Sperm adaptation in relation to salinity in three goby species

Received: 25 October 2020 Accepted: 4 April 2021 DOI: 10.1111/jfb.14749 FISH REGULAR PAPER Sperm adaptation in relation to salinity in three goby species | Jonathan Havenhand2,3 Kai Lindström1 Ola Svensson3,6,7 | Charlotta Kvarnemo3,6 | Erica Leder2,4,5 | Sofie Schöld6 | 1 Environmental and Marine Biology, Åbo Akademi University, Turku, Finland 2 Tjärnö Marine Laboratory, Department of Marine Sciences, University of Gothenburg, Strömstad, Sweden 3 Centre for Marine Evolutionary Biology, University of Gothenburg, Gothenburg, Sweden 4 Department of Biology, University of Turku, Turku, Finland 5 Natural History Museum, University of Oslo, Oslo, Norway 6 Department of Biological and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden 7 Department for Pre-School and School Teacher Education, University of Borås, Borås, Sweden Correspondence Kai Lindström, Environmental and Marine Biology, Åbo Akademi University, FIN – 20520 Turku, Finland. Email: [email protected] Funding information Assemble; Biotieteiden ja Ympäristön Tutkimuksen Toimikunta, Grant/Award Number: 136464; Vetenskapsrådet (SE), Grant/Award Numbers: 2016-03343, 621-2011-4004 Abstract In externally fertilizing species, the gametes of both males and females are exposed to the influences of the environment into which they are released. Sperm are sensitive to abiotic factors such as salinity, but they are also affected by biotic factors such as sperm competition. In this study, the authors compared the performance of sperm of three goby species, the painted goby, Pomatoschistus pictus, the two-spotted goby, Pomatoschistus flavescens, and the sand goby, Pomatoschistus minutus. These species differ in their distributions, with painted goby having the narrowest salinity range and sand goby the widest. Moreover, data from paternity show that the two-spotted goby experiences the least sperm competition, whereas in the sand goby sperm competition is ubiquitous. The authors took sperm samples from dissected males and exposed them to high salinity water (31 PSU) representing the North Sea and low salinity water (6 PSU) representing the brackish Baltic Sea Proper. They then used computer-assisted sperm analysis to measure the proportion of motile sperm and sperm swimming speed 10 min and 20 h after sperm activation. The authors found that sperm performance depended on salinity, but there seemed to be no relationship to the species' geographical distribution in relation to salinity range. The species differed in the proportion of motile sperm, but there was no significant decrease in sperm motility during 20 h. The sand goby was the only species with motile sperm after 72 h. KEYWORDS Gobiidae, Gobiusculus, salinity, sperm competition, sperm longevity, sperm motility 1 | I N T RO DU CT I O N Nissling et al., 2002; Nissling & Larsson, 2018; Nissling & Westin, 1997; Svensson et al., 2017), and can limit the distribution of To ensure maximal fertilization success, sperm need to be adapted to a species (Alavi & Cosson, 2006; Byrne et al., 2015; Green both abiotic and biotic factors. In external fertilizers, this means that et al., 2020; Svensson et al., 2017). In species with a geographical dis- sperm must survive and perform in the aquatic environment into tribution covering a range of salinities, or for estuarine and intertidal which they are released during mating. Salinity is a key abiotic factor species that routinely experience salinity fluctuations, individuals can affecting the viability and function of sperm in many organisms includ- be expected to show generally broad salinity tolerances (Tiersch & ing echinoderms (Allen & Pechenik, 2010), amphibians (Byrne Yang, 2012; Yang & Tiersch, 2009). Conversely, if gene flow is et al., 2015) and fish (Elofsson et al., 2003a; Elofsson et al., 2003b; restricted, subpopulations may be locally adapted to prevailing salinity This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles. J Fish Biol. 2021;1–7. wileyonlinelibrary.com/journal/jfb 1 2 LINDSTRÖM ET AL. FISH Upon release, sperm are activated by physiological reactions initi- levels (DeFaveri & Merilä, 2014; Serrao et al., 1996; Svensson et al., 2017). ated by the surrounding medium, often a change in osmolality, which Sperm adaptation is also strongly affected by a species mating is largely determined by salinity (Alavi & Cosson, 2006). Given the system. Sperm competition occurs when sperm from two or more large differences in geographical range among these three goby spe- males simultaneously try to fertilize the same group of eggs cies, this study's