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Gene 540 (2014) 7177

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Gene
journal homepage: www.elsevier.com/locate/gene

Molecular evolution of the androgenic hormone in terrestrial isopods


Nicolas Cerveau a,1, Didier Bouchon a, Thierry Bergs b, Pierre Grve a,
a
b

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, France
Laboratoire Signalisation et Transports Ioniques Membranaires (STIM)-ERL 7368, Universit de Poitiers, Ple Biologie Sant, 1 rue Georges Bonnet, F-86022 Poitiers cedex, France

a r t i c l e

i n f o

Article history:
Accepted 17 February 2014
Available online 20 February 2014
Keywords:
Crustacean
Isopod
Androgenic gland
Androgenic hormone
Male differentiation

a b s t r a c t
In crustaceans, the androgenic gland (AG), thanks to the synthesis of the androgenic gland hormone (AGH), controls the differentiation of the primary and secondary male sexual characters. In this study, we amplied 12 new
AGH cDNAs in species belonging to ve different families of the infra-order Ligiamorpha of terrestrial isopods. Putative essential amino acids for the production of a functional AGH protein exhibit signatures of negative selection
and are strictly conserved including typical proteolytic cleavage motifs, a putative N-linked glycosylation motif
on the A chains and the eight Cys positions. An insulin-like growth factor motif was also identied in
Armadillidium AGH sequences. The phylogenetic relationships of AGH sequences allowed one to distinguish
two main clades, corresponding to members of the Armadillidiidae and the Porcellionidae families which are congruent with the narrow specicity of AG heterospecic grafting. An in-depth understanding of the regulation of
AGH expression would help deciphering the interaction between Wolbachia, widespread feminizing endosymbiotic 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 of
Malacostraca. The role of the androgenic gland (AG) responsible for
the development of male sexual characters was rst established in the
amphipod Orchestia gammarellus by Charniaux-Cotton (1954). Since
this discovery, the AG was described in different malacostracan species
including 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 androgenic
gland hormone (AGH), controls the differentiation of the primary and
secondary male sexual characters (Charniaux-cotton, 1992; Khalaila
et al., 2001). In females, there is no AG and embryonic gonads develop
into ovaries (Katakura, 1989).
More than thirty years after the discovery of the AG, the proteinaceous nature of the sexual hormone has been elucidated in the isopod
Armadillidium vulgare (Hasegawa et al., 1987; Martin et al., 1990).
Abbreviations: AG, androgenic gland; AGH, androgenic gland hormone; RT-PCR, reverse transcriptase polymerase chain reaction; M-MLV, Moloney murine leukemia virus;
NTP, deoxy-ribonucleoside triphosphate; CBS, Center for Biological sequence analysis;
REL, random effect likelihood; IAG, insulin-like androgenic gland factor; IUB,
International Union of Biochemistry.
Corresponding author.
E-mail addresses: nicolas.cerveau@geo.uni-goettingen.de (N. Cerveau),
didier.bouchon@univ-poitiers.fr (D. Bouchon), thierry.berges@univ-poitiers.fr (T. Bergs),
pierre.greve@univ-poitiers.fr (P. Grve).
1
Present address: Courant Research Center Geobiology, Geomicrobiology and
Symbiosis Group, University of Gttingen, Goldschmidtstrae 3, 37077 Gttingen,
Germany.

http://dx.doi.org/10.1016/j.gene.2014.02.024
0378-1119/ 2014 Elsevier B.V. All rights reserved.

