|
|
Received: 26 August 2016 Revised: 21 November 2016 Accepted: 17 December 2016 DOI: 10.1002/ece3.2717
ORIGINAL RESEARCH
Environmental enrichment, sexual dimorphism, and brain size in sticklebacks Elisavet A. Toli1,2 | Kristina Noreikiene2 | Jacquelin DeFaveri2 | Juha Merilä2 1
Molecular Ecology & Conservation Genetics Lab, Department of Biological Applications & Technology, University of Ioannina, Ioannina, Greece 2
Ecological Genetics Research Unit, Department of Biosciences, University of Helsinki, Helsinki, Finland Correspondence Juha Merilä, Ecological Genetics Research Unit, Department of Biosciences, University of Helsinki, Helsinki, Finland. Email: [email protected] Funding information Biotieteiden ja Ympäristön Tutkimuksen Toimikunta, Grant/Award Number: 218343 and 286889; ERASMUS + Mobility Programme for Internship; Nordenskiöldsamfundet
Abstract Evidence for phenotypic plasticity in brain size and the size of different brain parts is widespread, but experimental investigations into this effect remain scarce and are usually conducted using individuals from a single population. As the costs and benefits of plasticity may differ among populations, the extent of brain plasticity may also differ from one population to another. In a common garden experiment conducted with three-spined sticklebacks (Gasterosteus aculeatus) originating from four different populations, we investigated whether environmental enrichment (aquaria provided with structural complexity) caused an increase in the brain size or size of different brain parts compared to controls (bare aquaria). We found no evidence for a positive effect of environmental enrichment on brain size or size of different brain parts in either of the sexes in any of the populations. However, in all populations, males had larger brains than females, and the degree of sexual size dimorphism (SSD) in relative brain size ranged from 5.1 to 11.6% across the populations. Evidence was also found for genetically based differences in relative brain size among populations, as well as for plasticity in the size of different brain parts, as evidenced by consistent size differences among replicate blocks that differed in their temperature. KEYWORDS
brain size, environmental enrichment, fish, phenotypic plasticity, sexual dimorphism
1 | INTRODUCTION
Hence, there appears to be a general consensus that intra- and inter-
Given the importance of the central nervous system to organismal
least to some extent, dictated by variation in the strength of positive
performance, and thereby also fitness, the size of the brain and its
natural selection acting on them.
specific variation in brain size and size of different brain parts is, at
different parts are likely to be traits under strong optimizing selec-
However, as maintenance of neural tissue is energetically
tion. Specifically, increased brain size can enhance individual fitness
expensive (Aiello & Wheeler, 1995; Isler & van Schaik, 2006, 2009;
through improved cognitive ability (Deaner, Isler, Burkart, & van
Mink, Blumenschine, & Adams, 1981; Nilsson, 1996; Soengas &
Schaik, 2007; Gibson, 2002; Kotrschal et al., 2013a,b; Striedter, 2005).
Aldegunde, 2002), these energetic costs are likely to generate selec-
Moreover, certain brain regions often show an increase in size that
tion pressures opposing increases in brain size (Isler & van Schaik,
is associated with specific ecological conditions likely to select for
2009; Kotrschal et al., 2013a). For instance, the fact that the two
this growth (Eifert et al., 2015; Gonzalez-Voyer & Kolm, 2010; Krebs,
sexes commonly differ in brain size and size of different brain parts
Sherry, Healy, Perry, & Vaccarino, 1989; de Winter & Oxnard, 2001).
(e.g., Jacobs, 1996; Kotrschal, Räsänen, Kristjánsson, Senn, & Kolm,
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. © 2017 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Ecology and Evolution. 2017;7:1691–1698.
www.ecolevol.org | 1691
|
TOLI
1692
et al
T A B L E 1 Descriptive information about study populations and samples. Age (in weeks) gives average of the individuals in each of the populations at time of brain measurements Country
Location
Sea area
Coordinates
Age salinity (ppt)
Germany
Sylt
North Sea
55°01′N, 08°25′E
26
Denmark
Mariager
Kattegat
56°38′N, 09°57′E
25
Finland
Kotka
Baltic Sea
60°33′N, 27°12′E
23
Finland
Oulu
Baltic Sea
65°07′N, 25°14′E
22
nFemales
nMales
28
21
16
37
20
15
21
36
6
8
21
29
3
18
19
37
62
77
139
Total
nTotal
N, sample size for brain measurements.
