New data on the presence of hemocyanin in Plecoptera: recomposing a puzzle

Valentina Amore1,2, Brunella Gaetani1, Maria Angeles Puig2, Romolo Fochetti1, *
1Environmental Sciences Department, University of Viterbo, 01100 Viterbo, Italy
2Centro de Estudios Avanzados de Blanes (CEAB-CSIC), 17300 Blanes, España
*Corresponding author ; fochetti@unitus.it
valamore@unitus.it; bgae85@libero.it; puig@ceab.csic.es
Abstract
So far, the specific role of hemocyanin in Plecoptera is still not completely understood, since none of the hypotheses advanced to this regard proved to be fully convincing. Previous data showed that mRNA hemocyanin sequences are not present in all Plecoptera and hemocyanin does not seem to be uniformly distributed within the order. All species possess hexamerins (hx), multifunction proteins that probably originated from hemocyanin.
 In order to obtain an increasingly detailed picture on the presence and distribution of hemocyanin across the order, we present new data regarding nymphs and adults of selected Plecoptera species.
The results confirm that the hemocyanin expression differs among nymphs in the studied stonefly species. As regards adults, even though hemocyanin has been found in the past in two stonefly species, in the present study we did not detect it, also in species where nymphs show hemocyanin, suggesting that the physiological need of this protein can change during life cycle.
The phylogenetic pattern obtained using hemocyanin sequences matches the accepted scheme of traditional phylogeny based on morphology, anatomy and biology. It is remarkable to note that the hemocyanin conserved region acts like a phylogenetic molecular marker within Plecoptera.

Key words: Stoneflies, Respiratory proteins, Phylogeny, cDNA

Introduction
The recent discovery of hemocyanin in many insect orders raises doubts about the common assumption that the tracheal system is sufficient for insects respiration, and thus respiratory proteins are not necessary. Based on a first report of hemocyanin in the perlid stonefly Perla marginata (Hagner-Holler et al. 2004), our research has been aimed at better understanding the presence, functional significance, and role of this protein in the Plecoptera (Fochetti et al. 2006; Amore et al. 2009; Amore and Fochetti 2009). In previous studies, we assessed the presence/absence of hemocyanin mRNA in the larval and adult stage of chosen species belonging to the seven European stonefly families (Amore et al. 2009). We also tested some selected Oriental and Afrotropical stonefly species living in rivers with different ecological features respect to those of the Palaearctic streams (Amore and Fochetti 2009; Amore et al. 2010). So far we have investigates 33 species  (present data included): 25 species belonging to the seven family of the two European superfamilies, 5 species belonging to Oriental Perlidae, 1 Oriental Peltoperlidae, 2 species of African Notonemouridae. We analyzed the target species during different phases of the life cycle (nymphs and adults), and from various streams and river tipologies (perennial temperate rivers, Mediterranean temporary streams, tropical rivers, high elevation rivers and lakes) (Amore and Fochetti 2009). Our data clearly show that mRNA hemocyanin sequences are not present in all Plecoptera (Fochetti et al. 2006; Amore et al. 2009; Amore and Fochetti 2009) and hemocyanin does not seem to be uniformly distributed within the order. All species possess hexamerins (hx), multifunction proteins that probably originated from hemocyanin. We hypothesized that the presence of hemocyanin could depend on the length of the life cycle (hence on the body size), the trophic role or environmental induction. None of these hypotheses proved to be fully convincing (Amore et al. 2010a  in press) and the specific role of hemocyanin in Plecoptera is still not completely understood. However, using liquid chromatography tandem mass spectrometry we proved that, regardless of its putative function (respiratory, immune defense, storage protein), hemocyanin is actually expressed in species in which its mRNA is present (Amore et al. 2010b submitted).
The hemocyanin expression pattern we have so far obtained for the entire Plecoptera order could also be explained by other functions besides respiration, but this investigation is beyond the scope of the present paper.
As far as nymphs are concerned, the present paper has the main goal to extend the study on the presence/absence of this pigment to other Plecoptera genera/species which have not been investigated so far (the genera Dyctiogenus, Perlodes, Besdolus, Arcynopteryx, Pachyleuctra). In order to obtain an increasingly detailed picture of the hemocyanin presence and distribution across the order and in the attempt to better understand functional significance and role of this protein in the Plecoptera we studied Italian stenoendemisms (Besdolus ravizzarum Zwick and Weinzierl, 1995), Pyrenean endemics (Pachyleuctra benllochi (Navás, 1917)) and species believed to have a very ancient origin, like the ercinic relict Arcynopteryx compacta (McLachlan, 1872).
As regards adults, hemocyanin has been recorded so far only in Perla marginata (Hagner-Holler 2004) and P. grandis (Fochetti et al. 2006); in our previous studies we never detected hemocyanin in adults of other species (Amore and Fochetti 2009), even in species where we could sequence hemocyanin in nymphs, suggesting that the physiological request of hemocyanin can change during the life cycle. Here we extend the study to other species, to cover a representative sample of the European biodiversity of the order at the family level (genera Pachyleuctra, Nemoura, Protonemura).

Material and methods
Sequence analysis
Specimens belonging to the following ten species (nymphs and adults) were collected and preserved in RNA later.

      Perlodidae
Dyctiogenus alpinum (Pictet, 1842) and Perlodes intricatus (Pictet, 1841) nymphs. Collected February 1st, 2009. Po river, Pian della Regina, Crissolo, m 1800 (Cuneo – Piemonte Region - Italy);
Besdolus ravizzarum nymphs. Collected February 3rd, 2009. Curone stream, Val Curone m 320 (Alessandria – Piemonte Region - Italy). 44°47′14″ N; 9°04′02″ E;
Arcynopteryx compacta, (McLachlan, 1872) nymphs. Collected June 6th, 2009, Blue Lake, Rosellón, m 2530. (Oriental Pyrenees department - Languedoc Region – France) N 42,61554; E 1,96704.
Isoperla acicularis (Despax, 1936) ssp. acicularis. Nymphs and adults. Collected July 2008. Vallarties river, m 1390. (Catalunya– Spain). 00º48'10,9"E; 42º39'24,07"N, 
Leuctridae
Leuctra alosi Navás, 1919. Adults. Collected July 2008. Vallarties river, m 1390. (Catalunya – Spain). 00º48'10,9" E; 42º39'24,07" N.
Pachyleuctra benllochi, (Navás, 1917). Nymphs and adults. Collected July 2008. Escita inlet, m 1790. (Catalunya – Spain). 01º00'56,0"E; 42º34'44,2"N.
Nemouridae
Amphinemura sulcicollis (Stephens, 1836). Adults. Collected July 2008. Vallarties tributary, m 1390. (Catalunya – Spain). 00º48'10,9" E; 42º39'24,07" N.
Nemoura cinerea (Retzius, 1783), and Protonemura tubercolata Kempny, 1888. Adults. Collected July 2008. Peguera river and tributaries,  m 2295. (Catalunya – Spain). 01º02'47,5"E; 42º32'43,9"N.

