Molecular profiling of proteolytic and lectin transcripts in Homalodisca vitripennis HemipteraAuchenorrhynchaCicadellidae feeding on sunflower and cowpea.код для вставкиСкачать
76 Coudron et al. Archives of Insect Biochemistry and Physiology 66:76–88 (2007) Molecular Profiling of Proteolytic and Lectin Transcripts in Homalodisca vitripennis (Hemiptera: Auchenorrhyncha: Cicadellidae) Feeding on Sunflower and Cowpea Thomas A. Coudron,1* Sandra L. Brandt,1 and Wayne B. Hunter2 Homalodisca vitripennis Germar 1821 (Hemiptera: Cicadellidae) [Takiya et al. (2006) Ann Entomol Soc Am 99:648–655; syn. H. coagulata (Say)] salivary gland and gut EST libraries were used to isolate cDNA fragments of the gene transcripts encoding for cathepsin L, asparaginyl endopeptidase, cathepsin B, metalloendopeptidase, cathepsin D, multicatalytic endopeptidase, and a sugar-binding C-type lectin. Transcript levels were evaluated in immature and adult H. vitripennis feeding on sunflower (Helianthus annuus) or cowpea (Vigna unguiculata). Northern blot hybridization results showed that expression of most of the transcripts were similar for all developmental stages and feeding on the two diets examined. However, the expression of the transcript for asparaginyl endopeptidase was less expressed in sunflower-fed adult females compared to sunflower-fed immatures. Also, the expression of the C-type lectin transcript was up-regulated in adults compared to immatures when fed on either diet. Documenting both the presence and variation of transcript expression involved in putative digestive proteolysis in this xylem-feeding leafhopper is noteworthy and aids efforts to design specific diet formulations for mass production of hosts and parasitoids to be used as effective biological control measures. Arch. Insect Biochem. Physiol. 66:76–88, 2007. Published 2007 Wiley-Liss, Inc.† KEYWORDS: digestion; glassy-winged sharpshooter; Homalodisca coagulata; Northern hybridization; Pierce’s disease; transcriptome; xylem feeder INTRODUCTION The glassy-winged sharpshooter (GWSS), Homalodisca vitripennis Germar 1821 (Hemiptera: Cicadellidae) (Takiya et al., 2006), is highly polyphagous, indigenous to the southern United States and to northern Mexico (Triapitsyn and Phillips, 2000; Redak et al., 2004) and is most frequently referred to as a xylem-fluid feeder. As such, this pest is cred- ited with transmitting the bacterium Xylella fastidiosa, a causal agent of several economically important diseases including Pierce’s disease of grapevines, citrus variegated chlorosis, and phony peach disease (Qin et al., 2001; Hopkins and Purcell, 2002). Undulating and seasonal populations and difficulties in rearing this insect have limited biological studies and, subsequently, the development of effective pest management strategies. 1 Biological Control of Insects Research Laboratory, USDA-Agriculture Research Service, Columbia, Missouri 2 Subtropical Insects Research Unit, USDA-Agriculture Research Service, Fort Pierce, Florida Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Contract grant sponsor: Pierce’s Disease Program, California Department of Food and Agriculture. Sandra L. Brandt’s present address is Department of Biochemistry, University of Missouri, Columbia, MO 65211. *Correspondence to: Thomas A. Coudron, Biological Control of Insects Research Laboratory, USDA-Agriculture Research Service, 1503 S. Providence Rd, Columbia, MO 65203. E-mail: firstname.lastname@example.org Received 22 November 2006; Accepted 6 March 2007 Published 2007 Wiley-Liss, Inc. † This article is a US Government work and, as such, is in the public domain in the United States of America. DOI: 10.1002/arch.20200 Published online in Wiley InterScience (www.interscience.wiley.com) Archives of Insect Biochemistry and Physiology October 2007 doi: 10.1002/arch. Molecular Profiling of Proteases in GWSS GWSS have a wide host range containing many plants that serve as effective reservoirs for X. fastidiosa, necessitating control strategies in urban and natural areas adjacent to cropping systems at risk of the disease (Phillips, 2000). Chemical control of GWSS on such a wide scale is neither an economically feasible nor an environmentally sustainable strategy. Therefore, the development of effective biological control agents for GWSS is an important part of an integrated pest management program (Hoddle, 2004). The mymarid wasps, Gonatocerus spp., are the most common egg parasitoids of GWSS in the southeastern