2012; Samuk, Iritani, & Schluter, 2014) is likely to be explainable
enrichment, but there remains little understanding of the causes
by sex differences in costs and benefits of maintaining certain sized
underlying this heterogeneity.
brain or brain regions. For instance, Nottebohm (1981) demon-
The main aims of this study were to test (1) whether environ-
strated that the song control nuclei in the telencephalon of canaries
mental enrichment leads to increased brain size (or size of different
(Serinus canarius) doubled in size during the breeding season, but
brain parts) in three-spined sticklebacks (Gasterosteus aculeatus),
only in males. Likewise, given the high energetic costs of maintaining
(2) whether the effect of enrichment is similar for the sexes, and (3)
neural tissue, the ability to reduce the size of the brain or a particular
whether the effects of enrichment and its interaction with sex are sim-
brain part through phenotypic plasticity when they are not critical
ilar across multiple populations. To this end, we conducted a common
for fitness should be favored by selection. It has been proposed that
garden experiment in which fish from four different populations were
the sexual dimorphism in the three-spined stickleback brain might
exposed to either a control (bare aquaria) or enriched (spatial com-
be subject to sex-specific plasticity such that males increase their
plexity generated with physical structures) treatment over a period of
brain size during the breeding season in response to the increased
2 months. Based on the results of an earlier experiment which found
cognitive demands imposed by mating, nest-guarding, and parental
that males developed larger brains in enriched tanks as compared
demands (Herczeg, Gonda, Balazs, Noreikiene, & Merilä, 2015). In
to females (Herczeg et al., 2015), we expected to see a similar sex-
this scenario, such plasticity could be adaptive, as the males would
specific response to environmental enrichment consistent across the
escape the energetic costs of maintaining large brains during the
four populations tested. Apart from assessing the treatment effects on
nonbreeding season.
brain size and size of different brain parts, we also investigated how
There is also evidence to suggest that various animals are capable of increasing their brain size in response to environmental enrichment
enrichment influenced growth (i.e., body size) and condition (i.e., residual mass) of the fish.
(Bennett, Diamond, Krech, & Rosenzweig, 1964; Bennett, Krech, & Rosenzweig, 1964; Bennett, Rosenzweig, & Diamond, 1969; Cummins, Walsh, Budtz-Olsen, Konstantinos, & Horsfall, 1973; Riege, 1971; Scotto Lomassese et al., 2000; Technau, 1984). However, the evidence for this effect from fish studies is conflicting. While some studies have
2 | MATERIAL AND METHODS 2.1 | Fish collecting and husbandry
supported this finding of positive effects of enrichment on brain size
Fish forming the parental generation were collected from four dif-
(DePasquale, Neuberger, Hirrlinger, & Braithwaite, 2016; Herczeg
ferent localities across the Baltic Sea between 5 and 24 June 2015
et al., 2015; Näslund, Aarestrup, Thomassen, & Johnsson, 2012), oth-
(Table 1). Live fish were transported to Helsinki and used to make arti-
ers have found either negative effects (Kotrschal, Sundström, Brelin,
ficial crosses between 6 June and 5 July 2015. Half of the Sylt and
Devlin, & Kolm, 2012; Turschwell and White, 2016) or none at all
Mariager fjord crosses were done in the field, and fertilized eggs were
(Burns, Saravanan, & Rodd, 2009; Kihslinger, Lema, & Nevitt, 2006).
