J. Anal. (1996) 188, pp. 129-135, with 8 figures Printed in Great Britain 129

Development of the vomeronasal organ in leschenaulti (Megachiroptera, Pteropodidae)

KUNWAR P. BHATNAGAR', JOHN R. WIBLEI AND K. B. KARIM2 I Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KY, USA, and 2 Institute of Science, Nagpur, Maharashtra, India

(Accepted 10 July 1995)

ABSTRACT A functional vomeronasal organ (VNO) is known to be lacking in adult of the suborder Megachiroptera, family Pteropodidae, studied to date. However, whether the VNO ever forms during ontogeny in megachiropterans has not been addressed. We report here on the development of the VNO in megachiropterans via study of 8 stages of the rousette fruit Rousettus leschenaulti, ranging from an early limb bud embryo to a young specimen attached to the nipple. A vomeronasal primordium appears in the 4 youngest stages (7-14 mm crown-rump length), but there is no sign of any of the components of the vomeronasal system (neuroepithelial tube, nerves, sinuses, glands, or trough-like cartilage) in the septal region of the 4 oldest stages examined, or in the adult. Given the number of genera investigated to date and their taxonomic diversity, a conclusion that a VNO is entirely lacking in Megachiroptera seems reasonable. However, final confirmation awaits study of the additional 27 genera not yet reported (out of a total of 41).

Key words: Vomeronasal organ; Rousettus leschenaulti; Megachiroptera; Pteropodidae; bats.

logical observations have not been published. To INTRODUCTION ascertain whether or not a primordial VNO appears in The vomeronasal (Jacobson's) organ (VNO) is a megachiropteran ontogeny, we report here on 8 stages paired chemosensory organ found in the anteroventral of the rousette fruit bat Rousettus leschenaulti ranging region of the nasal septum in most adult from an early limb bud embryo to a young specimen (Broom, 1897; Wysocki, 1979). In taxa lacking the attached to the nipple. VNO in the adult, it is not known, with few exceptions, whether the organ forms and then is lost in later development or fails to form altogether. The exceptions include catarrhine primates (Old World MATERIALS AND METHODS monkeys, apes, and humans) in which the VNO has Rousettus leschenaulti is a commonly available fruit been observed only as an atavistic vestige in embryonic bat in India. The females breed twice a year in quick stages (Maier, 1980) and cetaceans (whales) in which succession from the middle of November to March, the VNO apparently fails to form during development then soon after parturition until the end of July. (Oelschlager, 1989). Bats of the suborder Mega- Gestation lasts for about 125 d (Gopalakrishna & chiroptera (with a single family Pteropodidae) are Choudhari, 1977). In Nagpur, India, pregnant females among those mammals for which the VNO or its of Rousettus leschenaulti were collected at regular essential, functional relay centre in the central nervous intervals, anaesthetised, and perfused intracardially system, the accessory olfactory bulb, have been with aqueous Bouin's fluid. The uteri were removed; described as absent in the adult (Bhatnagar, 1980; embryos and fetuses were freed from the amniotic Frahm & Bhatnagar, 1980), but for which embryo- sacs, further fixed in Bouin's fluid for 4 d, transferred

Correspondence to Dr Kunwar P. Bhatnagar, Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KY 40292, USA. 130 K. P. Bhatnagar, J. R. Wible and K. B. Karim

