cacc17ce30902f889194c9057c4dcbcc pmc_10459852.pdf 05fc75fae765f56964b609fc37a4ea88385ba99a pmc_10459852.pdf 1a6c6a4f83f86a5f958750ada5ff6d79013bdbdeb3dc3639e44a4c5a01b65487 pmc_10459852.pdf Title: Exploring Gut Microbiome Variations between Popillia japonica Populations of Azores Subject: Popillia japonica (Coleoptera: Scarabaeidae), is an emerging invasive pest in Europe and America. In the Azores, this pest was first found on Terceira Island during the sixties and soon spread to other islands. The rate of infestation differs between islands, and we hypothesized that microbiome composition could play a role. Therefore, we sampled 3rd instar larvae and soil from sites with high and low infestation rates to analyze the microbiome using next-generation sequencing. We analyzed twenty-four 16S DNA libraries, which resulted in 3278 operational taxonomic units. The alpha and beta diversity of the soil microbiome was similar between sites. In contrast, the larvae from high-density sites presented a higher bacterial gut diversity than larvae from low-density sites, with biomarkers linked to plant digestion, nutrient acquisition, and detoxification. Consequently, larvae from high-density sites displayed several enriched molecular functions associated with the families Ruminococcaceae, Clostridiaceae and Rikenellaceae. These bacteria revealed a supportive function by producing several CAZyme families and other proteins. These findings suggest that the microbiome must be one drive for the increase in P. japonica populations, thus providing a checkpoint in the establishment and spread of this pest. Keywords: Popillia japonica; associated microbiome; bacterial diversity; enriched phyla; enriched pathways Author: Jorge Frias, Anna Garriga, Ángel Peñalver, Mário Teixeira, Rubén Beltrí, Duarte Toubarro and Nelson Simões Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Aug 1 06:25:44 2023 CEST ModDate: Tue Aug 1 07:44:34 2023 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 21 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 9779705 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- XKHPEK+VnURWPalladioL Type 1 Custom yes yes no 10 0 PYQQDV+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 EILXKB+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 AEOQIR+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 VGGBNJ+URWPalladioL-BoldItal Type 1 Custom yes yes no 28 0 CENDBP+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 SIXRFS+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 50 0 AAPCWM+CMSY10 Type 1 Builtin yes yes no 75 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 79 0 DCGFMA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 99 0 DCGFMB+PalatinoLinotype CID TrueType Identity-H yes yes yes 102 0 DCGFMC+PalatinoLinotype TrueType WinAnsi yes yes no 108 0 DCGFME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 111 0 DCGGKC+PalatinoLinotype TrueType MacRoman yes yes no 114 0 DCGFMA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 127 0 DCGFMB+PalatinoLinotype CID TrueType Identity-H yes yes yes 130 0 DCGFMC+PalatinoLinotype TrueType WinAnsi yes yes no 136 0 DCGFME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 139 0 DCGGKC+PalatinoLinotype TrueType MacRoman yes yes no 142 0 DCGHFA+PalatinoLinotype,Italic TrueType MacRoman yes yes no 158 0 DCGFMA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 161 0 DCGFMC+PalatinoLinotype TrueType WinAnsi yes yes no 164 0 DCGFME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 167 0 DCGGKC+PalatinoLinotype TrueType MacRoman yes yes no 170 0 DCGHFA+PalatinoLinotype,Italic TrueType MacRoman yes yes no 185 0 DCGFMA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 188 0 DCGFMB+PalatinoLinotype CID TrueType Identity-H yes yes yes 191 0 DCGFMC+PalatinoLinotype TrueType WinAnsi yes yes no 197 0 DCGFME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 200 0 DCGGKC+PalatinoLinotype TrueType MacRoman yes yes no 203 0 