expression proﬁles of cryptochrome genes in wing dimorphic

Insect Science (2016) 23, 805–818, DOI 10.1111/1744-7917.12256

ORIGINAL ARTICLE

Molecular characterization, tissue and developmentalexpression profiles of cryptochrome genes in wing dimorphicbrown planthoppers, Nilaparvata lugens

Jing-Jing Xu1,2, Gui-Jun Wan3, Ding-Bang Hu4, Juan He5, Fa-Jun Chen3, Xian-Hui Wang6,Hong-Xia Hua4 and Wei-Dong Pan1

1Beijing Key Laboratory of Bioelectromagnetics, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing; 2University of

Chinese Academy of Sciences, Beijing; 3Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing;4Collge of Plant Science & Technology, Huazhong Agricultural University, Wuhan; 5State College of Plant Protection, Northwest Agriculture

and Forest University, Yangling, Shaanxi and 6State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of

Zoology, Chinese Academy of Sciences, Beijing, China

Abstract Cryptochromes (CRYs) are blue and UV light photoreceptors, known to playkey roles in circadian rhythms and in the light-dependent magnetosensitivity of insects. Twonovel cryptochrome genes were cloned from the brown planthopper, and were given the des-ignations of Nlcry1 and Nlcry2, with the accession numbers KM108578 and KM108579 inGenBank. The complementary DNA sequences of Nlcry1 and Nlcry2 are 1935 bp and 2463bp in length, and they contain an open reading frame of 1629 bp and 1872 bp, encodingamino acids of 542 and 623, with a predicted molecular weight of 62.53 kDa and 70.60 kDa,respectively. Well-conserved motifs such as DNA-photolyase and FAD-binding-7 domainswere observed in Nlcry1 and Nlcry2. Phylogenetic analysis demonstrated the proteins ofNlcry1 and Nlcry2 to be clustered into the insect’s cryptochrome 1 and cryptochrome 2,respectively. Quantitative polymerase chain reaction showed that the daily oscillations ofmessenger RNA (mRNA) expression in the head of the brown planthopper were mild forNlcry1, and modest for Nlcry2. Throughout all developmental stages, Nlcry1 and Nlcry2exhibited extreme fluctuations and distinctive expression profiles. Cryptochrome mRNAexpression peaked immediately after adult emergence and then decreased subsequently.The tissue expression profiles of newly emerged brown planthopper adults showed higherexpression levels of CRYs in the head than in the thorax or abdomen, as well as signif-icantly higher levels of CRYs in the heads of the macropterous strain than in the headsof the brachypterous strain. Taken together, the results of our study suggest that the twocryptochrome genes characterized in the brown planthopper might be associated withdevelopmental physiology and migration.

Key words cryptochrome; migration; molecular characterization; Nilaparvata lugens;wing dimorphism

Correspondence: Wei-Dong Pan, Beijing Key Laboratoryof Bioelectromagnetics, Institute of Electrical Engineering,Chinese Academy of Sciences, No. 6 Beiertiao, Zhongguan-cun, Beijing 100190, China. Email: [email protected]

Hong-Xia Hua, Collge of Plant Science & Technology,Huazhong Agricultural University, No.1 Shizishan Street,Hongshan District, Wuhan 430070, China. Email: [email protected]

C© 2015 Institute of Zoology, Chinese Academy of Sciences805

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Introduction

Cryptochromes (CRYs), also known as blue lightphotoreceptors, are members of the DNA pho-tolyase/cryptochrome flavoprotein family that are widelydistributed across the kingdom of plants and animals(Cashmore et al., 1999; Todo, 1999; Todo et al., 1999).CRYs were first found in cryptogamae and hence calledcryptochrome (Gressel, 1979). In plants, CRYs mediateblue light photoreception for developmental signal trans-duction. A variety of responses to light including pho-totropism, chloroplast migration and stomatal openingare regulated by CRYs in plants (Ahmad & Cashmore,1993, 1998; Lin et al., 1998; Chang & Wen, 2004). Manystudies suggest that CRYs exist in insects (Wang et al.,2011; Cheng et al., 2012) and mammals (Kobayashi et al.,1998). Drosophila possesses one CRY (dCRY) (Emeryet al., 2000) and non-drosophilid insects such as Danausplexippus have two CRYs (dpCRY1 and dpCRY2) (Zhuet al., 2005). Type 1 CRYs (such as Drosophila CRY) arephotoreceptors, and Type 2 CRYs are light-independenttranscription repressors (Yuan et al., 2007).

