Pulmonary design in a microchiropteran bat (Pipistrellus subflavus) during hibernation

301

Respiration Physiology (1985) 59, 301-312 Elsevier

PULMONARY DESIGN IN A MICROCHIROPTERAN BAT (PIPISTRELLUS SUBFLAVUS) DURING HIBERNATION

ANDREW J. LECHNER Department of Physiology, St. Louis University School of Medicine, St. Louis, MO 63104, U.S.A.

Abstract. The Eastern pipistrelle (P/p/,m'e//ussubflams) is typical of exceptionally small bats capable of a 30-fold range in aerobic metabolism as they arouse from hypothermia and sustain foraging flight. This report describes their basic lung structure and the extent to which this organ is protected from protein depletion during hibernation. Bats were collected at the be#nning (Fall), middle (Winter), and end (Spring) of hibernation from a permanent overwintering cave, and analyzed within several days of capture. Regardless of whether bats were examined in the Fall (average body weight = 6.22 g) or in the Spring (4.58 g) no significantdifferences existed for total lung volume (237/A), alveolar surface area (338 cm2), harmonic mean septal thickness, ~ht (0.221/an), or membrane diffusing capacity (4.13 #l O2/sec/mbar). These parameters exceed predictions based on body weights for either season, and resemble published data for another highly active mammalian group, the insectivorous shrews. Both zht and the minimal septal thickness of 0.083/~m approach the anatomical limits for thinning of alveolar septa without loss of epithelial continuity. Although both the heart and lungs lost 13~ of their fresh weights during hibernation, compared to 25~ for the liver, the lung contents of DNA (0.14 nag) and blood-free protein (7.38 nag) were not altered significantly. These small bats possess lungs which are well suited for the high aerobic cost of flight. Those lungs are resistant to hibernation-induced proteolysis, and also resistant to the deterioration of alveolar membranes which occurs in nonkibernators subjected to starvation-induced weight losses of similar magnitude.

Lung morphometry Oxygen transport

Pipistrelle Starvation

Small bats possess exceptionally large aerobic scopes for activity, especially when the maximal rates of oxygen consumption associated with flight are compared to the minimal rates during torpor and hibernation (Hayward, 1968; Thomas et al., 1984). Nevertheless, many aspects of their oxygen transport systems remain poorly understood when compared to other small active mammals such as the insectivorous shrews (Snyder, 1976; Caire etal., 1981; Jurgens etal., 1981). Although a small bat of Accepted for publication 8 December 1984 0034-5687/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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unreported species was included in the original allometric description of mammalian pulmonary design (Tenney and Returners, 1963), the paucity of information on bat respiratory systems has only recently been recognized and addressed (Maina et al., 1982; Thomas et a/.,1984). By examining a common, small (4-8 g) bat of the American Midwest, the Eastern pipistrele (Pipistrellus subflavus, suborder Microchiroptera), this report has focused on two fundamental issues. First, do the lungs of a small flying mammal differ qualitatively or quantitatively from general mammalian models (Lechner, 1978; Gehr et al., 1981), or specifically from the lungs of other small nonflying mammals like the shrews (Gehr et al., 1980)? Second, since the body weight of this small hibernator fluctuates by at least 30~o during the course of a single winter, do lung components such as fresh weight and total DNA and protein contents also fluctuate? While it is generally agreed that the primary fuel for hibernation is stored fat, recent evidence indicates that certain organs in hibernating bats, including the heart and flight muscles, contribute substantial protein during periodic arousals (Ranch and Beatty, 1975; Yacoe, 1983a,b). Starvation of mature rats to a similar level of weight loss caused structural damage in their lungs which was not reversible with refeeding (Sahebjami and Wirman, 1981). Either the lungs of a small hibernator fike P. subflavus would be protected from protein depletion to a greater extent than other organs, or like muscle would have to be reconstructed each Spring during the animal's long normal lifespan (Davis, 1966; Keen and Hitchcock, 1980).

