Journal of Insect Physiology 55 (2009) 32–39 Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys 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, 1 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 20 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  10 cm  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 2 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.