BACKGROUND
the diamondback moth , plutella xylostella  , is a world-wide destructive pest of wild and cultivated crucifers  <cit> .
the larvae feed on cruciferous plants and may cause significant reductions in yield and quality of economically important crops such as canola and cabbage.
historical reliance on insecticides has led to the rapid development of resistance in p. xylostella populations  <cit> , making it difficult to control.
several studies have examined the potential mechanisms underlying the development of insecticide resistance in p. xylostella  <cit> .
one of the proposed mechanisms is metabolic resistance through the multifunctional glutathione s-transferases .
these enzymes can catalyze electrophilic compounds, making them water soluble and readily excreted  <cit> .
gsts are known more generally by insects to detoxify various xenobiotics, including insecticides and plant allelochemicals  <cit> .
the recent work has focused on the potential role of gsts in oxidative stress responses  <cit> .
insect gsts are classified as cytosolic and microsomal.
the number of microsomal gsts is much lower than that of cytosolic gsts, which have been grouped into six subclasses  <cit> .
delta and epsilon subclasses are insect specific, while the other four subclasses, omega, sigma, theta, and zeta, are found in various animal taxa  <cit> .
gsts are involved in the resistance of insects to organophosphate , chlorine, and pyrethroid insecticides  <cit> .
recombinant gst enzymes from p. xylostella and drosophila melanogaster have been shown to play a role in the metabolism of organophosphate insecticides  <cit> .
it has been suggested that, under elevated gst activity conditions, anopheles subpictus can detoxify fenitrooxon activation products, leading to organophosphate resistance  <cit> .
the silkworm zeta gst recombinant protein  has been found to initiate the dechlorination of permethrin and to be abundantly distributed in a permethrin-resistant strain  <cit> .
similarly, an omega gst is highly expressed in a fenitrothion-resistant strain of silkworm and its recombinant protein  shows high affinity with organophosphate insecticides, indicating that it may contribute to insecticide resistance and oxidative stress responses  <cit> .
the antennae-specific gst was found being involved with detoxification of xenobiotics and detection of sex pheromones in manduca sexta  <cit> .
the gsts were found to be one of the major enzyme families in the p. xylostella genome and to be linked to detoxification of plant defense compounds and insecticides  <cit> .
a recent study on the identification and characterization of multiple glutathione s-transferase genes  <cit>  based on the dbm transcriptome database  <cit>  provides a primary base for further investigation of this important gene family.
in the present study, the p. xylostella gsts  were identified and compared with the equivalent information from published insect genomes to better reveal their phylogenetic relationships and intron-exon organization.
we profiled and analyzed expression patterns of the pxgsts using the published transcriptome  <cit>  and reverse transcription-quantitative polymerase chain reaction  in different life stages and tissues from insecticide susceptible or resistant strains.
we then examined the major characteristics of gst subclasses and some particular gst genes in relation to their potential roles in p. xylostella insecticide resistance.
RESULTS
identification of the pxgsts
queries for pxgsts were done against the amino acid sequences from the other insects: drosophila melanogaster , culex quinquefasciatus , aedes aegypti , anopheles gambiae  , tribolium castaneum  , apis mellifera , nasonia vitripennis  , pediculus humanus , acyrthosiphon pisum  , and bombyx mori  .
twenty-two putative cytosolic gst genes with full-length sequence were identified from our p. xylostella  genome  <cit>   and further validated by cloning and sequencing.
using the listed gene ids in table  <dig>  the coding sequences , inferred amino acid sequences  and genomic dna sequences  can be found in the published dbm genomic database   <cit> .
compared to the previous dbm gsts  <cit>  identified from a published p. xylostella transcriptomic database, we identified three additional genes.
the pxgsts represented all six subclasses found in other insects  <cit> , plus two genes that could not be assigned to any one of the known subclasses, labeled as unclassified.
numbers of gsts varied greatly across insect species.
gsts were expanded in the diptera and coleoptera, with a relatively larger number of total genes than that in the species of lepidoptera, hymenoptera and exopterogota .
the number of gsts in p. xylostella was close to that of another lepidopteran species, b. mori.
the two insect-specific gst subclasses  were numerically superior, accounting for > 50% of the entire cytosolic gsts in diptera and coleoptera and ~ 50% in lepidoptera .