DCGFMA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 217 0 DCGFMC+PalatinoLinotype TrueType WinAnsi yes yes no 220 0 DCGFME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 223 0 DCGFMA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 238 0 DCGFMB+PalatinoLinotype CID TrueType Identity-H yes yes yes 241 0 DCGFMC+PalatinoLinotype TrueType WinAnsi yes yes no 247 0 DCGFME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 250 0 DCGGKC+PalatinoLinotype TrueType MacRoman yes yes no 253 0 DCGFMA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 269 0 DCGFMB+PalatinoLinotype CID TrueType Identity-H yes yes yes 272 0 DCGFMC+PalatinoLinotype TrueType WinAnsi yes yes no 278 0 DCGFME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 281 0 DCGGKC+PalatinoLinotype TrueType MacRoman yes yes no 284 0 DCGFMA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 301 0 DCGFMB+PalatinoLinotype CID TrueType Identity-H yes yes yes 304 0 DCGFMC+PalatinoLinotype TrueType WinAnsi yes yes no 310 0 DCGFME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 313 0 DCGFMA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 327 0 DCGFMC+PalatinoLinotype TrueType WinAnsi yes yes no 330 0 DCGFME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 333 0 DCGGKC+PalatinoLinotype TrueType MacRoman yes yes no 336 0 DCGFMA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 351 0 DCGFMB+PalatinoLinotype CID TrueType Identity-H yes yes yes 354 0 DCGFMC+PalatinoLinotype TrueType WinAnsi yes yes no 360 0 DCGFME+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 363 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-03-12 02:43:44 CET RepresentationInformation: pmc_10459852.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-03-11 02:07:58 CET Size: 9779705 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 629 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Collection and Processing of Insect and Soil Samples Destination: subsection.2.1 Item: Title: Soil and Climatic Parameters Destination: subsection.2.2 Item: Title: Next-Generation Sequencing and Processing of Reads Destination: subsection.2.3 Item: Title: Metabarcoding and Metagenomic Functional Analysis Destination: subsection.2.4 Item: Title: Results Destination: section.3 Children: Item: Title: Metadata of Amplicon 16S Sequence Destination: subsection.3.1 Item: Title: The Diversity and Composition of the Larval Microbiomes Differ between Insect Populations, While Soil Microbiome Remains Consistent Destination: subsection.3.2 Item: Title: Soil Microbiomes Revealed Minor Variations between Density Sites Destination: subsection.3.3 Item: Title: Some Bacterial Families Were More Enriched in the Microbiome of Larvae from HD than from LD Sites Destination: subsection.3.4 Item: Title: Metagenomic Analysis Uncovers Enhanced Functional Enrichment in Larvae from the HD Site Destination: subsection.3.5 Item: Title: The Core Microbiome Shared between Soil and Larvae in the HD Site, Is More Diverse and Abundant than in the LD Site Destination: subsection.3.6 Item: Title: Microbiome Biomarkers from the Two Density Sites Are Correlated with Soil Parameters Destination: subsection.3.7 Item: Title: Discussion Destination: section.4 Children: Item: Title: The Gut Microbiome of Larvae from HD Sites Is More Favorable to Insect than That of Larvae from LD Sites Destination: subsection.4.1 Item: Title: Metagenomic Analysis Uncovers Enhanced Functional Enrichment in the Larvae from the HD Site Destination: subsection.4.2 Item: Title: Larvae from the HD Site Share with Soil a Higher Number of Bacteria than Those from LD Site Destination: subsection.4.3 Item: Title: There Is a Correlation between Microbiome Biomarkers and Soil Parameters Destination: subsection.4.