Genes of the antioxidant system of the honey bee: Annotation and phylogeny

Abstract

Antioxidant enzymes perform a variety of vital functions including the reduction of life-shortening oxidative damage. We used the honey bee genome sequence to identify the major components of the honey bee antioxidant system. A comparative analysis of honey bee with Drosophila melanogaster and Anopheles gambiae shows that although the basic components of the antioxidant system are conserved, there are important species differences in the number of paralogs. These include the duplication of thioredoxin reductase and the expansion of the thioredoxin family in fly; lack of expansion of the Theta, Delta and Omega GST classes in bee and no expansion of the Sigma class in dipteran species. The differential expansion of antioxidant gene families among honey bees and dipteran species might reflect the marked differences in life history and ecological niches between social and solitary species.

Full text

Insect Molecular Biology (2006)
15
(5), 687–701
© 2006 The Authors
Journal compilation © 2006 The Royal Entomological Society
687
Blackwell Publishing Ltd
Genes of the antioxidant system of the honey bee:
annotation and phylogeny
M. Corona and G. E. Robinson
Department of Entomology, University of Illinois at Urbana-
Champaign, USA
Abstract
Antioxidant enzymes perform a variety of vital functions
including the reduction of life-shortening oxidative
damage. We used the honey bee genome sequence to
identify the major components of the honey bee anti-
oxidant system. A comparative analysis of honey bee
with
Drosophila melanogaster
and
Anopheles gambiae
shows that although the basic components of the anti-
oxidant system are conserved, there are important
species differences in the number of paralogs. These
include the duplication of thioredoxin reductase and
the expansion of the thioredoxin family in fly; lack of
expansion of the Theta, Delta and Omega GST classes
in bee and no expansion of the Sigma class in dipteran
species. The differential expansion of antioxidant gene
families among honey bees and dipteran species
might reflect the marked differences in life history and
ecological niches between social and solitary species.
Keywords: Antioxidant genes, honey bee genome
Introduction
Reactive oxygen species (ROS) are constantly generated
as by-products of aerobic metabolism. Accumulated
evidence suggests that oxidative damage to cellular com-
ponents induced by ROS is a major contributive cause of
degenerative diseases and ageing. ROS generation occurs
mainly in mitochondria in which more than 90% of the
oxygen used by the cell is consumed (Perez-Campo
et al
.,
1998). Aerobic organisms have evolved a complex network
of enzymatic and non-enzymatic antioxidant systems to
avoid oxidative damage. Key components of the antioxidant
defence system are conserved throughout evolution, but
there are unique adaptations among different groups. The
major changes in insects in comparison with vertebrates
and other phylogenetic groups include the loss of genes
encoding functional glutathione reductase (GR) and glutath-
ione peroxidase (GPX). Homologous genes for thioredoxin
reductase (TrxR) (Kanzok
et al
., 2001) and thioredoxin per-
oxidase (TPX) (Radyuk
et al
., 2001) activities, respectively,
act in their place.
There are both primary and secondary antioxidant
enzymes, which act directly or indirectly on ROS molecules.
The first line of defence against ROS attack is provided by
three different kinds of primary antioxidant enzymes that
act directly on ROS: (1) superoxide dismutases (SODs),
which rearrange superoxide to oxygen and hydrogen
peroxide; (2) catalase, which prevents free hydroxyl radical
formation by breaking down hydrogen peroxide into oxygen
and water; and (3) peroxidases, which catalyse an analog-
ous reaction in which hydrogen peroxide is reduced to
water by a reductant that acts as an electron donor, normally
reduced thioredoxin (TRX) or glutathione (GSH). In addition,
insects have three families of genes that encode antioxidant
enzymes that act as peroxidases: TPXs, also known as per-
oxiredoxins (Radyuk
et al
., 2001), phospholipid-hydroperoxide
GPX homologs with thioredoxin peroxidase activity (GTPX)
(Missirlis
et al
., 2003), and glutathione S-transferases (GSTs)
(Tang & Tu, 1994; Toba & Aigaki, 2000). Secondary antioxi-
dant enzymes that act indirectly on ROS include TrxR,
which recycles both TRX and GSH (Kanzok
et al
., 2001),
and methionine sulphoxide reductases (MsrA and MsrB),
which are involved in protein reparation by catalysing the
TRX-dependent reduction of methionine sulphoxide to
methionine (Moskovitz
et al
., 1996; Kumar
et al
., 2002).
Honey bee antioxidant enzymes are of particular interest
because of their potential involvement in some of the
exceptional biological characteristics of the queen honey
bee, especially its longevity relative to worker bees (10
×
longer; e.g. Page & Peng, 2001). Elevated expression of
several traditional antioxidant-encoding genes occurs
in young queens and old workers (Corona
et al
., 2005),
Received 21 June 2006; accepted after revision 18 July 2006. Correspond-
ence: M. Corona, Department of Entomology, University of Illinois at Urbana
Champaign, 505 S. Goodwin Ave, Urbana, IL, USA. Tel.: 217 2440895;
fax: 217 244 3499; e-mail: corona@life.uiuc.edu
Re-use of this article is permitted in accordance with the Creative Commons
Deed, Attribution 2·5, which does not permit commercial exploitation.

