DOI: 10.5061/dryad.k6djh9wc9
title: Male sexual signaling and expected effects of hatchery-induced sperm competition vary with water depth at which whitefish are caught

description: Salmonids like whitefish (Coregonus spp.) are often propagated in
 supportive breeding. Spawners are caught from their spawning locations,
 their gametes mixed, and the resulting offspring reared in a protected
 environment before being released into the wild. This procedure can affect
 sexual selection, for example, by enhancing the importance of sperm
 competition or by reducing the relevance of sexual signals. While it is
 often unclear how sperm competitiveness is affected by a male’s overall
 genetic quality, there is accumulating evidence that sexual signals reveal
 good genes and that mate choice based on such signals can increase
 offspring viability (Auld et al. 2019). Therefore, supportive breeding may
 affect the genetic variance and the mean genetic quality of next
 generations. We sampled whitefish from various locations along a depth
 gradient to test how male characteristics that are likely to affect sexual
 selection under natural conditions correlate with characteristics that
 affect hatchery-induced sperm competition. Whitefish are external
 fertilizers, and multi-male spawning and hence sperm competition is common
 under natural conditions. Mate choice is not sufficiently understood but
 could be based on breeding tubercles. These are small conical structures
 that grow on scales before the breeding sea- son and fall off shortly
 afterwards. The size of breeding tubercles varies much among males and has
 repeatedly been found to correlate positively with offspring viability
 (Wedekind et al. 2001; Keka ̈la ̈inen et al. 2010). Male dominance is
 typically depend- ent on body size (Auld et al. 2019) and could also be
 relevant in whitefish. Body size itself can reflect individual inbreeding
 coefficients (Su et al. 1996) and be an indicator of heritable genetic
 quality in small or structured populations (Neff and Pitcher 2008). In
 another fish with a somewhat comparable mating system, the size of
 breeding tubercles and male size was not correlated but could both be used
 to predict male reproductive success under close to natural conditions
 (Jacob et al. 2009). We study whitefish from Lake Hallwil (Switzerland).
 This lake has suffered so much from anthropogenic eutrophication that it
 is being artificially aerated since 1985. Three hatcheries around the lake
 are likely to have played a key role in maintaining the whitefish
 population, as concluded also from a recent mark–recapture experiment
 (Vonlanthen 2015). However, eutrophication combined with possible
 hybridization in hatcheries can have led to a speciation reversal
 (Vonlanthen et al. 2012) and may thereby have destroyed any genetic
 structure linked to water depth. Hatchery protocols now focus on
 maintaining over-all genetic variance by pooling milt of many males before
 adding the mix to eggs of multiple females. Milt volume varies among
 sires, for example, because males often lose milt when being pulled up
 from deep locations (Figure 1), an effect that likely depends on how much
 the swim bladder is inflated by the change in pressure. This variance in
 milt volume is likely to affect the genetic variance that, in combination
 with the average genetic quality, may then affect the long-term survival
 of a population. The extent to which hatchery protocols affect genetic
 quality can be estimated by the correlations between male quality
 indicators and traits that affect hatchery-induced sperm competition, that
 is, sperm number, velocity, and longevity (summarized here as “milt
 potency,” see also Supplementary Material). Many breeding protocols are
 likely to promote genetic quality if male attractiveness or dominance are
 positively correlated to milt potency. If there are no such correlations
 or negative ones because of life-history trade-offs, hatchery-induced
 sperm competition is likely to reduce the average genetic quality in
 future generations. We sampled fish from various depths and determined
 their age, size, breeding ornamentation, and milt potency (see methods in
 the Supplementary Material) to test whether and how different male
 characteristics affect reproductive success in supportive breeding in a
 heavily managed population.
description: Adult whitefish
 (<em>Coregonus suidteri</em>, n=104) were caught from Lake
 Hallwil (Switzerland; 47.2772° N, 8.2173° E) during their breeding season
 in January 2020 using gill nets set at 40, 25, 12, or 4 m depth along a
 line perpendicular to the bank (at decreasing distance to the shore,
 following the natural depth gradient). The nets were separated by
 distances of about 230, 50, and 40 m, respectively. At each depth, gill
 nets of 25, 27, 30, and 35 mm mesh size were used to ensure that adult
 whitefish of all size could be caught. Fish were immediately killed and
 kept in ice water until further processing. Standard body length (i.e.
 excluding the caudal fin) was determined, and scales were sampled above
 the lateral line from the caudal half of the body to later estimate fish
 age based on the number of annuli, a method offering same age estimates as
 methods based on otoliths or fin ray for fish younger than 5 years
 (Muir et al.