Around ten years later, the sequences of the prohormone (Martin


et al., 1999) and of the cDNA (Okuno et al., 1999) of A. vulgare AGH
were determined. The AGH precursor consists of a peptide of 123
amino acids, composed of a signal peptide, a B chain, a C peptide and
an A chain. The maturation of the hormone implies post-translational
modications, including the glycosylation of the A chain and the excision of the C peptide, leading to a heterodimer with the B and A chains
linked by disulde bridges (Martin et al., 1999). The masculinizing role
of the AGH has been determined by its ability to fully reverse the sex of
A. vulgare young females when AGs are grafted before sexual differentiation (Suzuki and Yamasaki, 1997). In adult females, AG grafting still induces the development of male secondary characters such as copulatory
organs (Suzuki and Yamasaki, 1997). Martin et al. (1999) conrmed that
this effect could be attributed to the AGH as repeated injections of puried
hormone also induced female sex reversal. In parallel, heterospecic
grafting of AGs showed that the AGH of one species displays a high specicity, complete efciency being retained only between species of the
same genus (Armadillidium, Porcellio or Oniscus) (Martin and Juchault,
1999). For example, heterospecic implantations of AGs within species
of the genus Porcellio (P. laevis, P. scaber and P. dispar) or the genus
Armadillidium (A. vulgare, A. depressum A. nasatum and A. maculatum) induce the masculinization of genetic females in all cases (Martin and
Juchault, 1999). Conversely, when AG graftings are realized between species from two different families, masculinization is most of the time partial or absent. This is the case when crossed graftings of AGs are realized
between A. vulgare and Porcellio gallicus which induce a masculinization
of A. vulgare but not of P. gallicus young females.
On the other hand, an antibody directed against a recombinant AGH
of A. vulgare was used to show that AGH from species of different

72

N. Cerveau et al. / Gene 540 (2014) 7177

families shares similar epitopes (Hasegawa et al., 2002). Indeed, immunostaining of AG from species of Armadillidiidae, Scyphacidae and
Porcellionidae families has been obtained using different dilutions
of the antibody. In particular in the Porcellionidae family, the AGs
of three different species including P. scaber showed positive
immunoreactions even if the signal was weaker than in the A. vulgare
AG. It suggested that the AGH of species from Armadillidiidae and
Porcellionidae families was not only close enough to be recognized by
the antibody but also divergent enough since A. vulgare AG implantations in P. scaber or P. gallicus do not induce masculinization of young females (Hasegawa et al., 2002; Martin and Juchault, 1999). This has been
conrmed by the cloning of AGH cDNA from P. scaber and Porcellio
dilatatus which revealed an amino acid identity of 88% between the
two hormone sequences and of ~ 82% between these sequences and
the one of A. vulgare (Greve et al., 2004; Ohira et al., 2003). The specicity of the AGH may be in accordance with the conservation of the peptide sequence of the hormone resulting in a specicity of the
masculinization effect at the family level. It is noteworthy that AGs are
the only tissue that synthesized the AGH, and no signal in other tissues
has been reported using either RT-PCR, Northern blot, in situ hybridization or immunohistochemical analysis (Greve et al., 2004; Hasegawa
et al., 2002; Ohira et al., 2003; Okuno et al., 1999).
Hence, the aim of this study was to clarify the molecular evolutionary patterns of isopod AGH at a larger scale. We developed a PCR strategy to amplify and sequence cDNAs encoding AGH of various species
belonging to different families of terrestrial isopods. The resulting sequences were compared and their phylogenetic relationships as well
as 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 laboratory
at 20 C under natural photoperiod on moistened soil with dead leaves
and carrots as food.
2.2. RNA extraction and RT-PCR amplication of AGH mRNA
Total RNA was isolated from AGs of 15 males (six glands per individual) using the RNeasy Mini kit according to the manufacturer's instructions (Qiagen). Single-stranded cDNA was synthesized by annealing
random hexanucleotide primers (80 ng) to 0.51 g of total RNA (previously heated for 5 min at 65 C and chilled on ice) and carrying out a

