1 Reproductive effort in ninespine stickleback (Pungitius pungitius): evidence of terminal

2 investment.

4 J. Gary Palmer

5 Department of Biology

6 Northern Michigan University

7 1401 Presque Isle Ave.

8 Marquette, MI 49855

9 jupalmer@nmu.edu

10

11 Word count: 2270

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12 Animals that mate more than once in their lifetimes (iteroparity) should optimize reproductive

13 effort for maximum lifetime reproductive success. Each bout of reproduction comes at the potential

14 expense of production of future offspring. This is known as the reproductive cost of mating. The other

15 component of reproductive effort is energetic cost, or the amount of energetic resources an individual

16 allocates to reproduction. Three separate hypotheses have been proposed as rules governing the

17 distribution of reproductive effort. This study sought to test which of these best explains the pattern of

18 reproductive effort in the life history of male ninespine stickleback. In an attempt to control costs of

19 reproduction among fish of varying ages, individuals were reared in isolation and fed a standardized

20 amount of food based on their body weight. Mass of sperm produced in individuals either one or three

21 years old was measured and standardized for body weight. This was used as an indicator of energetic

22 allocation to reproduction. It was found that on average three-year-old males produced nearly twice as

23 much sperm relative to their body weight as did one-year-old individuals. Two-year-old males

24 produced an intermediate amount of sperm. Three-year-old males also scored highest in a measure of

25 sperm motility and spent the most time fanning eggs in their nest. Results supported the terminal

26 investment hypothesis, while finding no evidence supporting senescence or the mating strategy-effort

27 hypothesis.

28

29 Keywords: Life history theory, ninespine stickleback, parental investment, Pungitius pungitius,

30 reproductive effort, senescence, terminal investment.

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31 There is substantial cost borne by parents when producing offspring. Consequently, heavy

32 investment in a parent's current brood may come at the expense of future reproduction (Trivers 1972).

33 This is considered the reproductive cost of breeding. Another factor in cost of breeding which is

34 suspected to be related with this is the energetic cost of breeding, or the amount of energy a parent

35 invests in a brood (Clutton-Brock 1984). Energetic cost is much more commonly studied, since it can

36 be measured more directly and over a shorter time period through quantifying the energy allocated to

37 production of gametes or measuring weight loss over the breeding season. The reproductive cost is

38 much more difficult to measure. It requires determining impact on a individual's future survival and any

39 change caused in future brood sizes, both of which would require considerable effort to make a record

40 of and be difficult to interpret.

41

42 Three hypotheses have been proposed to explain optimal allocation of reproductive effort over

43 an iteroparous animal's life history. First and perhaps most well known of these is the terminal

44 investment hypothesis. This trade-off between production of current and future offspring should favor

45 greater investment in current reproduction as parents age and chance of future reproduction decreases

46 (Clutton-Brock 1984). As an individual ages the proportion of it's lifetime remaining diminishes, and

47 consequently future reproductive possibilities decline. The result of this is a lowered reproductive cost

48 of breeding for older individuals since there are few future broods which could be negatively impacted

49 by current breeding efforts.

50

51 An alternative hypothesis is known as the senescence hypothesis. This states that as an animal

52 ages, reproductive effort will decrease due to loss of function associated with aging. leading to

53 decreased performance. This hypothesis refers specifically to the absolute amount of reproductive

54 effort an individual expends, whereas the terminal investment hypothesis specifies that the costs of

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55 reproduction must be taken into account. A decrease in ability could cause a decline in absolute output

56 even though output relative to ability may increase. It is likely that older individuals incur higher costs

57 of breeding, either due to poorer body condition entering the reproductive season (Mysterud et al.

58 2001) or decreased ability to defend a territory. These costs must be accounted for or held constant in

59 order to accurately determine whether a terminal decline in reproductive output is due to increased

60 costs or decreased investment. Because of this difference, the senescence and terminal investment

61 hypothesis are not mutually exclusive (Weladji et al. 2002).

62

63 A third hypothesis, proposed by Yoccoz et al. (2001), is the mating strategy-effort hypothesis.

64 This predicts a peak in reproductive effort in individuals just beyond maturity. This is based on the idea

65 that intrasexual competition plays a large role in reproductive success of males in many species, and

66 that investment should be highest when there is the greatest chance of success. In red deer it was found

67 that male reproductive effort increased quickly between maturity and peak age then slowly declined

68 (Yoccoz et al. 2001). Male reproductive success varies greatly based on number of mates (Trivers

69 1972) and investment during peak years should lead to greater benefit in this respect than toward the

70 end of the lifespan, when chance of success in these contests is low (Graves, in press).