authors hypothesize that sperm performance in dif- (Parker, 1970), and it is common in species with both internal and ferent salinities will depend on the range of salinities encountered in a external fertilization (Birkhead & Møller, 1998; Fitzpatrick, 2020). species' natural geographical distribution. In this study, they test this When sperm competition is common, it is expected that selection will prediction, predicting the painted goby to be least tolerant to salinity promote sperm traits that improve fertilization success, such as variation, the two-spotted goby intermediate and the sand goby to be increased longevity after release (Birkhead & Møller, 1998; most tolerant to salinity variation. Furthermore, it is predicted that Snook, 2005). The authors of this study therefore might expect that in sperm velocity and longevity will be affected by risk of sperm compe- systems where risk for sperm competition via parasitic spawning (i.e., tition (Liao et al., 2018). Based on existing information regarding multi- sneaking and other nest intrusions aiming to fertilize eggs; ple paternity for two of the three species, the authors therefore Taborsky, 1994) is high, not only sperm expenditure (Stockley predict that sand gobies have faster swimming and longer living sperm et al., 1997), but sometimes also sperm longevity should be higher than two-spotted gobies. (Fitzpatrick et al., 2009). Experimental and observational results on this are, however, somewhat variable (Morita et al., 2014), and recent studies show that different sperm motility traits could determine fer- 2 | M A T E R I A L S A N D M ET H O D S tilization success (Lymbery et al., 2018). In this study, the authors compare sperm performance (propor- All fish were caught at the Swedish west coast close to the tion of motile sperm and sperm swimming speed) with respect to Kristineberg Marine Research Laboratory, 28–29 May 2012. Sand salinity and levels of sperm competition in three different species of gobies and painted gobies were caught from shallow and sandy areas gobies, the painted goby, Pomatoschistus pictus (Malm, 1865), the (Bökevik and Smalsund) using a beach seine, whereas two-spotted two-spotted goby, Pomatoschistus flavescens (Fabricius, 1779), and gobies were caught from a habitat with more macroalgae (next to the sand goby Pomatoschistus minutus (Pallas, 1770). Of the three, the Råttholmen) using hand-nets while snorkelling. Natural salinity in this painted goby has the most restricted salinity distribution and is found area varies, but a long-term average, measured within 0–20 m, is 26.2 in full salinity waters from the Mediterranean to the North Sea but is PSU (min–max 7.3–34.5 PSU) (Table S1 in Leder et al., 2021). not found east of Öresund in the brackish waters of the Baltic Sea. The fish were sorted according to sex and species and kept in The two-spotted goby is similarly distributed along the coasts of the 50 l storage tanks overnight, before they were used in the study. All North Sea and also extends into the Baltic Sea to the south coast of storage tanks had a through-flow of seawater pumped directly from Finland where the salinity is only 5–6 PSU. The sand goby exhibits the adjacent bay at a depth of 7 m, with a temperature of 13.5 ± 1.7 C the widest geographical distribution in relation to salinity. It occurs (mean ± S.D.) and salinity of 23.0 ± 3.1 PSU. The bottom of each tank and breeds along most of the European coastline, from the Mediterra- had a 3 cm layer of sand for the fish to burrow in. The fish were fed a nean Sea to northern Norway, and the entire brackish water of the mix of frozen chopped mussel meat, brown shrimp and Alaska Baltic Sea, including its northernmost reaches (Bothnian Bay) where Pollock. salinity is only 3–5 ppm (Kullander et al., 2012; Miller, 1986). The sand Eight painted goby males, seven two-spotted goby males and goby also reproduces in estuaries and the tidal zone where it can be eight sand goby males, all in breeding colouration, were included in exposed to a range of salinities (Miller, 1984). the study. Each male was decapitated, pithed and dissected immedi- All three study species use a range of nesting substrates to which ately thereafter. From each male a sperm sample was obtained for females attach eggs. The sand and painted gobies build nests by exca- analysis of proportion of motile sperm and sperm velocity (swimming vating cavities with a single nest opening underneath empty bivalve speed) immediately after sampling, and for analysis of longevity (pro- shells, which they also cover with sand leaving a single narrow nest portion of motile sperm