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

Hematophagy has arisen independently many times across Metazoa, and recurrent anticoagulant protein families in blood-feeders are often read as convergent recruitment - the Kunitz/BPTI domain a paradigm case, with the leech an oft-cited low-Kunitz exception. We re-examine this at the gene level and ask whether a confirmatory cross-phylum test of this blood-feeding/anticoagulant association is feasible with public genomes. Applying an auditable gene-level protocol (one longest-isoform representative per gene; conservation-checked protein to gene mapping) to eight metazoan lineages, we find no consistent, universal elevation of whole-genome gene-level Kunitz-repertoire size in these blood-feeders (blood-feeder median 18 genes vs non-blood-feeder median 39; a descriptive comparison of non-independent taxa, not a formal test). Protein-entry counts inflate gene-level Kunitz counts by up to ~4.6x (mosquito 23 to 5), and neither this inflation nor proteome-wide isoform density (1.0-2.7x) tracks diet, so protein-entry comparisons are an unreliable basis for repertoire claims. Separately, deterministic bookkeeping under a fixed topology and a no-reversal rule counts 12 independent blood-feeding origins (11 if the ancestral lamprey is treated as parasitic with two losses); a non-exhaustive screen of annotated public genomes yielded only one candidate blood/non-blood pair (bedbug), and, under the pre-registered simulation scenario, only a cross-origin heterogeneity endpoint is attainable within a realistic origin ceiling, and only under strong heterogeneity (among-origin SD >= 3-4). An exploratory, feasibility-grade secretome-composition estimate did not meet the pre-registered criterion. We offer a gene-level, annotation-aware re-analysis, a caution about isoform/annotation bias in cross-phylum comparisons, and an account of what current data can and cannot support.