Table 1. Prenatal and juvenile specimens of Rousettus CRL human embryo of 6-7 wk (cf. Arey, 1974; leschenaulti examined Gasser, 1975). In the next stage, the 9.9 mm CRL embryo, the Series CRL HL No. no. (mm) (mm) Stage VNOP is seen alongside the now organised septal cartilage and the just-appearing paraseptal cartilages 1. R17 7.00 3.20 Nasal sac; VNOP; forelimb bud (Fig. 2). The VNOP is similar in thickness to that of 2. R22 9.90 5.80 VNOP the previous stage and extends about 100 antero- 3. R2 11.30 - VNOP gm 4. R4 14.05 8.45 VNOP posteriorly along the septum. 5. R12 18.50 13.50 VNO epithelial tube lacking in this The VNOP and neighbouring structures in the male and subsequent stages 11.3 mm CRL embryo are similar in development to 6. R24 34.10 - Female 7. R26 - Near term male those of the 9.9 mm stage (Fig. 3). The VNOP extends 8. R34 - Male young attached to nipple about 165 jim anteroposteriorly, and the entire epi- thelium of the nasal sac, including the vomeronasal CRL, crown-rump length; HL, head length; VNOP, vomeronasal and olfactory primordia, is proliferating. organ primordium. The VNOP appears to be at its maximum de- velopment in the 14.05 mm CRL specimen (Fig. 4), gradually to 80% ethyl alcohol, and stored. At the where it extends about 750 jm anteroposteriorly. As University of Louisville, USA, heads of embryos, in the preceding stages, the invaginating primordium fetuses, and 1 juvenile (Table 1) were prepared for has not separated from the surface epithelium and has paraffin sectioning, with those of older specimens not migrated towards the nasal septal tissue as it does treated with formic acid decalcifier (Bhatnagar & in other species where a definitive VNO forms. The Kallen, 1974). Serial coronal sections (6-10 gm) were VNOP consists of 6-8 layers of cells and is 0.04 gm in made. Sections were stained either with haematoxylin- height and 0.06 jm in width. A few mitotic figures are eosin or by the Gomori trichrome method (Gomori, discernible. 1950). The sections in Figures 1 a-4a and 5-8 were In the remaining 4 stages, 18.5 mm and 34.1 mm photographed with a Wild (M400) photomacroscope CRL, the term fetus, and juvenile (Figs 5-8), the and in the remaining figures with an Olympus Vanox VNOP is no longer identifiable, either as an microscope. invagination (cf. Figs 1-4) or as a separate epithelial Three additional adult megachiropterans, which tube as observed during VNO development, for were collected from Doi Inthanon (Chiang Mai) example, in the microchiropteran bat Miniopterus National Park, Thailand-male spelaea, schreibersii (Broom, 1895), the mole Talpa europaea, male sobrinus and female and man (Jacobson, 1928). The paraseptal cartilages niphanae-were perfused with Bouin's fluid in the field remain bar-like; when the vomeronasal epithelial tube and then processed for paraffin sectioning as above. develops in microchiropteran bats, the paraseptal Stained serial sections through their snouts were cartilages are not bar-like, instead they are 'C', 'J' or examined. 'U'-shaped (see Cooper & Bhatnagar, 1976: fig. 2). Straight and patent nasopalatine ducts are found in all stages of R. leschenaulti examined except the youngest RESULTS and are retained in the adult (Bhatnagar, 1980). No The youngest specimen in this study (Table 1) is a other components of the vomeronasal system are 7.0 mm crown-rump length (CRL) embryo of observable on the nasal septum. Consequently, there Rousettus leschenaulti featuring forelimb buds (not was no likelihood that the VNO's functional relay included in the figure) and nasal sacs (Fig. 1 a). It centre in the central nervous system, the accessory shows both an olfactory (OP) as well as a vomeronasal olfactory bulb, was present. primordium (VNOP, Fig. 1). The latter is globular, As stated in the Introduction, the VNO is reported bulging towards the presumptive septal region, and to be absent in all adult megachiropterans studied to extends about 150 gm anteroposteriorly. It is about date (Table 2). As additions to this list, we confirm 0.09 gm in height, thicker than the 0.05 gm high here the absence of the VNO in adult specimens from olfactory primordium. The nasal septum is not 3 more species-Eonycteris spelaea, Macroglossus organised at this stage. Fewer cells in the VNOP are sobrinus, and Megaerops niphanae. seen dividing at the luminal surface as compared with the olfactory primordium where more active mitosis is observed. This stage is comparable to a 12-14 mm Vomeronasal organ in Rousettus 131

a

Figures 1-8 are from 6 to 12 gm coronal sections through the heads of Rousettus leschenaulti stained either with haematoxylin-eosin or Gomori one-step trichrome procedure. Scale bars in all figures equal 1 mm, except in Figures 1 b-4b in which bars equal 50 Atm. Abbreviations in Figures 1 and 2 apply to all figures. The boxed areas in Figures 1 a-4a are shown enlarged in Figures 1 b-4b respectively. Fig. 1. The forelimb bud (not shown) and the nasal sac (NS) stage of a 7.0 mm CRL embryo. D, diencephalon; E, developing eye; OP, olfactory primordium; VNOP, vomeronasal primordium; III, third ventricle. Fig. 2. The developing septal cartilages (SC), paraseptal cartilages (PC), and nasopalatine ducts (NPD) are shown. A VNOP is invaginating toward the septal region. CRL 9.9 mm. 132 K. P. Bhatnagar, J. R. Wible and K. B. Karim

,| i A Nw L}J'sM.

K

a

Fig. 3. CRL 11.3 mm. Fig. 4. The VNOP is better organised, but the invaginating primordium does not separate from the surface epithelium. CRL 14.05 mm.