71

72 One problem encountered in past tests of these hypotheses is that parental investment is

73 specifically defined as effort relative to costs, and costs may differ among individuals. For example, in

74 species with indeterminate growth such as many fishes, older individuals will be larger than young

75 ones and may be able to make a much larger absolute investment in reproduction without necessarily

76 investing more relative to the costs. This is further complicated by the tendency for larger individuals to

77 be dominant, giving them greater access to resources.

78

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79 This study attempts to control for these factors by raising individuals from eggs under

80 controlled laboratory settings in order to maintain constant resource availability and by measuring

81 sperm production relative to the weight of each individual. This should eliminate biases found in

82 natural settings that favor older individuals and may exaggerate any evidence of terminal investment. It

83 is hypothesized that when energetic costs are held constant, relative investment will be found to

84 increase with age.

85

86 METHODS

87

88 To test these hypotheses data on sperm production was collected from male ninespine

89 stickleback (Pungitius pungitius), an iteroparous fish which can be easily bred in the laboratory. It has

90 been shown in male stickleback that individuals vary resource allocation based on reproductive

91 strategy, with production of sperm being a useful indicator of allocation (de Fraipoint et al. 1993). The

92 individuals used in this experiment were descended from a population of ninespine stickleback

93 captured in Lake Superior near Marquette, Michigan. A total of 50 individuals (25 male, 25 female)

94 were captured during September 2003 and placed in a large aquarium to establish a large freely

95 breeding population. A proportion of haphazardly selected offspring were reared in isolation, and

96 month and year of birth was recorded for each individual. This was done to control for differences in

97 energetic costs and availability of resources to an individual based on competitive interactions which

98 may change over the course of an individuals life in addition to facilitating separation of individuals

99 into age classes. These individuals were weighed bimonthly after reaching maturity and given food

100 daily in proportion to their weight. This was done to ensure all individuals had equal energetic

101 resources, controlled for differences in body mass. Individual fish were raised in 5 gallon aquaria

102 maintained at 20° C with a 12:12 light:dark cycle.

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104 Subjects for the experiment were chosen haphazardly from the available stock of healthy

105 individuals. Subjects were taken from three distinct age classes; one year old plus or minus 2 months

106 (n=20), two years old plus or minus 2 months (n=20), and three years old plus or minus 2 months

107 (n=20). To assess body condition a standard curve was created using data from all available males

108 raised in isolation (n=79). Body length was plotted against mass and a least squares linear regression

109 line was drawn. Individuals whose mass differed from the expected according to this curve by more

110 than 20% were not included in this experiment.

111

112 To measure sperm production the collection procedure from de Fraipoint et al. (1993) was used.

113 Each male was placed upside-down in a slit cut into a wet sponge and pressure was gently applied to

114 the abdomen. This resulted in secretion of milt and glue, which were easily distinguished and

115 separated. Sperm was collected from each individual three times, each time separated by 48 hours. The

116 masses recorded from these three collections were averaged for each individual then divided by body

117 mass and multiplied by 100 in order to obtain a standardized index of sperm production relative to

118 body mass. There was no apparent change in mass of sperm over the three collections for each

119 individual.

120

121 In addition to quantity, sperm quality was also assessed. Milt from the second sample taken

122 from each male was observed and assigned an ordinal motility score using a procedure similar to that

123 found in de Fraipoint et al. (1993). Each male's second sample was used in order to control for any

124 differences in motility that may occur as a function of time since last ejaculation. Briefly, sperm were

125 collected using a capillary tube then diluted in 5ml distilled water. One drop of this solution was then

126 placed on a slide and observed under a microscope. Because the value recorded reflects a proportion of

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127 sperm which are motile, it was not crucial to carefully control the volumes placed on the microscope

128 slides. Sperm motility values were assigned as follows: 0 if no motility is observed, 1 if most

129 spermatozoa are immotile, 2 if most spermatozoa are vibrating, 3 if all spermatozoa are vibrating, 4 if

130 most spermatozoa were motile with flagellar movements, and 5 if all spermatozoa were motile with

131 flagellar movements (Guest et al. 1976).