United States and appear to be a feasible control strategy for providing long-term, area-wide suppression of this pest (Pilkington et al., 2005). An increased comprehension of GWSS digestive physiology may lead to a more thorough understanding of the nutritional requirements, the host range, and possibly more effective rearing methods needed for the mass production of leafhopper hosts and parasitoids. Amino acids and soluble proteins are the primary nitrogen nutrients in xylem fluid where organic constituents include 19 amino acids, 7 organic acids, and 3 or 4 sugars (Andersen et al., 1992, 2003). The dietary nitrogen impacts survival, growth, and reproduction of phytophagous insects (Bi et al., 2005). Consequently, the nutritionally dilute chemistry of the xylem fluid is a probable cause of the extremely high rate of feeding by GWSS (Brodbeck et al., 2004). Equally important is that GWSS are able to assimilate at least 99% of the amino acids, organic acids, and sugars (Andersen et al., 1989; Brodbeck et al., 1993, 1996). Asparagine and glutamine or proline are the predominant amino acids detected in xylem fluid in most woody host plant species investigated (Andersen et al., 1992; Redak et al., 2004; Bi et al., 2005). Adult GWSS prefer and perform best on host plants with high amide content in the xylem fluid (Andersen et al., 1992), while immatures require lower levels of amides and a more balanced amino acid profile (Brodbeck et al., 1995, 1999). Substantial amounts of soluble proteins exist in xylem fluid of GWSS host plants. For example, Archives of Insect Biochemistry and Physiology October 2007 doi: 10.1002/arch. 77 total protein concentration in grape xylem extracts varies from 0.468 to 0.854% (Jain and Basha, 2003). However, soluble protein levels were higher in the xylem of orange trees during the times when GWSS adults were sexually active, while the levels of certain amino acids corresponded better to shifts in GWSS numbers (Bi et al., 2005). Also of interest is that several plants, including sunflower, show an increase in the concentration of xylem proteins in response to stress (Zhu and Zhang, 1997). The extent to which GWSS utilize host plant proteins is not understood. However, the ability to utilize xylem proteins as a nutrient source would depend on the presence and activity of the appropriate proteases within the digestive tract. Therefore, the current study was undertaken to test the hypothesis that suitable proteolytic enzymes exist within the digestive tract and consequently that finding could influence current tenets and advance our understanding of the underlying mechanisms of GWSS digestive physiology. The use of expression libraries is a timely approach to understanding the genetic basis of proteolytic activity as it relates to insect development (Hunter et al., 2003; Huang et al., 2006; Sabater-Munoz et al., 2006) and feeding and digestion (Colebatch et al., 2002; Abubakar et al., 2006). The focus of this study was to compare transcript levels of several proteases in immature and adult GWSS feeding on sunflower or cowpea plants. MATERIALS AND METHODS GWSS Rearing GWSS adults were originally obtained from a laboratory colony in Westlaco, Texas. Insects were maintained in a quarantine facility on cowpea (Vigna unguiculata) or sunflower (Helianthus annuus) (Chen et al., 2006) under high intensity lights and the following conditions: 28 ± 2°C, >70% relative humidity and 12 h light/12 h dark cycles. Specific developmental stages were selected based on head capsule width with the following assignment: 3rd instar 1.01–1.50 mm and 5th instar 1.98–2.20 mm (Sétamou and Jones, 2005). 78 Coudron et al. Library Construction Insects for library construction were collected off several citrus trees in Kern County, California. Dissected salivary glands and midguts from 50 adult GWSS were used in the construction of an expression library (Hunter B, Katsar CS, Dang PM, unpublished results). Tissues were ground in liquid nitrogen and total RNA was extracted using the guanidinium salt-phenol-chloroform procedure as previously described by Strommer et al. (1993). Poly (A) RNA was purified using Micromole (A) Pure™ according to the manufacturer’s instructions (Ambion, Austin, TX). A directional cDNA library was constructed in Lambda Uni-ZAP® XR vector using Stratagene’s ZAP-cDNA Synthesis Kit (Stratagene, La Jolla, CA). The resulting DNA was packaged into Lambda particles using Gigapack® III Gold Packaging Extract (Stratagene). An amplified library was generated with a titer of 1.0 × 109 plaque-forming units per mL. Mass excision of the amplified library was carried out using Ex-Assist® helper phage (Stratagene). An aliquot of the excised, amplified library was used for infecting XL1Blue MRF’ cells and subsequently plated on LB agar containing 100 µg/ml ampicillin. Bacterial clones containing excised pBluescript SK(+) phagemids were recovered by random colony selection. Sequencing of Clones pBluescript SK (+) phagemids were grown overnight at 37°C and 240 rpm in 96-well culture plates containing 1.7 ml of LB broth, supplemented with 100 µg/ml ampicillin. Archived stocks were prepared from the cell cultures using 75 µl of a LBamp-glycerol mixture and 75 µl of cells. These archived stocks are held at the United States Horticultural Research Lab (USHRL, Fort Pierce, FL) in an ultra low temperature freezer set at –80°C. Plasmid DNA was extracted using the Qiagen 9600 liquid handling robot and the QIAprep 96 Turbo miniprep kit according to the recommended protocol (Qiagen Inc., Valencia, CA). Sequencing reactions were performed using the ABI PRISM® BigDye™ Primer Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) along with a universal T3 primer. Reactions were prepared in 96-well format using the Biomek2000™ liquid handling robot (Beckman Coulter Inc., Fullerton, CA). Sequencing reaction products were precipitated with 70% isopropanol, resuspended in 15 µl sterile water and loaded onto an ABI 37300 DNA Analyzer (Applied Biosystems). Sequence Verification and Cloning Base confidence scores were designated using TraceTuner® (Paracel, Pasadena, CA). Low-quality bases (confidence score <20) were trimmed from both ends of sequences. Quality trimming, vector trimming, and sequence fragment alignments were executed using Sequencher® software (Gene Codes, Ann Arbor, MI). Sequences less than 100 nucleotides in length, after both vector and quality trimming were completed, were excluded from the analysis. Additional ESTs that corresponded to vector sequences were removed from the dataset. The potential identity of the gene was determined based on BLAST homology searches using the National Center for Biotechnology Information (NCBI) BLAST server (http://www.ncbi.nlm. nih.gov) with the sequence comparisons made to protein databases (BLASTX, BLASTP). To estimate the number of genes represented in the library and the redundancy of specific genes, ESTs were assembled into “contigs” using Sequencher®. Contig assembly parameters were set using a minimum overlap of 50 bases and 95% identity match. Vector and lowquality sequence were trimmed and the sequences filtered for a minimum length (200 bp), producing 4,853 high-quality ESTs. These ESTs were analyzed with the Sequencher® assembly program to identify those representing redundant transcripts. RNA Preparation for Northern Blots Total RNA was extracted from 100 mg of pooled GWSS nymphs (20–25 third instars, 3–5 fifth instars, or 2–4 adults) for each sample. Whole insect Archives of Insect Biochemistry and Physiology October 2007 doi: 10.1002/arch. Molecular Profiling of Proteases in GWSS 79 frozen samples were pulverized with a RNase-free pestle in a 1.5-ml RNase-free microcentrifuge tube, processed with 1.0 ml of TRIzol® reagent (Invitrogen Corporation, Carlsbad, CA) using a microhomogenizer, and held at room temperature for 10 min. Tubes were then centrifuged for 10 min at 12,000g at 4°C and the supernatant transferred to a new 1.5-ml RNase-free microcentrifuge tube. The RNA was purified following the manufacturer’s protocol. The isolated RNA pellet from each tube was stored under absolute ethyl alcohol at –70°C and then dissolved in 50 µl RNase-free water for Northern blot analysis. 95°C for 2 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s, followed by 72°C for 7 min. The amplified product was cloned into pCR2.1 vector using the TOPO TA Cloning® kit (Invitrogen), and the ligated product was transformed into DH5α Escherichia coli competent cells. Probes were labeled via PCR using the PCR DIG Probe Synthesis Kit™ (Roche Applied Science, Indianapolis, IN). Gene-specific primers (Table 1) and approximately 10–100 pg of each clone were used for each labeling reaction. Cloning of cDNA Fragments and Probe Labeling Five micrograms of total RNA per sample was separated on a 1.2% agarose glyoxal-based denaturing gel (NorthernMax™-Gly, Applied Biosystems). The RNA was then transferred onto a positively