Gene 540 (2014) 7177

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Gene

j ourna l homepage: www.e lsev ie r .com/ locate /geneMolecular evolution of the androgenic hormone in terrestrial isopodsNicolas Cerveau a,1, Didier Bouchon a, Thierry Bergs b, Pierre Grve a,a UMR CNRS 7267, cologie et Biologie des Interaction (EBI), quipe cologie, volution, Symbiose (EES), Universit de Poitiers, 40 avenue du Recteur Pineau, F-86022 Poitiers cedex, Franceb Laboratoire Signalisation et Transports Ioniques Membranaires (STIM)-ERL 7368, Universit de Poitiers, Ple Biologie Sant, 1 rue Georges Bonnet, F-86022 Poitiers cedex, FranceAbbreviations: AG, androgenic gland; AGH, androgenverse transcriptase polymerase chain reaction; M-MLV, MNTP, deoxy-ribonucleoside triphosphate; CBS, Center foREL, random effect likelihood; IAG, insulin-like anInternational Union of Biochemistry. Corresponding author.

E-mail addresses: nicolas.cerveau@geo.uni-goettingendidier.bouchon@univ-poitiers.fr (D. Bouchon), thierry.berpierre.greve@univ-poitiers.fr (P. Grve).

1 Present address: Courant Research Center GeobSymbiosis Group, University of Gttingen, GoldschmiGermany.

http://dx.doi.org/10.1016/j.gene.2014.02.0240378-1119/ 2014 Elsevier B.V. All rights reserved.a b s t r a c ta r t i c l e i n f oArticle history:Accepted 17 February 2014Available online 20 February 2014

Keywords:CrustaceanIsopodAndrogenic glandAndrogenic hormoneMale differentiationIn crustaceans, the androgenic gland (AG), thanks to the synthesis of the androgenic gland hormone (AGH), con-trols the differentiation of the primary and secondary male sexual characters. In this study, we amplified 12 newAGH cDNAs in species belonging tofivedifferent families of the infra-order Ligiamorpha of terrestrial isopods. Pu-tative essential amino acids for the production of a functional AGHprotein exhibit signatures of negative selectionand are strictly conserved including typical proteolytic cleavage motifs, a putative N-linked glycosylation motifon the A chains and the eight Cys positions. An insulin-like growth factor motif was also identified inArmadillidium AGH sequences. The phylogenetic relationships of AGH sequences allowed one to distinguishtwomain clades, corresponding tomembers of the Armadillidiidae and the Porcellionidae families which are con-gruent with the narrow specificity of AG heterospecific grafting. An in-depth understanding of the regulation ofAGH expressionwould help deciphering the interaction betweenWolbachia, widespread feminizing endosymbi-otic bacteria in isopods, and the sex differentiation of their hosts.

2014 Elsevier B.V. All rights reserved.1. Introduction

Crustacean sexual differentiation was largely studied in the class ofMalacostraca. The role of the androgenic gland (AG) responsible forthe development of male sexual characters was first established in theamphipod Orchestia gammarellus by Charniaux-Cotton (1954). Sincethis discovery, the AG was described in different malacostracan speciesincluding amphipods (Charniaux-cotton, 1992; Hasegawa et al., 1993),isopods (Juchault, 1966; Juchault and Legrand, 1964; Katakura, 1961,1989; Suzuki, 2000) and decapods (Hoffman, 1969; Sagi et al., 1997;Ventura et al., 2011a). This gland, which synthesizes the androgenicgland hormone (AGH), controls the differentiation of the primary andsecondary male sexual characters (Charniaux-cotton, 1992; Khalailaet al., 2001). In females, there is no AG and embryonic gonads developinto ovaries (Katakura, 1989).