reverse transcription reaction in 20 L containing 5 L of 5 M-MLV


buffer, 50 pmol of dNTP, and 200 U of Moloney murine leukemia virus
reverse transcriptase (M-MLV-RT, Promega), for 1 h at 42 C. PCR amplication was performed using several degenerated primer pairs designed 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 containing
0.5 L of the RT reaction, 12.5 pmol of each primer, 5 L of 5 GoTaq
buffer, 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 and
72 C for 1 min. PCR products were separated on a 1.5% agarose gel
and visualized after ethidium bromide staining.
2.3. Sequencing of AGH cDNA
PCR fragments were puried with the JetPure PCR purication kit
(Genomed) and directly sequenced on both strands using the BigDye
v3.1 terminator cycle sequencing reaction kit on an ABI 3130 Genetic
Analyzer (Perkin Elmer). New AGH sequences generated in this study
were deposited in GenBank/EMBL databases (Table 1). All sequences
have been checked using independent samples and several RT-PCR
and PCR reactions. The AGH sequences of A. vulgare, P. scaber and
Porcellio dilatatus dilatatus obtained in this study are 100% identical to
the ones previously deposited in GenBank database.
2.4. Amino acid sequence analyses
Amino acid sequence analyses were performed using bioinformatic
tools available on the CBS prediction servers (http://www.cbs.dtu.dk/
services/): SignalP predicts the presence and the localization of signal
peptide 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 cleavage
sites (Arg-X-X-Arg) on sequences of eukaryote prohormones (Duckert
et al., 2004). A pictogram of the two proteolytic cleavage sites and the putative N-linked glycosylation site was drawn using the Logo website
(http://weblogo.berkeley.edu/logo.cgi) (Crooks et al., 2004) (Supp. data
2). Finally, putative protein domains were identied using the Smart software (http://smart.embl-heidelberg.de) (Letunic et al., 2011).
AGH protein sequences including the 11 decapod insulin-like androgenic gland hormone (IAG) sequences available in GenBank (Ventura
et 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 calculate protein divergence between sequences of isopods and decapods

Table 1
List of woodlice species samples used in this study and accession numbers of AGH sequences. Underlined accession numbers correspond to sequences already available in GenBank. NA
(not amplied) means that the sequences could not be obtained for these species.
Sub-order

Infra-order

Family

Species

AH sequences accession #

Sampling location

Oniscidea

Ligiamorpha

Armadillidae
Armadillidiidae

Armadillo ofcinalis
Armadillidium vulgare
Armadillidium assimile
Armadillidium depressum
Armadillidium granulatum
Armadillidium maculatum
Armadillidium nasatum
Balloniscus sellowi
Cylisticus convexus
Oniscus asellus
Chaetophilosia elongata
Porcellio dilatatus dilatatus
Porcellio dilatatus petiti
Porcellio dispar
Porcellio gallicus
Porcellio laevis
Porcellio scaber
Porcellionides pruinosus
Helleria brevicornis

JQ304808
AB029615
NA
JQ304800
JQ304798
JQ304799
JQ304801
NA
JQ304809
JQ304810
NA
AB089811
JQ304802
JQ304803
JQ304804; JQ304805
JQ304806
AB089810; AY169973
JQ304807
NA

Montpellier, France
Nice, France
Cort, France
Sainte-Marie de R, France
Sousse, Tunisia
Ez, France
Mignaloux-Beauvoir, France
Caxias do Sul, Brasil
Villedaigne, France
Celles sur Belle, France
Thur, France
Rom, France
St Honorat island, France
Santa Maria del Sol, Spain
Chiz, France
Hammamet, Tunisia
Sassis, France
Nevers, France
Sainte-Marguerite island, France

Balloniscidae
Cylisticidae
Oniscidae
Phylosciidae
Porcellionidae

Tylomorpha

Tylidae

N. Cerveau et al. / Gene 540 (2014) 7177

and to construct the resulting phylogenetic tree. The maximum likelihood tree was created using the JonesTaylorThornton substitution
model with uniform rate among all the alignment sites. The tree was
generated with Nearest Neighbor Interchange heuristic method. Robustness of the tree was assessed with 1000 bootstrap replicates.
To detect signatures of positive and negative selection, analyses were
performed with the web-server of the HyPhy package (http://www.
datamonkey.org) (Pond and Frost, 2005). Pairwise estimates of the number of non-synonymous (dN) and synonymous (dS) substitutions per site
were calculated using maximum likelihood (Goldman and Yang, 1994).
The random effect likelihood (REL) analysis (Kosakovsky Pond and
Frost, 2005) was used to identify amino acid sites with signature of selection based on a Bayes factor of 95. Four different analyses were made. The
rst analysis was done with 16 sequences including the three available
AGH sequences ranging from positions 44 to 139 (Fig. 1). We then individually considered sequences of the B chain, the C peptide and the A
chain of the seven complete amino acid sequences among the data set.

