Dads matter, too: The behavioural and neurogenomic effects of receiving paternal care

Offspring are often influenced by the care they receive from their parents. However, we know little about the underlying neurogenomic mechanisms. Using a half-sib design, threespine stickleback fish were either raised by their fathers or hand-reared (“orphaned”). As offspring developed, they were tested in one of three behavioural assays: an open field assay with a simulated predator attack, a social behaviour assay with a simulated predator attack, and a scototaxis assay. Offspring that received parental care were bolder, more social, and less anxious. Fish in the open field assay had their brains sampled one hour following the simulated attack; brains were sampled at the same time from full-sib controls. These brains were processed for gene expression (via Tag-seq) and chromatin accessibility (via ATAC-seq). Experiencing paternal care affected brain gene expression, but sex was also a major factor, despite the fish being reproductively immature. The predator attack in the open field assay affected expression of fewer genes. Sex, and to lesser extent, paternal care, also influenced chromatin accessibility at a whole genome scale. Our findings further our understanding of the mechanistic basis for offspring response to variation in the care they receive from their parents. Overall design: Using a half-sib design, threespine stickleback fish were either raised by their wild-caught fathers (from Putah Creek, CA = Day 10) or hand-reared (“orphaned” = Day 0). Prior to sexual maturity, when these offspring were 6-8 months old, a subset were individually introduced to an open field assay (= open-field) and allowed to explore for 10 minutes. At 10 minutes, a model sculpin was moved through the pool in one complete circuit simulating a predatory attack. The subsequent behaviour of the juvenile fish was observed for an additional 10 minutes. Following this observation, the fish was left in the open field assay without any more disturbance. Sixty minutes after the attack, the juvenile fish was gently captured, euthanized via decapitation and its brain quickly dissected. Half of the brain was placed in RNAlater for subsequent RNA extraction for 3'TagSeq and the other half was placed in cold PBS and Roche cOmplete Mini Protease Inhibitor Cocktail (PIC) for processing for ATAC-seq. Each fish that went through the open field assay was paired with a full-sibling who was gently removed from its home tank and acted as a 'baseline control' (= baseline). This fish did not go into the pool, but had its brain dissected within 30 minutes of the fish with which it was paired. Fish were sexed with a DNA marker.

子代的发育往往会受到其获得的亲本抚育的影响，然而其背后的神经基因组学（neurogenomic）机制仍有待深入阐明。本研究采用半同胞设计，将三刺鱼（threespine stickleback）分为两组：一组由其父本抚育，另一组为人工抚育的"孤儿"个体。随着子代发育，研究人员对其开展三类行为学测试：带模拟捕食者攻击的旷场实验（open field assay）、带模拟捕食者攻击的社交行为实验，以及趋暗行为实验（scototaxis assay）。结果显示，获得亲本抚育的子代表现出更大的胆量、更强的社交性与更低的焦虑水平。

参与旷场实验的个体在模拟攻击后1小时被采集脑组织；同时采集与其配对的同窝全同胞对照（full-sib controls）的脑组织，采集时间保持一致。对采集的脑组织开展基因表达（Tag-seq）与染色质开放状态（ATAC-seq）检测。研究发现，父本抚育经历可显著影响子代脑组织的基因表达模式；尽管受试鱼尚未达到生殖成熟阶段，但性别同样是影响基因表达的关键因素。旷场实验中的模拟捕食者攻击仅影响少量基因的表达。此外，性别以及影响程度相对较弱的父本抚育行为，同样可在全基因组范围内影响染色质开放状态。本研究结果加深了人们对子代响应亲本抚育差异的分子机制的理解。

实验整体设计：本研究采用半同胞设计，将三刺鱼分为两组：一组由野外捕获的父本（采自美国加州普塔赫溪，培育天数记为第10天）抚育，另一组为人工抚育的"孤儿"个体（培育天数记为第0天）。在子代达到性成熟前（6~8月龄时），选取部分个体单独放入旷场实验装置中，使其自由探索10分钟。实验进行至第10分钟时，通过完整环绕受试水池的方式移动杜父鱼模型，模拟捕食者攻击行为。随后继续观察幼鱼的行为表现，时长为额外10分钟。观察结束后，将幼鱼留在旷场实验装置中，不再施加任何干扰。攻击行为发生后60分钟，轻柔捕获受试幼鱼，通过断头法实施安乐死，并快速采集其脑组织。将脑组织分为两半：一半置于RNAlater保存液中，用于后续通过3'TagSeq进行RNA提取；另一半置于添加了罗氏（Roche）完整迷你蛋白酶抑制剂混合物（PIC）的预冷磷酸盐缓冲液（PBS）中，用于ATAC-seq检测。每只参与旷场实验的个体，均匹配一只同窝全同胞个体作为"基线对照"（baseline）：将该对照个体从其饲养缸中轻柔取出，不放入受试水池，且在配对实验个体采样后的30分钟内完成脑组织采集。通过DNA分子标记对受试鱼进行性别鉴定。