The vomeronasal organ of Lemur catta

American Journal of Primatology 77:229–238 (2015) RESEARCH ARTICLE The Vomeronasal Organ of Lemur catta TIMOTHY D. SMITH1,2*, MAGDALENA N. MUCHLINSKI3, KUNWAR P. BHATNAGAR4, EMILY L. DURHAM5, CHRISTOPHER J. BONAR6, AND ANNE M. BURROWS2,5 1 School of Physical Therapy, Slippery Rock University, Slippery Rock, PA 2 Department of Anthropology, University of Pittsburgh, Pittsburgh, PA 3 Department of Anatomy and Neurobiology, University of Kentucky, College of Medicine, Lexington, Kentucky 40536 4 Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KY 5 Department of Physical Therapy, Duquesne University, Pittsburgh, PA 6 Dallas Zoo Management, Dallas, TX The vomeronasal organ (VNO), also known as the Jacobson’s organ, is a bilateral chemosensory organ found at the base of the nasal cavity specialized for the detection of higher‐molecular weight (non‐ volatile) chemostimuli. It has been linked to pheromone detection. The VNO has been well studied in nocturnal lemurs and lorises, but poorly studied in diurnal/cathemeral species despite the large repertoire of olfactory behaviors noted in species such as Lemur catta. Here, the VNO and associated structures were studied microanatomically in one adult female and one adult male L. catta. Traditional and immunohistochemical procedures demonstrate the VNO epithelium consists of multiple rows of sensory neurons. Immunoreactivity to Growth‐associated protein 43 (GAP43) indicates the VNO is postnatally neurogenic. In volume, the VNO neuroepithelium scales similarly to palatal length compared to nocturnal strepsirrhines. Numerous taste buds present at the oral opening to the nasopalatine duct, with which the VNO communicates, provide an additional (or alternative) explanation for the flehmen behavior that has been observed in this species. The VNO of L. catta is shown to be microanatomically comparable to that of nocturnal strepsirrhines. Like nocturnal strepsirrhines, the VNO of L. catta may be functional in the reception of high‐molecular weight secretions. Am. J. Primatol. 77:229–238, 2015. © 2014 Wiley Periodicals, Inc. Key words: olfactory; pheromonal; primates; ring‐tailed lemur; Jacobson’s organ INTRODUCTION The ring‐tailed lemur (Lemur catta) is a highly social strepsirrhine primate that uses olfactory cues extensively for interspecific and intraspecific signaling [Drea & Scordato, 2008; Gould & Overdorff, 2002; Kappeler, 1998; Palagi et al., 2003]. The amount of behavioral and experimental data on olfaction in L. catta is unrivaled compared to other diurnal or cathemeral strepsirrhines [e.g., Jolly, 1966; Mertl‐ Millhollen, 2006; Schilling, 1979]. The role of olfaction in their daily life has been well explored in field, captive, and experimental settings. By assessing conspecific scent marks, L. catta is able to glean information about individuals, such as sex, reproductive state, and even the identity of the signaler [Palagi & Dapporto, 2007; Scordato & Drea, 2007]. In addition, L. catta is able to use olfaction to assess food quality, with or without assistance from the visual system [Rushmore et al., 2012]. L. catta uses genital, brachial, and antebrachial scent glands in a wide array of scent marking behaviors [Jolly, 1966; Mertl‐Millhollen, 2006; Schilling, 1979]. Scent marking is often associated © 2014 Wiley Periodicals, Inc. with ritualized and exaggerated behaviors in which tail and body posture may form part of the signal [Evans & Goy, 1968; Jolly, 1966; Palagi & Norscia, 2009]. Behavioral responses to scent marking can be equally exaggerated (e.g., during male “stink” fights). Chemosensory investigation includes a wide range of behaviors [Evans & Goy, 1968]. L. catta approaches scent marks or urine by sniffing and/or licking [Crawford et al., 2011; Palagi et al., 2005]. One author has described the flehmen response in L. catta [Bailey, 1978]. The flehmen response is when an animal curls their upper lip back and draws fluid (and associated odorants) past the incisive papilla and into  Correspondence to: Timothy D. Smith, School of Physical Therapy, Slippery Rock University, Slippery Rock PA E‐mail: [email protected] Received 18 April 2014; revised 21 July 2014; revision accepted 5 August 2014 DOI: 10.1002/ajp.22326 Published online 12 September 2014 in Wiley Online Library (wileyonlinelibrary.com). 