 2008). Milt was collected
 by applying gentle bilateral abdominal pressure (see below). Fish were
 then scanned with a 3D optical scanner to determine mean breeding
 tubercles volume (see below). For age estimation, scales were first cleaned
 using a cloth and 60% ethanol and then rinsed in water. Three scales by
 fish were mounted onto microscope slides and photographed under a 1.6x
 magnification. Fish were then aged based on the number of annuli, except
 for 1 female caught at 25 m depth for which we had no readable
 scales. The milt of 22 males caught at 12 m and 4 m
 depths was stripped into large Petri dishes (145 × 20 mm, Greiner Bio-one,
 Frickenhausen, Germany; the discrepancy between this sample size and total
 males sample size (n=71) was due to organizational problems in the field.
 Care was taken to strip the milt drop by drop and to separate theses drop
 on the Petri dish, so that all drops of milt that were non-contaminated
 with urine or faeces (i.e. usually all except the last drop) could be
 collected and individually pooled, 20 µL was stored at a dilution ratio of
 1:9 in Storfish (IMV Technologies, l’Aigle, France), an isotonic
 inactivating medium, and kept on ice. The remaining milt was centrifugated
 to collect seminal plasma. Because the weight of the seminal plasma turned
 out to be not correlated to the sperm concentration (r = 0.18, d.f. = 18,
 p = 0.46), it was used to calculate the “milt potency”, here defined
 as  milt potency = sperm concentration × weight of
 seminal plasma × average sperm velocity × maximum sperm longevity         
                                (1) 
 The milt stored in Storfish
 were transported to the laboratory where sperm velocity and concentration
 were analysed within 48 h with CASA using the Qualisperm software (AKYmed
 AG, Cheseaux-sur-Lausanne, Switzerland) as in Nusbaumer et
 al. (2019), except that milt traits could mostly be
 summarised as a mean over 4 trials rather than over 2 trials. Briefly, 20
 µL of each sample were activated in standardized water (OECD, 1992) at a
 1:500 dilution ratio and measured at 20x magnification under phase
 contrast, at 6.5°C, 20s post-activation. Sperm longevity was measured as
 the time by which no more sperm motion could be observed. Our final sample
 size consisted of 20 males because the within sample measurement
 repeatability was too low in 2 samples (i.e. the standard error of sperm
 velocity and/or concentration exceeded 50% of the mean measurement; in the
 accepted samples, the standard errors of sperm velocity and concentration
 were on average 9 and 18 % of the means,
 respectively).  The average size of breeding tubercles per fish
 was determined with a 3D optical scanner (VR-5000,
 Keyence, Itasca, IL,
 USA). Each fish was first
 carefully dried before applying a thin layer of baby powder (Millette Baby
 powder, Migros, Zurich, Switzerland) on the skin to make the otherwise
 transparent breeding tubercles detectable by the optical scanner. The fish
 was then placed on a slightly angled plate so that its lateral line was
 approximately perpendicular to the laser beams in order to facilitate
 correction of the fish curvature in later analysis. Scans were then
 analysed using Keyence analysis software 3.1.0.56, with
 <em>curvature correction strength</em> set at 15,
 <em>reference plane</em> set as
 <em>continuous</em>, and measurements extracted using the
 <em>conplane</em> <em>method </em>to obtain the
 volume of individual breeding tubercles. All scans were carefully checked
 for anything that could potentially be wrongly identified as a breeding
 tubercle by the software. Only 3 males and 4 females could be caught at 40
 m depth. This depth category was therefore excluded from all statistical
 analyses. Also, because age classes varied from 1+ to 4+, but 89.5% of all
 fish where either 2+ or 3+, the fish were categorized as “young” (1+ or
 2+, n=51) and “older” (3+ or 4+, n=44). Fisher’s exact test were then used
 for frequency analyses. An ANOVA was used to test for differences in body
 length between the catches. Because no differences in body length was
 found among the catches, a Gaussian generalised multiple regression
 analysis (GLM) was then used on the mean breeding tubercles volume as
 response variable to test the effects of depth at catch (specified as
 factor), body length, and the interaction of these two potential
 predictors (after graphically verifying that the model assumptions were
 not significantly violated). Analogous models were used to test whether
 depth at catch, body length, mean breeding tubercles volume, or any
 interaction between these factors would explain variance in mean sperm
 velocity and milt potency. The AIC of full models was compared to the AIC
 of models lacking a variable or an interaction and models with the lowest
 AIC were retained as final models. Wilcoxon rank sum tests were used on
 milt potency and components of milt potency to test whether they would
 vary between samples. The same method was used to test whether milt
 potency or components would vary between age categories. Analyses were
 done in RStudio 4.0.2 (R
 Development Core Team 2015) and JMP14.0.0 (SAS Institute Inc., Cary,
 NC). <strong><em>References</em>
 <em>cited</em></strong>
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 structures for lake whitefish in Lake Michigan: selecting an aging method
 based on precision and a decision analysis. N Am J Fish Manag 28:1928-1940.
 Nusbaumer D, Marques da Cunha
 L, Wedekind C, 2019. Sperm
 cryopreservation reduces offspring growth. Proc R Soc B Biol Sci
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