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Rinnakkaistallenteet Luonnontieteiden ja metsätieteiden tiedekunta
2020
Offspring Phenotype Is Shaped by the Non-Sperm Fraction of Semen
Kekäläinen, Jukka
Wiley
Tieteelliset aikakauslehtiartikkelit
© 2020 European Society For Evolutionary Biology All rights reserved
http://dx.doi.org/10.1111/jeb.13592
https://erepo.uef.fi/handle/123456789/8197
Downloaded from University of Eastern Finland's eRepository
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Offspring phenotype is shaped by the non-sperm fraction of semen
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Jukka Kekäläinena,1, Annalaura Jokiniemia, Matti Janhunenb, Hannu Huuskonena 3
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aUniversity of Eastern Finland, Department of Environmental and Biological Sciences, P.O. Box 5
111, FI-80101, Joensuu, Finland 6
bNatural Resources Institute Finland (Luke), Yliopistokatu 6, FI-80130, Joensuu, Finland 7
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1Corresponding author: jukka.s.kekalainen@uef.fi, p. +358504674487 9
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Short title: Seminal plasma shape offspring phenotype 12
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2 Abstract
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In a large majority of animal species, the only contribution of males to the next generation has been 29
assumed to be their genes (sperm). However, along with sperm, seminal plasma contains a wide array 30
of extracellular factors that have many important functions in reproduction. Yet, the potential 31
intergenerational effects of these factors are virtually unknown. We investigated these effects in 32
European whitefish (Coregonus lavaretus) by experimentally manipulating the presence and identity 33
of seminal plasma and by fertilizing the eggs of multiple females with the manipulated and 34
unmanipulated semen of several males in a full-factorial breeding design. Presence of both own and 35
foreign seminal plasma inhibited sperm motility and removal of own seminal plasma decreased 36
embryo survival. Embryos hatched significantly earlier after both semen manipulations than in 37
control fertilizations; foreign seminal plasma also increased offspring aerobic swimming 38
performance. Given that our experimental design allowed us to control potentially confounding 39
sperm-mediated (sire) effects and maternal effects, our results indicate that seminal plasma may have 40
direct intergenerational consequences for offspring phenotype and performance. This novel source of 41
offspring phenotypic variance may provide new insights into the evolution of polyandry and 42
mechanisms that maintain heritable variation in fitness and associated female mating preferences.
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Keywords: Coregonus lavaretus, lek paradox, non-genetic inheritance, offspring fitness, paternal 45
effect, sperm, semen, transgenerational plasticity 46
47 48 49 50 51 52
3 Introduction
53
The sole function of the seminal plasma (non-sperm component of semen) has originally been thought 54
to serve as a transport medium for sperm cells (Sharkey et al., 2007; Bianchi et al., 2018). However, 55
in addition to sperm, semen contains a wide array of bioactive molecules and factors (Poiani, 2006;
56
Juyena & Stelletta, 2012; Bromfield et al., 2014; Siddique et al., 2016; Gombar et al., 2017), such as 57
salts, sugars, oligosaccharides, glycans, lipids, vitamins, hormones, enzymes, peptides, proteins, 58
DNA, and RNA (Keogan et al., 2016; Hopkins et al., 2017; Samanta et al., 2018; Dietrich et al., 59
2019). Many of these factors bind directly to sperm, whereas the others are packaged into small 60
extracellular vesicles (membrane-enclosed particles secreted by cells), such as exosomes, which are 61
capable of fusing with sperm and directly interacting with the female or their eggs (Robertson &
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Sharkey, 2016; Hopkins et al., 2017; Samanta et al., 2018). Consequently, seminal plasma has a much 63
larger array of functions than has traditionally been assumed (Perry et al., 2013).