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.A. Info: Title: Exploring Gut Microbiome Variations between Popillia japonica Populations of Azores Author: Jorge Frias, Anna Garriga, Ángel Peñalver, Mário Teixeira, Rubén Beltrí, Duarte Toubarro and Nelson Simões Subject: Popillia japonica (Coleoptera: Scarabaeidae), is an emerging invasive pest in Europe and America. In the Azores, this pest was first found on Terceira Island during the sixties and soon spread to other islands. The rate of infestation differs between islands, and we hypothesized that microbiome composition could play a role. Therefore, we sampled 3rd instar larvae and soil from sites with high and low infestation rates to analyze the microbiome using next-generation sequencing. We analyzed twenty-four 16S DNA libraries, which resulted in 3278 operational taxonomic units. The alpha and beta diversity of the soil microbiome was similar between sites. In contrast, the larvae from high-density sites presented a higher bacterial gut diversity than larvae from low-density sites, with biomarkers linked to plant digestion, nutrient acquisition, and detoxification. Consequently, larvae from high-density sites displayed several enriched molecular functions associated with the families Ruminococcaceae, Clostridiaceae and Rikenellaceae. These bacteria revealed a supportive function by producing several CAZyme families and other proteins. These findings suggest that the microbiome must be one drive for the increase in P. japonica populations, thus providing a checkpoint in the establishment and spread of this pest. Keywords: Popillia japonica; associated microbiome; bacterial diversity; enriched phyla; enriched pathways Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Aug 01 06:25:44 CEST 2023 ModDate: Tue Aug 01 07:44:34 CEST 2023 ID: 0xd611b4e7f7582fdf54e233028e379a6a, 0xd611b4e7f7582fdf54e233028e379a6a Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: CENDBP+VnURWPalladioL-Bold FontSubset: true FirstChar: 129 LastChar: 227 FontDescriptor: FontName: CENDBP+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: EILXKB+URWPalladioL-Roma FontSubset: true FirstChar: 1 LastChar: 246 FontDescriptor: FontName: EILXKB+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: AEOQIR+URWPalladioL-Ital FontSubset: true FirstChar: 3 LastChar: 151 FontDescriptor: FontName: AEOQIR+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: XKHPEK+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 243 FontDescriptor: FontName: XKHPEK+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: AAPCWM+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: AAPCWM+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: VGGBNJ+URWPalladioL-BoldItal FontSubset: true FirstChar: 80 LastChar: 112 FontDescriptor: FontName: VGGBNJ+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: PYQQDV+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 241 FontDescriptor: FontName: PYQQDV+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VVSYYB+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: VVSYYB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 281, 152, 290 Annotation: Subtype: Link Rect: 34, 269, 114, 278 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 197, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 96, 660, 107, 674 Annotation: Subtype: Link Rect: 394, 188, 401, 197 ActionDest: 377 Annotation: Subtype: Link Rect: 508, 163, 515, 172 ActionDest: 377 Annotation: Subtype: Link Rect: 192, 125, 199, 134 ActionDest: 377 Annotation: Subtype: Link Rect: 549, 126, 556, 134 ActionDest: 377 Annotation: Subtype: Link Rect: 141, 17, 322, 28 Annotation: Subtype: Link Rect: 379, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 366, 87, 385 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 392, 735, 399, 744 ActionDest: 377 Annotation: Subtype: Link Rect: 402, 735, 409, 744 ActionDest: 380 Annotation: Subtype: Link Rect: 272, 723, 284, 732 ActionDest: 380 Annotation: Subtype: Link Rect: 464, 648, 476, 657 ActionDest: 380 Annotation: Subtype: Link Rect: 386, 522, 398, 531 ActionDest: 380 Annotation: Subtype: Link Rect: 243, 509, 255, 519 ActionDest: 380 Annotation: Subtype: Link Rect: 255, 510, 267, 519 