Insect Molecular Biology (2006)

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Caste-specific cytochrome P450 in the damp-wood termite

Hodotermopsis sjostedti

(Isoptera, Termopsidae)

R. Cornette*†, S. Koshikawa*†, M. Hojo*, T. Matsumoto* and T. Miura†

*

Department of Biology, Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan

†

Laboratory of Ecology and Genetics, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan

Abstract

Termites are eusocial insects with a well-definedcaste system, which is an example of polyphenism.This polyphenism is based on hormonally controlleddifferential gene expression. In the damp-wood termite

Hodotermopsis sjostedti

, we induced differentiationinto the soldier caste by using juvenile hormoneanalogue treatment. We then investigated specificgene expression, which appeared during the hormonalresponse and triggered caste differentiation, usingfluorescent differential display. A candidate cDNAsequence with similarity to cytochromes P450, CYP6AM1,was characterized and its transcript shown to berepressed between 1 and 3 days after hormone treat-ment. CYP6AM1 was specifically expressed in the fatbody of pseudergates and soldiers. The putative func-tion of this P450 is discussed with respect to the castedifferentiation system.

Keywords: termite, caste differentiation, juvenilehormone, gene expression, cytochrome P450, fat body.

Introduction

Social insects form complex societies, with individualsassigned to different tasks, such as offspring care, foraging,and colony defense, known as division of labour (polyethism).With respect to their specific tasks or sex, individual insects

may develop different phenotypes, which define distinctcastes (Wilson, 1971). This caste polymorphism, observedin social insects, is interesting because all individuals in thecolony possess almost identical genotypes and thus theobserved morphological differences are largely based ondifferential gene expression (Miura, 2001). Caste polymor-phism is therefore a type of polyphenism (Noirot, 1991).Insect polyphenism is a complex phenomenon, in whichgenomes interact with environmental factors, inducingdevelopmental changes via hormonal responses (Nijhout,1999; Evans & Wheeler, 2001). In recent years, therehas been an increase in the number of molecular studiesattempting to characterize the specific gene expressionassociated with caste polyphenism in social insects (Ohashi

et al

., 1997; Evans & Wheeler, 1999; Scharf

et al

., 2003,2005a,b; Judice

et al

., 2004; Hojo

et al.

, 2005; Koshikawa

et al

., 2005; Robinson

et al

., 2005). In this context, termitesconstitute a good model for the study of polyphenismbecause they present a conspicuous and morphologicallydiverse caste system (Thorne, 1996; Roisin, 2000).

Contrary to social hymenopterans, which have aholometabolous postembryonic development, termites arehemimetabolous insects and consequently show a moreflexible caste differentiation system (Miura, 2004). Inparticular, caste differentiation in the lower termites (e.g.Kalotermitidae, Termopsidae and some Rhinotermitidae)appears at relatively late larval stages and can be reversible(Grassé, 1982; Roisin, 1990; Koshikawa

et al

., 2001). Thedamp-wood termite

Hodotermopsis sjostedti

belongs to thefamily Termopsidae, which is considered as relatively basalin the order Isoptera (Kambhampati

et al

., 1996; Kambhampati& Eggleton, 2000; Thompson

et al

., 2000).

H. sjostedti

presents a linear developmental pathway, depicted in Fig. 1,with seven apterous larval instars, followed by a singlenymphal stage, which can differentiate directly to the alateform (Miura

et al

., 2000; Miura

et al

., 2004). The worker rolein the society is mainly played by the seventh larval instar,which is thus called pseudergate (Grassé & Noirot, 1947).Pseudergates are totipotent and can differentiate into nymphsand alates, into soldiers through the transitional presoldierstage, or into neotenic secondary reproductives (Miura

et al

.,2000). The fate of pseudergates is controlled by environmental

doi: 10.1111/ j.1365-2583.2006.00632.x

Received 10 June 2005; accepted after revision 24 October 2005. Corre-spondence: Prof Toru Miura, Laboratory of Ecology and Genetics, GraduateSchool of Environmental Earth Science, Hokkaido University, Sapporo060–0810, Japan. Tel.: +81-11-706-4524; e-mail: miu@ees.hokudai.ac.jp.

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factors, such as temperature, food, pheromones or interac-tions with conspecifics (Noirot, 1991). These factors influencehormone levels and induce specific gene expression, which isresponsible for caste differentiation (Miura, 2001).

