Do all stoneflies nymphs have respiratory proteins? Further data on the presence of hemocyanin in the larval stages of plecoptera species

Abstract

Contrary to what was assumed regarding the presence of respiratory proteins in insects, a functional hemocyanin was recently found in larvae and adults of the stoneflies species Perla marginata, whereas in the close species Perla grandis, hemocyanin functionality was deduced from sequence data. In order to verify if the presence of this ancient trait is widespread within the order and to investigate why stoneflies have maintained it, we have extended the search for hemocyanin to species of other Plecoptera families. In particular, we assessed the presence of hemocyanin in the larval stage of nine Plecoptera species, belonging to six of the seven families of the European stonefly-fauna, and analyzed its potential functionality as deduced by sequence data. We cloned and sequenced the corresponding cDNAs and studied their expression with RT-PCR technique. Moreover, we performed homology studies using the deduced amino acid sequences. On the basis of our analysis, we hypothesized a functional role of the hemocyanin only for two species: Dinocras cephalotes and Isoperla grammatica (Perloidea). In all the investigated Nemouroidea and in Siphonoperla torrentium (Perloidea), this protein may have been lost. Larval size, life-cycle length, trophic role and environmental induction are discussed as possible explanations of these different physiological requirements.

Present knowledge on the presence of hemocyanin in stoneflies (Insecta: Plecoptera)

“…The results regarding most of them have been already published (Amore et al 2009); new data here presented concern Capnia bifrons (Newman, 1839), Guadalgenus franzi (Aubert, 1963), Afronemoura amatolae (Balinsky, 1956); and Aphanicercella bullata Stevens and Picker, 1999. RT-PCR cloning of stonefly hemocyanin Total RNA was extracted from larvae and adults, and degenerate oligonucleotide primers, targeting the hemocyanin conserved region (about 600 nucleotides), were used in an RT-PCR reaction.…”

“…On the contrary, RT-PCR reaction in adults of Dinocras cephalotes and Isoperla rivulorum did not result in PCR products or gave aspecific products. Siphonoperla torrentium (Chloroperlidae) and all the studied species of Nemouroidea (see Table 1 under Euholognatha) lack a functional hemocyanin because the conserved region sequence shows substitution of key amino acids involved in Cu þ bond and in stabilisation of the Cu þ -O 2 complex (see also Amore et al 2009). Moreover, these sequences have a certain similarity to insect hexamerins (hx).…”

“…Cloning and sequencing hemocyanin Based on cloning and sequencing RT-PCR products of expected size [see Genebank accession numbers DQ118369, DQ118370 (Fochetti et al 2006), EF218621, EU672885, EU672886, EU672887, EF218622, EU672888, EF218620, EU672889, EU672890, EU715327 (Amore et al 2009), FJ384672; FJ393060, FJ415315], we can conclude that hemocyanin is not homogeneously widespread across the order Plecoptera and that its presence can vary during the life cycle (Table 1). All studied larvae of Perlidae and Perlodidae species, except of Siphonoperla torrentium, harbour hemocyanin; this hemocyanin consists of two distinct subunit types (Hc1 and Hc2).…”

“…Amore and R. Fochetti a pGEM-T easy vector, and sequenced by a commercial service, as described elsewhere (Amore et al 2009). …”

“…Email: valamore@unitus.it Vol. 31, Supplement 1, 2009, 577-583 Gutie´rrez and Bastiani 1998); hemocyanin was found in Thermobia domestica and Lepisma saccarina (Zygentoma) (Pick, Hagner-Holler and Burmester 2008), and in larvae and adults of the stonefly Perla marginata (Hagner-Holler et al 2004), Perla grandis (Fochetti et al 2006) and other species of Perloidea (Amore et al 2009). Recently, Pick, Schneuer and Burmester (2009) have reported the presence of hemocyanin in many hexapod orders, like Collembola, Diplura, Archaeognata, Zygentoma, Orthoptera, Phasmida, Dermaptera, Isoptera and Blattodea.…”