BACKGROUND
the disaccharide trehalose consists of two α-glycosidically linked glucose units.
it is a non-reducing sugar found in many organisms as diverse as bacteria, yeast, fungi, nematodes, plants, insects and some other invertebrates, but is absent in mammals  <cit> .
trehalose may serve as a carbohydrate store and as an agent for protecting proteins and cellular membranes from a variety of environmental stress conditions, including desiccation, dehydration, heat, freezing and oxidation  <cit> .
in plants, trehalose not only has an impact on some metabolic processes and affects plant development as a signaling molecule, but also serves as an anti-stress substance to protect plants from drought, high salt and low temperature  <cit> .
in insects, unlike in mammals, trehalose is the main blood sugar and is present in the hemolymph of larvae, pupae and adults  <cit> .
it is the main reserve sugar in the hemolymph of flying insects and is also indispensable for thermotolerance in larvae.
trehalose is synthesized mainly in the insect fat body and is rapidly released into the hemolymph and other tissues.
to utilize blood trehalose, insect tissues contain trehalases  that catalyze the hydrolysis of one mole of trehalose to two moles of glucose.
thus, for uptake or utilization of trehalose in the blood, trehalases are essential enzymes in insects and are thought to be located on the cell membrane or within cells  <cit> .
the first insect trehalase, a soluble trehalase, was reported in  <dig>  <cit> .
although insects are believed to have two types, soluble trehalase  and membrane-bound trehalase   <cit> , the tre- <dig> gene was not reported until  <dig>  <cit> .
in bombyx mori, the tre- <dig> gene is expressed in the midgut; immunoblotting and immunohistochemical analyses showed that tre- <dig> is present mainly in goblet cell cavities and tre- <dig> penetrates the cell membrane and is predominantly evident on visceral muscle surrounding the midgut  <cit> .
although two trehalase genes have been cloned from b. mori,apis mellifera  <cit>  and spodoptera exigua, the different functions of these two trehalases in chitin biosynthesis in insects are not clear.
in addition, very little is known about the structure, tissue distribution and expression pattern of tre- <dig>
here, we report our findings regarding the gene  coding for a putative membrane-bound trehalase isolated from the fat body of s. exigua .
we observed that it is expressed not only in the midgut, but also in the fat body and malpighian tubules.
furthermore, its expression patterns differed between the midgut and fat body.
RESULTS
cloning of full-length setre- <dig> cdna
based on the conserved amino acid and nucleotide sequences of trehalases from b. mori , tenebrio molitor  and pimpla hypochondriaca , we designed three degenerate primers, setre-f <dig>  setre-f <dig> and setre-r, for pcr reactions.
a fragment of  <dig> bp was first obtained from pupal fat body cdna by a second pcr using setre-f <dig> and setre-r. the deduced amino acid sequence exhibited high similarity to insect trehalase sequences.
we then performed 5' and 3' rapid amplification of cdna ends  using several specific primers based on the sequence of the fragment and universal primers .
pcr products of  <dig> and  <dig> bp were amplified by 5' and 3' race, respectively.
assembly of the overlapping fragments revealed a full-length cdna of  <dig> bp.
the trehalase contained an open reading frame of  <dig> bp, encoding a protein of  <dig> amino acids  with a predicted molecular weight of approximately  <dig> kda and pi of  <dig> .
the deduced amino acid sequence of trehalase from s. exigua was aligned with the corresponding sequences of other insect trehalases .
setre- <dig> is most similar to lepidopteran trehalase- <dig> from b. mori  and ostrinia furnacalis .
it is also similar to setre- <dig> , sftre- <dig> , bmtre- <dig> , oftre- <dig> , phtre- <dig> , amtre- <dig> , amtre- <dig> , tctre- <dig> , nvtre- <dig> , agtre- <dig> , aatre- <dig> , dmtre- <dig> , dstre- <dig> , tmtre- <dig> , tctre- <dig> , rntre , mmtre  and hstre .
the insect tre- <dig> gene is highly conserved, particularly in the middle of the putative catalytic domain .
setre- <dig> cdna and protein sequence analysis
the deduced amino acid sequence of setre- <dig> contains two trehalase signature motifs, pggrfrefyywdsy  and qwdypnawpp   and five other conserved motifs: dsktfvdmk , ipnggriyy , rsqppll , gprpesykedv  and elkaaaesgwdfssrwfi .
residues 1– <dig> are a signal peptide leader and residues 530– <dig> correspond to a glycine-rich region .