this indicates that the gsts in the delta and epsilon subclasses have a greater general trend of duplication than the gsts in the other four subclasses as previously reported by friedman  <cit> .table  <dig>
description of  <dig> identified cytosolic gsts in the
p. xylostella
genome
gene name
orf
protein
gene size
scaffold/orientation
gene id
a
pxgstd1
b
pxgstd3b
pxgstd5
pxgste1
pxgste2
b
pxgste3
pxgste4
pxgste5
pxgsto1
pxgsto2
b
pxgsto3
pxgsto4
pxgsto5
pxgsts1
pxgsts2
pxgstt1
pxgstu1
b
pxgstu2
pxgstz1
pxgstz2
athe gene ids were obtained directly from the published dbm genomic database .
all the coding sequences  of the pxgst genes have been experimentally validated.
bcoding sequences of such genes were incomplete from the dbm-db, and have been experimentally completed by pcr, as explained in the methodology.
px: plutella xylostella.
comparison of gst gene numbers of various insect species*
insect species
delta
epsilon
omega
sigma
theta
zeta
unclassified
total
p. xylostella
b. mori
c. quinquefasciatus
d. melanogaster
a. gambiae
a. aegypti
t. castaneum
n. vitripennis
a. mellifera
a. pisum
p. humanus
*data were from cited literature: friedman   <cit> , oakeshott et al.
<cit> , yu et al.
<cit> , ding et al.
<cit>  and nair et al.
<cit> .
phylogenetic analysis of the pxgsts
the phylogenetic tree illustrated that the seven subclasses were well clustered into their relevant phylogenetic branches .
the unclassified subclass diverged from the delta subclass, suggesting that they may have similar functions.
in all the subclasses, the p. xylostella gsts were all clustered into the lepidoptera-specific branches.
within a specific subclass, the same genes in different species were first clustered into an upper branch within the phylogenetic tree, suggesting that specific gsts in different species might have same or similar functions  <cit> .figure  <dig>
unrooted phylogenetic tree of the cytosolic gsts in nine targeted insect species.
the tree was constructed using neighbor-joining approach with mega  <dig>   <cit>  on the basis of poisson correction amino acid model and pairwise deletion of gaps.
species acronym ; bm: bombyx mori; pp: papilio polytes; dp: danaus plexippus; px: plutella xylostella ; am: apis mellifera; nv: nasonia vitripennis ) was used right before each of the gst genes.
delta and epsilon gst subclasses are unique to insects and have been suggested to be implicated in insecticide resistance  <cit> .
the earlier diverging insects hymenoptera and exopterygota do not have epsilon subclass gsts.
hymenoptera has a few genes present in the delta subclass but none for exopterogota suggesting that these orders may be from an evolutionary older lineage  <cit> .
our tree suggests that delta and epsilon gsts have diverged more recently from the other subclasses.
the range of amino acid identities in the insect-specific gsts of p. xylostella are fairly variable, ranged from  <dig>  ~  <dig> % in delta and  <dig>  ~  <dig> % in epsilon .
except for pxgste <dig>  the remaining epsilon pxgsts were clustered in a monophyletic clade of lepidoptera , suggesting a lineage-specific expansion within the epsilon subclass in lepidopteran order.
characterization of the pxgst introns
a total of  <dig> introns were identified in the pxgsts.
except for one intronless gene , the intron numbers of individual pxgsts ranged from  <dig> to  <dig>  with an average of  <dig> .
these numbers are similar to those of b. mori gsts with an average of  <dig>   <cit>  and larger than those of dipteran  and coleopteran  gsts with averages of  <dig>  and  <dig> , respectively  <cit> .
the number of gsts introns has been shown to vary across insect species.
it is thought to be associated with the ability to respond to xenobiotics and endogenous compounds  <cit> .figure  <dig>
location of introns of the pxgst genes.
phase  <dig>   <dig> and  <dig> introns are shown by inverted filled triangle, arrow and inverted blank triangle, respectively.
phase  <dig> for a splice site lying between two codons, phase  <dig> for a splice site lying one base inside a codon in the 3’ direction, and phase  <dig> for a splice site lying two bases inside the codon in the 3’ direction.
in the pxgsts, the splice sites of introns were classified into three phases:  <dig> with  <dig> introns,  <dig> with  <dig> introns, and  <dig> with  <dig> introns, according to their positions in the codons.
the phase- <dig> introns were present only in the omega, zeta and sigma subclasses.
most phase- <dig> introns were found in insect-specific subclasses  as well as in zeta subclass.
most of the pxgst introns spliced in a given site tended to be from the same phase, suggesting that they might be relatively conserved .
intron sites are similar across different pxgst subclasses.
