University of Groningen
Dose-response effects of light at night on the reproductive physiology of great tits (Parus major)
Dominoni, Davide M.; de Jong, Maaike; Bellingham, Michelle; O'Shaughnessy, Peter; van Oers, Kees; Robinson, Jane; Smith, Bethany; Visser, Marcel E.; Helm, Barbara
Published in:
Journal of Experimental Zoology Part A: Ecological and Integrative Physiology
DOI:
10.1002/jez.2214
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Publication date: 2018
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Dominoni, D. M., de Jong, M., Bellingham, M., O'Shaughnessy, P., van Oers, K., Robinson, J., Smith, B., Visser, M. E., & Helm, B. (2018). Dose-response effects of light at night on the reproductive physiology of great tits (Parus major): Integrating morphological analyses with candidate gene expression. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, 329(8-9), 473-487.
https://doi.org/10.1002/jez.2214
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1
Dose-response effects of light at night on the reproductive physiology of
1
great tits (Parus major): integrating morphological analyses with candidate
2
gene expression
3
4 5
Davide M. Dominoni1,2 *, Maaike de Jong2, Michelle Bellingham1, Peter O’Shaughnessy1, 6
Kees van Oers2, Jane Robinson1, Bethany Smith1, Marcel E. Visser2, Barbara Helm1,3 7
8
1 Institute of Biodiversity, Animal Health and Comparative Medicine – University of
9
Glasgow, Glasgow, UK 10
2 Department of Animal Ecology, Netherlands Institute of Ecology (NIOO-KNAW),
11
Wageningen, The Netherlands 12
3 GELIFES - Groningen Institute for Evolutionary Life Sciences, University of Groningen,
13
Groningen, The Netherlands 14
15
*Correspondence to: Institute of Biodiversity, Animal Health and Comparative Medicine – 16
University of Glasgow, Glasgow, UK. E-mail: davide.dominoni@glasgow.ac.uk. 17
18
Total number of text figures, graphs and charts: 8 19
Running headline: Avian reproductive response to light at night 20
21
Funding information: Funding of DMD and of gene analysis was provided by a Marie-22
Curie Career Integration Grant to BH [EC CIG (618578) Wildclocks] and by the Wellcome 23
Trust [097821/Z/11/Z]. 24
2 Abstract
25
Artificial light at night (ALAN) is increasingly recognised as a potential threat to wildlife and 26
ecosystem health. Among the ecological effects of ALAN, changes in reproductive timing are 27
frequently reported, but the mechanisms underlying this relationship are still poorly 28
understood. Here, we experimentally investigated these mechanisms by assessing dose-29
dependent photoperiodic responses to ALAN in the great tit (Parus major). We individually 30
exposed photosensitive male birds to one of three nocturnal light levels (0.5, 1.5 and 5 lux), 31
or to a dark control. Subsequent histological and molecular analyses on their testes indicated 32
a dose-dependent reproductive response to ALAN. Specifically, different stages of gonadal 33
growth were activated after exposure to different levels of light at night. mRNA transcript 34
levels of genes linked to the development of germ cells (stra8 and spo11) were increased 35
under 0.5 lux compared to the dark control. The 0.5 and 1.5 lux groups showed slight 36
increases in testis size and transcript levels associated with steroid synthesis (lhr and hsd3b1) 37
and spermatogenesis (fshr, wt1, sox9 and cldn11), although spermatogenesis was not detected 38
in histological analysis. In contrast, all birds under 5 lux had 10 to 30 times larger testes than 39
birds in all other groups, with a parallel strong increase in mRNA transcript levels and clear 40
signs of spermatogenesis. Across treatments, the volume of the testes was generally a good 41
predictor of testicular transcript levels. Overall, our findings indicate that even small changes 42
in nocturnal light intensity can increase, or decrease, effects on the reproductive physiology 43
of wild organisms. 44
45
Keywords: ALAN, testis, timing of reproduction, HPG axis, urbanization, spermatogenesis 46
3
Introduction
48
Artificial light at night (ALAN) is one of the most evident anthropogenic modifications of the 49
natural environment (Falchi et al., 2016; Kyba et al., 2017). As natural and rural lands are 50
increasingly converted into urban areas globally, and in particular in developing countries, 51
the proportion of the Earth exposed to ALAN is also increasing (Gaston et al., 2015b; Falchi 52
et al., 2016; Kyba et al., 2017). This increase has been recently quantified as 6 % per year 53
worldwide (Falchi et al., 2016). The presence of light pollution alters natural regimes of light 54
and darkness (Davies et al., 2013), and this can have important consequences for human 55
health and economy (Gaston et al., 2014). The ecological consequences of ALAN were first 56
recognised in the early 20th century (Rowan, 1937), but have only recently become an 57
important focus of scientific research. ALAN is being increasingly associated with changes in 58
behaviour, physiology and life histories of wild organisms, from plants to invertebrates, 59
fishes, amphibians, reptiles, birds and mammals (Perry et al., 2008; Bruening et al., 2011; 60
Davies et al., 2012; Da Silva et al., 2015; Jones et al., 2015; Bennie et al., 2016; Knop et al., 61
2017; Spoelstra et al., 2017). 62
The effects of ALAN on seasonal cycles, for instance reproduction, have been a key 63
focus of light pollution research (Dominoni, 2015; Robert et al., 2015; Bruening et al., 2016; 64
Gaston et al., 2017). For example, clear evidence for a direct effect of ALAN on reproduction 65
comes from experimental studies on birds in controlled environments. European blackbirds 66
(Turdus merula) exposed to 0.3 lux of ALAN began to grow their gonads three weeks earlier 67
than conspecifics exposed to dark nights (Dominoni et al., 2013a). Birds have also been the 68
focus of experimental studies in the field, although these have not measured gonadal growth. 69
For instance, great tits (Parus major) living in areas polluted with white LEDs laid their eggs 70
5 days earlier than in dark areas, although only in cold springs (de Jong et al., 2015). In 71
addition, a correlational study has shown that female blue tits (Cyanistes careuleus) breeding 72
4
in proximity of street lights laid their first egg 1.5 days earlier than females breeding in dark 73
areas (Kempenaers et al., 2010). Other field studies have focused on the phenology of dawn 74
song and morning activity. These studies have shown that ALAN is associated with an 75
