© 2019. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2019) 222, jeb207605. doi:10.1242/jeb.207605

RESEARCH ARTICLE Nasal airflow in the pygmy slow (Nycticebus pygmaeus) based on a combined histological, computed tomographic and computational fluid dynamics methodology Timothy D. Smith1,‡, Brent A. Craven2,*, Serena M. Engel1, Christopher J. Bonar3 and Valerie B. DeLeon4

ABSTRACT genes (e.g. Rouquier et al., 2000). Moreover, in humans, the airflow ‘Macrosmatic’ have dedicated olfactory regions within their patterns differ greatly from those of mammals with a more primitive nasal cavity and segregated airstreams for olfaction and respiratory nasal cavity. In particular, humans and other haplorhines air-conditioning. Here, we examined the 3D distribution of olfactory (collectively, anthropoids and tarsiers) lack an olfactory recess, a surface area (SA) and nasal airflow patterns in the pygmy posterior cul-de-sac of the nasal fossa that is present in most (Nycticebus pygmaeus), a with primitive nasal cavities, terrestrial mammals (Van Valkenburgh et al., 2014; Smith et al., except for enlarged eyes that converge upon the posterodorsal nasal 2015). In mammals possessing this space, odorants migrate through region. Using the head of an adult loris cadaver, we co-registered the olfactory region more slowly and are thus more likely to be micro-computed tomography (CT) slices and histology sections to detected (Yang et al., 2007; Craven et al., 2010; Eiting et al., 2014). create a 3D reconstruction of the olfactory mucosa distribution. To date, nasal airflow has been studied in few . In this Histological sections were used to measure olfactory surface area study, we examined a nocturnal primate, the and to annotate CT reconstructions. The loris has a complex olfactory (Nycticebus pygmaeus), using computational fluid dynamics (CFD) recess (∼19% of total nasal SA) with multiple olfactory turbinals. simulations of airflow and odorant deposition. Nycticebus,likeother However, the first ethmoturbinal has a rostral projection that extends and , has a primitive nasal cavity with four medial far anterior to the olfactory recess, lined by ∼90% non-olfactory turbinals that are at least partially covered with olfactory mucosa. Like epithelium. Only one (of three) frontoturbinals bears olfactory other lorises and lemurs, a portion of the ethmoturbinal complex is mucosa. Computational fluid dynamics simulations of nasal airflow contained within the olfactory recess (DeLeon and Smith, 2014). and odorant deposition revealed that there is some segregation of Chemical communication via urine and glandular is respiratory and olfactory flow in the loris nose, but that it is not as important in territorial and other social interactions for these arboreal distinct as in well-studied ‘macrosmats’ (e.g. the dog). In the loris, primates (Fisher, 2003; Hagey et al., 2007). airflow is segregated medially and laterally to vertically elongated, All living strepsirrhine primates (lemurs and lorises) have a more plate-like first ethmoturbinals. Thus, lorises may be said to have complicated olfactory anatomy than living anthropoids (monkeys, certain macrosmatic anatomical characteristics (e.g. olfactory apes and humans). However, the entire order Primates has evolved recess), but not segregated nasal airflow patterns that are as visual specialists (Barton, 1998). Living primates possess one optimized for olfaction, as in canids. These results imply that a or more correlates of visual specialization that may physically binary ‘microsmatic/macrosmatic’ dichotomy does not exist. Rather, encroach on the interorbital (i.e. nasal) space during development. mammals appear to exhibit complex trends with respect to First, in many primates the eyes are relatively large compared with specialization of the turbinals and recesses. most other mammals (Ross and Kirk, 2007). Second, the eyes are convergent toward the midline in all primates, to a varying degree, KEY WORDS: Ethmoturbinal complex, Nasal airflow, Olfaction, for binocular vision (Heesy, 2009). Because eyes do not scale Primates, Strepsirrhine isometrically with the rest of the body (Ross and Kirk, 2007), these ocular traits create a bigger ‘packaging’ problem in small-bodied INTRODUCTION primates. In general, the eyes encroach on the nasal region to a To a varying degree, all primates employ olfaction in social greater degree than in large-bodied , but this is less interactions or in association with feeding functions (Nekaris, significant in strepsirrhines because the eyes are partially ectopic 2005). However, genetic evidence indicates that monkeys, apes and and may be positioned lateral or dorsolateral to the nasal fossa humans have undergone a functional reduction in certain olfactory (Cartmill, 1972). Nycticebus is a strepsirrhine with exceptionally large eyes and small body size. In the present study, we sought to determine the 1School of Physical Therapy, Slippery Rock University, Slippery Rock, PA 16057, degree to which airflow in the nasal fossa is optimized for odorant USA. 2Department of Mechanical and Nuclear Engineering, Pennsylvania State University, University Park, PA 16802, USA. 3Dallas Zoo, Dallas, TX 75203, USA. delivery to the olfactory recess. In particular, we investigated the 4Department of Anthropology, University of Florida, Gainesville, FL 32611, USA. degree to which nasal airflow patterns resemble those seen in the *Present address: Division of Applied Mechanics, Office of Science and dog and other macrosmatic . In the dog, olfactory airflow is Engineering Laboratories, Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, MD 20993, USA. shunted dorsally to the olfactory recess, rapidly bypassing more ventral structures such as the complex maxilloturbinal; respiratory ‡ Author for correspondence ([email protected]) streams are segregated ventrally (Craven et al., 2010; Lawson et al., T.D.S., 0000-0002-6883-8964 2012). We also considered the significance of relatively large eyes in lorises. These are known to influence bone resorption and medial