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One of these additional functions is to protect sperm from oxidative damage and, in internally 65
fertilizing species, immune attacks of the female’s reproductive tract (Crean et al., 2016; Hopkins et 66
al., 2017). Furthermore, seminal plasma is responsible for regulating sperm activation and swimming 67
activity prior to and after ejaculation; thus, it has an important role in determining the fertilization 68
ability of the sperm (Mochida et al., 1999; Locatello et al., 2013; Bromfield et al., 2014; Rudolfsen 69
et al., 2015; Borziak et al., 2016). Seminal plasma factors also have important consequences for the 70
reproductive fitness of females (Wigby et al., 2009; Crean et al., 2016). In internally fertilizing 71
species, seminal plasma, for example, stimulates ovulation, triggers females post-mating immune 72
response, and provides nutrition to females (Hopkins et al., 2017). Furthermore, seminal plasma 73
molecules are incorporated into the eggs (Sirot et al., 2006; Crean et al., 2016), which raises an 74
intriguing possibility that the cell-free fraction of the semen has some intergenerational consequences 75
for the developing embryo.
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Bromfield et al. (2014) tested this possibility in mice and demonstrated that the ablation of the 77
seminal plasma by surgical excision of seminal vesicle glands, affected the growth and health of male 78
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offspring. They concluded that the finding was a consequence of both sperm damage and the effect 79
of seminal fluid deficiency on female reproductive tract gene expression. Similarly, Crean et al.
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(2014) studied the influence of siring and non-siring males on offspring phenotype in the neriid fly, 81
Telostylinus angusticollis, and found that offspring body size was influenced by the female’s previous 82
mate (i.e. semen), which was not the sire of that offspring. Thus, the demonstrated ‘non-sire effect’
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was most likely mediated by either foreign seminal plasma or sperm (for example, via sperm 84
penetration into the somatic tissues of the female reproductive tract: Liu, 2011; Crean et al., 2014).
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Together, these studies indicate that seminal plasma may have important intergenerational 86
consequences for offspring phenotype. However, both these studies were conducted in internally 87
fertilizing species where females have the potential of altering the phenotype of their offspring via 88
various post-mating responses to different male phenotypes (Bonduriansky & Day, 2009; Crean &
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Bonduriansky, 2014; Guillaume et al., 2016; Pascoal et al., 2018; Evans et al., 2019). Thus, it is 90
unclear whether the effects observed were induced by the male (seminal plasma) or differential 91
female responses (maternal effects) to these male substances (see García-González & Simmons, 92
2007; Crean & Bonduriansky, 2014). Accordingly, the direct intergenerational effects of seminal 93
plasma remain equivocal. Furthermore, none of the earlier studies have attempted to separate the 94
relative importance of these effects, or paternal effects in general, from the total phenotypic effect of 95
the sires (Evans et al., 2019).
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Externally fertilizing salmonids, such as the European whitefish (Coregenus lavaretus L.), serve 97
as an ideal model system to study such effects independently of confounding sire effects and maternal 98
effects (Kekäläinen et al., 2018). Whitefish produce large quantities of eggs and semen, and neither 99
females nor males provide parental care for their offspring. Furthermore, earlier studies in salmonids 100
and other externally fertilizing fish have shown that, in these species, seminal plasma has a key role 101
in modifying both sperm phenotype and fertilization success (Locatello et al., 2013; Rudolfsen et al., 102
2015; Bartlett et al., 2017; Lewis et al., 2017 Hopkins et al., 2017). A recent study in whitefish also 103
indirectly indicated that sperm phenotypic plasticity may have important consequences for offspring 104
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fitness (Kekäläinen et al., 2015). However, the role of seminal plasma behind sperm phenotypic 105
plasticity and associated intergenerational effects remain unclear.
106
In the present study, we investigated the potential role of seminal plasma as a mediator of 107
intergenerational plasticity in whitefish. We manipulated the semen of 10 males by either 1) 108
experimentally removing seminal plasma, or 2) mixing the male’s own seminal plasma with the 109
seminal plasma of a foreign male (hereafter ‘seminal plasma treatment’). We then applied a modified 110
North Carolina II breeding design, where the eggs of five females were fertilized with both the 111
manipulated and unmanipulated (control) semen of all 10 males, in all possible male-female 112
combinations. The full-factorial breeding designs we utilized allowed us to partition the relative 113
contribution (i.e. the proportion of variance explained) of dams, sires, dam-sire interaction, and the 114
intergenerational effect of seminal plasma treatments. In this way, we were able to rule out potentially 115
confounding differences in maternal investments (see above) and differential female physiological 116
responses to different males, and thus disentangle intergenerational effects mediated by seminal 117
plasma from maternal effects and direct genetic (i.e. sperm-mediated) effects of males.