advancement of dawn song earlier in the morning (with the exception of Da Silva et al., 76
2017), and also earlier in the season, an effect that is suggestive of an earlier propensity to 77
breed (Miller, 2006; Kempenaers et al., 2010; Nordt and Klenke, 2013; Da Silva et al., 2015; 78
Dominoni and Partecke, 2015). In contrast to advanced reproductive behaviours in birds, 79
other taxa have been shown to delay reproduction in response to ALAN, presumably as a 80
consequence of short-day breeding. Examples include delayed average birth time of tammar 81
wallabies (Macropus eugenii) (Robert et al., 2015) or completely inhibited reproductive 82
physiology of adult perch (Perca fluviatilis) (Bruening et al., 2016). Similarly, in insects, 83
ALAN has been shown to disrupt mating behaviour and delay the time to pupation of moths 84
(van Geffen et al., 2014, 2015). 85
Effects of ALAN on reproduction are intuitive given that most organisms use 86
photoperiod to time the development of their reproductive system in anticipation of the 87
expected annual peak in resource availability (Bradshaw and Holzapfel, 2010; Helm et al., 88
2013). Use of reliable timing cues is particularly important for animals that substantially re-89
grow their reproductive organs on an annual basis, such as birds, where the process of 90
gonadal development requires several weeks to be completed. In temperate areas, for long-91
day breeders the increasing day length of late winter/early spring is the best proximate cue to 92
start gonadal development in advance of breeding (Dawson et al., 2001). This is because day 93
length, unlike temperature and food availability, shows little inter-annual variation. 94
Experimental exposure to long days causes birds to enter into reproductive state relatively 95
quickly (Rowan, 1938; Follett and Sharp, 1969; Follett et al., 1974; te Marvelde et al., 2012). 96
However, other environmental factors such as temperature and food availability fine-tune the 97
5
final breeding decisions, in particular the timing of egg-laying (Schoech and Hahn, 2007; 98
Ball, 2012; Schaper et al., 2012b). Therefore, variation in timing of reproductive physiology 99
does not necessarily predict variation in egg-laying dates, but defines its scope (Schaper et 100
al., 2012a; te Marvelde et al., 2012). 101
In birds, reproductive activation is mediated by the hypothalamus-pituitary-gonadal 102
(HPG) axis via photo-stimulation of deep-brain photoreceptors. Increasing day lengths 103
stimulate the secretion of gonadotropin-release hormone (GnRH) in the hypothalamus 104
(Nakane et al., 2010). GnRH then activates the pituitary gland that releases follicle-105
stimulating hormone (FSH) and luteinizing hormone (LH) into the blood stream (Dawson, 106
2002; Sharp, 2005) (Fig. 1). LH and FSH bind to their specific receptors (lhr and fshr). In the 107
males’ testes they activate Leydig and Sertoli cells, respectively (Brown et al., 1975). LH 108
stimulates Leydig cells to produce androgens, mediated by enzymes such as cyp11a1, 109
cyp17a1, as well as several dehydrogenases (for instance hsd3b1, required for progesterone
110
production, and hsd17b3, involved in the synthesis of testosterone) (Brown et al., 1975; 111
Purcell and Wilson, 1975). Sertoli cells are somatic cells that are essential for the 112
development of the testis and spermatogenesis (Fig. 1) (Thurston and Korn, 2000). They are 113
located in the seminiferous tubules of the testis and promote spermatogenesis through 114
formation of a blood-testis barrier and through direct interactions with the developing germ 115
cells. Sertoli cell activity depends on FSH stimulation and on androgens secreted by the 116
Leydig cells (da Silva et al., 1996; Thurston and Korn, 2000). In the absence of hormone 117
stimulation of the Sertoli cells, germ cell development does not progress beyond early 118
meiosis (O’Shaughnessy, 2014). 119
During gonadal development in birds transcript levels of glucocorticoid (nr3c1, also 120
referred to as gr) and mineralocorticoid receptors (nr3c2, also referred to as mr) also increase 121
(Kirby et al., 2009; Lattin et al., 2012; McGuire et al., 2013; Fudickar et al., 2017). This 122
6
provides a mechanistic basis for the widespread links between adrenal steroids and timing of 123
reproduction and reproductive investment reported in several bird species (Schoech et al., 124
2009; Deviche et al., 2010; Goutte et al., 2010a; Crespi et al., 2013; McGuire et al., 2013; 125
Lattin et al., 2016). Indeed, during stressful periods sex steroid production and 126
spermatogenesis can be suppressed via several routes, one of which is by increased 127
glucocorticoid levels (McGuire et al., 2013; Hazra et al., 2014; Blas, 2015; Witorsch, 2016). 128
Since ALAN has been linked to higher baseline corticosterone levels in birds (Ouyang et al., 129
2015), it could also influence reproductive activation, although it would be expected to slow, 130
not accelerate, testicular development. Alternatively, upregulation of gonadal receptors for 131
adrenal steroids could be a mechanism to enhance growth of the reproductive system, which 132
is an energetically costly process requiring metabolic activation by adrenal steroids 133
(Wingfield and Farner, 1993). 134
7
Fig. 1. Simplified scheme illustrating the structure and function of the avian HPG axis. Light 136
stimulates deep-brain photoreceptors which promote the release of GnRH from the 137
hypothalamus to the anterior pituitary. This releases FSH and LH into the blood circulation, 138
which then arrive at the testes binding to their receptors located on Sertoli and Leydig cells, 139
respectively. Sertoli cells stimulate the rapid and massive proliferation of germ cells which 140
then lead to sperm production, while Leydig cells stimulate the production of steroids. 141
Steroid hormones such as testosterone negatively feedback on the hypothalamus and pituitary 142
to stop further release of GnRH, FSH and LH. 143
144
Despite the increasing interest in the effects of light pollution on reproduction of wild 145
animals, we have still little understanding of the sensitivity of day length detection and of the 146
ensuing sequence of reproductive activation events to ALAN. The aim of our study was thus 147
to investigate the sensitivity of different stages of gonadal development to a realistic range of 148
light intensities, derived from recordings from free-living birds (Dominoni et al., 2013a). We 149