230 / Smith et al. a duct (the nasopalatine duct) located behind the incisors. This duct connects the oral cavity to the nasal cavity, and also provides a communication of the vomeronasal organ (VNO) to both spaces. Flehmen has been experimentally linked to VNO function in some non‐primate mammals such as goats [Ladewig & Hart, 1980; Melese‐d’Hospital & Hart, 1985], but no firm link has been made in primates. Although the repertoire of olfactory behavioral correlates in L. catta has been extensively investigated, our knowledge of pertinent anatomy is greatly limited. Anatomically, skin glands have been well studied, and advances are being made on chemical composition of glandular secretions [delBarco‐Trillo et al., 2012]. With regard to L. catta’s olfactory organs, there is ample potential surface area on their turbinals for olfactory epithelium [Cave, 1973], yet, the precise distribution of mucosa has yet to be mapped. Only two brief descriptions of the VNO in L. catta exist, indicating that a sensory epithelium is present in the VNO of this species [Evans 1984, 2003; Smith et al., 2007]. Although a VNO is clearly present in adult L. catta, no studies have compared it with that of other strepsirrhines. Based on the presence of a neuroepithelium in all strepsirrhines studied to date, it has been broadly accepted that the vomeronasal system (VNS) is functional in all strepsirrhines [e.g., Ankel‐Simons, 2007; Fleagle, 2013; Martin, 1990]. However, it is important to note that the mere presence of a neuroepithelium does not signify function. For example, in a recent survey VNO neuroepithelium has been reported in numerous New World monkeys [Smith et al., 2011a,b]; yet, in some species the VNO barely expresses neuronal markers of maturity [Smith et al., 2011a,b]. Genetic research indicates that many platyrrhines may have accumulated a large proportion of vomeronasal pseudogenes [Liman & Innan, 2003], which further supports the supposition that the mere presence of a VNO and/or VNO neuroepithelium does not indicate a functional VNO. Detailed quantitative data on the VNO have recently been published on nocturnal strepsirrhines [Garrett et al., 2013] and many platyrrhines [Smith et al., 2011a,b], but no studies have investigated cathemeral or diurnal strepsirrhines in detail. With the exception of a study on infant primates [Smith et al., 2007], no studies have compared the VNOs of nocturnal and other strepsirrhines. Similarly, molecular studies have focused heavily on data from nocturnal strepsirrhines [e.g., Garrett & Steiper, 2014; Hohenbrink et al., 2012, 2013; Young et al., 2010] with some exceptions [e.g., Liman & Innan, 2003]. Results of these studies uniformly indicate that strepsirrhine primates have less pseudogenization of vomeronasal receptor genes (V1R) and a signal transduction gene (TRP2). A recent study on V1R genes confirms this is likely true in a broad range of strepsirrhines, including both nocturnal and other forms [Yoder et al., 2014]. Am. J. Primatol. While the molecular results may seem expected based on the vast literature demonstrating olfactory behaviors in all strepsirrhines (e.g., see above), Yoder et al. [2014] also assert the V1R diversity is underestimated in strepsirrhines. Although V2R genes were thought to be completely pseudogenized in most mammals [Young & Trask, 2007], another recent study indicates that intact V2R genes are present in the mouse lemur (Microcebus murinus)[Hohenbrink et al., 2013]. The variability suggested by genetic studies underscores the need for more detailed microanatomical knowledge of the VNO in non‐nocturnal strepsirrhines. Here we investigate the VNO of L. catta in detail in order to place behavioral observations in a clearer context and for anatomical comparisons to the relatively better‐studied nocturnal lemurs and lorises [Garrett et al., 2013; Hedewig, 1980a,b; Schilling, 1970; Smith et al., 2007]. We test the hypothesis that the VNO of L. catta is functionally similar, as reflected by microanatomical organization and indicators of postnatal neurogenesis, to the VNO of nocturnal strepsirrhines. METHODS Two adult specimens of Lemur catta (one male, one female) were obtained after death by natural causes at the Cleveland Metroparks zoo. Specimens were placed in formalin shortly after death by the veterinarian (CJB). The use of these tissues was reviewed and approved by the Institutional Animal Care and Use Committee at Slippery Rock University, and adhered to the American Society of Primatologists principles for the ethical treatment of primates. Palatal length (prosthion to posterior midpalatal point) was collected on each specimen using digital calipers. Derived values were used for size‐corrections. Each specimen was