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Material and methods 120
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Experimental fish and gamete collection 122
Parental fish originated from River Koitajoki and River Kokemäenjoki (Finland) migratory European 123
whitefish populations. River Koitajoki whitefish were captured from their natural spawning area by 124
seining on the 1st and 2nd of November, 2016. River Kokemäenjoki whitefish were collected on 9th 125
November, 2017, from a hatchery-reared pedigreed population maintained at the Tervo Fish Farm by 126
the Natural Resource Institute Finland (Luke). In both of these years, we stripped the eggs from five 127
randomly selected females and semen from 10 males (River Koitajoki, 2016) or 20 males (River 128
Kokemäenjoki, 2017). After semen collection, we took 10 μl semen subsample from all the males 129
and measured the spermatocrits (sperm volumes) of the males by centrifuging the samples for 10 130
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minutes (11 000 rpm) in a micro-hematocrit centrifuge. The gametes collected were utilized in two 131
separate experiments, in which we studied the intergenerational effects mediated by seminal plasma 132
removal (Experiment 1, River Koitajoki whitefish) and seminal plasma identity (Experiment 2, River 133
Kokemäenjoki whitefish).
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Experiment 1: The effect of seminal plasma removal 136
After spermatocrit measurement (see above), the remaining semen of each 10 males was divided into 137
two batches (20 batches in total). Within each male, one batch was centrifuged for 10 minutes at 700 138
× g (+4°C) to remove the seminal plasma (supernatant), whereas the other batch acted as 139
uncentrifuged control sample. By using the highest male-specific spermatocrit as a reference value 140
(Kekäläinen et al., 2015), sperm volumes within control samples were equalized into the spermatocrit 141
value of 10% (i.e. equivalent to 0.1 μl of sperm in 1μl of semen) by diluting the original semen sample 142
of each male with the seminal plasma of the same male (own seminal plasma). We then conducted 143
artificial fertilization (see section “Artificial insemination and egg incubation”); the final sperm 144
volume in all fertilizations was either 0.8 μl of pure sperm (seminal plasma removed) or 8 μl of semen, 145
with a spermatocrit value of 10% (control samples). In other words, all the egg batches were fertilized 146
with the same volume of sperm. In order to rule out a potential influence of centrifugation on sperm 147
motility, we collected the semen from six additional males and removed the seminal plasma as 148
described above. Then we remixed seminal plasma with the sperm and compared sperm motility (see 149
section “Sperm motility measurements”) between centrifuged and uncentrifuged samples of the same 150
males.
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Experiment 2: The effect of seminal plasma identity 153
The 20 semen samples collected were divided into 10 pairs based on males’ genetic relationships.
154
The genetic relationships between individuals were calculated from the pedigree using RelaX2 - 155
pedigree analysis program (© Ismo Stranden, Natural Resources Institute Finland). Within each male 156
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pair, one male acted as a focal male and another as a ‘foreign’ male. The genetic relationship (a) 157
between a focal male and a foreign male was always zero, whereas the average a among males and 158
females was 0.021 (± 0.027 S.D.). Prior to the seminal plasma manipulations, we collected ca. 200 μl 159
of seminal plasma from all the males by taking a 250 μl subsample of semen from each of the 20 160
males and by centrifuging the samples for 10 minutes at 700 × g (+4°C). To ensure that all the seminal 161
plasma samples were free from sperm cells, only the clearly distinct (upper) layer of the supernatant 162
(seminal plasma) was collected for the experiment. According to our previous observations, the 163
above-mentioned centrifugation protocol will effectively purify seminal plasma from sperm cells 164
(pers. observ. by J. Kekäläinen). After the seminal plasma collection, the original (unmanipulated and 165
uncentrifuged) semen subsamples of the focal males were divided into two batches. One batch was 166
mixed (15 s vortexing) with the male’s own seminal plasma, whereas the other batch was mixed with 167
foreign seminal plasma. Otherwise the treatment of the two samples (own seminal plasma vs.
