Incubation attentiveness in zebra finches

In birds ambient temperature can influence adult incubation behaviour, energy budget, egg temperature, and embryonic development with downstream effects on offspring survival. Surprisingly, experimental manipulations of the whole nesting environment to test causes and consequences of variation in incubation pattern, energy balance, egg temperature, and the duration of development are lacking to date. Here, we bred pairs of Zebra Finches Taeniopygia guttataunder controlled conditions at 18°and 30°C and measured clutch size, temperature, hatching success, parental attentiveness and the length of the embryonic period. We found that when breeding at the higher temperature, males, but not females, increased the number of incubation bouts on the nest. Instead, females, but not males, reduced their attentiveness towards the clutch overall. Eggs showed no temperature differences between the two treatments and bigger clutches experienced lower temperatures. This suggests that parental behaviour may buffer the effect of ambient conditions on the thermal profile of eggs, including species with high rates of parental attentiveness. Warmer conditions yielded higher hatching rates but did not cause measurable differences in the length of embryonic development. Still, smaller clutches hatched earlier in accordance with the higher temperature experienced. Additionally, we used data from the literature to calculate parental energy expenditure and demonstrate that this was substantially different across the two treatments, although predicted energy savings from reduced attentiveness at 30°C appeared negligible. These results suggest that when food is available, ambient temperature and not energy trade-offs may explain variation in incubation behaviour. Methods Experimental design Twenty-three male and female zebra finches (domestically bred) were force-paired and reared in captivity under controlled conditions at Macquarie University (under Animal Ethics Committee approval ARA 2013/029). Each pair was housed in a cage with dimensions of 70 cm wide × 47 cm deep × 130 cm high containing two 13.5 × 15 cm rattan nest baskets and provided with November Grass Amphybromusspp., white cotton thread and Emu Dromaius novaehollandiaefeathers to line their cups. Cages were held inside temperature-controlled rooms set at either 18°C or 30°C (n= 12 pairs/temperature) where finches were acclimated for two weeks without nests before being allowed to breed. These two treatment temperatures are ecologically relevant because they reflect the average ambient temperatures recorded under natural conditions at Fowlers Gap Arid Research Station (our study field in Australia 31° S, 141° E), during the early cooler (July-August), and late hotter (December-Jannuary) months of the breeding season (Griffithet al.2016). Birds were maintained with dry millet finch seed mix (Panicum and Setaria spp.) and water available ad libitumplus a small daily supplement of Green Pea (Pisum sativum)-Spinach (Spinacia oleracea) mash with soft food, hard boiled Chicken (Gallus gallus domesticus) egg, and sprouted seed. During the egg-laying stage of the first reproductive attempt nests were monitored daily, with any new eggs collected for