ca82dc0543e477cc4ae94b36544de5b4 animals-15-01655.pdf f8d9771ad30e652e7491aabec000d1f59edc9a6a animals-15-01655.pdf b58e5275827572fd9a7d9ed8db157a0a367f0278e95a8444fd468e2e1354c525 animals-15-01655.pdf Title: The Administration of Heat Shock Protein-70 Bacterial Homolog (DnaK) Improves the Cumulative Survival and the Expression of Immune-Related Genes in Gnotobiotic Full-Sibling Sea Bass Larvae Challenged with Vibrio anguillarum Subject: Heat shock proteins (HSPs), particularly HSP70, play a vital role in fish immune defense against pathogens. The administration of DnaK (bacterial homolog of HSP70) may be a strategy to potentiate the immune response and survival of aquatic organisms. This study evaluates the effect of cells overexpressing DnaK on mortality and immune-related gene expression in gnotobiotic sea bass larvae challenged with Vibrio anguillarum. Larvae were subjected to different treatments: NB (no bacteria), YS0 (E. coli with no plasmid), YS1 (E. coli expressing truncated DnaK), and YS2 (E. coli expressing DnaK), and then infected with V. anguillarum at 7 days post-hatching (dph). Mortality was monitored, and RT-qPCR was used to evaluate immune gene expression at 0, 18, 24, 36, and 120 hpc. While no significant variations were recorded in the non-challenged larvae, constant and sustained mortality was observed in challenged larvae from 60 to 120 hpc. However, lower mortality was observed in the larvae treated with DnaK. DnaK treatment promoted the expression of antimicrobial (hepcidin, transferrin) and chemotaxis genes (ccl4), which was further enhanced after a challenge with V. anguillarum, in conjunction with the modulation of il1 and il-8 at 120 hpc. These findings suggest that DnaK induces a potent innate immune response, improving survival against V. anguillarum and supporting its potential use as a disease-preventive strategy in aquaculture. Keywords: aquaculture; HSP70; DnaK; immune response; sea bass larvae; Vibrio anguillarum Author: Eva Vallejos-Vidal, Camino Fierro-Castro, María Jesús Santillán-Araneda, Merari Goldstein, Sebastián Reyes-Cerpa, Joan Carles Balasch, Ali Reza Khansari, Kristof Dierckens, Peter Bossier, Lluis Tort and Felipe E. Reyes-López Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed Jun 4 10:17:26 2025 CEST ModDate: Wed Jun 4 10:53:44 2025 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 25 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2514283 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- MIKRSI+VnURWPalladioL Type 1 Custom yes yes yes 10 0 UVTOTY+URWPalladioL-Roma Type 1 Custom yes yes yes 16 0 DCZDQQ+URWPalladioL-Bold Type 1 Custom yes yes yes 22 0 HEXENT+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 TBVXYU+URWPalladioL-BoldItal Type 1 Custom yes yes yes 32 0 DUJUUF+PazoMath-Italic Type 1 Builtin yes yes yes 58 0 VVSYYB+EURM10 Type 1 Builtin yes yes yes 66 0 DFLSER+TeX-tipa10 Type 1 Builtin yes yes yes 74 0 OJYQXC+CMSY10 Type 1 Builtin yes yes yes 79 0 CDGJIA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 95 0 CDGJIC+PalatinoLinotype TrueType WinAnsi yes yes no 98 0 CDGJIE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 101 0 SHFVBA+TeX-tipa9 Type 1 Builtin yes yes yes 113 0 CDGJIC+PalatinoLinotype