Isoform inflation and annotation heterogeneity can confound Kunitz-repertoire comparisons in blood-feeding animals: a gene-level reappraisal

Hematophagy, or blood-feeding, has evolved independently multiple times throughout Metazoa. Families of anticoagulant proteins, such as the Kunitz/BPTI domain, are often associated with these blood-feeders, with the leech often cited as an exception. To examine this association further, researchers applied a gene-level protocol to eight metazoan lineages.

They discovered that while blood-feeders had a median of 18 Kunitz-repertoire genes compared to a median of 39 in non-blood-feeders, this did not represent a consistent, universal elevation in gene-level Kunitz-repertoire size. The researchers also found that protein-entry counts can inflate gene-level Kunitz counts by up to 4.6 times.

Additionally, neither protein-entry counts nor proteome-wide isoform density correlated with diet, making protein-entry comparisons an unreliable basis for repertoire claims. The researchers also found that 12 independent blood-feeding origins could be counted using deterministic bookkeeping, although this number may be lower if the ancestral lamprey is considered a parasitic species with two losses.

A screen of annotated public genomes yielded only one candidate blood/non-blood pair, the bedbug. Under the pre-registered simulation scenario, only a cross-origin heterogeneity endpoint was attainable within a realistic origin ceiling, and only under strong heterogeneity among origins. The researchers caution about isoform/annotation bias in cross-phylum comparisons and emphasize the need for a gene-level, annotation-aware re-analysis.

They also highlight the limitations of current data in supporting claims about cross-phylum associations.