after 20 h), in high and low salinity, as opening (Bouchereau et al., 2003; Hesthagen, 1979). In contrast, the described below. two-spotted goby does not cover its nest and often has a completely Before dissection, two tubes were prepared for each male, with open nest in an empty shell or a fold of macro algae 750 μl of either high salinity water (31 PSU, made from filtered natural (Amundsen, 2018). The male remains with the nest and guards and sea water, pumped from 32 m depth) representing the North Sea or cares for the eggs until hatching. Based on DNA analysis of eggs from low salinity water (6 PSU, made from the same water as above, but nests collected in the field, it is known that multiple paternity is rela- diluted with distilled water to the desired salinity) representing the tively rare in the two-spotted goby (Mobley et al., 2009; Monroe brackish Baltic Sea Proper. These tubes were kept in a rack sur- et al., 2016), but common in the sand goby (Jones et al., 2001a; Jones rounded by water of 14.0 ± 0.5 C. et al., 2001b), indicating a higher risk of sperm competition for nest The motility assay followed that of Havenhand and Schle- holding males of the latter species. For the painted goby, the gel (2009). Briefly, one testis was placed in an Eppendorf tube, the corresponding information does not yet exist. tissue was ruptured using a scalpel and 20 μl of Atlantic cod LINDSTRÖM ET AL. 3 FISH calcium-free Ringer's solution at pH 7.5 (150 mM NaCl, 5.2 mM KCl, and logit link function to analyse the proportion of motile sperm. VCL 1.8 mM MgSO4, 7.0 mM NaHCO, Karila was averaged over the technical replicates and then analysed using et al., 1993) was added. The tube was closed and shaken quickly by generalized linear mixed model with a gamma probability distribution hand. From this solution, 8 μl was suspended into the tube with high with log link function. 1.9 mM NaH2PO4; salinity water and 8 μl into the tube with low salinity water. The sus- The effect of time (longevity) on the proportion of motile sperm pension was mixed by turning the tubes upside down twice. A 40 μl and VCL for the three species was analysed using a repeated measures drop of the sperm suspension was placed between an albumin-coated design with times 10 min and 20 h as the within subjects factor and microscope slide and coverslip, separated by a 0.75 mm thick O-ring. species as the between subjects factor. The proportion of motile sperm Sperm movement was recorded for 0.5 s at the midpoint of the drop, was analysed using a generalized linear mixed model with a binomial at 30 frames per second, using a digital video camera (PixelLink 700 series, probability distribution and logit link function following the procedure PixeLink) mounted on an inverted microscope (Leica DM-IL, Leica Micro- described above. VCL was analysed using generalized linear mixed systems). Pilot experiments showed that illumination by the microscope model with a gamma probability distribution with log link function. lamp had no impact on the temperature inside the drop during videotaping For all analyses, the authors used Satterthwaite approximation (time of slide on microscope: c. 10 s). Nine technical replicates were taken for degrees of freedom because they had different sample sizes for from each combination of species and salinity, always with three samples the different treatment groups. All analyses were performed in on each of three microscope slides. All recordings were performed within SPSS (v 26). 30 s after placing the sample on the slide, and within 10 min of dissection. Pilot tests indicated that for all three species sperm longevity was exceptionally long. To assess this, the authors resampled each tube with (a) 8 0.3 They still had sufficient sperm suspension left in a few tubes for a third sampling after 72 h. Despite this data set being incomplete, these results are included in the current study to illustrate an exceptionally long sperm longevity found in one of the species. The tubes with sperm suspensions were kept in 14.0 ± 0.5 C water between the sampling times. Videos were post-processed and analysed with ImageJ using the Prop. moঞle sperm three to six 40 μl technical replicates per male and salinity after c. 20 h. computer-assisted sperm analysis (CASA) plugin (Wilson-Leedy & 0.2 8 7 0.1 8 0.0 Painted goby Ingermann, 2007). From the videos (see species-specific CASA settings in Supporting Information Table S1), the authors determined the 120 8 1 . The authors also determined the swimming 100 speed of the sperm, measured as the curvilinear path velocity (VCL), Data for these metrics were highly correlated (all r > 0.935, below. VCL was chosen as it is a standard metric for measuring sperm swimming speed as this best represents the speed of sperm along the swimming path and is a close proxy for fertilization success (Gage et al., 2004; Purchase & Moreau, 2012). Longevity was measured as proportion sperm that were motile (moving faster than 15 μm s 1 ) after 10 min, 20 and 72 h. The design to study the effect of salinity on the proportion of motile sperm and VCL follows a repeated measures design because individual sperm samples were divided between the two salinity treatments (high and low). Therefore, salinity was the within subjects factor, and species was the between subjects factor. For the analysis of the proportion of motile sperm, the authors summed up the number of motile sperm in the technical replicates to provide the target variable and the total number of sperm in the technical replicates to provide the number of trials variable. The authors then used a generalized linear mixed model with a binomial probability distribution VCL the straight line velocity (VSL) and the average path velocity (VAP). P < 0.001), and therefore the authors only include analyses of VCL Sand goby (b) currents, sperm were classified as swimming only if velocity was higher than 15 μm s Two-spo‚ed goby Species proportion of motile sperm relative to the total number of sperm in the video clip. To exclude non-swimming particles drifting in micro- 8 7 80 60 7 8 8 8 7 40 20 Painted goby Two-spo‚ed goby Sand goby Species F I G U R E 1 Box and whisker plots of sperm motility and swimming speed in relation to salinity in the painted goby (Pomatoschistus pictus), two-spotted goby (Pomatoschistus flavescens) and sand goby (Pomatoschistus minutus). (a) Proportion of motile sperm after 10 min for the three goby species in full marine water (31 PSU, white bar) and diluted marine water (6 PSU, grey bars). (b) Curvilinear path velocity (VCL) after 10 min for the three goby species in full marine water (white boxes) and diluted marine water (grey boxes). The box encloses the 25th and 75th percentiles with the median indicated by the horizontal line. The whiskers indicate minimum and maximum values. Numbers above boxes indicate sample sizes 4 LINDSTRÖM ET AL. FISH | 2.1 Ethical statement sand goby (Figure 1a) creating the above-mentioned interaction and resulting in significant differences among the species (species effect, The number of fish killed was kept to a minimum. This study was per- F2,32 = 7.76, P = 0.001). The proportion of motile sperm in painted formed under Swedish ethical permits 135-2010 and 143-2012. and two-spotted goby was very similar (for model parameter estimates, see Supporting Information Table S2). The three species exhibited a difference in curvilinear path veloc- 3 | RESULTS ity (species effect, F2,37 = 45.50, P < 0.001) with the sand goby having the fastest-moving sperm (Figure 1b). Salinity again had a strong and 3.1 | The effect of salinity similar effect on velocity in all three species (salinity effect, F1,37 = 202.70, P < 0.001; Figure 1b). There was no significant inter- The response to salinity in the proportion of motile sperm differed sig- action effect, F2,37 = 2.02, P = 0.15; for model parameter estimates, nificantly among species (generalized mixed linear model: species by see Supporting Information Table S3). salinity interaction effect, F2,32 = 4.76, P = 0.016). In all species, the proportion of motile sperm was higher in 31 PSU (salinity effect, F1,31 = 16.78, P < 0.001; Figure 1a), and this effect was biggest in the | 3.2 Sperm longevity There were large differences among the species in the overall proportion of motile sperm (species effect, F2,30 = 17.45, P < 0.001; (a) Prop. moঞle sperm Figure 2a). All three species had motile sperm after 20 h, but there 8 0.3 was no significant effect of time on the proportion of motile sperm (time effect, F1,31 = 0.97, P = 0.332; interaction effect, F2,30 = 1.95, 0.2 P = 0.160; Figure 2a). In the sand goby, live sperm could still be 8 7 5 8 0.1 painted goby sperm were motile at that point, although the sample 2 2 sizes for these species were very small (Figure 2a; for model parameter estimates, see Supporting Information Table S4). 0.0 Painted goby Two-spo‚ed goby Sand goby Species The curvilinear path velocity differed among the species (species effect, F2,29 = 19.36, P < 0.001; Figure 2b), but there were no significant effects of time (F1,29 = 3.22, P = 0.083) or of the interaction between species and time (F2,29 = 0.33, P = 0.724; Figure 2b; for (b) model parameter estimates, see Supporting Information Table S5). 