ActionDest: 380 Annotation: Subtype: Link Rect: 251, 484, 263, 493 ActionDest: 380 Annotation: Subtype: Link Rect: 460, 447, 472, 456 ActionDest: 380 Annotation: Subtype: Link Rect: 475, 447, 487, 456 ActionDest: 380 Annotation: Subtype: Link Rect: 270, 396, 282, 406 ActionDest: 380 Annotation: Subtype: Link Rect: 283, 396, 295, 406 ActionDest: 380 Annotation: Subtype: Link Rect: 231, 346, 243, 355 ActionDest: 380 Annotation: Subtype: Link Rect: 196, 321, 208, 330 ActionDest: 380 Annotation: Subtype: Link Rect: 208, 321, 220, 330 ActionDest: 380 Annotation: Subtype: Link Rect: 442, 283, 454, 293 ActionDest: 380 Annotation: Subtype: Link Rect: 454, 284, 466, 293 ActionDest: 380 Annotation: Subtype: Link Rect: 467, 283, 479, 293 ActionDest: 380 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 271, 644, 283, 653 ActionDest: 380 Annotation: Subtype: Link Rect: 319, 581, 331, 590 ActionDest: 380 Annotation: Subtype: Link Rect: 299, 455, 310, 464 ActionDest: 380 Annotation: Subtype: Link Rect: 402, 204, 414, 213 ActionDest: 380 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 374, 685, 386, 694 ActionDest: 380 Annotation: Subtype: Link Rect: 544, 673, 556, 682 ActionDest: 380 Annotation: Subtype: Link Rect: 502, 647, 514, 657 ActionDest: 380 Annotation: Subtype: Link Rect: 381, 597, 393, 606 ActionDest: 380 Annotation: Subtype: Link Rect: 228, 446, 240, 455 ActionDest: 380 Annotation: Subtype: Link Rect: 344, 434, 356, 443 ActionDest: 380 Annotation: Subtype: Link Rect: 425, 421, 437, 430 ActionDest: 383 Annotation: Subtype: Link Rect: 283, 371, 295, 380 ActionDest: 383 Annotation: Subtype: Link Rect: 374, 296, 386, 305 ActionDest: 383 Annotation: Subtype: Link Rect: 237, 195, 249, 204 ActionDest: 383 Annotation: Subtype: Link Rect: 252, 158, 264, 167 ActionDest: 383 Annotation: Subtype: Link Rect: 231, 133, 242, 142 ActionDest: 380 Annotation: Subtype: Link Rect: 420, 120, 432, 129 ActionDest: 383 Annotation: Subtype: Link Rect: 512, 95, 524, 104 ActionDest: 383 Annotation: Subtype: Link Rect: 506, 70, 518, 79 ActionDest: 383 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 371, 735, 383, 744 ActionDest: 383 Annotation: Subtype: Link Rect: 177, 685, 189, 694 ActionDest: 383 Annotation: Subtype: Link Rect: 224, 635, 236, 644 ActionDest: 383 Annotation: Subtype: Link Rect: 218, 244, 225, 256 ActionDest: 89 Annotation: Subtype: Link Rect: 260, 194, 267, 206 ActionDest: 89 Annotation: Subtype: Link Rect: 435, 168, 442, 180 ActionDest: 89 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 451, 71, 458, 83 ActionDest: 148 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 245, 720, 252, 732 ActionDest: 148 Annotation: Subtype: Link Rect: 299, 188, 307, 200 ActionDest: 148 Annotation: Subtype: Link Rect: 437, 150, 444, 162 ActionDest: 148 Annotation: Subtype: Link Rect: 422, 77, 428, 89 ActionDest: 175 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 543, 695, 550, 707 ActionDest: 175 Annotation: Subtype: Link Rect: 363, 120, 370, 132 ActionDest: 175 Annotation: Subtype: Link Rect: 481, 94, 488, 106 ActionDest: 175 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 276, 571, 283, 583 ActionDest: 207 Annotation: Subtype: Link Rect: 199, 546, 206, 558 ActionDest: 207 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 357, 507, 364, 519 ActionDest: 228 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 332, 720, 339, 732 ActionDest: 259 Annotation: Subtype: Link Rect: 199, 208, 206, 220 ActionDest: 291 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 488, 187, 495, 199 ActionDest: 291 Annotation: Subtype: Link Rect: 387, 161, 394, 173 ActionDest: 317 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 375, 198, 382, 210 ActionDest: 317 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 383, 708, 395, 720 ActionDest: 341 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 394, 720, 406, 732 ActionDest: 341 Annotation: Subtype: Link Rect: 236, 535, 247, 545 ActionDest: 383 Annotation: Subtype: Link Rect: 250, 535, 262, 545 ActionDest: 383 Annotation: Subtype: Link Rect: 382, 523, 394, 532 ActionDest: 380 Annotation: Subtype: Link Rect: 394, 523, 406, 532 ActionDest: 383 Annotation: Subtype: Link Rect: 229, 372, 241, 381 ActionDest: 380 Annotation: Subtype: Link Rect: 314, 347, 326, 356 ActionDest: 383 Annotation: Subtype: Link Rect: 329, 347, 341, 356 ActionDest: 383 Annotation: Subtype: Link Rect: 322, 297, 334, 306 ActionDest: 383 Annotation: Subtype: Link Rect: 335, 297, 347, 306 ActionDest: 383 Annotation: Subtype: Link Rect: 397, 297, 409, 306 ActionDest: 383 Annotation: Subtype: Link Rect: 410, 297, 422, 306 ActionDest: 383 Annotation: Subtype: Link Rect: 510, 297, 522, 306 ActionDest: 383 Annotation: Subtype: Link Rect: 270, 272, 282, 281 ActionDest: 380 Annotation: Subtype: Link Rect: 447, 247, 459, 256 ActionDest: 383 Annotation: Subtype: Link Rect: 544, 171, 556, 180 ActionDest: 383 Annotation: Subtype: Link Rect: 446, 146, 458, 155 ActionDest: 380 Annotation: Subtype: Link Rect: 386, 121, 398, 130 ActionDest: 383 Annotation: Subtype: Link Rect: 401, 121, 413, 130 ActionDest: 383 Annotation: Subtype: Link Rect: 413, 121, 425, 130 ActionDest: 383 Annotation: Subtype: Link Rect: 426, 121, 438, 130 ActionDest: 386 Annotation: Subtype: Link Rect: 365, 109, 377, 118 ActionDest: 386 Annotation: Subtype: Link Rect: 378, 109, 389, 118 ActionDest: 386 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 209, 735, 221, 744 ActionDest: 386 Annotation: Subtype: Link Rect: 187, 723, 199, 732 ActionDest: 386 Annotation: Subtype: Link Rect: 265, 536, 277, 545 ActionDest: 386 Annotation: Subtype: Link Rect: 423, 536, 434, 545 ActionDest: 386 Annotation: Subtype: Link Rect: 435, 536, 447, 545 ActionDest: 386 Annotation: Subtype: Link Rect: 351, 511, 363, 520 ActionDest: 386 Annotation: Subtype: Link Rect: 187, 424, 199, 432 ActionDest: 386 Annotation: Subtype: Link Rect: 194, 398, 206, 407 ActionDest: 386 Annotation: Subtype: Link Rect: 207, 398, 219, 407 ActionDest: 386 Annotation: Subtype: Link Rect: 190, 386, 202, 395 ActionDest: 386 Annotation: Subtype: Link Rect: 530, 386, 542, 395 ActionDest: 386 Annotation: Subtype: Link Rect: 320, 222, 332, 232 ActionDest: 386 Annotation: Subtype: Link Rect: 421, 198, 433, 206 ActionDest: 386 Annotation: Subtype: Link Rect: 256, 172, 268, 181 ActionDest: 386 Annotation: Subtype: Link Rect: 484, 72, 496, 81 ActionDest: 386 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 458, 685, 470, 694 ActionDest: 386 Annotation: Subtype: Link Rect: 210, 584, 222, 594 ActionDest: 380 Annotation: Subtype: Link Rect: 512, 559, 524, 568 ActionDest: 383 Annotation: Subtype: Link Rect: 314, 534, 325, 543 ActionDest: 380 Annotation: Subtype: Link Rect: 326, 534, 338, 543 ActionDest: 380 Annotation: Subtype: Link Rect: 338, 534, 350, 543 ActionDest: 386 Annotation: Subtype: Link Rect: 214, 509, 226, 518 ActionDest: 380 Annotation: Subtype: Link Rect: 226, 509, 238, 518 ActionDest: 383 Annotation: Subtype: Link Rect: 262, 384, 274, 393 ActionDest: 383 Annotation: Subtype: Link Rect: 343, 170, 354, 179 ActionDest: 380 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 198, 710, 210, 719 ActionDest: 380 Annotation: Subtype: Link Rect: 211, 710, 223, 719 ActionDest: 386 Annotation: Subtype: Link Rect: 536, 596, 560, 607 Annotation: Subtype: Link Rect: 165, 584, 424, 595 Annotation: Subtype: Link Rect: 187, 187, 224, 198 Annotation: Subtype: Link Rect: 459, 95, 497, 106 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 390, 722, 426, 733 Annotation: Subtype: Link Rect: 59, 688, 96, 698 Annotation: Subtype: Link Rect: 411, 652, 560, 664 Annotation: Subtype: Link Rect: 56, 642, 134, 652 Annotation: Subtype: Link Rect: 513, 629, 551, 640 Annotation: Subtype: Link Rect: 59, 607, 96, 617 Annotation: Subtype: Link Rect: 108, 584, 145, 594 Annotation: Subtype: Link Rect: 108, 561, 145, 571 Annotation: Subtype: Link Rect: 335, 537, 373, 548 Annotation: Subtype: Link Rect: 180, 504, 218, 514 