residues 585– <dig> were found to comprise a putative transmembrane domain.
n-terminal to this domain, residues 573– <dig>  are identical to amino acids 570– <dig>  in bmtre- <dig>  however, this is not identified as an omega site by the big-pi predictor used to predict glycosylphosphatidyl inositol modification sites  <cit> .
five potential n-glycosylation sites  are present in bmtre- <dig>  but six potential n-glycosylation sites were found in setre- <dig>  five sites  homologous to those in bmtre- <dig> and an additional site at amino acid  <dig>
structure of setre-2
we amplified the setre- <dig> genomic dna sequence, which is approximately  <dig> kb long.
the exon/intron composition of the gene was determined by comparing the genomic sequence with the setre- <dig> cdna sequence.
the setre- <dig> gene consists of  <dig> exons separated by  <dig> introns of different lengths and exon-intron splice junctions following the gt-ag rule .
the first intron is the longest, at  <dig>  kb.
exons 1– <dig> correspond to nucleotides 1– <dig>  5855– <dig>  6306– <dig>  7873– <dig>  8668– <dig>  9592– <dig>   <dig> – <dig> ,  <dig> – <dig> ,  <dig> – <dig> ,  <dig> – <dig> ,  <dig> – <dig> ,  <dig> – <dig> , and  <dig> – <dig>  in the genomic sequence, respectively.
compared to the seven exons and six introns of the a. mellifera tre- <dig> gene  <cit>  and the nine exons and eight introns of amtre- <dig> , setre- <dig> has more exons and introns.
southern blot analysis
gene copy number can be determined by southern blot analysis.
genomic dna was obtained from s. exigua pupae and approximately  <dig> μg of dna was digested with hindiii, sali and xhoi, electrophoresed and transferred to a nylon membrane, and then probed with a setre- <dig> cdna fragment  generated using setrefp and setrerp primers.
the probe was designed to be specific for a highly conserved region so that it would hybridize to both setre- <dig> and setre- <dig>  one strong and one faint band were detected when genomic dna was digested with sali and xhoi  whereas three strong and two faint bands were observed for hindiii treatment.
the latter is attributed to the presence of two hindiii sites in the setre- <dig> genomic sequence that correspond to the probe sequence.
a hindiii site may also be present in the setre- <dig> genomic sequence corresponding to the setre- <dig> probe.
setre- <dig> tissue distribution
tissue-specific expression setre- <dig> was determined by northern blotting.
setre- <dig> cdna was cloned from s. exigua fat body, suggesting that setre- <dig> mrna is expressed in this tissue.
this was confirmed by northern blot analysis .
in addition, setre- <dig> transcripts were also detected in the midgut, but not in the brain or cuticle .
setre- <dig> mrna may also be expressed in malpighian tubules, since a faint band was observed.
to determine the expression of setre- <dig> transcripts in malpighian tubules, rt-pcr was performed and a product of the size predicted for the setre- <dig> transcript was observed in malpighian tubules.
sequencing results confirmed the rt-pcr result , demonstrating that setre- <dig> is expressed in the fat body, midgut and malpighian tubules.
developmental setre- <dig> expression
semi-quantitative rt-pcr experiments were carried out to determine setre- <dig> expression patterns in the midgut and fat body during different developmental stages of s. exigua.
setre- <dig> transcripts were detected in the midgut of larvae throughout the feeding stage, with higher expression levels in day- <dig> fourth instar  and day- <dig> fifth instar larvae .
however, no setre- <dig> expression was observed in the midgut of day- <dig> and day- <dig> pupae .
furthermore, setre- <dig> expression was observed in the midgut of pre-pupae .
in fat body, setre- <dig> expression patterns were different.
setre- <dig> mrna was detected in the fat body of fifth instar larvae and pupae .
furthermore, higher setre- <dig> expression levels were observed in the fat body of day- <dig> fifth instar larvae , as well as day- <dig> and day- <dig> pupae .
lower setre- <dig> expression levels were observed in the fat body of day- <dig> and day- <dig> fifth instar larvae, pre-pupae, and day- <dig> and day- <dig> pupae.
however, setre- <dig> expression was not observed in the fat body of day- <dig> fifth instar larvae and day- <dig>  day- <dig> and day- <dig> pupae.
discussion
two types of trehalase, soluble  and membrane-bound trehalase , have been purified from a variety of organisms, and the corresponding genes have also been cloned.