charged nylon membrane (Nytran SuPerCharge, Whatman, Sanford, ME) using downward capillary action in 20× SSC transfer buffer (3 M NaCl, 0.3 M NaC6H5O7·2H2O, pH 7.0) for 3–4 h. After completion of the transfer, membranes were cross-linked at 12,000 µJ/cm2. Prehybridization and hybridization at 42°C overnight were carried out in DIG Easy Hyb® hybridization buffer (Roche). The digoxigenin- Gene-specific primers were designed using PrimerQuest software (Integrated DNA Technologies, Coralville, IA) and EST sequences generated from GWSS salivary gland (resulting in 3 probes) or GWSS gut (resulting in one housekeeping and 5 proteinase probes, Table 1). RNA was extracted from whole insects and DNase treated (DNAfree, Ambion, Austin, TX). cDNA was synthesized and then used with gene-specific primers (Table 1) for RT-PCR reactions under the following conditions: Northern Blotting TABLE 1. Gene-Specific Primer Sequences* GWSS EST Primer sequences (5′–3′) Housekeeping F: GACGAAGAAGTTGCTGCGTTGGTT R: TCCCAGTTGGTCACAATACCGTGT Mg1 F: TGTCCGAGGAAGATCTTGCCCAAT R: TGGCAAGCTCGATGTCTTCTGAGT Mg2 F: ATATGGATGCTTGTCACTCGGGCT R: TGTCACGTGATCGAAACCCAGACT Mg3 Expected size (bp) BLASTX homology and accession no. E value 236 β-Actin AAU84923 0.0 294 Cathepsin-L cysteine peptidase XP 970644 08E–109 311 Asparaginyl endopeptidase CAB71158 09E–27 F: TTACCCTGACTTGGCTTGGCCTTA R: TGAATGGACTGGACATCAGGTGGT 237 Cathepsin B XP 966663 07E–63 Mg4 F: TTGGCGAGGCCACAGAAATAGACT R: ACCTTGACAGGAGCGAAGACGAAA 275 Cathepsin-D aspartic protease XP 966517 06E–62 Mg5 F: AGGCCAACTCCTGTGTGACTTTCA R: GTGAACGCATCAGCGGCATAATGT 370 Metalloendo peptidase EAT36357 5E–30 Sg1 F: AGTGTTGCCATCGATGCGAGTCAT R: GCTTGCACAAGAAGCTATGCCACA 240 Cathepsin-L cysteine peptidase EAT45919 02E–40 Sg2 F: ATTTCACCAGCGTTCACAACGTCC R: ACTTTGACCTGCGGTTGTACAGGA 320 Multicatalytic endopeptidase complex B XP 394993 3E–30 Sg3 F: TCAGGATAACGCCACTTGCCAGAT R: ACCCATTGTCCCTGAGGAAAGTCA 345 Sugar-binding, C-type lectin EAT36282 5E–21 *F = forward; R = reverse. Archives of Insect Biochemistry and Physiology October 2007 doi: 10.1002/arch. 80 Coudron et al. labeled probes were detected using DIG High Prime DNA Labeling and Detection Start Kit II® (Roche) followed by 15–300-min exposure to autoradiography film (Classic BX, Molecular Technologies, St. Louis, MO). Films were imaged using a multi-imaging system equipped with a cooled CCD camera (VersaDoc 4000 Imaging System, BioRad, Hercules, CA). Densitometric analysis of the bands was performed using QuantityOne™ software (BioRad). The blots were stripped with two 15-min incubations at 37°C in a stripping buffer (0.2 M NaOH, 0.1% SDS). After stripping, the blots were rinsed twice for 5 min in 2× SSC and reprobed with β-actin in order to assure equal loading of RNA. Blots were probed no more than three times before being discarded. Transcript size was estimated by comparison to DIG-labeled RNA markers (Roche). Statistical Analysis Transcript levels of genes encoding for the seven proteinases and the C-type lectin were evaluated in immature and adult GWSS that had been reared on sunflower or cowpea plants. Three biological replicates were analyzed for each sample. The densitometric intensity for each transcript was divided by the intensity of the housekeeping transcript (βactin) for each corresponding sample. This value was then normalized to the value for cowpea-fed 3rd instar for each Northern blot. Average normalized intensity values were compared using the Student’s t-test (SAS Institute, 1990). RESULTS Identification of ESTs Transcripts which were ≥100 nucleotides subsequent to quality and vector trimming averaged 821 bases in length. Because multiple ESTs can be derived from a single gene, sequences were assembled into contiguous sequences (contigs) to estimate the number of genes giving rise to the ESTs. Contig alignment resulted in a dataset of 965 unique assembled sequences, including contigs and singlets. Of that total, 189 of the contigs and 379 of the sin- glets corresponded with a putative match in GenBank, while 397 of the cDNAs had “No Significant Homology” to any sequence currently listed in GenBank. “No Significant Homology” denoted either “no match” to GenBank’s database when similarity searches were performed using both BLASTX and BLASTN or an E value of > –5. About 40% of the cDNAs sequenced are potentially newly discovered or as yet undescribed insect genes, some of which may be specific to the leafhoppers. ESTs, identified as encoding enzymes, were