120 8 100 7 7 7 VCL observed after 72 h, although the proportion of motile sperm was markedly lower than after 10 min and 20 h. No two-spotted or 7 7 80 8 4 8 60 | DI SCU SSION The authors found clear differences in sperm motility and velocity among the three goby species. The sand goby tended to have the 40 highest values for all traits, especially in high salinity, which is the salinity level most similar to the marine location from which they 20 Painted goby Two-spo‚ed goby Sand goby Species F I G U R E 2 Box and whisker plots of longevity of sperm in the painted goby (Pomatoschistus pictus), two-spotted goby (Pomatoschistus flavescens) and sand goby (Pomatoschistus minutus). (a) Proportion of motile sperm after 10 min (white boxes), 20 h (grey boxes) and 72 h (dark grey boxes) for the three goby species in full marine water (31 PSU). (b) Curvilinear path velocity (VCL) after 10 min (white boxes) and 20 h (grey boxes) for the three goby species in full marine water (31 PSU). The box encloses the 25th and 75th percentiles with the median indicated by the horizontal line. The whiskers indicate minimum and maximum values. Numbers above boxes indicate sample sizes were caught. It is especially noteworthy that for all three species the proportion of motile sperm showed no or very slight decrease after 20 h. Even after 72 h, the authors found live sperm in the sand goby. In most fish sperm are reported to have a much shorter longevity, on the order of seconds to tens of minutes (Browne et al., 2015), but gobies appear to be an exception. For example, in the marine dusky frillgoby, Bathygobius fuscus sperm have been reported to have longevities of nearly 10 h (Nakanishi & Takegaki, 2019), and sand goby sperm still alive after 24 h has been documented using live/dead cell staining techniques (Green & Kvarnemo, 2019). The sand goby has the widest distribution occurring along Europe's coast (Miller, 1986) including full marine and low salinity areas. Low salinity areas include estuaries and the brackish Baltic Sea, LINDSTRÖM ET AL. FISH 5 where it occurs and breeds in near fresh water (Wiederholm, 1987). If no indication of such an effect as in all cases motility and speed were the range of salinities encountered by a species is reflected in the lower in the low salinity treatment, but the experiment of this study range of salinities its sperm can tolerate, then one would expect was not designed to test this particular observation. the sand goby to show sperm traits that are least affected by the In gobies sperm competition is often manifested as multiple salinity treatment. Similarly, the painted goby should exhibit the paternity arising from parasitic spawning in a single male's nest (Jones highest sensitivity to salinity because it has the most narrow salinity et al., 2001b). The authors found that the sand goby had the highest distribution, whereas the two-spotted goby should be intermediate sperm motility and velocity (and sperm still alive after 72 h), traits that (Kullander et al., 2012). Nonetheless, the results of this study indicate are argued to be shaped by sperm competition (Fitzpatrick, 2020; that there are no clear differences among the species in the degree to Snook, 2005). Parasitic spawning is common in the sand goby (Jones which salinity tolerance is related to the salinity ranges covered by et al., 2001b; Singer et al., 2006). In contrast, the two-spotted goby the species distribution. All three species showed a higher proportion had much lower sperm motility and velocity, and this species is known of motile sperm and higher sperm velocity in the high salinity treat- to have very low frequencies of multiple paternity suggesting low ment. A possible explanation for this result is that the activation of sperm competition levels (Mobley et al., 2009; Monroe et al., 2016). sperm is a plastic trait, as found, e.g., in three spine stickleback, Therefore, the ejaculate traits found in this study support the expecta- Gasterosteus aculeatus Linnaeus, 1758, in which the developmental tion of sperm competition theory and the authors' predictions. A environment determines the conditions under which sperm are acti- definitive weakness of this study is the lack of information about mul- vated (Taugbol et al., 2017). The authors of this study therefore sug- tiple paternity frequencies in the painted goby. Nonetheless, based on gest that in the two species (sand and two-spotted goby) that have the sperm trait findings of this study resembling the two-spotted invaded low salinity environments from a marine ancestry, the sperm goby, the authors predict that parasitic