Phd Thesis 33 - eCommons@USASK - University of Saskatchewan

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1. To gain a better understanding of the morphology, histology and formation of the PM throughout larval development. This included characterization of the enzymes responsible for chitin synthesis and degradation (Chapter 3). 2. To survey proteins associated with the PM (Chapter 4). 3. To characterize select PM proteins in detail (Chapter 5). 4. To expand the understanding of proteins with Peritrophin-A Domains (PPADs), of which peritrophins are members (Chapter 6). 5. To examine the interaction between IIMs and baculovirus and the baculoviral metalloprotease, enhancin (Chapter 7). 6. To evaluate RNAi as a means to inhibit the expression of specific midgut genes encoding PM components using both in vivo and in vitro systems (Chapter 8). 7. To develop a model of the lepidopteran PM (Chapter 9). 13
2. GENERAL MATERIAL AND METHODS This chapter presents the general material and common methodologies used in the entire study. Specific material and methods are presented in the appropriate chapters. 2.1 Research Material 2.1.1 Mamestra configurata The insect model chosen for this study was M. configurata, for which there is already some information on its digestive biochemistry (Hegedus et al., 2003; Erlandson et al., 2010). This species was also used to develop the current model of the lepidopteran Type I PM (Shi et al., 2004). M. configurata, commonly known as the bertha armyworm (BAW), is a noctuid moth found across western North America. It is a serious but sporadic pest of cruciferous oilseed rapes (Brassica napus and B. rapa) and several other crops (Turnock, 1985; Mason et al., 1998). The current control strategy for M. configurata consists of aerial application of chemical insecticides. In the last outbreak (in 1994 and 1995), 670,000 ha of canola were sprayed at a cost of $50 million and yet incremental yield losses of $60 million occurred in Canada (Mason et al., 1998). Larvae were reared at 21±1 ºC under a 16 h light/8 h dark photoperiod and fed ad libitum on artificial diet (Bucher and Bracken, 1976), artificial diet supplemented with 0.5% (w/v) soybean Kunitz-type trypsin inhibitor (Sigma, St Louis, MO, USA) or Brassica napus (AC Excel). The genetic diversity of the colony was maintained by annually mating colony insects with moths derived from field collected pupae. 2.2 Midgut cDNA Library Construction and Expressed Sequence Tag Generation Collection of larval midgut tissues and cDNA library construction were as described in Chamankhah et al. (2003). In total, 5797, 5866 and 4376 expressed sequence tags (ESTs) were generated from the midgut cDNA libraries from larvae feeding on diet, diet with soybean trypsin inhibitor and B. napus leaves, respectively. The data were warehoused using the software package APED (http://aped.sourceforge.net) (Links et al., 2007). DNA sequences were evaluated for quality and trimmed of vector sequences prior to assembly using Lucy (Chou and Holmes, 2001). Assembly of the data was performed using TGICL with a Cap3 (Huang and Madan, 1999) 14
Page 1 and 2: THE MOLECULAR ARCHITECTURE OF MAMES
Page 3 and 4: "Bugs are not going to inherit the
Page 5 and 6: DEDICATION This Ph.D thesis is dedi
Page 7 and 8: ACKNOWLEDGMENTS I owe my deepest gr
Page 9 and 10: 2.1.1 Mamestra configurata ........
Page 11 and 12: 4.3.3.3.1 Alkaline phosphatase ....
Page 13 and 14: 5.3.3.4 Localization...............
Page 15 and 16: 8.2.1 Preparation of dsRNA.........
Page 17 and 18: LIST OF FIGURES Figure 1.1 The alim
Page 19 and 20: Figure 5.12 Two dimensional gel ele
Page 21 and 22: LIST OF ABBREVIATIONS Ac...........
Page 23 and 24: Ld ................................
Page 25 and 26: wt.................................