trehalases facilitate the uptake and utilization of trehalose from food or blood  <cit> .
insects also have two types of trehalases  <cit> .
the presence of two trehalase genes in s. exigua was verified by southern blotting .
we cloned one trehalase gene and protein sequence analysis suggested that it codes for a soluble trehalase .
these results are consistent with studies in other insect species for which trehalase genes have been cloned  <cit> .
an insect trehalase gene was first cloned from tenebrio molitor  <cit> .
trehalase genes in b. mori  <cit>  and p. hypochondriaca  <cit>  have also been cloned and studied.
all of these insect genes code for soluble trehalases and are expressed mainly in the pupal midgut  <cit> , but also in larval midgut, malpighian tubules and ovary  <cit> .
although immunoblot analysis revealed that two trehalases exist in insects  <cit> , the second trehalase gene, tre- <dig>  was not reported until  <dig> for b. mori and  <dig> for a. mellifera  <cit> .
the bmtre- <dig> gene is completely different from the tre- <dig> gene of b. mori  <cit>  and t. molitor  <cit> .
bmtre- <dig> transcripts are expressed in the midgut of b. mori larvae  <cit> .
the tre- <dig> gene structure was first reported for a. mellifera  <cit> .
however, the tissue distribution, expression patterns and genomic structure of lepidopteran tre- <dig> are still unknown.
in the present study, northern blotting and rt-pcr results suggest that setre- <dig> is expressed not only in midgut, but also in the fat body and malpighian tubules .
moreover, setre- <dig> has different expression patterns in the midgut and fat body.
setre- <dig> is expressed in the fat body, with higher expression levels in day- <dig> fifth instar larvae, and day- <dig> and day- <dig> pupae .
setre- <dig> transcripts were also detected in midgut throughout the feeding stage, which is consistent with results for bmtre- <dig>  <cit> .
setre- <dig> expression levels before the wandering  larval stage were higher than those in day-1– <dig> fifth instar larvae in both the midgut and fat body.
a possible reason may be that more energy is needed for pupa development.
according to preliminary results for rnai experiments involving injection of dsrna of an ecdysteroid receptor gene in s. exigua, changes in setre- <dig> transcripts in the midgut and fat body are modulated by morphogenetic hormones .
insect trehalases have several common characteristics, namely, a signal peptide leader, a coiled-coil domain, a highly conserved glycine-rich  region, and two conserved signature motifs   <cit> .
in addition, tre- <dig> also has some unique characteristics, such as a transmembrane helical region and two conserved motifs , but tre- <dig> does not have a putative transmembrane region.
based on the genomic sequence of s. exigua obtained in this study, the exons and introns of tre- <dig> are reported for the first time.
trehalases are important enzymes in insects as they catalyze the hydrolysis of trehalose to glucose  <cit> .
it has been reported that b. mori midgut contains two trehalases, bmtre- <dig> and bmtre- <dig>  <cit> .
tre- <dig> is involved in incorporating trehalose from the blood into muscular cells and then providing the energy required by visceral muscles to support peristaltic movement of the midgut for active feeding  <cit> .
the chitin biosynthesis pathway starts with trehalose, which is mainly synthesized by trehalose-6-phosphate synthase in the fat body and released into the hemolymph in insects  <cit> .
according to the setre- <dig> expression patterns observed in the midgut and fat body , tre- <dig> may have different functions in these two tissues.
this is the first report of trehalase transcript expression in fat body, but its function in this tissue is still unknown.
we also cloned the trehalose-6-phosphate synthase gene  from s. exigua, which is expressed mainly in fat body and not in midgut, and found that its expression levels showed the same trend as trehalose levels in hemolymph of s. exigua .
this demonstrates that both tre- <dig> and trehalose-6-phosphate synthase are synthesized in the fat body.
thus, the tre- <dig> gene may have a crucial function in regulating the balance of trehalose in hemolymph.
the relative importance of tre- <dig> and tre- <dig> in the chitin biosynthesis pathway in s. exigua is currently being investigated in our laboratory.
CONCLUSIONS
we have demonstrated that two trehalase genes exist in s. exigua.
setre- <dig> transcripts are expressed not only in the midgut, but also in fat body and malpighian tubules.
furthermore, there are different setre- <dig> expression patterns between midgut and fat body.
this suggests that setre- <dig> may have different functions in these different tissues.