sorted into enzyme class and subclass by putative function based on homology to proteins that had been previously characterized in the NCBI database (Table 2, NCBI BLAST server http://www.ncbi.nlm. nih.gov using BLASTX, TBLASTX, TBLASTN). Enzyme classification of transcripts based on BLAST homology searches resulted in the majority of sequences being identified as belonging to the Hydrolases (50%), then Oxidoreductases (21%) and Transferases (18%). Transcripts with identity matches to digestive enzymes in other insects were selected and used to design the primers and molecular probes used in these studies. Northern Blot Analyses Northern blot analyses demonstrated transcripts of the following approximate sizes: 1,517 bp for β-actin, 1,049 bp for Calcium-dependent (C-type) lectin, 1,049 bp for multicatalytic endopeptidase, 1,560 bp for salivary gland cathepsin L, 1,250 bp for gut cathepsin L, 1,600 bp for asparaginyl endopeptidase, 1,250 bp for cathepsin B, 1,517 bp for cathepsin D, and 1,049 bp for metalloendopeptidase. With two exceptions, the expression levels of the transcripts did not vary significantly among developmental stages or diet treatments (Fig. 1). C-type lectin transcript expression was one exception. Expression of C-type lectin transcription was significantly greater (P < 0.05) in adult females and males compared to 3rd or 5th instar larvae feeding on cowpea. However, only in the sunflower treatment was the lectin expression significantly greater for adult males compared to the immatures. The other exception was the expression of asparaginyl endopeptidase transcript, which was 1.6-fold Archives of Insect Biochemistry and Physiology October 2007 doi: 10.1002/arch. Molecular Profiling of Proteases in GWSS 81 TABLE 2. Enzymatic Classification of Homalodisca vitripennis Midgut Sequences* EC no. Class Subclass 1 1.1 1.11 1.13 1.14 1.15 1.2 1.3 1.4 1.5 1.6 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 3.1 3.2 3.4 3.5 3.6 4.1 4.2 5.2 5.3 5.99 6.1 6.2 6.3 Oxidoreductase Oxidoreductase Oxidoreductase Oxidoreductase Oxidoreductase Oxidoreductase Oxidoreductase Oxidoreductase Oxidoreductase Oxidoreductase Oxidoreductase Oxidoreductase Oxidoreductase Transferase Transferase Transferase Transferase Transferase Transferase Transferase Transferase Hydrolases Hydrolases Hydrolases Hydrolases Hydrolases Lyases Lyases Isomerases Isomerases Isomerases Ligases Ligases Ligases Oxidoreductase Acting on the CH-OH group of donors Acting on a peroxide as acceptor Acting on single donors with incorporation of molecular oxygen (oxygenases) Acting on paired donors, with incorporation or reduction of molecular oxygen Acting on superoxide radicals as acceptor Acting on the aldehyde or oxo group of donors Acting on the CH-CH group of donors Acting on the CH-NH2 group of donors Acting on the CH-NH group of donors Acting on NADH or NADPH Acting on a sulfur group of donors Acting on a heme group of donors Transferase Transferring one-carbon groups Transferring aldehyde or ketonic groups Acyltransferases Glycosyltransferases Transferring alkyl or aryl groups, other than methyl groups Transferring nitrogenous groups Transferring phosphorus-containing groups Acting on ester bonds Glycosylases Acting on peptide bonds (peptidases) Acting on carbon-nitrogen bonds, other than peptide bonds Acting on acid anhydrides Carbon-carbon lyases Carbon-oxygen lyases cis-trans-Isomerases Intramolecular isomerases Other isomerases Forming carbon—oxygen bonds Forming carbon—sulfur bonds Forming carbon—nitrogen bonds Total No. of sequences 2 28 8 2 32 2 2 2 4 4 41 2 20 2 2 2 2 4 14 2 102 24 24 86 8 228 4 16 4 2 1 22 2 28 732 *Classification is hierarchial. All functional assignments of Homalodisca vitripennis (syn. H. coagulata) sequences described here are the “inferred from electronic evidence” (IEE) using the top 5 BLASTX hits with an E-value of ≤ –5 generated from NCBI’s nr database. The definition term associated with each sequence was defined according to The International Union of Biochemistry and Molecular Biology’s Enzyme classification system. (P = 0.022) and 1.8-fold (P = 0.028) greater in sunflower-fed 3rd and 5th instars, respectively, than was found in sunflower-fed adult females. DISCUSSION Within the order Hemiptera. it is widely recognized that many plant-feeding species in the suborder Heteroptera have high levels of proteases associated with the salivary gland and/or midgut to aid in the digestion of proteins (Colebatch et al., 