CRYs are well known for their crucial role in the gen-eration and maintenance of circadian rhythms (Klarsfeldet al., 2004). They may act as integral components of cir-cadian clocks (Klarsfeld et al., 2004; Ikeno et al., 2008;Wang et al., 2011). In mammals, CRYs act as transcrip-tion repressors within the circadian clockwork (Reppert& Weaver, 2002). Mouse cry1cry2 double mutant is ar-rhythmic and exhibit differentially altered free runningperiods (Vitaterna et al., 1999). Another, more recentlyobserved function of CRYs is fascinating: their partici-pation in magnetoreception (Heyers et al., 2007). Recentreports show that CRYs are associated with the sensingof magnetic fields in several species. Activation of CRYsmay affect the light sensitivity of retinal neurons, with theoverall result that the animal can, as it were, “see” themagnetic field (Solov’yov et al., 2012). Migratory birdsare known to orient themselves by using the direction andintensity of the local magnetic field as a magnetic com-pass, and CRYs are involved in this magnetic orientationduring birds’ migrations (Heyers et al., 2007). CRYs arealso essential for the light-dependent ability of Drosophilato sense the magnetic field (Gegear et al., 2008). It wasshown that cry-deficient cry0 and point-mutant cryb fliesdid not show either naive or trained responses to a mag-netic field under full-spectrum light, while wild-type fliesshowed significant naive and trained responses to the fieldunder ultraviolet-A/blue light (> 420 nm) (Gegear et al.,2008).

Circadian genes, including cryptochromes, were iden-tified as factors contributing to diapauses/reproductive

switch. They were found to be expressed in reproductiveorgans, such as mammalian ovaries, fallopian tubes andthe uterus (Johnson et al., 2002; Kennaway et al., 2003;Shimizu et al., 2012). Circadian genes including cry1/2were pointed out to be expressed in the uterus, placentaand fetal membranes of the mouse during late gestation(Ratajczak et al., 2010).

The genes period and cycle were shown to regulatephotoperiodic diapauses in bean bug Riptortus pedestrismales (Ikeno et al., 2011). In Pyrrhocoris apterus, cry2messenger RNA (mRNA) levels were much higher indiapause-promoting short days than in reproduction-promoting long days, and the expression of circadiangenes in the gut reflected the physiological state of fe-males with respect to diapause or reproduction (Bajgaret al., 2013).

Recently, bio-effects of near-zero magnetic fields werereported on the growth, development and reproductionof brown planthoppers (Wan et al., 2014), which sug-gests that brown planthoppers may also sense magneticfields. Given that CRYs of several species play a vitallyimportant role in magnetoreception, it stands to reasonthat CRYs may also be a factor in the navigation of migra-tory insects, which possibly orient themselves in relationto the Earth’s magnetic field. A thorough study of thenavigational capacity of migratory insect pests is an es-sential step toward their effective control. In this study weexamined the CRYs of brown planthoppers (Nilaparvatalugens), one such migratory insect, in which elaborateorientation mechanisms remain largely unexplored. Out-breaks of brown planthoppers, one kind of the most se-rious and destructive rice pests in large areas of South-east Asia, have resulted in catastrophically diminishedyields. Each year brown planthoppers undertake their sea-sonal migrations from Southeast Asia and across main-land China to the farthest north of Shandong Province.When the weather turns cold in the northeast, they havebeen known to migrate back to Southeast Asia. Brownplanthoppers show wing dimorphism, and they occur asmacropterous (long-winged) and brachypterous (short-winged) strains that differ in their migration abilities.Only newly emerged macropterous brown planthoppersare able to migrate over long distances (Kisimoto, 1979).Their migrating swarms may cause extensive damage torice crops. It has been observed in laboratory settings thatthe population ratio of macropterous brown planthoppersincreases annually around October, which is somehowconsistent with the observation that in the wild the brownplanthopper migrates northward around that time ofyear.

Although there have been studies on brown planthop-per’s migration-related genes like the flightin gene, the

C© 2015 Institute of Zoology, Chinese Academy of Sciences, 23, 805–818

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vitellogenin gene and neuroendocrine hormone genes(Tufail et al., 2010; Jia et al., 2013; Xue et al., 2013;Tufail et al., 2014), nothing has been published so far onthe role of CRYs in the insect’s wing dimorphism andpotential migratory mechanisms. This paper investigatesthe roles of CRYs in the wing-dimorphic brown planthop-pers and proceeds to construct a possible link betweenits CRYs and migratory mechanisms. The perspectivesof CRY expression in distinct tissues and developmentalstages are also discussed.

Materials and methods

Insect stocks

The original colony of laboratory brown planthopperswas collected from a rice field located in Wuhan, China,2011. The insects used in this experiment were reared at28 ± 1°C on TN1 rice seedlings under a photoperiod of14 : 10 h (L : D) with 70% ± 5% humidity. The first tofifth instar nymphs were sorted by appearance and bodysize. The developmental stages were synchronized at eachlarval molt. The first instar nymphs (< 12 h) were put intoa test tube (five nymphs/tube) and molting was recordedevery 12 h for the precise determination of developmentalstages.

The predominantly macropterous strain (MS) and pre-dominantly brachypterous strain (BS) were selected forsuccessive generations as described by Morooka and Tojo(1992). The percentage of brachypterous forms in BS was100%, the percentage of macropterous forms in MS wasmore than 80%. In this study, MS insects (Fig. 1A, 1B)and BS insects (Fig. 1C, 1D) were screened out by lowpopulation density plus rich nutrition and high popula-tion density plus poor nutrition, respectively (Hu et al.,2013). The laboratory was kept clean to prevent any in-fection with Beauveria bassiana or parasitic mites. Toobtain relatively stable populations, macropterous brownplanthoppers emerging in BS and brachypterous brownplanthoppers emerging in MS were removed from eachgeneration.