Juvenile hormone (JH) is one of the most importantfactors in the determination of caste polyphenism in socialinsects (Nijhout & Wheeler, 1982). In honeybees, increasedJH levels in larvae fed with royal jelly are responsible fordifferentiation into queen bees (Rachinsky

et al

., 1990). Theonset of foraging in worker bees is also JH-dependent (Amdam& Omholt, 2003). Similarly, queen determination in ants mayoccur as well during embryogenesis or in the developinglarvae, and JH sensitive periods were observed at variousstages (Nijhout, 1994). In the ant

Pheidole bicarinata

, thepolyphenism between minor and major workers is againunder JH control (Wheeler & Nijhout, 1984). The apterous/winged dimorphism was also revealed by JH treatment ona specific morph in some aphid species (Hardie, 1981).

In termites, the differentiation to presoldiers can be inducedby elevation of JH levels into the haemolymph of workers orpseudergates (Lüscher, 1958; Lüscher & Springhetti, 1960;Lenz, 1976; Park & Raina, 2005). The model proposedby Nijhout & Wheeler (1982), described three JH-sensitiveperiods responsible for the development of sexual characters,non-sexual adult characters and soldier characters. Presoldierdifferentiation was suggested to require a high JH levelthroughout the three JH-sensitive periods (Nijhout & Wheeler,1982). Treatment with juvenile hormone analogues (JHAs)can artificially trigger soldier differentiation in termites (Hrdy& Krecek, 1972; Howard & Haverty, 1979). However, thereis a paucity of information on the mechanism through whichJH and its analogues affect termite development. AlthoughJH regulates a large number of developmental processes ininsects, its mode of action is still unclear. Many proteins bindJH with different affinities, but all attempts to identify a singleJH receptor have failed. It was therefore suggested that JHmay act through both signal transduction and transcriptionregulation, in the context of a wide lipid signalling system(Wheeler & Nijhout, 2003). The molt to presoldier is directlyinducible by JH, and termites can thus be considered as a goodmodel with which to investigate the molecular mechanismsof responses to JH. The present study examined the effectof JH on differential gene expression, at the onset of soldiercaste differentiation in

H. sjostedti

, and characterized a cyto-chrome P450, which is expressed in a caste-specific manner.

Results

Differential display

The differential-display method was used to identify genesspecifically expressed during the early response to a juvenilehormone analogue, pyriproxyfen, inducing soldier caste differ-entiation. Total RNA was extracted from pseudergates andjuvenile hormone analogue (JHA)-treated termites at differenttimes (from 1 h to 7 days after treatment). Expression patternsfrom every stage were compared and the pseudergate stagewas used as a control for genes normally expressed. Differ-entially expressed genes identified reproducibly correspondedto less than 1% of the total number of bands (28/4027)observed on sequence gels with 20 combinations of arbitraryprimers (

Hin

dIII-1 to -20 primers). The majority of differencesin gene expression were observed after 24 h and until 3 daysafter treatment, and many of them (24 bands) were repressed.Among the total 28 observed candidates specific to JHA-induced termites, 5 were successfully cloned and sequencedfrom RNA extracted from termite heads, while 13 were sequ-enced from RNA extracted from termite bodies. B

LASTX

data-base searches showed similarities for some candidates fromtermite body sequencing (acceptable threshold:

e

-value < 1.0)with genes such as ribosomal proteins, the maternal effectprotein Staufen, D-titin and a cytochrome P450. From the 18sequenced candidates, a 462 bp fragment corresponding to

Figure 1. The caste differentiation system in damp-wood termite Hodotermopsis sjostedti. Note the seven larval instars; the seventh instar is the pseudergate (PE). A pseudergate can differentiate into alate (A) through one nymphal stage (N), to soldier (S) through the presoldier (PS) transitional stage, to neotenic reproductive (Neo), or undergo stationary molt (SM). (Miura et al., 2000, 2004).

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the cytochrome P450, was expressed in the body of pseuder-gates and until 24 h after treatment, but its expression wasthen repressed at 3 and 7 days post-treatment. This 462 bpfragment was the only cloned candidate, whose expressionpattern was clearly confirmed by Northern blot (data not shown).