TrueType WinAnsi yes yes no 123 0 CDGJIC+PalatinoLinotype TrueType WinAnsi yes yes no 134 0 CDGJIC+PalatinoLinotype TrueType WinAnsi yes yes no 151 0 CDGJIA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 165 0 CDGJIC+PalatinoLinotype TrueType WinAnsi yes yes no 168 0 CDGJIA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 196 0 CDGJIC+PalatinoLinotype TrueType WinAnsi yes yes no 199 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-09-18 03:08:48 CEST RepresentationInformation: animals-15-01655.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-09-17 13:36:49 CEST Size: 2514283 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 516 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: Bacterial Strain and Culture Conditions Destination: subsection.2.1 Item: Title: Induction and Synthesis of DnaK Destination: subsection.2.2 Item: Title: Protein Extraction, SDS-PAGE, and Western Blot to Verify DnaK Synthesis Destination: subsection.2.3 Item: Title: Gnotobiotic Full-Sibling Sea Bass Larvae Destination: subsection.2.4 Item: Title: Full-Sibling Sea Bass Larvae Incubated with E. coli Overexpressing DnaK Destination: subsection.2.5 Item: Title: Full-Sibling Sea Bass Larvae Incubated with E. coli Overexpressing DnaK and Challenged with Vibrio anguillarum Destination: subsection.2.6 Item: Title: Ethics Statement Destination: subsection.2.7 Item: Title: RNA Extraction and cDNA Synthesis of Single Full-Sibling Sea Bass Larvae Destination: subsection.2.8 Item: Title: Gene Expression Analysis by Real-Time PCR Destination: subsection.2.9 Item: Title: Statistical Analysis Destination: subsection.2.10 Item: Title: Results Destination: section.3 Children: Item: Title: DnaK Induction and Synthesis Destination: subsection.3.1 Item: Title: Cumulative Survival in Gnotobiotic Full-Sibling European Sea Bass Larvae Incubated with DnaK Destination: subsection.3.2 Item: Title: Innate Immune-Related Gene Expression Profile in Gnotobiotic Full-Sibling European Sea Bass Larvae Incubated with DnaK Destination: subsection.3.3 Item: Title: Innate Immune-Related Gene Expression Profile in Gnotobiotic Full-Sibling European Sea Bass Larvae Incubated with DnaK and Challenged with V. anguillarum Destination: subsection.3.4 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: Appendix A Destination: appendix.A. Item: Title: References Destination: appendix.B. Info: Title: The Administration of Heat Shock Protein-70 Bacterial Homolog (DnaK) Improves the Cumulative Survival and the Expression of Immune-Related Genes in Gnotobiotic Full-Sibling Sea Bass Larvae Challenged with Vibrio anguillarum Author: Eva Vallejos-Vidal, Camino Fierro-Castro, María Jesús Santillán-Araneda, Merari Goldstein, Sebastián Reyes-Cerpa, Joan Carles Balasch, Ali Reza Khansari, Kristof Dierckens, Peter Bossier, Lluis Tort and Felipe E. Reyes-López Subject: Heat shock proteins (HSPs), particularly HSP70, play a vital role in fish immune defense against pathogens. The administration of DnaK (bacterial homolog of HSP70) may be a strategy to potentiate the immune response and survival of aquatic organisms. This study evaluates the effect of cells overexpressing DnaK on mortality and immune-related gene expression