688
M. Corona and G. E. Robinson
© 2006 The Authors
Journal compilation © 2006 The Royal Entomological Society,
Insect Molecular Biology
,
15
, 687–701
suggesting that queen longevity is not related to higher
expression of these particular genes, a result consistent with
findings for Sod1 in
Lasius niger
ant queens (Parker
et al
.,
2004). However, traditional antioxidants likely play roles in
other processes. For example, Weirich
et al
. (2002) and
Collins
et al
. (2004) reported that catalase, GST and SOD
might contribute to the ability of queens to store sperm in
their spermatheca for several years without loss of viability.
The recent release of the honey bee genome sequence
provides the first opportunity to compare the whole set of
antioxidant genes between insect orders. In this report we
present the results of the manual annotation of the main
antioxidant genes of
Apis mellifera
, a hymenopteran social
insect, and a comparative analysis with the dipteran
Anopheles gambiae
and
Drosophila melanogaster
.
Results and discussion
We identified 38 antioxidant genes in the honey bee
genome, which include all major components of the
enzymatic antioxidant system. This report does not include
the annotation of genes encoding proteins thought to have
indirect antioxidant effects mediated by metal binding
capacities, such as vitellogenin (Seehuus
et al
., 2006),
transferrin (Kucharski & Maleszka, 2003; do Nascimento
et al
., 2004), ferritin (Dunkov & Georgieva, 1999; Geiser
et al
., 2003) and metallothioneins (Egli
et al
., 2006).
In general, antioxidant genes encode small proteins less
than 250 amino acids, with the exception of TrxR, catalase
and proteins of unknown function such as Rsod and Trx/
Grx-like proteins, which probably diverged by duplication of
ancestral
Cu/ZnSOD
and
Trx
/glutaredoxin (
Grx
) genes.
Most of the honey bee’s antioxidant genes have protein-
encoding regions with high A/T content (64% average,
Table 1), a characteristic that is not specific to antioxidant
genes, but rather is a general attribute of the honey bee
genome. The honey bee genome is reported to contain 67%
A/T, compared with 58% in
D. melanogaster
and 56% in
Anopheles gambiae
(Honey Bee Genome Consortium, 2006).
It has been postulated that genes from organisms with high
rates of metabolism use more A-ending codons than those
from organisms with lower rates (Xia, 1996). This hypothesis
has not yet been studied in insect species, which in general
have very high metabolic rates (Suarez
et al
., 2000).
Comparative analysis of
A. mellifera, D. melanogaster
and
A. gambiae
antioxidant genes
Superoxide dismutases.
SOD converts radical superoxide
to oxygen and hydrogen peroxide, providing the first line of
defence against ROS produced in the mitochondria. SODs
normally exist in two forms in eukaryotic cells; the two forms
differ in cellular localization and in the structure of their
active sites. MnSOD (SOD2) is present in the inner mito-
chondrial space and Cu/ZnSOD (SOD1) in the cytoplasm.
Like most eukaryotes, honey bees have a single mitochon-
drial MnSOD gene located on chromosome 11. Vertebrate
orthologs, including those in Tetraodon and human, have
higher overall identity with the honey bee ortholog (66.21
and 62.33% ID) than dipteran species (
Drosophila
, 59.09,
Anopheles
59.17). Possible explanations for this phylogenetic
discordance include rapid divergence of the dipteran orthologs
(Honey Bee Genome Sequencing Consortium, 2006).
The Cu/ZnSOD family includes five members in
Drosophila
and
Anopheles
and four members in
Apis
(Table 2). In
Drosophila
this group includes the canonical cytoplasmatic
Cu/ZnSOD (CG11793), extracellular SOD (
Sod3
, CG9027),
copper chaperone (CCS, CG17753), related to Sod (
Rsod
,
CG31028), and Sodesque (
Sodq
, CG5948). Extracellular
CuZnSODs are present in several animal groups, from
nematode to mammals. In insects, they have been identified
in
D. melanogaster
,
Anopheles gambiae
(Landis and
Tab le 1. Summary of honey bee antioxidant gene annotation. Gene
localization based on the scaffolds_assembly_2 database. Apis mellifera
GstO2 and Gstu1 are partial sequences
Gene aa Location introns ORF AT%
Sod2 218 Group11.11 2 65.3
Sod1 152 Group8.3 3 61.0
Sod3 178 GroupUn 2 64.2
CCS 266 GroupUn.5386 6 70.8
Rsod 1100 GroupUn.153 18 69.7
Cat 513 Group6.23 7 61.6
Gtpx1 168 GroupUn.5 3 68.4
Gtpx2 201 Group5.15 1 70.3
Tpx1 194 GroupUn.29 2 63.4
Tpx3 242 Group15.12 2 66.9
Tpx4 220 GroupUn.1374 4 61.8
Tpx5 220 Group12.14 3 67.0
Tpx6 219 Group9.2 1 57.2
GstT1 230 GroupUn.336 3 72.0
GstD1 217 Group15.2 4 56.1
GstS1 204 GroupUn.1306 3 66.0
GstS2 202 GroupUn.1306 2 63.4
GstS3 207 GroupUn.898 3 60.3
GstS4 206 Group4.16 3 58.6
GstZ1 217 Group5.15 4 63.3
GstO1 241 Group1.28 4 66.9
GstO2 partial GroupUn.264 4 ND
Gstu1 partial GroupUn.176 ND ND
Gstmic1 149 Group2.5 1 70.4
Gstmic2 156 Group1.56 1 53.6
Trxr-1 494 GroupUn.68 7 64.6
Trx-1 105 GroupUn.35 3 68.3
Trx-2 136 Group6.16 2 64.9
Trx-3 103 GroupUn.125 0 73.5
Trx-like1 287 Group14.6 3 66.1
Trx-like2 488 Group3.21 5 48.0
Trx-like3 411 Group13.2 5 65.6
Grx1 98 GroupUn.505 1 67.0
Grx2 133 Group11.6 2 65.2
Grx-like1 711 Group6.26 0 43.2
Tr x /Gtx 222 Group15.14 1 67.9
MsrA 217 GroupUn.104 3 65.7
MsrB 137 GroupUn.304 2 59.6
ND, not determined.