tested for the existence of dose-dependent, or alternatively, threshold responses, of the 150
reproductive system to increasing levels of ALAN. As study subject we chose a songbird, the 151
great tit, which was previously reported to show behavioural responses to similar levels of 152
ALAN (de Jong et al., 2016a). We used a between-animal design in which we exposed 153
captive male great tits to three different nocturnal light treatments (0.5, 1.5 and 5 lux) or to 154
dark nights in late winter. After three weeks of exposure birds were sacrificed and their testes 155
collected. We recorded testicular sizes for comparison with laparotomy data measured in 156
field and captivity studies of living birds (e.g., (Partecke et al., 2005)). These measures were 157
complemented by morphological, histological and molecular analyses. We assessed mRNA 158
transcript levels in the testis for 10 candidate genes implicated in morphological development 159
of the reproductive system, in synthesis of steroids and in promotion of spermatogenesis 160
(Table 1). More specifically, these genes are involved in germ cell development (stra8 and 161
spo11), Sertoli cell activation and spermatogenesis (fshr, wt1, sox9, cldn11), Leydig cell
162
activation and steroid synthesis (lhr and hsd3b1), and adrenal steroid function (gr and mr) 163
(Table 1, Fig. 1). 164
8 Overall, we hypothesised that:
165
1. Increasing light intensity at night would lead to larger testicular volume and advanced 166
morphological differentiation. 167
2. Changes in size and morphology would be paralleled by changes in transcript levels 168
of functionally related genes. Higher light levels should progressively activate early 169
stages of gonadal development, such as germ cells development (stra8 and spo11), 170
activation of Sertoli (fshr, wt1, sox9 and cldn11) and Leydig cells (lhR and hsd3b1), 171
and complete spermatogenesis. Whether or not a set of functionally related genes 172
would show increased mRNA levels would depend on light intensity. 173
3. Since reproductive activation has been linked to an increased expression of adrenal 174
steroid receptors, we also expect increased ALAN levels to be associated with 175
increased adrenal steroid receptors. Alternatively, reproductive activation could be 176
this association could be counteracted by elevated circulating CORT levels, which 177
have been associated to ALAN. 178
4. Levels of mRNA transcripts encoding cell-specific proteins would be related to testis 179
size at the individual level. 180
181
Materials and methods
182
Animals and experimental set-up
183
We conducted the experiment between Feb 1st and Feb 23rd 2014. We used 34 adult male
184
great tits that had been used in a previous experiment aimed at assessing the impact of 185
different levels of light intensity at night on daily activity and physiology (de Jong et al., 186
2016a). All birds had been hand-raised and housed at the Netherlands Institute of Ecology 187
(NIOO- KNAW), Wageningen, The Netherlands, in individual cages (90 cm × 50 cm × 40 188
cm). All birds were between one and four years of age (hatched in 2012 or before), but mean 189
9
age did not differ between treatment (P= 0.576). Temperature was maintained between 10 190
and 14 °C, and did not vary structurally between day- and night-time. Birds had access to 191
food and water ad libitum. 192
During the ALAN experiment, birds were kept under fixed natural daylength of 10h 193
light and 14h darkness. Each cage had two separate light sources for day- and night-time 194
illumination. We used dividers between the cages, so that birds could only hear but not see 195
each other, and light from one cage did not influence the light environment in adjacent cages 196
because we had placed a wooden plate in the front of the cage. During the daytime, all birds 197
were exposed to full spectrum daylight by high frequency fluorescent lights emitting ±1000 198
lux at perch level (Activa 172, Philips, Eindhoven, The Netherlands). During the night-time, 199
birds were assigned to different treatment groups that varied in the level of light intensity 200
used (warm white LED light; Philips, Eindhoven, The Netherlands). The spectral 201
composition of this light is shown in Fig. S1 as part of the description of the preceding 202
experiment mentioned in the introduction (de Jong et al., 2016a). In this earlier experiment, 203
the birds were exposed to five levels of ALAN (0.05; 0.15; 0.5; 1.5 and 5 lux) for one month 204
between Dec 10th 2013 and Jan 10th 2014 and otherwise kept under dark nights. The 205
experimental set-up we used here differed in that we used four and not five experimental 206
levels of ALAN, of which one was a dark control. The birds in the dark control were derived 207
from the two earlier treatment groups with the lowest light intensity (0.05 lux and 0.15 lux, 208
respectively), while birds in all other treatments were kept in the same treatment that they 209
were exposed to in the previous experiment. Thus, from the start of our present experiment 210
on 1st Feb 2014, the birds were exposed for the entire night to either one out of three 211
nocturnal light intensities measured at perch level in the cages: 0.5 lux (n=7), 1.5 lux (n=7) or 212
5 lux (n=7), or to dark control conditions (n=13). For details on the spectral composition of 213
lights, see the supplementary information of (de Jong et al., 2016a). 214
10
The four treatment groups were assigned to one of seven blocks of cages arranged 215
within two experimental rooms. Each block contained all treatment groups, distributed using 216
a Latin Squares design. The birds were kept under these conditions for three weeks, and were 217
then killed to collect tissues for morphological and molecular analyses. We sacrificed birds 218
under isoflurane anaesthesia (Forene, Abbott, Hoofddorp, The Netherlands) at midday (± 2 219
hours) on February 22nd or midnight (± 2 hours) on both February 22nd and 23rd, 2014. We 220
used two sampling times to assess day-night differences in transcript levels for a separate 221
study. Organs were extracted, snap-frozen on dry ice, and stored at -80oC within 10 min of 222
capture. Testes samples were then shipped to Glasgow, UK, in dry ice and stored at -80oC
223
until further use. All experimental procedures were carried out under licence NIOO 13.11 of 224
the Animal Experimentation Committee (DEC) of the Royal Netherlands Academy of Arts 225
and Sciences (KNAW). 226
227
Morphological and histological analyses
228
