The Jungle Histology Atlas of Gonad Stages in Coral‐Reef Fishes Second Edition Ken Longenecker and Ross Langston Honolulu, Hawaii May 2018 COVER Gonad sections produced using “Jungle Histology” methods. Left: mature female Acanthurus lineatus (100X). Right: mature male Caesio cuning (1000X). Images: Ross Langston. The Jungle Histology Atlas of Gonad Stages in Coral‐Reef Fishes Second Edition Ken Longenecker and Ross Langston Pacific Biological Survey Bishop Museum Honolulu, Hawaii 96817, USA Honolulu, Hawaii May 2018 1 This work is licensed under the Creative Commons Attribution‐Non‐commercial‐ShareAlike 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by‐ nc‐sa/4.0/. 2 CONTENTS LIST OF TABLES ....................................................................................................................................... 5 LIST OF FIGURES ..................................................................................................................................... 5 PREFACE TO THE SECOND EDITION ....................................................................................................... 6 ACKNOWLEDGMENTS ............................................................................................................................ 6 INTRODUCTION ....................................................................................................................................... 7 Purpose ............................................................................................................................................... 7 Organization ........................................................................................................................................ 7 Scoring Gonad Sections ....................................................................................................................... 8 An Iterative Process ......................................................................................................................... 8 Size Matters .................................................................................................................................... 8 SAMPLING GONADS ................................................................................................................................ 9 Whole Specimens ................................................................................................................................ 9 Dissection ............................................................................................................................................ 9 Processing Gonads ............................................................................................................................ 11 FEMALES ............................................................................................................................................... 14 Ovarian Anatomy .............................................................................................................................. 14 Oocyte Development ........................................................................................................................ 15 Oocyte Classification ......................................................................................................................... 16 Stage I Oocytes (Figure 6 &7) ............................................................................................................ 18 Stage II Oocytes (Figure 8 & Figure 9) ............................................................................................... 20 Stage III Oocytes (Figure 10 & 11) ..................................................................................................... 22 Stage IV Oocytes (Figure 12 & 13) .................................................................................................... 25 Post‐Ovulatory Follicles (Figure 14) .................................................................................................. 27 MALES ................................................................................................................................................... 28 Testicular Anatomy ........................................................................................................................... 28 Sperm Development ......................................................................................................................... 29 Male Germ‐Cell Classification ........................................................................................................... 30 Immature Testes (Figure 17 & Figure 18) ......................................................................................... 31 Mature Testes (Figure 19 & Figure 20) ............................................................................................. 33 UNDIFFERENTIATED GONADS (Figure 21) ............................................................................................ 35 OTHER FEATURES .................................................................................................................................. 36 Ovarian Lumen (Figure 22)................................................................................................................ 36 Atretic Oocyte (Figure 23) ................................................................................................................. 37 Melano‐Macrophage Centers (Figure 24) ......................................................................................... 38 OVOTESTES and Transitional Gonads (Figure 25) ................................................................................. 39 3 NON‐GONADAL TISSUES AND STRUCTURES ......................................................................................... 40 Fat (Figure 27) ................................................................................................................................... 41 Gut (Figure 28) .................................................................................................................................. 42 Blood (Figure 29) ............................................................................................................................... 43 STAINING ADVICE .................................................................................................................................. 44 Wet Cover‐Slipping ........................................................................................................................... 44 Hematoxylin and Eosin Staining ........................................................................................................ 45 Stage II and III Oocytes .................................................................................................................. 46 Immature and Mature Testes ....................................................................................................... 47 The Jungle Histology Approach to H & E Staining ............................................................................. 48 GLOSSARY.............................................................................................................................................. 49 ADDITIONAL RESOURCES ...................................................................................................................... 52 4 LIST OF TABLES Table 1. Oocyte‐stage classification. ..................................................................................................... 17 LIST OF FIGURES Figure 1. Idealized diagram of gonad location in a typical coral‐reef fish. ............................................. 9 Figure 2. Method for exposing the gonads in a typical coral‐reef fish. ................................................ 10 Figure 3. Idealized diagram of gonad location in a typical surgeonfish. ............................................... 11 Figure 4. Method for exposing the gonads in a typical surgeonfish ..................................................... 13 Figure 5. Idealized anatomy of a coral‐reef fish ovary. ........................................................................ 14 Figure 6. Stage I (primary growth) oocyte. .......................................................................................... 18 Figure 7. Examples of Stage I (primary growth) oocytes ...................................................................... 19 Figure 8. Stage II (yolk vesicle) oocyte. ................................................................................................ 20 Figure 9. Examples of Stage II (yolk vesicle) oocytes ........................................................................... 21 Figure 10. Stage III (vitellogenic) oocyte. ............................................................................................. 23 Figure 11. Examples of Stage III (vitellogenic) oocytes ........................................................................ 24 Figure 12. Stage IVa (maturation) and IVb (hydration) oocytes. ......................................................... 25 Figure 13. Examples of Stage IV (maturation and hydration) oocytes ................................................ 26 Figure 14. Post‐ovulatory follicle (POF). .............................................................................................. 27 Figure 15. Idealized anatomy of a coral‐reef fish testis. .....................................................................