transported to Helsinki in 50-ml tubes modified with a mesh bottom
This is true not only for overall brain size, but also in the size of cer-
for water circulation within a cool (10–14°C) and constantly aerated
tain brain regions such as the cerebellum, olfactory bulb, telenceph-
water bath. Fertilized eggs from 10 full-sib families from each popu-
alon, and optic tectum in which both positive (Herczeg et al., 2015;
lation were first kept in petri dishes until hatching and then trans-
Kihslinger & Nevitt, 2006; Kotrschal, Rogell, Maklakov, & Kolm, 2012;
ferred to 500-ml containers. Larvae were fed twice a day with live
Näslund et al., 2012) and negative (optic tectum; Herczeg et al., 2015)
Artemia salina nauplii. After 7 days, the families were transferred to
effects of environmental enrichment have been shown. However, in
1.2-L tanks in Allentown zebrafish racks (Allentown, San Diego, CA,
the cases where positive effects have been found, the effects have
USA), where they continued to be fed Artemia naupalii ad libitum for
been conditional to age (Näslund et al., 2012), sex, and social inter-
1–2 months. Each family was then divided into two separate 5-L tanks
actions (Herczeg et al., 2015; Kotrschal, Rogell et al., 2012) or other
on the zebrafish racks, each housing 20 fish. Finely chopped (frozen)
factors such as stress (DePasquale et al., 2016). Hence, there is a great
chironomid larvae were introduced into their diet by mixing with
deal of heterogeneity in the observed responses to environmental
Artemia for 1 month, after which the Artemia were eliminated and only
TOLI
|
1693
et al
(a)
(b)
F I G U R E 1 Frontal views of aquaria used in (a) control and (b) enriched treatments whole chironomid larvae were fed. Once the fish were 12–16 weeks
Experiments were terminated when the fish were between 22
old, four fish from each of the 10 families per population (i.e., a total
(Kotka and Oulu) and 26 (Sylt and Mariager fjord) weeks old by eutha-
of 160 fish) were randomly chosen to be used in the experiment (see
nizing the fish with an overdose (250 mg/L) of MS222 (tricaine meth-
below), which was conducted in a separate room.
ane sulphonate). In order to scale the size of the brain/different brain parts with individual variation in body size, the standard length (SL;
2.2 | Experiments The experiments were conducted in a room fitted with sixteen 38-L
from the tip of the mouth to the end of the tail base) of all individuals was measured with a digital caliper to the nearest 0.01 mm. Body weight was also recorded to the nearest 0.01 g.
aquaria (L:40 cm × H:30 cm × W:24 cm) distributed among two
Following measurements, fish were immediately placed in a solu-
shelves on two separate sides of the room (henceforth: blocks). Each
tion containing 4% paraformaldehyde and 2.5% glutaraldehyde in a
aquaria was assigned to one of two treatments, “enriched” or “control”.
phosphate-buffered saline solution and fixed for 5 days.
In the enriched treatment, the bottom of the aquaria was covered with ceramic pebbles, along with two artificial plants and a plastic cylinder (23 cm in height, 9 cm in diameter) to generate structural complexity. In the control treatment, the aquaria were left empty, except for
2.3 | Brain measurements and sexing Brains were dissected under a stereomicroscope by removing the top
the aerator, which was present in both treatments (Figure 1). In order
of the neurocranium and severing the cranial and optic nerves and
to control for possible aquaria and block effects, two replicate tanks
spinal cord. The caudal section of the medulla was cut between the
per population and treatment were used (i.e., four populations × two
second and the third vertebrae in each fish in order to standardize
treatments × two replicates = 16 aquaria). Each of the replicates was
the measurements. Hence, the “length” measurement for the medulla
placed on opposite sides of the room, such that each population and
did not represent the total length of the entire structure, but rather
treatment were represented (next to each other) on the same shelf
its length until a standardized cutoff point. Brains were kept in a
in both blocks. White plastic sheets were placed between aquaria to
phosphate-buffered saline solution until they were photographed
serve as visual partitions.
with a digital camera from the dorsal, lateral, and ventral sides, from a
The experiments started by introducing one fish from each of the
fixed distance. Width, height, and length of six different brain regions
10 full-sib families per population into a particular treatment aquaria.
(viz. olfactory bulb, optic tectum, telencephalon, cerebellum, dorsal
Hence, each aquaria housed 10 unrelated fish from a given population.
medulla, and hypothalamus) were measured with ImageJ (Abràmoff,
Fish were kept under constant light to mimic summer conditions at the
Magalhães, & Ram, 2004) using landmarks shown in Appendix S1, fol-
northern latitudes and fed with frozen chironomid larvae (ad libitum)
lowing Pollen et al. (2007). For bilateral brain parts, both sides were
twice daily.
measured and their average was used in all analyses. These data were
All fish were raised in freshwater, which was maintained at about
then fitted to the ellipsoid models (van Staaden, Huber, Kaufman, &
17°C (±1°C). However, due to temperature stratification in the exper-
Liem, 1994) to estimate the volume of total brain size and size of the
imental room, fish in the two blocks experienced slightly different
different brain parts. The ellipsoid-model approach is known to yield
temperatures (A block: x = 17.5°C; max–min = 17.2–17.9°C; B block:
reliable estimates of brain and brain part sizes, as verified by compari-
x = 16.5°C; max–min = 16.4–16.9°C).