More than thirty years after the discovery of the AG, the proteina-ceous nature of the sexual hormone has been elucidated in the isopodArmadillidium vulgare (Hasegawa et al., 1987; Martin et al., 1990).ic gland hormone; RT-PCR, re-oloney murine leukemia virus;r Biological sequence analysis;drogenic gland factor; IUB,

.de (N. Cerveau),ges@univ-poitiers.fr (T. Bergs),

iology, Geomicrobiology anddtstrae 3, 37077 Gttingen,Around ten years later, the sequences of the prohormone (Martinet al., 1999) and of the cDNA (Okuno et al., 1999) of A. vulgare AGHwere determined. The AGH precursor consists of a peptide of 123amino acids, composed of a signal peptide, a B chain, a C peptide andan A chain. The maturation of the hormone implies post-translationalmodifications, including the glycosylation of the A chain and the exci-sion of the C peptide, leading to a heterodimer with the B and A chainslinked by disulfide bridges (Martin et al., 1999). The masculinizing roleof the AGH has been determined by its ability to fully reverse the sex ofA. vulgare young females when AGs are grafted before sexual differenti-ation (Suzuki and Yamasaki, 1997). In adult females, AG grafting still in-duces the development ofmale secondary characters such as copulatoryorgans (Suzuki and Yamasaki, 1997). Martin et al. (1999) confirmed thatthis effect could be attributed to theAGHas repeated injections of purifiedhormone also induced female sex reversal. In parallel, heterospecificgrafting of AGs showed that the AGH of one species displays a high spec-ificity, complete efficiency being retained only between species of thesame genus (Armadillidium, Porcellio or Oniscus) (Martin and Juchault,1999). For example, heterospecific implantations of AGs within speciesof the genus Porcellio (P. laevis, P. scaber and P. dispar) or the genusArmadillidium (A. vulgare, A. depressum A. nasatum and A. maculatum) in-duce the masculinization of genetic females in all cases (Martin andJuchault, 1999). Conversely, when AG graftings are realized between spe-cies from two different families, masculinization is most of the time par-tial or absent. This is the case when crossed graftings of AGs are realizedbetween A. vulgare and Porcellio gallicuswhich induce a masculinizationof A. vulgare but not of P. gallicus young females.

On the other hand, an antibody directed against a recombinant AGHof A. vulgare was used to show that AGH from species of different

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72 N. Cerveau et al. / Gene 540 (2014) 7177families shares similar epitopes (Hasegawa et al., 2002). Indeed, immu-nostaining of AG from species of Armadillidiidae, Scyphacidae andPorcellionidae families has been obtained using different dilutionsof the antibody. In particular in the Porcellionidae family, the AGsof three different species including P. scaber showed positiveimmunoreactions even if the signal was weaker than in the A. vulgareAG. It suggested that the AGH of species from Armadillidiidae andPorcellionidae families was not only close enough to be recognized bythe antibody but also divergent enough since A. vulgare AG implanta-tions in P. scaber or P. gallicus do not inducemasculinization of young fe-males (Hasegawa et al., 2002;Martin and Juchault, 1999). This has beenconfirmed by the cloning of AGH cDNA from P. scaber and Porcelliodilatatus which revealed an amino acid identity of 88% between thetwo hormone sequences and of ~82% between these sequences andthe one of A. vulgare (Greve et al., 2004; Ohira et al., 2003). The specific-ity of the AGH may be in accordance with the conservation of the pep-tide sequence of the hormone resulting in a specificity of themasculinization effect at the family level. It is noteworthy that AGs arethe only tissue that synthesized the AGH, and no signal in other tissueshas been reported using either RT-PCR, Northern blot, in situ hybridiza-tion or immunohistochemical analysis (Greve et al., 2004; Hasegawaet al., 2002; Ohira et al., 2003; Okuno et al., 1999).

Hence, the aim of this study was to clarify the molecular evolution-ary patterns of isopod AGH at a larger scale. We developed a PCR strat-egy to amplify and sequence cDNAs encoding AGH of various speciesbelonging to different families of terrestrial isopods. The resulting se-quences were compared and their phylogenetic relationships as wellas their patterns of molecular evolution were analyzed.

2. Materials and methods

2.1. Animals

All animals used in this study (Table 1)were reared in the laboratoryat 20 C under natural photoperiod on moistened soil with dead leavesand carrots as food.