there are three highly conserved sites of introns within the delta and epsilon gst subclasses, except for the pxgstd <dig> and pxgste <dig>  these were between the 47th and 51st, the 114th and 118th and the 180th and 186th amino acids.
most of the pxgsts tended to have a nearby conserved site of the introns located between the 111th and 125th amino acids belonging to phase  <dig>  both of the intron sites and phases were strongly conserved within sigma and zeta subclasses , implying that these genes might have similar functions.
there appeared to be a correlation between the intron conservation and the phylogenetic cluster within a given pxgst subclass, indicating that gene structure evolution might be involved in the phylogenetic development of a specific subclass.
despite the conserved nature of intron sites and phases, the lengths were highly variable in the pxgsts ranging from  <dig> to  <dig>  bp with a larger proportion ranging from  <dig> to 399 bp  and an average of 918 bp.
the shortest intron was pxgstu <dig> , while the longest were pxgsto <dig>  and pxgstz <dig> .
a previous study has shown that long introns were considered to involve more functional elements than short introns and could effectively regulate gene expressions, possibly via the formation of pre-mrna secondary structures  <cit> .
however, the function of the longest introns in pxgsto <dig> and pxgstz <dig> needs to be further investigated.
gsh and substrate binding sites in the pxgsts
most of the insect gsts are composed of a conserved thioredoxin domain containing the gsh binding site  and a more variable α-helical domain containing the substrate binding site   <cit> , and can transfer gsh to a substrate by stabilization of the gsh thiolate  <cit> .
both g-sites and h-sites among the pxgsts were analyzed with the ncbi cd-search program, and the results showed that the g-sites appeared fairly conserved while the h-sites were variable among different subclasses .
the conserved g-sites indicate their important enzyme functions while the variable h-sites are related to their evolutionary divergence  <cit> .
no g-sites were found for all the genes in omega gsts as well as some genes in other subclasses in p. xylostella .
such gsts  may act as intracellular ligand transporters as documented in nilaparvata lugens and anopheles cracens  <cit> .figure  <dig>
gsh and substrate binding sites of glutathione
s
-transferase genes.
the short vertical lines represent functionally conserved residues of gst genes among insect species.
red vertical lines represent the gsh binding sites of gsts  and blue vertical lines represent the substrate binding sites gsts .
expression profiling of the pxgsts
stage-specific expression profiling
using our unpublished p. xylostella rna-seq data, expression patterns of the pxgsts at different developmental stages of the susceptible strain were characterized .
the results showed that all  <dig> pxgsts could be expressed at different developmental stages, and exhibited gene-differential and stage-specific patterns.
sixteen genes were found to be consistently expressed throughout different stages, two of which  tended to be expressed with high levels, four  with moderate levels  and ten with low levels .
those highly and moderately expressed genes may function as housekeeping genes with potential roles of protecting cells against endogenous oxidative stress or xenobiotics  <cit> .
four insect-specific pxgsts  were predominantly and highly expressed at in larval , indicating that these genes might play important roles in metabolizing plant secondary metabolites  <cit> .figure  <dig>
expression profiling of the pxgsts at different developmental stages based on rpkm value.
1st : first instar larva; 2nd : second instar larva; 3rd : third instar larva; 4th : fourth instar larva; f: female; m: male; gray denoted missing values.
the data were obtained from our unpublished rna-seq data.
the rpkm values are presented in additional file 7: table s <dig>  the data have been uploaded to the p. xylostella genomic database .
expression profiling with qrt-pcr confirmed that the  <dig> pxgsts genes could express at different developmental stages, but exhibited stage-specific patterns.
six genes were predominantly expressed at the larval stage, exhibiting the same patterns based on rpkm value  and qrt-pcr , which suggests that they might be associated with detoxification of plant defense compounds and insecticides  <cit> .
the insect-specific delta and epsilon gsts showed high expression in p. xylostella , while most of these gsts had little or no expression in the main detoxification organ  of domesticated b. mori with little exposure to insecticides for thousands years  <cit> , suggesting that these genes are associated with the evolution of insecticide resistance as proposed in previous reports  <cit> .
pxgstd <dig>  pxgste <dig>  pxgste <dig> and pxgsto <dig> were highly expressed in the p. xylostella adults , suggesting that these genes may be involved in odorant processing and/or xenobiotic metabolism  <cit> .
most of the pxgsts exhibited low gene expression at the egg stage .
such diversified expression patterns of the pxgsts imply that gsts may have multiple functions in p. xylostella, as documented in other insects  <cit> .figure  <dig>
expression profiling of selected preferentially expressed pxgsts at larval  and adult  stages based on qrt-pcr.