sons with histology and X-ray micro-computed tomography-based
|
TOLI
1694
et al
T A B L E 2 Linear model results of brain and body size traits. Tabled values are F-values from linear models for treatment (Tre), population (Pop), sex, block (Blo), standard length (SL), and for sex–treatment interaction. SSD(%) gives the degree of sexual dimorphism (in %), Tre (%) the degree of difference between treatment means as calculated from the back-transformed least-square means in the model. For SSD (%), positive values indicate male-biased SSD, and negative values, female; for Tre (%), positive values indicate larger trait mean in control and negative values indicate larger trait mean in treatment conditions. Tank refers to the proportion of total variance in a given trait explained by the random effect of tank Trait
Tre
Pop
Sex *
T × S ***
Blo
SL *
Tank
SSD(%)
Tre(%)
***
Brain (wgt)
0.18
5.86
35.05
0.45
5.59
276.72
0.0
10.2
−0.6
Brain (vol)
0.03
6.95**
22.87***
0.30
2.15
272.86***
6.3
8.0
0.3
***
0.1
5.1
−1.5
64.35***
2.4
11.6
−0.2
Dorsal medulla
0.20
3.74
Telencephalon
0.00
1.92
Optic tectum
0.12
8.41
Cerebellum
1.12
1.95
*
**
2.35
0.21
3.35°
18.35***
0.12
0.43
**
9.63
0.88
9.91**
0.39
101.49
4.29°
***
150.79
0.0
8.0
−0.9
7.04*
129.55***
0.0
9.1
2.7
**
Olfactory bulb
0.00
2.17
0.01
0.00
0.03
8.47
0.2
0.6
−0.2
Hypothalamus
0.10
1.66
8.91**
0.17
0.25
67.82***
25.4
10.3
−2.1
***
***
Standard length
0.29
13.56
19.29
0.17
4.42°
–
0.0
−6.3
−0.5
Body mass
0.64
2.47
31.07***
0.13
0.00
–
4.0
−28.4
−4.2
1.33
***
19.4
−9.3
−2.7
Condition
0.53
17.00
0.23
1.89
***
549.35
p
|
Received: 26 August 2016 Revised: 21 November 2016 Accepted: 17 December 2016 DOI: 10.1002/ece3.2717
ORIGINAL RESEARCH
Environmental enrichment, sexual dimorphism, and brain size in sticklebacks Elisavet A. Toli1,2 | Kristina Noreikiene2 | Jacquelin DeFaveri2 | Juha Merilä2 1
Molecular Ecology & Conservation Genetics Lab, Department of Biological Applications & Technology, University of Ioannina, Ioannina, Greece 2
Ecological Genetics Research Unit, Department of Biosciences, University of Helsinki, Helsinki, Finland Correspondence Juha Merilä, Ecological Genetics Research Unit, Department of Biosciences, University of Helsinki, Helsinki, Finland. Email: [email protected] Funding information Biotieteiden ja Ympäristön Tutkimuksen Toimikunta, Grant/Award Number: 218343 and 286889; ERASMUS + Mobility Programme for Internship; Nordenskiöldsamfundet
Abstract Evidence for phenotypic plasticity in brain size and the size of different brain parts is widespread, but experimental investigations into this effect remain scarce and are usually conducted using individuals from a single population. As the costs and benefits of plasticity may differ among populations, the extent of brain plasticity may also differ from one population to another. In a common garden experiment conducted with three-spined sticklebacks (Gasterosteus aculeatus) originating from four different populations, we investigated whether environmental enrichment (aquaria provided with structural complexity) caused an increase in the brain size or size of different brain parts compared to controls (bare aquaria). We found no evidence for a positive effect of environmental enrichment on brain size or size of different brain parts in either of the sexes in any of the populations. However, in all populations, males had larger brains than females, and the degree of sexual size dimorphism (SSD) in relative brain size ranged from 5.1 to 11.6% across the populations. Evidence was also found for genetically based differences in relative brain size among populations, as well as for plasticity in the size of different brain parts, as evidenced by consistent size differences among replicate blocks that differed in their temperature. KEYWORDS
brain size, environmental enrichment, fish, phenotypic plasticity, sexual dimorphism
1 | INTRODUCTION
Hence, there appears to be a general consensus that intra- and inter-
Given the importance of the central nervous system to organismal
least to some extent, dictated by variation in the strength of positive
performance, and thereby also fitness, the size of the brain and its
natural selection acting on them.