Materials and methods

Male Eastern pipistrelles (Pipistrellus subflavus) were collected from Mushroom Cave in Meramec State Park, Sullivan, MO (State Wildlife Colector Permit no. 0548). Collection dates were chosen to coincide with the animals' return to this cave in the Fall at the beginning of hibernation (November, 1982), the midpoint of the Winter hibernation period (February, 1983), and just prior to the onset of daily free flight in the Spring (May, 1983). The bats were collected by hand without causing arousal, placed immediately in dark containers over ice for the return trip to St. Louis (approximately 90 rain elapsed time), and then housed in a dark 5 °C humidified hibernaculum with access to water but no food. All animals were sacrificed within 3 days of colection as described below, with body weights (BW) recorded to + 0.01 g. Under sodium pentobarbital anesthesia (20 #g/animal, i.p.), and with body temperatures determined by thermistor to be less than 10 °C, groups of bats from the Fail and Spring collection dates were tracheostomized with 22-25 gauge needles fitted with tapered polyethylene tubing (Intramedic PE10 and PE50, Clay Adams, New Jersey). The lungs were then collapsed by puncture of the diaphragm from the abdominal side, and the lungs fixed/n situ by intratracheal instillation of buffered 2 ~o glutaraldehyde at 20 cm H20 of transpulmonary pressure for one hour. After fLxation, the left and right lungs were dissected free of the primary bronchi, and their volumes measured by fluid

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displacement with a reproducibility of 1~ at 100 #1. All fLxative and washes were isotonic with mammalian plasma. In this species of bat, each of the lungs is composed of a single large lobe. The entire left lung was processed in paraffin for some bats of each collection date for determination on 5-/am sections stained with Ehrlich's hematoxylin of the fraction of lung tissue composed of respiratory parenchyma, Frp (Lechner and Banchero, 1982). In these animals, the entire right lung was freely diced before dehydration and embedding in plastic for comprehensive morphometric analyses using electron microscopy (Lechner et al., 1982). The Frp was also estimated for these right lung blocks, as wel as their suitability for electron microscopy, using 'thick' sections (0.3/~m, stained with toluidine blue) examined on a Leitz Orthoplan Microscope. For other bats, both left and right lungs were diced for electron microscopy as described above, and the data compared for lateral differences. None was found, and the data for all animals were pooled by collection date. Thin, silver sections were cut of the plastic blocks, stained with uranyl acetate and lead citrate, mounted on 200-mesh copper grids, and photographed by a stratified random procedure on a Jeol 100S Electron Microscope. Using a photographic overlay grid, volume densities (Vvi) and surface densities (Svi) of lung tissue components were determined using the point counting and fnear intersection techniques, respectively (Weibel et al., 1966; Lechner et al., 1982). In practice this meant using a 168-endpoint lined grid on 50 electron photomicrographs from 5 blocks for each bat, at a final print magnification of about 5500 x. These volume and surface densities were then multiplied by the total respiratory lung volume (total lung volume, VL, multiplied by Frp) for each bat to obtain the absolute tissue volumes ~ul)or surface areas (cm2). The harmonic mean tissue barrier thickness, zht, was measured directly on a randomly selected subset of 10 photographs for each animal using a stratified array of calibrated lines (Weibel and Knight, 1964; Weibel, 1970/71). The harmonic thickness of the plasma layer between the inner endothelial surface and the erythrocytes was estimated to be 0.40 of the value for ~ht, an assumption which closely approximates its actual measured thickness in other small mammals (Gehr et al., 1980, 1981). The minimal barrier thickness (.#min) was also measured by arithmetically averaging the five smallest raw data entries used in the computations of zht. These measured and derived parameters were then used to compute the anatomical estimates of membrane and overall pulmonary diffusing capacity for oxygen (Dmo: and DLo2, respectively) for the entire lungs of each animal. The equations and minimal permeation and association coefficients (Kt = Kp = 4.1 x 10-1o cm2/sec/mbar and 0= 1.87 x 10 -2 ml OJml/sec/mbar) were used as described by Weibel (1970/71). In other groups of bats from the three collection dates, animals were anesthetized in similar fashion, and the left brachial artery exposed from the ventral surface. Blood samples of 80-120 #1 were then obtained by delicately severing the artery, and drawing the blood directly into heparinized capillary tubes. Samples which could not be collected within 30 sec were discarded. Subsequent analyses of hematocrit, hemoglobin concentration ([Hb]), and mean eel hemoglobin concentration (MCHC) were performed as previously described (Lechner et al., 1981). The thorax was then opened ventrally, and

