Characterization of Gene Expression Patterns During the Initiation And

Journal of Insect Physiology 55 (2009) 32–39

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Journal of Insect Physiology

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Characterization of gene expression patterns during the initiation and maintenance phases of diapause in the Colorado potato beetle, Leptinotarsa decemlineata§ George D. Yocum a,*, Joseph P. Rinehart a, Anitha Chirumamilla-Chapara b, Marnie L. Larson a a USDA-ARS Red River Valley Agricultural Research Center, Biosciences Research Laboratory, 1605 Albrecht Boulevard, Fargo, ND 58105, USA b Department of Entomology, North Dakota State University, Hultz Hall 202, Fargo, ND 58105, USA

ARTICLE INFO ABSTRACT

Article history: Using differential display, 55 differentially regulated transcripts were isolated from the Colorado potato Received 20 August 2008 beetle, Leptinotarsa decemlineata (Say). The insert sizes of the clones ranged from 114 to 795 bp. Fourteen Received in revised form 26 September 2008 of the transcripts were confirmed by northern blot analysis to be differentially regulated transcripts with Accepted 9 October 2008 respect to the diapause initiation and maintenance phases. Based on Blast search results, these 14 transcripts were assigned putative identities and placed into four broad categories of proteins: unknown Keywords: function, defensive, structural/glycine-rich, and digestive. The transcripts were highly expressed for the Leptinotarsa decemlineata first 13–15 days postemergence during the diapause initiation and early diapause maintenance phases Adult diapause and were then substantially down-regulated. These down-regulated transcripts were also highly Diapause initiation Diapause maintenance expressed for the first seven days postemergence in nondiapausing adults and their expression became more variable on day 9 or 11 in most individuals examined. The glycine-rich protein transcripts were all down-regulated by day 11 in the nondiapausing adults. A comparison of the transcript expression patterns between diapause initiation phase and nondiapausing adults showed that elevated levels of expression of the glycine-rich transcripts and two transcripts with unknown functions persisted for approximately four days longer in the diapause-programmed beetles. ß 2008 Elsevier LtdElsevier Ltd. All rights reserved.

1. Introduction Insects are challenged by both predictable and unpredictable fluctuations in environmental conditions that are unfavorable for The Colorado potato beetle, Leptinotarsa decemlineata (Say), is survival, development and reproduction. Dormancy in the form of the major defoliator of potato and also feeds on tomato and diapause or quiescence enables insects to survive these challenges eggplant. The center of origin of L. decemlineata is believed to be and synchronize their life cycles to the abiotic and biotic factors southern Mexico and the beetle experienced a rapid range needed for development and reproduction (Tauber et al., 1986; expansion in the mid-1800s. L. decemlineata was first reported Danks, 1987). Diapause is a dynamic physiological developmental as a pest of potato, tomato and eggplant in 1859–62 in the Iowa, process that can be divided into three general phases: prediapause, Kansas, and Nebraska regions of the central plains of North diapause, and postdiapause (Tauber et al., 1986; Danks, 1987; America (Tower, 1906); by 1877, it was discovered in potato fields Kostal, 2006). The diapause phase can be further subdivided into in Germany (Clark, 2007), and it was established in Europe by 1921 initiation and maintenance phases. Based on Kostal’s (2006) (de Wilde and Hsiao, 1981). The Colorado potato beetle is now morphogenesis classification system, the diapause initiation phase endemic in most of the potato-growing regions worldwide (IP) of the Colorado potato beetle begins at adult emergence and (Gauthier et al., 1981; Ferro, 1985; Weber and Ferro, 1994; EPPO, continues until the diapause maintenance phase (MP) starts which 2006). is characterized by a sharp decrease in the metabolic rate. Here we study the diapause IP and MP in the Colorado potato beetle: the period when the environmental and physiological signals inducing the diapause program are received and processed, § Mentioning of trade names or commercial products in this article is solely for physiological and behavioral changes occur that lead to the the purpose of providing specific information and does not imply recommendation selection of appropriate diapausing sites, metabolic reserves are or endorsement by the U.S. Department of Agriculture. * Corresponding author. Tel.: +1 701 239 1301; fax: +1 701 239 1348. stockpiled and finally metabolic rates are suppressed and E-mail address: [email protected] (G.D. Yocum). maintained at low levels even under conditions that would permit