Amplification of complementary DNA (cDNA) fragmentsand rapid amplification of cDNA ends

Total RNA was extracted from brown planthoppers us-ing TRIzol reagent (Invitrogen, Carlsbad, CA, USA). ThecDNA templates for the polymerase chain reaction (PCR)were synthesized from 1 µg of total RNA using Prime-ScriptTM RT reagent Kit with gDNA Eraser (TaKaRa,Tokyo, Japan). All the primers (showed in Table S1) were

Fig. 1 Wing dimorphic adults of brown planthoppers.Brown planthoppers used in this experiment were rearedat 28°C±1°C on TN1 rice seedlings under a 14 : 10 hlight : dark photoperiod 70% ± 5% humidity. Brown plan-thoppers undergo five instars to emerge into adults. Af-ter adult emergence, the insects were sorted by wing formsand sex: (A) macropterous female, (B) macropterous male,(C) brachypterous female and (D) brachypterous male.

designed using Primer Premier 5.0. PCR was performedon the Light Cycler instrument (Roche Applied Science,Mannheim, Germany) with the following cycling pro-gram: 94°C for 3 min followed by 30 cycles of 45 s at94°C, 45 s at 60°C and 60 s at 72°C. Amplification wasterminated with a 10 min final extension step at 72°C.After the PCR products were verified by 2% agarose gelelectrophoresis, they were ligated to the pGEM-T vec-tor (Promega, Madison, WI, USA) and the sequencesof cloned fragments were determined by BGI (Wuhan,China). The RNA ligase mediated rapid amplificationof cDNA ends (RLM-RACE) technique was applied toobtain the full-length CRY cDNAs. The 5′ and 3′ RLM-RACE templates were synthesized from 10 µg of totalRNA using FirstChoice R© RLM-RACE Kit (Invitrogen,USA) according to the manufacturer’s instructions. The


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universal primers for the 5′ and 3′ RACE were providedby the RACE kit, and the gene-specific primers were de-signed on CRY cDNA fragments (Table S1). To increasespecificity and yield, touchdown PCR was utilized (Kor-bie & Mattick, 2008). The cycling program was 94°C for3 min followed by an initial 10 cycles of 45 s at 94°C, 45s at 70°C (decreased 1°C each cycle), 60 s at 72°C, and30 final cycles of 45 s at 94°C, 45 s at 60°C, 60 s at 72°Cand 10 min at 72°C.

Phylogenetic inference and Bioinformatic analysis

Evolutionary relationships within the CRY family wereinvestigated by the neighbor-joining (NJ) method. TheNJ tree was constructed by MEGA6.0 (Tamura et al.,2013) using a complete deletion mode. The statisticalsignificance of the NJ tree topology was evaluated bybootstrap analysis (Felsenstein, 1985) with 5000 iterativeconstructions of the tree. Poisson correction distance wasadopted and rates among sites were set as uniform. Theamino acid sequences of CRYs were retrieved from THENational Center for Biotechnology Information (NCBI).Accession numbers used in the phylogenetic inference aregiven in Table S2.

The 5′ untranslated region (UTR) and 3′ UTR were de-termined with SIM4 online tools (Florea et al., 1998). Thealignment of multi-sequences was made using DNAMANand Weblogo (Crooks et al., 2004). The protein transla-tion and motif analysis were made with proteomics serverExpasy, Translation tool interface and Scan Prosite inter-face. Protein functional domains and secondary structureprediction were made with Smart online tools (Letunicet al., 2009) and the PBIL SOPMA program (King &Sternberg, 1996).

The two CRY proteins of the brown planthopper werealigned with the PDB database (ftp://ftp.wwpdb.org/pub/pdb/derived_data/pdb_seqres.txt.gz) by using BLAST+2.2.28 (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.2.28/). The most similar sequences were used astemplates for homology modeling in SWISS-MODEL(http://swissmodel.expasy.org/). The tertiary structureprediction and surface charge distribution of CRYs werecarried out by using Swiss-Pdb Viewer (http://spdbv.vital-it.ch/).

Expression profiles of CRYs established by quantitativereal-time PCR

To investigate circadian rhythmic expression patternsof cry, adult insects were collected at 4-h intervalsduring the second day after emergence, and total RNA

was extracted from their heads. The cDNAs of brownplanthopper nymphs and adults (from the first to eighthdays after emergence, namely A1 to A8) were used astemplates to investigate the expression profiles of cry ateach developmental stage. The first to fifth instar nymphsand the adults (A1 to A8) were collected on the duedate during the same time period (19:00–20:00 hours).Macropterous and brachypterous adults were collectedseparately on day 1 (A1), day 5 (A5) and day 7 (A7)after emergence for tissue expression analysis. The tissueswere dissected under a stereomicroscope in phosphatebuffered saline (PBS), treated with 0.1% diethylpyrocar-bonate (DEPC) and immediately frozen in liquid nitrogento be stored at -80°C for further operations. The individualbrown planthopper was dissected into three pieces, head,thorax (with legs and wings) and abdomen. Total RNAwas extracted by TRIzol reagent (Invitrogen). The qualityof samples was determined by spectrophotometric opticaldensity (OD)260/280 and 2% agarose gel electrophoresis.