Annotation and phylogeny
689
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Journal compilation © 2006 The Royal Entomological Society,
Insect Molecular Biology
,
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, 687–701
Tower, 2005) and
Lasius niger
(Parker
et al
., 2004). The
honey bee has an extracellular Cu/ZnSOD (SOD3) of 178
amino acids.
Phylogenetic analysis (Fig. 1) shows that the extracellular
SODs of insects and vertebrates form different mono-
phyletic clades. This suggests the possibility that they
evolved independently in each group, for example, by the
addition of a signal peptide to cytoplasmatic SOD (Landis
and Tower, 2005). Copper chaperone (CCS) has, in addition
to the SOD domain, a N-terminal heavy-metal-associated
domain (HMA) involved in the transport of copper to Cu/
ZnSOD. As insect and vertebrate homologs form a single
monophyletic clade, CCS proteins seem to have diverged
from cytoplasmatic SOD before the separation of these two
lineages.
A putative ortholog for the
Drosophila
Sodesque (
Sodq
)
gene is present in
Anopheles gambiae
; however, it
encodes a rapid evolving protein, with only 42% identity
between these dipteran species. As a Sodq-related protein
is also present in
Aedes aegypti
(EAT33630), but orthologs
for this gene are absent in honey bee, other insects, and
vertebrates, it is possible that this gene has diverged from
cytoplasmatic SOD only in dipteran species. Sodq function
in
Drosophila
is uncertain, because the fly ortholog lacks
several conserved residues essential for catalytic function
while possessing a signal peptide for extracellular targeting
(Landis and Tower, 2005).
The
Drosophila
related to Sod gene (
Rsod
) is an atypical
member in the Cu/ZnSOD family. It has a duplicated SOD
domain and an unusually high number (18) of introns
(Table 1). Homologous genes (with two or three SOD
domains) are present in
Anopheles
,
Apis
, protozoa (
Dicty-
ostelium discoideum
XP_639320 and XP_639300), fish
(
Tetraodon nigroviridis
, CAF89944), but not in mammals.
Rsod function is unknown in insects. However, a homolo-
gous protein (pernin, AAK20952) in
Per na canaliculus
(Mollusca) does not show SOD activity but might be
involved in the transport of divalent metal cations (Scotti
et al
., 2001).
Catalase.
Catalase prevents free hydroxyl radical formation
by breaking down hydrogen peroxide into oxygen and
water. A single catalase gene is normally present in eukary-
otes, with the exception of
C. elegans
, in which this gene is
duplicated. Honey bee catalase encodes a protein of 513
amino acids and is localized on chromosome 6. Catalase
in
Apis
, as in other eukaryotes, is located in the cytosol and
lacks a signal peptide necessary for secretion. Interest-
ingly, catalase activity has been reported to be present
in honey (White, 1975), which perhaps acts to keep H
2
O
2
levels in honey (produced by bees as a preservative) below
toxic levels. Since in the honey bee genome the only
catalase is not extracellular, the source of the catalase in
honey remains to be determined. It has been assumed that
Tab le 2.
Major components of the enzymatic antioxidant system of
Apis
mellifera
,
Drosophila melanogaster
and
Anopheles gambiae
. Gene
identification numbers: for bee, the BeeBase ID; for mosquito, the Genbank
accession number; for fly, the Flybase gene ID. NP indicates genes with no
automatic prediction in bees. For the
GST Delta
and
Epsilon
classes of
Drosophila
and
Anopheles
, only four representative members are shown
Gene Apis Anopheles Drosophila
Sod2
GB14346 AAS17758 CG8905
Sod1
GB10133 AAR90328 CG11793
Sod3
NP AAS17758 CG9027
CCS
GB14210 XP_308747 CG17753
Rsod
GB14567 EAA00894 CG31028
Sodq
Not identified EAA04552 CG5948
Cat GB11518 XP_314995 CG6871
Gtpx1 GB14138 XP_313166 CG12013
Gtpx2 GB18955 XP_562772 Not identified
Gpx-like Not identified Not identified CG15116
Tpx1 GB19380 XP_308081 CG1633
Tpx2 Not identified XP_308336 CG1274
Tpx3 GB10972 XP_565975 CG5826
Tpx4 GB10498 XP_320690 CG12405
Tpx5 GB10803 XP_308753 CG3083
Tpx6 NP Not identified CG6888
GstT1 GB12047 AAM61893,
AAM61892
CG1702,
CG30005
CG30000,
CG1681
GstD1 GB18045 AAC79995 CG10045
GstD2-12 Not identified CAA96104,
AAM53610
CG4181,
CG4381
AAM53607,
AAM53607
CG11512,
CG12242
GstS1 GB16959 AAA29358 CG8938
GstS2 NP Not identified Not identified
GstS3 GB19254 Not identified Not identified
GstS4 GB14372 Not identified Not identified
GstZ1 GB17672 AF515522 CG9363
GstZ2 Not identified Not identified CG9362
GstO1 GB11466 AAP13482 CG6781
GstO2 GB19678 CG6662
GstO3-4 Not identified Not identified CG6776
CG6673
GSTu1 GB15512 AAM61888 CG33546
GstE1-13 Not identified AAG45163,
AAG45164
CG5164
CG17524
AAL59653,
AAL59654
CG17523
CG17525
GSTmic1 GB12371 AAP37003 CG1742
GSTmic2 GB10566 AAP37005 CG33178
Trxr-1 GB14972 CAD30858 CG2151
Trxr-2 Not identified Not identified CG11401
Trx-1 GB17503 EAA04498 CG8993,
CG8517
Trx-2 GB15855 EAA14495 CG31884,
CG3315
CG4193,
CG13473
Trx-3 GB19972 EAA09650 CG3719
Trx1-like1 GB15457 EAA11972 CG5495
Trx1-like2 GB15572 XP_320264 CG14221
Trx1-like3 GB19276 XP_316887 CG9911
Grx1 GB10598 XP_309539 CG6852,
CG7975
Grx2 GB18700 XP_312440 CG14407
Grx-like1 GB11664 EAA06446 CG31559,
CG12206
Trx/Gtx GB12870 EAA07378 CG6523
MsrA GB10196 XP_320164 CG7266
MsrB GB15486 XP_311902 CG6584