Total RNA was extracted and degenerate oligonucleotide primers, designed according to hemocyanin conserved region (about 600 nucleotides), were used in a RT-PCR reaction. A β-actin fragment was used as control (P. marginata β-actin: HM991865, B. ravizzarum β-actin: HM991864). PCR fragments of expected size were cloned into pGEM-T easy vector, and sequenced by a commercial service, as described in Amore et al. 2009. The sequences thus obtained were translated with the tool provided by ExPASy Molecular Biology Server of the Swiss Institute of Bioinformatics (http://www.expasy.org).

Sequence data and multiple alignment
Two different multiple alignments of the proteins belonging to the hemocyanin superfamily (HcSF) were performed: the first one only for Plecoptera sequences, whereas the second one for sequences of Plecoptera and other groups of arthropods.
Multiple alignment: Plecoptera. Were deduced from our cDNA and from Genbank database the sequences in 29 stonefly species of 14 hemocyanins (6 of the subunit1 (hc1) and 8 of the subunit 2 (hc2)), and 27 hexamerins. Tab. 1 lists the sequences used for the alignment. Six Myriapoda hemocyanin sequences (i.e., Scutigera coleoptrata AJ344359, AJ344360, AJ431378, AJ431379, AJ512793 and Spirostreptus sp. AJ297738) were used in the alignment, since Myriapoda are in an ancestral position respect to Plecoptera (Kusche and Burmester 2001). The final alignment included 48 sequences, 520 nucleotides or 154 amino acids positions. 
Multiple alignment: Arthropod HcSF. The alignment of Plecoptera sequences was completed with others sequences of the arthropod hemocyanin superfamily, retrieved from the GenBank database. The alignment was composed of Crustacean prophenoloxidases (PPO), Insect prophenoloxidases (PPO), Crustacean cryptocyanins (CC) or pseudohemocyanins (Phc), Crustacean hemocyanins (hc), Myriapoda hemocyanins (hc), Insect hemocyanins (hc), and Insect hexamerins (hx).
The hexamerin receptors were ignored in this study because only a small part of the sequences aligned well with the hemocyanin conserved region we analyzed. A list of sequences, for the non-Plecopteran taxa used in this study is provided in Tab. 2. The final alignment comprises 102 sequences, 785 nucleotides and 161 amino acid positions.
Sequence alignment and phylogenetic inference. Multiple alignment of nucleotides and amino acid sequences was constructed with the MAFFT, online version (Katoh et al. 2005), matrix BLOSUM62. Long gap regions, as well as some highly divergent regions, were removed from the final data set and, in order to optimize the results of the phylogenetic analysis, we operated a selection of conserved blocks from multiple alignments with Gblocks server (Talavera and Castresana 2007) . The appropriate phylogenetic model for nucletidic sequences was selected with MrModeltest2 (Nylander et al. 2004) and the amino acid sequence evolution model was chosen by ProtTest (Abascal et al. 2005), using the Akaike Information Criterion (AICc). Nucleotidic tree constructions were performed by Bayesian analysis with MrBayes 3.1-2 (GTR model). The reliability of the trees was tested by bootstrap analysis (Felsenstein 1985) with 1000 replications.
Amino acidic trees were inferred by Maximum Likelihood (ML) methods. The phylogenetic analyses were performed with PhyML (http://www.atgc-montpellier.fr/phyml/) (Guindon and Gascuel 2003) with100 replications. Distances between pairs of protein sequences were calculated according to LG model (Le and Gascuel 2008) assuming a gamma distribution of substitution rate.