dissected using a fine saw, scalpel, and chisel to remove one half of the nasal cavity, including the midline nasal septum. Both specimens were prepared for histology similarly. Briefly, the half nasal chambers were re‐ fixed in 10% neutral buffered formalin (Fisher Scientific, Pittsburgh PA) after removal for at least an hour, decalcified using a formic acid‐sodium citrate solution, dehydrated in a graded series of ethanol, and embedded in paraffin. Blocks were sectioned serially at 10–12 mm and every 5th section was stained alternately with Gomori trichrome or hematoxylin‐eosin procedures. Intervening sections were saved for immunohistochemistry or for analysis of very small structures (see below). All histological preparations were studied using a Leica DMLB microscope at 25–630. Immunohistochemistry We used selected unstained sections to test the prediction that nocturnal strepsirrhines would Lemur catta VNO / 231 exhibit microanatomical vomeronasal neuroepithelial (VNNE) characteristics in common with nocturnal strepsirrhines [as seen in Garrett et al., 2013]. For the present analysis, we chose immunohistochemical markers that demonstrate neuronal growth. As immature neurons, vomeronasal sensory neurons (VSNs) are known to express markers such as Growth‐Associated Protein 43 (GAP43) [Ishii & Mombaerts, 2011; Weiler & Benali, 2005]. GAP43 is expressed in neuronal growth cones [Aigner and Caroni, 1995]. Although the specific function of GAP43 is still debated, it reliably demonstrates olfactory sensory neurons and VSNs at early stages of development in rodents and some other mammals [Garrett et al., 2013; Weiler & Benali, 2005]. Thus, in the present study we can determine if the VNNE of L. catta is neurogenic into adult ages. We also exposed randomly selected sections of the VNO of both specimens to neuron‐specific beta tubulin antibodies so we could describe the organization of the sensory epithelium through the anteroposterior axis of the VNO. These markers are expressed in a broader array of vomeronasal sensory neurons than those highlighted by GAP43 [Dennis et al., 2004; Garrett et al., 2013; Smith et al., 2005; Weiler & Benali, 2005]. Mounted tissue sections were deparaffinized in xylenes (Fisher Scientific, Pittsburgh PA) and hydrated to distilled water (dH2O). To abolish endogenous peroxidase‐like activity, the sections were incubated in 0.9% hydrogen peroxide (H2O2) in absolute methanol for 20 min at room temperature (23.5–25 C). Subsequently, the tissues were washed in dH2O then in 10 mM phosphate buffered saline (PBS) (2.7 mM KCl, 137 mM NaCl) (Sigma). Tissues were incubated 20 min in the appropriate blocking solution (5% normal serum (Sigma) of the species in which the secondary antibody was made and 2.5% bovine serum albumin (BSA, Sigma, St. Louis, MO) in phosphate buffered saline (PBS) then washed briefly in PBS. Sections were then incubated overnight at 4°C with in GAP43 primary antibody (NB300‐143, Novus) diluted to 1.25 mg/ml or anti‐beta tubulin primary antibody (MMS435P, Covance, Princeton, NJ) diluted to 1:4000. After this incubation, sections were treated with biotinylated secondary antibodies (Vector Labs, Burlingame, CA) diluted 1:200 then with ABC Elite reagent (Vector), reacted with diaminobenzidine (Vector), dehydrated, and mounted with Permount. Quantitative Methods For quantitative analyses, digital images from 50 to 400 were obtained using a Leica DMLB photomicroscope (Leica Microsystems, Wetzlar, Germany) equipped with an AxioCam MRc5 digital camera (Carl Zeiss Microscopy GmbH, Jena Germany). Images were acquired using ZEN software (Zeiss) Lower magnification (50) images were used to annotate taste buds on the incisive papilla (i.e., taste buds were counted at higher magnification and marked on the low magnification micrographs). These taste buds were observed during routine observations of the nasopalatine and vomeronasal duct systems. Additional unstained sections in this region were mounted and stained. Since the taste buds ranged from 30 to 45 mm in diameter, every other section was used to count taste buds. Adjacent sections were carefully compared to avoid counting the same taste bud twice if it was seen in two adjacent sections. Measurements of the VNO (one side only) were carried out as follows using higher magnification micrographs (200 to 400). Every 10th section of the nasal fossa was examined for the presence of VNNE. Due to the small length of the VNNE in some previously studied species, every 5th section was examined near the anterior and