in gnotobiotic sea bass larvae challenged with Vibrio anguillarum. Larvae were subjected to different treatments: NB (no bacteria), YS0 (E. coli with no plasmid), YS1 (E. coli expressing truncated DnaK), and YS2 (E. coli expressing DnaK), and then infected with V. anguillarum at 7 days post-hatching (dph). Mortality was monitored, and RT-qPCR was used to evaluate immune gene expression at 0, 18, 24, 36, and 120 hpc. While no significant variations were recorded in the non-challenged larvae, constant and sustained mortality was observed in challenged larvae from 60 to 120 hpc. However, lower mortality was observed in the larvae treated with DnaK. DnaK treatment promoted the expression of antimicrobial (hepcidin, transferrin) and chemotaxis genes (ccl4), which was further enhanced after a challenge with V. anguillarum, in conjunction with the modulation of il1 and il-8 at 120 hpc. These findings suggest that DnaK induces a potent innate immune response, improving survival against V. anguillarum and supporting its potential use as a disease-preventive strategy in aquaculture. Keywords: aquaculture; HSP70; DnaK; immune response; sea bass larvae; Vibrio anguillarum Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed Jun 04 10:17:26 CEST 2025 ModDate: Wed Jun 04 10:53:44 CEST 2025 ID: 0x243dbd9dc541d9c344aa35919758f86d, 0x243dbd9dc541d9c344aa35919758f86d Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: UVTOTY+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 250 FontDescriptor: FontName: UVTOTY+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: TBVXYU+URWPalladioL-BoldItal FontSubset: true FirstChar: 86 LastChar: 117 FontDescriptor: FontName: TBVXYU+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: SHFVBA+TeX-tipa9 FontSubset: true FirstChar: 207 LastChar: 207 FontDescriptor: FontName: SHFVBA+TeX-tipa9 Flags: Symbolic FontBBox: -76, -296, 833, 769 FontFile: true ToUnicode: true Font: BaseFont: VVSYYB+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: VVSYYB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: DCZDQQ+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 250 FontDescriptor: FontName: DCZDQQ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: MIKRSI+VnURWPalladioL FontSubset: true FirstChar: 225 LastChar: 225 FontDescriptor: FontName: MIKRSI+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: DUJUUF+PazoMath-Italic FontSubset: true FirstChar: 97 LastChar: 98 FontDescriptor: FontName: DUJUUF+PazoMath-Italic Flags: Symbolic FontBBox: -70, -277, 902, 733 FontFile: true ToUnicode: true Font: BaseFont: DFLSER+TeX-tipa10 FontSubset: true FirstChar: 207 LastChar: 207 FontDescriptor: FontName: DFLSER+TeX-tipa10 Flags: Symbolic FontBBox: -70, -296, 812, 771 FontFile: true ToUnicode: true Font: BaseFont: HEXENT+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 150 FontDescriptor: FontName: HEXENT+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: OJYQXC+CMSY10 FontSubset: true FirstChar: 0 LastChar: 14 FontDescriptor: FontName: OJYQXC+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 108, 177, 152, 186 Annotation: Subtype: Link Rect: 34, 165, 127, 174 Annotation: Subtype: Link Rect: 36, 75, 152, 84 Annotation: Subtype: Link Rect: 34, 63, 