Characterization of CYP6AM1 sequence

Specific primers were designed from this 462 bp fragmentand the corresponding full-length sequence was determined

using the RACE-PCR method. The obtained full-lengthcDNA sequence (accession number: AB194532) was 1977nucleotides long and contained an open reading frameof 1587 nucleotides, followed by a 3

′

-untranslated regionof 288 nucleotides presenting a polyadenylation signal; 102nucleotides from the 5

′

-untranslated region were sequenced(Fig. 2). The DNA fragment obtained by differential displaywas found in the nucleic acid sequence from position 1364to position 1825. The protein was composed of 528 amino

Figure 2. cDNA and amino acid sequences of CYP6AM1. Start codon (atg), termination codon (tga) and polyadenylation signal (aataaa) appear in bold letters. Sequences in grey indicate the DNA fragment obtained by differential display. The 1587-nucleotide long ORF encodes a putative 528-amino acid polypeptide. The predicted transmembrane region is underlined at the N-terminus and cytochrome P450 cysteine heme-iron ligand signature is shown double underlined.

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acids with a calculated molecular weight of 61.1 kDa andan isoelectric point (pI) of 7.58. A putative transmembranedomain was predicted at position 8–27 in the amino acidsequence with the TMpred program (Hofmann & Stoffel, 1993).

When submitted to the B

LAST

program (Altschul

et al

.,1997), our sequence showed significant similarity withdifferent P450 cytochromes which belong to the family six.The highest percentage of identity (42%) was observedwith CYP6X1, from the true bug

Lygus lineolaris

(Zhu &Snodgrass, 2003). According to the current nomenclature,cytochromes P450 are assigned to the same family whenthey have similarity of greater than 40% at the aminoacid sequence level, and to the same subfamily when itis greater than 55% (Nelson, 1998; Feyereisen, 1999). Our

Hodotermopsis

P450 belonged to family six, but differedfrom all the defined subfamilies (up to CYP6AL whenthe sequence was obtained). It was most similar to a cDNAfragment (G

EN

B

ANK

accession number: BQ476023) fromthe beetle

Platystomus albinos

, which was unnamed(Theodorides

et al

., 2002). Thus, our Hodotermopsiscytochrome P450 sequence was named CYP6AM1.

The alignment of the N-terminal amino acid sequencesfrom CYP6AM1 and other microsomal cytochromes P450showed that the hydrophobic putative transmembrane

domain was followed by a cluster of positively chargedresidues and then a proline rich motif (Fig. 3A). The presenceof this proline rich motif associated with glycine residues,which also function as alpha-helix breakers, seems tobe important for the correct folding of active microsomalenzymes (Yamazaki

et al

., 1993; Chen & Kemper, 1996).CYP6AM1 sequence contained only a few of theseproline/glycine residues, but the general structure of the N-terminus was conserved and thus CYP6AM1 is apparentlya typical microsomal P450. The P450 consensus sequencecorresponding to the heme-binding loop was particularlywell conserved in the alignment (Fig. 3B). This region con-tains 5 conserved residues (F-x-x-G-x-R-x-C-x-G) and isorganized around the invariant cysteine, which participatesin the heme iron binding (Nelson

et al

., 1996; Werck-Reichhart & Feyereisen, 2000). This characteristic P450heme-iron ligand signature was identified in CYP6AM1 aminoacid sequence (FGEGPRMCIG), by comparison with thePROSITE database (Bairoch

et al

., 1997). Finally, anotherconsensus sequence, corresponding to the central part ofthe I-helix was found in CYP6AM1. This sequence wasparticularly well conserved among CYP6 enzymes (Fig. 3C).The conserved central glutamic acid-threonine and thesurrounding sequence in the I-helix of cytochromes P450 are

Figure 3. Amino acid sequence alignments of CYP6AM1 with other insect microsomal cytochromes P450s. (A) Alignment of N-terminal amino acid sequences, showing the hydrophobic transmembrane region and the Proline/Glycine rich region with underlined residues. Between these regions, the amino acid sequences present positively charged residues (shown in grey). (B) Alignment of the heme-binding domain sequences. (C) Alignment of the conserved central region of the I-helices. Position of the first residue in each sequence is indicated by the number between brackets. Amino acids identical to CYP6AM1 are highlighted in grey and conserved residues from the characteristic P450 consensus sequence are indicated with an asterisk. CYP6X1, bug pyrethroid-inducible P450 (AAL15173); CYP6K1, cockroach P450 (AAK57914); CYP6H1, locust ecdysone 20-hydroxylase (AF115777); CYP6F1, pyrethroid-resistant mosquito P450 (AB001324); CYP6B1, black swallowtail caterpillar furanocoumarin metabolizing P450 (CAA82732); CYP4C1, cockroach JH-regulated P450 (P29981); CYP312A1, fruit fly male-specific P450 (Q9VVN6); CYP18A1, fruit fly ecdysteroid-regulated P450 (U44753).