Annotation: Subtype: Link Rect: 108, 469, 145, 479 Annotation: Subtype: Link Rect: 152, 469, 188, 479 Annotation: Subtype: Link Rect: 342, 445, 380, 456 Annotation: Subtype: Link Rect: 386, 445, 422, 456 Annotation: Subtype: Link Rect: 113, 412, 151, 422 Annotation: Subtype: Link Rect: 157, 412, 193, 422 Annotation: Subtype: Link Rect: 437, 388, 475, 399 Annotation: Subtype: Link Rect: 450, 365, 488, 376 Annotation: Subtype: Link Rect: 332, 342, 370, 353 Annotation: Subtype: Link Rect: 357, 319, 395, 330 Annotation: Subtype: Link Rect: 112, 297, 150, 307 Annotation: Subtype: Link Rect: 185, 273, 347, 284 Annotation: Subtype: Link Rect: 59, 239, 96, 249 Annotation: Subtype: Link Rect: 498, 227, 535, 238 Annotation: Subtype: Link Rect: 59, 182, 96, 192 Annotation: Subtype: Link Rect: 131, 147, 168, 157 Annotation: Subtype: Link Rect: 175, 147, 211, 157 Annotation: Subtype: Link Rect: 478, 123, 515, 134 Annotation: Subtype: Link Rect: 521, 123, 557, 134 Annotation: Subtype: Link Rect: 189, 78, 227, 88 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 236, 676, 274, 687 Annotation: Subtype: Link Rect: 280, 676, 316, 687 Annotation: Subtype: Link Rect: 59, 653, 96, 664 Annotation: Subtype: Link Rect: 117, 630, 155, 640 Annotation: Subtype: Link Rect: 204, 607, 241, 617 Annotation: Subtype: Link Rect: 415, 583, 453, 594 Annotation: Subtype: Link Rect: 337, 560, 374, 571 Annotation: Subtype: Link Rect: 501, 537, 538, 548 Annotation: Subtype: Link Rect: 324, 514, 361, 525 Annotation: Subtype: Link Rect: 341, 491, 378, 502 Annotation: Subtype: Link Rect: 354, 457, 392, 468 Annotation: Subtype: Link Rect: 454, 434, 491, 445 Annotation: Subtype: Link Rect: 178, 400, 216, 410 Annotation: Subtype: Link Rect: 476, 376, 514, 387 Annotation: Subtype: Link Rect: 470, 330, 507, 341 Annotation: Subtype: Link Rect: 513, 330, 549, 341 Annotation: Subtype: Link Rect: 195, 308, 232, 318 Annotation: Subtype: Link Rect: 239, 308, 275, 318 Annotation: Subtype: Link Rect: 388, 284, 425, 295 Annotation: Subtype: Link Rect: 140, 251, 177, 261 Annotation: Subtype: Link Rect: 184, 251, 220, 261 Annotation: Subtype: Link Rect: 100, 216, 137, 226 Annotation: Subtype: Link Rect: 215, 192, 252, 203 Annotation: Subtype: Link Rect: 485, 169, 522, 180 Annotation: Subtype: Link Rect: 165, 136, 202, 146 Annotation: Subtype: Link Rect: 153, 101, 189, 111 Annotation: Subtype: Link Rect: 108, 78, 146, 88 Annotation: Subtype: Link Rect: 152, 78, 188, 88 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 364, 722, 402, 733 Annotation: Subtype: Link Rect: 454, 711, 491, 721 Annotation: Subtype: Link Rect: 520, 699, 557, 710 Annotation: Subtype: Link Rect: 511, 641, 548, 652 Annotation: Subtype: Link Rect: 112, 619, 150, 629 Annotation: Subtype: Link Rect: 156, 619, 192, 629 Annotation: Subtype: Link Rect: 196, 596, 233, 606 Annotation: Subtype: Link Rect: 239, 596, 275, 606 Annotation: Subtype: Link Rect: 360, 550, 396, 560 Annotation: Subtype: Link Rect: 354, 526, 392, 537 Annotation: Subtype: Link Rect: 398, 526, 434, 537 Annotation: Subtype: Link Rect: 200, 503, 237, 514 Annotation: Subtype: Link Rect: 465, 480, 503, 491 Annotation: Subtype: Link Rect: 108, 458, 146, 468 Annotation: Subtype: Link Rect: 113, 435, 151, 445 Annotation: Subtype: Link Rect: 158, 400, 195, 410 Annotation: Subtype: Link Rect: 131, 377, 168, 387 Annotation: Subtype: Link Rect: 348, 342, 386, 353 Annotation: Subtype: Link Rect: 301, 319, 338, 330 Annotation: Subtype: Link Rect: 226, 297, 264, 307 Annotation: Subtype: Link Rect: 270, 297, 306, 307 Annotation: Subtype: Link Rect: 421, 273, 459, 284 Annotation: Subtype: Link Rect: 465, 273, 501, 284 Annotation: Subtype: Link Rect: 479, 250, 516, 261 Annotation: Subtype: Link Rect: 521, 250, 557, 261 Checksum: 0eb69797 Type: CRC32 Checksum: cacc17ce30902f889194c9057c4dcbcc Type: MD5 Checksum: 05fc75fae765f56964b609fc37a4ea88385ba99a Type: SHA-1 Checksum: 1a6c6a4f83f86a5f958750ada5ff6d79013bdbdeb3dc3639e44a4c5a01b65487 Type: SHA-256