690 M. Corona and G. E. Robinson
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Journal compilation © 2006 The Royal Entomological Society, Insect Molecular Biology, 15, 687–701
it comes from plants (White, 1975), but extracellular cata-
lases are apparently only found in some bacteria and fungi.
An intriguing possibility is that catalase in honey originates
from endosymbiotic bacteria.
Thioredoxin peroxidases. TPXs, also known as peroxire-
doxins, are a type of peroxidase that reduces H2O2 using
electrons provided by TRX (Chae et al., 1994). Based on
the number of conserved cysteins, TPXs are classified into
two subfamilies: 1-Cys and 2-Cys. In contrast to the 1-Cys,
the 2-Cys subfamily has a second conserved Cys in the
C-terminus (Trivelli et al., 2003) (Fig. 3A). The TPX family
has five members in humans, which include cytosolic,
mitochondrial and extracellular forms (Chang et al., 2004).
The Drosophila genome also contains five TPX homologs
(Radyuk et al., 2001) that comprise three cytosolic
variants (Tpx1, CG1633, Tpx4, CG12405, Tpx5, CG3083),
one mitochondrial (Tpx3, CG5826) and one secretable
(Tpx2, CG1274).
We identified a new putative TPX homolog in Drosophila
(DmTpx6, CG6888), five Tpx members in Anopheles and
five homologs in Apis (Table 2). Compared with dipteran
species, honey bee seems to have lost the secretable
variant (Tpx-2). AmTpx6 and DmTpx6 are the more diverged
members of the Cys-1 subfamily; there is no mosquito
homolog (Fig. 2A and 3A). Phylogenetic analysis (Fig. 3A),
showed that the different insect and human homologs are
grouped in separate phylogenetic groups. Three of them
are included in the 2-Cys subfamily and two in the 1-Cyst
subfamily. This distribution suggests that the major members
of the TPX family could have diverged before the separation
of the insect and vertebrate metazoan ancestor. Consistent
with this analysis is the finding that each of the phylogenetic
groups contain members that seem to have conserved
their particular subcellular localization. Clades A, D and E
contain cytoplasmic, clade B contains mitochondrial, and
clade C contains extracellular variants (as inferred in Apis
mellifera and Anopheles gambiae by the presence of
predicted mitochondrial targeting and signal peptides).
Glutathione peroxidase homologs. GPX catalyses the re-
duction of hydrogen peroxide and organic hydroperoxides.
Figure 1. Neighbour joining tree showing the
relationships of the CuZn SOD family. The
GenBank accession number (Anopheles
gambiae), Flybase ID (Drosophila melanogaster)
and BeeBase ID (Apis mellifera) are shown for
each sequence. Values above the branches
represent bootstrap support.

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Figure 2. Apis mellifera and Drosophila
melanogaster thioredoxin-dependent peroxidase
homologs. (A) Thioredoxin peroxidase family
(peroxiredoxins). Predicted signal peptide for Dm
Tpx-2 (Dpx4156) and mitochondrial targeting
peptide of AmTpx3 and DmTpx-3 (Dpx5037) are
underlined. Asterisks mark the peroxiredoxin
domain. Conserved cysteins are highlighted.
(B) Glutathione peroxidase homologs with
thioredoxin peroxidase activity. Predicted signal
peptides (AmGtpx2, DmGtpx1C) and
mitochondrial targeting peptides (DmGtpx1D) are
shown underlined. Amino acids of the catalytic
site (Ursini et al., 1995) are highlighted. Amino
acid colour follows the ClustalW code.

692 M. Corona and G. E. Robinson
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Journal compilation © 2006 The Royal Entomological Society, Insect Molecular Biology, 15, 687–701
In mammals, GPX catalyses the reduction of hydroxy-
peroxides utilizing GSH as an electron donor (Ursini et al.,
1995). Early work (Smith & Shrift, 1979) found that insects
lack GPX activity. However, the Drosophila genome con-
tains two GPX homologs. One of these genes encodes for
an enzyme that uses TRX, rather than GSH, as an electron
donor and was therefore referred to as a GPX homolog with
TPX activity, Gtpx-1 (CG12013) (Missirlis et al., 2003). This
gene also is known as DmPHGpx and has been shown to
be highly expressed in testis (Li et al., 2003). The second
Drosophila GPX homolog remains to be biochemically
characterized and is referred to as GPX-like gene (Gpxl,
CG15116).
We found that both Apis mellifera and Anopheles gambiae
also have a pair of GPX homologs (Table 2), although one
of the honey bee homologs (AmGtpx-2, GB18955) lacks
one of the three conserved residues of the catalytic site
(Fig. 2B) (Ursini et al., 1995). Homologs in each species
share more identity with each other than with homologs
in other species, suggesting that they are paralogs that
diverged after speciation. As might be expected, the
dipteran homologs are more closely related to each other
(Fig. 3B, clade A) compared with those of the honey bee,
which form a monophyletic group (clade B) with human
Gpx4. Our phylogenetic analysis also shows that each pair
of homologous genes in mosquito and bee are more
Figure 3. Neighbour joining tree showing the
phylogenetic relationships of Apis mellifera (Am),
Anopheles gambiae (Ag), Drosophila
melanogaster (Dm) and Homo sapiens (Hs)
peroxidases homologs. (A) Thioredoxin family. (B)
Glutathione peroxidase homologs. Values above
the branches represent bootstrap support.