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thought to be involved in the proton delivery for catalysis(Hasemann

et al

., 1995; Gorokhov

et al

., 2003).

RNA expression profile of CYP6AM1

The 462 bp cDNA fragment that was obtained first wasused as a probe to investigate the expression pattern ofCYP6AM1 by Nothern-blot analysis. As shown in Fig. 4,a single mRNA band was detected in the body (thorax +abdomen) with a size of approximately 2.0 kb, which matchedthe size of the sequenced full-length CYP6AM1 cDNA.Under experimental conditions, CYP6AM1 was expressedin pseudergates and soldiers. In JHA-treated pseudergates,CYP6AM1 expression persisted until 24 h after the applica-tion. On the third day after treatment, the transcript wasstill weakly detected, but subsequently, it was completelyrepressed (Fig. 4A). Later, CYP6AM1 was still not expressedeven in induced presoldiers. In pseudergates, the transcriptfor CYP6AM1 was not detected in transcripts extractedfrom termite heads, and was consequently localized mainlyin those from the thorax and abdomen. We also verifiedthe expression in unmanipulated individuals from differentcastes (Fig. 4B). The CYP6AM1 transcript was faintly detectedin second instar larvae, appeared clearly in fourth instarlarvae, and reached its highest level of expression inpseudergates. CYP6AM1 was not constitutively expressedin presoldiers, a finding consistent with observationsmade in experimental conditions. However, the CYP6AM1transcript reappeared at its highest level in the terminalsoldier caste, but was not expressed in the reproductivecastes (i.e. nymphs, alates and neotenics). Northernblot analysis showed no gender-based differences in theexpression patterns (data not shown).

Tissue localization of CYP6AM1 transcript

The localization of CYP6AM1 expression was investigatedby real-time quantitative PCR in different tissues dissectedand treated separately (Fig. 5). As expected from the Northernblot results, the expression of CYP6AM1 was extremelyweak in the presoldiers. In pseudergates, CYP6AM1 transcriptwas present in significant quantities only in the fat body.Levels of expression were significantly higher (up to 200fold) in the fat body than in all other tissues investigated(one-way

ANOVA

followed by Tukey’s HSD test). The expres-sion of CYP6AM1 in the midgut and salivary glands wasweakly detected. Although in small amount, the CYP6AM1transcript was also found in pseudergate legs, probablybecause legs also contain little fat body.

Discussion

We used a JHA, pyriproxyfen, to induce presoldier differen-tiation in damp-wood termite

H. sjostedti

in order to observethe early changes in gene expression.

JH III, which is considered as the natural form of JH intermites, was unable to induce soldier differentiation inthis species even at high dose (data not shown), whereaspresoldier formation is naturally induced by JH III in othertermite species. Pseudergates of

H. sjostedti

may havehigh levels of JH degrading enzymes in their haemolymphand pyriproxyfen, which is different in structure from JH III,is probably not affected by the major enzymes responsiblefor JH degradation (Zhang

et al

., 1998). Consequently, onlypyriproxyfen may be allowed to exhibit its juvenoid effects.

Only a few variations in gene expression were detected(less than 1% of the total number of transcripts observed),

Figure 4. Northern hybridization analysis of Hodotermopsis sjostedti RNA with CYP6AM1 cDNA. Northern blot was performed using total RNA (20 µg) from the bodies (thorax + abdomen). 28S rRNA bands stained with ethidium bromide indicate that approximately equal amount of RNA were loaded in each lane. (A) Pseudergates (PE), individuals sampled 1 h, 3 h, 6 h, 24 h, 3 days (3 d), 7 days (7 d) and 14 days (14 d) after JHA treatment, induced presoldiers (PS), natural soldiers (S) and heads of pseudergates (PEh). (B) Individuals sampled from natural colonies: second (L2) and fourth (L4) instar larvae, pseudergates (PE), presoldiers (PS), soldiers (S), nymphs (N), alates (A) and neotenics (Neo).

