Abstract
Helicoverpa armigera is a globally-important crop pest with a WZ (female)/ZZ (male) sex chromosome system. The absence of discernible sexual dimorphism in its egg and larval stages makes it impossible to address any sex-related theoretical and applied questions before pupation unless a W-specific sequence marker is available for sex diagnosis. To this end, we used one pair of morphologically pre-sexed pupae to PCR-screen 17 non-transposon transcripts selected from 4855 W-linked candidate reads identified by mapping a publicly available egg transcriptome of both sexes to the male genome of this species and detected the read SRR1015458.67499 only in the female pupa. Subsequent PCR screenings of this read and the previously reported female-specific RAPD (random amplified polymorphic DNA) marker AF18 with ten more pairs of pre-sexed pupae and different annealing positions and/or temperatures as well as its co-occurrence with the female-specific transcript splicing isoforms of doublesex gene of H. armigera (Hadsx) and amplification and sequencing of their 5′ unknown flanking sequences in three additional pairs of pre-sexed pupae verified that SRR1015458.67499 is a single copy protein-coding gene unique to W chromosome (named GUW1) while AF18 is a multicopy MITE transposon located on various chromosomes. Test application of GUW1 as a marker to sex 30 neonates of H. armigera yielded a female/male ratio of 1.14: 1.00. Both GUW1 and Hadsx splicing isoforms assays revealed that the H. armigera embryo cell line QB-Ha-E-1 is a male cell line. Taken together, GUW1 is not only a reliable DNA marker for sexing all stages of H. armigera and its cell lines, but also represents the first W-specific protein-coding gene in lepidopterans.
Highlights
Sex has profound effects on almost all aspects of animal life
The laboratory colony of H. armigera used in this study was established with about 1200 larvae collected in tobacco fields in Xuchang (Henan, China) in June 2016 The colony had been maintained in a growth chamber at 26 ± 0.5◦C with a photoperiod of 16 h light: 8 h dark and a relative humidity of 75 ± 5% or 50 ± 5%
Sequencing of the two AF18 bands reveals that they miss the first 11 and 45 bp of the reported AF18 sequence respectively, and share no significant similarity in their 5 flanking sequences (Figure 5C and Supplementary Figure S1). These results indicate that the two bands represent two different copies of AF18 located on Z chromosome or autosome, whereas the previously reported female-specific copy (Niu et al, 2007) locates on W chromosome
Summary
Sex has profound effects on almost all aspects of animal life. This is manifested by the ubiquity of sexually-dimorphic morphological, physiological, behavioral, and life-history traits (Allen et al, 2011; Mowrey and Portman, 2012). Less universal dimorphic traits include higher tolerance to insecticides (Li et al, 1992), heat stress (Gruntenko et al, 2016), and pathogen infection (Vincent and Sharp, 2014) as well as lower tendency to enter diapuse and shorter post-diapause period (Shimizu and Fujisaki, 2002) in females Some of these secondary sexually-dimorphic traits, such as larger females (greater fecundity) and male protandry (better chance to mate with females), are largely associated with different reproductive roles, whereas others have a mix of sexual and non-sexual functions [e.g., arctiid moth auditory apparatus (Weller et al, 1999)] or function exclusively in non-reproductive contexts [e.g., female-limited mimetic color patterns (Kunte, 2009; Stillwell et al, 2010; Allen et al, 2011)]
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