3. Results
In this study, among the 16 tested species we amplied the cDNA of
13 new AGH sequences from 12 species belonging to ve different families of the infra-order Ligiamorpha using several degenerated primer
pairs (Table 1; Supp. data 1). We obtained nine complete and four partial AGH coding sequences from four species of the Armadillidium genus,
ve species of the Porcellio genus and one species of the Armadillo,
Cylisticus, Oniscus and Porcellionides genus respectively (Fig. 1). In each
case except for P. gallicus, only one sequence was obtained suggesting
1

10

90

20

100

that there is only one gene encoding the AGH in these species. In
P. gallicus, the AGH mRNA seems to be transcribed from two different
genes since the Long (P. gallicus_L) and the Short (P. gallicus_S) identied sequences differ in size and in sequence (Fig. 1). The presence of
the two copies was conrmed using specic P. gallicus AGH primers
(AGH-Pg_S and L in Supp. data 1).
The protein sequence alignment (Fig. 1) of 16 sequences including
the 13 sequences obtained in this study and the three available AGH sequences showed that the organization observed in the A. vulgare AGH
sequence is preserved among all species. It consists of a signal peptide,
a B chain, a C peptide and an A chain. Prediction algorithms dened a
signal peptide excised after the 21st amino acid (Table 2). For each sequence, a typical proteolytic cleavage motif which is composed of two
Arg separated by two variable amino acids (Arg-X-X-Arg; Duckert
et al., 2004) was found at two positions between the B chain and the C
peptide, and between the C peptide and the A chain. The motif of the
rst predicted cleavage site (positions 65 to 70; Fig. 1) is conserved, except in the AGH sequence of Porcellionides pruinosus where methionine
was identied at the rst position of the cleavage site (Met-Glu-ArgArg). Nevertheless, Prop algorithm still predicts a cleavage at this modied site (Table 2). At the second putative cleavage site (positions 124 to
127; Fig. 1), the predicted motif is conserved in all sequences. As already
identied in A. vulgare, P. scaber and P. dilatatus AGH sequences (Greve
et al., 2004; Ohira et al., 2003; Okuno et al., 1999), an N-linked glycosylation motif (Asn147-X-Thr/Ser) was predicted in the A chain of the AGH
sequences 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 possibilities at position II: an Arg in nine sequences out of 14 (64.29%), a Lys
in four sequences out of 14 (28.57%) and a Ser in one sequence (7.14%).

30

110

73

40

120

50

130

60

140

70

80

150

Fig. 1. Alignment of AGH amino acid sequences. Orange frames highlight the eight conserved Cys residues, blue frames the two proteolytic cleavage sites and the green frame the putative
N-linked glycosylation site. The different AGH chains and the positions of the main primers used in this study are indicated under the sequences.

P. scaber

21
nd
/
6970
125126
21
nd
146
6970
125126
21
nd
146
6970
125126
/
nd
146
6970
125126

P. gallicus_L P. gallicus_S P. laevis

21
nd
146
6970
125126
21
nd
146
6970
125126
21
nd
146
6970
125126
21
nd
146
6970
125126
21
nd
/
6970
125126
n.d
nd
146
6970
125126
21
IlGF
146
6970
125126
21
IlGF
146
6970
125126
21
IlGF
146
6970
125126
21
IlGF
146
6970
125126
21
IlGF
146
6970
125126