The cDNA templates were synthesized with 1 µg oftotal RNA using PrimeScriptTM RT reagent Kit withgDNA Eraser (TaKaRa, Japan). Each cDNA product wasdiluted from 20 µL to 60 µL with sterilized double dis-tilled water. For fluorescence-based quantitative real-timePCR (qPCR) (Bustin et al., 2009), 2 µL cDNA dilutionand 0.2 µmol/L primers were used in 1× SYBR R© PremixEx TaqTM (TaKaRa, Japan) with 7300 Real-Time PCRSystem (Applied Biosystems, Foster City, CA, USA) ac-cording to the supplier’s instructions. Primers used forcrys and control gene (actin1) are shown in Table S1. ThePCR amplification efficiency was established by meansof calibration curves (Bustin et al., 2009). The optimizedthermal program was set according to the instructions forthe kit. Quantification of the transcript level or relativecopy number of genes was conducted according to the��Cq method (Livak & Schmittgen, 2001). RNA sam-ples were analyzed independently for three times.

Data analysis

The quantitative PCR was repeated three times, eachtime with independently extracted total RNA. Each re-action was performed in three replicates to minimizeintra-experiment variations. All statistical analyses wereperformed by using SPSS 20.0 (SPSS Inc., Chicago, IL,USA). The relative transcript levels of crys during Zeit-geber Times (ZTs) and developmental stages were ana-lyzed using Scheffe correction for multiple comparisons atP < 0.05. The circadian rhythmic mRNA expression lev-els of crys between female and male adults were analyzedby repeated measures at P < 0.05. The relative transcript



levels of crys in corresponding tissues of MS or BS wereanalyzed using two-tailed Student’s t-test. The significantdifferences between tissues of MS or BS were analyzedusing Bonferroni correction for multiple comparisons atP < 0.05. Moreover, the relative transcript levels of eachgene were analyzed using two-way analysis of variance(ANOVA) with wing dimorphism (WD) as main factor(macropterous vs. brachypterous) and tissues (head vs.thorax vs. abdomen) as sub-factors.

Results

cry cDNAs

Two cDNA clones of cry genes with full length se-quences were obtained. Determination of their putativeamino acid sequences showed that both of them are mem-bers of the CRY family. The two genes were designatedas Nlcry1 and Nlcry2, and the corresponding proteinswere designated as NLCRY1 and NLCRY2, respectively.The complete nucleotide sequence length of Nlcry1 was1934 bp, which contained an open reading frame (ORF)of 1629 bp and encoded a protein with 542 amino acidresidues (Fig. S1A). The deduced molecular weight andisoelectric point of Nlcry1 were 62.5 kDa and pI 5.72, re-spectively. The nucleotide sequence of Nlcry2 is 2463 bpin full length (Fig. S1B). It contained an ORF of 1872 bpthat encoded a protein with 623 amino acid residues anda predicted molecular weight of 70.6 kDa.

Domains of DNA photolyase and FAD binding 7,and muti-domains of PhrB (deoxyribodipyrimidine pho-tolyase), photolyase 8HDF and PRK10674 were identi-fied in both presumptive amino acid sequences of Nlcry1and Nlcry2. These domains, used as a signature pattern inthe CRY family (Brautigam et al., 2004), appeared highlyconserved in the brown planthopper, as they are in otherinsects.

Phylogenetic inference

The results showed that the total 55 CRY proteins weredivided into five clusters: vertebrata CRY1, vertebrataCRY2, insect CRY1, insect CRY2 and plant CRY (con-taining CRY1 and CRY2). The constructed NJ tree (Fig. 2)was similar in topology to the tree that had been previouslyreported (Cheng et al., 2012). On the tree, NLCRY1 di-verged earlier than other insects except for Acyrthosiphonpisum, suggesting that NLCRY1 can be recognized as anancestor gene in the evolution of CRY proteins. How-ever, NLCRY2 bifurcated in a later evolutionary era andclustered together with the CRY in Acyrthosiphon pisum,

indicating a close relationship of CRY origin between thetwo species.

Bioinformatic analysis

The protein secondary structures of NLCRY1 and NL-CRY2 consist of four structural units: alpha helix (Hh),extended strand (Ee), random coil (Cc) and beta turn (Tt).The percentages of Hh, Ee, Cc and Tt in NLCRY1 and NL-CRY2 are 37.45% versus 32.74%, 11.25% versus 9.95%,44.83% versus 50.72% and 6.46% versus 6.58%, respec-tively. Although the Hh and Cc are the main structuralforms in both CRYs, the Cc in NLCRY2 shows a higherpercentage than that in NLCRY1.