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TABLE l Morphometric description of lungs from male bats (1). subflavus) collected at the beginning (Fall) and end (Spring) of hibernation. Values are means 4-SEM. Symbol

Definition

Fall (N = 5)

Spring (N = 6)

P*

BW VL VL/BW LVL Va Ve Vi Ven Vt Vc Sva Sa Sa/BW S% Sc Sc/Sa ~ht Zmi, Dmo2 DLo2

Body weight, g Total lung volume, #1 Weight-specificlung volume,/d/g Lei~ lung volume, 7eeL Alveolar air space volume,/d Epithelial tissue volume, p.l Interstitial tissue volume, gl Endothelialtissue volume, gl Total septal tissue volume, #1 Pulmonary capillary volume, pl Alveolar epithefial surface density, cm2/#l Total epithelial surface area, cm2 Weight-specificalveolar surface area, cm2/g Capillary endothelial surface density, cm2//d Total endothelial surface area, cm2 Capillary/alveolarsurface ratio, % Harmonic mean septal thickness, #m Minimal septal thickness,/~m Membrane diffusing capacity, #10z/sec/mbar Pulmonary diffusing capacity, #10z/sec/mbar

6.22 4. 0.05 351.0 _+20.5 56.4 ± 3.1 41.0 _+ 1.4 292.0 4- 19.3 5.8 4- 0.6 2.4 4- 0.3 5.0 ± 0.2 13.2 ± 1.0 27.6 4- 4.0 0.989 4- 0.047 326.2 _ 9.0 52.4 ± 3.2 0.871 4- 0.056 286.8 4- 11.4 87.8 ± 2.0 0.224 4- 0.006 0.084 ± 0.006 3.93 4- 0.12 0.454 4- 0.059

4.58 ± 0.07 307.8 4- 17.3 67.0 4- 3.2 36.2 ± 1.3 250.8 ± 14.1 8.8 4- 0.9 2.9 4- 0.6 5.0 4- 0.5 16.7 ± 1.8 22.9 + 2.9 1.178 ± 0.066 347.0 4- 39.6 75.8 ± 2.7 1.016 4- 0.066 300.0 4- 37.0 86.1 ± 0.8 0.2194- 0.008 0.082± 0.003 4.30 ± 0.62 0.389± 0.050

0;001 n.s. 0.05 n.s. n.s. 0.05 n.s. n.s. n.s. n.s. 0.05 n.s. 0.01 n.s. n.s. n.s. n.s. n.s. n.s. n.s.

* Significance determined with Student's t-test.

the lungs, heart, and liver removed en bloc. Tissues were separated, rinsed with saline, and then blotted before determining fresh weight. The left lungs as well as hearts and livers were frozen at - 80 ° C until lyophilized to constant dry weight. F r o z e n right lungs were homogenized in cold saline, and assayed for total D N A content using the diphenylamine reaction o f Burton 0956), and total protein content using a modified microbiuret procedure (Ohnishi and Barr, 1978). By also measuring the hemoglobin concentration o f the lung homogenate, and by estimating total plasma protein to be 7 g/dl (unpublished data for this species), a blood-free estimate o f total lung protein was obtained. Results for both blood-free protein and D N A contents o f the right lungs were then computed for the entire lung based on the ratio o f the fresh right lung weight to total fresh lung weight. Similarly, total dry lung weight was computed by assuming that the dry weight/fresh weight ratio o f the right lungs was the same as that measured for the left lungs. F o r all animals used for fresh weights and biochemistry, the entire humerus was carefully disartieulated at its proximal and distal connections, and its length measured to the nearest 0.02 mm using vernier calipers (Mitutoyo Corp., Japan).