specific variation in brain size and size of different brain parts is, at
different parts are likely to be traits under strong optimizing selec-
However, as maintenance of neural tissue is energetically
tion. Specifically, increased brain size can enhance individual fitness
expensive (Aiello & Wheeler, 1995; Isler & van Schaik, 2006, 2009;
through improved cognitive ability (Deaner, Isler, Burkart, & van
Mink, Blumenschine, & Adams, 1981; Nilsson, 1996; Soengas &
Schaik, 2007; Gibson, 2002; Kotrschal et al., 2013a,b; Striedter, 2005).
Aldegunde, 2002), these energetic costs are likely to generate selec-
Moreover, certain brain regions often show an increase in size that
tion pressures opposing increases in brain size (Isler & van Schaik,
is associated with specific ecological conditions likely to select for
2009; Kotrschal et al., 2013a). For instance, the fact that the two
this growth (Eifert et al., 2015; Gonzalez-Voyer & Kolm, 2010; Krebs,
sexes commonly differ in brain size and size of different brain parts
Sherry, Healy, Perry, & Vaccarino, 1989; de Winter & Oxnard, 2001).
(e.g., Jacobs, 1996; Kotrschal, Räsänen, Kristjánsson, Senn, & Kolm,
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. © 2017 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Ecology and Evolution. 2017;7:1691–1698.
www.ecolevol.org | 1691
|
TOLI
1692
et al
T A B L E 1 Descriptive information about study populations and samples. Age (in weeks) gives average of the individuals in each of the populations at time of brain measurements Country
Location
Sea area
Coordinates
Age salinity (ppt)
Germany
Sylt
North Sea
55°01′N, 08°25′E
26
Denmark
Mariager
Kattegat
56°38′N, 09°57′E
25
Finland
Kotka
Baltic Sea
60°33′N, 27°12′E
23
Finland
Oulu
Baltic Sea
65°07′N, 25°14′E
22
nFemales
nMales
28
21
16
37
20
15
21
36
6
8
21
29
3
18
19
37
62
77
139
Total
nTotal
N, sample size for brain measurements.
2012; Samuk, Iritani, & Schluter, 2014) is likely to be explainable
enrichment, but there remains little understanding of the causes
by sex differences in costs and benefits of maintaining certain sized
underlying this heterogeneity.
brain or brain regions. For instance, Nottebohm (1981) demon-
The main aims of this study were to test (1) whether environ-
strated that the song control nuclei in the telencephalon of canaries
mental enrichment leads to increased brain size (or size of different
(Serinus canarius) doubled in size during the breeding season, but
brain parts) in three-spined sticklebacks (Gasterosteus aculeatus),
only in males. Likewise, given the high energetic costs of maintaining
(2) whether the effect of enrichment is similar for the sexes, and (3)
neural tissue, the ability to reduce the size of the brain or a particular
whether the effects of enrichment and its interaction with sex are sim-
brain part through phenotypic plasticity when they are not critical
ilar across multiple populations. To this end, we conducted a common
for fitness should be favored by selection. It has been proposed that
garden experiment in which fish from four different populations were
the sexual dimorphism in the three-spined stickleback brain might
exposed to either a control (bare aquaria) or enriched (spatial com-
be subject to sex-specific plasticity such that males increase their
plexity generated with physical structures) treatment over a period of
brain size during the breeding season in response to the increased
2 months. Based on the results of an earlier experiment which found
cognitive demands imposed by mating, nest-guarding, and parental
that males developed larger brains in enriched tanks as compared
demands (Herczeg, Gonda, Balazs, Noreikiene, & Merilä, 2015). In
to females (Herczeg et al., 2015), we expected to see a similar sex-
this scenario, such plasticity could be adaptive, as the males would
specific response to environmental enrichment consistent across the
escape the energetic costs of maintaining large brains during the
four populations tested. Apart from assessing the treatment effects on
nonbreeding season.
brain size and size of different brain parts, we also investigated how
There is also evidence to suggest that various animals are capable of increasing their brain size in response to environmental enrichment
enrichment influenced growth (i.e., body size) and condition (i.e., residual mass) of the fish.