89, 72 Annotation: Subtype: Link Rect: 34, 773, 133, 809 Annotation: Subtype: Link Rect: 515, 773, 560, 809 Annotation: Subtype: Link Rect: 262, 621, 273, 634 Annotation: Subtype: Link Rect: 422, 621, 433, 634 Annotation: Subtype: Link Rect: 522, 621, 533, 634 Annotation: Subtype: Link Rect: 253, 607, 264, 620 Annotation: Subtype: Link Rect: 221, 593, 232, 606 Annotation: Subtype: Link Rect: 424, 27, 560, 37 Annotation: Subtype: Link Rect: 34, 415, 87, 434 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 406, 468, 413, 477 ActionDest: 224 Annotation: Subtype: Link Rect: 414, 468, 421, 477 ActionDest: 224 Annotation: Subtype: Link Rect: 280, 440, 287, 449 ActionDest: 224 Annotation: Subtype: Link Rect: 369, 398, 376, 407 ActionDest: 224 Annotation: Subtype: Link Rect: 379, 398, 386, 407 ActionDest: 224 Annotation: Subtype: Link Rect: 549, 384, 556, 393 ActionDest: 224 Annotation: Subtype: Link Rect: 259, 328, 266, 338 ActionDest: 224 Annotation: Subtype: Link Rect: 269, 329, 281, 338 ActionDest: 224 Annotation: Subtype: Link Rect: 393, 315, 404, 324 ActionDest: 224 Annotation: Subtype: Link Rect: 355, 287, 362, 296 ActionDest: 224 Annotation: Subtype: Link Rect: 520, 273, 532, 282 ActionDest: 224 Annotation: Subtype: Link Rect: 400, 259, 412, 268 ActionDest: 224 Annotation: Subtype: Link Rect: 354, 231, 361, 240 ActionDest: 224 Annotation: Subtype: Link Rect: 362, 231, 369, 240 ActionDest: 224 Annotation: Subtype: Link Rect: 425, 203, 437, 212 ActionDest: 224 Annotation: Subtype: Link Rect: 438, 203, 450, 212 ActionDest: 224 Annotation: Subtype: Link Rect: 313, 175, 324, 184 ActionDest: 224 Annotation: Subtype: Link Rect: 486, 175, 498, 184 ActionDest: 224 Annotation: Subtype: Link Rect: 409, 133, 421, 143 ActionDest: 224 Annotation: Subtype: Link Rect: 323, 120, 330, 129 ActionDest: 224 Annotation: Subtype: Link Rect: 294, 78, 306, 87 ActionDest: 224 Annotation: Subtype: Link Rect: 307, 78, 318, 87 ActionDest: 224 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 459, 759, 471, 767 ActionDest: 224 Annotation: Subtype: Link Rect: 383, 731, 395, 740 ActionDest: 224 Annotation: Subtype: Link Rect: 250, 703, 262, 712 ActionDest: 227 Annotation: Subtype: Link Rect: 235, 675, 247, 684 ActionDest: 224 Annotation: Subtype: Link Rect: 247, 675, 259, 684 ActionDest: 227 Annotation: Subtype: Link Rect: 201, 605, 213, 614 ActionDest: 227 Annotation: Subtype: Link Rect: 508, 564, 515, 572 ActionDest: 224 Annotation: Subtype: Link Rect: 516, 563, 528, 572 ActionDest: 227 Annotation: Subtype: Link Rect: 322, 522, 334, 531 ActionDest: 227 Annotation: Subtype: Link Rect: 292, 480, 299, 489 ActionDest: 224 Annotation: Subtype: Link Rect: 300, 480, 312, 489 ActionDest: 227 Annotation: Subtype: Link Rect: 199, 410, 211, 419 ActionDest: 224 Annotation: Subtype: Link Rect: 527, 396, 539, 405 ActionDest: 224 Annotation: Subtype: Link Rect: 276, 354, 288, 364 ActionDest: 227 Annotation: Subtype: Link Rect: 480, 299, 492, 308 ActionDest: 227 Annotation: Subtype: Link Rect: 315, 215, 327, 224 ActionDest: 224 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 463, 545, 475, 555 ActionDest: 227 Annotation: Subtype: Link Rect: 202, 295, 214, 304 ActionDest: 227 Annotation: Subtype: Link Rect: 