Results
Sequence analysis. The designed primers were applied to cDNAs reverse transcribed from the investigated species. When these primers were applied on nymphs, they produced fragments of the expected size. We amplified two sequences for D. alpinum a.n. GU121395, GU121396, one sequence for B. ravizzarum a.n. GU121394, A. compacta a.n. GU121393, P. intricatus a.n. GU121397, I. acicularis acicularis a.n. GU121398, and P. benllochi a.n. GU121399. The amplified fragments were about 600 nucleotides long; the translated amino acid sequences resulted in about 195 amino acids, except for P. intricatus whose amplified fragment were of 893 nucleotides and the translated sequence of 297 aminoacids (Tab. 2). The same primers applied to adult specimens gave no band in PCR experiments.
Both BLAST (Blastn and Blastp) and phylogenetic analyses (see below) unequivocally identified the sequences of D. alpinum (Dyc_al.hc1; Dyc_al.hc2), B. ravizzarum (Bes_ra.hc2) and A. compacta, (Arc_co.hc2) as insect hemocyanins. The five histidines (His) of the studied fragment, crucial for the O2-binding function, were present in all subunits (Fig. 1), while the sequence of P. intricatus (Per_in.hx) and P. benllochi (Pac_be.hx) were identified as hexamerins. In order to compare and describe the amino acid sequences of nymphs they were further compared to the hemocyanins known from the Perlodidae Isoperla grammatica (Iso_gr.hc1 and Iso.gr.hc2; Amore et al. 2009), the Chloroperlidae Siphonoperla torrentium (Sip_tor.hx) and the Leuctridae Leuctra fusca (Leu_fus.hx) (Tab. 3). As expected, D. alpinum hemocyanin subunit 1 (Dyc_al.hc1) showed the highest degree of identity with the type 1 hemocyanin subunits (0.90 amino acidic and 0.85 nucleotidic), whereas lower scores were obtained when comparing to type 2 subunits (0.53-0.55 aminoacidic and 0.61-0.63 nucleotidic). Subunit 2 of D. alpinum (Dyc_al.hc2), B. ravizzarum (Bes_ra.hc2), A. compacta (Arc_co.hc2), I. acicularis acicularis (Iso_ac.hc2) and stonefly hemocyanin subunit 2 of I. grammatica displayed 0.86-0.95 identical amino acids, and 0.86-0.94 identical nucleotides, while lower identity scores were observed with other type 1 subunits (0.53-0.54 aminoacidic and 0.60-0.62 nucleotidic). Only one of the four Cu-binding histidines are conserved in the P. intricatus and P. benllochi hexamerins. Comparison with hc1 and hc2 were in the range of 0.38-0.46 for aminoacids and 0.53-0.58 for nucleotides, whereas identity values were higher among Plecoptera hexamerins (0.58-0.83 aminoacids and 0.69-0.86 nucleotides). P. benllochi showed a close relationship with L. fusca (0.83 amino acid and 0.86 nucleotide), both species belong to Leuctridae.
Phylogenetic analysis. Both types of analyses (Bayes and ML) gave similar tree topologies (Figs 2 and 3).
Plecoptera. The Myriapoda sequences were used to root the tree for graphics purposes. ML analysis (Fig 2) resulted in three well supported monophyletic clades. Dyc_al.hc1 joined the clade with the previously identified Plecoptera hemocyanin subunit 1 (100% bootstrap support) (Fig. 2). Dyc_al.hc2; Bes_ra.hc2, Iso_ac.hc2, and Arc_co.hc2 grouped with hemocyanin subunit 2; (100% bootstrap support). Hexamerins, where Per_in.hx and Pac_be.hx grouped, formed a third clade; (100% bootstrap support). Within hemocyanin subunit type 1, the Perlodidae sequences (Iso_gr.hc1; Guad_fr.hc1; Dyc_al.hc1) resulted to be monophyletic and derived from Perlidae (87% bootstrap support), whereas the Perlidae clade is more disordered. Within the clade of hemocyanin subunit 2, Perlidae and Perlodidae formed two distinct clades, even if the Perlodidae clade was supported by a 50% bootstrap value. The phylogenetic analysis indicated that the hemocyanin subunit 2 shares a common ancestor with all Plecoptera hexamerins. Within this clade the systematic relationship among groups were less resolved. 
The results of Bayesian inference (Fig. 3) generate a posteriori distribution starting from a a priori probability: the Plecoptera tree shows an unresolved node when examining hc1, hc2 and hexamerins genetic affinity. 
HcSF: Within the hemocyanins, three distinct clades emerged in accordance to the  phylogeny of arthropod subphyla. Both ML and Bayesian inference (Fig. 4 and Fig. 5) resulted in a branch representing Chelicerata hemocyanin (97-100% bootstrap support), a second branch representing Myriapoda hemocyanin (100% bootstrap support), and a third branch for crustacean and insect hemocyanins, insect hexamerins and crustacean cryptocyanins (95-100% bootstrap support). Myriapoda is the sister group respect to crustacean and insect hemocyanins. All insect hexamerins formed a clade (82-98% bootstrap support). The results of ML analysis showed that there is one clade for insect hemocyanin subunit 1, one clade for insect hemocyanin subunit 2, and one for crustacean hemocyanins and cryptocyanin. This macro-clade had a low bootstrap support (<80%). At any rate, all hexapod hexamerins joined in the same clade in all analyses. In subunit 1, the hemocyanins from Zygenthoma (Ter_do.hc1 and Lep_sa.hc1) formed the sister group of the pterygote proteins (97% bootstrap support) and Collembola (Sin_cu.hc1 and Fol_ca.hc1) was basal to the ectognathan subunits (38% bootstrap support). Within the hemocyanin subunit types 2, phylogeny resembled that of subunit types 1, and Machilis germanica (Mac_ge.hc1) was in an ambiguous position and clustered within hexamerins.
In Bayesian inference (Fig 5), there is an unresolved node in the cluster including sequences of hexapod hc1 and hc2, crustacean hc and cryprocyanin.