168
own+foreign seminal plasma) was identical. In all cases, the seminal plasma volume added was based 169
on the measured spermatocrit value of the focal male and represented exactly 50% of the original 170
seminal plasma volume. In other words, each of the 10 males had two different seminal plasma 171
treatments: 1) semen batch with 100% own seminal plasma, and 2) semen batch with 50% own and 172
50% foreign seminal plasma. As described above, sperm volumes were again equalized to a 173
spermatocrit value of 10% allow all the egg batches to be fertilized with identical volumes of both 174
sperm (0.8 μl) and seminal plasma (7.2 μl).
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Sperm motility measurements 177
Sperm motility in all four seminal plasma treatments (with vs. without and own vs. own+foreign) was 178
measured using Computer Assisted Sperm Analysis, CASA (Integrated Semen Analysis System, 179
ISAS v1: Proiser, Valencia, Spain) with a B/W CCD camera (capture rate 60 frames/s) and a negative 180
phase contrast microscope (100× magnification). Sperm motility analyses were performed after 181
vortexing the samples for 5 s and then by adding semen or pure sperm to Leja 2-chamber (chamber 182
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height 20 μm) microscope slides (Leja, Nieuw-Vennep, The Netherlands) and by activating the sperm 183
with 3 μl of 4°C water collected from River Koitajoki (experiment 1) or Tervo Fish Farm (experiment 184
2). Sperm motility parameters (straight-line velocity, VSL; linearity of sperm swimming tracks, LIN 185
and proportion of static sperm cells) were measured 10 seconds after activation (at least two replicate 186
measurements/male).
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Artificial insemination and egg incubation 189
In both experiments, artificial fertilizations were conducted in all possible combinations (n = 50 190
families) between 10 males and five females (North Carolina II design) (Supporting Information, 191
Figure S1). Within each family, the eggs were divided into two batches. One batch was fertilized with 192
untreated (control) semen and the other with manipulated semen (with/without seminal plasma or 193
own/own+foreign seminal plasma), resulting in 100 male-female combinations (split-clutch + split- 194
ejaculate design) in both experiments. In order to minimize the potential time effects for the sperm 195
and offspring parameters measured, all the fertilizations (and CASA measurements, see above) were 196
always performed sequentially for both seminal plasma treatments within each male. Furthermore, in 197
order to ensure balanced fertilization design, the first replicate was fertilized in the following order:
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female 1, female 2,…, female 5, whereas the second was fertilized in the opposite order: female 5, 199
female 4,…, female 1.
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All the fertilizations were replicated twice (n = 200 egg batches in total). Fertilizations were 201
performed on Petri dishes by injecting the semen with a micropipette directly on freshly stripped eggs 202
(n = 106 ± 1.3 S.E. and 155 ± 1.0 S.E. eggs per male-female -combination, in 2016 and 2017, 203
respectively). Immediately thereafter, 50 ml of 4°C natural water was poured on the Petri dish and 204
each dish was gently shaken for three seconds. To allow the eggs to be fertilized, they were left 205
undisturbed in the dishes for at least five minutes, ensuring that all the sperm had lost their motility.
206
Fertilized eggs were then randomly divided into individual incubating containers (two replicate 207
containers per family within each seminal plasma treatment) in four 600 l temperature-controlled 208
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water tanks filled with 4°C non-chlorinated tap water. Eggs were incubated in these same containers 209
until hatching. Offspring hatching time was determined by counting the time taken from fertilization 210
day to the date when all the individual embryos in each of the 200 egg containers had hatched. Dead 211
embryos were counted and removed, both on a weekly basis, during the whole incubation period.
