J. Anat. (1990), 168, 143-166 143 With 17 figures Printed in Great Britain The megachiropteran pineal organ: a comparative morphological and volumetric investigation with special emphasis on the remarkably large pineal of Dobsonia praedatrix KUNWAR P. BHATNAGAR, HEIKO D. FRAHM*t AND H. STEPHAN* Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Health Sciences Center, Louisville, Kentucky 40292, USA and * Max-Planck-Institut fur Hirnforschung, Vergleichende Neurobiologie, Deutschordenstrasse 46, D-6000 Frankfurt a.M. 71, FRG (Accepted 4 July 1989) INTRODUCTION In a recent comparative study, morphological and volumetric data were presented on the pineal organs of 19 species of megachiropteran fruit bats of the family Pteropodidae (Bhatnagar, Frahm & Stephan, 1986). Since that time brains of several other species of fruit bats have become available in our laboratory. The relatively and absolutely large pineal of Dobsonia species, especially that of Dobsonia praedatrix, forms part of this report. While absolutely large pineal organs have been reported among birds in the emu Dromaeus novaehollandiae (Cobb & Edinger, 1962) and among mammals in neonates of the antarctic seals Mirounga leonina and Leptonychotes weddelli (Bryden, Griffiths, Kennaway & Ledingham, 1986), the shape, size, and topography of the Dobsonia pineal is unlike any that has been described so far, at least among bats. Furthermore, instead of remaining deeply situated either under or between the cerebral hemispheres, as in other pteropodids, the pineal organ in Dobsonia extends to the brain surface and covers the cerebellum anteriorly. This report deals not only with the morphology of the large pineal organ of the New Guinean naked-backed bat Dobsoniapraedatrix Andersen, but also documents further the comparative pineal morphology of 16 additional species of megachiropteran bats and compares this with the available data on other megachiropteran pineals. MATERIALS AND METHODS This investigation is based on 45 pineal organs from adult animals of both sexes belonging to 17 species of the family Pteropodidae (Table 1). The specimens were collected in Africa, Papua New Guinea, Australia and South-East Asia by Heinz Stephan. Under Nembutal anaesthesia, the animals were weighed, measured, photographed, and then perfused through the left ventricle with normal saline followed by freshly made Bouin's fluid. Brains were carefully removed from the cranial vault, weighed, measured (see Table 3) and immersed in Bouin's fluid for four days, then transferred to 70 % alcohol which was changed several times during the first few weeks. Detailed notes on preparation in the field are given in Stephan, Frahm & Baron (1981). (These can be obtained from the authors upon request.) Morphological t Present address: Anatomisches Institut der Universitat K6ln, Joseph-Stelzmann-Str. 9, D-5000 Koin 41, FRG. 144 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN Table 1. Selected data on the pineal organ of 17 species of megachiropteran bats (Family Pteropodidae) Body Brain Mean Pineal + Size Pineal Code Sex weight weight dimensions volume CV index type no. Spec:-s ,y (g) (mg) (1) (2) (3) (4) (5) (6) Subfamily Pteropodinae Tribe Pteropini Subtribe Rousettina 1319 Myonycteris torquata 2,1 36-4 1160 037 055 0-0818 508 72 A Subtribe Pteropodina 1439 Pteropus tonganus 2,1 435 0 6025 1 70 1-04 1-4883 13-3 212 AB Subtribe Dobsoniina 1485 Dobsonia inermis 3,0 150-0 2700 3 65 1 45 0 7596 8-0 237 a,fC* 1489 Dobsonia moluccensis 1,0 465 0 5300 4 58 1-92 2 3000 313 1490 Dobsonia spec. 1,1 230-0 3775 4-22 2 35 4-9226 9-8 1123 a,8C*a,flC* 1495 Dobsonia praedatrix 1,2 184-0 3030 4-22 4-51 16-3447 19-6 4393 a,dC* Tribe Epomophorini 1503 Hypsignathus monstrosus 1,0 310-0 3480 0-62 091 0-4100 75 A 1509 Epomops franqueti 1,2 120-0 2215 0-83 0 93 0 5360 13-1 197 A 1539 Scotonycteris zenkeri 1,1 214 710 038 0.44 0-0361 3-1 47 A 1541 Casinycteris argynnis 2,2 27-0 840 0 35 0-62 0-0768 245 85 A Tribe Cynopterini Subtribe Cynopterina 1611 Paranyctimene raptor 2,1 26-1 731 0-42 0 50 0-0778 6-8 88 A Subfamily Macroglossinae Tribe Macroglossini 1621 Megaloglossus woermanni 0,3 181 680 0-42 059 0-0927 33.5 137 A 1625 Macroglossus minimus 1,2 14-5 560 0-28 0-37 0 0190 39.3 33 A 1631 Syconycteris crassa 1,2 17-6 623 0 33 0-38 0-0421 44.3 64 A Tribe Notopterini 1635 Melonycteris melanops 1,1 47-6 1285 0-42 047 0-0820 21 6 60 A 1639 Nesonycteris woodfordi 0,3 356 1020 0-35 055 0-0671 27-0 60 A 1641 Notopteris macdonaldi 1,2 704 1456 050 0-62 0-1280 21 7 70 A * See Discussion. The standard values of body and brain weights were taken by H. Stephan in the field. (1) The anteroposterior extent (in mm) of pineal was calculated from serial coronal sections. The expression of this