2.2. RNA extraction and RT-PCR amplification of AGH mRNA

Total RNAwas isolated fromAGs of 15males (six glands per individ-ual) using the RNeasy Mini kit according to the manufacturer's instruc-tions (Qiagen). Single-stranded cDNA was synthesized by annealingrandomhexanucleotide primers (80 ng) to 0.51 g of total RNA (previ-ously heated for 5 min at 65 C and chilled on ice) and carrying out aTable 1List of woodlice species samples used in this study and accession numbers of AGH sequences.(not amplified) means that the sequences could not be obtained for these species.

Sub-order Infra-order Family Species

Oniscidea Ligiamorpha Armadillidae Armadillo officinalisArmadillidiidae Armadillidium vulgar

Armadillidium assimArmadillidium depreArmadillidium granuArmadillidium macuArmadillidium nasat

Balloniscidae Balloniscus sellowiCylisticidae Cylisticus convexusOniscidae Oniscus asellusPhylosciidae Chaetophilosia elongPorcellionidae Porcellio dilatatus dil

Porcellio dilatatus pePorcellio disparPorcellio gallicusPorcellio laevisPorcellio scaberPorcellionides pruino

Tylomorpha Tylidae Helleria brevicornisreverse transcription reaction in 20 L containing 5 L of 5 M-MLVbuffer, 50 pmol of dNTP, and 200 U of Moloney murine leukemia virusreverse transcriptase (M-MLV-RT, Promega), for 1 h at 42 C. PCR am-plification was performed using several degenerated primer pairs de-signed based on the consensus sequences of AGH cDNAs of A. vulgare,P. scaber and P. dilatatus (Supp. data 1) (Greve et al., 2004; Ohira et al.,2003; Okuno et al., 1999). PCR was carried out in 25 L containing0.5 L of the RT reaction, 12.5 pmol of each primer, 5 L of 5 GoTaqbuffer, 10 mM of dNTP and 1 U of Taq DNA polymerase (Promega).The cyclic parameters were 94 C for 1 min, 5060 C for 1 min and72 C for 1 min. PCR products were separated on a 1.5% agarose geland visualized after ethidium bromide staining.

2.3. Sequencing of AGH cDNA

PCR fragments were purified with the JetPure PCR purification kit(Genomed) and directly sequenced on both strands using the BigDyev3.1 terminator cycle sequencing reaction kit on an ABI 3130 GeneticAnalyzer (Perkin Elmer). New AGH sequences generated in this studywere deposited in GenBank/EMBL databases (Table 1). All sequenceshave been checked using independent samples and several RT-PCRand PCR reactions. The AGH sequences of A. vulgare, P. scaber andPorcellio dilatatus dilatatus obtained in this study are 100% identical tothe ones previously deposited in GenBank database.

2.4. Amino acid sequence analyses

Amino acid sequence analyses were performed using bioinformatictools available on the CBS prediction servers (http://www.cbs.dtu.dk/services/): SignalP predicts the presence and the localization of signalpeptide cleavage site on peptide sequence of eukaryotes (Petersen et al.,2011), NetNGlyc detects putative glycosylation sites (Asn-Xaa-Ser/Thr)(Blom et al., 2004) and ProP predicts the presence of peptide cleavagesites (Arg-X-X-Arg) on sequences of eukaryote prohormones (Duckertet al., 2004). A pictogram of the two proteolytic cleavage sites and the pu-tative N-linked glycosylation site was drawn using the Logo website(http://weblogo.berkeley.edu/logo.cgi) (Crooks et al., 2004) (Supp. data2). Finally, putative protein domainswere identified using the Smart soft-ware (http://smart.embl-heidelberg.de) (Letunic et al., 2011).