In the PDB, NLCRY1 shared the greatest amino acididentity with the Cryptochrome-A (PDB ID: 4jzy) inDrosophila (56.6%). NLCRY2 shared the greatest aminoacid identity with the Cryptochrome-A (PDB ID: 4k0r) inMus musculus (56.6%). Therefore, the Cryptochrome-Ain Drosophila and Mus musculus were used as tem-plates for homology modeling in NLCRY1 and NLCRY2,respectively (Fig. S2). The FAD-binding domain was de-termined based on the method as previously described byDym and Eisenberg (2001). The cavity structures wrappedin the alpha helix were labeled as FAD-binding domain(Fig. S2 A1 and Fig. S2 B1). The surface charge of thetwo proteins appeared to be different with more negativecharge deployed in NLCRY1 and more positive chargedeployed in NLCRY2. The calculation showed the pI ofNLCRY1 and NLCRY2 to be 5.72 and 8.96, respectively.The surface charge of NLCRY1 remained mostly negative,and only the area near the FAD-binding domain displayedsome positive charge enrichment. Such a positive chargeenrichment near the FAD-binding domain occurs in bothproteins, and the positive charge ratio in the FAD bind-ing pocket of NLCRY1 appeared to be less than that ofNLCRY2.

Four highly similar sequences were selected for thesequence alignment (Fig. S3). NLCRY1 displayed aminoacid identity with the CRY1 in Bombyx mori (56.55%), inHelicoverpa armigera (60.31%), in Mythimna separata(59.92%) and in Acyrthosiphon pisum (55.22%). On theother hand, NLCRY2 displayed higher amino acid identitywith the CRY2s in Apis mellifera (72.08%), in Bombusimpatiens (72.44%), in Pyrrhocoris apterus (75.43%) andRhyparobia maderae (72.49%).

The multiple sequence alignment with 14 CRYs showedthat the N-terminal appeared more conservative than theC-terminal except for the difference in length of the tworegions (Fig. S4). Further analysis of CRY protein motifsshowed that there were 12 motifs in the 14 CRY proteins.


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Fig. 2 The phylogenetic tree organized with the CRYl and CRY2 proteins. The tree was constructed by MEGA6.0 with a completedeletion mode according to the neighbor joining (NJ) method. The statistical significance of the NJ tree topology was evaluated bybootstrap analysis with 5000 iterative constructions. Poisson correction distance was adopted and rates among sites were set as uniform.The bootstrap probability for each node was enclosed with probability � 90.0% as the criterion for statistical significance. The aminoacid sequences of CRY were retrieved from NCBI. Accession numbers used in the phylogenetic inference are given in Table S2. TheCRY1 and CRY2 of the brown planthopper are marked by triangles.

As shown in Fig. 3, all of the 14 CRY proteins possessedthe protein kinase C phosphorylation site, the casein ki-nase II phosphorylation site, the N-myristoylation site, andthe photolyase/cryptochrome alpha/beta domain profile.The motifs of both NLCRY1 and NLCRY2 showed enor-mous similarity with the CRYs of Acyrthosiphon pisum,except that NLCRY1 and Acyrthosiphon pisum CRY2both contained the amidation site, whereas NLCRY2 alonecontained the cell attachment sequence.

Circadian rhythmic expression profiles

Quantitative PCR analysis revealed that the relativemRNA level of Nlcry1 in the heads of adults on day 2did not exhibited any daily oscillations (Fig. 4A–4D).No significant differences among ZTs were detected byANOVA (P > 0.05). However, the expression levels ofNlcry1 were significantly different between female andmale adults (repeated measures, P < 0.05, for both wing



Fig. 3 The motif alignment with other CRY proteins. The height of columns indicates the number of motifs. The scale for columnheight is indicated with different colors, as shown in the legend. The CRY protein accession numbers from the left to the right are asfollows: Anopholes gambiae CRY1: DQ219482.1; Acyrthosiphon pisum CRY1: 283476380; Bombyx mori CRY1: NM_001195699.1;Nilaparvata lugens CRY1: KM108578; Danaus plexippus CRY1: AY860425.1; Mus musculus CRY1: BC085499.1; A. gambiae CRY2:DQ219483.1; Apis mellifera CRY2: NM_001083630.1; A. pisum CRY2-1: 283806649; Bombus impatiens CRY2: NM_001280122.1;B. mori CRY2: NM_001195698.1; N. lugens CRY2: KM108579; D. plexippus CRY2: DQ184682.1; M. musculus CRY2: AF156987.1.

forms). Significant differences between female and maleadults were detected only at ZT8 for macropterous brownplanthoppers and brachypterous brown planthoppers(t-test, P < 0.05). Modest and consistent daily oscilla-tions were observed in Nlcry2 mRNA levels. As shown inFig. 4E–4H, Nlcry2 expressed at significantly differentlevels among ZTs (ANOVA, P < 0.05, in all four ex-periments). Nlcry2 transcript levels were low during theday, started to increase in the evening, and reached theirmaxima late at night (ZT20) or early in the morning (ZT0).For the four experimental groups, peak/trough ratios were3.4, 4.5, 3.9 and 3.0 in turn. Generally, the temporal ex-pression pattern of Nlcry2 was similar in female and maleadults (repeated measures, P > 0.05, for both wing forms).Neither cry expressed differences in mRNA levels be-tween female and male adults at ZT12 (t-test, P > 0.05).To sum up, the four group experiments revealed no dailyoscillations but different expression patterns for Nlcry1

as well as modest and consistent daily oscillations forNlcry2.