On the 4th of June 2014 one frozen testis from each bird was placed into Neutral Buffered 229
Formalin overnight to fix and preserve the tissue. Testes were weighed and their length and 230
width measured before being placed into 70% ethanol. Testes were then embedded in wax, 231
sectioned and stained with hematoxylin and eosin for visualisation of tissue structure. Tubule 232
diameter and tubule area (where round tubular sections could be seen) were measured using 233
ImageJ (NIH Image, https://imagej.net) and the development of spermatogenesis assessed. 234
We suspected that freezing might have caused the testes to change in size from the 235
original, freshly measured state. We used measurements on fresh and frozen testes obtained 236
from male great tits in another experiment to test this hypothesis (data are courtesy of Irene 237
Verhagen). Testes length and width were highly correlated between fresh and frozen 238
11
measurements (percentage change ± s.d.: width = 8.43 ± 9.84, length = 6.43 ± 11.13; P < 239
0.001 and r= 0.98 in both cases; N = 36; Fig. S1). 240
12 Table 1: List of gene transcripts measured and function of the relevant protein. 241
General function
Gene
acronym Specific function References
Germ cells development
Germ cells development stra8 Regulation of meiotic initiation (Krentz et al., 2011; Zhang et al., 2015) Germ cells development spo11 Involved in meiotic recombination (Oréal et al., 2002; Guioli et al., 2014) Sertoli cells activity (spermatogenesis)
Sertoli cells activity fshr Gonadotropin receptor, stimulates spermatogenesis (Yamamura et al., 2000; Akazome et al., 2002) Sertoli cells activity wt1 Marker of Sertoli cells activity (Kent et al., 1995; Oréal et al., 2002)
Sertoli cells activity sox9 Required for Sertoli cell differentiation and marker of adult Sertoli cell (da Silva et al., 1996; Lee et al., 2007) Sertoli cells activity cldn11 Involved in the formation of tight junctions (blood testis barrier) (Gunzel and Yu, 2013)
Leydig cells activity (steroid synthesis)
Leydig cells activity lhr Leydig cell gonadotropin receptor, stimulates steroid synthesis (Yamamura et al., 2000; Akazome et al., 2002) Leydig cells activity hsd3b1 Enzyme required for androgen synthesis (Lee et al., 2007; London and Clayton, 2010) Adrenal steroid receptors
Stress and metabolism nr3c1 (gr) Glucocorticoid receptor (Landys et al., 2004; Liebl and Martin, 2013) Stress and metabolism nr3c2 (mr) Mineralocorticoid receptor (Landys et al., 2004; Liebl and Martin, 2013)
242 243 244 245 246 247 248
13
Transcript analysis
249
The second testis was used for assessing mRNA transcript levels. Tissue was homogenised 250
with a ribolyzer, and RNA extracted using 0.5 ml of Trizol (Thermofisher, UK) per sample. 251
During the extraction, 5ng of luciferase mRNA (Promega U.K., Southampton, UK) was 252
added to each sample to serve as an external control (Rebourcet et al., 2014). Total RNA was 253
reverse transcribed to generate cDNA using random hexamers and Moloney murine 254
leukaemia virus reverse transcriptase (Superscript III, Life Technologies) as described 255
previously (O’Shaughnessy and Murphy, 1993). 256
Primers for the 10 candidate genes (Table S1) were designed using the great tit 257
genome version 1.03 (Laine et al., 2016), and intron/exon boundaries were identified through 258
BLAST against the zebra finch (Taeniopygia guttata) genome (Warren et al., 2010). Primers 259
were designed using Primer ExpressTM 2.0.0 (Applied Biosystems) using parameters 260
described previously (Czechowski et al., 2004; O’Shaughnessy et al., 2008). In order to avoid 261
genomic DNA (gDNA) amplification, every primer pair was designed to span an intron of 262
more than 1,000 base pairs. 263
Real-time PCR used the SYBR green method with a Stratagene MX3000 cycler 96-264
well plates.Reactions contained 5 µl 2xSYBR mastermix (Agilent Technologies, 265
Wokingham, UK), primers (100 nM) and template in a total volume of 10 µl. The thermal 266
profile used for amplification was 95oC for 8 min followed by 40 cycles of 95oC for 25 s,
267
63oC for 25 s, and 72 oC for 30 s. At the end of the amplification phase a melting curve 268
analysis was carried out on the products formed. We ran one transcript on each plate. Each 269
sample was run in duplicate, and in each plate we also included duplicate negative control 270
wells (with RNA instead of cDNA). None of the primer pairs amplified gDNA and the 271
efficiency of the qPCR reactions was always between 95 and 103 %. Levels of transcripts 272
14
encoding genes of interest were quantified relative to the luciferase external standard 273
(luciferase) using the delta Ct method levels (Baker and O’Shaughnessy, 2001). 274
275
Statistical analyses
276
We ran all analyses in the statistical environment R (R Development Core Team, 2015). All 277
models were linear mixed effects (LMMs) with block nested into room as random effect, to 278
account for possible effects of location of the cage, and a Gaussian error structure. Treatment 279
(continuous variable with four levels: dark (=0 lux), 0.5, 1.5 and 5 lux), time (factor with two 280
levels: day and night), the interaction of treatment and time, and age were always included as 281
explanatory variables in the initial maximal models, unless specified otherwise. In the 282
preliminary models we also always included the second order polynomial effect of treatment 283
to test for potential quadratic effect of increasing light intensities. Model selection was done 284
by backward stepwise deletion of non-significant terms. When treatment or time were found 285
to be significant, post-hoc tests were done by comparing estimated marginal means and 286
confidence intervals (CI) of the estimates for each level, using the function emmeans in the R 287
package emmeans (https://cran.r-project.org/web/packages/emmeans/index.html). Two levels 288
were considered to be significantly different if the estimated marginal mean (Tukey’s 289
corrected) for one level was not included in the CI of another level. Assumptions for using 290
linear models (normality and homogeneity of residuals) were met. 291
We first ran two separate LMMs to assess changes in morphology, with either testis 292
volume or tubule diameter as response variables. In these models we neither included time 293
nor the interaction of time and treatment as explanatory variables, because we did not expect 294
any change between day and night on the weekend that birds were sacrificed. We then ran 295
