Centro de Estudos de Insetos Sociais-CEIS, Instituto de Biociências, Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP-SP), Rio Claro, SP, Brasil.
Instituto Tecnológico Vale-Desenvolvimento Sustentável (ITVDS), Instituto Tecnológico Vale-Belém II, Belém, PA, Brasil.
PLoS One. 2019 Mar 20;14(3):e0213109. doi: 10.1371/journal.pone.0213109. eCollection 2019.
Brazil has the highest biodiversity of native stingless bees in the world. However, Brazilian regulations are based on protocols standardized by the Organization for Economic Cooperation and Development (OECD), which uses Apis mellifera as a model organism. The safety of the use of an exotic species as a substitute for a native species is a problem that concerns members of the academy and the government agencies responsible for studies of this nature in the neotropical regions where there are occurrences of stingless bee species. Regarding the exposure of larvae to pesticides, several indicators suggest that the same rearing method for A. mellifera cannot be applied to stingless bees, mainly because of their different feeding systems. Thus, it is necessary to establish an in vitro rearing method for native social bees. We developed a larval rearing method for the stingless bee species Melipona scutellaris and evaluated parameters such as the defecation rate, pupation, emergence, mortality and morphometry of the newly emerged workers. The control was represented by the morphometry of individuals that emerged from natural combs (in vivo). In addition, we determined the average lethal concentration (LC50) of the insecticide dimethoate, the standard active ingredient used for the validation of toxicity tests. Procedures conducted prior to the in vitro bioassays allowed us to obtain the actual dimensions of the rearing cells for making acrylic plates for use in establishing how much each larva consumes during its development, that is, determining how much larval food should be placed in every artificial cell. Tests performed with M. scutellaris indicated an average of 80.2% emergence of individuals relative to the larvae, 92.61% relative to the pupae and a mean of 7.42% larval mortality. The mean of the intertegular distance, head width and wing asymmetry parameters were not significantly different between individuals from the in vitro and in vivo rearing methods. The LC50 value determined was 27.48 ng dimethoate / μL diet. The method described for M. scutellaris showed development rates above OECD standards, which requires at least 75% emergence, and produced newly emerged workers with similar dimensions to those produced under natural conditions; thus these results enable their use as a rearing protocol for this species (or genus) and, consequently, their use in toxicity tests. The results produced with M. scutellaris are the first steps for a proposed toxicity test protocol for stingless bee larvae that can be standardized and included as a protocol in the OECD.
巴西拥有世界上生物多样性最高的本土无刺蜜蜂。然而，巴西的法规是基于经济合作与发展组织（OECD）标准化的协议，该组织将 Apis mellifera 作为模式生物。使用外来物种替代本地物种的安全性是学术界成员和负责新热带地区此类研究的政府机构关注的问题，因为这些地区存在无刺蜜蜂物种。关于幼虫接触杀虫剂的问题，有几个指标表明，不能将用于 Apis mellifera 的相同饲养方法应用于无刺蜜蜂，主要是因为它们的喂养系统不同。因此，有必要为本土社会性蜜蜂建立一种体外饲养方法。我们开发了一种针对无刺蜜蜂物种 Melipona scutellaris 的幼虫饲养方法，并评估了粪便排泄率、化蛹、羽化、死亡率和新羽化工蜂的形态测量等参数。对照组由天然蜂巢中羽化的个体的形态测量值（体内）表示。此外，我们还确定了杀虫剂二甲基硫醚的平均致死浓度（LC50），二甲基硫醚是用于验证毒性测试的标准活性成分。体外生物测定之前进行的程序使我们能够获得饲养细胞的实际尺寸，以便制作用于确定每个幼虫在其发育过程中消耗多少的丙烯酸板，即确定每个人工细胞中应放置多少幼虫食物。对 M. scutellaris 进行的测试表明，与幼虫相比，个体的羽化率平均为 80.2%，与蛹相比为 92.61%，幼虫死亡率平均为 7.42%。体外和体内饲养方法的个体间的跗节间距离、头宽和翅膀不对称性参数平均值没有显著差异。确定的 LC50 值为 27.48ng 二甲基硫醚/μL 饮食。描述的 M. scutellaris 方法显示出高于 OECD 标准的发育率，该标准要求至少 75%的羽化率，并产生与自然条件下相似尺寸的新羽化工蜂；因此，这些结果使其能够作为该物种（或属）的饲养方案，并随后在毒性测试中使用。用 M. scutellaris 产生的结果是为无刺蜜蜂幼虫提出的毒性测试方案的第一步，可以标准化并包含在 OECD 协议中。