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closely related to each other compared with the pair of GPX
homologs in Drosophila. This could be due to several
causes, including the possibilities that the duplication event
occurred early in Drosophila or there was rapid sequence
divergence of the Gpxl gene.
Humans have six GPX homologs, some with cytosolic,
mitochondrial or extracellular localization. In Drosophila
there are four Gpx-1 isoforms, two of them with putative
cytosolic (CG12013-PA, CG12013-PB), one with mitochon-
drial (CG12013-PD) and one with extracellular localization
(CG12013-PC), as inferred by computational identification
of putative mitochondrial targeting sequences and signal
peptides. This suggests that diversity in subcellular locali-
zation in Drosophila is achieved via alternative splicing
rather than gene duplication, and honey bee may share a
similar gene expression strategy (Table 3).
Like Gpx-1, the second Drosophila GPX homolog (Gpxl)
also has a splicing variant with a putative signal peptide
sequence (CG15116-PB), and a splice variant with a
putative signal peptide sequence occurs for at least one
of the Apis (AmGtpx1, GB18955) and Anopheles (Ag Gtpx-
1, XP_313166) Gpx-like genes (Table 3). Thus, it is likely
that at least one of the two paralogs in each species have
an extracellular function, as it is the case for four of six
human Gpx genes (Lee et al., 2005). At present the func-
tion of the putative extracellular GPX-like proteins in insects
is unknown. Interestingly, an extracellular GPX homolog
with no enzymatic activity was found in the parasitic wasp
Venturia canescens that is included in a virus-like particle
injected with the eggs into the host, and is probably
involved in protection of the egg (Li et al., 2003).
Thioredoxin reductase. TrxR is an essential enzyme that
in insects transfers reducing equivalents from NADPH
to thioredoxin (TrxS2) and GSH disulphide (GSSG). The
resulting products, Trx (SH)2 and GSH, respectively, act as
thiol-based reductants and powerful intracellular anti-
oxidants (Holmgren, 1989). Mammal TrxR carries a distinc-
tive COOH-Terminal extension that includes a tetrapeptide
motif (Gly-Cys-Sec-Gly-OH) containing a selenium in the
form of selenocysteine (s residue) involved in TRX
reduction. This motif distinguishes TrxR proteins from other
structural and functionally closely related flavoprotein
disulphide oxidoreductases such as lipoaminede hydroge-
nases and ferredoxin reductases (Nordberg & Arner, 2001).
The Drosophila ortholog (Trxr-1) has a cysteine instead of
selenocysteine, with equivalent function (Kanzok et al., 2001).
As Anopheles orthologs also have a cysteine residue in
this site (Bauer et al., 2003) the absence of selenium-
containing TrxR might be general characteristic of dipteran
species.
In contrast with human, which has three Trxr genes, and
Drosophila, which has two, Apis and Anopheles have only
a single Trxr gene (Table 2, Fig. 4A). In Drosophila, Trxr-1
encodes three splice variants that include one mitochon-
drial and two cytoplasmic forms (Missirlis et al., 2002). The
functional significance of the second Drosophila Trxr gene
(Trxr2) is unknown, but it encodes a protein with a potential
mitochondrial targeting peptide. Anopheles has a single
Trxr gene, and, as in the Drosophila ortholog has three
splice variants encoding for one mitochondrial and two
cytoplasmic forms (Bauer et al., 2003). Apis also has a
single Trxr gene (Table 2); we identified two putative splice
variants, but none of them appear to encode a mitochon-
drial variant. We were unable to localize an alternative 5′
Tab le 3. Predicted subcellular localization and available expression data for
honey bee antioxidant genes. Putative mitochondrial and extracellular
variants were inferred by computational identification of predicted
mitochondrial targeting and secretory signal peptides
Gene Localization W Q
Sod2 MBTABTA
Sod1 CBTABTA
Sod3 EB
CCS C
Rsod E
Cat CBTABTA
Gtpx1 CBTABTA
Gtpx2 E
Tpx1 CB
Tpx3 MBTABTA
Tpx4 CB
Tpx5 C
Tpx6 C
GstT1 C
GstD1 CBTABTA
GstD2-12 C
GstS1 CB
GstS2 CB
GstS3 CB
GstS4 C
GstZ1 C
GstZ2 C
GstO1 MB
GstO2 Unk
GstO3-4 C
Gstu1 C
GstE1-13 C
Gstmic1 Mic
Gstmic2 Mic
Trxr-1 CBTABTA
Trx-1 M
Trx-2 C
Trx-3 C
Trx1-like1 C
Trx1-like2 C
Trx1-like3 CB
Grx1 C
Grx2 M
Grx-like1 NB
Tr x /Gtx CB
MsrA CBTABTA
MsrB C
Cellular localization: C, cytosolic; M, mitochondria; E, extracellular. N,
nuclear; Mic, microsomal; Unk, unknown (5′ truncated genes). Honey bee
castes: W, workers; Q, queens. Tissues: B, brain; T, thorax; A, abdomen.

694 M. Corona and G. E. Robinson
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Journal compilation © 2006 The Royal Entomological Society, Insect Molecular Biology, 15, 687–701
Figure 4. Alignments for thioredoxin reductases
and thioredoxins from Apis mellifera (Am),
Drosophila melanogaster (Dm) and Anopheles
gambiae (Ag). (A) Thioredoxin reductase family.
The sequences of redox-active centres are
highlighted. (B) Fragment of an alignment of
thioredoxin family proteins. The conserved active
site (CXXC) (Holmgren, 1989) is highlighted.