Received 23 May 2019; Accepted 5 November 2019 drift at shared boundaries of the orbital and nasal cavities during Journal of Experimental Biology

1 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb207605. doi:10.1242/jeb.207605 development (Smith et al., 2014), and thus affect nasal fossa epithelium in the nasal fossa was ‘mapped’ by annotating configuration and possibly respiratory and olfactory function. olfactory portions of the ethmoturbinals in each section where olfactory mucosa is present. Olfactory epithelium was identified MATERIALS AND METHODS based on the presence of olfactory receptor neurons, which usually Specimen information and imaging methods occur in multiple rows and whose nuclei are larger and rounder than An 11 year old male Nycticebus pygmaeus Bonhote 1907 that died those of adjacent respiratory epithelium (see Smith et al., 2007, for in captivity at the Cleveland Metroparks Zoo was studied. After further information). necropsy and removal of the brain for an unrelated study, the With this information, we annotated the segmented bitmap remainder of the head was immersed in formalin. Use of images that were used in the airflow simulations according to tissues was reviewed and approved by the IACUC at Slippery Rock olfactory/respiratory mucosa boundaries on ethmoturbinals. In University. The head was scanned at Northeast Ohio Medical addition, we created mucosal ‘masks’ to import into Amira. University (NEOMED) using the Scanco vivaCT micro-computed Briefly, this involved aligning binary images of ethmoturbinal tomography (CT) scanner (parameters: 25 µm cubic voxels; contours with CT slices using Adobe Photoshop. This was done 70 kVp, 114 µA). Subsequently, the head was processed for separately for total mucosal contours and then for olfactory mucosa paraffin histology. Serial sections at 12 µm thickness were made and only. Using Amira software, we reconstructed the bony anatomy of alternating sections were stained with Gomori Trichrome or the internal nasal fossa and then superimposed all mucosa and hematoxylin–eosin procedures. olfactory mucosa. The final results of these procedures were two The CT image stack was then imported into Amira software to models: (1) a segmented image stack of the right nasal airway lumen generate a 3D reconstruction of the bony nasal fossa. The CT data of with the contours delineated by epithelium type (squamous, the were reconstructed and resliced to match the cross- olfactory or respiratory), and (2) a 3D surface model of the sectional plane of histology. The full details of this method are skeletal anatomy of the nasal cavity, color coded to denote the described elsewhere (DeLeon and Smith, 2014). Briefly, we olfactory mucosa distribution on the ethmoturbinal complex. observed corresponding anatomical features in the CT and Finally, surface areas of mucosa type, including olfactory and histological data, and used these to adjust the plane of section in non-olfactory, were quantified using ImageJ for a comparison to the CT data. The CT dataset was then digitally re-sliced, so that the previous reports on primates. The annotated images of histology plane of CT slices corresponded to the histological sections. sections were used. After setting the scale based on a micrograph of Next, we segmented the lumen of the nasal airway based on a stage micrometer at the same magnification as the sections, two histological sections of the right nasal fossa. Micrographs of every measurements were taken using the freehand tracing tool: the total tenth histological section were segmented using Adobe Photoshop perimeter of the nasal mucosa and the perimeter of the olfactory and saved as a stack of bitmap images. In cases where a histological mucosa. These perimeter measurements (in mm) were multiplied by section was damaged, an adjacent stained section was prepared the distance between adjacent sections to compute an approximate from archived paraffin sections. The series of segmented images segmental surface area. All segmental areas were summed to were auto-aligned using ImageJ with manual alignment used as compute the total surface area of the right nasal fossa. The surface necessary. Achieving congruity of cross-sectional planes in CT and areas of individual turbinals and spaces were also calculated (more histology allowed us to correct artefactual distortions that are details are provided in Smith et al., 2011). All 3D reconstructions attributable to the decalcification and ethanol baths used for paraffin and quantifications are based on corrected rostrocaudal dimensions histology. In particular, the free margins of the ethmoturbinals, of all ethmoturbinals as described above. which project far rostral from their basal lamellae, undergo shrinkage (DeLeon and Smith, 2014). To transform the histology CFD simulations images to correspond to the CT images, each segmented histology Given the segmented histological sections that were annotated by image from the ethmoturbinal region was registered to the epithelium type (squamous, olfactory and other), a 3D surface model corresponding CT slice using Adobe Photoshop. Whenever a of the right nasal airway was reconstructed as in previous work turbinal structure was observed in the CT slice but not in the (Craven et al., 2007; Ranslow et al., 2014; Coppola et al., 2014; Pang corresponding segmented histology image, the image file of a more et al., 2016). The anatomical reconstruction extends from the posterior histology section containing that turbinal structure was first histology section, located in the nasal vestibule approximately also imported, and the turbinal was cut and pasted into alignment 2.8 mm from the tip of the nose, to the nasopharynx (Fig. 1). corresponding to the CT image. This process of expanding the Importantly, the reconstruction includes a specific delineation of the rostrocaudal dimension of the turbinal, as visualized through nasal epithelium, allowing us to investigate airflow and odorant histology, to correspond to the CT images resulted in CT slices with deposition in sensory and non-sensory regions of the nose. no corresponding histology for the turbinal. Additional intervening Given the reconstructed surface model, two unstructured hexahedral histological sections were mounted, stained and photographed computational meshes (coarse and fine) were generated using the to provide the corresponding histology for those CT slices. This snappyHexMesh utility available in the open-source computational avoided excessive interpolation of interslice distance as the turbinal continuum mechanics library OpenFOAM (version 2.4). The coarse length was corrected. and fine meshes (Fig. 1C,D) contain approximately 5.6 million and We used the histological sections to determine olfactory mucosa 11.2 million computational cells, respectively, including five wall- distribution. This was done by microscopic observations on sections normal layers to resolve the large velocity and odorant concentration with a Leica DMLB photomicroscope at ×50 to ×200 magnification. gradients that occur at the wall. Digital micrograph images of each section were simultaneously CFD simulations of quasi-steady inspiratory airflow and odorant projected on a second screen to annotate according to epithelium deposition were performed using OpenFOAM as described by type. First, the levels where only squamous epithelium exists (e.g. in Rygg et al. (2017). Briefly, the SIMPLE (Semi-Implicit Method for the vestibule) were indicated anteriorly, because this epithelium Pressure-Linked Equations) algorithm was used to numerically is comparatively impermeable to odorant. Second, the olfactory solve the governing equations for the steady-state incompressible Journal of Experimental Biology