 the disaccharide trehalose consists of two α-glycosidically linked glucose units. it is a non-reducing sugar found in many organisms as diverse as bacteria, yeast, fungi, nematodes, plants, insects and some other invertebrates, but is absent in mammals. trehalose may serve as a carbohydrate store and as an agent for protecting proteins and cellular membranes from a variety of environmental stress conditions, including desiccation, dehydration, heat, freezing and oxidation. in plants, trehalose not only has an impact on some metabolic processes and affects plant development as a signaling molecule, but also serves as an anti-stress substance to protect plants from drought, high salt and low temperature. in insects, unlike in mammals, trehalose is the main blood sugar and is present in the hemolymph of larvae, pupae and adults. it is the main reserve sugar in the hemolymph of flying insects and is also indispensable for thermotolerance in larvae. trehalose is synthesized mainly in the insect fat body and is rapidly released into the hemolymph and other tissues. to utilize blood trehalose, insect tissues contain trehalases that catalyze the hydrolysis of one mole of trehalose to two moles of glucose. thus, for uptake or utilization of trehalose in the blood, trehalases are essential enzymes in insects and are thought to be located on the cell membrane or within cells. the first insect trehalase, a soluble trehalase, was reported in. although insects are believed to have two types, soluble trehalase and membrane-bound trehalase, the tre- gene was not reported until. in bombyx mori, the tre- gene is expressed in the midgut; immunoblotting and immunohistochemical analyses showed that tre- is present mainly in goblet cell cavities and tre- penetrates the cell membrane and is predominantly evident on visceral muscle surrounding the midgut. although two trehalase genes have been cloned from b. mori,apis mellifera and spodoptera exigua, the different functions of these two trehalases in chitin biosynthesis in insects are not clear. in addition, very little is known about the structure, tissue distribution and expression pattern of tre- here, we report our findings regarding the gene coding for a putative membrane-bound trehalase isolated from the fat body of s. exigua. we observed that it is expressed not only in the midgut, but also in the fat body and malpighian tubules. furthermore, its expression patterns differed between the midgut and fat body.  cloning of full-length setre- cdna based on the conserved amino acid and nucleotide sequences of trehalases from b. mori, tenebrio molitor and pimpla hypochondriaca, we designed three degenerate primers, setre-f setre-f and setre-r, for pcr reactions. a fragment of bp was first obtained from pupal fat body cdna by a second pcr using setre-f and setre-r. the deduced amino acid sequence exhibited high similarity to insect trehalase sequences. we then performed 5' and 3' rapid amplification of cdna ends using several specific primers based on the sequence of the fragment and universal primers. pcr products of and bp were amplified by 5' and 3' race, respectively. assembly of the overlapping fragments revealed a full-length cdna of bp. the trehalase contained an open reading frame of bp, encoding a protein of amino acids with a predicted molecular weight of approximately kda and pi of. the deduced amino acid sequence of trehalase from s. exigua was aligned with the corresponding sequences of other insect trehalases. setre- is most similar to lepidopteran trehalase- from b. mori and ostrinia furnacalis. it is also similar to setre-, sftre-, bmtre-, oftre-, phtre-, amtre-, amtre-, tctre-, nvtre-, agtre-, aatre-, dmtre-, dstre-, tmtre-, tctre-, rntre, mmtre and hstre. the insect tre- gene is highly conserved, particularly in the middle of the putative catalytic domain. setre- cdna and protein sequence analysis the deduced amino acid sequence of setre- contains two trehalase signature motifs, pggrfrefyywdsy and qwdypnawpp and five other conserved motifs: dsktfvdmk, ipnggriyy, rsqppll, gprpesykedv and elkaaaesgwdfssrwfi. residues 1– are a signal peptide leader and residues 530– correspond to a glycine-rich region. residues 585– were found to comprise a putative transmembrane domain. n-terminal to this domain, residues 573– are identical to amino acids 570– in bmtre- however, this is not identified as an omega site by the big-pi predictor used to predict glycosylphosphatidyl inositol modification sites. five potential n-glycosylation sites are present in bmtre- but six potential n-glycosylation sites were found in setre- five sites homologous to those in bmtre- and an additional site at amino acid structure of setre-2 we amplified the setre- genomic dna sequence, which is approximately kb long. the exon/intron composition of the gene was determined by comparing the genomic sequence with the setre- cdna sequence. the setre- gene consists of exons separated by introns of different lengths and exon-intron splice junctions following the gt-ag rule. the first intron is the longest, at kb. exons 1– correspond to nucleotides 1– 5855– 6306– 7873– 8668– 9592– –, –, –, –, –, –, and – in the genomic sequence, respectively. compared to the seven exons and six introns of the a. mellifera tre- gene