Discussion
Hemocyanin in nymphs. Our results suggest that the hemocyanin expression differs among nymphs of different stonefly species. We sequenced the hemocyanin conserved region in all nymphs, except in those of P. intricatus and P. benllochi, where we found only hexamerins. These results confirm that hemocyanin is not expressed in all Plecoptera species. It is worthy to note that P. intricatus and D. alpinum were collected in the same river, sampling site, and sampling date. Both species belong to Perlodidae, are medium-size, semivoltine, and are mainly predators (Fochetti and Tierno de Figueroa 2008), but they display a different physiological response on hemocyanin production. On the other hand, B. ravizzarum, a Perlodidae living at lower altitude, in the potamal river zone, expresses hemocyanin in its mRNA repertory.
Summarizing all the data regarding stoneflies published so far (Tab. 4) (Hagner-Holler et al. 2004; Fochetti et al. 2006; Amore et al. 2009; Amore and Fochetti 2009; Amore et al. 2010a), we can confirm that hemocyanin expression in Plecoptera does not depend on size or trophic role. Environmental adaptation to ecological condition might have led to the loss of the protein in some lineages. It is conceivable that independent adaptations to local conditions caused a decrease in hemocyanin requirement, a precondition to generate variability. Cumulative mutations and divergent evolution probably caused significant change in hemocyanin domain II, to the point of disabling copper-binding sites and oxygen affinity, thus leading to ancestor-like hexamerin proteins. 
Hemocyanin in adults. Plecoptera are hemimetabolous insects whose habitat completely changes when they become adults. While nymphs live in aquatic habitats, adult stoneflies emerge from the streams, lakes or rivers. They have reduced flight ability and in some cases, males are brachypterous (for the species investigated in the present paper, this condition occurs in D. cephalotes and I. viridinervis). They can generally be found on the banks next to the emergence area. Although the amount of oxygen in the air is much higher compared to the oxygen dissolved in water, it was proven that even insects which are terrestrial in all their developmental phases possess respiratory proteins. In fact, hemoglobin genes were found in holometaboulos insects such as Drosophila (Hankeln et al. 2002) and Apis (Hankeln et al. 2006), and some Hemiptera, Coleoptera and Lepidoptera that live in normoxic conditions (Burmester and Hankeln 2007).
Adults and nymphs have very different activities in the Plecoptera: the nymphal stage is mainly devoted to feeding and molting, thus undergoing considerable physiological stress (Fochetti and Tierno de Figueroa 2008; Tierno de Figueroa et al. 2003). Adults are mainly devoted to mating (Tierno et al. 2006), and in some cases, they do not feed at all (for instance Perla marginata, P. grandis, D. cephalotes) (Tierno De Figueroa and Fochetti 2001). 
Preliminary data on the presence of hemocyanin in adults were reported in Amore and Fochetti, 2009. In the present study we extended the number of investigated species to a representative of all the European families of the order. So far, hemocyanin had been recorded for Perla marginata (Hagner-Holler 2004) and P. grandis (Fochetti et al. 2006), but in our previous and present studies, we never detected hemocyanin in adults, even in species where hemocyanin was sequenced in nymphs, suggesting that the physiological need of hemocyanin may change during life cycle.
Plecoptera hexamerins. Hexamerins were sequenced in nymphs and in the adult of Capnia bifrons, an ovoviviparous species (Hynes 1941; Fochetti and Tierno de Figueroa 2008). It is interesting to note that hexamerins are proteins usually expressed at high concentrations in larval and nymphal stages and rarely in adults (Beintema et al. 1994). Insect hexamerins show significant similarities in structure and sequence to arthropod hemocyanins (Markl et al. 1992; Beintema et al. 1994; Burmester and Scheller 1996) and it has been suggested that hexamerins changed their function to storage proteins after losing the ability to bind oxygen (Markl and Winter 1989). Hexamerins serve mainly as sources of aminoacids during non-feeding periods, in larval molting or adult development (Telfer and Kunkel 1991; Haunerland 1996; Beintema et al. 2004), but can also work as carrier proteins for small organic compounds, like steroid hormones, riboflavin and juvenile hormones (Enderle et al. 1983; Magee et al. 1994; Braun and Wyatt 1996), or may be involved in immune response (Hayakawa 1994; Beresford et al. 1997). 
Phylogenetic implications. 
Plecoptera HcSF. Starting from the hypothesis that a common ancestor of all modern Plecoptera possessed hemocyanin, this character was lost several times during the evolution of the order. A first loss might have happened in the Nemouroidea ancestor, since we never found hemocyanin in any of the Nemouroidea species we analysed. Secondarily, hemocyanin might have been independently lost in some Perloidea lineages, such as in Chloroperlidae or in the genus Perlodes. This idea is in agreement with the accepted theory that, even if Plecoptera is a very ancient order (fossil stoneflies date from the early Permian), the existing families do not seem to be very old, and recent and repeated phenomena of speciation and extinction have been described (Zwick 2000). In species where we did not sequenced hemocyanin, we only found hexamerins. Hexamerins evolved from hemocyanins in the early steps of insect evolution, so they are paralogous proteins. Our data would indicate that hexamerins evolved from subunit 2 (hc2), even though the analysis of a different dataset led Burmester and Hankeln (2007) to hypothesize hc1 as the probable closest subunit.
It is remarkable to note that the hemocyanin conserved region acts like a phylogenetic molecular marker within Plecoptera. Two branches of hemocyanin subunits (hc1 and hc2) are always evident in the topology of the trees, and the phylogenetic pattern obtained using hemocyanin conserved fragment matches the accepted scheme of traditional phylogeny based on morphology, anatomy and biology, even when examining taxonomy of subfamilies (e.g. Perlodinae, Isoperlinae and Arcynopteryginae within Perlodidae). Hexamerins follow more loosely the accepted systematic arrangement, indicating a lower evolutionary pressure that allowed them to accumulate mutations and distinct types of aminoacids (Telfer and Kunkel 1991; Burmester et al. 1998). The use of hemocyanin as molecular marker could be interesting to study in detail taxa whose systematic position within Plecoptera in still uncertain (for instance the relationships between Perlidae, Perlodidae and Chloroperlidae), and to analyse phenomena of speciation and adaptation.
Arthropoda HcFS. 
Chelicerata hemocyanins form a separate clade. It has been noted a phenoloxidase activity of some subunit of chelicerata hemocyanin (Decker et al. 2001), therefore these subunit types may be considered as transitional structures between phenoloxidases and hemocyanins. 
The Myriapoda hemocyanins clade is the sister group of insect and crustacean hemocyanin and their derivates (insect hexamerins and crustacean cryptocyanins), according to Kusche and Burmester, 2001. Assuming that protein phylogeny reflects species evolution, the presence of a unique clade for crustacean and hexapod hemocyanins and descendents strongly supports the Pancrustacea hypothesis, where all crustaceans and hexapods are included in a unique monophyletic taxon, in contrast to the Atelocerata hypothesis in which Myriapoda and Hexapoda are sister taxa, and Crustacea are more distantly related (see for instance Brusca and Brusca 2002).
Hexamerins and cryptocyanins underwent a parallel evolution. The hexamerins form a monophyletic clade which is the sister group of the known Insect and Crustacean hemocyanin, while cryptocyanins derived from crustacean hemocyanins (Beintema et al. 1994; Burmester and Scheller 1996; Durstewitz and Terwillinger 1997; Burmester et al. 1998; Burmester, 1999a, 1999b, 2001; Pick et al. 2009).

Further considerations
The study of hemocyanin in insects is at the center of an ongoing scientific debate. Several studies have explored the functional properties of Arthropod hemocyanins and have led to a plethora of hypothetical functions, included the role as oxygen carrier (Markl et al. 1979a, 1979b; Markl and Decker 1992) or non respiratory functions as phenoloxidase and antimicrobial activity (Terwilliger 1998; Bridges 2001; Decker and Jaenicke 2004; Jaenicke and Decker 2004). 
Recent studies on Chelicerates, which have no phenoloxidases, stressed that evolution has developed a double function for this molecule, suggesting that hemocyanin acquires a phenoloxidase activity after proteolitic cleavage at the amino-terminal part (Decker and Rimbke 1998, Decker and Tuczek 2000). Hemocyanins of Eurypelma californicum, Limulus polyphemus and Tachypleus tridentatus are comparable to phenoloxidases based on activation mechanisms, substrate specificity and inhibition (Nagai and Kawabata 2000; Nagai et al. 2001).
The role of hemocyanins in immune response seems to be present in chelicerates and also in crustaceans. Under normal conditions the hemocyanin functions as oxygen carrier, but it may be converted to phenoloxidase after microbial infections. In some crustacea (Penaeus vannamei and P. stylirostris), antimicrobial and antifungal peptides can be cleaved from the C-terminal domain of hemocyanin.(Destoumieux-Garzò et al. 2001, Lee et al. 2003) and hemocyanin concentration is associated with the molting cycle, suggesting a specific utilization during starvation (Depledge and Bjeregaard, 1989). Under special circumstances, hemocyanin is metabolically recycled and employed as a source of energy from aminoacids (Zuckerkandl 1960; Hagerman 1983).
What remains to be determined is if hemocyanin might have functions other than respiration in the Hexapoda as well (hence in the Plecoptera). In vitro or in vivo studies on functions other than respiration have not yet been carried out in Hexapoda.

