A non-invasive method to genotype cephalopod sex by quantitative PCR

Coleoid cephalopods (cuttlefish, octopus, and squid) are emerging model organisms in neuroscience, development, and evolutionary biology, and are of major economic importance in global fisheries. However, they are notoriously difficult and expensive to culture. The ability to determine sex early in development would enable more efficient and sustainable population management in both lab and wild settings. Here, we present a non-invasive method to genotype the sex of dwarf cuttlefish ( Ascarosepion bandense ) as young as three hours post-hatching using a skin swab and quantitative PCR assay, which detects a two-fold dosage difference between ZZ and Z0 sex chromosomes of males and females, respectively. Furthermore, we designed and validated primers for four additional cephalopod research species with assembled genomes ( Octopus bimaculoides, Sepia officinalis, Euprymna berryi, Doryteuthis pealeii ), and for a wild-caught species of economic value ( Illex illecebrosus ) for which we generated low-coverage whole genome sequencing data. This sex-genotyping method enables accurate sex determination from hatchlings to adults across cephalopods, independent of genome quality or availability.