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305

Fig. 1. Light photomicrographs of bat lungs, as 0.3 #m plastic sections stained with toluidine blue. Fixation occurred with a transpulmonary pressure of 20 cm H20. Alveolar surface density is relatively uniform, whether tissue is examined from medial regions (A; note large ciliated bronchiole), or near the peripheral regions of the lung (B). Original print magnification was 510 x.

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Whenever data were analyzed for all three collection dates, a one-way analysis of variance (ANOVA) was used with the Student-Neuman-Keuls a posteriori test to determine significant differences (Sokal and Rohlf, 1969). Sitmificance was estabfished between the two groups of morphometric data using Student's t-test. Differences were judged to be significant when P < 0.05. Results are presented as means + SE.

Results Lung histology. Complete morphometric data were obtained for five bats collected in the Fall and for six animals collected in the Spring (table 1). Despite a 27~ reduction in BW during hibernation, pulmonary dimensions remained nearly constant between the two groups. In general, approximately 92~o of VL below the primary bronchi contained respiratory parenchyma. Of that parenchymatous volume, the volume density of alveolar air space (Vva) comprised 86-88 ~o, septal tissue volume density (Vvt) equalled 4-6 ~o, and the volume density of pulmonary capillary blood (Vv¢) was 8~o. There were no si~ificant differences in these parameters due to season. These constituent volumes are in excellent agreement not only with those for other small mammals such as shrews (Gehr et al., 1980) but for a fruit-eating bat weighing 96 g examined by Maina et al. (1982). The actual values for total VL in P. subflavus are considerably larger than predicted for mammals of either the Fall or Spring body weights, although alveolar and capillary surface areas (Sa and So, respectively) are similar to predicted values for shrews of similar BW to the Fall bats (Gehr etaL, 1980, 1981). For the Eastern Pipistrelle, dat was consistently about 70~o of the expected thickness for a small mammal, and the minimal septal thickness zmin of about 0.083 pm is among the thinnest ever reported (Weibel and Knight, 1964; Meban, 1980). Although very small shrews (2-4 g) have been found to have lower VL'S and higher alveolar surface densities

TABLE 2 Body dimensions and hematology in P. subflavus collected during the hibernation season. Values are means _+SEM for (N) animals. Season

Body weight (g)

Right humerus length (mm)

Hematocrit (~ )

[Hemoglobin] (g/100 ml)

Mean red cell [Hemoglobin] (~o)

Fall

6.22 + 0.20 (9) 5.19 _ 0.14 a (11) 4.61 _ 0.08 a,b (13)

21.3 _+ 0.2 (9) 21.9 _ 0.2 (11) 20.9 _ 0.2b (13)

53.44 _+ 1.31 (9) 54.77 ± 1.99 (9) 56.84 _ 2.67 (8)

19.84 + 0.78 (9) 20.93 + 0.78 (8) 20.90 _ 1.13 (10)

37.09 _+ 0.93 (9) 37.98 _+ 0.45 (8) 38.36 _+ 0.90 (8)

Winter Spring

Significance tested by analysis of variance and the Student-Neuman-Keuls test: a p < 0.05 vs Fall; b p < 0.05 vs Winter.

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307

Fig. 2. Random electron photomicrograph of giutaraldehyde-fixed lung from a 4.71 g P. subflavus collected in the Spring. The alveolar air spaces (a) are divided by several converging capillaries containing erythrocytes (e) and plasma (p). An endothelial cell nucleus (en), alveolar macrophage (m) and an epithelial Type 2 cell (ep2) are also evident. (4720 x ).