Open questions
The present study focuses on Arctoperlaria species (the Northern hemisphere Plecoptera suborder), mainly on European fauna. The only sequence of the Antarctoperlaria (the Southern hemisphere Plecoptera suborder), Dhiamphipnopsis samali, included in our phylogenetic analysis, derives from a specific study on Plecoptera hexamerin (Hagner-Holler et al. 2007). Enlarging the study to Antactoperlaria would give a wider general indication to the problematic investigation of hemocyanin distribution in Plecoptera.
Another issue concerns the plasticity of hemocyanin with respect to the environmental context. Changes in hemocyanin expression can affect the total concentration of hemocyanin in the hemolymph or can modify the level of expression of a single subunit with respect to the others. Experiments aimed to monitoring adaptive physiology of Plecoptera in response to environmental stimuli, at the level of protein expression modulation and subunit ratio, are in progress with quantitative real-time PCR (qRT-PCR). If oxygen affinity and cooperativity of hemocyanin, and consequently the capacity of oxygen-transport are adapted to environmental conditions, possessing hemocyanin represents, for animals, a potential adaptive capacity in the context of global warming. In this future context the presence of hemocyanin, and its variability in subunits type and multimeric formation, may represent a focal aspect to be analyzed also from perspective of ecological selection (Schluter 2001).

Acknowledgement
This research was supported by the Autonom Organisms of Spanish National Parcs of "Medio Ambiente" Ministry (MIMAM), MAYSTONS projects (10/2007) and by the Spanish Ministry of Reseach and Innovation (MICIIN), GRACCIE project (CSD2007-00067). Cristina Bruno (IASMA, Trento, Italy) is thanked for having revised the English form.

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Iso_gr.hc1      VAYLGEDVGLNSHHSHWHMDFPFWWKAAEYGIEKDRKGELFYYMHHQMIARYDLERLSNH
Iso_gr.hc2      VAYLGEDIGVNSHHAHWHMDFPFWWKRT-YDVTKDRRGELFFYMHHQMVSRFDAERLSNN
Iso_ac.hc2      VAYLGEDIGVNSHHAHWHMDFPFWWKRT-YDVTKDRRGELFFYMHHQMVNRFDAERLSND
Bes_ra.hc2      VAYLGEDIGVNSHHAHWHMDFPFWWKKT-YDVTKDRRGELFFYMHHQMVNRFDAERLSNN
Arc_co.hc2      VAYFGEDIGVNSHHAHWHMDFPFWWKAT-YDVTKDRRGELFFYMHHQMTNRFDAERLSNN
Dyc_al.hc1      VAYLGEDVGLSSHHAHWHMDFPFWWKATEYGIEKDRKGELFYYMHHQMIARYDLERLSNH
Dyc_al.hc2      VAYLGEDIGVNSHHAHWHMDFPFWWKKT-YDITKDRRGELFFYMHHQMVNRFDAERLSNN
Per_int.hx      ISYFTEDVGLNAFHTYWNLDYPFWANSKNYNLKFDRRGELFYYTQHQLMARYYLERLSNG
Sip_tor.hx      VAYFGEDVGVNTFNTYWHLDYPFWMNSAKYNMHFDRRGELFYYTQHQLLARYYLERISNG
Pac_ben.hx      VAYLGEDVGLSTFHTYWNMDYPFWANHKTYGIKWDRTGELFYYTQHQILARYYLERLSNG
Leu_fus.hx      VAYLGEDVGLSTFHTYWNMDYPFWANSKYYNLKFDRDGELFYYTQDQILARYYLERLSNG
                ::*: **:*:.:.:::*::*:*** :   *.:  ** ****:* :.*:  *:  **:** 

Iso_gr.hc1      LPFVEPLSFE--EEIEHGFYPQTTYRVGGEFPSRPDNFEFHDLEHIKIQDMLDYTRRIRN
Iso_gr.hc2      LPQVEPLDWH--HEIVEGFAPGAMYHNGQEFPMRPDGMYFHDLPWLTIKDNEEFEGRIRD
Iso_ac.hc2      LPQVEPLDWH--HEIVEGFAPGAIYHNGQEFPMRPDGMYFHDLPWLTTKDNEEFEGRIRD
Bes_ra.hc2      LPQVEPLDWH--HEITEGFAPGAMYYNGQEFPMRPDGMHFHDLPWSTVKDNEELEGRIRD
Arc_co.hc2      LPQVEPLDWQ--NEIVEGFAPGAMYHNGQEFPMRPDDMHFHDLPWLTVKENEEFEGRIRD
Dyc_al.hc1      LPVVKPLNFE--EKIEHGFYPQTTYRVGGEFPSRPDNFEFHDLEHIKIKDMIDYTRRIRE
Dyc_al.hc2      LPQVEPLDWH--HEITEGFAPGAMYYNGKEFPMRPDGMHFHDLPWSTVKDNEELEGRIRD
Per_int.hx      LGEVKAFSY--FDSEIVGYQPSLRYQNGKEFPMRPEGMSVVYN--RQTEELYTLERRIQD
Sip_tor.hx      LGEIKHFDWSDRKTLMVGYEPMMRYQNGQEFTMRPEGSTFSRN--YRSEDAMIFERRIVD
Pac_ben.hx      LGEIKPFSYS-YKTPISGFEPSLRYQNGKEFPMRPEGVRFSNS--YKTEEALAYERRIFD
Leu_fus.hx      LGEIKPFSYT-FKT-IAGFEPSLRYQNGKEFPMRPEGVRFSNN--YKSEEAYAYERRIFD
                *  :: :.:   .    *: *   *  * **. **:.  .        ::      ** :