215, 295, 227, 304 ActionDest: 227 Annotation: Subtype: Link Rect: 480, 171, 492, 180 ActionDest: 224 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 544, 620, 556, 629 ActionDest: 227 Annotation: Subtype: Link Rect: 314, 579, 326, 588 ActionDest: 224 Annotation: Subtype: Link Rect: 474, 466, 489, 480 ActionDest: 71 Annotation: Subtype: Link Rect: 434, 358, 448, 370 ActionDest: 71 Annotation: Subtype: Link Rect: 502, 235, 514, 244 ActionDest: 227 Annotation: Subtype: Link Rect: 239, 205, 246, 217 ActionDest: 87 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 264, 190, 276, 199 ActionDest: 227 Annotation: Subtype: Link Rect: 230, 176, 242, 185 ActionDest: 227 Annotation: Subtype: Link Rect: 242, 176, 254, 185 ActionDest: 227 Annotation: Subtype: Link Rect: 412, 148, 424, 157 ActionDest: 227 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 183, 730, 195, 740 ActionDest: 227 Annotation: Subtype: Link Rect: 396, 475, 403, 487 ActionDest: 110 Annotation: Subtype: Link Rect: 492, 461, 499, 473 ActionDest: 110 Annotation: Subtype: Link Rect: 304, 447, 311, 459 ActionDest: 110 Annotation: Subtype: Link Rect: 199, 419, 206, 431 ActionDest: 110 Annotation: Subtype: Link Rect: 487, 405, 494, 418 ActionDest: 110 Annotation: Subtype: Link Rect: 392, 378, 399, 390 ActionDest: 110 Annotation: Subtype: Link Rect: 405, 364, 411, 376 ActionDest: 110 Annotation: Subtype: Link Rect: 441, 270, 449, 282 ActionDest: 110 Annotation: Subtype: Link Rect: 541, 256, 548, 268 ActionDest: 110 Annotation: Subtype: Link Rect: 542, 144, 549, 157 ActionDest: 110 Annotation: Subtype: Link Rect: 486, 117, 493, 129 ActionDest: 110 Page: Sequence: 8 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 388, 644, 395, 656 ActionDest: 143 Annotation: Subtype: Link Rect: 469, 644, 476, 656 ActionDest: 143 Annotation: Subtype: Link Rect: 542, 644, 549, 656 ActionDest: 143 Annotation: Subtype: Link Rect: 222, 630, 229, 642 ActionDest: 143 Annotation: Subtype: Link Rect: 327, 630, 334, 642 ActionDest: 143 Annotation: Subtype: Link Rect: 473, 588, 480, 600 ActionDest: 143 Annotation: Subtype: Link Rect: 327, 546, 334, 558 ActionDest: 143 Annotation: Subtype: Link Rect: 472, 518, 479, 530 ActionDest: 143 Annotation: Subtype: Link Rect: 332, 504, 339, 516 ActionDest: 143 Annotation: Subtype: Link Rect: 416, 463, 423, 475 ActionDest: 143 Annotation: Subtype: Link Rect: 520, 393, 527, 405 ActionDest: 143 Annotation: Subtype: Link Rect: 266, 365, 280, 377 ActionDest: 208 Annotation: Subtype: Link Rect: 300, 365, 315, 377 ActionDest: 211 Annotation: Subtype: Link Rect: 316, 229, 323, 241 ActionDest: 157 Annotation: Subtype: Link Rect: 452, 202, 459, 214 ActionDest: 157 Annotation: Subtype: Link Rect: 297, 160, 304, 172 ActionDest: 157 Annotation: Subtype: Link Rect: 435, 132, 442, 144 ActionDest: 157 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 378, 201, 385, 213 ActionDest: 157 Annotation: Subtype: Link Rect: 286, 160, 293, 172 ActionDest: 157 Annotation: Subtype: Link Rect: 199, 132, 206, 144 ActionDest: 157 Annotation: Subtype: Link Rect: 285, 104, 292, 116 ActionDest: 157 Annotation: Subtype: Link Rect: 373, 104, 380, 116 ActionDest: 157 Annotation: Subtype: Link Rect: 419, 62, 426, 74 ActionDest: 