2001; Zhu et al., 2003). In contrast, plant-feeding species in the suborder Auchenorrhyncha, especially xylem-feeders like GWSS, have Archives of Insect Biochemistry and Physiology October 2007 doi: 10.1002/arch. long been assumed to lack digestive proteases because the protein content of xylem under normal circumstances is extremely low (Terra et al., 1996). Yet, plants under environmental stress, especially drought stress (Zhu and Zhang, 1997) and possibly heavy insect infestation, can have a significant level of protein in the xylem. Additionally, adult GWSS have been reported to occasionally feed on phloem of transitional host plants (Katsar et al., 2007). The use of the GWSS expression datasets provided a means for profiling the presence of transcripts for cysteine and aspartic proteinases as well as other endopeptidases and, thus, for providing support for our hypothesis that GWSS express gene Coudron et al. Fig. 1. (continues on following pages) 82 Archives of Insect Biochemistry and Physiology October 2007 doi: 10.1002/arch. 83 Fig. 1. (continues on overleaf) Molecular Profiling of Proteases in GWSS Archives of Insect Biochemistry and Physiology October 2007 doi: 10.1002/arch. Fig. 1. Northern blots of GWSS RNA from 3rd instar, 5th instar, adult female (AF), and adult male (AM) feeding on cowpea (cp) or sunflower (sf) and densitometric comparison of the average normalized intensity values from 3 replicates. Each lane was loaded with 5 µg total RNA. β-actin was used as an internal loading reference and is shown below each respective blot. Probes are indicated in the left margin. Significance levels in the average blot intensities were determined by Student’s t-test. 84 Coudron et al. Archives of Insect Biochemistry and Physiology October 2007 doi: 10.1002/arch. Molecular Profiling of Proteases in GWSS transcripts encoding proteolytic polypeptides that play a role in GWSS feeding and overall nitrogen nutrition, especially when feeding on stressed, heavily infested, or transitional plants. Furthermore, demonstrating that GWSS express genes that are involved in proteolytic degradation supports the testing of artificial diets that contain protein. Recent developments in the area of nutrigenomics have demonstrated how nutrition alters global gene expression patterns (Coudron et al., 2006). It is plausible that one or more of the proteolytic enzymes expressed by GWSS can be used as a biomarker for, and an early indicator of, the insect’s response to different nutritional quality. Digestive proteolytic enzymes, as described in different orders of economically important insect pests, belong predominantly to one of the major classes of proteinases. Coleopteran and hemipteran species tend to utilize cysteine proteinases while lepidopteran, hymenopteran, orthopteran, and dipteran species mainly use serine proteinases (Lawrence and Koundal, 2002). Cathepsin D-like aspartic proteinases were identified along with cysteine proteinases in species representing six families in the order Hemiptera (Houseman and Downe, 1983). The low pH of midguts in many coleopteran and hemipteran species provides a more favorable environment for aspartic and cysteine proteinases than the high pH found in other insect guts (Lawrence and Koundal, 2002). Thus, if any digestive proteinase activity were to be present in GWSS, it might be expected to be of the cysteine or aspartic classes. Also, plant xylem fluid is slightly acidic (Andersen et al., 1992). Specifically, cowpea and sunflower xylem pH in our experiments were estimated as between 5.0 and 6.0 using pH indicator strips (data not shown), which would be optimal for cysteine proteinases. Digestive proteolysis has been reported for other sap-feeding insects. Putative digestive cysteine proteinases have been characterized for the phloemfeeding insects Aphis gossypii and Nilaparvata lugens (Deraison et al., 2004; Foissac et al., 2002). Foissac et al. (2002) theorized that digestive proteolysis may be widespread in sap-sucking hemipterans and makes a significant contribution to nutrition. Archives of Insect Biochemistry and Physiology October 2007 doi: 10.1002/arch. 85 The occurrence of a sugar-binding C-type lectin with higher expression levels in GWSS adults than in immatures is intriguing. C-type lectins are a group of calcium-dependent carbohydrate-binding extracellular proteins. The majority of the Ctype lectins characterized from insects are involved in immune responses, although involvement