Influence of Protein supplements on thermoregulation of stingless bees

The research hypothesis was that protein-rich supplementation would improve the nutritional status of Tetragonula iridipennis, reflected by increased free amino acid content in honey and worker-bee tissue, and would enhance brood-chamber thermoregulation through an indirect, treatment-mediated pathway. The study tested six plant-protein formulations against a sugar control using 21 colonies, with three colonies per treatment. The experiment ran for 121 days, from 1 December 2025 to 31 March 2026. Brood-chamber temperature and relative humidity were recorded daily at seven daytime intervals, while honey and pooled worker-bee samples were collected at Day 0 and at 30-day intervals up to Day 120. Total free amino acids were quantified using the ninhydrin colorimetric method and expressed as L-leucine equivalents. The data support the hypothesis strongly for biochemical responses but only partially for thermoregulation. Protein supplementation significantly affected brood-chamber temperature and relative humidity, but the biological magnitude was small. The total difference among treatments was only 0.65 °C. The chickpea plus wheat-germ formulation, T6, produced the highest brood-chamber temperature, 30.39 °C, and the highest thermal differential, +4.31 °C, compared with 29.74 °C and +3.65 °C in the sugar control. The treatment effect was strongest under higher thermal load and became smaller as ambient temperature approached the preferred range. The strongest response occurred in free amino acid content. By Day 120, T6 increased honey free amino acids to 43.9 mg LE/100 g, approximately 2.4 times its baseline value. Worker-bee tissue reached 18,185 µg LE/g fresh weight, approximately 2.2 times baseline. Treatment, sampling time and their interaction were significant in both matrices, showing progressive and treatment-dependent enrichment. Feed disappearance also supported the superior performance of T6. It recorded 10.54 g colony⁻¹ week⁻¹, the highest among the solid formulations. The evaporative control indicated that differences in feed disappearance were not explained by moisture loss alone. A notable finding was the strong positive association between free amino acids and thermoregulatory parameters, reaching r = +0.953 for honey free amino acids and ΔRH at Day 120. However, treatment-adjusted correlations were weak and non-significant. This indicates that the strong overall correlation arose mainly because both responses changed according to the dietary treatment, rather than because increased free amino acids directly controlled thermoregulation. Therefore, the data support an indirect effect of protein supplementation on nest climate, while the biochemical response is the stronger and more consistent outcome.本研究提出的假设为：富含蛋白质的补饲可改善亮胸无刺蜂（Tetragonula iridipennis）的营养状况，具体体现为蜂蜜与工蜂组织内游离氨基酸含量提升，并可通过处理介导的间接通路增强育幼室体温调节能力。本研究以21个蜂群为实验对象，设置6种植物蛋白配方组与1组蔗糖对照组，每组对应3个蜂群；实验周期为121天，自2025年12月1日至2026年3月31日。研究人员每日在日间的7个时间节点记录育幼室温度与相对湿度，并于第0天及第每30天的节点（直至第120天）采集蜂蜜与混合工蜂样本。总游离氨基酸含量采用茚三酮比色法（ninhydrin colorimetric method）进行定量，结果以L-亮氨酸当量（L-leucine equivalents）表示。 实验数据在生化响应层面强烈支持本研究假设，但在体温调节层面仅部分支持该假设。蛋白质补饲对育幼室温度与相对湿度存在显著影响，但生物学效应幅度较小；各组间的总温差仅为0.65℃。鹰嘴豆+小麦胚芽配方组（T6）的育幼室平均温度达30.39℃、温度差达+4.31℃，均为各组最高；而蔗糖对照组的对应数值分别为29.74℃与+3.65℃。该处理效应在更高热负荷条件下最为显著，且随着环境温度接近蜂群偏好温度区间时逐渐减弱。 游离氨基酸含量的响应最为显著。至第120天时，T6组的蜂蜜游离氨基酸含量升至43.9 mg LE/100 g，约为基线值的2.4倍；工蜂组织游离氨基酸含量达18185 µg LE/g 鲜重，约为基线值的2.2倍。处理方式、采样时间及其交互作用对两种样本基质均存在显著影响，体现出游离氨基酸含量随采样时间递增且依赖于处理组的富集趋势。 饲料消耗量数据同样印证了T6组的优异表现，其饲料消耗量达10.54 g·群⁻¹·周⁻¹，为所有固体配方组中最高。蒸发对照实验表明，饲料消耗量的差异并非仅由水分蒸发导致。 一项值得关注的发现为：游离氨基酸含量与体温调节参数间存在显著正相关关系，其中第120天时蜂蜜游离氨基酸含量与相对湿度变化量（ΔRH）的相关系数r达+0.953。但经处理校正后的相关系数则较弱且无统计学显著性。这表明整体层面的强相关关系主要源于两项响应均随日粮处理方式发生变化，而非游离氨基酸含量提升直接调控了体温调节过程。因此，实验数据支持蛋白质补饲对蜂巢微气候存在间接影响，而生化响应则是更为显著且稳定的实验结果。