2 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb207605. doi:10.1242/jeb.207605

A Fig. 1. Computational fluid dynamics (CFD) geometry and mesh. Olfactory (A) Lateral view of the anatomical reconstruction of the right nasal airway of the pygmy slow loris that includes an accurate delineation of the nasal epithelium from segmented histological sections that were Vestibule annotated by epithelium type (squamous, olfactory and transitional/ respiratory). The olfactory epithelium is shown here as yellowish- brown, the respiratory epithelium is pink and the squamous epithelium is gray. (B) Medial view of the reconstructed anatomical model with the septum digitally resected. The jagged line indicates that the rostral part of the nasal fossa is not shown. (C,D) A transverse slice through the coarse (C) and fine (D) CFD meshes at the location shown in A. The coarse and fine meshes contain approximately 5.6 million and 11.2 million computational cells, respectively, Naris including five wall-normal layers. Respiratory Nasopharynx BC

D

flow of air in the nose using second-order accurate spatial and insoluble, respectively, in the mucus layer that lines the nasal discretization schemes. A uniform velocity inlet boundary cavity. The required odorant properties were obtained as described condition was specified at the naris to obtain physiologically by Rygg et al. (2017) and CFD simulations of quasi-steady odorant realistic flow rates for two conditions: quiet breathing and a quasi- deposition were performed as in previous work (Rygg et al., 2017; steady sniff. The airflow rate for breathing through a single nostril Coppola et al., 2017, 2019). (7.9 ml s−1) was approximated based on the average adult body To investigate the numerical accuracy of the computed solutions, mass for the species (0.42 kg; Rowe and Myers, 2013) and the we performed a mesh refinement study at the sniffing flow rate. allometric equation for mean inspiratory flow rate provided by Applying the same inlet flow rate boundary condition, the overall Frappell et al. (1992). The airflow rate through a single nostril pressure drop between the naris and nasopharynx for the coarse and during a quasi-steady sniff was estimated by comparing respiratory fine meshes differed by only 1.4%, indicating that the CFD solution and sniffing flow rate measurements for an animal that is of similar is fairly insensitive to mesh resolution at this level of refinement. For size to the loris, the rat. Specifically, we compared the average all of the simulations reported in this study, we utilized the fine mesh inspiratory flow rate measurements of Frappell et al. (1992) for that contains approximately 11.2 million computational cells. breathing in the rat with the measurements of Youngentob et al. (1987) for the peak flow rate during sniffing in the rat, yielding RESULTS a multiplicative factor of 1.6 between the two physiological The mucosal maps revealed that most olfactory mucosa on the conditions (breathing and sniffing). That is, based on available ethmoturbinals is restricted, approximately, to the posterior third of experimental data, the maximum flow rate during sniffing in the rat this entire complex of turbinals. Almost all of the olfactory mucosa is about 1.6 times greater than the average flow rate for respiration. on the ethmoturbinals is restricted to the olfactory recess, defined as We then utilized this multiplicative factor of 1.6 to calculate an the space dorsal to the transverse lamina (Fig. 2). The first estimate for the sniffing flow rate in the loris of 12.7 ml s−1.To ethmoturbinal is primarily covered with non-olfactory epithelium further explore the sensitivity of our results to these flow rate (∼90%; Table 1). It has a free projection that extends rostrally to estimates, we also simulated two other flow rates: 4.1 and overlap the maxilloturbinal; none of this part of ethmoturbinal 8.4 ml s−1. I bears olfactory mucosa. As previously observed (Le Gros Clark, Converged steady-state airflow simulations were obtained by 1959; Smith et al., 2007), this turbinal also extends deeply ensuring that the normalized solution residuals were less than inferiorly, near the palate. Our reconstruction of N. pygmaeus 1×10−6 and by monitoring the iterative convergence of various shows the medial lamina of the first ethmoturbinal descends near computed quantities (e.g. volumetric flow rate, maximum velocity, the floor of the nasal fossa, and parallel to the posteroinferior-most minimum and maximum pressure). Given the computed steady- limit of the maxilloturbinal. Both structures are visible when state airflow solutions for breathing and sniffing, simulations of looking through the nasopharyngeal ducts from the posterior odorant deposition were performed for three odorants: heptanoic side (Fig. 2). acid, isoamyl acetate and nonane. These three specific odorants The olfactory recess is complex in that all four ethmoturbinals are were chosen because they are highly soluble, moderately soluble located at least partially within it. In addition, one interturbinal Journal of Experimental Biology