posterior points, for greater accuracy. The anterior start points and posterior end points were recorded. The difference between the start and stop‐point section numbers was multiplied by section thickness in each specimen to obtain the anteroposterior length of the VNNE. To obtain neuroepithelial volumes, the VNNE was manually traced in every 10th section (every 5th section at rostral and caudal limits) using ImageJ software (NIH). Cross‐sectional area was obtained based on calibration to a stage micrometer photographed at the same magnification. Based on the distance between sections, the segmental volume was obtained. Segment volumes were summed to obtain the total volume for the VNNE. These data were compared to previously acquired data on nocturnal strepsirrhines [Garrett et al., 2013]. The VNNE volume in L. catta was compared to mean VNNE volume of nocturnal strepsirrhines, using a t‐test described in Sokal and Rohlf (1981, p 231), considered significantly different if P < 0.05. In addition, L. catta VNNE volume was plotted against palatal length. Previously, VNNE volume in nocturnal strepsirrhines was shown to have a linear relationship to palatal length [Garrett et al., 2013]. Individual VNNE volumes for L. catta were therefore examined relative to a linear regression line of the data for nocturnal strepsirrhines. RESULTS The entrance to the VNO occurs through a short vomeronasal duct near the oral entrance to the nasopalatine duct (Fig. 1a). In both the female and male, numerous taste buds are observed near the entry, lining the lateral sides of the incisive papilla (Fig. 1b–d). These spherical assemblages of sensory cells are found near the surface of the epithelium on the lateral margin of the incisive papilla (Fig. 1d). At their apical side, taste buds open into the oral cavity via minute openings, taste pores (Fig. 1d shows two taste buds at approximately the level of the taste pore, Am. J. Primatol. 232 / Smith et al. Fig. 1. a) a short vomeronasal duct ( ) is shown near the mid‐point of the nasopalatine duct (open arrow). In both the female (1b,c) and male (1d,e), numerous taste buds (TB) are observed near the entry, lining the lateral sides of the incisive papilla. In b, numerous beta tubulinþ axons leading away from taste bud on lateral edge of incisive papilla. The inferior side of the papilla (1f), facing the tongue, is devoid of taste buds, bud contains numerous nerves (NN), and some collections of cells resembling Merkel cells (open arrows). A beta‐ tubulin preparation in this region indicates numerous axons communication with the base of the epithelium (g). Scale bars: a, 1mm; b, 20 mm; c, 0.5 mm; d, 50 mm; e, 250 mm; f, g, 30 mm. Am. J. Primatol. Lemur catta VNO / 233 although the section thickness is too thick to see the actual opening). At their basal side, these taste buds communicate with afferent axons (Fig. 1b). In the adult female, 23 taste buds were counted on the right side of the incisive papilla, all situated along the anterior portion of the papilla, adjacent to the nasopalatine duct opening. It was not possible to count the taste buds in the incisive papilla of the male since some sections were damaged, but based on the available sections, taste buds may be similarly numerous. The connective tissue core of the incisive papilla has numerous nerves, and clusters of cells resembling Merkel cells are seen at the base of the epithelium on the inferior aspect of the papilla (i.e., the surface facing the tongue (Fig. 1f). Beta tubulin preparations indicate numerous axons are in contact with the base of the epithelium at these locations (Fig. 1g). Both the female (Fig. 2a–c) and male (Fig. 2d–f) specimens possess a VNO with a large lumen, surrounded by a highly vascular lamina propria and protected by a J‐shaped vomeronasal cartilage (Fig. 2a, d), which is partially ossified in the male. A relatively thick ventromedial VNNE and a thinner dorsolateral nonsensory epithelium are observed throughout the length of the VNO (Fig. 2a–f). No regions that are only lined with VNNE are seen, as described by Evans [1984], although there is only scant nonsensory epithelium near the anterior limits of the VNNE (Fig. 2c, f). Multiple rows of VSNs are visible (Fig. 2g), most of which are BTþ. Intraepithelial nerve bundles are also observed (Fig. 2h). Some Gap43þ VSNs are visible, restricted near the basal aspect of the VNNE (Fig. 2i). Gap43þ axons are found in the lamina propria just deep to the VNNE, and some intraepithelial nerves are Gap43þ. Volume of the VNNE was nearly