Iso_gr.hc1      AILKHSVLTKTGEHIALDNEHGIDILGDLMEPSMETLHHDYYGSLHNYAHILLGQITDPL
Iso_gr.hc2      IIASGFVKMTDGHLVYLNTTEGIDILGLIVETLDHSYNRDFFGKFHANSHVMLGRITDPM
Iso_ac.hc2      IIASGFVKMTDGHLVYLNTTEGIDILGLIVETLDHGYNRDFFGKFHANSHVVLSRITDPM
Bes_ra.hc2      TIAAGFVKMNDGHLAYLNTTEGINILGLIVESLDHDYNRVFFGKYHASAHTMLSRITDPM
Arc_co.hc2      AIAAGFVRMDDGHFAYLNTTEGIDILGLIVESLDHEYHHDLFGKYHSNTHVMLSRITDPQ
Dyc_al.hc1      AIFKKSVLTKTGDHIALDNIHGIDILGDLMEPSVETAHHDYYGSLHNYAHILLGQITDPK
Dyc_al.hc2      TIAAGFVKMTDGHLAYLNTTEGINILGLIVESLDHDYSRVFFGKYHANAHVMLSKITDPM
Per_int.hx      AIDLGFVFGKEGQKVSLKEKEGISILGEMIEGTADSANKNFYGSVYNMMKTVFGHVTDPT
Sip_tor.hx      AIDAGYIVSFDGQKLSLKDKEGITLLGELIMSTGDSPNKEFYGKIYTNLCTIFGHVMDHT
Pac_ben.hx      AIDLGFVVSKDGTKIPLKEKEGINILGEIIKGTTDTVNEHFYGTIYNMMRGIFGHVTDPN
Leu_fus.hx      AIDLGFVIAKDGSKIALKEKDGINILGELIKGTTDTVNENFYGAIYNMMRGIFGHVTDPN
                 *    :    *    *.  .** :** ::    .   .  :*  :     ::.:: *  

Iso_gr.hc1      GKFNMPPGVMEHFETP---
Iso_gr.hc2      LKFGMPPGVMEHFETAT--
Iso_ac.hc2      LKFGMPPGVMEHFETATIT
Bes_ra.hc2      LKFGMPPGVMEHFETA---
Arc_co.hc2      FKFGMPPGVMEHFETAT--
Dyc_al.hc1      GKFNMPPGVMEHFETAT--
Dyc_al.hc2      LKFGMPPGVMEHFETA---
Per_int.hx      FQFQVAPSALEHFETA---
Sip_tor.hx      FAFDTVPSVLEHSKPP---
Pac_ben.hx      FQYGIAPGVMEHFETAT--
Leu_fus.hx      FQYGVAP-CSGTLRNRH--
                  :   *      .     

Fig. 1.  Multiple sequence alignment (BLOSUM62) of hemocyanins conserved amino acid sequences (hc) and correspondent hexamerins sequences (hx). His (yellow) and Phe (green) residues involved in the oxygen-binding site are indicated. The residues involved in the trimer (blue) and dimer (red) contacts are also shown.

Fig. 2. ML analysis of the Plecoptera hemocyanin superfamily (HcSF): hemocyanins (hc) and hexamerins (hx). The numbers represent the bootstrap support. The bar equals 0.1 substitutions per site.

Fig. 3. Bayesian analysis of the Plecoptera hemocyanin superfamily (HcSF): hemocyanins (hc) and hexamerins (hx). The numbers represent the bootstrap support. The bar equals 0.1 substitutions per site.

Fig. 4. ML analysis of Arthropoda hemocyanin superfamily (HcSF): prophenoloxidases (PPO), hemocyanins (hc), hexamerins (hx) and cryptocyanins (CCI and PHc). The numbers represent the bootstrap support. The bar equals 0.1 substitutions per site.

Fig. 5. Bayesian analysis of the Arthropoda hemocyanin superfamily (HcSF): prophenoloxidases (PPO), hemocyanins (hc), hexamerins (hx) and cryptocyanins (CCI and PHc). The numbers represent the bootstrap support. The bar equals 0.1 substitutions per site.

Table 1. List of stoneflies species included in phylogenetic analysis, with acronyms and GenBank accession number (a.n.).

Superfamily	Family	Subfamily	Species	type	a.n.	acronym	
Perloidea	Perlodidae	Isoperlinae	Isoperla grammatica
	hc1	EU672885	Iso_gr.hc1	
				hc2	EU672886	Iso_gr.hc2	
			Isoperla acicularis acicularis	hc2	GU121398	Iso_ac.hc2	
		Perlodinae	Dyctiogenus alpinum 	hc1	GU121395	Dyc_al.hc1	
				hc2	GU121396	Dyc_al.hc2	
			Arcynopteryx compacta 	hc2	GU121393 	Arc_co.hc2	
			Guadalgenus franzi	hc1	FJ393060	Gua_fr.hc1	
			Besdolus ravizzarum 	hc2	GU121394 	Bes_ra.hc2	
			Perlodes intricatus 	hx	GU121397	Per_int.hx	
	Perlidae 	Perlinae
 	Perla marginata
	hc1	AJ555403	Per_ma.hc1	
				hc2	AJ555404	Per_ma.hc2	
				hx1	AJ690365	Per_ma.hx1	
				hx2	AJ690366	Per_ma.hx2	
				hx3	AJ690367	Per_ma.hx3	
				hx4	AJ690368	Per_ma.hx4	
			Perla grandis	hc1	DQ118369	Per_gr.hc1	
				hc2	DQ118370	Per_gr.hc2	
			Dinocras cephalotes
	hc1	FJ415315	Din_ce.hc1	
				hc2	EF218621	Din_ce.hc2	
			Caroperla sp.	hx1	GU121400 	Car_sp.hx	
			Tetropina sp.	hx	GU121388	Tetr_sp.hx	
			Togoperla sp.
	hx1	GU121389	Tog_sp.hx1	
				hx2	HM346532	Tog_sp.hx2	
			Neoperla sp. 	hx	GU121390	Neo_sp.hx	
			Etrocorema sp. 	hx	GU121391	Etr_sp.hx	
			Phanoperla sp.	hx	GU121392	Pha_sp.hx	
	Chloroperlidae	Chloroperlinae	Siphonoperla torrentium	hx	EU6772887	Sip_sp.hx	
Pterocarcyoidea	Peltoperlidae	 	Cryptoperla sp.	hx	GU121387 	Cry_sp.hx	
Nemouroidea	Taeniopterygidae	Taeniopteryginae	Taeniopteryx stanchovitchi	hx	EF218622	Tae_st.hx	
			Taeniopteryx stanchovitchi	hx	EF617598	Tae_bur.hx	
			Brachyptera risi	hx	EU6772888	Bra_ris.hx	
	Nemouridae
 	Amphinemurinae
 	Amphinemoura sulcicollis	hx	EU715327	Amp_su.hx	
			Protonemura ausonia	hx	EU6772890	Pro_aus.hx	
		Nemourinae
 	Nemoura hesperiae	hx	EU6772889	Nem_hes.hx	
			Nemoura sp.
	hx1	AM690369	Nem_sp.hx1	
				hx2	AM690370	Nem_sp.hx2	
				hx3	AM690371	Nem_sp.hx3	
	Capniidae
 	 