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exon encoding a mitochondrial targeting peptide. However,
as a mitochondrial TrxR is necessary to provide reduced
TRX for mitochondrial peroxidases (including at least Tpx3)
and given that catalase is not expressed in mitochondria to
reduce H2O2, a mitochondrial variant should be present.
Thioredoxins. TRXs are small, highly conserved oxidore-
ductase proteins required to maintain the redox homeostasis
of the cell. TRX is reduced by TrxR through NADPH
(Holmgren et al., 2005). In mammals seven TRX/TRX-like
proteins have been identified, including tissue-specific
and ubiquitously expressed forms with cytoplasmic,
mitochondrial and Golgi apparatus-associated variants
(Spyrou et al., 1997; Miranda-Vizuete et al., 2001; Jimenez
et al., 2004, 2006). In Drosophila three Trx genes have been
characterized: Trx-1 (deadhead gene, CG4193) (Pellicena-
Palle et al., 1997; Kanzok et al., 2001), Trx-2 (CG31884)
(Bauer et al., 2002) and TrxT (CG3315) (Svensson et al.,
2003). Whereas Trx-1 and TrxT are localized in the nucleus
and are ovary- and testis-specific, respectively, Trx-2 is
localized in the cytoplasm of somatic tissues. This distribution
suggests that Trx-2 plays a major part in whole-body redox
homeostasis. Accordingly, Trx-2 but not Trx-1, functions as a
substrate for TrxR (Bauer et al., 2002).
The Drosophila genome contains four additional genes
(CG8993, CG8517, CG3719, CG13473) that contain both
an overall TRX-like fold domain (Martin, 1995) and the con-
served motif Cys-X1X2-Cys of the active site (Holmgren
et al., 2005). Two of these genes (CG8993 and CG8517)
encode for proteins with probable mitochondrial targeting
peptides. The Anopheles genome contains at least three
putative Trx genes, one with cytoplasmic localization (Txr-1,
EAA14495) (Bauer et al., 2002) and two with probable
mitochondrial localization (Trx-2, EAA04498 and Trx-3,
XP_314234).
As in Anopheles, the Apis genome contains three genes
encoding putative TRX homologs: Am Trx-1 (GB17503)
with predicted mitochondrial localization and an apparent
ortholog of Drosophila CG8993 and Anopheles Trx-2
(clade C, Fig. 5); AmTrx-2 (GB15855), a putative ortholog
Figure 5. Phylogenetic tree of the thioredoxin/
glutharedoxin protein family. ‘M’ and ‘N’ after the
accession number indicate mitochondrial or
nuclear predicted subcellular localization. Values
above the branches represent bootstrap support.

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of Drosophila Trx-2 (60.38% ID) and Anopheles Trx-1 (56.6%
ID) (clade E) and the intronless gene Am Trx3 (GB19972),
putative ortholog of Drosophila CG3719 and Anopheles
Trx3, suggesting that it is not of bacterial origin (clade D).
Thus, each TRX homolog in honey bee and mosquito has
a corresponding putative ortholog in fly. But Drosophila
melanogaster has four additional genes with no apparent
ortholog in honey bee and mosquito. These genes include
CG8517, which seems to have duplicated from CG8993,
Trx-1, TrxT and CG13473, which possibly diverged from
Drosophila Trx-2 after fly and mosquito diverged from the
common dipteran ancestor. Thus, compared with Apis and
Anopheles, the TRX subfamily in Drosophila was clearly
expanded.
As in other organisms, insect genomes also contain a
large group of genes encoding TRX-related proteins con-
taining one or multiple TRX domains, which include protein
disulphure isomerases (Arner & Holmgren, 2000) and other
proteins of unidentified function. One group of these pro-
teins, which have higher identity to bona fide TRX, contain
a single N-terminal TRX domain, but have an additional C-
terminal extension of unknown function. One homolog of
this protein in humans, TRX-like-1 (TXL-1), is a substrate
for the cytosolic selenoprotein TrxR-1 (Jimenez et al., 2006).
We identified three genes encoding this kind of TRX-like
protein with homologs in Apis, Anopheles and Drosophila
genomes (Table 2, Fig. 5 clades A, B and F). Only two of
them (Trx-like-1 and Trx-like 2) have a TRX domain with a
conserved CXXC active site (Fig. 4B).
Glutaredoxin. GRXs are both structurally and functionally
related to TRXs. Insect genomes contain genes encoding
GRX homologs, although at present their products have not
been characterized. In most organisms oxidized GRX pro-
teins are regenerated by reduced GSH, and the resulting
oxidized GSH (GSSG) is reduced by GSH reductase
(Holmgren et al., 2005). However, in insects the reduction
of GSSG is performed by TrxR (Kanzok et al., 2001). In
vertebrates, the products of three Grx genes have been
characterized: GRX1, GRX2 (Johansson et al., 2004) and
the more distantly related, GRX5 (Wingert et al., 2005). In
humans, GRX1 is localized primarily in the cytoplasm,
whereas Grx2 encodes for both nuclear and mitochondrial
variants (Johansson et al., 2004; Holmgren et al., 2005). In
zebrafish GRX5 is primarily localized in mitochondria
(Wingert et al., 2005), although in human the reported
uncharacterized homolog (NP_057501) lacks a potential
mitochondrial targeting peptide.
In Apis, we identified two GRX homologs that we named
Grx1 (GB10598) and Grx2 (GB18700), with predicted
cytoplasmic and mitochondrial localizations, respectively.
Grx1 forms a monophyletic group (Clade I, Fig. 5) with one
human (Grx2, NP_057150), one Anopheles (XP_309539)
and two Drosophila (CG6852, CG7975) homologs. This
suggests that Grx1 was duplicated only in flies. Grx2 has
putative orthologs in human (Grx5, NM_016417), Drosophila
(GCG14407) and Anopheles (XP_312440). Although this
group of proteins shares a clear common evolutionary
origin with other GRX proteins, members of this group
contain a single cysteine residue at the putative active site
(Rodriguez-Manzaneque et al., 1999).
Insect genomes contain two additional groups of genes
encoding GRX-related proteins of unknown function
(Grx-like genes). The first group contains a GRX domain in
the C-terminal of the predicted protein and has a predicted
nuclear localization. In honey bees this group is represented
by Grx-like-1, which forms a monophyletic group with two
Drosophila and one Anopheles homologs (Clade G, Fig. 5).
The other group of Grx-like genes, with orthologs in honey
bee (GB12870), fly (CG6523) and mosquito (EAA07378),
is interesting because it encodes proteins that contain a
TRX domain in the N-terminal region and a GRX domain in
C-terminal region.
Glutathione S-transferases. GSTs are multifunctional pro-
teins essential for xenobiotic metabolism and protection
against peroxidative damage. The GST superfamily can be
divided into several structurally and functionally classes
that show unique variations among different phylogenetic
groups. Plants have exclusive Tau and Phi classes, whereas
mammalian have the mitochondrial Kappa class. In insects
eight different classes have been identified: Epsilon
(GSTe), Delta (GSTd), Theta (GSTt), Zeta (GSTz), Omega
(Gsto) and Sigma (GSTs), the structurally unrelated micro-
somal class (GSTmic) and the denominated unclassified
class (u), so designated for the lack or precise immunological
or biochemical data (Ding et al. 2003). Most studies of GSTs
in insects have been focused on their role in conferring
insecticide resistance. (Claudinos et al. in press) have
recently analysed the GST family in honey bees from this
perspective. GST can be considered a primary antioxidant
enzyme, given the fact that at least the Delta (Tang & Tu,
1994), microsomal (Toba & Aigaki, 2000), and Sigma
classes (Singh et al., 2001) exhibit GPX activity with
cumene hydroperoxide.
The GST superfamily includes 43 members in Dro-
sophila and 37 in Anopheles. (Ding et al., 2003). In contrast,
we only identified 12 genes in the Apis genome (two of them
with partial sequences, Table 1) Compared with dipteran
species, which experienced considerable expansion of
the Delta and Epsilon GTS subfamilies, the bee genome
contains a single ortholog of the Delta class and no
members of the Epsilon class. Another difference includes
double and single duplications in the Omega and Zeta
classes that occurred only in fly. In addition, the Theta class
ortholog that experienced two duplications in fly and one
in mosquito was apparently not duplicated in bee (Table 1
and Fig. 6).