four LMMs using grouped transcript levels as response variables. Genes were grouped based 296
on their function as in Table 1, resulting in four groups: germ cells development (stra8 and 297
15
spo11), Sertoli cells activity (fshr, wt1, sox9 and cldn11), Leydig cells activity (lhr and
298
hsd3b1) and adrenal steroid receptors (gr and mr). In these models individual ID was nested
299
into block nested into room as random effect to correct for multiple transcript measurements 300
of individuals in each model. Transcript levels were standardized within each group by 301
calculating Z-scores. To do so, we first calculated the mean and standard deviation of the 302
entire vector of transcript levels within a functional group. Then, each value was re-303
calculated by subtracting the mean and then divided by the standard deviation. Finally, we 304
took a closer look at transcript levels of individual target genes by running 10 additional 305
LMMs with the 10 target genes as response variables, log-transformed. 306
To test for a potential relationship between testes size and gene transcript levels, we 307
selected a single trait for testes size as response variable. Since testis volume was highly 308
correlated to tubule diameter (r = 0.95 and P < 0.001), we decided to select testis volume 309
because it is commonly taken on live birds through laparotomy, therefore enabling 310
comparison with other studies. We then ran independent LMMs for each gene where 311
transcript levels were the response variable, and testis volume, treatment, the quadratic effect 312
of treatment, age, mass and the interaction between testis volume and treatment were 313
modelled as the explanatory variables. 314
315
Results
316
Morphology and histology
317
All our models showed a significant increase in testis volume and tubule diameter with 318
increasing light intensity (P < 0.001 in both cases, Table S2). For testis volume this 319
relationship was quadratic, while for tubule diameter this was linear. Post-hoc comparisons of 320
estimated means and confidence intervals indicated that there was a slight, non-significant 321
increase in testis volume between the dark group and the 0.5 lux group, and a much larger, 322
16
highly significant increase between 0.5-1.5 lux and between 1.5-5 lux (Fig. 2a, Table S2). 323
Indeed, the testes of the 5 lux birds were 6 times larger than those of the birds in the 1.5 lux 324
group (Fig. 2a and Table S2). Tubule diameter was found to be significantly different 325
between all treatment groups, with a particularly strong increase between 1.5 and 5 lux, 326
although with a smaller effect size compared to the testis volume (Fig. 2b, Fig. 3, Table S2). 327
Spermatogenesis was detected in all 5 lux testes, but not in the testes of any other treatment 328
group (Fig. 3). Age was not a significant predictor of either testis volume (P=0.79) or tubule 329
diameter (0.94). 330
331
332
Figure 2. Light intensity at night (continuous variable) affects testis volume (a) and tubule 333
diameter (b). Data are presented as mean ± s.e.m. Sample sizes were: dark = 13; 0.5 lux = 7; 334
1.5 lux = 7; 5 lux = 7. 335
17 337
Figure 3. Testis histology. Photomicrographs of representative testes sections for each 338
treatment. A marked increase in the size of the seminiferous tubules (diameter and total area 339
inside the tubule, also called lumen) can be seen in the 5 lux example. The small dots visible 340
in all treatments are germ cells. Complete spermatogenesis can be seen in the 5 lux group by 341
the presence of all stages of germ cell development including elongated spermatids (dark 342 dots). 343 344 Gene expression 345
The analysis of functionally grouped transcripts revealed progressive activation on different 346
stages of gonadal growth with increasing light intensity. Transcript levels of all functional 347
groups analysed were significantly increased by light exposure (P < 0.001 in all cases, Table 348
2). However, for the germ cell development group, we found significant differences between 349
all levels of the variable treatment, whereas for all other functional groups we found 350
significantly increased transcript levels only at 5 lux (Table 2). In addition, for the germ cell 351
development and the corticoid receptor groups, transcript levels were significantly higher 352
when birds were sacrificed during the day compared to when they were euthanized during the 353
night (Table 2). However, for the corticoid receptors the effect of time of sacrifice depended 354
on the treatment level (treatment*time interaction, P < 0.001): daytime and night-time 355
18
transcript levels were similar in the dark control group and in the 0.5 lux group, while at 1.5 356
lux and 5 lux, daytime levels were significantly higher than night-time ones (Table 2). 357
358
19
Figure 4. Testicular transcript levels in birds exposed to different levels of light based on 360
real-time PCR (mean ± SEM). Treatment was a continuous variable in all models. Open and 361
closed bars represent birds culled at noon or midnight respectively. Sample sizes: dark: 362
day=6, night=7; 0.5 lux: day=2, night=5; 1.5 lux: day=2, night=5; 5 lux: day=2, night=5. 363
364
The individual analyses of transcript levels of all genes revealed similar patterns to 365
the functional groups analysis. Transcript levels were significantly increased by light 366
exposure (P < 0.001 in all cases, Fig. 4 and Table S3). For spo11, we detected significant 367
changes over all light levels and both at day and at night, while this was not true for stra8, the 368
other marker of germ cell development, for which we only found significant changes between 369
0.5-1.5 lux and 1.5-5 lux. 370
The general pattern for the other genes indicated that mRNA transcript levels were 371
low and not different between the dark group and the 0.5 lux group. Then, transcript levels 372
increased slightly but significantly between the 0.5 and the 1.5 lux group (except for hsd3b1), 373
and finally peaked at 5 lux, with a minimum increase of 20 % compared to the 1.5 lux 374
treatment for all genes (Fig. 4 and Table S3). The time at which the gonads were harvested 375
had little effect on these transcript levels. The variable time was only found to be significant 376
for stra8, spo11 and gr, which showed reduced transcript levels at night compared to daytime 377
(Table S3), similarly to what we found in the functional group analysis. In addition, for 5 out 378
of the 10 transcripts measured (gr, lhr, fshr, hsd3b1 and sox9), there was a positive, 379
