Ontogeny of hemocyanin in the ovoviviparous cockroach Blaptica dubia suggests an embryo-specific role in oxygen supply.

For a long time it had been assumed that specific oxygen transport proteins are absent in insects. Only recently it has been demonstrated that hemocyanins occur in the hemolymph of many ametabolous and hemimetabolous insect taxa, but not in the Eumetabola (Hemiptera+Holometabola). Therefore, the loss of respiratory hemocyanin in insects is not correlated with the evolution of an efficient tracheal system. The specific contribution of hemocyanin to oxygen supply in insects, however, has remained uncertain. Here we investigate the stage-specific expression of hemocyanin in the ovoviviparous cockroach Blaptica dubia (Blattaria), which consists of two distinct subunit types (Hc1 and Hc2). Employing quantitative real-time RT-PCR and Western blotting, we showed that the expression of hemocyanin is restricted to late embryos, thus being detectable also in whole female extracts and oothecae. Hemocyanin protein is also present in 1st instar nymphs, but not in later developmental stages. The ontogeny of hemocyanin in cockroaches is distinct from that known from Zygentoma and Plecoptera, in which hemocyanin occurs in both nymphal and adult stages. Our findings suggest a specific role of hemocyanin in embryonic cockroaches, which may be related to an enhanced oxygen supply in the oothecae. For some reason, the fundamental physiological changes associated to the evolution of holometaboly have made hemocyanin unnecessary.

The occurrence of hemocyanin in Hexapoda.

Hemocyanins are copper-containing, respiratory proteins that have been thoroughly studied in various arthropod subphyla. Specific O(2)-transport proteins have long been considered unnecessary in Hexapoda (including Insecta), which acquire O(2) via an elaborate tracheal system. However, we recently identified a functional hemocyanin in the stonefly Perla marginata (Plecoptera) and in the firebrat Thermobia domestica (Zygentoma). We used RT-PCR and RACE experiments to study the presence of hemocyanin in a broad range of ametabolous and hemimetabolous hexapod taxa. We obtained a total of 12 full-length and 5 partial cDNA sequences of hemocyanins from representatives of Collembola, Archeognatha, Dermaptera, Orthoptera, Phasmatodea, Mantodea, Isoptera and Blattaria. No hemocyanin could be identified in Protura, Diplura, Ephemeroptera, Odonata, or in the Eumetabola (Holometabola + Hemiptera). It is not currently known why hemocyanin has been lost in some taxa. Hexapod hemocyanins usually consist of two distinct subunit types. Whereas type 1 subunits may represent the central building block, type 2 subunits may be absent in some species. Phylogenetic analyses support the Pancrustacea hypothesis and show that type 1 and type 2 subunits diverged before the emergence of the Hexapoda. The copperless insect storage hexamerins evolved from hemocyanin type 1 subunits, with Machilis germanica (Archeognatha) hemocyanin being a possible 'intermediate'. The evolution of hemocyanin subunits follows the widely accepted phylogeny of the Hexapoda and provides strong evidence for the monophyly of the Polyneoptera (Plecoptera, Dermaptera, Orthoptera, Phasmatodea, Mantodea, Isoptera, Blattaria) and the Dictyoptera (Mantodea, Isoptera, Blattaria). The Blattaria are paraphyletic with respect to the termites.