Figure 5. Quantification of CYP6AM1 expression by real-time quantitative PCR in the body of presoldiers (PS) and legs (LEG), fat body (FB), midguts (MG) and salivary glands (SG) of pseudergates (PE) of Hodotermopsis sjostedti. The relative abundance of CYP6AM1 transcript in total RNA from each tissue is indicated by arbitrary units, corresponding to the ratio of CYP6AM1 transcripts against 18S rRNA values. 18S rRNA was used as an endogenous control of constitutive expression. Data are mean ± SD of 6 samples each. ***P < 0.001 for the whole group, and compared to other samples (one-way ANOVA followed by Tukey’s HSD test).

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but there was a general trend towards gene repressionbetween 24 h and 3 days after JHA application. During thisperiod, there should be an important switch in gene expres-sion responsible for soldier differentiation.

Differential display is a PCR-based method used toidentify specific expression in different tissues; this techniquesometimes amplifies non-specific products, the so called ‘falsepositives’ (Liang & Pardee, 1992; Livesey & Hunt, 1996).The relatively low sensitivity of this method could explainwhy we did not succeed in identifying genes with lowexpression levels, such as transcription factors, which shouldappear rapidly after induction by the JHA. Nevertheless,some candidate genes were successfully cloned andsequenced. Those genes belonged to families, which werealso described partly in a previous study concerninggene expression, later during soldier morphogenesis(Koshikawa

et al

., 2005). For example, the candidate genesimilar to Staufen was found to be a false positive in thepresent study, but Koshikawa

et al

. (2005) shown that thesame gene was up-regulated during the following soldiermorphogenesis. Most of the other candidate genes werealso false positives, but we confirmed the specific expressionpattern for one of them by Northern blot. This candidategene showed sequence similarity with cytochromes P450,and its expression was repressed between 24 h and 3 daysafter JHA treatment.

Cytochromes P450 (P450s) are heme-containing oxida-tive enzymes, which require the presence of an electrondonor, NAD(P)H, to catalyse the oxidation of their substrates(Feyereisen, 1999; Scott & Wen, 2001). In eukaryotes,P450 enzymes can be located in the endoplasmic reticulumor in the mitochondria (Feyereisen, 1999). In insects,P450 genes constitute a large superfamily, including a largenumber of gene copies (Scott & Wen, 2001). For example,even in a single organism such as

Drosophila melanogaster

,up to 90 P450 genes were identified (Tijet

et al

., 2001). P450sfulfil a variety of functions in insect physiology includingtheir involvement in the metabolism of juvenile hormone,ecdysteroids and other endogenous substrates, and in thedegradation of xenobiotic compounds (Feyereisen, 1999).

According to the current P450 nomenclature based onprotein sequence identity (Nelson, 1998), the

H. sjostedti

sequence indicated that it was member of family 6 butbelonged to a new subfamily and was consequently namedCYP6AM1. CYP6 family members have only been found ininsects and many of them are associated with insecticideresistance (Nelson

et al

., 1996; Ranson

et al

., 2002). How-ever, CYP6AM1 is more likely to be involved in a metabolicpathway specific to pseudergates and soldiers rather thanin the degradation of JHA itself, because its expression isrepressed after treatment, whereas xenobiotic metaboliz-ing P450s are generally induced in response to thepresence of their specific substrates (Feyereisen, 1999).Furthermore, the repression of the CYP6AM1 transcript

during soldier differentiation was also observed in naturalcolonies (data from presoldiers), and morphometric analysisshowed that JHA-induced presoldiers were normal, whencompared to their naturally occurring counterparts (Koshikawa

et al

., 2002). The repression of CYP6AM1 expression wasconsequently demonstrated not to be an artifact of JHA.However, the possibility that pyriproxyfen may be a substratefor CYP6AM1 cannot be ruled out.