0022-1910/$ – see front matter ß 2008 Elsevier LtdElsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2008.10.003 G.D. Yocum et al. / Journal of Insect Physiology 55 (2009) 32–39 33 direct development. The underlying molecular aspects of these directly to a preheated (94 8C) thermocycler. 10 ml of each sample critical processes governing insect diapause are still undeﬁned was separated on a 2% agarose gel containing ethidium bromide. (Denlinger, 2002). The aim of this investigation is threefold: (1) to Bands of potentially differentially ampliﬁed amplicons were determine the start of the diapause MP in the Red River Valley of excised from the gels and the cDNA isolated using GENECLEAN1 the North strain of the Colorado potato beetle, (2) to characterize II Kit (MP Biomedicals), cloned into pCR 2.1-TOPO (Invitrogen) and gene expression patterns during the IP and MP phases of diapause sequenced. and (3) to examine the expression patterns of genes of interest from objective two during the early gonotrophic cycles in 2.5. Northern blot analysis nondiapausing adults. Five micrograms of total RNA per sample was separated on a 1% 2. Materials and methods denaturing agarose gel (0.41 M formaldehyde, 1Â MOPS–EDTA–

NaC2H3O2). All samples contained ethidium bromide, and after 2.1. Insects electrophoresis a photograph of the gel was taken to compare the intensity of the rRNA bands to insure equivalent loading of L. decemlineata was obtained from colonies maintained at the samples. The RNA was transferred overnight onto a positively Red River Valley Agricultural Research Center, USDA-ARS, Fargo, charged nylon membrane (Roche) using 20Â SSC DEPC-treated

ND. To obtain nondiapausing adults, larvae and pupae were buffer (3 M NaCl, 0.3 M NaC6H5O7Á2H2O, pH 7.0). The RNA was then maintained at 16 h light:8 h dark:24 Æ 2 8C and 65% relative UV cross-linked to the membrane at 12,000 mJ/cm3 and stored at humidity. The larvae were fed potato (Solanum tuberosum L. ‘Luther À20 8C. Prehybridization and hybridization were carried out in DIG Burbank’) plants. Once the adults emerged they were transferred Easy Hyb1 hybridization buffer (Roche). The DIG High Prime DNA 1 from the emergence cage to 10 cm Â 10 cm Â 21 cm plastic contain- Labeling and Detection Start Kit II (Roche) was used to detect the ers and provided potato leaf bouquets held in water bottles and digoxigenin-labeled probes. Membranes were stripped by washing maintained under nondiapause-inducing conditions. Diapause IP and the membranes twice at 80 8C for 1 h in stripping buffer (50% MP adults were obtained by rearing larvae and pupae at 8 h light, 16 h molecular grade formamide, 5% SDS, 50 mM Tris–HCl pH 7.5). dark, 24 Æ 2 8C and 65% relative humidity. Adults were reared as Following stripping, the membranes were washed twice for 5 min above except they were held under diapause-inducing conditions. On in 2Â SSC. Membranes were only stripped three times; after the day 20 after adult emergence the diapausing adults were placed in third stripping, the membranes were probed with a control gene to moist vermiculite, double-bagged in resealable plastic bags and insure equivalent transfer of RNA onto the membranes. stored at 5 8C in darkness. 2.6. Bioinformatics 2.2. Respirometry The Blast program was used to search the GenBank sequence At selected intervals during the first 20 days postemergence, repository for possible sequence identities (Altschul et al., 1997). If constant volume respirometry was used to measure O2 consump- the BlastX search of the non-redundant protein sequences (nr) tion and CO2 production in diapause IP and early MP beetles. database failed to find a possible significant hit on a sequence, the Oxygen consumption was measured using a Sable System search was rerun with the low complexity region filter turned off. If