AGH protein sequences including the 11 decapod insulin-like andro-genic gland hormone (IAG) sequences available in GenBank (Venturaet al., 2011a) were manually aligned using the software BioEdit v7.0.9(Hall, 1999). MEGA software v5.0 (Tamura et al., 2007) was used to cal-culate protein divergence between sequences of isopods and decapodsUnderlined accession numbers correspond to sequences already available in GenBank. NA

AH sequences accession # Sampling location

JQ304808 Montpellier, Francee AB029615 Nice, Franceile NA Cort, Francessum JQ304800 Sainte-Marie de R, Francelatum JQ304798 Sousse, Tunisialatum JQ304799 Ez, Franceum JQ304801 Mignaloux-Beauvoir, France

NA Caxias do Sul, BrasilJQ304809 Villedaigne, FranceJQ304810 Celles sur Belle, France

ata NA Thur, Franceatatus AB089811 Rom, Francetiti JQ304802 St Honorat island, France

JQ304803 Santa Maria del Sol, SpainJQ304804; JQ304805 Chiz, FranceJQ304806 Hammamet, TunisiaAB089810; AY169973 Sassis, France

sus JQ304807 Nevers, FranceNA Sainte-Marguerite island, France

http://www.cbs.dtu.dk/services/)http://www.cbs.dtu.dk/services/)http://weblogo.berkeley.edu/logo.cgi)http://smart.embl-heidelberg.de)ncbi-n:JQ304808ncbi-n:AB029615ncbi-n:JQ304800ncbi-n:JQ304798ncbi-n:JQ304799ncbi-n:JQ304801ncbi-n:JQ304809ncbi-n:JQ304810ncbi-n:AB089811ncbi-n:JQ304802ncbi-n:JQ304803ncbi-n:JQ304805ncbi-n:JQ304805ncbi-n:JQ304806ncbi-n:AY169973ncbi-n:AY169973ncbi-n:JQ304807

73N. Cerveau et al. / Gene 540 (2014) 7177and to construct the resulting phylogenetic tree. The maximum likeli-hood tree was created using the JonesTaylorThornton substitutionmodel with uniform rate among all the alignment sites. The tree wasgenerated with Nearest Neighbor Interchange heuristic method. Ro-bustness of the tree was assessed with 1000 bootstrap replicates.

To detect signatures of positive and negative selection, analyses wereperformed with the web-server of the HyPhy package (http://www.datamonkey.org) (Pond and Frost, 2005). Pairwise estimates of the num-ber of non-synonymous (dN) and synonymous (dS) substitutions per sitewere calculated using maximum likelihood (Goldman and Yang, 1994).The random effect likelihood (REL) analysis (Kosakovsky Pond andFrost, 2005) was used to identify amino acid sites with signature of selec-tion based on a Bayes factor of 95. Four different analysesweremade. Thefirst analysis was done with 16 sequences including the three availableAGH sequences ranging from positions 44 to 139 (Fig. 1). We then indi-vidually considered sequences of the B chain, the C peptide and the Achain of the seven complete amino acid sequences among the data set.3. Results

In this study, among the 16 tested species we amplified the cDNA of13 new AGH sequences from 12 species belonging to five different fam-ilies of the infra-order Ligiamorpha using several degenerated primerpairs (Table 1; Supp. data 1). We obtained nine complete and four par-tial AGH coding sequences from four species of the Armadillidium genus,five species of the Porcellio genus and one species of the Armadillo,Cylisticus, Oniscus and Porcellionides genus respectively (Fig. 1). In eachcase except for P. gallicus, only one sequence was obtained suggesting1 10 20 30

90 100 110

Fig. 1. Alignment of AGH amino acid sequences. Orange frames highlight the eight conserved CyN-linked glycosylation site. The different AGH chains and the positions of the main primers usthat there is only one gene encoding the AGH in these species. InP. gallicus, the AGH mRNA seems to be transcribed from two differentgenes since the Long (P. gallicus_L) and the Short (P. gallicus_S) iden-tified sequences differ in size and in sequence (Fig. 1). The presence ofthe two copies was confirmed using specific P. gallicus AGH primers(AGH-Pg_S and L in Supp. data 1).