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(Sv,) than these bats, calculated values for anatomical Dmo2 and DLo2for both groups are more similar to each other (Gehr et al., 1980) than to values which can be predicted based on data primarily for larger mammals (Gehr etal., 1981). Morphologically, the lungs ofP. subflavus resemble those of other mammals, although the relative density of alveolar membranes is greater than in larger animals (fig. 1). The very thin septal membranes only occasionally contained epithelial type 2 cells, and interstitial fibroblasts even less frequently (fig. 2).

Organ weights and lung biochemistry. Bats collected for analyses of blood and organ size also showed a progressive reduction in body size throughout the course of hibernation (table 2). Based on humerus length this weight loss of the population was not due to the presence of younger animals in the Spring versus Fall and thus it is likely that a relatively homogenous age group was studied throughout. Values for hematocrit, [Hb] and MCHC reported here (table 2) all substantiate previous reports that microchiropterans are relatively polycythemic mammals, with erythrocytes containing high internal hemoglobin concentrations (Caire et aL, 1981; Jurgens et aL, 1981; Bassett and Wiederhielm, 1982). The absolute values for fresh or dry lung and heart weights were only affected to minor degrees by hibernation, and then only in the latter part of this yearly cycle (table 3). For both tissues, their relative size as a percentage of BW increased siLmificanfly from Fall to Spring. In agreement with values for left lung volumes (table 1), fresh weights of the

TABLE 3 Fresh and dry organ weights in P. subflavus during hibernation. Values are means _+SEM.See table 2 for animal body weights. Season

N

Fresh organ weight

Dry organ weight

(mg)

(% BW)

(mg)

(% Fresh)

Heart Fall Winter Spring

6 11 10

53.5 + 1.0 50.2 + 2.0 46.3 + 0.9 a

0.85 + 0.02 0.97 + 0.03 a 1.03 + 0.03 a

13.6 + 0.1 13.0 + 0.4 12.0 + 0.4

25.5 + 0.5 26.0 + 0.4 25.9 + 0.6

Lungs Fall Winter Spring

9 11 10

49.7 + 2.6 50.6 + 2.4 43.1 _+ 2.9

0.80 + 0.04 0.98 + 0.04 a 0.94 _+ 0.05 a

11.6 + 0.5 12.2 + 0.4 9.8 _+ 0.3 a'b

23.4 + 0.4 24.4 + 0.6 23.2 _+ 1.3

Liver Fall Winter Spring

6 11 10

269.2 + 14.2 230.0 + 6.5" 200.4 + 15.3"

4.25 + 0.13 4.44 + 0.10 4.39 _+ 0.27

87.6 + 4.1 73.2 + 2.2" 68.1 + 5.5"

32.6 + 0.6 31.8 + 0.3 33.9 + 0.6 b

Significance determined by ANOVA and post hoc tests: " P < 0.05 vs Fall; b p < 0.05 vs Winter.

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LUNG STRUCTURE IN HIBERNATING BATS

left lung comprised 39.6%, 40.2% and 36.7% of total fresh lung weights for bats in the Fall, Winter and Spring, respectively (no si~ificant differences among collection dates). Conversely, the fresh and dry liver weights decreased si~ificantly during hibernation in direct proportion to the overall reductions in BW occurring from November to May. In general, the water content of each of the three organs remained constant during the study period. Although dry lung weight was sitmificantly reduced in the bats collected in May, neither total lung DNA nor blood-free protein content were significantly altered compared to animals examined from the November collection date (table 4).