To our knowledge, CYP6AM1 is the first cytochromeP450 from family 6, to have a fully characterized sequencein a termite species. Until now, only partial P450 sequenceswere determined in

Mastotermes darwiniensis

, mainlywith respect to pesticide resistance (Falckh

et al

., 1997; Wu

et al

., 2001). In other social insects, caste-specific P450expression was also recorded in several bee and antspecies (Evans & Wheeler, 2000; Judice

et al

., 2004; Liu &Zhang, 2004). In these studies, P450s were found to beoverexpressed in the workers compared to the queens,suggesting the implication in the physiology of non-reproductive castes.

CYP6AM1 expression was repressed after JHA treatment,but the information available regarding JH concentrationsamong the different castes of termites is fragmented(Greenberg & Tobe, 1985). Until recently, JH levels intermites were only indirectly inferred from the size of thecorpora allata, which are the JH-producing glands (Gäde

et al

., 1997). Some studies on different genera like

Kaloter-mes

,

Cubitermes

or

Macrotermes

, demonstrated that intermites, the volume of the corpora allata was importantin the presoldier, alate and neotenic castes, but less so inworkers/pseudergates and soldiers (Kaiser, 1956; Okot-Kotber, 1977, 1980; Grassé, 1982). Direct quantification in

Coptotermes formosanus

confirmed that JH III levelswere higher in presoldiers than in workers and soldiers(Park & Raina, 2004). Those results suggest, that CYP6AM1is expressed in castes with a low JH titre.

In addition, expression of the CYP6AM1 transcript seemsto be associated with a low level of fat body development.Whereas the abdomen of presoldiers is filled with fat body,the abdominal volume of soldiers contains only a smallproportion of fat body. In

Kalotermes flavicollis

, fat bodywas weakly developed in elder larvae and soldiers, butreached a maximal development in nymphs with longwing buds. Fat body was also well developed in alates(Gharagozlou, 1965). In queens, kings and neotenicindividuals, fat body undergoes significant modificationsin relation to protein synthesis and reaches a high level ofdevelopment, being called royal fat body (Grassé, 1982;Costa-Leonardo

et al

., 1999).Some P450s were identified as ecdysone 20-hydroxylases,

and one of them belonged to the family 6 (Winter

et al

.,1999; Petryk

et al

., 2003). The implications of CYP6AM1 in20-hydroxyecdysone synthesis could be considered, butthe absence of the CYP6AM1 transcript in the pseudergates

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before molt to presoldiers and in the premolting nymphsdoes not support this function. In contrast, several P450enzymes are known to act in JH biosynthesis (Sutherland

et al

., 2000; Helvig

et al

., 2004) and CYP6AM1 may beinvolved in the activation of JH by epoxidadion, for example.CYP6AM1 could also be involved in JH degradation, assuggested for CYP6A1 in the house fly (Andersen

et al

.,1997), or in the metabolism of JH acid, which was demon-strated to present a hormonal effect on vitellogenesis(Ismail

et al

., 1998).Cytochromes P450 are also known to take part in the

oxidation of fatty acids (Feyereisen, 1999). For example,CYP4C1,from cockroach fat body, was proposed to partic-ipate in fatty acid oxidation (Lu

et al

., 1999). CYP6AM1 mayalso be involved in the oxidation of some fatty acid in

H.sjostedti and

thus potentially participate to the modulationof a lipid based metabolic pathway, specific to pseudergateand soldier castes.

Only

in vitro

tests using purified or recombinant CYP6AM1protein may permit identification of its substrate and clarifythe role of this P450 in termite physiology. However, ourdata clearly demonstrated that CYP6AM1 is an indicatorof early caste differentiation in

H. sjostedti

. The earlyphysiological response to JH is the first step triggeringcaste differentiation. For the investigation of differentiallyexpressed gene, the development of new molecularmethods such as RNAi, will advance our understanding ofthis important phenomenon.

Experimental procedures

Insects and JH analogue treatment

Damp-wood termite

H. sjostedti

(Isoptera, Termopsidae) colonieswere collected from decayed logs of pine trees from the evergreenforests of Yakushima Island (Kagoshima prefecture, Japan) inMay 2002 and May 2003. Colonies were kept in the laboratoryat approximately 25

°

C under constant darkness. Pseudergateswere isolated in groups (n = 10) into petri dishes and fed with filterpaper. They were treated by topical application of 5 µg pyriproxyfen(JH analogue, JHA) diluted in 5 µl acetone. Presoldier molts werenormally induced 2 weeks after initiation of treatment.