International Oxzilla II oxygen analyzer and CO2 production was the BlastX search strategy failed to yield a possible identity, a measured using a Li-Cor Model LI-6252 CO2 analyzer. Respiro- BlastN search was carried out of the nucleotide collection (nr/nt) metry analysis was carried out in differential mode with a ﬂow rate database. All the sequences were deposited in GenBank and of 100 ml/min at 24 8C. Seven to 14 beetles were measured per assigned accession numbers (Table 1). time point with duration of measurements varying from 10 min to 2 h depending on the age of the beetles. The data were collected 3. Results and analyzed using the Sable data acquisition program ExpeData version 1.0.15. For purpose of comparison, the data were adjusted 3.1. Respirometry by the individual weights of beetles and presented as ml/g/h. To more accurately determine the timing of the transition 2.3. RNA isolation between the diapause IP and MP of the Red River Valley of the North (USA) strain of Colorado potato beetle under our laboratory Total RNA was isolated from days 1, 3, 5, 7, 9, and 11 conditions, respirometry was employed to measure overall nondiapausing, days 1–14 diapause IP, and days 15, 20, and 102 metabolic rate. Changes in the respiration rate during the IP postemergence diapause MP adult beetles using Trizol1 (Mole- formed a general inverted ‘‘U’’ shaped curve (Fig. 1). Oxygen cular Research Center) following the manufacturer’s protocol. The consumption was at 0.4–0.35 ml/g/h in days 1 and 2 postemer- isolated RNA pellets were stored under absolute ethyl alcohol at gence respectively, then increased to 0.52 ml/g/h by day 3, À70 8C until needed. The pellets were dissolved in either 60 ml followed by a sharp increase to 1.1 ml/g/h by day 4. Oxygen DEPC-treated water for use in differential display or 60 ml consumption then ﬂuctuated around 1.0 ml/g/h until day 7 when it formamide for northern blot analysis. dropped to 0.08 ml/g/h by day 15. The CO2 production curve generally paralleled that of the O2 consumption curve, although 2.4. Differential display CO2 production was elevated as compared to O2 consumption. The greatest differences between CO2 production and O2 consumption Differential display was carried out using the GeneFishing DEG occurred on days 4–7 (0.13–0.57 ml/g/h, respectively) (Fig. 1). Premix Kits (Seegene) following the manufacturer’s protocol. Four developmental stages were screened: nondiapausing day 1, IP days 3.2. Clones 1 and 7, and early MP beetles (day 20 postemergence). All reagents were kept on ice and the PCR tubes were kept on a chilled Screening nondiapausing day 1, IP days 1 and 7, and early MP aluminum plate at all times during the setup and then transferred beetles (day 20 postemergence) with differential display resulted 34 G.D. Yocum et al. / Journal of Insect Physiology 55 (2009) 32–39

Table 1 Leptinotarsa decemlineata diapause maintenance phase down-regulated transcripts.

Clone Size (bp) Predicted Acc. no. Putative identity Score, % identity, organism, Acc. no. Blast transcript size (kb)

Unknown function 4941 453 0.82 FG591161 Unknown X 5513 753 0.69 FG591167 Unknown X 5611 390 1.3 FG591168 Unknown X 6131 427 0.69 FG591177 Diapause-associated transcript-2 4eÀ48, 76%, L. decemlineata, AAN05630 X 6922 315 3.4 FG591182 Unknown X

Defensive 3313 543 0.7 FG591145 C-type lectin, mannose-binding 8eÀ14, 38%, Anopheles gambiae, XP001688639 X 4131 451 2.0 FG591151 Gamma interferon inducible 2eÀ07, 29%, Tribolium castaneum, XP_975539 X lysosomal thiol reductase

Structural 3612 309 0.51 FG591147 Glycine-rich protein 8eÀ31, 87%, L. decemlineata, ABF60889 X 4831 383 0.54 FG591159 Glycine-rich protein 3eÀ49, 94%, L. decemlineata, ABF60889 X 4921 627 0.73 FG591160 Chitin-binding peritrophin-A domain 6eÀ16, 28%, A. gambiae, XP_001237083 X 5012 430 0.55 FG591163 Glycine-rich protein 7eÀ06, 100%, L. decemlineata, DQ515965 N 7811 470 0.5 FG591186 Glycine-rich protein 1eÀ14, 84%, L. decemlineata, ABF60888 X