significant relationship between age of a bird and transcript levels (Table S3), with older 380
birds showing higher gene expression. 381
In our separate analysis of testis size, we found highly significant, positive 382
relationships between testis volume and transcript levels for all genes analysed (Table S4 and 383
Fig. 5). However, the slope of these relationships was not different between treatments. 384
385 386
20
Main model Post-hoc test
Germ cells development (stra8 and spo11) Germ cells development (stra8 and spo11)
Estimate SEM df t p Treatment Estimate SEM lower CI upper CI sig
Intercept -0.37 0.13 30 -2.86 0.008 dark -0.57 0.09 -0.76 -0.38 a Treatment 0.43 0.04 30 11.40 <0.001 0.5 -0.35 0.08 -0.52 -0.18 b Time -0.40 0.15 30 -2.66 0.012 1.5 0.08 0.08 -0.07 0.24 c 5 1.60 0.16 1.28 1.92 d Sertoli cells activity (fshr, wt1, sox9, cldn11) Sertoli cells activity (fshr, wt1, sox9, cldn11)
Estimate SEM df t p Treatment Estimate SEM lower CI upper CI sig
Intercept -0.29 0.10 130 -2.89 0.004 dark -0.29 0.10 -0.50 -0.09 a Treatment 0.20 0.04 130 4.83 <0.001 0.5 -0.19 0.09 -0.37 -0.01 a 1.5 0.01 0.08 -0.15 0.18 b 5 0.72 0.17 0.37 1.06 c Leydig cells activity (lhr and hsd3b1) Leydig cells activity (lhr and hsd3b1)
Estimate SEM df t p Treatment Estimate SEM lower CI upper CI sig
Intercept -0.93 0.20 30 -4.73 <0.001 dark -0.35 0.12 -0.61 -0.10 a Treatment 0.29 0.05 30 5.56 <0.001 0.5 -0.21 0.11 -0.44 0.02 a Age 0.25 0.07 30 3.45 0.002 1.5 0.08 0.10 -0.13 0.28 b 5 1.08 0.21 0.65 1.51 c
Corticoid receptors (gr and mr) Corticoid receptors (gr and mr)
Estimate SEM df t p Treatment Time Estimate SEM lower CI upper CI sig
Intercept -0.44 0.16 62 -2.80 0.007 dark day -0.44 0.16 -0.76 -0.12 a Treatment 0.59 0.07 62 8.06 <0.001 0.5 day -0.14 0.14 -0.43 0.14 a Time -0.09 0.20 62 -0.45 0.654 1.5 day 0.45 0.13 0.17 0.72 b Treatment*Time -0.35 0.09 62 -3.90 <0.001 5 day 2.52 0.31 1.88 3.15 c dark night -0.53 0.13 -0.79 -0.27 a 0.5 night -0.41 0.11 -0.64 -0.18 a 1.5 night -0.17 0.10 -0.37 0.04 d 5 night 0.69 0.20 0.28 1.09 e 387
Table 2. Summary of model outputs for gene expression data with transcripts grouped by 388
functional traits. All models were linear mixed models with Gaussian error structure with 389
treatment, time, age, mass and the interaction treatment*time as explanatory variables, and 390
individual ID nested into block (position of a cage within the wall of an experimental room) 391
as random variable, to correct for multiple transcript measurements per individual. All 392
transcript levels were standardised (z-scores) to enable using them as grouped response 393
variable. Post-hoc tests were done by comparing the confidence intervals (CI) of the 394
estimates (marginal means). Two levels were considered to be significantly different if the 395
mean estimate for one level was not included in the CI of the other level, and such differences 396
are indicated in the column “sig”. Reference level for treatment is the dark group, for time it 397
is daytime. Sample sizes: dark: day=6, night=7; 0.5 lux: day=2, night=5; 1.5 lux: day=2, 398
night=5; 5 lux: day=2, night=5. 399
21 400
401
Figure 5. Relationship between testicular volume and relative mRNA transcript levels. Each 402
point in the figures represents one individual bird. Lines and shaded areas represent mean 403
predicted values ± confidence intervals obtained from linear mixed models. Sample sizes 404
were: dark = 13 (crosses), 0.5 lux = 7 (triangles), 1.5 lux = 7 (squares), 5 lux = 7 (circles). 405
22
Discussion
406
In this study we show that all investigated intensities of ALAN affect the reproductive system 407
of male great tits. Our lowest treatment level, an intensity of 0.5 lux, is comparable to 408
measurements obtained from individual wild birds living in light-polluted areas (Dominoni et 409
al., 2013a; de Jong et al., 2016b). It thus represents realistic levels that birds may encounter in 410
the wild. In particular, we found evidence that different stages of gonadal growth are 411
activated at different levels of light at night. Indeed, mRNA transcript levels of genes linked 412
to the development of germ cells (stra8 and spo11) were already increased under 0.5 lux 413
compared to the dark control group. Germ cell development depends on Sertoli cells activity, 414
but we detected a significant change in the markers of Sertoli cells only under light levels 415
higher than the 0.5 lux group. The explanation for this apparent contradiction might be partly 416
biological and partly methodological. When Sertoli cells are first activated, their response is 417
to induce the proliferation of germ cells. Such proliferation rapidly leads to a much higher 418
number of germ cells, implying a very strong response of germ cell markers compared to 419
those of Sertoli cells. Our method for measuring activation was probably more sensitive to 420
the cumulative transcripts of an increasing numbers of germ cells, as compared to the more 421
gradual activation of Sertoli cells. 422
mRNA transcript levels associated with markers of Leydig cell activity were found to 423
be increased only under light levels equal to or higher than 1.5 lux. Indeed, we also found that 424
testes of birds under 1.5 lux were larger than those of birds under 0.5 lux or under dark 425
nights, confirming that a second stage of gonadal development that includes increase in size 426
and the initiation of sperm and steroid production was initiated. However, we only found full 427
spermatogenesis in the histological analyses of birds under 5 lux, which indicates that only 428
birds in this treatment group had functional testes and were thus potentially ready to breed 429
(Fig. 6). These birds also showed a dramatic increase in testes size, as they had 10 to 30 times 430
23
larger testes than birds kept at lower light intensities, as well as greatly increased mRNA 431
levels of all transcripts analysed (Fig. 6). Therefore, while exposure to ALAN as low as 0.5 432
lux during our experiment already induced a photoperiodic response in great tits, only levels 433
equal to 5 lux led to full spermatogenesis. However, despite the fact that birds in the 5 lux 434
group had far larger testes than birds in any other treatment group and also showed full 435
spermatogenesis, at the time of sampling the birds were still far from having reached the 436
average and maximum testis volume of this species (130 and 150 mm3, Fig. 6 and (Schaper et 437
al., 2012b; a)). 438
439
440
