Background: Reproductive stages are key to low-salinity adaptation

A study reports that genetic changes related to sperm function, egg-membrane strengthening, and membrane breakdown at hatching contribute to Baltic herring’s adaptation to reproduction in low salinity. Published in the Proceedings of the National Academy of Sciences (PNAS), the study tracked adaptation in reproduction and early development using population-genome comparisons and protein and enzyme analyses. The proposed mechanism that prevents sperm from swelling remains a hypothesis requiring further testing.

Atlantic herring (Clupea harengus) colonized the Baltic Sea within approximately the past 8,000 years. Salinity is 34–35‰ in the North Atlantic, compared with as low as 2–3‰ in the inner Gulf of Bothnia in the northern Baltic. Because sperm and eggs of externally fertilizing fish are directly exposed to the surrounding water, adaptation must be examined during fertilization and embryonic development as well as in adults.

The researchers reanalyzed existing whole-genome data from 69 population samples. In addition to Baltic populations, they compared a population that lives in the sea as an adult and spawns in the brackish Ringkøbing Fjord in Denmark. They narrowed down candidates for reproductive adaptation by looking for variants shared by the two brackish-water spawning populations, then investigated four genomic loci using mass-spectrometry-based proteomics and experiments on egg membranes and enzymes.

Key findings: A membrane that resists swelling and a hatching enzyme that opens it

In sperm, the researchers detected LRRC8C2, a type of volume-regulated anion channel (VRAC). Combinations of amino-acid variants in this protein showed a clear association with spawning-site salinity. Based on VRAC’s known function and the protein’s sperm-specific detection, the researchers proposed that Baltic-type LRRC8C2 may help prevent excessive sperm swelling by promoting the release of ions and osmoregulatory molecules. They did not directly establish the protective effects of individual variants.

In the egg membrane, two key candidates emerged: the structural protein ZPBA1 and the FTG1-3 gene family, which encodes fish transglutaminases (FTG) that form crosslinks between proteins. The most common Baltic-type ZPBA1 sequence had 12 crosslinking target sites, compared with five in the Atlantic type. FTG activity in unfertilized-egg extracts was higher in the Baltic type at 6‰ and in the Atlantic type at 35‰.

The protein density of Baltic herring egg membranes was more than twice that of Atlantic herring. At 6‰ salinity, the diameter of fertilized Atlantic herring eggs increased by more than 30%, while Baltic herring eggs generally resisted swelling under the same conditions. The results link changes in egg-membrane proteins and enzymes with denser membranes and resistance to swelling in low salinity.

Even a sturdy egg membrane must break down at hatching. Analysis of long-read genome assemblies from eight Atlantic herring and seven Baltic herring found that, with one exception, Baltic-type haplotypes carried about 20 copies of the hatching enzyme gene HE1C. Here, a haplotype means a combination of genetic variants linked together on the same chromosome. Atlantic-type haplotypes had at most two copies at this locus, and most had none. The activity of partially purified hatching enzyme peaked at low salinity for the Baltic type and at high salinity for the Atlantic type.

Meaning and outlook: Linked adaptations, with functional validation still needed

These findings support an adaptation in which changes that strengthen the egg membrane are matched by changes that break it down at hatching. Population and phylogenetic analyses indicated that adaptive variants at the FTG1-3 and HE1C loci were introduced from the sister species, Pacific herring. The results are interpreted as an example of genetic variants introduced from another species contributing to adaptation to a new environment.

However, the study did not isolate and verify the causal effects of every individual variant. In vitro fertilization experiments showed differences in fertilization rates by salinity, but the sample was too small to establish statistical significance. The experiments were also not designed to compare fertilization rates in natural spawning grounds, where waves and currents act. The findings therefore should not be extended to claim quantitative evidence of increased reproductive success or greater stock abundance.

The methods and results can be found in the public full text of the PNAS paper “Sperm, egg, and embryo proteins critical for genetic adaptation of herring to low salinity in the Baltic Sea.”