in cellular adhesion and developmental regulation has also been reported (Zelensky and Gready, 2005). A C-type lectin expressed in mouthparts of the flesh fly Sarcophaga peregrina is thought to play a novel biological role in the food intake of this insect (Yamamoto-Kihara and Kotani, 2004). Considered important in detection and neutralization of pathogenic materials, C-type lectins expressed in the salivary gland of the mosquito Aedes aegypti are postulated to have a role in controlling bacterial growth in ingested meals (Valenzuela et al., 2002). Determination of the role of C-type lectin in GWSS feeding, digestive physiology or immunity, possibly relating to X. fastidiosa interactions, merits further study. A comparison of preliminary results of caseinolytic activity in homogenates of the salivary gland and midgut of GWSS included as part of this study with that of Lygus Hesperus Knight (Hemiptera: Miridae), the tarnished plant bug (Wright et al., 2006), in the suborder Heteroptera, further supports the hypothesis that substantial protease activity is present within the digestive track of GWSS. Caseinolytic activity in the salivary gland of L. hesperus increased with an increase in pH and time, with the highest activity observed at pH 8.5, achieving 11,000 RFU/IE in 6 h. A much slower activity was recorded in L. hesperus over a pH range of 6.5– 8.0. That pH profile suggested the presence of a trypsin-like serine protease and a chymotrypsin protease, which was confirmed using protease inhibitors and peptide digestion studies. In comparison, the caseinolytic activity in the salivary gland of GWSS detected in our study decreased with an increase in pH but increased with time, with the highest activity observed at pH 5.0–6.0, achieving 1,000 RFU/IE in 18 h. Caseinolytic activity in the midgut of L. hesperus varied with pH and increased 86 Coudron et al. with time, with the highest activity observed at pH 4.5, achieving 7,000 RFU/IE in 18 h. The pH profile suggested the presence of aspartic and cysteine proteases, which was confirmed using protease inhibitors and peptide digestion studies. In comparison, the caseinolytic activity in the midgut of GWSS detected in our study also decreased with an increase in pH and increased with time, with the highest activity observed at pH 5.0–6.0, achieving 8,000 RFU/IE in 18 h. That pH profile suggests the prominence of aspartic and cysteine protease in the midgut as well as the salivary gland of GWSS. Overall, the caseinolytic digestion demonstrated that GWSS have the ability to digest protein and showed major differences in proteolytic activity between the salivary gland and midgut, and that the activity was affected by pH, as was the case for L. hesperus. For GWSS, the greatest activity was found at a low pH in midgut tissues suggesting the presence of aspartic and cysteine activity whereas the greatest activity was found at a higher pH in the salivary gland of L. hesperus, indicative of serine and chymotrypsin activity, and at a low pH in the midgut of L. hesperus, indicative of aspartic and cysteine activity. Further work to determine the mechanistic classes of proteolytic activity (Wright et al., 2006) in GWSS will add clarification and confirmation of the class of protease activity suggested by our preliminary data. Expression levels of the putative digestive enzyme transcripts in this study changed very little over development or in response to the two plant diets examined. Future studies plan to explore expression levels with specific dietary modifications presented through an artificial membrane. Clearly, the enzymatic changes observed herein are linked to the feeding and digestive ability of GWSS to utilize specific host plant proteins. These enzymes are critically linked to interactions that also affect GWSS development and performance on different hosts and/or artificial diets. As we begin to elucidate more of the variables underlying GWSS feeding and digestion, we come closer in our efforts to produce an efficacious system for the mass production of GWSS and its parasitoids. ACKNOWLEDGMENTS We thank the following ARS employees: Drs. Joe Patt and Roger Leopold for providing GWSS, Maureen Wright, James Smith. and John Willenberg for their creative contributions to rearing the GWSS, and Laura Hunnicutt for providing bioinformatic and technical assistance. LITERATURE CITED Abubakar LU, Bulimo WD, Mulaa FJ, Osir EO. 2006. 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