A. ofcinalis A. vulgare A. depressum A. granulatum A. maculatum A. nasatum C. convexus O. asellus P. dilatatus dilatatus P. dilatatus petiti P. dispar

At position III, the Ser residue was never found (Fig. 1; Supp. data 2c).
Finally, eight Cys residues were located at identical positions in all the
six complete AGH sequences compared with those of the A. vulgare sequence, all of them being involved in disulde bridges in the mature
hormone of this species (Martin et al., 1999).
Signatures of purifying selection were identied on the Arg of the two
typical proteolytic cleavage motifs (positions 65 & 70, 123 & 126; Fig. 1),
on the Asn of the putative N-linked glycosylation site (position 146) and
on ve of the eight Cys (positions 34, 43, 45, 60 and 154). Three other
amino 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 addition, two long stretches, one in the A chain between amino acids 30
and 53 and the second one in the B chain between amino acids 119
and 146, are strictly conserved among the Armadillidium genus.
Apart from these conserved patterns, there are a lot of differences between the sequences. An insulin-like growth factor motif was identied
with Smart in the Armadillidium sp. AGH sequences but not in the ones of
other species (Table 2). There are also many differences in the size of
both A and B chains and of the C peptide sequences which correspond insertions or deletions (Fig. 1). Indeed, one or more additional amino acids
are observed in the A chain of Armadillo ofcinalis and Porcellio sp. AGHs
and in the B chain of Oniscus asellus and P. pruinosus AGHs or in the C
peptide of Armadillidium granulatum, Cylisticus convexus and O. asellus
AGHs. Size variations are also due to deletions like in the C peptide of
P. scaber, P. gallicus_S/L and A. ofcinalis AGHs (Fig. 1).
Despite these variations, the amino acid identity percentage between
sequences, calculated with patristic distance considering pairwise deletion in the full length alignment, ranges from 40.5% (A. ofcinalis/
P. pruinosus) to 97.9% (P. dilatatus dilatatus and Porcellio dilatatus petiti)
with a mean at 68.1% (Supp. data 3). The highest values are observed between sequences within the Armadillidium genus (mean at 93.3%, Supp.
data 3). These relationships are illustrated by the phylogenetic tree based
on full length alignment (Fig. 2). Indeed, all the Armadillidium genus AGH
sequences form a monophyletic group in contrast with the Porcellio
genus AGH sequences which are more distantly related (Fig. 2). The
tree obtained with the largest common AGH sequences in all isopod species (positions 44 to 139; Fig. 1) presents the same topology (data not
shown). AGH sequences of isopods and IAG sequences of decapods
branch in two distinct clusters. This pattern is in accordance with the
low sequence similarity observed between these two groups.

4. Discussion

SignalP
nd
Smart
nd
Glycosylation 146
ProP
125126

P. pruinosus

N. Cerveau et al. / Gene 540 (2014) 7177


Table 2
Bioinformatics analysis of the peptide AGH sequences. The SignalP algorithm allows one to predict the excision of the signal peptide. It identies the amino acid position after which the cleavage is putatively realized. The NetNGlyc algorithm allows
one to predict where N-linked glycosylation motifs are localized, including the Asn position which is potentially glycosylated. The proteolytic cleavage site positions were predicted with Prop algorithm, including the two amino acid positions which
are putatively removed after the excision. These three programs are hosted by the CBS server (http://www.cbs.dtu.dk/services/). Positions refer to the amino acid alignment numbering (Fig. 1). Smart software (http://smart.embl-heidelberg.de/)
allows one to predict the presence of protein domains.