Developmental expression profiles

The expression of both Nlcry1 and Nlcry2 clearly ex-hibited up-and-down fluctuations during the entire periodof growth and development (Fig. 5). Both crys showedhigh expression levels in the first instar nymph, with asubsequent drop. Besides the expression levels in the firstinstar, fifth instar and on A1, the expression of Nlcry1showed significantly higher levels on A2 (relative level= 1.12, P < 0.05) than at other developmental stages.The oscillating expression pattern of Nlcry2 was similarto that of Nlcry1. The peak expression of Nlcry2 occurredon A1 (relative level = 1.50, P < 0.05), except for the ex-pression level in the first instar. The expression of Nlcry1


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Fig. 4 The circadian rhythmic messenger RNA (mRNA) expression of Nlcry1 (A–D) and Nlcry2 (E–H) in the head of brown planthopperadults. The relative mRNA levels of Nlcry1 and Nlcry2 (the ratios to actin expression) are plotted. Lines represent the means of threeindependent samples. For each assay, the value of mRNA expression at Zeitgeber times (ZT)0 was set at 1.0 as the calibrator. Thedifferent lowercase letters indicate significant differences among ZTs (ANOVA, P < 0.05; Scheffe post hoc test, P < 0.05).

and Nlcry2 decreased to the marginally lowest level of0.34 on A5 and 0.22 on A3, respectively. During the lateradult stages, the expression of Nlcry1 increased slowlyfrom 0.34 to 0.67 (A5 to A8). For Nlcry2, the expressionlevel remained relatively steady from A4 to A8.

Tissue expression profiles

More precise expression patterns were determined forthe tissues (head, thorax and abdomen) of both macropter-ous and brachypterous brown planthoppers (Fig. 6). On



A1, the expression levels of both Nlcry1 and Nlcry2 inthe head of macropterous brown planthoppers were sig-nificantly higher than those in the heads of brachypterousbrown planthoppers (P < 0.01 and P < 0.05, respectively).No significant difference was observed on A5 in the ex-pression of either of the two CRYs between the differentwing forms or tissues of the brown planthopper. On A7,significant differences were detected in the abdomen forboth crys between macropterous and brachypterous brownplanthoppers (P < 0.05 and P < 0.01, respectively). ForNlcry1, the expression levels in the head of macropterousbrown planthoppers were higher than in other tissues onA1, A5 and A7 (P < 0.05). In the heads of the brachypter-ous brown planthoppers, the expression levels of Nlcry1were higher than that in other tissues only on A1 andA5 (P < 0.05). For Nlcry2, the expression levels in theheads of macropterous brown planthoppers were higherthan in other tissues only on A1, albeit the difference wasnot significant. The abdomen expressed the highest levelof Nlcry2 among the three tissues on A7 (P < 0.05). Asshown in Table 1, the expression levels of Nlcry1 in themacropterous brown planthoppers on A1 and A5 weresignificantly different from those in the brachypterousbrown planthoppers (P < 0.001 and P < 0.05). On A7,the expression levels of Nlcry2 in the macropterous brownplanthoppers were significantly different from those inthe brachypterous brown planthoppers (P < 0.001). Theexpression of either cry showed significant differencesbetween tissue types at all sampling time points (A1, A5,and A7) (P < 0.01). Interaction between wing dimor-phism and type of tissue was found on A1 for Nlcry1(P < 0.001).

Discussion

CRYs are evolutionarily ancestral proteins that belong tothe flavorprotein superfamily (Brautigam et al., 2004).During evolution, some species lost certain types ofcry genes so that only one type of CRY protein re-mained in their genome (Wang et al., 2011). Although theCRYs appear to be ubiquitous in living organisms, dif-ferent species have different types of CRYs. Drosophilapossesses only CRY1, while Hymenoptera insects such asApis mellifera, Bombus terrestris and Coleoptera insectssuch as Tribolium castaneum have only CRY2. Conserveddomain analysis revealed that the putative proteins exam-ined in this study belong to the CRY family, which gener-ally contains PhrB, DNA photolyase, and FAD binding 7.Previous research has shown that another Hemiptera in-sect, Acyrthosiphon pisum, also possesses two cry genes

Fig. 5 The relative expression of Nlcry1 (A) and Nlcry2 (B)at different developmental stages. The expression levels wereexpressed as mean ± SE (n = 3), with the first instar nymphas the calibrator. Different lowercase letters indicate signifi-cant differences among developmental stages by Scheffe test atP < 0.05.

(Cortes et al., 2010). Whether all Hemiptera insects con-tain two cry genes remains to be determined.

In the NJ tree, CRYs in plants and animals are alignedseparately. They may originate from diverse ancestors:CRYs in plants have apparently evolved from type I pho-tolyases, while CRYs in animals have evolved from 6–4photolyases (Cashmore et al., 1999). Phylogenetic anal-ysis confirmed that the two CRY proteins in the brownplanthopper were clustered into the insect CRY1 andCRY2. Unlike Cheng et al.’s report (2012), our studyshowed that insect CRY1 could be clustered by or-der. This difference may stem from the fact that twoHemiptera species were considered in establishing theNJ tree of CRY1 and CRY2, while Cheng et al.’s reportdealt with only one species (Cheng et al., 2012). ForCRY2, the bootstrap values (<75) were such that some


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of the nodes may not completely represent the evolu-tionary history. It is also worth noting that the CRY1/2distinction is not restricted to insects, as other inver-tebrates such as molluscs share the same phylogeneticgroupings.