Discussion

Lung morphometry. In the earliest morphometric description of a microchiropteran lung, a 10 g bat of unreported type possessed lungs with VL equal to approximately 0.5 ml and Sa equal to about 450 cm e (Tenney and Returners, 1963). More recently, detailed pulmonary data were reported for the 96 g fruit bat, Epomophoru~ wahlbergi (Maina et al., 1982). In that species, the qualitative arrangement of the respiratory parenchyma and the fractional composition of the entire lung were similar to other mammals, but total VL, Sa, and DLo, were several times greater than predicted for a nonflying mammal of the same size (Gehr et ai., 1981). Despite the larger size of the fruit bat, its alveolar surface density (Sv~) of 1.21 cm2//d and ~ t of 0.27/an were similar to average values for smaller shrews (Gehr et al., 1980; Maina et al., 1982). Pulmonary design in P#~istrellus subflams is also typically mammalian, but VL, S~, So and DLo~ always exceed predicted values by factors of 1.5-3.0, based on data in table 1 for Fall or Spring animals (Gehr et al., 1981). Thus, regardless of the actual body weight which can be considered normal for this bat, its lung morphometry is similar to that for other highly active mammals vs sedentary ones (Leclmer, 1978; Gehr et al., 1980, 1981). As expected for a very small mammal, ¢ht in P. subflams is exceptionally low (table 1), and similar to values of 0.27/an for the shrew Suncus etruscus weighing 2.5 g (Gehr et al., 1980). The major differences between this small bat and shrews of similar BW

TABLE 4 Biochemical evaluation of lungs from hibernating Eastern pipistrelles.Values are means _+SEM. See table 3 for other organ data. Season

N

Dry lung weight (rag)

DNA content (mg)

Blood-free protein content (rag)

Protein/DNA

Fall Winter Spring

8 9 10

11.2 ± 0.4 12.2 ± 0.4 9.8 ± 0.3"b

0.14 ± 0.01 0.16 ± 0.01 0.13 ± 0.01

7.52 ± 0.47 8.08 ± 0.41 6.63 ± 0.47

56.30 ± 2.89 51.43 _+ 2.60 53.55 ± 3.98

Significance determined by ANOVA and post hoc tests: * P < 0.05 v s Fall; b p < 0.05 vs Winter.

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appear to be the smaller VL and larger Sv~ of the latter group. Like the lungs of shrews (Gehr et al., 1980), alveolar septa in the Eastern pipistrelle contained few epithelial and endothelial nuclei or interstitial fibers and cells (figs. 1, 2). The calculation of a minimal tissue border thickness in this report was done for descriptive purposes only (table 1). However, it seems clear that the values for this term of about 0.083 #m represent the anatomical limit to septal thickness which can be achieved without disruption of epithelial and endothelial continuity (cf Weibel and Knight, 1964; Meban, 1980). Regions of the alveolar septa occasionally approach this thickness in other mammals (personal observations). However, it is the pervasive degree of septal thinning, apparently without loss of structural stability, that is remarkable. In overview, the Microchiroptera probably all possess lungs of a complexity hitherto only appreciated in the small insectivores, and this complexity is achieved without an excessive increase in lung weight compared to other mammals (table 3, and Snyder, 1976; Jurgens etal., 1981). Furthermore, the resultant high estimates of DLo2 are consistent with recent observations of oxygen extraction efficiencyduring activity in this suborder of bats which approach values seen only in birds (Thomas et aL, 1984).