The protein sequence alignment (Fig. 1) of 16 sequences includingthe 13 sequences obtained in this study and the three available AGH se-quences showed that the organization observed in the A. vulgare AGHsequence is preserved among all species. It consists of a signal peptide,a B chain, a C peptide and an A chain. Prediction algorithms defined asignal peptide excised after the 21st amino acid (Table 2). For each se-quence, a typical proteolytic cleavage motif which is composed of twoArg separated by two variable amino acids (Arg-X-X-Arg; Duckertet al., 2004) was found at two positions between the B chain and the Cpeptide, and between the C peptide and the A chain. The motif of thefirst predicted cleavage site (positions 65 to 70; Fig. 1) is conserved, ex-cept in the AGH sequence of Porcellionides pruinosuswhere methioninewas identified at the first position of the cleavage site (Met-Glu-Arg-Arg). Nevertheless, Prop algorithm still predicts a cleavage at this mod-ified site (Table 2). At the secondputative cleavage site (positions 124 to127; Fig. 1), the predictedmotif is conserved in all sequences. As alreadyidentified in A. vulgare, P. scaber and P. dilatatus AGH sequences (Greveet al., 2004; Ohira et al., 2003; Okuno et al., 1999), anN-linked glycosyl-ation motif (Asn147-X-Thr/Ser) was predicted in the A chain of the AGHsequences in all species (Marshall, 1972) (Fig. 1; Table 2; Supp. data 2c).An Asn is always present at position I whereas there are only three pos-sibilities at position II: an Arg in nine sequences out of 14 (64.29%), a Lysin four sequences out of 14 (28.57%) and a Ser in one sequence (7.14%).40 50 60 70 80

120 130 140 150

s residues, blue frames the two proteolytic cleavage sites and the green frame the putativeed in this study are indicated under the sequences.

http://www.datamonkey.org)http://www.datamonkey.org)

Table2

Bioinformaticsan

alysisof

thepe

ptideAGHsequ

ences.Th

eSign

alPalgo

rithm

allowson

eto

pred

ictthe

excision

ofthesign

alpe

ptide.Itiden

tifies

theam

inoacid

position

afterwhich

thecleava

geispu

tative

lyrealized

.The

NetNGlycalgo

rithm

allows

oneto

pred

ictw

here

N-linke

dglycosylationmotifs

arelocaliz

ed,inc

luding

theAsn

position

which

ispo

tentially

glycosylated

.The

proteo

lyticcleava

gesite

position

swerepred

ictedwithProp

algo

rithm,inc

luding

thetw

oam

inoacid

position

swhich

arepu

tative

lyremov

edaftertheex

cision

.The

sethreeprog

ramsareho

sted

bytheCB

Sserver

(http://www.cbs.dtu.dk/services/).P

ositions

referto

theam

inoacid

alignm

entnu

mbe

ring

(Fig.1

).Sm

artsoftware(h

ttp://sm

art.e

mbl-heide

lberg.de

/)allowson

eto

pred

ictthepresen

ceof

proteindo

mains

.

A.officina

lisA.

vulgare

A.de

pressum

A.g

ranu

latum

A.maculatum

A.na

satum

C.conv

exus

O.a

sellu

sP.

dilatatusdilatatus

P.dilatatuspe

titi

P.dispar

P.ga

llicus_L

P.ga

llicus_S

P.laevis

P.scab

erP.

pruino

sus

Sign

alP

nd21

2121

2121

n.d

2121

2121

21/

2121

21Sm

art

ndIlG

FIlG

FIlG

FIlG

FIlG

Fnd

ndnd

ndnd

ndnd

ndnd

ndGlycosylation

146

146

146

146

146

146

146

/14

614

614

614

614

614

614

6/

ProP

6970

6970

6970

6970

6970

6970

6970

6970

6970

6970

6970

6970

6970

6970

6970

125

126

125

126

125

126

125

126

125

126

125

126

125

126

125

126

125

126

125

126

125

126

125

126

125

126

125

126

125

126

125

126

74 N. Cerveau et al. / Gene 540 (2014) 7177At position III, the Ser residue was never found (Fig. 1; Supp. data 2c).Finally, eight Cys residues were located at identical positions in all thesix complete AGH sequences compared with those of the A. vulgare se-quence, all of them being involved in disulfide bridges in the maturehormone of this species (Martin et al., 1999).