identical in the two specimens, measuring at 0.59 and 0.57 mm3 for female and male, respectively. T tests comparing VNNE volumes of individual L. catta to mean VNNE volume of nocturnal strespsirrhines reveals significantly larger (female, t ¼ 2.66, df ¼ 24, P < 0.02; male, t ¼ 2.49, df ¼ 24, P < 0.05) VNNE volume in L. catta. However, if VNNE volume is scaled to palatal length, no significant differences are observed (female, t ¼ 0.665, df ¼ 24, P ¼ 0.5; male, t ¼ 0.559, df ¼ 24, P ¼ 0.5). Thus, differences in absolute VNNE volume likely reflect an allometric relationship. This is further supported by a graphical comparison (Fig. 3). When cube root of the VNNE volume is log10 transformed and plotted against log10 palatal length, VNNE volume of Lemur catta plots well with data for numerous nocturnal strepsirrhines, residing just above the linear regression line for these data (Fig. 3). logical observations of the nasal fossa. Conversely, haplorhines can be viewed as microsmatic [olfactory function reduced ‐ Negus, 1958; Cave, 1973; or applied to only apes and humans – Turner, 1891]. However, not all authors view the primate olfactory system as being so dichotomous, either structurally or functionally [e.g., Heymann, 2006; Laska et al., 2000; Smith & Bhatnagar, 2004; Van Valkenburgh et al., 2014]. There appears to be a clearer distinction among primates when it concerns the VNS. The VNS is present and is neurally intact in all strepsirrhines, tarsiers, and all New World anthropoids that have been studied to date [Hedewig, 1980a, b; Schilling, 1970; Smith et al., 2011a,b]. It is important to note that although the VNS is present in many haplorhines, there is genetic and microanatomical variation in the VNS [e.g., Liman & Innan, 2003; Smith et al., 2011a,b]. Most notably, the VNNE is greatly variable in its maturational state and relative size, which suggests possible functional differences [Smith et al., 2011a,b]. However, because anatomical work on strepsirrhines is biased towards nocturnal forms, it is unclear whether strepsirrhines possess a similar amount of variation as seen in haplorhines that possess the VNS. The precise role of the VNS remains unclear in haplorhines that retain it. Despite the anatomical variability of the VNO observed in New World monkeys (see above), the experimental removal of the VNO (VNX) has an impact on endocrine physiology of at least one species, Callithrix jacchus [Barrett et al., 1993]. However, these effects are also observed when the main olfactory epithelium is lesioned (MOX) and perhaps most profound with both VNX and MOX in the same animals. Moreover, chemical cues are likely insufficient by themselves, as visual or other behavioral cues are implicated for suppression of ovulation in subordinate C. jacchus [Barrett et al., 1993]. There are clearer data available for mouse lemurs. In mouse lemurs, experimental evidence via removal of the vomeronasal organ (VNX) suggests the VNS has some functional similarities to that of rodents. For example, VNX males have reduced aggression toward other males, and exhibit fewer sexual behaviors (e.g., mounting attempts) [Aujard, 1997]; both of these phenomena have been observed in VNX mice [Clancy et al., 1984a]. No other strepsirrhines have been similarly studied; experimental ablation on other species seems difficult, impractical, and undesirable. However, there may be clues available to us through behavioral (existing and future) studies that could elucidate the role of VNS. We discuss the details of our anatomical results in light of this. DISCUSSION Anatomy of the Peripheral Vomeronasal System in Lemur catta Strepsirrhine primates are considered the more macrosmatic (or “keen‐scented”) suborder of Primates, largely based on gross anatomical or osteo- The VNS has been linked to reception of a particular class of chemosignals, those of higher Am. J. Primatol. 