212 213
Offspring swimming performance and size 214
Offspring post-hatching aerobic swimming performance was determined using a swimming tube 215
system with gravity-driven flow and a constant water velocity of 5.7 cm s-1. Offspring swimming 216
performance of whitefish predicts predator avoidance ability of offspring and, thus, is strongly linked 217
to fitness (Huuskonen et al., 2009; Kekäläinen et al., 2010a). In our experiments, individual larvae 218
were forced to swim against a current at 6°C water temperature and their fatigue time, i.e. the time 219
taken to drift against a net placed at the rear end of the tube, was recorded. For each of the 200 male- 220
female combinations (in both experiments), three randomly selected individuals were used in the 221
experiments. After the swimming experiments, the larvae were sacrificed in an overdose of tricaine 222
methanesulfonate (MS-222, Sigma®, Sigma Chemical Co., Perth, Australia) and preserved in a 223
solution of 70% ethanol and 1% neutralized formalin. The larvae were later measured for total length 224
and fresh mass. All these experiments were based on a license by the Finnish Animal Experiment 225
Board (ESAVI/3443/04.10.07/2015, modified in ESAVI/8062/04.10.07/2015).
226 227
Statistical analyses 228
The effect of seminal plasma treatments on sperm swimming velocity (straight line velocity, VSL), 229
linearity of the sperm swimming trajectory (LIN) and sperm motility (proportion of static sperm cells) 230
were studied by using paired t-tests (n = 10 males or 10 male pairs). The effects of male (n = 10), 231
female (n = 5), male-female interaction, and seminal plasma treatments (n = 2), on embryo mortality 232
(proportion of dead embryos), offspring hatching time (number of days), offspring swimming 233
performance and offspring size were tested in linear mixed-effects models or generalized linear 234
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mixed-effects models (with negative binomial distribution). In both models, seminal plasma 235
treatment was used as a fixed factor; male, female, and male-female interaction were used as random 236
factors. All of these random factors were specified and modelled as fully crossed random effects 237
terms (see Bates et al., 2015: Table 2). Furthermore, our initial (full) models included all two- and 238
three-level interactions between random factors (males and females), and fixed factor (seminal 239
plasma treatment). All of these interactions were modelled as random effects (Magezi, 2015). Models 240
were simplified, based on a likelihood ratio test, by removing non-significant interaction terms. In 241
order to estimate the relative contribution of both parents (and their interaction) and seminal plasma 242
treatments on the measured offspring traits, we calculated marginal R2 (i.e. variance explained by the 243
fixed factor) and conditional R2 (i.e. variance explained by both fixed and random factors) (Nakagawa 244
and Schielzeth, 2013). Then we partitioned the variance of random factors further into female, male, 245
and male-female interaction variance (observational variance components). Finally, we calculated the 246
relative proportions of variance explained by the fixed factor (seminal plasma treatment) and random 247
factors (female, male, and male-female interaction). Model assumptions were graphically verified by 248
using Q-Q plots and residual plots. All P-values presented are from two-tailed tests, with α = 0.05.
249
LMM was conducted by using lmerTest package, GLMM analyses with the glmmTMB package and 250
R2 calculations with the MuMIn package in R (version 3.5.1). Both the lmerTest and the glmmTMB 251
packages are capable of handling fully crossed random effects and thus capture the non-independence 252
of data points in full factorial experimental designs (see e.g. Bates et al., 2015).
253 254 255 256 257 258 259 260
11 Results
261 262
Sperm motility 263
Sperm swimming velocity (VSL) was higher and the swimming trajectory (LIN) more linear when 264
sperm were activated without seminal plasma (experiment 1), compared to when it was present 265
(paired t-test, VSL: t9 = 4.60, P = 0.001; LIN: t9 = 5.25, P = 0.001) (Fig. 1a and b). Similarly, the 266
proportion of motile sperm cells was higher without seminal plasma (paired t-test, t9 = 4.30, P = 267
0.009). Foreign seminal plasma (50% of the total volume, experiment 2) reduced sperm VSL, in 268
comparison to 100% own seminal plasma (paired t-test, t9 = 2.35, P = 0.043) (Fig. 1c). Origin of 269
seminal plasma had no effect on the linearity of the sperm swimming trajectory although sperm 270
tended to have a more linear swimming trajectory in their own seminal plasma (paired t-test, t9 = 271
1.84, P = 0.100) (Fig. 1d). The proportion of motile sperm cells did not differ between treatments 272
(paired t-test, t9 = 1.13, P = 0.29). Sperm centrifugation (CF) did not affect sperm swimming velocity 273
or swimming trajectory (paired t-test, t5 = 2.13, P = 0.31 and t5 = 0.32, P = 0.76, respectively).