157 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 519, 686, 526, 698 ActionDest: 157 Annotation: Subtype: Link Rect: 372, 76, 379, 88 ActionDest: 157 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 353, 756, 360, 768 ActionDest: 157 Annotation: Subtype: Link Rect: 195, 714, 209, 726 ActionDest: 214 Annotation: Subtype: Link Rect: 229, 714, 244, 726 ActionDest: 221 Annotation: Subtype: Link Rect: 392, 576, 404, 585 ActionDest: 224 Annotation: Subtype: Link Rect: 405, 576, 417, 585 ActionDest: 227 Annotation: Subtype: Link Rect: 544, 562, 556, 571 ActionDest: 227 Annotation: Subtype: Link Rect: 366, 535, 378, 544 ActionDest: 224 Annotation: Subtype: Link Rect: 424, 493, 436, 502 ActionDest: 224 Annotation: Subtype: Link Rect: 437, 493, 449, 502 ActionDest: 227 Annotation: Subtype: Link Rect: 389, 465, 401, 474 ActionDest: 227 Annotation: Subtype: Link Rect: 201, 437, 208, 446 ActionDest: 224 Annotation: Subtype: Link Rect: 208, 437, 220, 446 ActionDest: 227 Annotation: Subtype: Link Rect: 322, 353, 334, 363 ActionDest: 224 Annotation: Subtype: Link Rect: 272, 256, 284, 265 ActionDest: 227 Annotation: Subtype: Link Rect: 285, 256, 297, 265 ActionDest: 227 Annotation: Subtype: Link Rect: 517, 242, 529, 251 ActionDest: 227 Annotation: Subtype: Link Rect: 305, 200, 317, 209 ActionDest: 227 Annotation: Subtype: Link Rect: 233, 172, 245, 181 ActionDest: 227 Annotation: Subtype: Link Rect: 531, 117, 543, 126 ActionDest: 227 Annotation: Subtype: Link Rect: 544, 117, 556, 126 ActionDest: 227 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 299, 647, 311, 656 ActionDest: 224 Annotation: Subtype: Link Rect: 441, 633, 453, 642 ActionDest: 227 Annotation: Subtype: Link Rect: 395, 577, 407, 586 ActionDest: 227 Annotation: Subtype: Link Rect: 486, 550, 493, 559 ActionDest: 224 Annotation: Subtype: Link Rect: 333, 508, 345, 517 ActionDest: 227 Annotation: Subtype: Link Rect: 222, 480, 234, 489 ActionDest: 224 Annotation: Subtype: Link Rect: 383, 466, 395, 475 ActionDest: 227 Annotation: Subtype: Link Rect: 395, 466, 407, 475 ActionDest: 227 Annotation: Subtype: Link Rect: 444, 438, 456, 447 ActionDest: 227 Annotation: Subtype: Link Rect: 208, 354, 220, 364 ActionDest: 227 Annotation: Subtype: Link Rect: 220, 354, 232, 364 ActionDest: 230 Annotation: Subtype: Link Rect: 221, 299, 233, 308 ActionDest: 227 Annotation: Subtype: Link Rect: 211, 243, 223, 252 ActionDest: 227 Annotation: Subtype: Link Rect: 223, 243, 235, 252 ActionDest: 230 Annotation: Subtype: Link Rect: 452, 215, 464, 224 ActionDest: 227 Annotation: Subtype: Link Rect: 464, 215, 476, 224 ActionDest: 230 Annotation: Subtype: Link Rect: 294, 159, 305, 169 ActionDest: 227 Annotation: Subtype: Link Rect: 543, 159, 555, 169 ActionDest: 230 Annotation: Subtype: Link Rect: 258, 146, 270, 155 ActionDest: 230 Annotation: Subtype: Link Rect: 362, 146, 374, 155 ActionDest: 230 Annotation: Subtype: Link Rect: 384, 104, 396, 113 ActionDest: 227 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 237, 703, 249, 712 ActionDest: 227 Annotation: Subtype: Link Rect: 249, 703, 261, 712 ActionDest: 227 Annotation: Subtype: Link Rect: 281, 647, 293, 656 ActionDest: 230 Annotation: Subtype: Link Rect: 268, 577, 280, 586 ActionDest: 