Signatures of purifying selectionwere identified on theArg of the twotypical proteolytic cleavage motifs (positions 65 & 70, 123 & 126; Fig. 1),on the Asn of the putative N-linked glycosylation site (position 146) andon five of the eight Cys (positions 34, 43, 45, 60 and 154). Three otheramino acids of the C peptide also exhibit signatures of negative selection:His in position 93, Pro in position 94 and Leu in position 97 (Fig. 1). In ad-dition, two long stretches, one in the A chain between amino acids 30and 53 and the second one in the B chain between amino acids 119and 146, are strictly conserved among the Armadillidium genus.

Apart from these conserved patterns, there are a lot of differences be-tween the sequences. An insulin-like growth factor motif was identifiedwith Smart in theArmadillidium sp. AGH sequences but not in the ones ofother species (Table 2). There are also many differences in the size ofboth A andB chains and of the C peptide sequenceswhich correspond in-sertions or deletions (Fig. 1). Indeed, one ormore additional amino acidsare observed in the A chain of Armadillo officinalis and Porcellio sp. AGHsand in the B chain of Oniscus asellus and P. pruinosus AGHs or in the Cpeptide of Armadillidium granulatum, Cylisticus convexus and O. asellusAGHs. Size variations are also due to deletions like in the C peptide ofP. scaber, P. gallicus_S/L and A. officinalis AGHs (Fig. 1).

Despite these variations, the amino acid identity percentage betweensequences, calculated with patristic distance considering pairwise dele-tion in the full length alignment, ranges from 40.5% (A. officinalis/P. pruinosus) to 97.9% (P. dilatatus dilatatus and Porcellio dilatatus petiti)with amean at 68.1% (Supp. data 3). The highest values are observed be-tween sequences within the Armadillidium genus (mean at 93.3%, Supp.data 3). These relationships are illustrated by thephylogenetic tree basedon full length alignment (Fig. 2). Indeed, all theArmadillidium genus AGHsequences form a monophyletic group in contrast with the Porcelliogenus AGH sequences which are more distantly related (Fig. 2). Thetree obtainedwith the largest commonAGH sequences in all isopod spe-cies (positions 44 to 139; Fig. 1) presents the same topology (data notshown). AGH sequences of isopods and IAG sequences of decapodsbranch in two distinct clusters. This pattern is in accordance with thelow sequence similarity observed between these two groups.4. Discussion

To gain insight into the specificity of the AGHs, we assessed the di-versity and evolution of the hormone sequences in 16 terrestrial isopodspecies belonging to five different families. We succeeded in amplifyingAGH cDNA of species mostly belonging to the Armadillidium andPorcellio genera. This result was expected since degenerated primershave been designed according to A. vulgare, P. scaber and P. dilatatusAGH available sequences. However, PCR failed when using AGHcDNAs of Balloniscus sellowi, Chaetophilosia elongata and Helleriabrevicornis, three species that are phylogenetically distant of theArmadillidium and Porcellio genera. This may indicate a more variableprimer site sequence than expected. The organization of the resulting16 AGH sequences is highly conserved and consists of a signal peptide,a B chain, a C peptide normally excised during the post-transcriptionalprocesses, and an A chain as already established for the AGH ofA. vulgare, P. scaber and P. dilatatus precursors (Greve et al., 2004;Martin et al., 1999; Ohira et al., 2003; Okuno et al., 1999). For theAGHs of Armadillidium species, a motif search revealed the presence ofan insulin-like growth factor domainwhich is consistentwith the struc-tural similarity of the protein and the insulin superfamily peptides andwith identical maturation processes of the two proteins (Steiner et al.,1985). However, both hormones are clearly different in terms ofamino acid sequence and peptide chain length.