other work (Andrew et al. 2018), and replaced with dummy eggs (made from white Mont Marte hardening modeling clay, Mont Marte, Australia) until clutch completion. This first attempt was interrupted on day 9 of incubation when nests were removed for another study looking at temperature-dependent variation in nest size and building material (Campbell et al.2018). At this point, new rattan nesting baskets and material were provided and birds were allowed a second attempt where they could rebuild their nest, lay and incubate a new clutch, and raise a brood of chicks to independence.Pairs were all then switched into the alternative temperature treatment and following another two-week acclimation period with no nest baskets or material, the entire process was repeated(seeAndrew et al.2017for further methodological details). As a result, each pair had two reproductive attempts and completed a full successful breeding cycle (from nest building to fledging) in the two different temperature treatments. In this way, over the experimental period, half of the pairs bred first at 18°C then 30°C and the other half bred first at 30°C then 18°C. This design allowed us to take a within-individual analytical approach, reducing the confounding effects of differences across individuals, and also the effects of the first and second breeding attempt by each pair. Measurements of parental behaviour, clutch temperature and embryonic period length Between days 5 and 7 of the incubation of dummy eggs, average egg temperature was quantified using an iButton (DS1921G Thermochron iButton: Maxim Integrated, San Jose, CA) set to record temperature (±0.1°C) every minute. The device was placed in the bottom of the nest cup, alongside the eggs before 09:00 (GMT+10) in the morning and removed at the same time two days later. We decided to measure temperature in nests with dummy eggs for two main reasons. The first was to prevent the iButton from damaging the actual eggs. The second was to avoid increasing the total mass in the real nest by adding the iButton. Indeed this could dissipate the amount of heat received by the eggs and influence timing of embryonic development as found in other studies (Moreno & Carlson 1989; Thompson et al. 1998). The morning following the first day of egg temperature measurements we video recorded parental incubation behaviour at the nest for six hours starting between 08:00 and 09:00 am. Overall, the majority of videos were recorded on day 7 of incubation, with seven occurring on day 8. The total video duration of six hours was split in three two-hour sections called early, mid and late morning, respectively. One random hour per section was watched thereby providing three hours of parental incubation attentiveness for each pair. Measuring clutch temperature with the iButton while also video recording parental behaviour allowed us to match the timing of parental on and off bouts with the temperature recorded in the nest. The temperature data recorded were then averaged to obtain mean incubation temperature for each sex. This process was repeated for each randomly selected one-hour interval of incubation (early, mid and late morning) and for both breeding cycles. Therefore we ended up analyzing a dataset of 276 data points