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Medial view Posterior view

FR

Orb

ET I ET I II III IV OR MT OR

ML NPD

5 mm ML 4 mm MT

Fig. 2. Computed tomography (CT) reconstruction of a segment of the right medial orbit and nasal fossa in an adult pygmy slow loris. The olfactory (red) and non-olfactory (blue) mucosa that covers the ethmoturbinal complex is superimposed on the images. On the left and right is a hemisected portion of the right nasal fossa (septum removed) including most of the ethmoturbinal complex, but with the posterior part removed. The inset (center) shows the entire nasal fossa, with the posterior parts highlighted to show the olfactory recess (green) and nasopharyngeal duct (NPD; orange). These two spaces are separated by a horizontal plate of bone, the transverse lamina (blue arrow). The left image is a medial view of the lateral nasal wall; the right image is a posterior view at the level of the olfactory recess (cut open to view the internal space). Note, the rostral extent of the first ethmoturbinal (ET I) projects far rostrally from the olfactory recess (OR). Also note the medial lamina (ML) of this turbinal hangs far ventrally, below the level of the transverse lamina (blue arrow). This lamina (ML) descends so far that it is visible next to the maxilloturbinal (MT, right) within the nasopharyngeal duct. FR, frontal recess; Orb, medial wall of orbit. begins anterior to the olfactory recess and then tucks within it, the dorsal meatus is directed to the olfactory recess. However, some between ethmoturbinals II and III. In sum, about 19% of the total flow streams outside of the dorsal meatus also reach the olfactory nasal surface area is within the olfactory recess. Moving posteriorly, recess (Fig. 3B). Thus, while there is some segregation of the ethmoturbinals bear progressively more olfactory mucosa respiratory and olfactory flow in the loris nose, it is not as distinct (Fig. 2). Ethmoturbinal IV is lined with 24.2% olfactory mucosa. as in the dog (e.g. Craven et al., 2010). The frontal recess has three frontoturbinals within it, but only the CFD simulations of odorant deposition showed that there is not a inferior-most of them bears any olfactory mucosa (46%; Table 1). large difference in the deposition patterns for respiration and sniffing CFD simulations of airflow revealed similar gross inspiratory flow conditions, particularly within the olfactory recess (Fig. 4). The nasal flow patterns for both respiration and sniffing. In both cases, highly soluble odorant heptanoic acid is deposited in the respiratory the highest flow speeds in the nasal cavity are primarily along the region and in the anterior olfactory recess (Fig. 4A,B). The insoluble septum in the common meatus (Fig. 3A). Generally, the flow within odorant nonane is deposited more uniformly in both regions (Fig. 4E,F). There is a larger gradient of odorant flux in the nose Table 1. Comparative distribution of olfactory mucosa (as a percentage for the moderately soluble isoamyl acetate (Fig. 4C,D). However, of the total surface area) from selected turbinals in pygmy slow loris within the olfactory region, most of the isoamyl acetate vapor is and mouse deposited within the anterior half of the recess, along the septum and Olfactory % Olfactory on the medial side of the ethmoturbinals. Comparing the gross Turbinal Total SA (mm2) SA (mm2) mucosa deposition patterns for heptanoic acid and isoamyl acetate in the olfactory recess (Fig. 4A,B versus C,D), there is a modest difference, Nycticebus pygmaeus ET I 190.3 20.11 10.5 but it is not as substantial as the marked difference in deposition Free projection of ET I 118 0 0 patterns observed between highly and moderately soluble chemicals ET IV 136.24 32.96 24.2 in the dog (Lawson et al., 2012) and more recently in the mouse FT* 31.51 14.49 46.0 (Coppola et al., 2017). Microcebus murinus Our flow rate sensitivity study confirmed that the present nasal b ET I 40.47 13.89 34.3 airflow and odorant deposition patterns are insensitive to our Free projection of ET Ia 39.4 ET IVb 20.03 9.03 45.1 estimates of the respiratory and sniffing flow rates for the loris. FTb 11.72 8.16 69.6 Specifically, the additional CFD simulations performed at 4.1 and 8.4 ml s−1 revealed extremely similar nasal airflow and odorant SA, surface area; ET, ethmoturbinal; FT, frontoturbinal. Data for pygmy slow loris (Nycticebus pygmaeus) are from this study. *Two other frontoturbinals deposition patterns to those observed for respiration and sniffing. lacked any olfactory mucosa (thus, an even lesser percentage of total FT Thus, the present results are expected to be generally representative surface area is lined with olfactory mucosa). Data for mouse lemur for the loris across a wide range of nasal flow rates, from quiet a b (Microcebus murinus) are from Smith et al. (2007) and Smith et al. (2011). breathing to the higher flow rates that occur during sniffing. Journal of Experimental Biology