1st : first instar larva; 2nd : second instar larva; 3rd : third instar larva; 4th : fourth instar larva.
strain- and tissue-specific expression profiling
based on the dbm transcriptome, all of the pxgsts were either up- or down-regulated in the insecticide resistant strains when compared to the susceptible strain  .
seven pxgsts were up-regulated in both of the chlorpyrifos- and fipronil-resistant strains  .
they are mostly insect specific gsts  with the potential function of detoxification.
however, seven pxgsts were down-regulated in crs and frs .
most of the rest were up-regulated in frs, but down- regulated in crs, possibly reflecting different mechanisms of detoxification between the two strains.figure  <dig>
differential expressions of the pxgsts in different resistant strains based on rpkm value.
crs: chlorpyrifos resistant strain; frs: fipronil resistant strain.
differential expressions are illustrated by different colors compared to the expression in susceptible strain , with the red representing up-regulated, the green down-regulated and the black no difference with ss.
the gray denotes missing values.
the data were obtained from our published transcriptome data.
the rpkm values are presented in additional file 8: table s <dig>  the data have been uploaded to the p. xylostella genomic database .
the qrt-pcr-based analysis showed that four of the pxgsts  showed significantly greater expression in both crs and frs.
pxgstd <dig>  pxgsts <dig>  pxgsts <dig> and pxgstz <dig> in frs and pxgsto <dig> and pxgsto <dig> in crs also had greater gene expressions than in ss.
interestingly, pxgste <dig> and pxgstt <dig> exhibited higher expressions in frs but lower expressions in crs when compared to the ss .
expression of the other  <dig> pxgsts were not significantly different among strains.
the qrt-pcr-based analysis could not confirm the transcriptome-based expression profiling patterns of all the pxgsts, which might result from different sampling times when the resistant dbms  were collected for transcriptome sequencing in december  <dig>  and collected three years later for qrt-pcr.
the diversified patterns of strain-specific expressions suggest that the pxgsts might involve a functionally complex system in response to detoxifying different classes of insecticides  <cit> .figure  <dig>
expression patterns of pxgsts in three strains as determined by qrt-pcr.
ss, insecticide susceptible strain; frs, fipronil resistant strain; crs, chlorpyrifos resistant strain; error bars indicate standard errors of the mean.
statistically significant differences were labeled with different letters as evaluated with one-way anova .
twelve pxgsts with significantly high expression in resistant strains were further analyzed using qrt-pcr in different tissues of fipronil-resistant strain.
the results showed that  <dig> genes  were significantly more highly expressed in malpighian tubules,  <dig>  both in malpighian tubules and midguts and  <dig>  in epidermis, except for pxgsto <dig> with no significant different expression in the various tissues .
these results further validate the association of the pxgsts with insecticide detoxification because these tissues were documented to play important roles in digestion and metabolism of xenobiotics in insects  <cit> .figure  <dig>
expression patterns of pxgsts in multiple tissues as determined by qrt-pcr.
frs, fipronil resistant strain; ep, epidermis; fb, fat body; h, head; mg, midgut; mt, malpighian tubules; error bars indicate standard errors of the mean.
statistically significant differences were labeled with different letters as evaluated with one-way anova .
CONCLUSIONS
to date, this is the most comprehensive study on genome-wide identification, characterization and expression profile of the gsts in p. xylostella.
twenty-two gsts were found in p. xylostella, which is similar in number to another lepidopteran species, b. mori.
variable features and different expression patterns of the genes reveal that the p. xylostella gsts are evolutionary and functionally diversified, and may be involved in the evolution of adaptive capacity in response to environmental variation.
because gst enzymes are considered to be important in insecticide resistance, many of these newly identified genes are potential candidates for inhibiting the pathway of insecticide resistance and targeting lepidopteran-selective insecticides.
thus, further functional research on the pxgsts is essential to identify the key genes and their roles in xenobiotic detoxification of insects, and understand the mechanisms underlying the insecticide resistance.