dimension, therefore, should not be taken as the pineal 'length'. (2) The greatest width (in mm) of pineal was calculated at its widest point. Both expressions of size (1,2) are from serial sections and therefore they include a shrinkage artefact of approximately 20%. (3) Pineal volume (in mm3), corrected to fresh volume (see Materials and Methods). (4) CV, the coefficient of variation, i.e. the standard deviation in percentage of the mean (see Results), (5) The formula for the calculation of pineal size indices (SI) is: actual pineal size SI, expressed in percent = . x 100, computed pineal size where the actual pineal size is calculated from serial sections, and the computed pineal size is derived from the allometric formula, antilog [-2094 + (0 735 x log body weight)]. The value, -2-094 is the y-intercept of the reference baseline running through the family Vespertilionidae and the value, 0 735 is the mean ascent of regression lines of bat families and/or subfamilies. Pineal size here refers to pineal volume. For further explanation see Materials and Methods. (6) Classification ofpineal types is based on Vollrath (1979). For further explanation see Results and Table 3 in Bhatnagar et al. (1986). observations were made on 20 ,um serial coronal brain sections stained with cresyl violet, gallocyanin, or by the Heidenhain-Woelcke procedure. Selected sections were stained with the one-step Gomori trichrome or Masson trichrome. One brain of Dobsonia praedatrix was cut sagittally. The anteroposterior extent and greatest width of the pineal organ (Table 1) were obtained in each species from enlarged photomicrographs. Megachiropteran pineal organ 145 The volume of the pineal organ was calculated in all 45 specimens. Even though detailed description for this determination is provided in Bhatnagar et al. (1986), a brief explanation of the procedure is given again. Equidistant serial sections through the epiphysis were photographed directly from the slides, the borders delineated and the pineal cut out. Volumes were calculated according to the formula: V = APx WSx D/MS where V = epiphyseal volume in mm3; AP = average area of the photographic paper in mm2/mg; WS = total weight of the cut-out pineal photographs in mg; D = distance between measured sections in mm x section thickness; M2 = square of linear magnification of the photographs. A conversion factor to account for the shrinkage due to fixation was applied to correct the pineal volumes using the formula: Corrected epiphyseal volume = serial section epiphyseal volume (V) x conversion factor (CF), volume of fresh brain where CF = (VFB) serial section brain volume (VSB)' where VFB = weight of fresh brain obtained in the field specific gravity of the brain (= 1 036) and VSB = volume determined through the photographic method (see above and Stephan et al. 1981 for details). The standard values of body and brain weights were derived by H. Stephan in the field from several specimens of each species. Regression line analyses of megachiropteran pineal volumes against body weight result in similar slopes, as reported by Bhatnagar et al. (1986) (a = 0735). The average ascent for the five tribes Pteropini, Epomophorini, Cynopterini, Macroglossini and Notopterini was a = 0767; the average slope for the two subfamilies Pteropodinae and Macroglossinae was a = 0 730. To make a direct comparison possible between the chiropteran pineal indices in Bhatnagar et al. (1986) and the additional species reported in the present paper, and taking into consideration the quite similar slopes mentioned above, it was considered unnecessary to change from the one used in Bhatnagar et al. (1986). This slope is now confirmed and stabilised by the additional data on 17 species. As reported in Bhatnagar et al. (1986) the reference line is drawn through the Vespertilionidae. Any value on the reference line is considered to represent the average volume of the pineal for a typical vespertilionid and has an index of 100 (%). The distances from the reference line are expressed by deviations from 100 and are a measure of the degree of deviation of the pineal volumes from those of typical Vespertilionids. The indices are listed in Tables 1 and 2 and scaled in Figures 14-17. The allometric formula used for determining the pineal size indices was: log pineal volume =-2 094 + 0735 x log body weight. Data previously reported on 19 species of megachiropteran pineal organs (Bhatnagar et al. 1986) were combined with that on the 17 new species of megachiropterans of this study (Tables 1 and 2). Thus, the conclusions encompass a total of 36 pteropodid species belonging to the two subfamilies and five of the six tribes of the family Pteropodidae, excluding the tribe Harpyionycterini.