Digestive X 3211 274 1.3 FG591141 Endo-polygalacturonase 4eÀ13, 50%, Colletotrichum gloeosporioides, CAC14022 X 4612 795 1.3 FG591157 Digestive cysteine proteinase 8eÀ116, 98%, L. decemlineata, AAS20589 X

The Blast program was used to search GenBank sequence repository for possible sequence identities. A three-step search strategy was employed: (1) A BlastX search of the non-redundant protein sequences (nr) was done. (2) On those sequences failing to yield a significant hit (eÀ5), the BlastX search was repeated with the low complexity region filter turned off. (3) For the sequence failing the two BlastX searches, a BlastN search of the nucleotide collection (nr) database was conducted. in 55 putative differentially regulated transcripts. The insert sizes conflicting results. Only those transcripts that gave evidence of ranged from 114 to 795 bp. Differential expression of 14 of these differential regulation were investigated in further detail. transcripts was confirmed by northern blot (Figs. 2–5). The 14 differentially regulated transcripts were placed into four general 3.3. Diapause initiation and maintenance developmental northern categories of proteins (Table 1): unknown function (4941, 5513, blot analysis 5611, 6131 and 6922), defensive (3313 and 4131), structural/ glycine-rich (3612, 4831, 4921, 5012 and 7811), and digestive To examine expression of the transcripts in more detail during (3211 and 4612). Thirteen transcripts were expressed at equiva- the diapause IP and MP, days 1–15, 20 and 102 Postemergence lent levels in all developmental stages. These transcripts were were screened by northern blots. Two rounds of northerns were placed in four general categories (Table 2): unknown function performed. All the transcripts followed the same general (4141, 5411, 5641 and 8212), protein synthesis and maturation expression pattern, being highly expressed in the diapause (1111, 3222, 4112, 4332, 5712 and 8611), structural (1621) and initiation samples up to day 13–15 postemergence and substan- metabolic (3224 and 5811). The rest of the transcripts were either tially down-regulated in diapause MP samples (i.e., days 20 and below the level of detection by northern blot analysis or gave 102) (Figs. 2A–5A).

Fig. 1. Mean Æ S.E. (n = 7–14) O2 consumed (^) and CO2 produced (~)byL. decemlineata diapause-programmed adults during days postemergence at 24 8C. Data were collected by the Sable data acquisition program ExpeData following the manufacturer’s protocol. G.D. Yocum et al. / Journal of Insect Physiology 55 (2009) 32–39 35

Fig. 2. Temporal expression patterns of ﬁve genes with unknown function during the diapause initiation phase (IP) and maintenance phase (MP), and early nondiapausing adults. (A) Day 1–15, 20 and 102 postemergence diapause-programmed adults. Day 15, 20 and 102 postemergence adults are in diapause maintenance phase. (B) Day 1, 3, 5, 7, 9 and 11 postemergence nondiapausing adults. The total RNA (5 mg) was separated on a 1% formaldehyde–agarose gel and screened with digoxigenin PCR-labeled probes. A representative control gene (ribosomal protein L18a) blot is shown at the bottom of the ﬁgure.

Fig. 3. Temporal expression patterns of two defensive gene transcripts during the diapause initiation phase (IP) and maintenance phase (MP), and early nondiapausing adults. See Fig. 2 caption for description. 36 G.D. Yocum et al. / Journal of Insect Physiology 55 (2009) 32–39

Fig. 4. Temporal expression patterns of ﬁve structural gene transcripts during the diapause initiation phase (IP) and maintenance phase (MP), and early nondiapausing adults. See Fig. 2 caption for description.