R. leschenaulti progresses only to a primordial stage, DISCUSSION but never develops into an epithelial tube. Other The development of the vomeronasal and olfactory components of the vomeronasal system in the septal primordia from the nasal placode is a well established region (nerves, sinuses, glands, or trough-like car- fact of amniote embryology (see, e.g. Rathke, 1838, as tilage) apparently fail to form altogether in R. cited by Beard, 1889). The olfactory primordium leschenaulti, because they are not found in any of the forms in the roof of the nasal sac and the VNOP 8 specimens examined or in the adults studied by invaginates from its medial epithelium. Therefore, the Bhatnagar (1980). The VNO's functional relay centre bulging epithelial tissue found in the medial wall of in the central nervous system, the accessory olfactory the developing olfactory region in the 4 youngest bulb, is reported as absent in adults of the closely specimens of Rousettus leschenaulti can only be the related species R. aegyptiacus (Frahm & Bhatnagar, VNOP (Figs 1-4). Furthermore, the gradual increase 1980). in height and anteroposterior elongation ofthe VNOP Based upon the study of the anterior nasal region, in the first 4 specimens are consistent with the pattern the VNO is known to be absent in adults of 11 species of VNO growth in other forms. However, decline in of Megachiroptera including representatives of both active cell division in the VNOP and the failure to subfamilies, Pteropodinae and Macroglossinae (Table separate from the surface epithelium are indicative of 2). A tiny invagination of the nasal epithelium in the the VNOP's early arrest in development and eventual medial wall of the nasal fossa where the VNO appears loss in R. leschenaulti, as confirmed in the 4 oldest in other mammals has been reported in an adult specimens (Figs 5-8). It is concluded that the VNO in macroglossine Macroglossus minimus (Bhatnagar, Vomeronasal organ in Rousettus 133

Fig. 5. The VNOP is no longer visible. A VNO epithelial tube does not develop in this or any of the successive stages (Figs 6-8). VNGP, vomeronasal gland primordium. CRL 18.5 mm. Fig. 6. The paraseptal cartilages have lengthened and are bar-like. NC, nasal cavity, CRL 34.1 mm. Fig. 7. T, tongue; near term fetus. Fig. 8. In the juvenile male, the paraseptal cartilages are even longer; the nasopalatine ducts are vertically oriented. The adult configuration of the nasal region is reached.

1980). However, without studies of a suitable on- authors. Yet, because the absence of an accessory togenetic series, it is not known whether this is olfactory bulb also indicates the absence of vomero- retained from or is entirely unrelated to the VNOP. nasal neuroepithelium, it can be concluded that none In a comparative study of the accessory olfactory of these megachiropterans would possess a functional bulb in bats (Frahm & Bhatnagar, 1980), this neural VNO. However, as a remote possibility, a highly structure is reported as absent in 15 species of cystic and vestigial VNO may be present in some of megachiropterans, again including both pteropodines these adult megachiropterans (for examples of cystic and macroglossines (Table 2). The anterior nasal VNOs in Microchiroptera, see Bhatnagar, 1980). region of these species was not examined by these Despite the fact that a VNO has not yet been 134 K. P. Bhatnagar, J. R. Wible and K. B. Karim

Table 2. Adult pteropodid speciesa for which either the microchiropterans (Bhatnagar, 1980) and primates vomeronasal organ or an accessory olfactory bulb (indicated (Maier, 1980), a highly developed VNO is said by by an asterisk *) are reported as absent these authors to be present primitively in both groups. Male, Reference Species female (see below) ACKNOWLEDGEMENTS Pteropodinae Balionycteris maculata* 0,1 4 The authors are grateful to Louise Bond and Lucinda argynnis* 0,1 4 Shultz for histological preparations, to Cathie Caple, Chironax melanocephalus* 1,1 4 Carol Bergman, and Elizabeth Klar for photographic brachyotis* 1 ? 4 Cynopterus horsfieldi* 1?,1 4 assistance, and to Susan Hodge for secretarial help. Cynopterus sphinx 0,1 3 We also thank Dr H. Stephan, Max Planck Institute wahlbergi 0,1 2 for Brain Research, Frankfurt, Germany, for help in Hypsignathus monstrosus 0,2 4 collecting bats in Thailand and acknowledge the Megaerops niphanae 0,1 6 logistic support of the scientists and staff of the Megaerops ecaudatus* 1,0 4 robinsoni* 1,1 4 Thailand Institute of Scientific and Technological alecto 0,2 4 Research, Bangkok. The comments of two anony- Pteropus conspicillatus* 1,0 4 mous reviewers improved an earlier version of the Pteropus edwarsi (= P. rufus) - 5 manuscript. This research was supported in part by Pteropus giganteus 1,0 3 National Science Foundation grant BSR 91-19212 (to Pteropus lylei* 2,2 4 Pteropus poliocephalus* 1,1,1? 1,4 J. R. W.). Pteropus scapulatus* 2,0 4 Pteropus vampyrus* 1,1 4 Rousettus aegyptiacus* 0,2 4 REFERENCES Rousettus leschenaulti 0,1 3 Macroglossinae 1,1 4 AREY WB (1974) Developmental Anatomy, 7th edn. Philadelphia: Eonycteris spelaea* 1,? 4 W. B. Saunders. 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