DOI: 10.5061/dryad.x3ffbg7rs
title: Genotypic data from: Lab-based evaluation of the reproductive performance of trojan (MYY) brook trout (Salvelinus fontinalis)

description: Evaluating the efficacy of the use of trojan male brook trout with two Y
 chromosomes (MYY) requires a better understanding of reproductive
 performance. We measured the reproductive performance of hatchery age-0
 and age-1 MYY brook trout compared to hatchery XY males using laboratory
 crosses. Offspring of XY males had higher survival than offspring of age-1
 MYY one day post-fertilization but not offspring of age-0 MYY. We found no
 detectable differences in survival from eyed-egg to the juvenile-fry
 stage. However, size-at-age differed, where offspring of age-0 MYY were
 3.6% smaller in length and 25.2% smaller in weight than those of XY males.
 For crosses fertilized by both MYY and XY males, we found that a
 significantly higher proportion of offspring within families were sired by
 MYY versus XY males. These results show, under controlled conditions,
 evidence for possible fitness advantage for MYY under sperm competition,
 but a possible fitness disadvantage associated with early growth of their
 offspring. Overall, our results hold promise for the use of MYY brook
 trout to serve as an effective eradication tool. 
description: M<sub>YY</sub> brook trout were obtained as eyed-eggs
 from Hayspur Hatchery (Idaho) and reared at Abernathy Fish Technology
 Center. Females were assessed for ripeness weekly to determine when to
 begin spawning. Females that were considered ripe (eggs readily released
 when light pressure was applied to the abdomen) were then placed into a
 separate holding area to prepare for spawning. Before spawning, all three
 male fish (XY male, age-0 M<sub>YY</sub>, and age-1
 M<sub>YY</sub>) to be used for each cross were anesthetized
 using MS-222, then weighed (g) and measured for fork length (mm). The
 female was euthanized using MS-222, wiped dry, and a ventral incision was
 made and all eggs were manually expelled into a colander to remove excess
 fluid. The overall weight (g) of the eggs was determined, and eggs were
 divided into four evenly weighted groups and placed into four large Petri
 dishes. Each group was then fertilized using milt from one male type: XY
 male (cross-type 1), age-0 M<sub>YY</sub> (cross-type 2),
 age-1 M<sub>YY</sub> (cross-type 3), or a mixture of milt from
 the XY and age-0 M<sub>YY </sub>(cross-type 4). For each
 female, we randomized the order of fertilization across each cross-type.
 The collection of milt and fertilization of eggs for each cross was as
 follows: One male was randomly chosen, anesthetized, and wiped dry. Milt
 was then expressed into a Petri dish, collected using a 200 to 1000µL
 pipette, and deposited directly into the Petri dish containing the eggs
 for that cross. We standardized milt volume, but not sperm concentration,
 for each cross-type for each female by determining the male of the three
 male types with the smallest total volume of milt and halving it. For
 cross-type 4, the same volume of milt from the XY and age-0
 M<sub>YY</sub> used for each of the other crosses for that
 female was combined within a separate 0.5 mL microcentrifuge tube and
 gently mixed using the pipette. Half of that mixture was then used to
 fertilize the eggs so that the same volume of milt was used to fertilize
 each egg group for each female. For this cross-type, we chose to combine
 milt between the two male types before fertilization to increase the
 effects of sperm competition (Compton 2004). Once milt was added to all
 groups, water was poured onto each cross-type, then gently mixed and
 allowed to fertilize for 10 minutes. After fertilization, all males were
 euthanized. Tissue samples were collected from all adults, including
 females, and stored in 95% ethanol for subsequent parentage analysis. 