Fig. 6. Stages of testis growth under the investigated light intensities, superimposed on a 441
curve of full testicular development. Different processes appear to be activated at different 442
stages of growth due to exposure to ALAN. Germ cell proliferation starts partly already 443
under 0.5 lux, although little changes in testis volume are observed. Under 1.5 lux most of the 444
genes analysed showed increased transcript levels, and testis tubules started to enlarge, 445
indicating that steroid synthesis and spermatogenesis had started. Full spermatogenesis was 446
only achieved under 5 lux, in parallel with a large increase in testis size. Note that at the time 447
of culling, birds in the 5 lux group had only 1/3 of the maximal testis size known from great 448
tits, suggesting that our birds had not yet completed gonadal growth. 449
450
A gradual increase of effects with increasing levels of light at night for gonadal 451
development parallels our findings for daily rhythms (de Jong et al., 2016a). When the same 452
24
individual birds were exposed to similar levels of ALAN during the preceding experiment, 453
activity patterns and nocturnal melatonin concentration were also affected in a dose-454
dependent manner. The earlier study also reported slight but significant effects at lower light 455
intensities, and more marked effects under 5 lux (de Jong et al., 2016a). In particular, the 456
strong advancement of the morning onset of activity found under 5 lux in the previous study 457
(Fig. 2a in (de Jong et al., 2016a)) is comparable to the strong increase in testes size and gene 458
expression that we show here for the 5 lux group. Over the course of these two studies the 459
birds were exposed twice to their respective ALAN conditions, interrupted for three weeks by 460
exposure to dark nights. It is therefore possible that the reproductive activation we measured 461
had been primed before the start of our experiment (Sockman et al., 2004; te Marvelde et al., 462
2012). However, given the intermittent pausing of the ALAN treatments, we consider it likely 463
that the main effects detected in our present study had developed during our 3-week 464
experiment in February. We also need to stress that our experimental findings are specific to 465
the photosensitive phase of the annual cycle. It is currently unclear what effects temporary 466
exposure to ALAN will have at other phases of the annual cycle. In a previous experiment, in 467
which we exposed blackbirds to ALAN for an entire annual cycle, we found that ALAN 468
advanced reproductive development, as in this study. Under continued exposure to ALAN, 469
blackbirds regressed testes at similar times as dark-night controls. However, under ALAN, 470
birds did not recover photosensitivity and remained locked in the photorefractory phase until 471
at least the following summer. Associated physiological processes, i.e. postbreeding moult, 472
were also impaired by persistent exposure to ALAN (Dominoni et al., 2013b). 473
A limitation of our sampling design is that we have obtained only a single-point 474
measurement during the gonadal growth of great tits. It is therefore impossible to obtain 475
information about the growth curve of each individual’s reproductive system, which would 476
have been helpful to establish more precisely the shape of the dose-dependent response of the 477
25
reproductive system to ALAN. On the other hand, our terminal experiment allowed us to 478
obtain tissue samples for histological and molecular analyses. Future studies should attempt 479
to use more treatment groups and repeatedly measure gonadal size in the same individuals, to 480
understand the exact shape of the dose-response of reproductive growth to ALAN. In our 481
experiment we found that testis volume was correlated at the individual level to mRNA 482
transcript levels, suggesting that it is possible to infer from consecutive measures of testicular 483
size of living birds the underlying molecular processes (Partecke et al., 2005). 484
Interestingly, the increase in testis volume due to ALAN was also strongly linked to 485
increased mRNA levels of two adrenal steroid receptors, GR and MR. In birds, 486
glucocorticoids have been suggested to mediate timing of reproduction via the regulation of 487
steroid hormone synthesis (Schoech et al., 1997, 2009; Kirby et al., 2009; Deviche et al., 488
2010; Goutte et al., 2010a; b; Crespi et al., 2013; McGuire et al., 2013). Indeed, recent studies 489
have shown that acute stressors and the resulting heightened circulating corticosterone levels 490
are able to lower testosterone and estradiol release in Rufous-winged sparrows (Peucaea 491
carpalis) and European starlings (Sturnus vulgaris) (Deviche et al., 2010; McGuire et al.,
492
2013). However, these manipulations did not result in changes in the gonadal expression of 493
GnRH in the starling study (McGuire et al., 2013). Moreover, recent work in captive dark-494
eyed juncos (Junco hyemalis thurberi) has also questioned the direct effect of glucocorticoids 495
on gonadal processes (Fudickar et al., 2017). While we did not measure corticosterone, the 496
ratio of MR/GR, which is often used to test for altered stability of the stress axis (Marasco et 497
al., 2016), did not vary between the four treatments (Fig. S2a), and individual gr and mr 498
transcript levels were highly correlated (Fig. S2b). These data seem to indicate that the birds 499
in our experiment were not stressed, and thus provide little support for a negative influence of 500
ALAN on the HPA axis. Rather, they suggest that higher levels of adrenal steroid receptors 501
are likely a by-product of major metabolic changes accompanying, or even preparing for, 502
26
important life-history transitions, as shown for avian migration (Piersma et al., 2000) and for 503
reproduction (this study). 504
One additional factor that affected mRNA levels was the age of birds. An 505
advancement of reproductive activation with increasing age is common in birds and has also 506
been linked to age-related changes in photic sensitivity (Sockman et al., 2004). However, in 507
our birds only mRNA levels of few genes were related to age, and both testis volume and 508
tubule diameter were not. Thus, whether the effect of ALAN on reproductive development of 509
wild birds might be age-specific, and particularly affects older birds, remains to be 510
established. 511
From previous work on avian species, exposure to ALAN is known to advance both 512