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A Fig. 3. Flow patterns in the pygmy slow –1 loris nasal cavity during inspiration from a Dorsal meatus Velocity magnitude (m s ) 0 1234 CFD simulation of a quasi-steady sniff. (A) Velocity distribution in the nose visualized as contours of velocity magnitude on transverse planes. (B) Streamlines extracted from the CFD simulation results. Respiratory streamlines (blue) are those that do not enter the olfactory recess, while olfactory streamlines (red) correspond to those that enter the olfactory recess. The olfactory epithelium is shown here as yellowish-brown and all non-sensory epithelium is gray. The view in both A and B is from the lateral perspective.

Flow

B Respiratory streamlines Olfactory streamlines Olfactory epithelium

DISCUSSION showed that a larger functional recess is potentially advantageous Much of our detailed knowledge of primate olfactory anatomy is for olfactory odorant transport (Eiting et al., 2014). In most rooted in studies on humans or other anthropoid primates, all of mammalian orders, at least some species possess this specialized which possess relatively less-complex ethmoturbinal , chamber. In Primates, all extant strepsirrhines and perhaps some and less surface area of olfactory neuroepithelium compared with haplorrhine ancestors possess it (Smith and Rossie, 2006; Kirk most other mammals (Harkema et al., 2006). These traits have et al., 2014). relegated primates such as ourselves to so-called microsmatic In the mouse lemur (Microcebus murinus), this space is animals, or those with a comparatively poor sense of smell (Turner, demonstrably dedicated to olfactory function in that ∼69% of the 1891; Negus, 1958; but see Laska et al., 2000). Strepsirrhine olfactory recess is lined with olfactory mucosa, whereas only ∼28% primates have not been as closely studied. Although their internal of the remainder of the nasal cavity (excluding the recess) is lined nasal architecture is more complex and more extensively covered with olfactory mucosa (Smith et al., 2011). Similarly, our 3D with olfactory mucosa compared with that of anthropoids (Smith reconstruction shows that the majority of the olfactory mucosa on et al., 2011, 2016), a broader comparative understanding is lacking. ethmoturbinals is contained within the olfactory recess in Because their nasal anatomy more closely resembles that of early N. pygmaeus (Fig. 1). In this way, they are both similar to rodents primates, a detailed analysis of their nasal physiology may provide and dogs. However, a separate question is whether the strepsirrhine clues for the evolution of the primate nose and face. nasal apparatus has segregated respiratory and olfactory airflow Rodents and dogs are among the most commonly studied streams, as in the dog (Craven et al., 2010). The present CFD mammals regarding nasal airflow and olfaction. In both, the nasal simulations reveal that there is some segregation of respiratory and cavity includes a dorsoposterior cul-de-sac, termed the olfactory olfactory flow in the loris nose (Fig. 3B), but that it is not as distinct recess. This recess communicates anteriorly with the main nasal as in the dog (Craven et al., 2010; Rygg et al., 2017) and mouse chamber. Computer simulations reveal that air enters this recess via (Coppola et al., 2017). Moreover, the simulations show that, unlike the dorsal meatus and then slowly filters through the convoluted the dog, the dorsal meatus in the loris does not contain high-speed airways between ethmoturbinals (Yang et al., 2007; Craven et al., airflow (Fig. 3A). In dogs, the dorsal meatus is the only route by 2010; Coppola et al., 2017). Computer modeling has also been used which air enters the olfactory recess and high flow speeds minimize to study the influence of the functional size of the olfactory recess odorant deposition in the upstream respiratory region (Craven et al., by virtually altering the extent of the transverse lamina, which 2009; Rygg et al., 2017). In contrast, the lack of high-speed flow in Journal of Experimental Biology

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A Heptanoic acid Medial µ –2 –1 Lateral Odorant flux ( mol m s ) Medial 0 0.005 0.01 0.015 0.02 Respiration