The right-handed Hh was a major secondary structureof the CRY protein. In agreement with a previous report(Lovell, 2000), we speculated that Hh was the basis ofCRY functional structure. CRYs are known to derive fromphotolyases, which are known to be involved in the re-pair of UV-induced DNA damage. In eukaryotes, CRYsno longer retain this original enzymatic activity (Weber,2005). The predicted ditch at the FAD-binding domain ofNLCRY1 and NLCRY2 could possibly be connected withthe fact that CRY proteins in mammals and plants lackphotolyase activity (Malhotra et al., 1995). NLCRY1 andNLCRY2 showed some difference in their surface chargedistribution, especially in the FAD binding pocket. Thismay be related with differences in the functions of the twoCRYs.

Compared with the CRYs of other insects, the deducedamino acid sequences of Nlcry1 and Nlcry2 showed somedifference. In general, the identity of NLCRY2 (identity> 70%) is higher than that of NLCRY1 (identity<65%).Therefore, NLCRY2 is more conservative than NLCRY1among different insects. The motifs of the protein ki-nase C phosphorylation site, the casein kinase II phos-phorylation site, the N-myristoylation site, and the pho-tolyase/cryptochrome alpha/beta domain profile exist inall the CRYs, and these four motifs can thus be consideredto be more conservative than other motifs, which suggeststhat they may be closely connected with the functions ofCRYs.

CRYs have been studied as circadian clock genes insome agricultural pests, including Agrotis ipsilon (Chenget al., 2012), Acyrthosiphon pisum (Cortes et al., 2010)and Riptortus pedestris (Ikeno et al., 2008). In this study,

Nlcry1 displayed no daily oscillations. That would beconsistent with the idea that dCRY is the primary circadianphotoreceptor and that it is not necessary for rhythm gen-eration (Rubin et al., 2006). In contrast, Nlcry2 expressedmodest rhythmic oscillations. The clock genes, such asper mRNAs of D. melanogaster and A. mellifera and vrimRNA of D. melanogaster, are down-regulated in thephotophase and up-regulated in the scotophase, exhibitingapparent daily oscillations (So & Rosbash, 1997; Cyranet al., 2003; Rubin et al., 2006). For cry-m of A. mellifera,the expression level in the scotophase is up to 6.6 timesthe level in the photophase (Rubin et al., 2006). Sincebrown planthoppers migrate soon after adult emergence,we took samples at different developmental stages to de-tect the expressions of Nlcry1 and Nlcry2. The expressionof Nlcry1 was significantly up-regulated on the secondday, that of Nlcry2 significantly up-regulated on the firstday after emergence. The expression levels of Nlcry1 andNlcry2 in the head of macropterous brown planthop-per was significantly higher than that in the head ofbrachypterous brown planthopper on the first day (P <

0.01 and P < 0.05, respectively), while approximatelyequal expression levels were observed on the fifth andseventh days. The expression levels of Nlcry1 and Nlcry2in the abdomen of macropterous brown planthoppers weresignificantly lower than in the abdomen of brachypter-ous brown planthoppers (P < 0.05 and P < 0.01, re-spectively). Two-way ANOVA analysis showed a signif-icant difference in the expression levels of Nlcry1 be-tween macropterous and brachypterous brown planthop-pers on A1 and A5 (P = 0.000 and P = 0.02, respec-tively) (Table 1), while the expression levels of Nlcry2 inbrachypterous brown planthoppers showed significantlyhigher values than in brachypterous brown planthoppersonly on A7. These different expression patterns suggestthat the two crys possess different functions. Generallyon the seventh day after emergence, macropterous brown

Table 1 The two-way ANOVA results (F/P values) with wing dimorphism (WD) as main factor (macropterous vs. brachypterous) andtissues (head vs. thorax vs. abdomen) as sub-factor on the relative expression of Nlcry1 and Nlcry2.

WD Tissues WD × tissues

Relative expression of Nlcry1First day after emergence 173.01/0.000*** 249.64/0.000*** 68.363/0.000***

Fifth day after emergence 7.12/0.020* 71.688/0.000*** 3.330/0.071Seventh day after emergence 0.273/0.611 13.822/0.001** 0.501/0.618

Relative expression of Nlcry2First day after emergence 4.183/0.063 38.811/0.000*** 0.699/0.516FIfth day after emergence 0.849/0.375 10.406/0.002** 1.097/0.365Seventh day after emergence 32.659/0.000*** 61.879/0.000*** 2.165/0.157

*P < 0.05; **P<0.01; ***P< 0.001.