234 / Smith et al. Fig. 2. Female (Fig. 2a–c) and male (Fig. 2d–f) L. catta VNOs. The lamina propria is highly vascular lamina, with multiple venous sinus (VS) channels, protected by a J‐shaped vomeronasal cartilage (VNC, Fig. 2a, d); note partial ossification in the male. A relatively thick ventromedial VNNE and a thinner dorsolateral nonsensory epithelium (NE) is observed throughout the length of the VNO (Fig. 2a–f). Multiple rows of vomeronasal sensory neurons are visible (Fig. 2g), most of which are BTþ; intraepithelial nerve bundles are also observed (IN, Fig. 2h). Some Gap43þ vomeronasal sensory neurons are visible (open arrows). Gap43þ axons (small arrows) are found in the lamina propria just deep to the VNNE, and Gap43þ intraepithelial nerve is shown (large arrow). Fig. 2g and h show closely adjacent sections stained with hematoxylin‐eosin and BT immunohistochemistry preparation, respectively. Fig. 2i and insets show closely adjacent sections stained with hematoxylin‐eosin and Gap‐43 immunohistochemistry preparation, respectively. Scale bars: a, d, 300 mm; b,c,e,f, 200 mm; g‐i, 50 mm. molecular weight [Clancy et al., 1984b; Evans, 2003; Eisthen & Park, 2005]. Lighter molecular weight (volatile) signals are preferentially detected by the main olfactory system [Schilling et al., 1990]. The flehmen behavior, a sterotypical facial movement with a number of subtle variants [at least five according to Evans, 2003] is known to facilitate stimulus access in some mammals. Based on experiments using goats, chemostimulus transport to the VNO lumen is facilitated by this behavior [Ladewig & Hart, 1980; Melese‐d’Hospital & Hart, 1985]. A flehmen response was described in Lemur catta by Bailey [1978], though it has not been described in any other lemurs or lorises since. The behavioral evidence for flehmen involvement in primate VNO function is complicated since a flehmen‐like response has been described in the mandrill, most instances occurring in Am. J. Primatol. one dominant male [Charpentier et al., 2013]. Given that the VNO has yet to be located in any adult Old World monkey [Smith et al., 2001; Smith et al., 2002], this catarrhine primate likely lacks a functional VNO. Regardless of the role of flehmen response, there is strong direct and indirect evidence for VNS functionality in strepsirrhine primates. However, all experimental work [Aujard, 1997; Schilling et al., 1990] and the vast majority of microanatomical studies have centered on nocturnal species [Garrett et al., 2013; Hedewig, 1980a,b; Hunter et al., 1984; Schilling, 1970; Smith et al., 2005]. L. catta has been studied previously, but only in infants [Smith et al., 2007] and a cursory description of the adult VNO [Evans, 1984]. Previously, expression of Olfactory Marker Protein (expressed in terminally differentiated olfactory sensory neurons) was demonstrated at the Lemur catta VNO / 235 Fig. 3. Volume of the VNNE in L. catta compared with that of numerous nocturnal strepsirrhines [data from Garrett et al., 2013]. Log10‐transformed cube root of the VNNE volume is plotted against log10 palatal length. VNNE volume of Lemur catta resides just above the linear regression line. perinatal age, suggesting L. catta is broadly similar to many nocturnal strepsirrhines [Garrett et al., 2013] in precocious maturation of the VNNE. Here, we show that adult L. catta has a postnatally GAP43þ (i.e., neurogenic), thick sensory epithelium with numerous rows of VSNs. In L. catta the volume of the VNO neuroepithelium relative to palatal length (an excellent proxy for size adjustment for midfacial dimensions) is similar to a large number of nocturnal strepsirrhines [studied by Garrett et al., 2013]. There are no sexual differences evident in our small sample. Our data show there are some microanatomical similarities in the VNO of the diurnal [or perhaps cathemeral, Parga, 2011] L. catta and the nocturnal strepsirrhines. This indicates that VNS is functional in L. catta and may be important behaviorally. An autonomic vasomotor mechanism, functionally present in some non‐primates [Meredith & O’Connell, 1979] may well contribute to chemoinvestigation in rodent‐sized primates. A “vomeronasal pump” was proposed as the main stimulus‐uptake/expulsion system for the mouse lemur’s VNO (Schilling, 1970; Evans & Schilling, 1995). The presence of several large venous sinuses within the lamina propria (Fig. 2d) suggests a means for autonomic vascular expansion, which could assist in stimulus‐presentation to the VNNE in L. catta. On the other hand, it is conceivable that flehmen response [see Bailey, 1978] facilitates higher molecular weight odorant access to the VNO. In L. catta, as in other studied strepsirrhines [Hofer, 1977; Smith et al., 2002, 2005; Wöhrmann‐ Repenning & Bergmann, 2001], stimulus access to the VNO occurs near the middle of the nasopalatine duct, which has both oral and nasal openings. The oral entrance of these paired duct opens occurs beside the bulbous incisive papilla. The papilla is well innervated with afferent nerves, as confirmed by beta tubulin immunohistochemistry (although the presence of Merkel cells requires confirmation of more specific staining or electron microscopy) and a