274
However, the proportion of motile sperm cells was higher in centrifuged samples than control samples 275
(paired t-test, t9 = 4.04, P = 0.010), indicating that centrifugation did not have negative impact on 276
sperm viability.
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Embryo mortality and offspring size 279
Absence of seminal plasma (experiment 1) increased embryo mortality, which was also affected by 280
female identity, male identity, and a male-female interaction (Table 1). The addition of foreign 281
seminal plasma (experiment 2) had no influence on embryo mortality, but again, mortality was 282
affected by females, males and a male-female interaction. The presence or origin of seminal plasma 283
had no effect on offspring post-hatching size (seminal plasma removal, length: P = 0.51; body mass:
284
P = 0.95; seminal plasma identity, length: P = 0.57; body mass: P = 0.68). In all models, the two- and 285
three-level interaction terms between seminal plasma treatment and males/females (treatment × male, 286
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treatment × female, treatment × male × female) were statistically non-significant and, thus, were 287
removed from the final models. In other words, the effect of males, females, or male-female 288
combinations, on embryo mortality or offspring size was similar in both seminal plasma treatments.
289
Thus, embryo development was not affected by differential egg responses (i.e. egg-mediated maternal 290
effects) to males in different seminal plasma treatments (nonsignificant treatment × male × female 291
effect) or by differential egg responses to seminal plasma manipulations (treatment × female) at the 292
gamete level (see differential egg responses to sperm of different males: Crean & Bonduriansky, 293
2014).
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Offspring hatching time and swimming performance 296
Offspring hatched significantly earlier when eggs were fertilized without seminal plasma (experiment 297
1) than when it was present (Table 2, Fig. 2a). Seminal plasma removal explained 1.7% of total 298
phenotypic variation in offspring hatching time. On the other hand, foreign seminal plasma 299
(experiment 2) accelerated hatching by 9.24 days (7%) in comparison to control treatment (100%
300
own seminal plasma: 132.67 ± 0.81 (S.E) days; 50% foreign seminal plasma: 123.43 ± 0.64 (S.E.) 301
days), explaining 27.5% of total phenotypic variation in hatching time (Table 3, Fig. 2c). Given that 302
the combined influence of both parents and their interaction (male × female effect) explained 26.5%
303
of phenotypic variation, seminal plasma identity was of approximately equal magnitude to the 304
combined effects of male, female and their interaction. Hatching time was also affected by females 305
(in both seminal plasma treatments) and males (seminal plasma removal only). Furthermore, origin 306
of seminal plasma modified offspring post-hatching performance (Table 3, Fig. 2d): offspring that 307
had been fertilized in the presence of foreign seminal plasma had better swimming performance than 308
did offspring that had been fertilized only in the presence of a male’s own seminal plasma. As above, 309
the non-significant two- and three-level interaction terms between seminal plasma treatment and 310
males/females (or male-female combinations) indicate that offspring hatching time and swimming 311
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performance were unaffected by differential egg responses (egg-mediated maternal effects) at the 312
gamete level.
313 314
Discussion 315
Our results show that sperm swimming velocity was higher in the absence of both own and foreign 316
seminal plasma, but own seminal plasma had a positive effect on embryo survival. Furthermore, both 317
seminal plasma treatments accelerated the embryo hatching rate, in comparison to control treatments, 318
and foreign seminal plasma was also found to improve offspring aerobic swimming ability.
319
Importantly, seminal plasma identity (presence of foreign seminal plasma) was the most important 320
determinant of embryo hatching time. Given that our experimental design allowed us to exclude 321
confounding maternal investments and differential egg responses (reviewed by Kekäläinen & Evans, 322
2018) to seminal plasma treatments and to control sperm-mediated effects on offspring, these results 323
indicate that seminal plasma may have direct intergenerational consequences for offspring phenotype 324
and fitness.
325
Several studies have shown that, in many species, seminal plasma has an important role in 326
regulating the viability and motility of sperm (den Boer et al., 2010; Simmons & Beveridge, 2011;
327
Locatello et al., 2013; Rudolfsen et al., 2015; Bartlett et al., 2017; Lewis & Pitcher, 2017; Poli et al., 328
2018). In salmonids, sperm remain immotile in seminal fluid and motility is activated only when the 329
seminal plasma is diluted into the surrounding water (Dzyuba & Cosson 2014). Our results indicate 330
that both the male’s own and foreign seminal plasma have capability to inhibit sperm motility.