spawning and sperm competi- responses to salinity have evolved through local adaptation rather tion in this species are relatively rare. than facilitated through exaptation. For the sand goby there is further Another possible reason for long-lived sperm is a need to fertilize evidence to support this conclusion, as males sampled from a low eggs over an extended time (Scaggiante et al., 1999). Egg laying in salinity site (3 PSU at Härnösand in the Bothnian Bay) showed gobies generally is a slow process where the female attaches her eggs markedly better sperm performance in low than in high salinity to the nest substrate one at a time, sometimes resulting in a total (Leder et al., 2021; Svensson et al., 2017) and show strong genome spawning time of several hours (Marconato et al., 1996). For example, differentiation along the salinity gradient between these sites (Leder in the sand goby a female needs an hour or more to deposit her egg et al., 2021). clutch (CK, KL, OS, pers. obs.). The sand and painted gobies build Sticklebacks are an interesting group of fish for comparison to the closed cavities as nest sites, which may aid in keeping the sperm near authors' findings on the gobies. Three spine sticklebacks are widely fertilizable eggs for an extended time as compared to free spawning distributed, in both marine and freshwater habitats. Three spine stick- species (Liao et al., 2018; Morita et al., 2014) or species spawning in lebacks that are sympatric to the gobies studied here show much turbulent water (Neat et al., 2003). A successful sand goby male can shorter sperm longevity in low salinity (Elofsson et al., 2003a). The mate with several females in sequence (Jones et al., 2001b). In a 15 spine stickleback, Spinachia spinachia (Linnaeus, 1758), extensively closed nest cavity, a single long-lived sperm ejaculation could remain overlaps the distribution of the two-spotted goby and is similarly in the nest and fertilize eggs from several females. Recently, in the limited by minimum salinity (Gross, 1978). Fifteen spine stickleback dusky frillgoby with similar nesting behaviour as the sand goby, nest sperm are more sensitive and short-lived in brackish water (Elofsson guarding males were shown to use tail fanning to remove sperm from et al., 2003b) than three spine stickleback sperm. The authors found other males (Takegaki et al., 2020). Furthermore, in many gobies males the same pattern in the gobies, the species with a more salinity prepare the spawning surface with a sperm containing mucus trail restricted distribution showing more limited sperm survival at lower from which new sperm are activated over time, as the mucus dissolves salinities. (Marconato et al., 1996; Scaggiante et al., 1999; Svensson & Salinity tolerance of sperm is likely to reflect a species' ability to Kvarnemo, 2005), creating a further reason for increased longevity of invade novel environments (Green et al., 2020), and the fertilizing ability sperm. If extended sperm longevity is adaptive, then the authors pre- of sand goby sperm is a function of a male's native salinity (Svensson dict that it will be correlated with the nest structure and the mating et al., 2017). In the current study, the proportion of motile sperm was system of the species. Specifically, in mating systems where success- always lower in the low than the high salinity treatment, suggesting ful males spawn with many females sequentially, and in relatively that the sperm performance of all three species was indeed better enclosed microenvironments, the authors expect sperm longevity to adapted to high salinity. Given that all three species are of marine ori- be extended (Morita et al., 2014). gin, and the sampled fish came from a marine site, this is perhaps not surprising. It is thought that sperm of marine fish species are activated AC KNOWLEDG EME NT S upon exposure to a hyperosmotic medium (Morisawa, 1985), but in This study was performed within the Centre for Marine Evolutionary some cases, it has been shown that the life span of sperm is longer if Biology at the University of Gothenburg (https://www.gu.se/en/ the water is slightly diluted as compared to water of native salinity cemeb-marine-evolutionary-biology), supported by a Linnaeus-grant (Groison et al., 2010; Lahnsteiner & Patzner, 1998). The authors found from the Swedish Research Councils VR and Formas (grant nr 6 FISH 217-2008-1719). Additional funding was given by the Swedish Research Council VR (LK, 2016-03343 & 621-2011-4004), the Academy of Finland (EL, grant nr 136464) and the ASSEMBLE programme (KL and EL). 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