generated from 23 pairs × two sexes (male or female) × three distinct part of the day (early, mid and late morning) × two treatment temperature, × two breeding cycles that yielded 276 observations (see complete data set for temperature and attentiveness analysis Dryad). Because Zebra Finches in captivity start incubating as soon as they lay eggs, rather than at clutch completion (Gilby et al. 2013), the duration of the embryonic period was calculated as the number of days between the laying and the hatch date of the first egg in the clutch. To capture hatch date nests were monitored every day only in the morning between 07:00 and 8:00 to avoid interferences with other tests and measurements performed during the day. Timing of hatch was assigned to the previous or same day based on presence of eggshell fragments in the nest, appearance of the nestling (wet versus dry), and mass of the nestling compared to the mass of the eggs during late incubation. Note that the developmental time was measured for the second reproductive attempt at each temperature treatment, while the egg temperatures and behavioural videos were taken while the parents incubated dummy eggs that replaced the first clutch that was laid. None of the pairs we used in our experiment died so that we could use the same adults in both breeding cycles at two different temperatures. Data analysis We tested for the effects of the temperature treatment on the amount of time spent in the nest, and on number of incubation bouts by fitting a linear mixed model with treatment (18°or 30°C), breeding cycle (first or second), order of exposure to treatment (18à30 or 30à18°C), and sex (male or female) as independent variables. Individual pair identity was included as a random factor. We also built a model to examine differences between temperature treatments on clutch temperature and percent of eggs hatched with treatment, sex, breeding cycle, clutch size and order as independent variables and pair identity as random factor. Finally, we evaluated the effect of experimental temperature on length of embryonic period using treatment, breeding cycle, clutch size, and order as independent variables and pair identity as a random factor. Marginal and conditional R2for our generalized mixed-effects models were calculated using the r squaredGLMM function MuMIn that implements the method described by Nakagawa and Schielzeth (2013). We tested for two- and three-way interactions between all independent variables included in each model. We dropped all interactions when not significant. Descriptive statistics (mean andSD) are reported in the Results section while model outputs are summarized in the tables. We used a threshold α = 0.05 to test the significance of our models. All statistical analyses were performed using packages lme4 and in program R version 3.4.2 for Mac (R Development Core Team 2008). P-valueswere obtained using package lmerTest (Kuznetsova et al. 2016).All data and codes used in our study are available in Dryad and in Appendix S1 of Supporting Information, respectively). To quantify the differences in energy expenditure (kJ) between incubating and non incubating birds at 30°C and between birds breeding at the two treatment temperatures we used the equations provided by Vleck (1981). To translate the amount of energy calculated from those equations into equivalent gr....see Dryad link for full text本研究探讨环境温度对鸟类的影响：环境温度可调控成鸟孵卵行为、能量收支、卵温及胚胎发育，并对子代存活产生级联效应。