete time point when the fish that the image belongs to was sampled, same as hours since fish were released to cages  * Filament: Gill filament upon which the image was taken * Replicate: Replicate analysis of the same image * Blind_ID: Blind ID given to image to avoid bias during analysis * Location: Location that the fish that the image was collected from was rereleased to * Blind_ID2: An additional Blind ID given to image to avoid bias during analysis * Mag: Magnification of SEM imaging * Area: Total area of the imaged cell * Perim: Total perimeter of the analyzed cell  * Avg_T1_Len: : Average length of the type 1 microvilli on the surface of the cell, analyzed using Image J * Avg_T1_Dia: Average diameter of the type 1 microvilli on the surface of the cell, analyzed using Image J * Avg_T2_Len: Average length of the type 2 microvilli on the surface of the cell, analyzed using Image J * Avg_T2_Dia: Average diameter of the type 2 microvilli on the surface of the cell, analyzed using Image J * Avg_T3_Len: Average length of the type 3 microvilli on the surface of the cell, analyzed using Image J * Avg_T3_Dia: Average diameter of the type 3 microvilli on the surface of the cell, analyzed using Image J * T1_Count: Total count of type 1 microvilli * T2_Count: Total count of type 2 microvilli * T3_Count: Total count of type 3 microvilli * T1_SA: Total surface area of type 1 microvilli * T2_SA: Total surface area of type 2 microvilli * T3_SA: Total surface area of type 3 microvilli * Avg_T1_Rad: Average radius of type 1 microvilli  * Avg_T2_Rad: Average radius of type 2 microvilli  * Avg_T3_Rad: Average radius of type 3 microvilli  * T1_side: Measurement of type 1 microvilli used to improve accurate estimation of surface area * T2_side: Measurement of type 2 microvilli used to improve accurate estimation of surface area * T3_side: Measurement of type 3 microvilli used to improve accurate estimation of surface area * Total_SA: Total SA of the ionocyte in the image calculated by combining Area, and T1, T2, and T3 SA Please note that some cells in DS_SEM_Final5.csv were left empty in columns pertaining to various microvilli type surface area measurements. This was done intentionally when no microvilli of that type were observed in the sample, to ensure that our model would not test for differences in microvilli surface area in types that were not present in the sample. Cells in the column "Location" for the Control samples (rows 710-738) were also left intentionally blank, as the factor "Location" did not apply for these samples. R treats all empty cells at n/a when the data is read in. Please note that in column "Sample_ID", .tif files are referenced as samples names. These represent the particular gill ionocyte images analyzed by the blinded readers. All associated data for each image is included in the .csv, and this data can be used to replicate all aspects of our data analysis. Request of specific images can be made to the corresponding author if desired.  ## Code/software All code used to analyze the attached data files are written in R. These scripts can be accessed and used by downloading the latest version of R, and if desired, R studio. Relevant packages needed are listed and loaded within each script.