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The Sigma class is the only GST lineage larger in honey
bees in comparison with dipteran species. There are four
members of this group in bee and a single ortholog in fly
and mosquito. This is also the group with the higher con-
servation in intron position (Fig. 7). In addition, two members
of this group (GstS1–2) are the only antioxidant genes so
far found to be physically located close to each other
(Table 1). Both findings suggest that in bees the GST
Sigma class could have been expanded by a recent
duplication event, as seems to be the case for the Delta
and Epsilon classes in Drosophila (Sawicki et al., 2003)
and Anopheles (Ding et al., 2003). Lack of knowledge of
endogenous insect GST substrates makes it difficult to
interpret the functional consequences deriving from the
differential expansion of GST subfamilies between dipteran
species and honey bees. Perhaps they reflect both dif-
ferences in metabolic activity and variation in the quantity of
pro-oxidant molecules ingested with the food. For example,
the Epsilon class (expanded in dipteran but lost in bees)
is involved with DDT resistance (Ranson et al., 2000;
Lumjuan et al., 2005) and is expected to be related to the
detoxification of xenobiotics in general. It is reasonable to
expect a higher quantity of xenobiotics in the food of a
solitary species, with no parental care or sociality, compared
Figure 6. Phylogenetic relationships of GST
family. GSTs belonging to the unclassified (Ding
et al., 2003) class were not included. Values
above the branches represent bootstrap support.
Each entry has a species name (Am, for A.
mellifera; Ag, for A. gambiae; Dm, for D.
melanogaster), GST class, number if assigned,
and accession number.

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with the food received by honey bees, especially during the
larval stages and the first 2 weeks of adulthood, when their
food is restricted to honey, pollen and glandular secretions
provided by other members of the colony (Winston, 1987).
In addition, honey bees feed on angiosperms in a highly
mutualistic relationship; angiosperms have evolved many
traits to attract bees for pollination purposes. Bees are much
less likely to be exposed to naturally occurring feeding
deterrents or toxins.
The expansion of the Sigma class, which occurred only
in bees, seems to be involved with protection against
oxidants produced by aerobic metabolism, rather than
xenobiotics. In flies, these proteins are primarily located in
the indirect flight muscles (Franciosa & Berge, 1995) and
have been reported to play an important part in the detoxi-
fication of lipid peroxidation products (Singh et al., 2001).
Honey bees take foraging trips that may last up to 1 h and
they carry heavy loads of nectar and pollen during this time
(Winston, 1987), so they likely produce a high level of free
radicals (Young & Robinson, 1983). Perhaps this aspect of
their life-style exerted selection on these detoxification genes.
Methionine-R-sulphoxide reductases. Methionine-R-
sulphoxide reductases (Msr) are secondary antioxidant
enzymes involved in protein repair, catalysing the TRX-
dependent reduction of methionine sulphoxide to methio-
nine (Moskovitz et al., 1996). Methionine sulphoxides can
be reduced to methionines by methionine-S-sulphoxide
reductase (MsrA) and methionine-R-sulphoxide reductase
(MsrB), two structurally unrelated proteins (Kumar et al.,
2002). A single gene for each of these enzymes is present
in the analysed insect species (Table 2).
Validation by gene expression
The expression of 16 of the 38 antioxidant genes annotated
in this paper (Sod2, Sod3, Cat, Gtpx1, Tpx1, Tpx3, Tpx4,
GstD1, GstS1, GstS2, GstS3, GstO1, Trxr-1, Trx-like 3, Trx/
Gtx and MsrA) was validated by their identification in a brain
expressed sequence tag library (Whitfield et al., 2002). In
addition, age and tissue specific expression profiles for
eight of these genes (Sod1, Sod2, Cat, Tpx3, Trx-1, GstD1,
Gtpx-1 and MsrA) encoding representative members of the
main antioxidant families were reported for both workers
and queens (Corona et al., 2005) (Table 3).
Bacterial genes
During the annotation of honey bee antioxidant genes, we
also found several genes encoding putative bacterial-like
antioxidant enzymes, including catalase, Mn SOD, TPX,
GST and TRX (Supplementary material, Table 1). In the
case of the catalase gene, a fragment was amplified by
PCR only in samples from the thorax and abdomen of
worker pupae and adult (but not larvae), and was not
detectable in worker heads or any body part of adult
queens (data not shown). These results suggest that this
gene is not integrated into the bee’s genomic DNA and
might therefore come from endosymbiotic bacteria
infecting the digestive tract of the larva. This gene is distinct
from the bona fide Apis catalase gene discussed above.
We also identified a bacterial-like gene encoding a
putative TRX (XP_561198) in the Anopheles genomic
sequence, which is also presumably the product of
bacterial DNA contamination. These examples show that
contamination from endosymbiotic bacterial genomes are
a common phenomenon present in insect genomic
sequence projects, as has been shown for Wolbachia in
Drosophila species (Salzberg et al., 2005).
Conclusions
We presented the results of manual annotation of the main
component of the enzymatic antioxidant system of Apis
mellifera and a comparative analysis with Anopheles gam-
biae and Drosophila melanogaster. This report represents
the first systematic comparison of antioxidant genes
between insect orders and between social vs. solitary
insects. We found that although the basic components of
the antioxidant system are conserved, there are important
differences in the number of paralogs between species.
The main differences include the absence of one of the
five members of CuZn SOD family (Sodesque) in bee;
duplication of TrxR in fly; expansion of the TRX family in fly;
expansion of the Theta, Delta and Omega GST classes in
fly and mosquito, and expansion of the Sigma GST class
in bee. We have also speculated on how the differential
expansion of antioxidant gene families among these
species could reflect both differences in their life-style and
the quantity of pro-oxidant molecules ingested with the food.
Experimental procedures
Annotation of Apis mellifera antioxidant genes
Identification of putative orthologs. We initially identified genes
encoding known components of the enzymatic antioxidant system
Figure 7. Intron position in Apis mellifera GST family members. With the
exception of the third intron of GstS2, intron positions are conserved
between the members of the Gst Sigma class. GstO2 genomic sequences
is truncated toward the deduced C terminal region.