 the diamondback moth, plutella xylostella, is a world-wide destructive pest of wild and cultivated crucifers. the larvae feed on cruciferous plants and may cause significant reductions in yield and quality of economically important crops such as canola and cabbage. historical reliance on insecticides has led to the rapid development of resistance in p. xylostella populations, making it difficult to control. several studies have examined the potential mechanisms underlying the development of insecticide resistance in p. xylostella. one of the proposed mechanisms is metabolic resistance through the multifunctional glutathione s-transferases. these enzymes can catalyze electrophilic compounds, making them water soluble and readily excreted. gsts are known more generally by insects to detoxify various xenobiotics, including insecticides and plant allelochemicals. the recent work has focused on the potential role of gsts in oxidative stress responses. insect gsts are classified as cytosolic and microsomal. the number of microsomal gsts is much lower than that of cytosolic gsts, which have been grouped into six subclasses. delta and epsilon subclasses are insect specific, while the other four subclasses, omega, sigma, theta, and zeta, are found in various animal taxa. gsts are involved in the resistance of insects to organophosphate, chlorine, and pyrethroid insecticides. recombinant gst enzymes from p. xylostella and drosophila melanogaster have been shown to play a role in the metabolism of organophosphate insecticides. it has been suggested that, under elevated gst activity conditions, anopheles subpictus can detoxify fenitrooxon activation products, leading to organophosphate resistance. the silkworm zeta gst recombinant protein has been found to initiate the dechlorination of permethrin and to be abundantly distributed in a permethrin-resistant strain. similarly, an omega gst is highly expressed in a fenitrothion-resistant strain of silkworm and its recombinant protein shows high affinity with organophosphate insecticides, indicating that it may contribute to insecticide resistance and oxidative stress responses. the antennae-specific gst was found being involved with detoxification of xenobiotics and detection of sex pheromones in manduca sexta. the gsts were found to be one of the major enzyme families in the p. xylostella genome and to be linked to detoxification of plant defense compounds and insecticides. a recent study on the identification and characterization of multiple glutathione s-transferase genes based on the dbm transcriptome database provides a primary base for further investigation of this important gene family. in the present study, the p. xylostella gsts were identified and compared with the equivalent information from published insect genomes to better reveal their phylogenetic relationships and intron-exon organization. we profiled and analyzed expression patterns of the pxgsts using the published transcriptome and reverse transcription-quantitative polymerase chain reaction in different life stages and tissues from insecticide susceptible or resistant strains. we then examined the major characteristics of gst subclasses and some particular gst genes in relation to their potential roles in p. xylostella insecticide resistance.  identification of the pxgsts queries for pxgsts were done against the amino acid sequences from the other insects: drosophila melanogaster, culex quinquefasciatus, aedes aegypti, anopheles gambiae, tribolium castaneum, apis mellifera, nasonia vitripennis, pediculus humanus, acyrthosiphon pisum, and bombyx mori. twenty-two putative cytosolic gst genes with full-length sequence were identified from our p. xylostella genome and further validated by cloning and sequencing. using the listed gene ids in table  the coding sequences, inferred amino acid sequences and genomic dna sequences can be found in the published dbm genomic database. compared to the previous dbm gsts identified from a published p. xylostella transcriptomic database, we identified three additional genes. the pxgsts represented all six subclasses found in other insects, plus two genes that could not be assigned to any one of the known subclasses, labeled as unclassified. numbers of gsts varied greatly across insect species. gsts were expanded in the diptera and coleoptera, with a relatively larger number of total genes than that in the species of lepidoptera, hymenoptera and exopterogota. the number of gsts in p. xylostella was close to that of another lepidopteran species, b. mori. the two insect-specific gst subclasses were numerically superior, accounting for > 50% of the entire cytosolic gsts in diptera and coleoptera and ~ 50% in lepidoptera. this indicates that the gsts in the delta and epsilon subclasses have a greater general trend of duplication than the gsts in the other four subclasses as previously reported by friedman.table description of identified cytosolic gsts in the p. xylostella genome gene name orf protein gene size scaffold/orientation gene id a pxgstd1 b pxgstd3b pxgstd5 pxgste1 pxgste2 b pxgste3 pxgste4 pxgste5 pxgsto1 pxgsto2 b pxgsto3 pxgsto4 pxgsto5 pxgsts1 pxgsts2 pxgstt1 pxgstu1 b pxgstu2 pxgstz1 pxgstz2 athe gene ids were obtained directly from the published dbm genomic database. all the coding sequences of the pxgst genes have been experimentally validated. bcoding sequences of such genes were incomplete from the dbm-db, and have been experimentally completed by pcr, as explained in the methodology. px: plutella xylostella. comparison of gst gene numbers of various insect species* insect