132

133 The third portion of the experiment involved observation of egg fanning behavior displayed by

134 males. After sperm quantity and motility had been obsesses each of the males included in the study was

135 allowed to mate. Males were given 10g (wet weight) of Spyrogyra algae with which to construct a nest.

136 Mates were gravid females, all two years old plus or minus four months. Females were introduced into

137 the aquarium of each male 48 hours after the third sperm sample was obtained and allowed to remain

138 until eggs were deposited, usually occurring within 90 minutes. For the next seven days a group of

139 student volunteers observed each male for a 15 minute period between 1400 and 1600 hours each day.

140 Time spent fanning eggs was carefully observed and recorded as a proportion of the entire 15 minute

141 observation period.

142

143 RESULTS

144

145 All data analysis was performed using SPSS 16.0 for Windows. The data collected show a

146 significant correlation between length and body mass of a fish (figure 1) (Pearson correlation r = 0.996,

147 P < 0.001). This relationship was used to judge the body conditions of individuals used in the study,

148 with those falling below the regression line considered in poor condition, and those above in good

149 condition. Age was also found to be significantly correlated with body mass (Pearson correlation r =

150 0.985, P < 0.001). Body mass also correlated significantly with mass of sperm produced (Pearson

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151 correlation r = 0.954, P < 0.001). This supports the assumption that older males will be larger and

152 produce more sperm, therefore cost of sperm production must be standardized by body size.

153 Sperm mass index data for individuals of all three age classes were assumed to be normally

154 distributed according to a Shapiro-Wilk test (one year: T20 = 0.925, P =0.124; two year: T20=0.952,

155 P=0.406, three year: T20 = 0.948, P = 0.340). Data for sperm mass were also assumed to have equal

156 variance (Levene's Test for Homogeneity of Variance F = 0.673, P = 0.514). Using one-way ANOVA a

157 significant difference was found in in the mean sperm production index among the age classes (F2,59 =

158 196.029, p < 0.001). A Tukey's HSD post-hoc test showed that sperm mass was significantly higher for

159 second-year than first-year males (P<0.001), for third-year than second-year males (P<0.001), and for

160 third-year than first-year males (P<0.001). Mean value for sperm production index among first-year

161 males was 5.840±0.802 (all mean values given as mean ± standard deviation). This value was

162 8.011±0.688 for second-year individuals and 10.479±0.729 for third-years (figure 2).

163 Sperm motility data were also assumed to be normally distributed based on results of a Shapiro-

164 Wilk test (one year: T20 = 0.914, P =0.076; two year: T20=0.918, P=0.091, three year: T20 = 0.910, P =

165 0.056). Results of a one-way ANOVA showed that significant differences existed among the three age

166 classes (F2,59 =23.615, P<0.001). Mean values were 2.050±0.944 for first-year, 3.050±0.999 for second

167 year, and 4.00±0.725 for third-year males (figure 3). A Tukey's HSD post-hoc test showed that mean

168 motility was significantly greater for second-year males than first-year (P=0.002), for third-year than

169 second-year (P=0.004), and for third-year than first-year males (P<0.001).

170 The third set of data, time spent fanning, was also found to follow a normal distribution for each

171 age class (one year: T20 = 0.976, P =0.867; two year: T20=0.928, P=0.144, three year: T20 = 0.936,

172 P=0.197). Mean values for proportion of time spent fanning were 0.110±0.034 for first-year,

173 0.200±0.022 for second year, and 0.288±0.028 for third-year males (figure 4). Using a one-way

174 ANOVA it was found that a significant difference in proportion of time spent fanning eggs existed

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175 among the age classes (F2,59=198.834, P<0.001). A Tukey's HSD post-hoc test showed that mean time

176 spent fanning eggs was significantly greater for second-year males than first-year (P<0.001), for third-

177 year than second-year (P<0.001), and for third-year than first-year males (P<0.001).

178

179 DISCUSSION

180

181 The data collected show a significant difference in sperm production relative to body weight in

182 male ninespine stickleback of different ages. Three-year-old males produced a significantly greater

183 mass of sperm relative to body weight than did one-year-old individuals, as seen when index of sperm

184 production was compared between the two classes. This supports the hypothesis that male stickleback

185 increase their reproductive effort toward the end of their life spans. Similar results were found for

186 measures of sperm motility and fanning behaviour, with both showing a positive linear relationship

187 with age.