74

To gain insight into the specicity of the AGHs, we assessed the diversity and evolution of the hormone sequences in 16 terrestrial isopod
species belonging to ve different families. We succeeded in amplifying
AGH cDNA of species mostly belonging to the Armadillidium and
Porcellio genera. This result was expected since degenerated primers
have been designed according to A. vulgare, P. scaber and P. dilatatus
AGH available sequences. However, PCR failed when using AGH
cDNAs of Balloniscus sellowi, Chaetophilosia elongata and Helleria
brevicornis, three species that are phylogenetically distant of the
Armadillidium and Porcellio genera. This may indicate a more variable
primer site sequence than expected. The organization of the resulting
16 AGH sequences is highly conserved and consists of a signal peptide,
a B chain, a C peptide normally excised during the post-transcriptional
processes, and an A chain as already established for the AGH of
A. vulgare, P. scaber and P. dilatatus precursors (Greve et al., 2004;
Martin et al., 1999; Ohira et al., 2003; Okuno et al., 1999). For the
AGHs of Armadillidium species, a motif search revealed the presence of
an insulin-like growth factor domain which is consistent with the structural similarity of the protein and the insulin superfamily peptides and
with identical maturation processes of the two proteins (Steiner et al.,
1985). However, both hormones are clearly different in terms of
amino acid sequence and peptide chain length.

N. Cerveau et al. / Gene 540 (2014) 7177

75

92 Porcellio dilatatus dilatatus


69

Porcellio dilatatus petiti

86

Porcellio laevis
Porcellio scaber

43

Porcellio gallicus L

20

Porcellio gallicus S

93
48

Porcellio dispar
Porcellionides pruinosus

17

Armadillidium maculatum
77

Armadillidium depressum

16
25
29
33

79

Armadillidium nasatum
Armadillidium vulgare
Armadillidium granulatum
Oniscus asellus

99

Cylisticus convexus
Armadillo officinalis
Palaemon pacificus

60

Palaemon paucidens
Macrobrachium lar

87

Macrobrachium rosenbergii

62

Penaeus monodon

73
89

Litopenaeus vannamei
Marsupenaeus japonicus
Callinectes sapidus

32

Cherax destructor
56

Cherax quadricarinatus
98

Portunus pelagicus

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.

Putative essential sites for the production of a functional AGH protein are strictly conserved and carry signatures of negative selection.
The eight Cys residues of which ve are under negative selection are localized at the same positions as in the A. vulgare AGH sequence (Martin
et al., 1999). In this species, it was previously reported that four Cys residues of each chain form two intrachain (one more than in insulin) and
two interchain disulde bridges (Martin et al., 1999). This result suggests that these Cys residues are also involved in disulde bridges in
the mature hormone of AGH of all isopod species. Furthermore, correct
disulde bond arrangements are required for the activity of the
A. vulgare AGH (Katayama et al., 2010). Indeed, a semisynthetic AGH
folded into non-native form with wrong disulde linkages does not display any biological activity (Katayama et al., 2010). It has also been
demonstrated that A. vulgare recombinant AGH composed of the B
chain, the C peptide and the A chain does not show any AGH activity
(Okuno et al., 2002). AGH activity was recovered after lysyl endopeptidase digestion leading to a heterodimeric peptide linked by disulde
bridges and that lacks most of the C peptide (Okuno et al., 2002). In addition, both Arg residues of the two typical proteolytic cleavage motifs
have also signatures of purifying selection. Excision of the C peptide
seems also to be essential for insulin since the activity of proinsulin is
lower than insulin by about two orders of magnitude (Gross et al.,
1989). Finally, the glycosylation site of the A chain in which the Asn residue carries signature of negative selection, is another important feature
of the AGH which also differs from insulin (Greve et al., 2004; Martin