species delta epsilon omega sigma theta zeta unclassified total p. xylostella b. mori c. quinquefasciatus d. melanogaster a. gambiae a. aegypti t. castaneum n. vitripennis a. mellifera a. pisum p. humanus *data were from cited literature: friedman, oakeshott et al., yu et al., ding et al. and nair et al.. phylogenetic analysis of the pxgsts the phylogenetic tree illustrated that the seven subclasses were well clustered into their relevant phylogenetic branches. the unclassified subclass diverged from the delta subclass, suggesting that they may have similar functions. in all the subclasses, the p. xylostella gsts were all clustered into the lepidoptera-specific branches. within a specific subclass, the same genes in different species were first clustered into an upper branch within the phylogenetic tree, suggesting that specific gsts in different species might have same or similar functions.figure unrooted phylogenetic tree of the cytosolic gsts in nine targeted insect species. the tree was constructed using neighbor-joining approach with mega on the basis of poisson correction amino acid model and pairwise deletion of gaps. species acronym; bm: bombyx mori; pp: papilio polytes; dp: danaus plexippus; px: plutella xylostella; am: apis mellifera; nv: nasonia vitripennis ) was used right before each of the gst genes. delta and epsilon gst subclasses are unique to insects and have been suggested to be implicated in insecticide resistance. the earlier diverging insects hymenoptera and exopterygota do not have epsilon subclass gsts. hymenoptera has a few genes present in the delta subclass but none for exopterogota suggesting that these orders may be from an evolutionary older lineage. our tree suggests that delta and epsilon gsts have diverged more recently from the other subclasses. the range of amino acid identities in the insect-specific gsts of p. xylostella are fairly variable, ranged from  ~  % in delta and  ~  % in epsilon. except for pxgste the remaining epsilon pxgsts were clustered in a monophyletic clade of lepidoptera, suggesting a lineage-specific expansion within the epsilon subclass in lepidopteran order. characterization of the pxgst introns a total of introns were identified in the pxgsts. except for one intronless gene, the intron numbers of individual pxgsts ranged from to with an average of. these numbers are similar to those of b. mori gsts with an average of and larger than those of dipteran and coleopteran gsts with averages of and, respectively. the number of gsts introns has been shown to vary across insect species. it is thought to be associated with the ability to respond to xenobiotics and endogenous compounds.figure location of introns of the pxgst genes. phase and introns are shown by inverted filled triangle, arrow and inverted blank triangle, respectively. phase for a splice site lying between two codons, phase for a splice site lying one base inside a codon in the 3’ direction, and phase for a splice site lying two bases inside the codon in the 3’ direction. in the pxgsts, the splice sites of introns were classified into three phases: with introns, with introns, and with introns, according to their positions in the codons. the phase- introns were present only in the omega, zeta and sigma subclasses. most phase- introns were found in insect-specific subclasses as well as in zeta subclass. most of the pxgst introns spliced in a given site tended to be from the same phase, suggesting that they might be relatively conserved. intron sites are similar across different pxgst subclasses. there are three highly conserved sites of introns within the delta and epsilon gst subclasses, except for the pxgstd and pxgste these were between the 47th and 51st, the 114th and 118th and the 180th and 186th amino acids. most of the pxgsts tended to have a nearby conserved site of the introns located between the 111th and 125th amino acids belonging to phase both of the intron sites and phases were strongly conserved within sigma and zeta subclasses, implying that these genes might have similar functions. there appeared to be a correlation between the intron conservation and the phylogenetic cluster within a given pxgst subclass, indicating that gene structure evolution might be involved in the phylogenetic development of a specific subclass. despite the conserved nature of intron sites and phases, the lengths were highly variable in the pxgsts ranging from to  bp with a larger proportion ranging from to 399 bp and an average of 918 bp. the shortest intron was pxgstu, while the longest were pxgsto and pxgstz. a previous study has shown that long introns were considered to involve more functional elements than short introns and could effectively regulate gene expressions, possibly via the formation of pre-mrna secondary structures. however, the function of the longest introns in pxgsto and pxgstz needs to be further investigated. gsh and substrate binding sites in the pxgsts most of the insect gsts are composed of a conserved thioredoxin domain containing the gsh binding site and a more variable α-helical domain containing the substrate binding site, and can transfer gsh to a substrate by stabilization of the gsh thiolate. both g-sites and h-sites among the pxgsts were analyzed with the ncbi cd-search program, and the results showed that the g-sites appeared fairly conserved while the h-sites were variable among different subclasses. the conserved g-sites indicate their important enzyme functions while the variable h-sites are related to their evolutionary divergence. no g-sites were found for all the genes in omega gsts as well