Rapid assessment of the sex of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) eggs and larvae

Two different methods were tested to identify the sex of the early developmental stages of the codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) with a WZ/ZZ (female/male) sex chromosome system. First, it was shown that the sex of all larval stages can be easily determined by the presence or absence of sex chromatin, which is formed by the female-specific W chromosome in interphase nuclei. This trait can also be used to identify the sex of newly hatched larvae but it does require care and accuracy. Secondly, a new sexing technique was developed based on a molecular marker of the codling moth W chromosome. Flanking regions of an earlier described W-specific sequence (CpW2) were isolated and sequenced and a 2.74 kb sequence (CpW2-EcoRI), specific for the W chromosome, was obtained. Several PCR tests were conducted, which confirmed that the CpW2-EcoRI sequence is a reliable marker for the sex identification in codling moth samples of different geographical origin. In addition, a fragment of a codling moth gene, period (Cpper) was isolated and sequenced. Results of southern hybridization of the Cpper probe with female and male genomic DNA suggested that the Cpper gene is located on the Z chromosome. Then a multiplex PCR assay was developed, which co-amplified the CpW2-EcoRI sequence to identify the W chromosome and the Z-linked Cpper sequence, which served as a positive control of accurate processing of tested samples. The multiplex PCR provides an easy and rapid identification of the sex of embryos and early larval instars of the codling moth.