然而截至目前，学界仍缺乏通过操控完整巢环境，以探究孵卵模式、能量平衡、卵温与发育时长变异的成因及后果的相关研究。 在此研究中，我们于18℃与30℃的可控条件下人工配对饲养斑胸草雀（Zebra Finch, *Taeniopygia guttata*），并记录了窝卵数、卵温、孵化成功率、亲鸟投入度以及胚胎发育时长。结果发现，在较高温度环境下繁殖时，雄性斑胸草雀会增加巢内孵卵轮次，而雌性则整体降低了对窝卵的投入度；两组处理间卵温无显著差异，但较大窝卵的卵温更低。这表明亲鸟行为可缓冲环境条件对卵温特征的影响，即便在亲鸟投入度较高的物种中亦是如此。 较温暖的环境可提升孵化成功率，但未对胚胎发育时长产生可检测的影响；不过较小窝卵会因所处环境温度更高而更早孵化。此外，我们通过文献数据估算了亲鸟的能量消耗，结果显示两组处理间的能量消耗存在显著差异，尽管30℃下亲鸟投入度降低所带来的理论能量节约微乎其微。上述结果表明，在食物充足的前提下，环境温度而非能量权衡，或许可解释孵卵行为的变异。 ## 实验设计 本研究共使用23对人工饲养的斑胸草雀，于麦考瑞大学（Macquarie University）动物伦理委员会批准编号ARA 2013/029的框架下开展实验。每对亲鸟被饲养于尺寸为70 cm宽 × 47 cm深 × 130 cm高的笼舍中，笼内配备两个13.5 × 15 cm的藤编巢篮，并提供十一月草（*Amphybromus* spp.）、白色棉线以及鸸鹋（*Dromaius novaehollandiae*）羽毛用于巢材铺垫。笼舍置于控温室内，分别设置18℃与30℃两个处理组（每组12对亲鸟），在允许繁殖前，亲鸟需在无巢材的条件下适应两周。这两个处理温度具有生态相关性，分别对应澳大利亚Fowlers Gap干旱研究站（本研究野外样地，南纬31°，东经141°）繁殖季早期凉爽的7-8月，与后期炎热的12月至次年1月的平均环境温度（Griffith et al. 2016）。 亲鸟每日可自由采食小米雀类混合种子（*Panicum*属与*Setaria*属植物种子）与饮水，并辅以每日少量的青豆（*Pisum sativum*）-菠菜（*Spinacia oleracea*）泥、煮熟的家鸡（*Gallus gallus domesticus*）蛋以及发芽种子。在首次繁殖尝试的产卵阶段，我们每日监测巢箱，将新产出的卵用于其他研究（Andrew et al. 2018），并使用由澳大利亚Mont Marte公司生产的白色硬质塑形黏土制作的假卵替代，直至窝卵完成。首次繁殖尝试在孵卵第9天时被中断，此时巢箱被移除用于另一项关于巢尺寸与筑巢材料温度依赖性变异的研究（Campbell et al. 2018）。随后我们为亲鸟提供新的藤编巢篮与巢材，允许亲鸟进行第二次繁殖尝试：重建巢穴、产卵、孵卵并将雏鸟抚育至独立。 随后，所有亲鸟被切换至另一温度处理组，在无巢篮与巢材的条件下再适应两周后，重复上述整个繁殖流程（详细方法参见Andrew et al. 2017）。最终每对亲鸟均完成两次繁殖尝试，在两种不同温度处理下各完成一次完整的繁殖周期（从筑巢至雏鸟离巢）。其中一半亲鸟先在18℃下繁殖，再转移至30℃；另一半则先在30℃下繁殖，再转移至18℃。该设计允许我们采用个体内分析方法，减少个体间差异以及每对亲鸟首次与第二次繁殖尝试的混杂效应。 ## 亲鸟行为、卵温与胚胎发育时长的测量 在假卵孵卵的第5至7天，我们使用iButton（DS1921G Thermochron iButton: Maxim Integrated, San Jose, CA）记录平均卵温，该设备设置为每分钟记录一次温度（精度±0.1℃）。于每日09:00（GMT+10）前将设备放置于巢杯底部、卵群旁，并于两天后的同一时间取出。我们选择使用假卵测量卵温主要有两个原因：一是避免iButton损伤真实卵粒；二是避免在真实巢中增加总重量，进而影响卵粒接收的热量以及胚胎发育时序，这与此前其他研究的发现一致（Moreno & Carlson 1989; Thompson et al. 1998）。 在首次卵温测量的次日清晨，我们于08:00至09:00间开始，对巢内亲鸟的孵卵行为进行6小时的视频录制。绝大多数视频录制于孵卵第7天，另有7次录制于第8天。6小时的总录制时长被分为清晨早、中、晚三个两小时时段，每个时段随机选取1小时进行观测，最终每对亲鸟获得3小时的亲鸟孵卵投入度数据。通过在测量卵温的同时录制亲鸟行为，我们可将亲鸟的进出巢轮次与巢内记录的温度进行匹配。随后对温度数据取平均，得到每只亲鸟的平均孵卵温度。该流程在每个随机选取的观测时段（早、中、晚）以及两次繁殖周期中均重复进行。最终我们共获得276组数据，来自23对亲鸟 × 2个性别（雄性/雌性） × 3个时段（早、中、晚） × 2个处理温度 × 2个繁殖周期，总计276个观测值（卵温与投入度分析的完整数据集参见Dryad数据库）。 由于圈养斑胸草雀会在产卵时即开始孵卵，而非待窝卵完成后才启动（Gilby et al. 2013），因此胚胎发育时长被定义为窝中首枚卵的产卵日期至孵化日期之间的天数。为准确记录孵化日期，我们仅在每日07:00至08:00间监测巢箱，以避免干扰当日开展的其他测试与测量。孵化时间根据巢内卵壳碎片、雏鸟外观（湿态/干态）以及孵化末期雏鸟体重与卵重的对比，被归为前一日或当日。需要说明的是，胚胎发育时长的测量针对每个温度处理下的第二次繁殖尝试，而卵温与行为视频的录制则针对替换了首次产卵窝的假卵孵卵阶段。本实验中所用亲鸟均未死亡，因此我们可在两种温度处理的两次繁殖周期中使用同一批成鸟。 ## 数据分析 我们构建线性混合模型，检验温度处理对亲鸟巢内停留时长与孵卵轮次的影响，模型中的自变量包括处理（18℃或30℃）、繁殖周期（首次或第二次）、处理暴露顺序（18℃→30℃或30℃→18℃）以及性别（雄性/雌性），并将亲鸟对的个体标识作为随机因子。此外，我们还构建模型以检验温度处理间的卵温与孵化率差异，自变量包括处理、性别、繁殖周期、窝卵数以及处理顺序，同样以亲鸟对个体标识作为随机因子。最后，我们评估实验温度对胚胎发育时长的影响，自变量包括处理、繁殖周期、窝卵数以及处理顺序，以亲鸟对个体标识作为随机因子。我们使用MuMIn包中的r.squaredGLMM函数计算广义线性混合模型的边际与条件R²，该函数实现了Nakagawa与Schielzeth（2013）提出的计算方法。我们检验了每个模型中所有自变量间的二阶与三阶交互作用，当交互作用不显著时将其剔除。结果部分报告了描述性统计量（均值与标准差），模型结果汇总于表格中。我们以α=0.05作为模型显著性检验的阈值，所有统计分析均通过Mac系统下R 3.4.2版本（R Development Core Team 2008）的lme4包完成，P值通过lmerTest包计算得到（Kuznetsova et al. 2016）。本研究所用的全部数据与代码分别可于Dryad数据库与附录S1支持信息中获取。 为量化30℃下孵卵与非孵卵亲鸟的能量消耗（kJ）差异，以及两种温度处理下繁殖亲鸟的能量消耗差异，我们使用Vleck（1981）提供的计算公式。为将通过上述方程计算得到的能量值转换为等效质量……完整文本请参见Dryad数据库链接。