Protocol for the in vitro rearing of Frieseomelitta varia workers (Hymenoptera: Apidae: Meliponini)

ABSTRACT In vitro rearing protocols already established for honeybees are currently being adapted to assess the risk of pesticides, and to conduct comparative developmental biology studies on stingless bees. However, differences in critical life-history traits (development time and the type of larval nutrition leading to caste differentiation process) among social bees require the development of an in vitro rearing protocol for each species and caste. We generated a protocol to produce workers of Frieseomelitta varia (Lepeletier, 1836), a non-endangered and highly eusocial pollinator species with wide geographical distribution. We tested the viability of using either the eggs or the first instar larvae as the starting point for in-vitro transfer. In vitro rearing was performed in acrylic plates at 30 °C and 99% relative humidity during the larval feeding phase. The humidity was subsequently reduced to 75% during the following days of development. The experimental larvae were offered either 25 µL or 27 µL of larval food. The development time, emergence and mortality rates, and morphological parameters of the emerged workers were assessed. In the process of validating the protocol, the adults that emerged after in vitro rearing were compared with colony-reared adults. In our results, 27 µL of larval food allowed 90% of workers to emerge. No significant differences were found between the emerging workers reared in vitro and those reared in the colony. The described protocol is a useful method for rearing F. varia workers in vitro, which can be used for diverse types of experimental approaches.

摘要 当前，已建立的蜜蜂体外（in vitro）饲养方案正被优化改造，用于评估农药风险，并开展无刺蜂的比较发育生物学研究。然而，群居蜂类的关键生活史性状（发育时长及调控级型分化的幼虫营养类型）存在显著差异，因此需针对每个物种及级型开发专属的体外饲养方案。本研究开发了一套用于繁育弗氏无刺蜂（Frieseomelitta varia (Lepeletier, 1836)）工蜂的标准化方案，该物种为无危且高度真社会性的传粉昆虫，分布范围广泛。我们测试了以卵或一龄幼虫作为体外转移起始材料的可行性。幼虫取食阶段，体外饲养在亚克力培养板中进行，饲养条件设置为30℃、相对湿度99%；后续发育阶段则将相对湿度降至75%。实验幼虫分别被饲喂25 μL或27 μL的幼虫专用饲料。研究对发育时长、羽化率、死亡率以及羽化工蜂的形态参数进行了评估。在方案验证环节，本研究将体外饲养羽化的成虫与蜂巢内繁育的成虫进行了对照比较。实验结果显示，饲喂27 μL幼虫专用饲料时，工蜂羽化率可达90%。体外饲养的羽化工蜂与蜂巢内繁育的工蜂之间未发现显著差异。本研究描述的这套体外繁育弗氏无刺蜂工蜂的方案实用性较强，可适用于多种类型的实验研究。