Page 27 and 28: 1.2 The Peritrophic Matrix The pres
Page 29 and 30: Now that many PM proteins have been
Page 31 and 32: 1.2.3.1 Mechanical protection and s
Page 33 and 34: 1.2.3.3 Compartmentalization of dig
Page 35 and 36: the anterior midgut and disintegrat
Page 37: of larvae reared on the leaves of r
Page 41 and 42: Blots were prehybridized, hybridize
Page 43 and 44: samples were mixed with loading buf
Page 45 and 46: over the m/z range 50 to 1900. LC-M
Page 47 and 48: acetylglucosamine (UDP-GlcNAc) as t
Page 49 and 50: For SEM analyses, PMs were dissecte
Page 51 and 52: Chapter 2) was conducted using tota
Page 53 and 54: significant difference was detected
Page 55 and 56: The PMs from all three stages studi
Page 57 and 58: amino acids (Figure 3.4) including
Page 59 and 60: 1 McCHS-B MATKPKTPGFTGLGDDSEDESEYTP
Page 61 and 62: 36 1 70 AiCHI MKAILATLAVLAVVTTAIEAD
Page 63 and 64: 38 1 70 AiNAG MWLQKYSLCAVYITLLSVICV
Page 65 and 66: 3.3.4 Expression Analysis of the Ge
Page 67 and 68: Figure 3.10 Expression of Mamestra
Page 69 and 70: H. virescens (Ryerse et al., 1992)
Page 71 and 72: 3.4.3 Tissue Specific Expression of
Page 73 and 74: (Chamankhah et al., 2003), McSerpin
Page 75 and 76: 4. SURVEY AND PRELIMINARY CHARACTER
Page 77 and 78: Table 4.1 RT-PCR primers used in ex
Page 79 and 80: Table 4.2 Proteins identified from
Page 81 and 82: Figure 4.2 Two dimensional gel elec
Page 83 and 84: Figure 4.3 Expression of Mamestra c
Page 85 and 86: Trypsins Chymotrypsins Elastase 57
Page 87 and 88: (Figure 4.5 continued) 351 420 McAP
Page 89 and 90:
(Figure 4.5 continued) 1051 1102 Mc
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4.3.3.2.3 Insect intestinal lipases
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4.3.3.2.5 α-Amylase Mascot analysi
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4.3.4.3 REPAT Mascot analysis ident
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Appendix A). All proteins were pred
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eported to be restricted to midgut
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and Ellar, 2007; Angelucci et al.,
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the M. configurata PM. McALP1 is pr
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larvae; whereas HMG176 is expressed
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4.4.4 Proteins without Orthologs Th
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5.2 Material and Methods 5.2.1 Rapi
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Table 5.2 (continued) Primer 1 Sequ
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uffer (without β-mercaptoethanol)
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5.3.2 Insect Intestinal Mucins (McI
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proline (9.3%), histidine (11.1%),
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molting larvae (Figure 5.4B). A low
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Figure 5.6 One dimensional (1D) and
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(Figure 5.7 continued) 421 429 BmCD
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meliloti (RhiNODB) and Colletotrich
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5.3.3.5 Demonstration of chitin dea
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similar colour changes, indicating
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5.3.4.3 Gene expression Expression
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transfer to a gelatin impregnated p
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produced and stored in secretory ve
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The results of CDA activity assays
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catalytically active. Of the 10 lip
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6. SURVEY, CHARACTERIZATION AND EVO
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PAD, conserved aromatic amino acids
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6.2.5 Antisera Production, Protein
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McPPAD1 MIAKFLTTVL LLNVVLTAEI PQKNA
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with the 951 bp cDNA. The McPPAD2 p
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The anti-rMcPPAD1 antiserum reacted
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Four types of PPADs were identified
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Figure 6.7 (legend is on the next p
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Figure 6.8 Model showing the propos
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Figure 6.9 Modeling of Mamestra con
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6.4.1.2 Expression Genes encoding P
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Table 6.4 (continued) Species Prote
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however, this is not present in McP
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that bonds could form between C2 an
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to form a bond with C3 in the eight
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Mamestra configurata nucleopolyhedr
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solution. In parallel, PMs without
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Figure 7.1 Analysis of strongly ass
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Figure 7.3 Analysis of Mamestra con
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kDa representing the degradation pr
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8. TARGETING GENES INVOLVED IN PERI
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Table 8.1 RT-PCR primers used for a
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50 µl 0.1% Triton-100 in nuclease
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Figure 8.1 RT-PCR analysis to detec
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clear as the same dosages and dsRNA
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RNAi to be initiated by dsRNA feedi
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2003), which could inhibit the acti
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in vitro (Cho et al., 2000), which
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glycan, two GlcNAc residues are lin
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The current study is also the first
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TcCHS2 are specialized for synthesi
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Blair, D.E., Hekmat, O., Schuttelko
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Chapman, R.F., 1985. Structure of t
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Dong, D-J., He, H-J., Chai, L-Q., W
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Filho, B.P.D., Lemos, F.J.A., Secun
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Grossi de Sa, M.F., Chrispeels, M.J
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Huang, X., Madan, A., 1999. CAP3: A
Page 215 and 216:
Kim, M.G., Shin, S.W., Bae, K.S., K
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Lepore, L.S., Roelvink, P.R., Grana
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novel, very large fluorescent lipoc
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Palli, S.R., Locke, M., 1987. Hemol
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Ponnuvel, K.M., Nakazawa, H., Furuk
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Schorderet, S., Pearson, R.D., Vuoc
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Strobl, S., Maskos, K., Betz, M., W
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Tomoyasu, Y., Miller, S.C., Tomita,
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Wang, S., Jayaram, A.S., Hemphala,
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Zhou, H-X., Tan, X-M., Li, C-Y., Wa
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Appendix A. (continued) Protein Acc
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Page 239 and 240:
Page 241 and 242:
Page 243 and 244:
218 Appendix B. Amino acid composit
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220 Appendix D. Data from serine pr
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Appendix F. Domain organization and
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Appendix F. (continued) Spodoptera
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Appendix F. (continued) The colour
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Appendix G. (continued) 27. McPM1 C
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Appendix G. (continued) 81. HaIIM4
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Appendix G. (continued) 135. SeCBP6
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234 Appendix I. Tandem repeats with
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