http://smart.embl-heidelberg.de/http://smart.embl-heidelberg.de/

Porcellio dilatatus dilatatus

Porcellio dilatatus petiti

Porcellio laevis

Porcellio scaber

Porcellio gallicus L

Porcellio gallicus S

Porcellio dispar

Porcellionides pruinosus

Armadillidium maculatum

Armadillidium depressum

Armadillidium nasatum

Armadillidium vulgare

Armadillidium granulatum

Oniscus asellus

Cylisticus convexus

Armadillo officinalis

Palaemon pacificus

Palaemon paucidens

Macrobrachium lar

Macrobrachium rosenbergii

Penaeus monodon

Litopenaeus vannamei

Marsupenaeus japonicus

Callinectes sapidus

Cherax destructor

Cherax quadricarinatus

Portunus pelagicus98

87

62

60

8973

56

32

99

79

77

25

29

9269

86

9348

43

20

16

17

33

0.2

Fig. 2.Maximum likelihood phylogenetic tree of protein AGH sequences based on full length alignment. IAG sequences from decapods were obtained from the GenBank database.

75N. Cerveau et al. / Gene 540 (2014) 7177Putative essential sites for the production of a functional AGH pro-tein are strictly conserved and carry signatures of negative selection.The eight Cys residues of which five are under negative selection are lo-calized at the same positions as in the A. vulgare AGH sequence (Martinet al., 1999). In this species, it was previously reported that four Cys res-idues of each chain form two intrachain (one more than in insulin) andtwo interchain disulfide bridges (Martin et al., 1999). This result sug-gests that these Cys residues are also involved in disulfide bridges inthe mature hormone of AGH of all isopod species. Furthermore, correctdisulfide bond arrangements are required for the activity of theA. vulgare AGH (Katayama et al., 2010). Indeed, a semisynthetic AGHfolded into non-native formwithwrong disulfide linkages does not dis-play any biological activity (Katayama et al., 2010). It has also beendemonstrated that A. vulgare recombinant AGH composed of the Bchain, the C peptide and the A chain does not show any AGH activity(Okuno et al., 2002). AGH activity was recovered after lysyl endopepti-dase digestion leading to a heterodimeric peptide linked by disulfidebridges and that lacks most of the C peptide (Okuno et al., 2002). In ad-dition, both Arg residues of the two typical proteolytic cleavage motifshave also signatures of purifying selection. Excision of the C peptideseems also to be essential for insulin since the activity of proinsulin islower than insulin by about two orders of magnitude (Gross et al.,1989). Finally, the glycosylation site of the A chain inwhich the Asn res-idue carries signature of negative selection, is another important featureof the AGH which also differs from insulin (Greve et al., 2004; Martinet al., 1999). This glycosylation seems to be obligatory for the activityof the AGH since it was shown that the injection of a recombinant AGHlacking the glycan moiety does not induce masculinization of A. vulgarefemales (Okuno et al., 2002). The sizes of the A and B chains are con-served compared with the one of the C peptide where there are a lot ofsize variations, particularly in the A. officinalis AGH sequence. A similarstructural divergence has been also observed in the insulin peptide su-perfamily, while the amino acid sequences of A and B chains were con-versely highly conserved among various species (Steiner et al., 1985).

More recently, an insulin-like factor (Cq-IAG) has been identified inthe AG of the red-claw crayfish Cherax quadricarinatus (Manor et al.,2007). The gene encoding this peptide is exclusively expressed in theAG and Cq-IAG is thought to promote male growth that leads to sexualdimorphism. The sequence similarity of Cq-IAGwith the isopod AGH se-quences is very low, although six of the eight Cys residues of Cq-IAG areconserved. Two putativeN-linked glycosylationmotifs both in theA andB chainswere also identified in Cq-IAG, whereas only onewas identifiedin the A chain in isopod AGHs. This result marked the beginning of ele-gant studies realized in decapods including the identification of 11 otherinsulin-like hormones and the utilization of RNAi which allowed one toshow that IAG regulates male sex differentiation (Rosen et al., 2010;Ventura et al., 2009, 2011b).