 Fertilized eggs were placed in four circular screened rearing vessels (one
 for each cross-type) within Heath trays, such that each tray contained the
 four crosses for a single female. This procedure was
 replicated across all 43 females and 43 males that were spawned so that a
 separate male of each type was used to fertilize one cross-type for each
 female. Each Heath stack was supplied with ~12<sup>o </sup>C
 well water at a flow rate of 11.4 L per minute. The placement of
 individual rearing vessels within each Heath tray was randomized by
 cross-type to account for potential variability in flow from front to
 back. Twenty-four hours after fertilization, unfertilized eggs were
 removed and enumerated.  Early Rearing
 Developing embryos were examined daily, and mortalities were
 removed and enumerated. At the eyed-egg stage, eggs were physically
 shocked by dropping them into a 600 mL glass beaker, and non-viable eggs
 were removed twenty-four hours later. Surviving eggs from cross-type 4
 were incubated until the eyed-egg stage (Figure 1), whereupon eggs were
 removed and preserved in 95% ethanol for subsequent dissection and
 parentage analysis. Surviving eggs from cross-type 3 (age-1
 M<sub>YY</sub> sire) were incubated until hatching (Figure 1)
 and were then removed and enumerated. Eggs from cross-types 1 (XY) and 2
 (age-0 M<sub>YY</sub> sire) were incubated until the swim-up
 fry stage with continued daily checks for non-viable eggs. Once the
 swim-up fry stage was reached (after yolk-sac depletion), individuals from
 crosses 1 and 2 were transferred into 57 L fiberglass tanks supplied with
 12°C well water at 1.9 L per minute. The fry was then fed a daily ration
 of 3.5% of body weight (biomass)/day. Tanks were monitored and cleaned
 daily, and all mortalities were removed and enumerated. After
 approximately one month (post swim-up fry stage), we standardized fish
 density by selecting a random sample of 80 individuals from each cross
 (i.e., tank) for continued rearing. The remaining fish within each cross
 (in excess of 80) were removed, euthanized with an overdose of MS-222, and
 enumerated. The 80 individuals remaining in each tank were reared to the
 juvenile-fry stage (Figure 1) with continued daily tank cleaning and
 mortality checks. At the juvenile-fry stage, all fish were removed from
 tanks and euthanized with an overdose of MS-222. All crosses were then
 photographed for subsequent measurement of length using the ImageJ
 software package (Schneider et al. 2012). We also weighed (g) each
 offspring for a subset of six of the crosses: cross-types 1 and 2 from
 three randomly chosen females. Survival and Growth
 Analysis Survival was calculated for six stages of
 development: 1-day post-fertilization (1 dpf; the measure of fertilization
 success), eyed-egg (30 dpf), hatch (40 dpf), swim-up fry (65 dpf), post
 swim-up fry (105 dpf), and juvenile-fry (150 dpf). For survival 1-day
 post-fertilization, we were unable to differentiate between mortalities
 that occurred post-fertilization and eggs that were unfertilized.
 Therefore, we classified all dead eggs as eggs that were unfertilized and
 then used the surviving eggs as a measure of fertilization success. For
 cross-types 1 and 2, survival to each stage was calculated. For cross-type
 3, only survival to 1 day post-fertilization, the eyed-egg stage, and
 hatch stage were calculated. This was due to limited space for
 post-hatching rearing vessels within the laboratory, and individual
 families for cross-types 1 and 2 needed to be reared separately after
 hatching. Survival to the first five stages of development was calculated
 by determining the number of individuals per cross at fertilization, then
 subtracting the number of mortalities observed to the designated
 developmental stage (Figure 2). We then calculated the proportion survival
 of each cross to each stage by taking the survival of each cross at each
 life stage and dividing by the total number of individuals per cross at
 fertilization. Survival to juvenile-fry stage for cross-types 1 and 2 was
 determined by subtracting the number of individuals that survived to 150
 days post-fertilization from the number of individuals at post-swim-up fry
 stage (determined upon subsampling to 80 fish per cross). We then
 calculated the proportion survival of each cross to the juvenile-fry stage
 by taking the total number survived for each cross at the juvenile-fry
 stage and dividing it by the total number of individuals per cross at the
 post-swim-up fry stage. Differences in the proportion
 of offspring survival to each developmental stage for cross-types 1-3 were
 analyzed using a Bayesian model with a beta-binomial distribution in Stan
 (Stan Development Team 2023) using the package "brms" in R
 (Bürkner 2018) with the female as a random intercept. Four parallel chains
 were run. The number of MCMC iterations was set at 5,000 and burn-in was
 set at 1,000. All priors were drawn from uniform distributions.