as some genes in other subclasses in p. xylostella. such gsts may act as intracellular ligand transporters as documented in nilaparvata lugens and anopheles cracens.figure gsh and substrate binding sites of glutathione s -transferase genes. the short vertical lines represent functionally conserved residues of gst genes among insect species. red vertical lines represent the gsh binding sites of gsts and blue vertical lines represent the substrate binding sites gsts. expression profiling of the pxgsts stage-specific expression profiling using our unpublished p. xylostella rna-seq data, expression patterns of the pxgsts at different developmental stages of the susceptible strain were characterized. the results showed that all pxgsts could be expressed at different developmental stages, and exhibited gene-differential and stage-specific patterns. sixteen genes were found to be consistently expressed throughout different stages, two of which tended to be expressed with high levels, four with moderate levels and ten with low levels. those highly and moderately expressed genes may function as housekeeping genes with potential roles of protecting cells against endogenous oxidative stress or xenobiotics. four insect-specific pxgsts were predominantly and highly expressed at in larval, indicating that these genes might play important roles in metabolizing plant secondary metabolites.figure expression profiling of the pxgsts at different developmental stages based on rpkm value. 1st: first instar larva; 2nd: second instar larva; 3rd: third instar larva; 4th: fourth instar larva; f: female; m: male; gray denoted missing values. the data were obtained from our unpublished rna-seq data. the rpkm values are presented in additional file 7: table s the data have been uploaded to the p. xylostella genomic database. expression profiling with qrt-pcr confirmed that the pxgsts genes could express at different developmental stages, but exhibited stage-specific patterns. six genes were predominantly expressed at the larval stage, exhibiting the same patterns based on rpkm value and qrt-pcr, which suggests that they might be associated with detoxification of plant defense compounds and insecticides. the insect-specific delta and epsilon gsts showed high expression in p. xylostella, while most of these gsts had little or no expression in the main detoxification organ of domesticated b. mori with little exposure to insecticides for thousands years, suggesting that these genes are associated with the evolution of insecticide resistance as proposed in previous reports. pxgstd pxgste pxgste and pxgsto were highly expressed in the p. xylostella adults, suggesting that these genes may be involved in odorant processing and/or xenobiotic metabolism. most of the pxgsts exhibited low gene expression at the egg stage. such diversified expression patterns of the pxgsts imply that gsts may have multiple functions in p. xylostella, as documented in other insects.figure expression profiling of selected preferentially expressed pxgsts at larval and adult stages based on qrt-pcr. 1st: first instar larva; 2nd: second instar larva; 3rd: third instar larva; 4th: fourth instar larva. strain- and tissue-specific expression profiling based on the dbm transcriptome, all of the pxgsts were either up- or down-regulated in the insecticide resistant strains when compared to the susceptible strain. seven pxgsts were up-regulated in both of the chlorpyrifos- and fipronil-resistant strains. they are mostly insect specific gsts with the potential function of detoxification. however, seven pxgsts were down-regulated in crs and frs. most of the rest were up-regulated in frs, but down- regulated in crs, possibly reflecting different mechanisms of detoxification between the two strains.figure differential expressions of the pxgsts in different resistant strains based on rpkm value. crs: chlorpyrifos resistant strain; frs: fipronil resistant strain. differential expressions are illustrated by different colors compared to the expression in susceptible strain, with the red representing up-regulated, the green down-regulated and the black no difference with ss. the gray denotes missing values. the data were obtained from our published transcriptome data. the rpkm values are presented in additional file 8: table s the data have been uploaded to the p. xylostella genomic database. the qrt-pcr-based analysis showed that four of the pxgsts showed significantly greater expression in both crs and frs. pxgstd pxgsts pxgsts and pxgstz in frs and pxgsto and pxgsto in crs also had greater gene expressions than in ss. interestingly, pxgste and pxgstt exhibited higher expressions in frs but lower expressions in crs when compared to the ss. expression of the other pxgsts were not significantly different among strains. the qrt-pcr-based analysis could not confirm the transcriptome-based expression profiling patterns of all the pxgsts, which might result from different sampling times when the resistant dbms were collected for transcriptome sequencing in december and collected three years later for qrt-pcr. the diversified patterns of strain-specific expressions suggest that the pxgsts might involve a functionally complex system in response to detoxifying different classes of insecticides.figure expression patterns of pxgsts in three strains as determined by qrt-pcr. ss, insecticide susceptible strain; frs, fipronil resistant strain; crs, chlorpyrifos resistant strain; error bars indicate standard errors of the mean. statistically significant differences were labeled with different letters as evaluated with