The reconstructed phylogeny showed that the AGH and IAG se-quences of both isopods and decapods group in two distinct clusters.This result was expected considering that the closest decapod IAG

76 N. Cerveau et al. / Gene 540 (2014) 7177sequence shares 32.3% identity at the most with isopod AGH sequences(Fig. 2). Within the isopod AGH sequence group, two main clusters arealso observed, thefirst one including thefiveAGHsequences from speciesof the Armadillidiidae family and the second one the eight AGH sequencesfrom species of the Porcellionidae family. The three remaining sequencesbelong to species of three other families. This pattern is congruent withprevious terrestrial isopod phylogenetic analyses (Michel-Salzat andBouchon, 2000; Schmalfuss, 2003). The amino acid identity obtainedwithin the AGH sequences of species from the Porcellio genus is onaverage 81.4% and 71.48% between species of Porcellio and Porcellionidesgenera. The genetic distance between the AGH sequences of these twogroups is also illustrated during heterospecific graftings (Martin et al.,1999). Indeed, whereas grafting of P. dispar, P. scaber and P. laevis AG inP. pruinosus young females induces a male phenotype, the opposite isnot true. Within the Porcellio genus, AG grafting is efficient in both di-rections, as well as within the Armadillidium genus (Martin et al.,1999). This result is consistent with the phylogenetic relationships be-tween the AGH sequences and seems to confirm that the Porcellionidaefamily which is one of the largest oniscidean families is more diversethan the Armadillidiidae family. Nevertheless, some amino acid frag-ments are still conserved in the AGH sequences of the different speciesas shown by immunohistochemistry experiments (Hasegawa et al.,2002) that may explain heterospecific effects of the AG graftings(Martin et al., 1999).

In isopods, the AGHmay also be the target of the intracellular bacte-riaWolbachia (Bouchon et al., 2008; Negri et al., 2010). These verticallytransmitted alpha-Proteobacteria infect many arthropod species andare considered as reproductive parasites because of their capacity tomanipulate host reproduction that increases the fitness of infectedfemales, thereby enhancingWolbachia transmission in host populations(Werren et al., 2008). In A. vulgare and several other isopod species,Wolbachia induce the feminization of genetic males (Bouchon et al.,2008; Cordaux et al., 2011). In infected genetic males, the developmentof the AG is not observed and individuals developed as functional fe-males. Wolbachia transinfections between different isopod speciesshowed that the resulting phenotype dependsmore on the host phylog-eny than on the bacterial phylogeny (Bouchon et al., 1998, 2008; Rigaudet al., 1997). For example, feminizingWolbachia of A. vulgare induces thefeminization A. nasatummales whereas it is not efficient when injectedin P. pruinosusmales (Bouchon et al., 1998; Juchault et al., 1974; Rigaudand Juchault, 1995; Rigaud et al., 2001). These results are in accordancewith AG grafting experiments which showed that A. vulgare AG graftinginducesmasculinization ofA. nasatum young females but not the ones ofP. pruinosus (Martin et al., 1999).

Therefore, it seems that the AGH receptor and the hormone co-evolved likely at the same time (Negri et al., 2010). An in-depthunderstanding of the regulation of AGH expression would help indeciphering the interaction between Wolbachia and A. vulgare sexdifferentiation.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.gene.2014.02.024.Conflict of interest

There is no conflict of interest.Acknowledgments

We are grateful to C. Debenest, C. Delaunay and M. Raimond fortechnical assistance. We thank Joanne Bertaux for comments on anearlier version of the manuscript and for improving the English. Thisresearchwas funded by the CentreNational de la Recherche Scientifique(CNRS) and the French Ministre de l'Education Nationale, del'Enseignement Suprieur et de la Recherche.References

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Molecular evolution of the androgenic hormone in terrestrial isopods1. Introduction2. Materials and methods2.1. Animals2.2. RNA extraction and RT-PCR amplification of AGH mRNA2.3. Sequencing of AGH cDNA2.4. Amino acid sequence analyses

3. Results4. DiscussionConflict of interestAcknowledgmentsReferences