331
Furthermore, we also demonstrate that removal of male’s own seminal plasma prior to sperm 332
activation led to higher sperm motility than when seminal plasma was present during the activation.
333
Given that seminal plasma can be expected to beneficial for sperm performance this finding may 334
indicate that the absence of seminal plasma led to more effective sperm activation.
335
The intergenerational consequences of seminal plasma identity and associated sperm phenotypic 336
plasticity have remained ambiguous. Bromfield et al. (2014) demonstrated in mice that the absence 337
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of seminal plasma hindered conception rates and caused pathological changes in male offspring.
338
Similarly, we found that the absence of seminal plasma increased embryo mortality, indicating that 339
seminal plasma may play an important role in the fertilization process, embryo development, or both.
340
The observed negative effects of seminal plasma removal on embryo viability may be partly 341
attributable to oxidative stress-induced sperm DNA damage (Bromfield et al., 2014; Kekäläinen et 342
al., 2018). However, since the absence of seminal plasma (or the seminal plasma extraction protocol) 343
did not have any effect on sperm motility or offspring aerobic swimming performance, it is possible 344
that other (sperm damage-independent) mechanisms also play a role in the process. Along with 345
seminal plasma removal, seminal plasma identity was also found to have potentially important 346
intergenerational consequences. Earlier studies have shown that both the identity and composition of 347
seminal plasma have a key role in modifying the fertilization success and metabolism of sperm and 348
additionally regulate early embryo development (Pesch et al., 2006; Finseth et al., 2013; Bromfield, 349
2014; Crawford et al., 2015; Yamane et al., 2015; Bartlett et al., 2017; Hopkins et al., 2017; Nederlof 350
et al., 2017). Our results revealed that seminal plasma composition shape the hatching time and 351
aerobic swimming performance of offspring. Accordingly, seminal plasma may additionally modify 352
the metabolism and/or aerobic capacity of offspring (Bromfield et al., 2014; Lane et al., 2014;
353
McDonnell & Chapman, 2016; Metcalfe et al., 2016; Watkins et al., 2018).
354
Molecular-level mechanisms underlying above-mentioned effects (or intergenerational effects of 355
seminal plasma in general) have remained unclear (Morgan et al., 2019). However, it is known that 356
seminal plasma contains a complex mixture of potentially bioactive molecules. It has been envisaged 357
that, particularly RNAs, proteins, and lipids, may function as mediators of intergenerational effects 358
(Locatello et al., 2013; Crean et al., 2014; Sharma et al., 2016; Castillo et al., 2018; Samanta et al., 359
2018). In semen, these molecules are frequently transported within exosomes and other extracellular 360
vesicles (Vojtech et al., 2014; Du et al., 2016; Jodar et al., 2016; Morgan et al., 2019). Extracellular 361
vesicles of seminal plasma bind to the sperm membrane and, thus, deliver associated molecules into 362
sperm cells, which have an important role in modifying sperm function and fertilization success (Du 363
15
et al., 2016; Machtinger et al., 2016; Samanta et al., 2018). After fusion with sperm, semen-borne 364
molecules can transfer into oocytes and later regulate the gene expression of developing embryos 365
(Chen et al., 2016). Sciamanna & Spadafora (2012) also showed that virtually all animal species can 366
spontaneously bind exogenous RNA and DNA molecules via sperm plasma membrane receptors 367
(Lanes & Marins 2012). Together these findings suggests that males (i.e. sperm) potentially can shape 368
the phenotype of their offspring via extra-nuclear DNA or RNA. Alternatively, foreign seminal 369
plasma may penetrate directly into the eggs during the fertilization process. Supporting this 370
possibility, earlier studies have demonstrated that the surface of the salmonid eggs is highly 371
permeable to extra-cellular chemical factors (e.g. Poisson et al., 2017) and that the exposure of 372
embryos to conspecific chemical signals (alarm cues) increase both the development rate of the 373
embryos and the post-hatching performance of the offspring (Mourabit et al., 2010). Thus, it is 374
possible that salmonid eggs may also be capable of ‘sensing’ chemical cues of the seminal plasma, 375
which may trigger plastic (and possibly adaptive) changes in the developing offspring.