3.4. Nondiapause developmental northern blot analysis screened. This interval covers the period of rapid increase in the ovipositioning rate for these beetles (Peferoen et al., 1981). All the To examine the possible expression of the MP down-regulated transcripts were expressed within the ﬁrst 11 days of adult life transcripts in nondiapausing adult beetles, four replicates of days (Figs. 2B–5B). The days 1–7 samples gave consistent results, with 1, 3, 5, 7, 9, and 11 postemergence nondiapausing beetles were initiation of expression by day 3 depending on the transcript and

Fig. 5. Temporal expression patterns of two digestive gene transcripts during the diapause initiation phase (IP) and maintenance phase (MP), and early nondiapausing adults. See Fig. 2 caption for description. G.D. Yocum et al. / Journal of Insect Physiology 55 (2009) 32–39 37

Table 2 Leptinotarsa decemlineata transcripts equivalently expressed during the diapause initiation and maintenance phases.

Clone Size (bp) Acc. no. Putative identity Score, % identity, organism, Acc. no. Blast

Unknown function 4141 196 FG591152 Unknown X 5411 164 FG591166 Unknown X 5641 679 FG591170 Unknown X 8212 254 FG591190 Unknown X

Protein synthesis and maturation 1111 583 FG591137 Ribosomal protein L13e 4eÀ13, 50%, Tribolium castaneum, XP_967899 X 3222 540 FG591143 Ribosomal protein S15e 5eÀ60, 96%, T. castaneum, XP_973139 X 4112 260 FG591149 Ribosomal protein L29e 1eÀ27, 86%, Timarcha balearica, CAJ17407 X 4332 653 FG591155 Heat shock cognate 70 1eÀ75, 85%, T. castaneum, XP_966611 X 5712 211 FG591173 16S ribosomal RNA 4eÀ60, 89%, Paraleptusa ripicola, EF989015 N 8611 568 FG591192 Ribosomal protein L22e 1eÀ39, 83%, T. castaneum, XP_973501 X

Structural 1621 752 FG591140 Tubulin alpha-1 chain 4eÀ106, 100%, Culex pipiens quinquefasciatus, X XP_001849099

Metabolic 3224 596 FG591144 Adenine nucleotide translocase 3eÀ10, 89%, Monochamus alternatus, ABU68467 X 5811 221 FG591174 ATP synthase F0 subunit 6 7eÀ14, 75%, Culicoides oxystoma, BAF80214 X