(Bennett, Diamond, Krech, & Rosenzweig, 1964; Bennett, Krech, & Rosenzweig, 1964; Bennett, Rosenzweig, & Diamond, 1969; Cummins, Walsh, Budtz-Olsen, Konstantinos, & Horsfall, 1973; Riege, 1971; Scotto Lomassese et al., 2000; Technau, 1984). However, the evidence for this effect from fish studies is conflicting. While some studies have
2 | MATERIAL AND METHODS 2.1 | Fish collecting and husbandry
supported this finding of positive effects of enrichment on brain size
Fish forming the parental generation were collected from four dif-
(DePasquale, Neuberger, Hirrlinger, & Braithwaite, 2016; Herczeg
ferent localities across the Baltic Sea between 5 and 24 June 2015
et al., 2015; Näslund, Aarestrup, Thomassen, & Johnsson, 2012), oth-
(Table 1). Live fish were transported to Helsinki and used to make arti-
ers have found either negative effects (Kotrschal, Sundström, Brelin,
ficial crosses between 6 June and 5 July 2015. Half of the Sylt and
Devlin, & Kolm, 2012; Turschwell and White, 2016) or none at all
Mariager fjord crosses were done in the field, and fertilized eggs were
(Burns, Saravanan, & Rodd, 2009; Kihslinger, Lema, & Nevitt, 2006).
transported to Helsinki in 50-ml tubes modified with a mesh bottom
This is true not only for overall brain size, but also in the size of cer-
for water circulation within a cool (10–14°C) and constantly aerated
tain brain regions such as the cerebellum, olfactory bulb, telenceph-
water bath. Fertilized eggs from 10 full-sib families from each popu-
alon, and optic tectum in which both positive (Herczeg et al., 2015;
lation were first kept in petri dishes until hatching and then trans-
Kihslinger & Nevitt, 2006; Kotrschal, Rogell, Maklakov, & Kolm, 2012;
ferred to 500-ml containers. Larvae were fed twice a day with live
Näslund et al., 2012) and negative (optic tectum; Herczeg et al., 2015)
Artemia salina nauplii. After 7 days, the families were transferred to
effects of environmental enrichment have been shown. However, in
1.2-L tanks in Allentown zebrafish racks (Allentown, San Diego, CA,
the cases where positive effects have been found, the effects have
USA), where they continued to be fed Artemia naupalii ad libitum for
been conditional to age (Näslund et al., 2012), sex, and social inter-
1–2 months. Each family was then divided into two separate 5-L tanks
actions (Herczeg et al., 2015; Kotrschal, Rogell et al., 2012) or other
on the zebrafish racks, each housing 20 fish. Finely chopped (frozen)
factors such as stress (DePasquale et al., 2016). Hence, there is a great
chironomid larvae were introduced into their diet by mixing with
deal of heterogeneity in the observed responses to environmental
Artemia for 1 month, after which the Artemia were eliminated and only
TOLI
|
1693
et al
(a)
(b)
F I G U R E 1 Frontal views of aquaria used in (a) control and (b) enriched treatments whole chironomid larvae were fed. Once the fish were 12–16 weeks
Experiments were terminated when the fish were between 22
old, four fish from each of the 10 families per population (i.e., a total
(Kotka and Oulu) and 26 (Sylt and Mariager fjord) weeks old by eutha-
of 160 fish) were randomly chosen to be used in the experiment (see
nizing the fish with an overdose (250 mg/L) of MS222 (tricaine meth-
below), which was conducted in a separate room.
ane sulphonate). In order to scale the size of the brain/different brain parts with individual variation in body size, the standard length (SL;
2.2 | Experiments The experiments were conducted in a room fitted with sixteen 38-L
from the tip of the mouth to the end of the tail base) of all individuals was measured with a digital caliper to the nearest 0.01 mm. Body weight was also recorded to the nearest 0.01 g.
aquaria (L:40 cm × H:30 cm × W:24 cm) distributed among two
Following measurements, fish were immediately placed in a solu-
shelves on two separate sides of the room (henceforth: blocks). Each
tion containing 4% paraformaldehyde and 2.5% glutaraldehyde in a
aquaria was assigned to one of two treatments, “enriched” or “control”.
phosphate-buffered saline solution and fixed for 5 days.