RNA extraction

JHA-induced pseudergates and individuals from different castes(50 each) were dissected. The heads and bodies were treatedseparately, endosymbionts-containing guts were removed andsamples were frozen in liquid nitrogen and preserved at −80 °C.Total RNA was isolated from frozen tissues with the RNAgent® totalRNA isolation system (Promega, Madison, WI, USA), following theinstructions provided by the manufacturer.

Differential display

RNA samples (3 µg) from the head and body of pseudergatesand individuals taken 1 h, 3 h, 6 h, 24 h, 3 days and 7 days afterJHA treatment were reverse-transcribed using SuperScriptII™

first-strand synthesis kit (Invitrogen, Carlsbad, CA, USA). Briefly, RNAsamples were at first treated with 1.5 units of RNase-free DNase Iin 20 mM Tris-HCl (pH 8.4) containing 50 mM KCl and 2 mM MgCl2in a total volume of 30 µl. Samples were then reverse-transcribedwith or without 150 units of SuperScriptII™, with 30 pmol of ananchored oligo(dT) primer with a BamHI site (Bam-TG primer,5′-CCC GGA TCC T15 G-3′) and 1 mM dNTPs in 20 mM Tris-HCl(pH 8.4), 50 mM KCl, 2.5 mM MgCl2 and 10 mM DTT (Miura et al.,1999). The resulting products were finally treated with RNase H.

The resulting samples were amplified by PCR, using 20 com-binations of arbitrary 12-mers with a HindIII site (HindIII-1 to-20 primers, 5′-CGGGAAGCTTN12-3′, where N is any base)(Linskens et al., 1995) and a Rhodamine-labelled Bam-TG primer.The reaction mixtures (20 µl) contained 1 µl of the cDNA obtainedbefore, 2 µl of 10x PCR buffer, 0.1 mM dNTP, 8 pmol of each primerand 0.5 U of AmpliTaq Gold™ polymerase (Applied Biosystems,Foster City, CA, USA). The PCR conditions were 1 cycle of 94 °Cfor 10 min, 37 °C for 5 min, and 72 °C for 5 min, 35 cycles of 94 °Cfor 30 s, 55 °C for 1 min, 72 °C for 1 min, followed by 72 °C for5 min. PCR products were separated on a denaturing 6% polyacr-ylamide gel and the migration profile was read with a fluorescentimage analyser FMBIO® II Multi-View (Hitachi, Tokyo, Japan),using the FMBIO® Analysis software. Reactions without reversetranscriptase were used as control experiments to detect bandsamplified from genomic DNA contamination. To ensure the repro-ducibility of the band pattern, each reaction was duplicated.

Subcloning and sequencing

Bands of interest were excised, the gel was boiled in deionizedwater and the obtained DNA was reamplified by PCR using Pfupolymerase (Stratagene, La Jolla, CA, USA), with the same primercombination used in the differential display method, but without flu-orescent marking. The reamplified DNA was subcloned into thepGEM-3Zf(+) vector (Promega) at the SmaI site and transfectedinto competent Escherichia coli JM109 (Takara, Otsu, Japan). Thenucleotide sequence of each strand was determined by the DNA-sequencing reaction with dideoxynucleotide cycle-sequencingprocedure, using the ThermoSequenase II™ dye terminator cyclesequencing kit (Amersham Pharmacia Biotech Inc., Piscataway,NJ, USA). Electrophoresis was performed on 6% polyacrylamidegels (Super Reading DNA Sequence solution, Toyobo, Osaka, Japan),and the data were obtained with an automatic DNA sequencer(model 373S. Applied Biosystems). Database searches forhomologies were performed using BLASTX at the NCBI server(www.ncbi.nlm.nih.gov/BLAST/). Protein deduced molecular weightand isoelectric point were calculated with Genetyx-Mac ver. 12.0.6software (Software development co., Italo, Tokyo, Japan).