B 0 0.01 0.02 0.03 0.04 0.05 Sniff

C Isoamyl acetate

0 0.005 0.01

D 0 0.004 0.008 0.012 Sniff

E Nonane

0 6.5e–5 0.00013 Respiration

F 0 6.5e–5 0.00013 Sniff Respiration

Fig. 4. CFD predictions of odorant deposition in the pygmy slow loris nasal cavity during inspiration. Contours of odorant flux on the nasal airway walls for (A,B) heptanoic acid, (C,D) isoamyl acetate and (E,F) nonane, which are highly soluble, moderately soluble and insoluble, respectively, in the mucus layer that lines the nasal cavity. Results are shown for inspiratory flow rates corresponding to respiration (A,C,E) and sniffing (B,D,F) with the nasal airway model viewed from the lateral and medial perspectives. The middle column shows odorant deposition along the nasal septal mucosa. In the right column, the model is viewed from the medial perspective with the septum digitally resected to reveal odorant deposition patterns on the medial side of the ethmoturbinals. the dorsal meatus of the loris may explain why there is not a These findings suggest that strepsirrhine primates do not have the substantial difference in odorant deposition patterns between degree of macrosmatic specialization seen in dogs, in which internal respiration and sniffing, or between the highly soluble heptanoic nasal anatomy is organized to deliver a functionally distinct acid and the moderately soluble isoamyl acetate (Fig. 4). We airstream optimized for odorant delivery to the olfactory recess. speculate that this might influence olfactory function by reducing Dedicated respiratory and olfactory flow streams in the dog’s nose the amount of ‘imposed’ patterning in the loris nose for different correspond to segregated nasal anatomy: respiratory and olfactory odors compared with the dog, which is thought to be used in concert turbinals are in relatively non-overlapping regions (Craven et al., with the ‘inherent’ patterning of olfactory receptors for odor 2010; Pang et al., 2016). Thus, a distinction between the most

recognition (Lawson et al., 2012). extreme olfactory specialists and other mammals may be Journal of Experimental Biology