and the nine exons and eight introns of amtre-, setre- has more exons and introns. southern blot analysis gene copy number can be determined by southern blot analysis. genomic dna was obtained from s. exigua pupae and approximately μg of dna was digested with hindiii, sali and xhoi, electrophoresed and transferred to a nylon membrane, and then probed with a setre- cdna fragment generated using setrefp and setrerp primers. the probe was designed to be specific for a highly conserved region so that it would hybridize to both setre- and setre- one strong and one faint band were detected when genomic dna was digested with sali and xhoi whereas three strong and two faint bands were observed for hindiii treatment. the latter is attributed to the presence of two hindiii sites in the setre- genomic sequence that correspond to the probe sequence. a hindiii site may also be present in the setre- genomic sequence corresponding to the setre- probe. setre- tissue distribution tissue-specific expression setre- was determined by northern blotting. setre- cdna was cloned from s. exigua fat body, suggesting that setre- mrna is expressed in this tissue. this was confirmed by northern blot analysis. in addition, setre- transcripts were also detected in the midgut, but not in the brain or cuticle. setre- mrna may also be expressed in malpighian tubules, since a faint band was observed. to determine the expression of setre- transcripts in malpighian tubules, rt-pcr was performed and a product of the size predicted for the setre- transcript was observed in malpighian tubules. sequencing results confirmed the rt-pcr result, demonstrating that setre- is expressed in the fat body, midgut and malpighian tubules. developmental setre- expression semi-quantitative rt-pcr experiments were carried out to determine setre- expression patterns in the midgut and fat body during different developmental stages of s. exigua. setre- transcripts were detected in the midgut of larvae throughout the feeding stage, with higher expression levels in day- fourth instar and day- fifth instar larvae. however, no setre- expression was observed in the midgut of day- and day- pupae. furthermore, setre- expression was observed in the midgut of pre-pupae. in fat body, setre- expression patterns were different. setre- mrna was detected in the fat body of fifth instar larvae and pupae. furthermore, higher setre- expression levels were observed in the fat body of day- fifth instar larvae, as well as day- and day- pupae. lower setre- expression levels were observed in the fat body of day- and day- fifth instar larvae, pre-pupae, and day- and day- pupae. however, setre- expression was not observed in the fat body of day- fifth instar larvae and day- day- and day- pupae. discussion two types of trehalase, soluble and membrane-bound trehalase, have been purified from a variety of organisms, and the corresponding genes have also been cloned. trehalases facilitate the uptake and utilization of trehalose from food or blood. insects also have two types of trehalases. the presence of two trehalase genes in s. exigua was verified by southern blotting. we cloned one trehalase gene and protein sequence analysis suggested that it codes for a soluble trehalase. these results are consistent with studies in other insect species for which trehalase genes have been cloned. an insect trehalase gene was first cloned from tenebrio molitor. trehalase genes in b. mori and p. hypochondriaca have also been cloned and studied. all of these insect genes code for soluble trehalases and are expressed mainly in the pupal midgut, but also in larval midgut, malpighian tubules and ovary. although immunoblot analysis revealed that two trehalases exist in insects, the second trehalase gene, tre- was not reported until for b. mori and for a. mellifera. the bmtre- gene is completely different from the tre- gene of b. mori and t. molitor. bmtre- transcripts are expressed in the midgut of b. mori larvae. the tre- gene structure was first reported for a. mellifera. however, the tissue distribution, expression patterns and genomic structure of lepidopteran tre- are still unknown. in the present study, northern blotting and rt-pcr results suggest that setre- is expressed not only in midgut, but also in the fat body and malpighian tubules. moreover, setre- has different expression patterns in the midgut and fat body. setre- is expressed in the fat body, with higher expression levels in day- fifth instar larvae, and day- and day- pupae. setre- transcripts were also detected in midgut throughout the feeding stage, which is consistent with results for bmtre-. setre- expression levels before the wandering larval stage were higher than those in day-1– fifth instar larvae in both the midgut and fat body. a possible reason may be that more energy is needed for pupa development. according to preliminary results for rnai experiments involving injection of dsrna of an ecdysteroid receptor gene in s. exigua, changes in setre- transcripts in the midgut and fat body are modulated by morphogenetic hormones. insect trehalases have several common characteristics, namely, a signal peptide leader, a coiled-coil domain, a highly conserved glycine-rich region, and two conserved signature motifs. in addition, tre- also has