Fig. 6 The relative expression of Nlcry1 (A, B, C) and Nlcry2 (D, E, F) in respective tissues with different developmental stages. Thecolumns represent averages with vertical bars indicating SE. Different lowercase and uppercase letters indicate significant differencesamong different tissues of brachypterous and macropterous adults, respectively, by Bonferroni test at P < 0.05; Significant differencesbetween brachypterous adults and macropterous adults for the same tissue were measured by t-test at P < 0.05 and P < 0.01.

planthoppers accomplish their migratory flight and landon new habitats, while brachypterous brown planthoppersremain close to their place of emergence, hence the resultssuggest that Nlcry1 possesses a type of constitutional ex-pression in the migratory population of brown planthop-per, while the Nlcry2 shows more dynamic changes in theadaptation of circadian rhythms.

Conclusion

To sum up, two different types of cryptochrome genes,Nlcry1 and Nlcry2, were cloned with their full lengthand analyzed with regard to the structural features andevolutionary position of the brown planthopper. More-over, the expression profiles of both crys at different ZT


816 J. J. Xu et al.

points, developmental stages and in different tissues inmacropterous versus brachypterous brown planthopperswere investigated. Based on our results, it is likely thatthe two CRYs might play different roles in the develop-mental physiology of the brown planthopper. However, itwould be premature to relate our findings directly to thefunction of brown planthopper cryptochromes as magne-toreceptors for orientation and navigation. Further workshould focus on the function of cryptochromes as mag-netoreceptors in the context of the brown planthopper’smigratory behavior.

Acknowledgments

We thank the staff in the Beijing READ BIO Bioin-formatic Technology Company for their assistance inthe phylogenetic inference and bioinformatic analysis ofbrown planthopper CRY proteins. This research was sup-ported by the Key Program of National Natural Science ofChina (51037006), the National Basic Research Programof China ‘‘973’’ (2010CB126200) and the National Na-ture Science Foundations of China (31170362, 31272051,31470454 and 31070755).

Disclosure

All authors declare no conflict of interest.

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Accepted July 20, 2015

Supporting Information

Additional Supporting Information may be found in theonline version of this article at the publisher’s web-site:

Fig. S1 The cDNA sequences and deduced aminoacid sequences of Nlcry1(A) and Nlcry2 (B) inbrown planthoppers. The start codon, stop codon, andpolyadenylation signal were marked by gray shadow.The DNA-photolyase domains and the FAD-binding-7

domains were marked by red boxes and yellow boxes,respectively.

Fig. S2 Protein tertiary structure and surface chargedistribution of NLCRY1 and NLCRY2. DrosophilaCryptochrome-A (PDB ID: 4jzy) and Mus muscu-lus Cryptochrome-A (PDB ID: 4k0r) were used astemplates to homology modeling in SWISS-MODEL(http://swissmodel.expasy.org/) respectively. The 3Dstructures were observed and the surface charge dis-tribution was calculated by using Swiss-PdbViewer(http://spdbv.vital-it.ch/). A1: Protein tertiary structureof NLCRY1; A2: surface charge distribution of NL-CRY1; B1: Protein tertiary structure of NLCRY2;B2: surface charge distribution of NLCRY2. FADsof protein tertiary structure in were marked by redshadows. For A2 and B2, the blue and red colordescribed positive and negative electrostatic potential,respectively.

Fig. S3 Amino acid sequence alignment of CRY1(A) and CRY2 (B). The sequence alignment was madeusing DNAMAN. Gaps have been introduced to per-mit alignment, and the gap penalty was 3. Identi-cal amino acids (100%) were highlighted in dark blueboxes. Amino acids of 75% similarity were shownin red boxes, and amino acids of 50% similaritywere shown in light blue boxes. The CRY proteinsaccession numbers are as followed. B. mori CRY1:NM_001195699.1; H. armigera CRY1: JQ713131.1;M. separata CRY1: JX077108.1; A. pisum CRY1:283476380; A. mellifera CRY2: NM_001083630.1;B. impatiens CRY2: NM_001280122.1; P. apterusCRY2: AGI17567.1; R. maderae CRY2: AGA01579.1.N. lugens CRY1: KM108578; N. lugens CRY2:KM108579.

Fig. S4 The Weblogo analysis with 14 kinds of CRYproteins. The multiple sequence alignment of 14 se-lected CRYs was performed by Weblogo online anal-ysis tool. The conservatism of one residue was di-rectly related to its height. The CRY proteins ac-cession numbers are as followed. A. gambiae CRY1:DQ219482.1; A. gambiae CRY2: DQ219483.1; A. pisumCRY1: 283476380; A. pisum CRY2-1: 283806649;B. mori CRY1: NM_001195699.1; B. mori CRY2:NM_001195698.1; D. plexippus CRY1: AY860425.1;D. plexippus CRY2: DQ184682.1; M. musculus CRY1:BC085499.1; M. musculus CRY2: AF156987.1; A. mel-lifera CRY2: NM_001083630.1; B. impatiens CRY2:NM_001280122.1. N. lugens CRY1: KM108578; N. lu-gens CRY2: KM108579.

Table S1 Primers used in the study.Table S2 Accession numbers of CRY proteins used in

the study.


expression proﬁles of cryptochrome genes in wing dimorphic

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