Background: Water fleas that do not produce males despite environmental cues

A study published in PNAS links the production of only female offspring by some lineages of the water flea Daphnia pulex, even under male-inducing conditions, to chromosomal structural variation and reduced expression of the Dfh gene. The findings identify features resembling early sex-chromosome evolution in a system where environmental and genetic factors both contribute to sex determination.

Female water fleas produce female offspring by parthenogenesis but can produce male offspring in response to environmental cues such as photoperiod or population density. This is called environmental sex determination (ESD). It is distinct from a mother herself changing into a male. In nature, non-male-producing (NMP) lineages that produce only female offspring even under male-inducing conditions coexist with male-producing (MP) lineages. The researchers used this difference to investigate early steps in the emergence of genetic sex determination (GSD) from ESD.

Key findings: Suppressed recombination and Dfh function

The team assembled high-quality genomes from one MP clone and three NMP clones obtained from different populations, and analyzed genomes from 100 MP and 67 NMP individuals at the population level. On chromosome I of NMP clones, structural variants—including inversions that reverse the orientation of a DNA segment—were concentrated on one of the two homologous chromosome copies. This copy was 13.8–17.0 Mb long, larger than the other copy, which was about 9.0 Mb. The researchers suggested that structural variation may have suppressed recombination, the exchange of genetic information between chromosomes, and contributed to the accumulation of differences between the two copies.

Expression analyses narrowed the focus to Dfh as a key candidate. Under short-photoperiod conditions that induce males, total Dfh expression in embryos 60 hours after ovulation was more than 3.7 times as high in MP as in NMP. The Dfh allele on the structurally variant chromosome copy in NMP showed lower expression than the allele on the other copy. The change associated with loss of male-producing ability was reduced expression, not constitutive activation of Dfh.

Functional experiments also supported Dfh’s involvement in male development. Lowering its expression by RNA interference (RNAi) reduced the proportion of mothers producing male offspring in both MP clones tested. Eight lines generated by CRISPR gene editing also showed reduced Dfh expression and lower male production rates when male production was induced with the hormone analog methyl farnesoate (MF). In particular, the two lines in which expression was almost absent had male production rates of about 30%, compared with about 90% in controls. These results were obtained under specific induction conditions and cannot be generalized to mean that a fixed sex ratio was maintained across all environments.

Significance and outlook: A model for studying early sex-chromosome differentiation

The researchers found that lowering expression of the hormone-responsive transcriptional regulators E75 and HR3 also reduced Dfh expression. This supports a role for both regulators upstream of Dfh. However, the interpretation that they bind directly to Dfh regulatory regions relies in part on structural predictions and should be distinguished from experimental confirmation of direct binding. Analysis of maternal carapace tissue also supports the possibility that Dfh connects molting and reproductive processes with the transmission of male-inducing signals.

The overall results support a model in which existing hormone-responsive genes and neighboring genes were retained together in a region of suppressed recombination, strengthening genetic control of sex. However, manipulating Dfh alone did not fully reproduce the natural NMP phenotype. Contributions from other candidate genes or incomplete suppression of expression in edited lines may explain the remaining differences. The relative contributions of natural selection and genetic drift to maintaining recombination suppression also remain unresolved. There is no basis for concluding that this system is progressing stably and irreversibly toward complete GSD.

The findings and experimental conditions are available in the public full text of the PNAS paper.