 	Capnia bifrons	hx	FJ384672	Cap_bif.hx	
			Allocapnia vivipara	hx	EF617597	All_vi.hx	
	Leuctridae
 	 Leuctrinae
 	Leuctra fusca	hx	EF218620	Leu_fus.hx	
			Pachyleuctra benllochi	hx	GU121399	Pac_ben.hx	
Eusthenioidea	Diamphipnoidae		Diamphipnopsis samali	hx	EF620538	Dia_sam.hx	

Table 2. List of arthropod species, other than Plecoptera, included in the Plecoptera and Arthropod HcSF multiple alignment. Protein type, systematic position (subphylum and species) and Genbank accession number are shown. PPO: prophenoloxidases; hc: hemocyanin; hx: hexamerin; CC: cryptocyanins or pseudo-hemocyanin.

tiype	Subphylum	Specie	Acronym	accession number	
PPO	Crustacea	Peneus monodon	Pen_mo.PPO	AF099741	
		Fenneropenaeus chinensis	Fen_ch.PPO	EU015060	
		Pacifastacus leniusculus	Pac_le.PPO	X83494	
	Hexapoda	Tenebrio molitor	Ten_mo.PPO	AB020738	
		Locusta migratoria
	Loc_m.PPO1	FJ771025	
			Loc_m.PPO2	FJ771024	
		Bombix mori
	Bom_m.PPO1	D49370	
			Bom_m.PPO2	D49371	
hc	Chelicerata	Eurypelma californicum
	Eur_ca.hcb	AJ290429	
			Eur_ca.hcc	AJ277489	
			Eur_ca.hcd	AJ290430	
			Eur_ca.hcf	AJ277491	
			Eur_ca.hcg	AJ277492	
		Limulus polyphemus
	Lim_po.hc2	AM260213	
			Lim_po.hc3	AM260214	
			Lim_po.hc4	AM260215	
			Lim_po.hc6	AM260216	
	Crustacea	Cancer magister	Can_ma.hc6	U48881	
		Peneaus vannamei	Pen_va.hc	X82502	
		Fenneropenaeus chinensis	Fen_ch.hc	FJ594414	
		Homarus americanus
	Hom_am.hcb	EF095142	
			Hom_am.hca	AJ272095	
		Pacifastacus leniusculus
	Pac_le.hc1	AF522504	
			Pac_le.hc2	AY193781	
	Myriapoda	Scutigera coleopatra 
 	Scu_co.hcA	AJ344359	
			Scu_co.hcD	AJ344360	
			Scu_co.hcB	AJ512793	
			Scu_co.hcC	AJ431379	
			Scu_co.hcX	AJ431378	
		Spirostreptus sp. 	Spi_sp.hc	AJ297738	
	Hexapoda	Folsomia candida	Fol_ca.hc1	FM242650	
		Sinella curviseta 	Sin_cu.hc1	FM242638	
		Lepisma saccharina
	Lep_sa.hc1	FM165291	
			Lep_sa.hc2	FM165292	
		Thermobia domestica
	The_do.hc1	FM165288	
			The_do.hc2	FM165289	
		Machilis germanica	Mac_ge.hc1	FM242639	
		Schistocerca americana 	Sch_am.EHP	AF038569	
		Locusta migratoria	Loc_mi.hc1	FM242651	
		Carausius morosus	Car_mo.hc1	FM242640	
		Chelidurella acanthopygia
	Che_ac.hc1	FM242641	
			Che_ac.hc2	FM242654	
		Hierodula membranacea
	Hie_me.hc2	FM242643	
			Hie_me.hc1	FM242642	
		Blaptica dubia
	Bla_du.hc1	FM242646	
			Bla_du.hc2	FM242647	
		Periplaneta americana
	Per_am.hc2	FM242649	
			Per_am.hc1	FM242648	
		Shelfordella lateralis
	She_la.hc2	FM242653	
			She_la.hc1	FM242652	
		Cryptotermes secundus	Cry_se.hc2	FM242645	
			Cry_se.hc1	FM242644	
CC	Crustacea	Homarus americanus
	Hom_a.Phc1	AJ132141	
			Hom_a.Phc2	AJ132142	
		Cancer magister 	Can_ma.CC1	AF091261	
hx	Hexapoda	Thermobia domestica	The_do.hx	FM165290	
		Locusta migratoria	Loc_m.JHBP	U74469	
		Periplaneta americana	Per_am.hx	L40818	
		Blaberus discoidalis	Bla_di.hx	U31328	
		Tenebrio molitor	Ten_mo.hx	AB021700	

Table 3. Nucleotidic (above) and amino acidic (below) identity. Species acronyms are as in the phylogenetic analysis. Seq.: sequences.

Seq	Iso_gr.hc1	Iso_gr.hc2	Iso_ac.hc2	Bes_ra.hc2	Arc_co.hc2	Dyc_al.hc1	Dyc_al.hc2	Per_int.hx	Sip_tor.hx	Pac_ben.hx	Leu_fus.hx	
Iso_gr.hc1	ID	0,62	0,60	0,60	0,60	0,85	0,59	0,55	0,54	0,57	0,57	
Iso_gr.hc2	0,54	ID	0,94	0,86	0,84	0,62	0,86	0,55	0,54	0,58	0,55	
Iso_ac.hc2	0,53	0,95	ID	0,84	0,82	0,60	0,83	0,55	0,53	0,57	0,54	
Bes_ra.hc2	0,53	0,89	0,87	ID	0,84	0,61	0,95	0,54	0,54	0,56	0,54	
Arc_co.hc2	0,54	0,86	0,85	0,86	ID	0,63	0,84	0,54	0,53	0,57	0,54	
Dyc_al.hc1	0,90	0,55	0,54	0,53	0,55	ID	0,61	0,54	0,53	0,57	0,56	
Dyc_al.hc2	0,54	0,88	0,87	0,96	0,85	0,54	ID	0,53	0,53	0,56	0,55	
Per_int.hx	0,38	0,39	0,39	0,39	0,38	0,39	0,38	ID	0,69	0,73	0,76	
Sip_tot.hx	0,35	0,39	0,37	0,38	0,38	0,34	0,37	0,59	ID	0,71	0,70	
Pac_ben.hx	0,45	0,43	0,42	0,42	0,42	0,46	0,43	0,66	0,59	ID	0,86	
Leu_fus.hx	0,40	0,36	0,35	0,36	0,35	0,40	0,36	0,66	0,58	0,83	ID	