the development of the reproductive system and egg-laying (Dominoni, 2015). Indeed, in 513
blackbirds, experimental exposure to 0.3 lux of ALAN for eight weeks in captivity caused 514
birds to develop fully functional testes (Dominoni et al., 2013a). Although exposure was 515
longer and methods were not directly comparable to those in our study, it is possible that 516
blackbirds are particularly sensitive to ALAN, matching reports of species-specific 517
differences (Kempenaers et al., 2010; Da Silva et al., 2014). To explore possible differences 518
in sensitivity to ALAN, future work could empirically compare the physiological responses 519
of different avian species, both closely and distantly related, to increasing levels of light at 520
night. 521
In an ecological context, it is still unclear whether earlier development of the 522
reproductive system due to ALAN would lead to earlier breeding. In wild great tits the 523
advancement of lay date due to ALAN is limited to only a few days. Moreover, such an effect 524
seems to be modulated by temperature, being stronger in cold and late springs compared to 525
warmer ones (de Jong et al., 2015). This refines reported relationships between warmer 526
temperatures in urban areas (the “heat island effect”) and avian reproductive timing (Deviche 527
27
and Davies, 2014). In any case, in our experiment temperature was kept equal across all 528
treatments and rooms, thus the observed variation in reproductive timing is mostly 529
attributable to the light treatments. However, although ALAN can be perceived as a long 530
photoperiod (Dominoni and Partecke, 2015) and lead to earlier gonadal development, 531
individual variation in reproductive physiology is not necessarily related to individual 532
variation in egg-laying dates. Indeed, recent experimental work in great tits has shown that 533
although long photoperiods lead to increased secretion of reproductive hormones and larger 534
gonads, this was unrelated to subsequent egg-laying dates (Salis et al. in review; Schaper et 535
al., 2012a; te Marvelde et al., 2012). To our knowledge no study has attempted to 536
simultaneously measure the timing of both gonadal growth and egg-laying in response to 537
ALAN, and this remains a considerable research gap. 538
Further comparisons can be made also to studies performed in other taxa. Indeed, in a 539
similar experiment with a freshwater fish species (European perch), Bruening and 540
collaborators (Bruening et al., 2016) also showed intensity-dependent effects of ALAN on the 541
expression of gonadotropins (LH and FSH), although with two key differences compared to 542
our study. Firstly, they only found an effect in females but not males, andsecondly, they 543
found that gonadotropin expression decreased, rather than increased, with higher light 544
intensity at night. This latter difference can be explained by the different reproductive 545
strategies of the two species. Our work in great tits was conducted in late winter when great 546
tits are photosensitive and may be expected to show reproductive activation under ALAN, as 547
the presence of light at night may be interpreted as a stimulating photoperiod in long-day 548
avian breeders (Dominoni et al., 2013a; Dominoni and Partecke, 2015). Conversely, perch 549
are short-day breeders, which require shortening photoperiods to initiate reproductive activity 550
(Migaud et al., 2006, 2010). ALAN-induced perceived long photoperiods are known to 551
suppress gonadotropins in this species and other short-day breeders (Bruening et al., 2015; 552
28
Robert et al., 2015). Overall, the studies suggest that the photoperiodic response of birds and 553
fish to ALAN is dose-dependent. 554
The presence of ALAN is globally increasing in both spatial extent and radiance, despite the 555
recent switch to LED technology in developed countries (Gaston et al., 2015a; Falchi et al., 556
2016; Kyba et al., 2017). This suggests that exposure to ALAN of wild animals is likely also 557
increasing, and thus there is impellent need to understand what the ecological consequences 558
of ALAN will be in the coming years (Davies and Smyth, 2017). Our study adds to the 559
increasing evidence that the exposure to artificial light can affect the reproductive system of 560
animals, and in particular of birds. Our study was aimed at understanding in detail the 561
physiological pathways involved in the stimulation of the reproductive system by ALAN, as 562
well as potential thresholds above which the reproductive response is triggered. It remains to 563
be established whether not only seasonal processes, but also diel ones, can be profoundly 564
affected at the tissue level by low levels of light at night (de Jong et al., 2016a). In our 565
experiment we also collected other tissue samples, such as brain, liver, and spleen, which are 566
key regulators of daily rhythms in sleep, metabolism and immunity, but these data will be 567
published separately (Dominoni et al in prep). From this present work we conclude that even 568
light levels as low as 0.5 lux can produce early gonadal activation, although only higher light 569
intensities of at least 5 lux were able to strongly increase testes size and lead to full 570
spermatogenesis within our experimental period. Wild birds are likely to be exposed to such 571
levels in light polluted areas (Dominoni et al., 2013a, 2014; de Jong et al., 2016b). For some 572
species exposure as in our captive experiment might not last for the entire night. However, 573
others, in particular animals living in open areas, will be less able to “escape” light pollution, 574
as shown in wallabies (Robert et al., 2015). Thus, we argue that ALAN should be limited to 575
minimal levels wherever possible to avoid chronically high exposure for wildlife. However, 576
to build a stronger case for the negative effects of light pollution on wildlife, and thus to 577
29
support the implementation of novel policies aimed at limiting ALAN, future work should 578
not only examine the behavioural and physiological effects of light pollution, but also clarify 579
whether these come with health and fitness consequences (Kempenaers et al., 2010; de Jong 580
et al., 2015; Swaddle et al., 2015; Dominoni et al., 2016; McLay et al., 2017; Ouyang et al., 581 2017). 582 583 Acknowledgements 584
We thank all animal caretakers, Lisa Trost and Pietro D’Amelio for their wonderful support 585
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