227 Annotation: Subtype: Link Rect: 255, 508, 267, 517 ActionDest: 227 Annotation: Subtype: Link Rect: 267, 508, 279, 517 ActionDest: 227 Annotation: Subtype: Link Rect: 374, 424, 386, 433 ActionDest: 230 Annotation: Subtype: Link Rect: 197, 313, 209, 322 ActionDest: 224 Annotation: Subtype: Link Rect: 210, 313, 222, 322 ActionDest: 230 Annotation: Subtype: Link Rect: 442, 215, 454, 224 ActionDest: 230 Annotation: Subtype: Link Rect: 457, 215, 469, 224 ActionDest: 230 Annotation: Subtype: Link Rect: 356, 201, 368, 210 ActionDest: 230 Annotation: Subtype: Link Rect: 369, 201, 381, 210 ActionDest: 230 Annotation: Subtype: Link Rect: 515, 201, 527, 210 ActionDest: 230 Annotation: Subtype: Link Rect: 475, 173, 487, 182 ActionDest: 230 Annotation: Subtype: Link Rect: 239, 132, 251, 141 ActionDest: 227 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 496, 758, 503, 767 ActionDest: 224 Annotation: Subtype: Link Rect: 221, 744, 233, 754 ActionDest: 230 Annotation: Subtype: Link Rect: 531, 675, 543, 684 ActionDest: 224 Annotation: Subtype: Link Rect: 544, 675, 556, 684 ActionDest: 227 Annotation: Subtype: Link Rect: 492, 633, 504, 642 ActionDest: 227 Annotation: Subtype: Link Rect: 362, 536, 374, 545 ActionDest: 227 Annotation: Subtype: Link Rect: 357, 438, 369, 447 ActionDest: 224 Annotation: Subtype: Link Rect: 446, 410, 458, 419 ActionDest: 227 Annotation: Subtype: Link Rect: 227, 368, 239, 377 ActionDest: 227 Annotation: Subtype: Link Rect: 394, 299, 406, 308 ActionDest: 230 Annotation: Subtype: Link Rect: 263, 257, 270, 266 ActionDest: 224 Annotation: Subtype: Link Rect: 299, 159, 311, 169 ActionDest: 224 Annotation: Subtype: Link Rect: 227, 118, 239, 127 ActionDest: 230 Annotation: Subtype: Link Rect: 240, 118, 252, 127 ActionDest: 230 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 358, 700, 365, 712 ActionDest: 188 Annotation: Subtype: Link Rect: 438, 332, 531, 344 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 237, 608, 246, 619 ActionDest: 205 Page: Sequence: 18 Page: Sequence: 19 Page: Sequence: 20 Page: Sequence: 21 Page: Sequence: 22 Page: Sequence: 23 Annotations: Annotation: Subtype: Link Rect: 86, 716, 124, 726 Annotation: Subtype: Link Rect: 130, 716, 166, 726 Annotation: Subtype: Link Rect: 414, 702, 452, 713 Annotation: Subtype: Link Rect: 131, 652, 168, 662 Annotation: Subtype: Link Rect: 175, 652, 211, 662 Annotation: Subtype: Link Rect: 381, 638, 418, 649 Annotation: Subtype: Link Rect: 59, 613, 96, 624 Annotation: Subtype: Link Rect: 422, 587, 459, 598 Annotation: Subtype: Link Rect: 262, 562, 300, 573 Annotation: Subtype: Link Rect: 59, 537, 96, 547 Annotation: Subtype: Link Rect: 102, 537, 138, 547 Annotation: Subtype: Link Rect: 59, 499, 96, 509 Annotation: Subtype: Link Rect: 499, 472, 536, 483 Annotation: Subtype: Link Rect: 104, 435, 142, 445 Annotation: Subtype: Link Rect: 148, 435, 184, 445 Annotation: Subtype: Link Rect: 519, 408, 557, 419 Annotation: Subtype: Link Rect: 59, 396, 95, 407 Annotation: Subtype: Link Rect: 59, 358, 96, 368 Annotation: Subtype: Link Rect: 460, 345, 497, 356 Annotation: Subtype: Link Rect: 280, 306, 317, 317 Annotation: Subtype: Link Rect: 508, 281, 545, 292 Annotation: Subtype: Link Rect: 59, 243, 96, 253 