1. Incidence of parasitism is often underestimated because ‘ghost’ parasitoids (dead unemerged parasitoids or those that have emerged leaving the host carcasses) are difficult to detect and identify. This study demonstrates that the use of melting curve analysis (MCA) of host carcasses can detect and identify DNA of ghost parasitoids even a month after host death.
2. The coccinellid hosts Cycloneda sanguinea, Eriopis connexa, Harmonia axyridis and Hippodamia convergens were sampled from cole crops in 2017 and 2018 in the Midwest of Brazil, and reared and observed daily for parasitoid emergence. Dead coccinellids were held for 30 days after death before storage and only host carcasses with parasitoid emergence observed were analysed. Species-specific primers were designed for the identification of the parasitoid species that emerged during host rearing: Dinocampus coccinellae, Homalotylus mirabilis, Ho. terminalis, Strongygaster triangulifera and Phalacrotophora sp. The melting temperatures (T_m) of their amplicons were used as positive controls in MCA post-amplification in qPCR.
3. Detection of parasitoid DNA in host carcasses was possible for D. coccinellae, Ho. mirabilis and Phalacrotophora sp. with a limit of detection (LOD) for all the parasitoids <1 pg of DNA, except for Phalacroptopthora sp. (LOD = 1.9 ng). Parasitoids were detected in 31 out of 70 host carcasses (44.3%) with detection ranging from 0% to 71%, depending on the parasitoid species.
4. Synthesis and applications. This work demonstrates that MCA could be used to detect and rapidly identify the DNA of some parasitoid species (e.g. Ho. mirabilis, D. coccinellae and Phalacrotophora sp.) in host carcasses up to a month after parasitoid emergence with high sensitivity and specificity. MCA can be used on any field-collected host and, for some parasitoid species, it should lead to more accurate estimates of the incidence of parasitism. Our approach can be easily adapted and applied to study other parasitoid–host interactions.

中文翻译：

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寄主死亡一个月后寄主尸体中幽灵寄生蜂检测鉴定的熔解曲线分析

1. 寄生的发生率通常被低估，因为“幽灵”寄生蜂（死亡的未融合寄生蜂或那些离开宿主尸体的寄生蜂）难以检测和识别。这项研究表明，即使在宿主死亡一个月后，使用宿主尸体的熔解曲线分析 (MCA) 也可以检测和识别幽灵寄生蜂的 DNA。
2. 瓢虫寄主Cycloneda sanguinea、Eriopis connexa、Harmonia axyridis和Hippodamia converens于 2017 年和 2018 年从巴西中西部的油菜作物中取样，并每天饲养和观察寄生蜂的出现。死后的球虫在储存前保持 30 天，仅分析观察到出现寄生蜂的寄主尸体。物种特异性引物被设计用于鉴定在宿主饲养期间出现的寄生物种：Dinocampus coccinellae、Homalotylus mirabilis、Ho。Terminalis , Strongygaster triangulifera和Phalacrotophorasp. 的熔融温度（Ť_米其扩增子）用作在qPCR中MCA后扩增的阳性对照。
3. D. coccinellae , Ho可以检测寄主尸体中的寄生物 DNA 。mirabilis和Phalacrotophora sp。除Phalacroptopthora sp.外，所有寄生虫的检测限 (LOD) <1 pg DNA 。（LOD = 1.9 纳克）。在 70 个寄主尸体中的 31 个（44.3%）中检测到了寄生蜂，检测范围从 0% 到 71%，具体取决于寄生蜂种类。
4. 合成与应用。这项工作表明，MCA 可用于在寄生物出现后长达一个月的时间内检测和快速鉴定寄主尸体中某些寄生物物种（例如奇异奇异果、D. coccinellae和Phalacrotophora sp.）的 DNA，具有高灵敏度和特异性。MCA 可用于任何野外采集的宿主，对于某些寄生物种，它应该可以更准确地估计寄生发生率。我们的方法可以很容易地适应和应用于研究其他寄生蜂 - 宿主相互作用。