Salinity-induced phenotypic plasticity in threespine stickleback sperm activation
Phenotypic expression may be and often is influenced by an organism's developmental environment, referred to as phenotypic plasticity. The sperm cells of teleosts have been found to be inactive in the seminal plasma and are activated by osmotic shock for most fish species, through release in either hypertonic (for marine fish) or hypotonic (for freshwater fish) water. If this is the case, the regulatory system of sperm mobility should be reversed in salt- and freshwater fish. We tested this hypothesis by first activating sperm of salt- and freshwater populations of threespine stickleback in salt- and freshwater. The sperm from saltwater stickleback could be activated in either salinity, which matches the freshwater colonization history of the species, whereas the sperm from the freshwater population acted as predicted by the osmotic shock theory and was activated in freshwater only. As the freshwater population used here was calculated to be thousands of years old, we went on to test whether the trait(s) were plastic and sperm from freshwater males still could be activated in saltwater after individuals were exposed to saltwater. After raising freshwater stickleback in saltwater, we found the mature males to have active sperm in both saltwater and freshwater. Further, we also found the sperm of wild-caught freshwater stickleback to be active in saltwater after exposing those mature males to saltwater for only 2 days. This illustrates that the ability for stickleback sperm to be activated in a range of water qualities is an environmentally induced plastic trait.
Selection can favour flexible individuals or genotypes that are able to modify their development, physiology or behaviour in response to environmental cues, a phenomenon broadly referred to as phenotypic plasticity . Although phenotypic plasticity is usually thought of as adaptive, it can also be neutral or maladaptive with regards to individual fitness, depending on the environment in which the individual finds itself . Adaptive plasticity can affect evolutionary dynamics because it has the potential to promote colonization and rapid divergent evolution in newly founded populations . If the founding population persists and the environment is stable, these plastic traits might become genetically assimilated and expressed constitutively, rather than being plastic . This loss of plasticity, also called canalization, can then represent a reproductive barrier between populations, if the isolated populations are differently canalized.
It seems phenotypic plasticity could have played an important role in the evolutionary dynamics of the threespine stickleback, Gasterosteus aculeatus (hereafter stickleback). Originally a marine species, the stickleback successfully colonized many newly established freshwater systems at the end of the last glacial period . Most of these freshwater stickleback populations display evidence of parallel adaptation in shape and other morphological traits compared with the ancestral marine form, notably in the morphology of the bony lateral plates covering each side of the stickleback body, which are reduced in most freshwater populations . Yet, despite physical adaptation, all stickleback populations investigated thus far, not only those that are anadromous, have an extremely wide salinity tolerance, and can be exposed directly to a new salinity without inducing any known stress responses [6,7].
The possibility for gametic isolation has largely been overlooked in studies of adaptation to freshwater, and little is known about actual fertilization success in nature between stickleback inhabiting different water salinities. Stickleback reproduce externally, and have gametes that might experience a dramatically different environment after being released into the surrounding water/nest . There are studies on stickleback indicating that sperm are unable to activate in highly contrasting salinities, which suggested the possibility of a gametic barrier between fresh- and saltwater populations [8,9]. The objective of this study was to test whether sperm mobility could be a potential mating barrier in the threespine stickleback.
2. Material and methods
Details concerning sampling, sample sites and keeping fish in the laboratory can be found in electronic supplementary material, S1. This study consists of four parts as seen in figure 1. At the end of the holding period, males were immediately killed by a swift blow to the head, and the testes were removed surgically. Each testis was cut open and diluted in either naturally occurring fresh (approx. 0 ppt) or artificially made saltwater (30 ppt), mixed and immediately transferred to a chambered microscope where sperm movement was recorded. For further details, see electronic supplementary material, S1.
(a) Statistical analysis
Data were analysed using R . The total number (n) of males analysed for each salinity treatment is given in figure 1. For each experimental set-up, we tested for differences in sperm velocity between the two treatments, fresh- and saltwater, using a two-tailed t-test on mean curvilinear velocity (VCL) across treatments and within individual.