one-way anova. twelve pxgsts with significantly high expression in resistant strains were further analyzed using qrt-pcr in different tissues of fipronil-resistant strain. the results showed that genes were significantly more highly expressed in malpighian tubules, both in malpighian tubules and midguts and in epidermis, except for pxgsto with no significant different expression in the various tissues. these results further validate the association of the pxgsts with insecticide detoxification because these tissues were documented to play important roles in digestion and metabolism of xenobiotics in insects.figure expression patterns of pxgsts in multiple tissues as determined by qrt-pcr. frs, fipronil resistant strain; ep, epidermis; fb, fat body; h, head; mg, midgut; mt, malpighian tubules; error bars indicate standard errors of the mean. statistically significant differences were labeled with different letters as evaluated with one-way anova.  to date, this is the most comprehensive study on genome-wide identification, characterization and expression profile of the gsts in p. xylostella. twenty-two gsts were found in p. xylostella, which is similar in number to another lepidopteran species, b. mori. variable features and different expression patterns of the genes reveal that the p. xylostella gsts are evolutionary and functionally diversified, and may be involved in the evolution of adaptive capacity in response to environmental variation. because gst enzymes are considered to be important in insecticide resistance, many of these newly identified genes are potential candidates for inhibiting the pathway of insecticide resistance and targeting lepidopteran-selective insecticides. thus, further functional research on the pxgsts is essential to identify the key genes and their roles in xenobiotic detoxification of insects, and understand the mechanisms underlying the insecticide resistance. 
the resulting phylogenetic tree showed that the p. xylostella gsts were all clustered into lepidoptera-specific branches. the diversified features and expression patterns of the gsts are inferred to be associated with the capacity of this species to develop resistance to a wide range of pesticides and biological toxins. glutathione s-transferases are multifunctional detoxification enzymes that play important roles in insects. to date, this is the most comprehensive study on genome-wide identification, characterization and expression profiling of the gst family in p. xylostella. this study presents a genome-wide investigation of the diamondback moth, plutella xylostella, a species in which the gsts are of special importance because this pest is highly resistant to many insecticides. a total of putative cytosolic gsts were identified from a published p. xylostella genome and grouped into subclasses. intron sites and phases as well as gsh binding sites were strongly conserved within each of the subclasses in the gsts of p. xylostella. most of the highly expressed genes in insecticide resistant strains were also predominantly expressed in the malpighian tubules, midgut or epidermis. the completion of several insect genome projects has enabled the identification and characterization of gst genes over recent years. transcriptome-, rna-seq- and qrt-pcr-based analyses showed that the gst genes were developmental stage- and strain-specifically expressed. our findings provide a base for functional research on specific gst genes, a better understanding of the evolution of insecticide resistance, and strategies for more sustainable management of the pest. delta, epsilon and omega gsts were numerically superior with genes for each of the subclasses.
 glutathione s-transferases are multifunctional detoxification enzymes that play important roles in insects. the completion of several insect genome projects has enabled the identification and characterization of gst genes over recent years. this study presents a genome-wide investigation of the diamondback moth, plutella xylostella, a species in which the gsts are of special importance because this pest is highly resistant to many insecticides.  a total of putative cytosolic gsts were identified from a published p. xylostella genome and grouped into subclasses. delta, epsilon and omega gsts were numerically superior with genes for each of the subclasses. the resulting phylogenetic tree showed that the p. xylostella gsts were all clustered into lepidoptera-specific branches. intron sites and phases as well as gsh binding sites were strongly conserved within each of the subclasses in the gsts of p. xylostella. transcriptome-, rna-seq- and qrt-pcr-based analyses showed that the gst genes were developmental stage- and strain-specifically expressed. most of the highly expressed genes in insecticide resistant strains were also predominantly expressed in the malpighian tubules, midgut or epidermis.  to date, this is the most comprehensive study on genome-wide identification, characterization and expression profiling of the gst family in p. xylostella. the diversified features and expression patterns of the gsts are inferred to be associated with the capacity of this species to develop resistance to a wide range of pesticides and biological toxins. our findings provide a base for functional research on specific gst genes, a better understanding of the evolution of insecticide resistance, and strategies for more sustainable management of the pest. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords transcriptome analysisqrt-pcrphylogenetic analysisinsect pestlepidopteraissue-copyright-statement© the author 2015 