some unique characteristics, such as a transmembrane helical region and two conserved motifs, but tre- does not have a putative transmembrane region. based on the genomic sequence of s. exigua obtained in this study, the exons and introns of tre- are reported for the first time. trehalases are important enzymes in insects as they catalyze the hydrolysis of trehalose to glucose. it has been reported that b. mori midgut contains two trehalases, bmtre- and bmtre-. tre- is involved in incorporating trehalose from the blood into muscular cells and then providing the energy required by visceral muscles to support peristaltic movement of the midgut for active feeding. the chitin biosynthesis pathway starts with trehalose, which is mainly synthesized by trehalose-6-phosphate synthase in the fat body and released into the hemolymph in insects. according to the setre- expression patterns observed in the midgut and fat body, tre- may have different functions in these two tissues. this is the first report of trehalase transcript expression in fat body, but its function in this tissue is still unknown. we also cloned the trehalose-6-phosphate synthase gene from s. exigua, which is expressed mainly in fat body and not in midgut, and found that its expression levels showed the same trend as trehalose levels in hemolymph of s. exigua. this demonstrates that both tre- and trehalose-6-phosphate synthase are synthesized in the fat body. thus, the tre- gene may have a crucial function in regulating the balance of trehalose in hemolymph. the relative importance of tre- and tre- in the chitin biosynthesis pathway in s. exigua is currently being investigated in our laboratory.  we have demonstrated that two trehalase genes exist in s. exigua. setre- transcripts are expressed not only in the midgut, but also in fat body and malpighian tubules. furthermore, there are different setre- expression patterns between midgut and fat body. this suggests that setre- may have different functions in these different tissues. 
these findings provide new data on the tissue distribution, expression patterns and potential function of membrane-bound trehalase. alignment of setre- with other insect trehalases showed that it contains two trehalase signature motifs and a putative transmembrane domain, which is an important characteristic of tre- comparison of the genomic dna and cdna sequences demonstrated that setre- comprises exons and introns. to gain an insight into trehalase functions in insects, we investigated a putative membrane-bound trehalase from spodoptera exigua cloned from the fat body. southern blot analysis revealed that s. exigua has two trehalase genes and that setre- is a single-copy gene. setre- transcripts were detected in the midgut of feeding stage larvae, but not in pupae, whereas setre- mrna was detected in the fat body of fifth instar larvae and pupae. northern blot analyses showed that the setre- transcript is expressed not only in the midgut, as previously reported for bombyx mori, but also in the fat body and malpighian tubules, although expression patterns differed between the midgut and fat body. the chitin biosynthesis pathway starts with trehalose in insects and the main functions of trehalases are hydrolysis of trehalose to glucose. the results suggest that the setre- gene may have different functions in the midgut and fat body. the deduced amino acid sequence of setre- contains residues and has a predicted molecular weight of ~ kda and pi of. although insects possess two types, soluble trehalase and membrane-bound trehalase, very little is known about tre- and the difference in function between tre- and tre- 
 the chitin biosynthesis pathway starts with trehalose in insects and the main functions of trehalases are hydrolysis of trehalose to glucose. although insects possess two types, soluble trehalase and membrane-bound trehalase, very little is known about tre- and the difference in function between tre- and tre-  to gain an insight into trehalase functions in insects, we investigated a putative membrane-bound trehalase from spodoptera exigua cloned from the fat body. the deduced amino acid sequence of setre- contains residues and has a predicted molecular weight of ~ kda and pi of. alignment of setre- with other insect trehalases showed that it contains two trehalase signature motifs and a putative transmembrane domain, which is an important characteristic of tre- comparison of the genomic dna and cdna sequences demonstrated that setre- comprises exons and introns. southern blot analysis revealed that s. exigua has two trehalase genes and that setre- is a single-copy gene. northern blot analyses showed that the setre- transcript is expressed not only in the midgut, as previously reported for bombyx mori, but also in the fat body and malpighian tubules, although expression patterns differed between the midgut and fat body. setre- transcripts were detected in the midgut of feeding stage larvae, but not in pupae, whereas setre- mrna was detected in the fat body of fifth instar larvae and pupae.  these findings provide new data on the tissue distribution, expression patterns and potential function of membrane-bound trehalase. the results suggest that the setre- gene may have different functions in the midgut and fat body. 