376
Observed intergenerational effects can have important evolutionary consequences, especially in 377
numerous species, in which the only provision of the males to females and offspring is their semen.
378
In these non-resource-based mating systems, all the benefits females gain from mating have 379
traditionally been thought to be mediated by paternal genes. This, in turn, has been assumed to lead 380
to the evolution of directional female mating preferences for certain male genes (or alleles) and 381
associated phenotypic indicators of male condition and genetic quality (secondary sexual ornaments) 382
(Fisher, 1930; Zahavi, 1975). However, consistent female preference for such ‘good genes’ is 383
expected to deplete additive genetic variance in fitness, which in turn should eliminate all the benefits 384
of being choosy (Head et al., 2016). This results in the lek paradox, a conundrum of how additive 385
genetic variation is maintained under directional female mate choice. Numerous potential resolutions 386
to the paradox have been presented, but none of them are entirely satisfactory (Bonilla et al., 2016).
387
However, Bonduriansky & Day (2013) showed theoretical evidence that non-genetic paternal effects 388
may provide a novel resolution to this long-standing evolutionary enigma.
389
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Supporting this view, seminal plasma composition and quality exhibit considerable variation 390
between males (e.g. Robertson, 2010) and a high degree of environmental- and male condition- 391
dependent plasticity (Ramm et al., 2015; Hopkins et al., 2017; Macartney et al., 2019). Accordingly, 392
seminal fluid quality is expected to vary independently of sperm quality or male genetic quality (Perry 393
et al., 2013; Crean et al., 2016). These findings, together with the present results, clearly indicate that 394
males have the potential to affect offspring fitness independently of the sperm nuclear genotype. If 395
offspring fitness is dependent on both genetic (sperm) and non-genetic (or extra-nuclear genetic) 396
components of the semen, females may have evolved mating strategies that allow optimizing both of 397
these heritable components of fitness (see e.g. Kekäläinen et al., 2010b). Consequently, female mating 398
preference for male genes and their phenotypic indicators may turn out to be less directional than we 399
have traditionally been assumed. Thus, seminal plasma-mediated non-genetic benefits could help to 400
understand both the maintenance of heritable variation in fitness and associated female mating 401
preferences. Furthermore, our results raise a possibility that the benefits of multiple mating (sperm 402
competition) may not be transmitted exclusively genetically (García-González & Simmons, 2007;
403
Kekäläinen et al., 2010b). Thus, the present results may also have novel insights into the deeper 404
understanding of the evolution of polyandry.
405
In conclusion, our results demonstrate that seminal plasma has potentially important 406
intergenerational consequences for offspring phenotype and fitness. Our experimental design allowed 407
us to manipulate seminal plasma identity independently of sperm and rule out potential differences 408
in maternal investments (maternal effects) in offspring. Thus, the intergenerational effects observed 409
are likely largely mediated by seminal plasma-borne factors that are incorporated into the sperm prior 410
to fertilization, and/or the direct impact of these factors on unfertilized eggs. Irrespectively of the 411
underlying mechanisms, our results suggest that all the benefits of mating even in non-resource-based 412
mating systems may not necessarily have a fully genetic basis (Kekäläinen et al., 2010b). Thus, we 413
envisage that a more comprehensive understanding of the above-mentioned effects can offer 414
important new insights into the evolution of polyandry and mating strategies.
415
17 Acknowledgements
416
We thank Anastasia Savolainen, Annika Inha, Annika Huupponen, Aurora Hatanpää, and Pyry 417
Pihlasvaara, for their help in experiments and staff at the Tervo Fish Farm (Luke) and Reijo 418
Piitulainen for providing whitefish gametes. Gerald Netto checked the language of the manuscript.
419
This study was financially supported by Academy of Finland (grant 308485) and Kone foundation 420
(to J.K.). The authors declare that they have no conflict of interest.
421 422
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