188

189 The results of the current study are in contrast to the findings of a similar study conducted by de

190 Fraipoint et al. (1993). The previous study differed however in its goals. De Fraipoint et al. showed that

191 three-spined stickleback males are able to change their reproductive strategy conditionally whether they

192 held a territory or not. They found significantly higher sperm production in younger, non-territorial

193 individuals, but these males also employed a different reproductive strategy. These males often

194 attempted to 'steal' fertilizations from the older, territorial males for whom the eggs were intended. This

195 strategy likely benefits more from increased sperm production than the typical strategy. It is certainly

196 possible that older males in their experiment were in fact increasing reproductive effort through the

197 costs of maintaining a territory.

198

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199 It would be of interest to test these hypotheses through measurement of sperm production in

200 other species, particularly ones in which this is the only means of male parental investment.

201 Stickleback males provide a great deal more investment than just gametes, they construct and defend a

202 nest as well as fanning eggs during brooding. It is possible due to the relatively low cost of sperm

203 production that this is only a minor component of a male stickleback's parental investment and different

204 effects would be seen if other measures of parental investment were used.

205

206

207 References

208

209 Clutton-Brock, T. H. 1984. Reproductive effort and terminal investment in iteroparous animals.

210 American Naturalist, 123, 212-229.

211 De Fraipont, M., Fitzgerald, G. J., & Guderly, H. 1993. Age-related differences in reproductive

212 tactics in three-spined stickleback, Gasterosteus aculeatus. Animal Behaviour, 46, 961-968

213 Graves, B. M. 2008. Ritualized Fighting and Life History Evolution in Human Males. In press.

214 Mysterud, A., Yoccoz, N.G., Stenseth, N.C., & Langvatn, R. 2001. Effects of age, sex, and density

215 on body weight of Norweigan red deer: evidence of senescence. Proceedings of the Royal Society

216 of London B, 268, 911-919.

217 Guest, W.C., Avault, J.W., & Roussel, J.D. 1976. A spermatology study of channel catfish, Ictalurus

218 punctatus. Transactions of the American Fisheries Society, 105, 463-468.

219 Trivers, R. L. 1972. Parental investment and sexual selection. In: Sexual Selection and the

220 Descent of Man 1871-1971. (Ed. by B. Campbell), pp. 136-179. Chicago, Illinois: Aldine.

221 Weladji, R. B., Mysterud, A., Holand, Ø., & Lenvik, D. 2002. Age-related reproductive effort in

222 reindeer (Rangifer tarandus): evidence of senescence. Oecologia, 131, 79-82

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223 Yoccoz, N. G., Mysterud, A., Langvatn, R., & Stenseth, N. C. 2002. Age- and density-dependent

224 reproductive effort in male red deer. Proceedings of the Royal Society of London B, 269, 1523-

225 1528

226

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228 Figure 1: Length vs. body mass of male stickleback. Males of three different ages were measured and

229 weighed to construct a standard curve in order to assess body condition. Data from all individuals

230 available (n=79) are included.

231

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Age vs. average sperm production index

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10

Average sperm production index value

8

0
1 2 3
Age of individuals (years)

234 Figure 2: Average sperm production by age. The sperm production index, calculated for each individual

235 by dividing mass of sperm by body mass, was averaged for each age group. The data show that on

236 average this index was twice as high for three-year-old individuals as one-year-olds.

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 Age vs. average sperm motility score
4.5

3.5

3
Average sperm motility score

2.5

1.5

0.5

0
1 2 3
Age of individuals (years)

238 Figure 3: Average sperm motility score by age. Sperm motility was ranked on a scale of 0-5, with 0

239 representing no motility observed and 5 representing all spermatozoa motile with flagellar movement

240 (Guest et al. 1976).

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 Age vs. proportion of time spent fanning
4.5

3.5

Proportion of time spent fanning eggs 3

2.5

1.5

0.5

0
1 2 3
Age of individuals (years)

242 Figure 4: Average proportion of time fanning eggs. The proportion of time during a 15 minute

243 observation window that a male spent fanning eggs was recorded daily for seven days immediately

244 following fertilization of eggs by the male.

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