6 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb207605. doi:10.1242/jeb.207605 recognizable in the extent to which ethmoturbinals are spatially ethmoturbinals participate in non-olfactory functions. For example, segregated from the main chamber, and from the maxilloturbinal. In about 2/3 of the surface area of ethmoturbinal I is non-olfactory in this sense, all strepsirrhines studied to date diverge from the extreme M. murinus, compared with 90% in N. pygmaeus (Table 1). Our macrosmatic form in important ways. First, there is far more overlap observations also reveal a contrast in the olfactory recess in these of maxilloturbinals and ethmoturbinals in strepsirrhine primates species. Much less of the ethmoturbinal complex is contained in the (Smith et al., 2007; 2016) than has been observed in rodents and recess in M. murinus (ethmoturbinal IV only) compared with dogs (Adams, 1972; Craven et al., 2007). Second, ethmoturbinal I is N. pygmaeus (parts of all ethmoturbinals). Microcebus murinus enlarged and largely overlapping the maxilloturbinal. The overlap likely relies more heavily on airflow in the main chamber for of ethmoturbinal I and the maxilloturbinal is also characteristic of odorant detection compared with N. pygmaeus; indeed, the scandentians and dermopterans, which, with primates, comprise the olfactory mucosa projects far beyond the reaches of the recess in living euarchontans (Smith et al., 2015). This suggests that the the former. These variations suggest that the presence of an arrangement may be primitive for primates. olfactory recess alone does not imply complete segregation of The majority of ethmoturbinal I in all primates is known to be respiratory and olfactory flow streams in the nose. non-olfactory (Smith et al., 2007, 2011, 2016), indicating its most The primitive arrangement for mammals is not completely clear as important functions may be humidifying inspired air or directing yet. The olfactory recess is an early innovation in mammals, present airstreams. In lorisoids and some other strepsirrhines, ethmoturbinal in at least some mammalian cynodonts (Ruf et al., 2014). However, I has a large medial plate which is vertically oriented (Smith et al., the delicate nature of the turbinal lamellae, as well as their initially 2007). This may explain medial versus lateral differences in airflow cartilaginous composition, make it uncertain how far rostrally the and the associated disruption of respiratory and olfactory flow ethmoturbinals projected from within the recess (Crompton et al., segregation observed here. The air medially adjacent to the septum 2017). Spatial overlap of the maxilloturbinal and the ethmoturbinal is likely segregated by the vertical plate of ethmoturbinal I (Fig. 3B), complex is minimal in some groups of mammals, including canids which is large and non-olfactory in N. pygmaeus (Fig. 2, Table 1). (Craven et al., 2007), many ungulates (Negus, 1958; Ranslow et al., The paranasal spaces are also distinctive in the loris compared 2014), and rodents (e.g. Adams, 1972). However, the ethmoturbinals with those described in other strepsirrhines. In particular, we (particularly the first) project far rostrally from the olfactory recess in suggest that the frontal recess may be diminished developmentally a diverse array of mammals, including known felids (Pang et al., by the encroaching orbits (see Fig. 2). There is empirical 2016), at least some marsupials (Rowe et al., 2005), as well as many developmental evidence for how a trade-off may occur between living euarchontans (Smith et al., 2015) and at least some the shape of the orbital and nasal cavities. Bone modeling patterns primates (Lundeen and Kirk, 2019). The widespread nature of this were recently examined in a fetal (Loris tardigradus); arrangement across mammals is suggestive of a primitive condition. patterns of osteoclast activity revealed medial resorption within the If so, then those mammals in which the ethmoturbinals are more developing fetal orbit (Smith et al., 2014). Specifically, bone cell limited to the olfactory recess, with less projection of ethmoturbinal activity indicates that the frontal bone, a shared skeletal boundary of I, possess a derived morphology. The macrosmatic segregation of the orbit and frontal recess, drifts medially during growth. This respiratory and olfactory regions might have occurred convergently suggests that the enlarged eyes of this nocturnal species may in living rodents, ungulates and canids via rearrangement and spatial constrain the size of the paranasal spaces during development. segregation of turbinals. Similar constraints may explain a relatively reduced frontal recess in Finally, we note a limitation of this study and prospects for future N. pygmaeus because the orbit is greatly enlarged at its interface work. Because the present CFD model was reconstructed from with the dorsolateral nasal cavity (Fig. 2). segmented histology sections, the model did not include the external Compared with the remainder of the nasal fossa, olfactory nose. The neglect of the external nose and the use of a uniform function of the paranasal spaces is less well understood. Yet, many velocity boundary condition at the truncated naris located mammals possess olfactory turbinals within this space. The results approximately 2.8 mm from the tip of the nose may have slightly of the present study concur with a recent study by Rygg et al. (2017) influenced the downstream flow field, but is unlikely to have on the coyote (Canis latrans). In N. pygameus and C. latrans, the influenced the gross nasal airflow and odorant deposition patterns solubility of odorants determines where deposition occurs. The least reported here. Even so, future work should leverage high-resolution soluble among the tested odorants (nonane) was the only one to magnetic resonance imaging (MRI) or CT data to reconstruct the reach the frontal recess in both species. This suggests that the frontal tissue-lined nasal cavity that includes the external nose (e.g. Craven paranasal space may only be sensitive to some odorant types that are et al., 2009; Ranslow et al., 2014). Diffusible iodine-based contrast- relatively insoluble. Despite the similarity, N. pygmaeus possesses enhanced CT (diceCT) (Gignac et al., 2016) might be used to relatively little olfactory mucosa in the paranasal space compared resolve the nasal epithelium, but further work is needed to verify with the olfactory recess; only one of the three frontoturbinals bears that it can be used to reliably delineate sensory from non-sensory olfactory mucosa. An interesting implication is that N. pygmaeus nasal epithelium. Alternatively, tissue thickness has been used by and perhaps some other strepsirrhines may have a reduction in Yee et al. (2016) to delineate sensory and non-sensory epithelium, olfactory surface area for detecting some odorants as a consequence which may also be explored as a means to map the nasal epithelium of orbital growth patterns. from high-resolution CT data alone. This approach, however, will Broader comparative evidence may be needed to fully understand need to be validated by comparison with histology data in the same whether olfactory specialization reflects a process of segregating the specimen. maxilloturbinal and ethmoturbinals from one another. Whereas strepsirrhine primates have more complex turbinals than humans, Conclusions the so-called ‘olfactory’ turbinals (ethmoturbinals) are not The findings on nasal morphometry and airflow patterns in specialized for olfaction to the degree seen in dogs or rodents. N. pygmaeus indicate that this strepsirrhine primate does not have Furthermore, comparison of N. pygmaeus and M. murinus shows the degree of macrosmatic specialization seen in canids, in which that there may be considerable variability in the degree to which internal nasal anatomy is organized to deliver a functionally distinct Journal of Experimental Biology