Effects of hibernation. Since small bats such as P. subflavus hibernate each Winter during a lifespan of several decades (Davis, 1966; Keen and Hitchcock, 1980), it would seem unlikely that the yearly cycle of fattening and depletion would compromise organ function. This is particularly true if it is assumed that the energy requirements of hibernation are met with stored fats. However, Yacoe (1983a,b) recently reported that although periodic arousals occupied only 2~o of the total elapsed hibernation time in big brown bats (Eptesicus fuscus, prehibernatory body wt = 19 g), these arousals represented periods of intense proteolysis for gluconeogenesis. As a result, the weights of both the liver and pectoralis muscle in that bat decreased by 27~o during 110 days of hibernation, and the total protein contents of these organs decreased by 54~ and 46~o, respectively. The reduction of pectoralis mass may have been of sufficient magnitude to alter flight mechanics in the Spring (Yacoe, 1983a). Other muscles, as well as the heart, probably also contribute significantly to this protein catabolism (Rauch and Beatty, 1975). The fresh or dry weights of the lungs, heart and liver were all significantly reduced in P. subflavus by the end of hibernation, with the effects on the liver being the greatest (table 3). Although the decrease in dry lung weight by Spring was not associated with significant changes in lung DNA or blood-free protein contents, a trend toward reduced protein content was evident (table 4). However, the essential features of pulmonary morphology were preserved, most notably the Vt, V¢, Sa, So, dat, and the anatomicallyestimated Dmo2 (table 1). Although VL, Va, and the volume densities Vv~, Vvt, and Vv¢ were not reduced significantly, the actual values for VL and Va plus the significant increase in Sv~ suggest that the bat lungs collected in the Spring were underinflated at the same transpulmonary pressure used for bats in the Fall. Thus, chest wall compliance may have been altered, rather than any lung dimensions per se. Interestingly, when the food intake of mature rats was restricted until the animals lost about 40Yo of their initial

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311

body weight, Svl was pegmanently decreased, despite ad libitum refeeding (Sahebjami and Wirman, 1981). Thus, while the proteolytic cost of hibernation may resemble starvation to a degree only recently appreciated CYacoe 1983a,b), the lungs of hibernating species may be better protected from catabolism than the lungs of species which do not.

Acknowledgements The author would like to thank Dr. J o s e p h M. Puccinelli for invaluable field assistance. The late Dr. Louis S. D'Agrosa was instrumental in initiating this project due to his past experience with the native Missouri Chiroptera. This research was supported by the American Lung Association of Eastern Missouri and by grant no. HL-29640 from the National Heart, Lung, and Blood Institute.

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Ranch, J.C. and D.D. Beatty (1975). Comparison of regional blood distribution in Eptesicusfuscus (Big brown bat) during torpor (summer), hibernation (winter), and arousal. Can. J. Zool. 53: 207-214. Sahebjami, H. and J.A. Wirman (1981). Emphysema-like changes in the lungs of starved rats. Am. Rev. Resp/r. D/s. 124: 619-624. Snyder, G.K. (1976). Respiratory characteristics of whole blood and selected aspects of circulatory physiology in the common short-nosed fruit bat, Cynopterus brachyo~. Respir. Physiol. 28: 239-247. Sokal, R.R. and F.J. Rohlf (1969). Biometry. First Edn. San Francisco, W.H. Freeman Co., pp. 239-242. Tenney, S.M. and J.E. Returners (1963). Comparative quantitative morphology of the mammalian lung: diffusing area. Nature (London) 197: 54-56. Thomas, S.P., M.R. Lust and H.J. Van Riper (1984). Ventilation and oxygen extraction in the bat Phyllostomus hastutus during rest and steady flight. Physiol. Zool. 57: 237-250. Weibel, E.R. and B.W. Knight (1964). A morphometrie study on the thickness of the pulmonary air-blood barrier. J. Ceil Biol. 21: 367-384. Weibel, E.R., G.S. Kistler and W.F. Scbefle (1966). Practical stereological methods for morpbometric cytology. J. CeU Biol. 30: 23-38. Weibel, E.R. (1970/71). Morphometrie estimation of pulmonary diffusion capacity. I. Model and method. Respir. Physiol. 11: 54-75. Yacoe, M.E. (1983a). Maintenance of the pectoralis muscle during hibernation in the big brown bat, Eptesicus fuscus. J. Comp. Physiol. 152: 97-104. Yaeoe, M.E. (1983b). Protein metabolism in the pectoralis muscle and liver of hibernating bats, Eptesicus fuscus. J. Comp. Physiol. 152: 137-144.