5′- and 3′-Rapid Amplification of cDNA Ends (RACE)

In order to obtain the 5′ end sequence of the CYP6AM1 cDNA,RACE PCR was performed using the SMART™ RACE amplifica-tion kit (Clontech Laboratories, Palo Alto, CA, USA). Total RNA(5 µg) from pseudergates bodies was used as a template to syn-thesize first-strand cDNA with an oligo(dT) primer and theSMART™ II A oligonucleotide included in the Clontech kit. Theobtained first-strand cDNA was used as a template for the 5′-RACE PCR reaction with the universal primer A (5′-CTAATAC-GACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′)and an internal 5′-specific primer (5′-CGCGAACCTCATTCCTAT-GCACATCC-3′). A second nested PCR was then performed with

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the nested universal primer A (5′-AAGCAGTGGTATCAACGCA-GAGT-3′) and a second 5′-specific primer (5′-TTAGGGTCGTGAT-GAAGCCCTAAGAC-3′).

To obtain the 3′ end sequence of the CYP6AM1 cDNA, synthe-sis of the first-strand cDNA was performed as described beforebut using only the 3′CDS primer A from the Clontech kit. Thisfirst-strand cDNA was used as a template for the 3′-RACE PCRreaction with the universal primer A (5′-CTAATACGACTCACTATAG-GGCAAGCAGTGGTATCAACGCAGAGT-3′) and a 3′-specific primer(5′-CAAAAACCCTGTGGTATGACTTG-3′). The amplified DNAfragments were subcloned into a pGEM-T vector (Promega) andtransfected into E. coli JM109. Nucleotide sequences were deter-mined on both strands, with the BigDye™ Terminator v3.0 kit(Applied Biosystems) on the capillary DNA sequencer ABI PRISM™310 Genetic Analyser (PE Biosystems, Foster City, CA, USA).

Northern blotting

Total RNA was extracted from gut-removed bodies of pseudergatestreated with JHA for 1 h, 3 h, 6 h, 24 h, 3 days, 7 days, 14 days andinduced presoldiers. Total RNA was also obtained from secondand fourth instar larvae, pseudergates, presoldiers, soldiers, nymphs,alates and neotenics taken from natural colonies. Total RNA (20 µg)was run on a denaturing formaldehyde-agarose gel and trans-ferred to a nylon membrane (Hybond-N +; Amersham, Piscata-way, NJ, USA). Hybridization was performed using the AlkPhosDirect Labeling and Detection kit (Amersham). Briefly, the 462 bpfragment from CYP6AM1 cDNA was used as a probe andlabelled with alkaline phosphatase. Prehybridization and hybridi-zation were carried out according the instructions recom-mended by the manufacturer at 55 °C. After membrane washing,the fluorescent CDP-Star signal was detected with a Hyperfilm™ECL chemiluminescence film (Amersham).

Real-time quantitative PCR

Different tissues (legs, fat body, midguts with Malpighi tubes andsalivary glands) were dissected from pseudergates (50 individuals);decapitated bodies were obtained from presoldiers as controlCYP6AM1 non-expressing tissues. Total RNA was extracted fromeach tissue using the SV total RNA isolation system kit (Promega).Extracted total RNA was then reverse transcribed under the con-ditions described above, with the exception that random hexamerprimers were used instead of Bam-TG primer. Relative quantifica-tion of cDNAs was performed using a SYBR™ Green I chemistryand a sequence detection system ABI PRISM 7000 (AppliedBiosystems). As endogenous control of constitutive expression,we used 18S rRNA gene sequence (accession number AF220567)(Lo et al., 2000), because this housekeeping gene is convenientlyassayed and highly expressed. Primers of both target and endog-enous controls were designed using the Primer Express software(Applied Biosystems). Data analysis was performed according tothe user’s manual. Statistical tests for multiple comparisonswere preformed using One-way ANOVA and Tukey’s HSD test. Allstatistical analyses were performed using SYSTAT (SPSS Inc.,Chicago, IL, USA) statistical software.

Acknowledgements

We are grateful to David R. Nelson for his kind assistancein the naming of CYP6AM1 in accordance with current P450

nomenclature. We also thank Kiyoto Maekawa, Shin-yaSameshima, Ai Fujita, Hiroshi Katoh, Ayako Gotoh, andSakiko Hongo for their help during the field and laboratorystudies. Sumitomo chemicals (Osaka, Japan) generouslyprovided the juvenile hormone analogue. This work wassupported by a postdoctoral fellowship for foreign research-ers from the Japanese Society for the Promotion of Science(JSPS) and by Grants-in-Aid for Scientific Research(Nos. 15687001, 16370011 and 17207003), the 21st CenturyCOE program.

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