In the enriched treatment, the bottom of the aquaria was covered with ceramic pebbles, along with two artificial plants and a plastic cylinder (23 cm in height, 9 cm in diameter) to generate structural complexity. In the control treatment, the aquaria were left empty, except for
2.3 | Brain measurements and sexing Brains were dissected under a stereomicroscope by removing the top
the aerator, which was present in both treatments (Figure 1). In order
of the neurocranium and severing the cranial and optic nerves and
to control for possible aquaria and block effects, two replicate tanks
spinal cord. The caudal section of the medulla was cut between the
per population and treatment were used (i.e., four populations × two
second and the third vertebrae in each fish in order to standardize
treatments × two replicates = 16 aquaria). Each of the replicates was
the measurements. Hence, the “length” measurement for the medulla
placed on opposite sides of the room, such that each population and
did not represent the total length of the entire structure, but rather
treatment were represented (next to each other) on the same shelf
its length until a standardized cutoff point. Brains were kept in a
in both blocks. White plastic sheets were placed between aquaria to
phosphate-buffered saline solution until they were photographed
serve as visual partitions.
with a digital camera from the dorsal, lateral, and ventral sides, from a
The experiments started by introducing one fish from each of the
fixed distance. Width, height, and length of six different brain regions
10 full-sib families per population into a particular treatment aquaria.
(viz. olfactory bulb, optic tectum, telencephalon, cerebellum, dorsal
Hence, each aquaria housed 10 unrelated fish from a given population.
medulla, and hypothalamus) were measured with ImageJ (Abràmoff,
Fish were kept under constant light to mimic summer conditions at the
Magalhães, & Ram, 2004) using landmarks shown in Appendix S1, fol-
northern latitudes and fed with frozen chironomid larvae (ad libitum)
lowing Pollen et al. (2007). For bilateral brain parts, both sides were
twice daily.
measured and their average was used in all analyses. These data were
All fish were raised in freshwater, which was maintained at about
then fitted to the ellipsoid models (van Staaden, Huber, Kaufman, &
17°C (±1°C). However, due to temperature stratification in the exper-
Liem, 1994) to estimate the volume of total brain size and size of the
imental room, fish in the two blocks experienced slightly different
different brain parts. The ellipsoid-model approach is known to yield
temperatures (A block: x = 17.5°C; max–min = 17.2–17.9°C; B block:
reliable estimates of brain and brain part sizes, as verified by compari-
x = 16.5°C; max–min = 16.4–16.9°C).
sons with histology and X-ray micro-computed tomography-based
|
TOLI
1694
et al
T A B L E 2 Linear model results of brain and body size traits. Tabled values are F-values from linear models for treatment (Tre), population (Pop), sex, block (Blo), standard length (SL), and for sex–treatment interaction. SSD(%) gives the degree of sexual dimorphism (in %), Tre (%) the degree of difference between treatment means as calculated from the back-transformed least-square means in the model. For SSD (%), positive values indicate male-biased SSD, and negative values, female; for Tre (%), positive values indicate larger trait mean in control and negative values indicate larger trait mean in treatment conditions. Tank refers to the proportion of total variance in a given trait explained by the random effect of tank Trait
Tre
Pop
Sex *
T × S ***
Blo
SL *
Tank
SSD(%)
Tre(%)
***
Brain (wgt)
0.18
5.86
35.05
0.45
5.59
276.72
0.0
10.2
−0.6
Brain (vol)
0.03
6.95**
22.87***
0.30
2.15
272.86***
6.3
8.0
0.3
***
0.1
5.1
−1.5
64.35***
2.4
11.6
−0.2
Dorsal medulla
0.20
3.74
Telencephalon
0.00
1.92
Optic tectum
0.12
8.41
Cerebellum
1.12
1.95
*
**
2.35
0.21
3.35°
18.35***
0.12
0.43
**
9.63
0.88
9.91**
0.39
101.49
4.29°
***
150.79
0.0
8.0
−0.9
7.04*
129.55***
0.0
9.1
2.7
**
Olfactory bulb
0.00
2.17
0.01
0.00
0.03
8.47
0.2
0.6
−0.2
Hypothalamus
0.10
1.66
8.91**
0.17
0.25
67.82***
25.4
10.3
−2.1
***
***
Standard length
0.29
13.56
19.29
0.17
4.42°
–
0.0
−6.3
−0.5
Body mass
0.64
2.47
31.07***
0.13
0.00
–
4.0
−28.4
−4.2
1.33
***
19.4
−9.3
−2.7
Condition
0.53
17.00
0.23
1.89
***
549.35
p