Annotation and phylogeny 699
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in organisms with well-characterized genomes, primarily human
and Drosophila melanogaster. Searches were performed using
both key-word searches or protein queries vs. translated DNA
databases (tblastn) at NCBI (http://www.ncbi.nlm.nih.gov/),
ENSEMBL (http://www.ensembl.org/index.html), and Flybase
(www.flybase.indiana.edu). Then, we searched the Apis mellifera
genome for candidate antioxidant genes using the tblastn
program with the scaffolds_assembly_2 database at BEEBASE
(http://racerx00.tamu.edu/bee_resources.html). This database
included a number of gene prediction sets as well as a combined
prediction data set (Glean3). Identification of putative antioxidant
gene orthologs was completed by multiple protein sequence
alignments followed by phylogenetic analysis (see details in next
section). As in some cases overall protein homology does not
always determine similar function and therefore the identity of an
ortholog, additional bioinformatics support for the identification of
putative orthologs were performed using the Conserved Domain
Architecture Retrieval Tool (CDART) (http://www.ncbi.nlm.nih.gov/
Structure/lexington/lexington.cgi?cmd=rps) and by identifying
reported conserved residues of the catalytic site for each predicted
enzyme.
Ve r ification and correction of gene predictions. Verification of
automatic gene predictions derived from the honey bee genome
project (Honey Bee Genome Sequencing Consortium, 2006) were
performed using protein alignments with existing gene prediction
sets, selected orthologs (including known isoforms) and if available,
EST sequences (http://titan.biotec.uiuc.edu/cgi-bin/ESTWebsite/
estima_blastui?seqSet = bee). When conflicts in gene structure
were detected between existing gene predictions or with respect to
homologs across species, they were resolved using a combination
of protein alignments, splice prediction algorithms (http://
www.fruitfly.org/seq_tools/splice.html) and manual verification
of splicing consensus sequences. A similar approach was followed
to build the structure of genes with no automatic predictions
(Sod3, Tpx6).
Classification and nomenclature of Apis mellifera antioxidant genes.
After the identification of a putative Apis ortholog, the gene was
named following the closest Drosophila ortholog. In the case of
genes with no assigned names in this Drosophila (as in the case
of several members of the GST family) we followed the Anopheles
classification (Ding et al., 2003). In the case of bee genes with no
identified orthologs in other species, we assigned a name using
the family and subfamily abbreviation plus a number (for example,
GstS2–4). When members of a gene family have both conserved
structural domains and conserved residues of the catalytic site, but
are atypical family members (for example, by containing other
structural domains) we used in addition the term ‘like’ as in
Trx-like1 and Trx-like2.
Phylogenetic analysis. Initial protein alignments were performed
using CLUSTALW and then edited using the jalview program (http://
www.ebi.ac.uk/clustalw/). We removed the predicted N-term and
C-term regions when they were extended relative to other
homologs in the alignment. Edited sequences were re-aligned
using the ClustalX 1.81 program (Thompson et al., 1997) with the
following parameters. Pair-wise: gap opening = 35.0, gap
extension = 0.75; Alignment: gap opening = 15, gap extension =
0.3, protein weight matrix, Gonnet series. Phylogenetic trees were
made with the Neighbour Joining method (Saitou & Nei, 1987)
using the PAUP 4.0 b10 program (Swofford, 2002). Trees were
rooted using as outgroup the most divergent sequence in each
group. The statistical significance of branch order was estimated
by the generation of 1000 replications of bootstrap re-sampling of
the original aligned amino acid sequences.
Prediction of subcellular localization
Prediction of subcellular protein localization was performed for
all identified antioxidant genes using four programs: PSORT II
(http://psort.ims.u-tokyo.ac.jp/form2.html), iPSORT (http://
hc.ims.u-tokyo.ac.jp/iPSORT/) (Bannai et al., 2002); TargetP
(http://www.cbs.dtu.dk/services/TargetP/) (Emanuelsson et al.,
2000) and SignalP (http://www.cbs.dtu.dk/services/SignalP/)
(Bendtsen et al., 2004).
Acknowledgements
We thank Hugh Robertson for assistance with phylogenetic
analysis, Hilary Ranson for collaboration on the annotation
of the GST family, and Axel Brockmann, David Nanney,
Rodrigo Velarde, James Whitfield, and anonymous review-
ers for reviewing the manuscript. Supported by R01 AG
022824–04 (GER).
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Supplementary material
The following material is available for this article online:
S1 Deduced protein sequences of bacterial-like anti-
oxidant genes found in the honey bee genomic sequence
databases.
This material is available as part of the online article from
http://www.blackwell-synergy.com