et al., 1999). This glycosylation seems to be obligatory for the activity


of the AGH since it was shown that the injection of a recombinant AGH
lacking the glycan moiety does not induce masculinization of A. vulgare
females (Okuno et al., 2002). The sizes of the A and B chains are conserved compared with the one of the C peptide where there are a lot of
size variations, particularly in the A. ofcinalis AGH sequence. A similar
structural divergence has been also observed in the insulin peptide superfamily, while the amino acid sequences of A and B chains were conversely highly conserved among various species (Steiner et al., 1985).
More recently, an insulin-like factor (Cq-IAG) has been identied in
the AG of the red-claw craysh Cherax quadricarinatus (Manor et al.,
2007). The gene encoding this peptide is exclusively expressed in the
AG and Cq-IAG is thought to promote male growth that leads to sexual
dimorphism. The sequence similarity of Cq-IAG with the isopod AGH sequences is very low, although six of the eight Cys residues of Cq-IAG are
conserved. Two putative N-linked glycosylation motifs both in the A and
B chains were also identied in Cq-IAG, whereas only one was identied
in the A chain in isopod AGHs. This result marked the beginning of elegant studies realized in decapods including the identication of 11 other
insulin-like hormones and the utilization of RNAi which allowed one to
show that IAG regulates male sex differentiation (Rosen et al., 2010;
Ventura et al., 2009, 2011b).
The reconstructed phylogeny showed that the AGH and IAG sequences 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) 7177

sequence shares 32.3% identity at the most with isopod AGH sequences
(Fig. 2). Within the isopod AGH sequence group, two main clusters are
also observed, the rst one including the ve AGH sequences from species
of the Armadillidiidae family and the second one the eight AGH sequences
from species of the Porcellionidae family. The three remaining sequences
belong to species of three other families. This pattern is congruent with
previous terrestrial isopod phylogenetic analyses (Michel-Salzat and
Bouchon, 2000; Schmalfuss, 2003). The amino acid identity obtained
within the AGH sequences of species from the Porcellio genus is on
average 81.4% and 71.48% between species of Porcellio and Porcellionides
genera. The genetic distance between the AGH sequences of these two
groups is also illustrated during heterospecic graftings (Martin et al.,
1999). Indeed, whereas grafting of P. dispar, P. scaber and P. laevis AG in
P. pruinosus young females induces a male phenotype, the opposite is
not true. Within the Porcellio genus, AG grafting is efcient in both directions, as well as within the Armadillidium genus (Martin et al.,
1999). This result is consistent with the phylogenetic relationships between the AGH sequences and seems to conrm that the Porcellionidae
family which is one of the largest oniscidean families is more diverse
than the Armadillidiidae family. Nevertheless, some amino acid fragments are still conserved in the AGH sequences of the different species
as shown by immunohistochemistry experiments (Hasegawa et al.,
2002) that may explain heterospecic effects of the AG graftings
(Martin et al., 1999).
In isopods, the AGH may also be the target of the intracellular bacteria Wolbachia (Bouchon et al., 2008; Negri et al., 2010). These vertically
transmitted alpha-Proteobacteria infect many arthropod species and
are considered as reproductive parasites because of their capacity to
manipulate host reproduction that increases the tness of infected
females, thereby enhancing Wolbachia 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 development
of the AG is not observed and individuals developed as functional females. Wolbachia transinfections between different isopod species
showed that the resulting phenotype depends more on the host phylogeny than on the bacterial phylogeny (Bouchon et al., 1998, 2008; Rigaud
et al., 1997). For example, feminizing Wolbachia of A. vulgare induces the
feminization A. nasatum males whereas it is not efcient when injected
in P. pruinosus males (Bouchon et al., 1998; Juchault et al., 1974; Rigaud
and Juchault, 1995; Rigaud et al., 2001). These results are in accordance
with AG grafting experiments which showed that A. vulgare AG grafting
induces masculinization of A. nasatum young females but not the ones of
P. pruinosus (Martin et al., 1999).
Therefore, it seems that the AGH receptor and the hormone coevolved likely at the same time (Negri et al., 2010). An in-depth
understanding of the regulation of AGH expression would help in
deciphering the interaction between Wolbachia and A. vulgare sex
differentiation.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.gene.2014.02.024.

Conict of interest
There is no conict of interest.

Acknowledgments
We are grateful to C. Debenest, C. Delaunay and M. Raimond for
technical assistance. We thank Joanne Bertaux for comments on an
earlier version of the manuscript and for improving the English. This
research was funded by the Centre National de la Recherche Scientique
(CNRS) and the French Ministre de l'Education Nationale, de
l'Enseignement Suprieur et de la Recherche.

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