Superfamily	Family	Species		Size (mm) animal	diet
	altitudal range (m)	habitat	Life cycle	ecological category	Stream type	Corology	hc	hx	
Pteronarcyoidea	Peltoperlidae	Cryptoperla sp.	nymph		detritivourus	?	?	semivoltine	?	permanent	OR	-	yes	
Systellognatha	Perlidae	Perla marginata 	1st, 2nd, 3rd	16 -33	detritivourus and predator	160-2800	rhithron,	semivoltine	rheophilous, stenotherm	permanent	M-S-EU; MAG	hc1; hc2	yes	
			adult		no feeding				-			hc1; hc2	-	
		Perla grandis 	1st, 2nd, 3rd	23 -31	predator	465-2500	hyporhithron	semivoltine	rheophilous, stenotherm	permanent	M-S-EU	hc1; hc2	-	
			adult		no feeding				-			hc1; hc2		
		Dinocras cephalotes	nymph	14 - 31	predator	40-2800	hyporhithron	semivoltine	rheophilous, stenotherm	permanent	EU	hc1; hc2	yes	
			adult		no feeding				-			no	-	
		Neoperla sp.	nymph		?	?	?	semivoltine	?	permanent	OR	-	yes	
		Togoperla sp.	nymph		?	?	?	semivoltine	?	permanent		-	yes	
		Etrocorema sp.	nymph		?	?	?	semivoltine	?	permanent		-	yes	
		Phanoperla sp.	nymph		?	?	?	semivoltine	?	permanent		-	yes	
		Caroperla sp.	nymph		?	?	?	semivoltine	?	permanent		-	yes	
	Perlodidae	Isoperla grammatica	nymph	11 – 16	predator	10-2000	rhithron	univoltine	rheophilous. stenotherm	permanent	EU	hc1; hc2	-	
		Isoperla rivulorum 	adult	10 - 15	predator		rhithron	univoltine	orophilous	permanent	M-S-EU	no		
		Isoperla viridinervis	adult male	10 – 12	predator	1000-2400	rhithron	semivoltine	rheophilus	permanent-	PYR	no		
		Isoperla acicularis acicularis	nymph	14 - 16	phytophagous and detritivourus	1620-2152	rithron	univoltine	rheophilous. stenotherm	permanent		hc2		
			adult						-			no		
		Guadalgenus franzi	nymph	11 – 18	predator	100-1660	rhithron and mountain lakes	semivoltine	thermophilous	temporal	IB	hc1	-	
		Perlodes intricatus	nymph	15 – 25	predator	800-2700	rhithron	semivoltine	orophilous	permanent	EU	-	yes	
		Dyctiogenus alpinum	nymph	16 - 24	detritivourus and predator	570-2700	hyporhithron	semivoltine	rheophilous - orophilous	permanent	EU	hc1; hc2		
		Besdolus ravizzarum	nymph	15 - 19	phytophagous	220-520	potamon	univoltine		temporal	M-S-EU	hc2		
		Arcynopteryx compacta	nymph	15 – 22	predator	1950-2400	rhithron and mountain lakes	semivoltine	orophilous - stenotherm	permanent	OL	hc2		
	Clhoroperlidae	Siphonoperla torrentium
	nymph	7 – 9	predator and phytophagous	30-2000	rhithron	univoltine	rheophilous -orophilous	permanent	M-EU	-	yes	
			adult		predator and phytophagous				-			-	-	
Eulognatha	Taeniopterygidae	Taeniopteryx stanckovitchi	nymph	8 – 12,5	phytophagous	250-1800	rhithron	univoltine	rheophilous.	permanent	S-EU	-	yes	
		Brachyptera risi	nymph	8 – 12	phytophagous	100-1100	rhithron	univoltine	rheophilous - orophilous	permanent		-	yes	
		Brachyptera vera	nymph	8.5 – 10.5	phytophagous	640-1000	rhithron	univoltine	thermophilous	temporal	IB	-	-	
	Leuctridae	Leuctra fusca	nymph	6 – 8	phytophagous	400-1800	ubiquitous	univoltine	rheophilous, mesotherm	permanent	EU-AS	-	yes	
		Leuctra aloisi	adult	5 – 7	phytophagous	1400-2245	rhithron	univoltine	rheophilous,	permanent	PYR	-	-	
		Pachyleuctra benllochi	nymph	11 – 12	phytophagous and detritivourus	1000-2500	rhithron	semivoltine	stenotherm	permanent	PYR	-	yes	
			adult						-			-	-	
	Nemouridae	Nemoura hesperiae	nymph	6 - 9	phytophagous		rhithron	univoltine	rheophilous	permanent	IT	-	yes	
		Nemoura cinerea	adult	6 – 10	detritivourus	85-2410	ubiquitous	univoltine	reophilous, eurytherm	-	PAL	-	-	
		Protonemura ausonia	nymph	7 -11	phytophagous	500-2000	crenon	univoltine	stenotherm	permanent	IT	-	yes	
		Protonemura tuberculata	adult	7.5 – 10,5	phytophagous and detritivourus	1000-2350	Crenon	univoltine	reophilous, stenotherm	-	PYR	-	-	
		Amphinemura sulcicollis	nymph	4 – 8	phytophagous	240-2100	rhitron	univoltine	reophilous, eurytherm	permanent	EU	-	yes	
			adult						-			-	-	
	Capniidae	Capnia bifrons	nymph	6 - 9	phytophagous and detritivourus			univoltine	reophilous, stenotherm	permanent	OL	-	yes	
			adult		phytophagous				-			-	yes	
	Notonemuridae	Afronemura anhatolae
	nymph			-	-	univoltine		-	S-AF	no	-	
			adult									no	-	
		Aphanicella bullata	adult					univoltine		-	S-AF	no	-	
Tab. 4 : Systematic position, autoecology (size, trophic role, altitudinal range, habitat type and stream type, ecological category, corology), hemocyanin (hc) and hexamerins presence in nymph and adult stage of studied species. AF: Africa; PAL: Palaeartic ; PYR: Pyrenees ; EU: Europe; EU-AS: euroasiatica; OL: Oloartic; OR: Oriental; MAG: Maghreb; M: medium; S: South; N:North.