The mean velocities (±standard deviation) for marine and freshwater stickleback are shown in table 1 and illustrated in figure 2. Sperm from both marine and freshwater threespine stickleback could be activated by either fresh- or saltwater. However, freshwater males must acclimate to saltwater conditions before their sperm can be activated by saltwater, while sperm from saltwater males can be activated immediately by either fresh- or saltwater.
|acclimated salt 2 days||57.9||3.47||0.72||5||0.50|
|acclimated fresh 2 days||54.7||3.59|
|acclimated salt 7 days||54.9||1.94||0.31||5||0.77|
|acclimated fresh 7 days||53.9||3.47|
|acclimated combined salt||56.46||1.95||0.79||11||0.44|
|acclimated combined fresh||54.38||2.01|
The ability of sperm to have a somewhat wide osmotic activation tolerance has also been observed in other fish species, such as the medaka (Oryzias latipes) , and Gulf killifish (Fundulus grandis) , where the sperm of Gulf killifish and tilapia also had the ability to be activated by both hypotonic and hypertonic osmolarities. Osmolarity is not the only mechanism by which sperm are activated, as both the concentration of specific ions and/or pH levels have also been found to be of importance in some species [13–15]. Again, it is interesting that the saltwater stickleback sperm can be activated in both salt- and freshwater, as salt- and freshwater have the opposite concentration of critical ions such as calcium and potassium .
Details of sperm activation in stickleback are not known, and the sperm characteristics found in this study seem more similar to those of internally fertilizing fishes, e.g. ceasing to move quite quickly in water, regardless of salinity. In internally fertilizing fish such as ocean pout (Macrozoares americanus L.)  and spotted wolfish (Anarhichas minor) , the milt contains highly active sperm that are immobilized immediately upon dilution in saltwater, but stay active for days in the ovarian fluid of the females. In stickleback, the male builds a nest where the female deposits her eggs, which are covered in gelatinous ovarian fluid. The ovarian fluid contains a variety of ions [8,18], and as the stickleback male spawns directly on the eggs, which are within the nest, the ejected sperm are exposed to the ovarian fluid or a dilution of that fluid rather than the surrounding water. This presumably increases the longevity of the sperm cells by several hours , an important effect as fertilization of a stickleback egg clutch has been found to take several minutes .
Sperm from freshwater males failed to activate when exposed to saltwater in our experimental conditions. However, when offspring of the same freshwater population were raised in saltwater, and more surprisingly, when wild-caught freshwater males were acclimated for just 2 days to saltwater, the freshwater males' sperm could be activated by either fresh- or saltwater. This indicates that phenotypic plasticity in sperm activation has been maintained during the almost 8000 years that this population has been living in freshwater, and there has not been environmental assimilation causing sperm to be activated in only freshwater. The degree of phenotypic plasticity in sperm of saltwater males is unclear and was not tested, given their robust activation in both salinity treatments. Phenotypic plasticity in sperm activation has also been observed in tilapia , and the range of osmolarity that activated sperm motility shifted higher and broadened as the acclimation salinity of the fish increased for Gulf killifish . Spermatogenesis usually starts in adult stickleback males immediately after the reproductive season, meaning the sperm can be mature (thought to be a fixed state) for several months prior to the spawning season . It is, therefore, quite surprising that the sperm of mature, wild-caught freshwater stickleback have the ability to adapt to sudden changes in osmolarity and go from almost zero activation in saltwater when being freshwater adapted, to having fully active sperm in saltwater after being acclimated to saltwater for only a few days. One likely explanation for the plastic activation of the sperm cells could be a change of the ion concentration in the seminal plasma that is surrounding the sperm while in storage —if the seminal plasma increases in osmolarity when the fish is exposed to saltwater, the spermatozoa could change such that they become active already in the testis, as is the case for internally fertilizing fish [16,17]. However, while sperm of internally breeding fishes loses the ability to move instantly in pure saltwater (not tested in freshwater) , the sperm of saltwater stickleback and saltwater-acclimated freshwater stickleback are motile also in both salt- and freshwater. Spermatozoa mobility signalling is a complex and highly orchestrated process that has not been extensively studied in fish . More work is needed to determine which physiological acclimation mechanisms the stickleback uses to change the interpretation of the osmotic stress signals from hypo- to hyperosmolarity, and vice versa, to activate the spermatozoa mobility.
This research was approved by the National Animal Research Authority in Norway, permit number 4442.
A.T., A.B.M. and T.L. designed the study. All authors contributed to data collection. E.R.A.C. analysed the sperm velocity videos, A.T. analysed the data and wrote the first draft of the paper. All authors contributed towards subsequent drafts critically and approved the final version of the manuscript. All authors agree to be held accountable for the content in this manuscript.
The authors declare no competing interests.
This article was funded by the Norwegian Research Council.
We thank Anders Herland and Haaken Hveding Christensen for assistance in the fish laboratory, Sondre Ski, Martin Malmstrøm and Arthur Bass for help in the field, and Asbjørn Vøllestad for constructive comments on the manuscript.
Published by the Royal Society. All rights reserved.
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