7 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb207605. doi:10.1242/jeb.207605 airstream optimized for odorant delivery to the olfactory recess speculations on the evolution of mammalian endothermy. J. Vert. Paleontol. (Craven et al., 2010; Rygg et al., 2017). In N. pygmaeus, this doi:10.1080/02724634.2017.1269116 DeLeon, V. B. and Smith, T. D. (2014). Mapping the nasal airways: using histology appears to be due to the large vertical lamina of ethmoturbinal I that to enhance CT-based three-dimensional reconstruction in Nycticebus. Anat. Rec. partitions airflow vertically into medial and lateral streams. Whether 297, 2113-2120. doi:10.1002/ar.23028 the large degree of overlap of the ethmoturbinal complex with the Eiting, T. P., Smith, T. D., Perot, J. B. and Dumont, E. R. (2014). The role of the maxilloturbinals is primitive for mammals remains a critical olfactory recess in olfactory airflow. J. Exp. Biol. 217, 1799-1803. doi:10.1242/jeb. 097402 unanswered question. In addition, primate trends in evolution of Fisher, H. S. (2003). Countermarking by male pygmy lorises (nycticebus the frontal recess, which may detect a specific range of odorants, pygmaeus): do females use odor cues to slect mates with high competitice require elucidation. ability. Behav. Ecol. Sociobiol. 53, 123-130. Frappell, P., Lanthier, C., Baudinette, R. V. and Mortola, J. P. (1992). Metabolism Finally, our results imply that in N. pygmaeus and other and ventilation in acute hypoxia: a comparative analysis in small mammalian strepsirrhines, the ethmoturbinal complex is multifunctional to a species. Am. J. Physiol. Reg. Integr. Comp. Physiol. 262, R1040-R1046. doi:10. degree not seen in canids. While in canids, nasal morphology may 1152/ajpregu.1992.262.6.R1040 be said to reflect an optimized design for distinct respiratory and Gignac, P. M., Kley, N. J., Clarke, J. A., Colbert, M. W., Morhardt, A. C., Cerio, D., Cost, I. N., Cox, P. G., Daza, J. D., Early, C. M. et al. (2016). Diffusible iodine- olfactory airflow streams (Craven et al., 2010), in strepsirrhines the based contrast-enhanced computed tomography (diceCT): an emerging tool for ethmoturbinals presumably play more significant roles in directing rapid, high-resolution, 3-D imaging of metazoan soft tissues. J. Anat. 228, airflow and in humidifying and warming inspired air (the latter in 889-909. doi:10.1111/joa.12449 combination with the maxilloturbinal). These results imply that a Hagey, L. R., Fry, B. G. and Fitch-Snyder, H. (2007). Taking defensively, a duesl use for the brachial gland exudate of slow and pygmy lorises. In Primate Anti- binary microsmatic/macrosmatic dichotomy does not exist. Rather, Predator Strategies (ed. S. L. Gursky and K. A. I. Nekaris), pp. 253-272. New York: mammals appear to exhibit a diversity in nasal morphology that lies Springer. along a continuum characterized by the degree to which respiratory Harkema, J. R., Carey, S. A. and Wagner, J. G. (2006). The nose revisited: a brief and olfactory anatomy and function are segregated in the nose. review of the Comparative structure, function, and toxicologic pathology of the nasal epithelium. Toxicol. Pathol. 34, 252-269. doi:10.1080/01926230600713475 Future work should consider the evolutionary origins of this Heesy, C. P. (2009). Seeing in stereo: the ecology and evolution of primate binocular diversity. vision and stereopsis. Evol. Anthropol. 18, 21-35. doi:10.1002/evan.20195 Kirk, E. C., Daghighi, P., Macrini, T. E., Bhullar, B.-A. S. and Rowe, T. B. (2014). Acknowledgements Cranial anatomy of the Duchesnean primate Rooneyia viejaensis: new insights The authors thank C. J. Vinyard for scanning the cadaveric head used in the present from high resolution computed tomography. J. Hum. Evol. 74, 82-95. doi:10.1016/ study. j.jhevol.2014.03.007 Laska, M., Seibt, A. and Weber, A. (2000). ‘Microsmatic’ primates revisited: Competing interests olfactory sensitivity in the squirrel monkey. Chem. Senses 25, 47-53. doi:10.1093/ chemse/25.1.47 The authors declare no competing or financial interests. Lawson, M. J., Craven, B. A., Paterson, E. G. and Settles, G. S. (2012). A computational study of odorant transport and deposition in the canine nasal Author contributions cavity: Implications for olfaction. Chem. Senses 37, 553-566. doi:10.1093/ Conceptualization: T.D.S., B.A.C., V.B.D.; Methodology: T.D.S., B.A.C., C.J.B., chemse/bjs039 V.B.D.; Formal analysis: T.D.S., B.A.C., S.M.E., V.B.D.; Investigation: T.D.S., Le Gros Clark, W. E. (1959). The Antecedents of Man: An Introduction to the B.A.C.; Resources: C.J.B.; Writing - original draft: T.D.S., B.A.C.; Writing - review & Evolution of the Primates, p. 374. Edinburgh: Edinburgh University Press. editing: T.D.S., B.A.C., S.M.E., V.B.D.; Funding acquisition: T.D.S., B.A.C., V.B.D. Lundeen, I.K. and Kirk, E.C. (2019). Internal nasal morphology of the Eocene primate Rooneyia viejaensis and extant Euarchonta: Using μCT scan data to Funding understand and infer patterns of nasal fossa evolution in primates. J Hum Evol This study was supported by the US National Science Foundation [grant numbers: 132, 137-173. doi:10.1016/j.jhevol.2019.04.009 BCS-1231717, BCS-1231350, IOS-1120375]. Negus, V. (1958). The Comparative Anatomy and Physiology of the Nose and Paranasal Sinuses. London: E. & S. Livingstone. References Nekaris, K. A. I. (2005). Foraging bahvior of the slender loris (Loris lydekkarianus Adams, D. R. (1972). Olfactory and non-olfactory epithelia in the nasal cavity of the lydekkarianus): implications for theories of primate origins. J. Hum. Evol. 49, mouse, Peromyscus. Am. J. Anat. 133, 37-50. doi:10.1002/aja.1001330104 289-300. doi:10.1016/j.jhevol.2005.04.004 Barton, R. A. (1998). Visual specialization and brain evolution in primates. Proc. Pang, B., Yee, K. K., Lischka, F. W., Rawson, N. 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