Zebrafish imprint to amino acids: Evidence from attraction and avoidance tests

Imprinting to environmental cues is behind some of the most critically important behaviours on the planet. In certain fishes, olfactory imprinting is believed to direct return migration to natal areas. In this, amino acids are putative imprintable odorants. We tested the ability of a model fish (Danio rerio) to imprint to amino acids. We found that developmental exposure to some amino acids could result in attraction, avoidance or indifference later at adult stage. Imprinting to a binary mixture of amino acids made from attracting and repelling amino acids resulted in imprinting, which was surprising. However, the response was avoidance, not attraction. These data suggest avoidance is a more powerful driver of behavioural response. Our data highlight the specificity with which a single amino acid can have in the imprinting response, as well as the complexity that even the most basic of odorant mixtures can encode.

Methods
Zebrafish were raised from fertilization to 7 dpf in either embryo media alone or embryo media spiked with amino acids (L-leucine, L-lysine, or L-valine, or a mixture of L-leucine and L-lysine) or a positive control (phenylethyl alcohol [PEA]). They were then transferred to regular tank water and grown to adulthood. At 90 days post-fertilization, zebrafish were placed in an avoidance-attraction trough, which is a two choice arena. Zebrafish were tested for their tendency to either spend more or less time in the area where odorants were delivered. The odorants consisted of the same odorants they were exposed to as embryos (or blank). Data consists of time spent in the odorant zone 10 min before odorant introduction and 10 min after odorant introduction. A positive difference between the two indicates attraction (or preference).

环境线索印记是地球上部分至关重要生命行为背后的核心调控机制。在部分鱼类中，嗅觉印记被认为可引导其洄游返回原生栖息地，而氨基酸被推定为此过程中的可印记气味物质。本研究以模式生物斑马鱼（Danio rerio）为对象，测试其对氨基酸的印记能力。结果发现，发育阶段暴露于部分氨基酸，会在其成年阶段引发趋性、避性或无反应三种行为。当暴露于由吸引性与驱避性氨基酸组成的二元氨基酸混合物时，同样会产生印记效应，这一结果出乎预料，但最终的行为响应为避性而非趋性。上述数据表明，避性是驱动行为响应的更强效因素。本研究结果凸显了单一氨基酸在印记响应中的特异性，以及即便最基础的气味混合物也可能编码的复杂行为调控机制。

实验方法
本研究将斑马鱼从受精后至受精后7天（days post-fertilization, dpf）的发育阶段，分别饲养于仅含胚胎培养基的环境，或添加了氨基酸（L-亮氨酸、L-赖氨酸、L-缬氨酸，或L-亮氨酸与L-赖氨酸的混合物）的胚胎培养基中，同时设置阳性对照组（苯乙醇[PEA]）。随后将幼鱼转移至常规养殖水体中饲养至成年。在受精后90天时，将斑马鱼置于趋避槽（双选择实验装置）中，测试其在气味投放区域停留时长的偏好性。气味物质采用与幼鱼胚胎阶段暴露的同种物质（或空白对照）。实验数据采集气味投放前10分钟与投放后10分钟内，斑马鱼在气味区的停留时长。两组时长的正向差值代表趋性（或偏好）。