Myriapod Metallothioneins conserve the ancestral architecture of Arthropods while displaying lineage-specific adaptations in metal binding

Abstract

Metallothioneins (MTs) are central to metal metabolism and contribute to organismal adaptation to variable metal bioavailability across ecosystems. Although well studied in chordates and mollusks, MTs remain poorly investigated in many arthropod lineages, particularly within the Myriapoda subphylum. Myriapods, comprising thousands of millipede (Diplopoda) and centipede (Chilopoda) species, are especially relevant for evolutionary studies because they are the sister group to Pancrustacea (crustaceans and insects), and they are some of the earliest arthropods to colonize land. Their MTs therefore provide critical insights into the origin and evolution of arthropod MTs and into the molecular adaptations underlying the colonization of new environments. In this work, we have identified 48 putative MTs from 30 myriapod species, all classified as type 1 (MT1) and occurring in two configurations: the bidomain MT1S (S for short) or the multidomain MT1L (L for long) variants. Evolutionary analyses suggest that MT1S represents the ancestral type not only in myriapods but across Arthropoda, whereas MT1L likely arose during chilopod diversification, probably restricted to the order Glomerida. Despite shared structural features, metal-binding characterization of three myriapod MTs–GminMT1Sa, GminMT1La from <em>Glomeridella minima</em>, and LforMT1S from <em>Lithobius forficatus</em>–revealed marked functional differences. The diplopod proteins GminMT1Sa and GminMT1La displayed a Cd-thionein character, while the chilopod LforMT1S was a multipurpose protein, binding cadmium, zinc, and copper without a clear metal preference. These differences likely reflect distinct metal uptake, retention, and excretion strategies in diplopods and chilopods, associated with their ecological adaptations as peaceful decomposers and voracious predators, respectively.