large number of taste buds as observed in both the female and male L. catta. These have been previously described in primates [Hofer, 1977, 1980], and it is notable that their presence was not detected in a previous study of L. catta [Schmidt & Wöhrmann‐Repenning, 2004; Borcherding & Wöhrmann‐Repenning, 2007]. They may therefore be difficult to detect in some specimens or aberrantly absent. Their presence and density deserve further study in a broader array of species, and functional considerations, including possible involvement in the flehmen response. The role of flehmen response in the VNS (as opposed to gustatory) of L. catta is not self‐evident given that flehmen‐like behaviors may exist in primates without a functional VNS [Charpentier et al., 2013]. Moreover, the behavior can occur whether or not the oral opening of the nasopalatine duct exists [e.g., in horses – Salazar et al., 1997; Evans, 2003]. Chemical composition of glandular secretions in L. catta has recently received much study [Hayes et al., 2004; Scordato et al., 2007]. Brachial (from glands near the axillary region of male L. catta) secretions contain squalene and cholesterol derivatives. Antebrachial glands have received less scrutiny. A large body of literature has discussed the function of these secretions as social signals. Lower molecular weight volatile compounds of antebrachial glands may be more useful for conveying social status, often as a part of a multimodal display (e.g., stink fights of male L. catta). Labial and scrotal secretions contain organic acids and esters. Scordato et al. [2007] found the secretions to contain high‐molecular weight compounds. Further, they found that the genital gland secretions were chemically more complex than brachial or antebrachial glands. Scordato et al. [2007] also suggest that the chemical composition of secretions of the genital and brachial glands may provide for long lasting signals (Fig. 4), perhaps for advertising reproductive or territorial information. The authors further note that these high molecular weight compounds are relatively less volatile than compounds derived from antebrachial glands. Secretions derived from samples obtained from the genital region of anaesthetized lemurs contain a greater number of chemical compounds in female compared to male L. catta. This is the reverse of what is typically expected compared to that observed in other species of lemur [delBarco‐Trillo et al., 2012]. This array of chemical signals suggests important roles for both main olfactory system and VNS detection. In light of the microanatomy described Am. J. Primatol. 236 / Smith et al. Further detailed work should consider the vomeronasal organ of other cathemeral or diurnal species. In part due to the relative rarity of well‐preserved primate materials, and the difficulty in preparation, few observations on the VNS of medium and large‐ bodied strepsirrhines have been offered. Here, we show that the VNO of L. catta is broadly similar in neuronal characteristics and size compared to nocturnal species of strepsirrhines. Along with taste buds that border opening of the nasopalatine delivery system, an important role for non‐volatile signals is indicated. ACKNOWLEDGMENTS We thank K Jankord for help with histological sectioning and staining of one of the specimens. We also thank JC Dennis for helpful advice on immunohistochemical procedures. REFERENCES Fig. 4. Lemur catta at the Pittsburgh Zoo engaging in the “handstand” scent marking posture described in both sexes by Scordato et al. (2007), in which genital gland secretions are deposited on vertical substrates. (Photograph by TD Smith). here, and licking investigative behaviors noted in some previous studies [Bailey, 1978; Crawford et al., 2011; Dugmore et al., 1984; Palagi & Norscia, 2009], we suggest a synergistic chemosensory reception is important regarding nonvolatile signals by L. catta, one which also involves gustation. Such a close link between “vomerolfaction” and gustation was previously suggested by some other authors [Evans, 2003; Borcherding & Wöhrmann‐Repenning, 2007]. It has been postulated that mammals with oral communications to the VNO employ direct lip and rhinarial contact to access chemostimuli [Evans, 2003; Poran et al., 1993; Poran, 1998]. Evans [2003] further postulated that tongue pressure on the palate facilitates entry to the VNO. Our observations of the well‐ innervated inferior surface of the incisive papilla are consistent with this suggestion. 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