See Table 1 caption for details. high levels of expression to day 7. The individual transcripts’ Differences in expression of closely related members of a gene family expression patterns were somewhat variable in the days 9 and 11 in relationship to diapause have also been observed for members of samples. The transcripts for the glycine-rich proteins (3612, 4831, the heat shock 70 family of the Colorado potato beetle (Yocum, 2001). 5012 and 7811) and two unknown transcripts (4941 and 6922) were all down-regulated by day 11. A comparison of the diapause 4.2. Defensive IP and nondiapause northern blots shows that the elevated level of expression of the glycine-rich proteins transcripts and the two Two transcripts (3313 and 4131) for putative defensive genes – unknown transcripts (4941 and 6922) persisted for approximately a C-type lectin mannose-binding protein (C-MBP) and a gamma 4 days longer in the diapause IP beetles (Figs. 2 and 4). interferon inducible lysosomal thiol reductase (GILT) – were isolated. The expression of the C-MBP transcript in nondiapausing 4. Discussion adults is highly variable. Expression of the C-MBP transcript in day 1 nondiapausing adults was at trace levels or not detectable. In IP Using differential display, 14 clones were isolated and adults, the expression of this transcript continued until day 13–15 confirmed to be differentially regulated between the diapause IP (Fig. 3A and B). GILT expression was detected in both the and MP of the Colorado potato beetle. These 14 clones were divided nondiapausing and IP adults. In the IP adults, GILT expression into four tentative functional groups based on BlastX and BlastN was detected until day 13–15 postemergence (Fig. 3A and B). results: proteins of unknown function, defensive, structural/ C-MBPs are calcium-dependent pattern recognition receptors glycine-rich, and digestive. for the innate immune response in invertebrates and vertebrates (Fujita, 2002). Immune-challenging Heliothis virescens (Fabricius) 4.1. Unknown function by injecting live bacteria induced the expression of a C-MBP gene (Ourth et al., 2005), indicating that these proteins also have an Clones of five genes (4941, 5513, 5611, 6131 and 6922) with no immune function in insects. GILT breaks disulfide bonds of known functions were isolated. All five genes are expressed in both proteins, leading to lysosomal degradation. In organisms with nondiapausing adults and beetles in the diapause IP and are down- adaptive immune systems, GILT may be involved in antigen regulated in the diapause MP adults by day 15 or 20 (Fig. 2A and B). processing in antigen-presenting cells (Arunachalam et al., 2000). Day 15 postemergence was the day of the lowest recorded Recently, a role for GILT in the innate immune response has been respiration rate, indicating that the beetles have entered the suggested as well (Liu et al., 2007; Zoysa and Lee, 2007). diapause MP by this time. The expression of genes 4941 and 6922 GILT is constituently expressed in the digestive tract of the adult persisted 4–6 days longer in the diapause IP adults than in the amphioxus Branchiostoma belcheri (Gray) (Liu et al., 2007) and is nondiapausing adults, suggesting that their expression is differen- induced by an immunological challenge in the digestive tract of the tially regulated with respect to nondiapausing and diapause IP abalone Haliotis discus discus (Reeve) (Zoysa and Lee, 2007). Giving individuals. A BlastX search using clone 6131 yielded a significant hit insects plant antinutritional factors or non-infectious microorgan- (76%) to diapause-associated transcript 2 (DAT-2) from the Colorado isms induces a defensive response (Evans and Lopez, 2004; Freitak potato beetle (Yocum, 2003), suggesting that 6131 may be a member et al., 2007; Moon et al., 2004). If these two genes are induced by of a gene family with DAT-2. The expression patterns of DAT-2 and stresses caused by feeding, it would explain why their expression is 6131 are similar in IP adults except that while DAT-2 is barely not detectable in diapausing beetles. detectable on day 3 before increasing its expression on the following days (Yocum, 2003), 6131 is highly up-regulated on day 2. In early 4.3. Structural diapause MP adults, DAT-2 expression remains high, while 6131 is down-regulated but still clearly detectable. The biggest difference in The four putative glycine-rich protein transcripts (3612, 4831, the expression of these transcripts is that DAT-2 is not expressed in 5012 and 7811) clearly seem to be co-regulated in nondiapausing nondiapausing adults (Yocum, 2003) and 6131 is expressed (Fig. 2B). adults, being down-regulated on day 9 and at low levels or not 38 G.D. Yocum et al. / Journal of Insect Physiology 55 (2009) 32–39 detectable by day 11 (Fig. 4B). In contrast, in diapause IP adults key enzymes involved in meeting that challenge. Polygalactur- elevated levels of expression of the glycine-rich transcripts onase is an enzyme used to break down pectin found in cell walls in persisted until day 13 or 15, indicating differential regulation of the higher non-woody plants. Polygalacturonase activity is these transcripts between early nondiapausing and IP adults commonly found in bacteria, plants and fungi; within insects, (Fig. 4A). Glycine-rich proteins perform a diverse range of polygalacturonase activity has been found in only a few species, physiological functions including structural support in plant cell