Annotation: Subtype: Link Rect: 102, 243, 138, 253 Annotation: Subtype: Link Rect: 486, 217, 523, 228 Annotation: Subtype: Link Rect: 413, 179, 450, 190 Annotation: Subtype: Link Rect: 460, 153, 498, 164 Annotation: Subtype: Link Rect: 416, 115, 453, 126 Annotation: Subtype: Link Rect: 460, 115, 496, 126 Annotation: Subtype: Link Rect: 393, 89, 431, 100 Annotation: Subtype: Link Rect: 437, 89, 473, 100 Page: Sequence: 24 Annotations: Annotation: Subtype: Link Rect: 104, 731, 142, 742 Annotation: Subtype: Link Rect: 192, 705, 229, 716 Annotation: Subtype: Link Rect: 171, 680, 208, 690 Annotation: Subtype: Link Rect: 520, 654, 557, 665 Annotation: Subtype: Link Rect: 59, 616, 96, 627 Annotation: Subtype: Link Rect: 409, 590, 446, 601 Annotation: Subtype: Link Rect: 380, 552, 418, 563 Annotation: Subtype: Link Rect: 159, 526, 196, 537 Annotation: Subtype: Link Rect: 414, 501, 451, 512 Annotation: Subtype: Link Rect: 458, 501, 494, 512 Annotation: Subtype: Link Rect: 454, 475, 492, 486 Annotation: Subtype: Link Rect: 498, 475, 534, 486 Annotation: Subtype: Link Rect: 482, 450, 519, 461 Annotation: Subtype: Link Rect: 247, 411, 284, 422 Annotation: Subtype: Link Rect: 290, 411, 326, 422 Annotation: Subtype: Link Rect: 59, 374, 96, 384 Annotation: Subtype: Link Rect: 312, 335, 349, 346 Annotation: Subtype: Link Rect: 59, 310, 96, 320 Annotation: Subtype: Link Rect: 324, 284, 361, 295 Annotation: Subtype: Link Rect: 131, 246, 168, 256 Annotation: Subtype: Link Rect: 520, 207, 557, 218 Annotation: Subtype: Link Rect: 315, 182, 352, 193 Annotation: Subtype: Link Rect: 358, 182, 394, 193 Annotation: Subtype: Link Rect: 249, 144, 287, 154 Annotation: Subtype: Link Rect: 293, 144, 329, 154 Annotation: Subtype: Link Rect: 202, 118, 240, 129 Annotation: Subtype: Link Rect: 246, 118, 282, 129 Annotation: Subtype: Link Rect: 206, 79, 244, 90 Page: Sequence: 25 Annotations: Annotation: Subtype: Link Rect: 314, 731, 351, 742 Annotation: Subtype: Link Rect: 59, 668, 96, 678 Annotation: Subtype: Link Rect: 456, 641, 494, 652 Annotation: Subtype: Link Rect: 520, 616, 557, 627 Annotation: Subtype: Link Rect: 499, 565, 537, 576 Annotation: Subtype: Link Rect: 113, 527, 150, 537 Annotation: Subtype: Link Rect: 156, 527, 192, 537 Annotation: Subtype: Link Rect: 393, 488, 431, 499 Annotation: Subtype: Link Rect: 437, 488, 473, 499 Annotation: Subtype: Link Rect: 59, 450, 96, 461 Annotation: Subtype: Link Rect: 102, 450, 138, 461 Annotation: Subtype: Link Rect: 325, 411, 362, 422 Annotation: Subtype: Link Rect: 59, 374, 96, 384 Annotation: Subtype: Link Rect: 59, 336, 96, 346 Annotation: Subtype: Link Rect: 520, 309, 557, 320 Annotation: Subtype: Link Rect: 95, 272, 132, 282 Annotation: Subtype: Link Rect: 520, 245, 557, 256 Annotation: Subtype: Link Rect: 291, 194, 328, 205 Annotation: Subtype: Link Rect: 282, 156, 319, 167 Annotation: Subtype: Link Rect: 325, 156, 361, 167 Annotation: Subtype: Link Rect: 122, 119, 160, 129 Annotation: Subtype: Link Rect: 166, 119, 202, 129 Checksum: daaf7a32 Type: CRC32 Checksum: ca82dc0543e477cc4ae94b36544de5b4 Type: MD5 Checksum: f8d9771ad30e652e7491aabec000d1f59edc9a6a Type: SHA-1 Checksum: b58e5275827572fd9a7d9ed8db157a0a367f0278e95a8444fd468e2e1354c525 Type: SHA-256