 Convergence between chains was assessed using the statistic Ȓ (Brooks and
 Gelman 1998) where Ȓ values of less than &lt; 1.01 suggest appropriate
 convergence of the model (Gelman et al. 2014). Growth
 differences at the juvenile-fry stage were examined by comparing length
 (mm) between all offspring of XY and all offspring of age-0
 M<sub>YY</sub> males and comparing weight (g) for the subset
 of six crosses using a glmm. For the growth analyses, the total number of
 individual juvenile-fry within each cross was used as a random intercept
 to account for the density of tanks with either length or weight as the
 dependent variable and cross-type as a covariate. All glmm’s were fit
 using the R package <em>glmmTMB</em> (R v 4.1.1). 
 Parentage of Milt-Mixture Crosses (Cross-Type 4)
 The parentage of cross-type 4 offspring was determined through
 genetic analysis of eyed-eggs. We allowed offspring from cross-type 4 to
 develop to the eyed-egg stage to ensure we would be able to successfully
 extract enough embryonic DNA to determine parentage. DNA was extracted
 from 100 randomly chosen eggs from each cross using a modified version of
 the extraction method found in Ali et al. (2016). Extracted DNA was then
 sent to the Idaho Fish and Game (IDFG) Eagle Fish Genetics Lab for library
 preparation and sequencing analysis. Library preparation was completed
 following the GTSeq approach developed by Campbell et al. (2015). GTSeq
 was performed for each individual using an established 240 GTSeq SNP
 panel. This SNP panel has been used to determine the sire type (either XY
 or M<sub>YY</sub>) of offspring sampled from experimental
 systems into which M<sub>YY</sub> brook trout have been
 released (Kennedy et al. 2018). Screening of
 SNPs We conducted a locus-specific analysis of
 conformation to Hardy–Weinberg proportions in a companion brook trout
 population genomic analysis (Davenport et al. <em>in
 prep</em>). Loci that showed significant deviation
 (<em>P</em> &lt; 0.05) from HW proportions across large
 numbers of population samples (≥ 7) in this analysis were removed (Waples
 2015). We did not screen for linkage disequilibrium among loci as that is
 not an assumption of exclusion-based parentage assignments.
 Parentage Estimation for Milt-Mixture Crosses
 The parentage of cross-type 4 offspring at the eyed-egg stage was
 estimated from genotypes based on the exclusion approach implemented by
 the R package <em>Hiphop</em> (Cockburn et al. 2020). Adults
 were excluded from possible parentage if the offspring possessed two
 identical copies of alleles at a given locus and adults possessed two
 identical copies of the alternate allele at that same locus (i.e. they
 were alternate homozygotes) or if both possible parents were homozygous
 for the same allele while the putative offspring was heterozygous at that
 locus. Each cross was examined separately, and the putative parent with
 the fewest number of mismatches was the assigned parent. We expected high
 accuracy of parent assignments for each cross because the female parent
 was known and there were only two possible male parents (the XY and
 M<sub>YY </sub>used<sub> </sub>for that cross).
 Differences in the proportion of offspring that were assigned to an XY
 compared to an M<sub>YY</sub> were tested using a
 beta-binomial glmm (Gelman and Hill 2007) in R with sire type as the fixed
 effect and female as a random intercept.
description: # Genotypic data from: Lab-based evaluation of the reproductive
 performance of trojan (MYY) brook trout (*Salvelinus fontinalis*)
 [https://doi.org/10.5061/dryad.x3ffbg7rs](https://doi.org/10.5061/dryad.x3ffbg7rs) Genotypic data of the offspring as well as potential parents (both male and female) for mixture crosses (Cross-type 4) spawned. Includes individual ID, female parent ID, percent of genotype that was successfully extracted, and 240 loci with 1 column per locus. ## Description of the data and file structure Variables: * FieldID1- unique individual identifier * Age- Young-of-year (YOY) or adult * sex- sex of adult (missing for YOY) * Female #- the ID number of the female parent (missing for adult individuals) * GenotypeSuccess- percent of genotype that was successfully extracted * Columns F-IK - individual loci with 1 column per locus Missing data =0 ## Sharing/Access information NA ## Code/Software NA