including the Coleopteran sister families of Curculionoidae and walls and insect cuticle (Mousavi and Hotta, 2005; Anderson et al., Chrysomeloidea (Shen et al., 2003). Shen et al. (2003) proposed 1995), antifreeze proteins (Graham and Davies, 2005), and that the polygalacturonase gene in these sister families is the result antibacterial peptides (Otvos, 2000). Some have RNA-binding of horizontal transfer from a fungus into a common ancestor of motifs, suggesting a possible regulatory function (Mousavi and these two beetle families. An active polygalacturonase gene would Hotta, 2005). BlastX searches using sequence data from the four greatly increase feeding efficiency of any plant-feeding beetle. clones found significant identity to the characterized glycine-rich Once proteins are extracted from plant cells, the Colorado potato cuticular genes (Ld-GRP-1, -2, and -3) from L. decemlineata beetle possesses at least nine forms of cysteine proteinases that aid (Table 1)(Zhang et al., 2008). Our deduced proteins also contained in breaking down dietary proteins into the free amino acids needed the common G(Y/L) motif of glycine-rich cuticular proteins (Willis for development and reproduction (Michaud et al., 1995). Clone et al., 2005), suggesting that these glycine-rich genes also serve a 4612 represents a member of this functional group. cuticular function. In mature adult beetles the expression of the Ld- In this investigation we found: (1) Some genes are expressed at GRP-1 and -2 genes can be induced by insecticide treatment or equivalent levels in diapause IP and MP beetles and can serve as desiccation, suggesting that cuticular protein synthesis has an control genes for future studies (data not shown). (2) Peak environmental component to its regulation (Zhang et al., 2008). respiration rate occurred on day 7 postemergence in the diapause The prolonged expression of the four glycine-rich genes under IP adults and then decreased to its lowest level on day 15 short-day rearing conditions strengthens the argument for postemergence. This indicates that by day 15 postemergence, the environmental regulation of cuticular protein synthesis, and beetles had entered the diapause MP. (3) The differentially regulated indicates that the cuticle protein composition of diapausing genes were down-regulated between days 13 (late IP) and 20 (early beetles is distinct from that of nondiapausing beetles. MP) postemergence in the diapause-programmed beetles. (4) Expression of the peritrophin-A domain transcript (4921) was Elevated levels of expression of the glycine-rich protein genes detected in IP and nondiapausing adults and was down-regulated and two transcripts with unknown functions persisted longer in the from day 15 to 20, and not detectable in day 102 postemergence diapause IP beetles than in the nondiapause beetles. This observa- short-day adults (Fig. 4A and B). The peritrophic-A domain is found tion substantiates Denlinger (2002) conjecture that preparation for in chitin-binding proteins primarily in the peritrophic membrane, entering diapause may be characterized by the simple extension of and in chitinases. Chitinases normally have only one copy of the expression of certain genes. The characterization of gene expression peritrophic-A domain whereas structural chitin-binding proteins patterns provides potential biomarkers for monitoring the intrinsic have multiple copies (Tellam et al., 1999). A BlastP search of the changes occurring during diapause that will help to clarify the Swiss-Prot repository identified two putative peritrophin-A transitional phases involved in diapause development (Kostal, domains, indicating that the transcript encodes a structural 2006). As previously suggested (Denlinger’s, 2002), biomarkers chitin-binding protein. In light of the fact that two putative delineating the phases of diapause also hold promise as tools for defensive transcripts are up-regulated during feeding, the possible monitoring the developmental status of pest species. immune function of the chitin-binding proteins is intriguing (Du et al., 2006; Gorman and Paskewitz, 2000; Leu et al., 2007). Acknowledgements Infecting the fleshy prawn, Fenneropenaeus chinensis (Osbeck), with bacteria induces a transcript encoding a peritrophin-like We thank Lisa B. Yocum for her editing of the various drafts of this protein (FCP) with four peritrophin-A domains. Recombinant FCP manuscript. We also extend our gratitude to Dr V. Kostal for his binds chitin and Gram-negative bacteria (Du et al., 2006). correspondence on diapause terminology and the journal reviewers The chitin-binding proteins are best known for their role in for their helpful suggestions and comments that strengthen our helping to form the stable matrix of the peritrophic membrane, manuscript. which serves as a porous barrier that protects the exposed cells of References the midgut from mechanical damage due to abrasive food material, parasites, pathogens and toxins. The peritrophic membrane travels Altschul, S.F., Madden, T.L., Scha¨ffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., down the alimentary canal with the food material and is excreted, 1997. Gapped Blast and PSI-Blast: a new generation of protein database search necessitating its replacement during periods of active feeding programs. Nucleic Acids Research 25, 3389–3402. Anderson, S.O., Hojrup, P., Roepstorff, P., 1995. 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