Gas exchange and habitat selection in the aquatic ... · Gas exchange and habitat selection in the aquatic salamanders ... Oxygen consumption ... ing their ability to regulate the

Oecologia (1990) 83:250-258 Oecologia 9 Springer-Verlag 1990

Gas exchange and habitat selection in the aquatic salamanders Necturus maculosus and Cryptobranchus Mlegam'ensis Gordon R. Ultsch and Jeffrey T. Duke Department of Biology, The University of Alabama, Tuscatoosa, AL 35487, USA

Received December 15, 1989 / Accepted January 12, 1990

Summary. The standard metabolic rate (SMR) and criti- cal 02 tension (Pc) of water-breathing mudpuppies and hellbenders were determined at 20 ~ C using open-system respirometry. Both species are metabolic 02 regulators, although the Pc of hellbenders (90 mmHg) is much high- er than that of mudpuppies (40 mmHg). The SMR of the two species in water saturated with air was similar (19.5 and 20.0 gl O2/g" h for Cryptobranchus and Nectur- us, respectively) and not different from that of salaman- ders in general. Both species were able to survive for at least 5-11 days in severely hypoxic water (9- 10 mmHg) by breathing air, indicating that the lungs are functional accessory respiratory structures.

We conclude that hellbenders are restricted to rela- tively cool and flowing waters because of their limited gas exchange capabilities, particularly with regard to their limited aerobic scope for activity and slow recovery from exercise. Necturus maculosus is much more tolerant of hypoxia, but it is not known if they can inhabit areas were hypoxia is combined with hypercarbia.

Key words: Cryptobranchus alleganiensis - Necturus ma- culosus - Oxygen consumption - Metabolic rate - Criti- cal oxygen tension

The giant salamanders of North America include the genera Siren, Amphiuma, Cryptobranchus (hellbenders), and Necturus (i.e.N. maculosus, most other species being smaller stream-dwelling forms). All exhibit cutaneous gas exchange, all have lungs, and Necturus and Siren have external gills. The group is of particular interest because of the differing degree of development of each of these modes of gas exchange and the resultant impli- cations for their respiratory physiology, acid-base bal- ance, energetics, and habitat selection. In particular, none of the species has all three modes of gas exchange highly developed. Amphiuma, with no gills, and Siren,

Offprint requests to: G.R. Ultsch

with poorly developed gills, both have highly developed lungs.

Adult Siren lacertina and Amphiuma means are obli- gate air-breathers at temperatures that routinely occur in the summer (e.g. 25 ~ C) within their habitats (Gui- mond and Hutchison 1976; Ultsch 1973a, 1976a). In contrast, mudpuppies and hellbenders can tolerate con- tinuous (at least two weeks) submergence in aerated water at temperatures up to 25 ~ C (Guimond and Hutch- ison 1972, 1973, 1976). Necturus maculosus is much the smallest of the group (200-300 g), its relatively small size and gas-permeable integument increasing the effi- cacy of cutaneous gas exchange; in addition, it has high- ly arborized external gills that can be actively ventilated. Cryptobranchus alleganiensis is much larger (over 1 kg) and does not have gills, but has a flattened body form and highly vascularized skin folds that in the aggregate function as a gill. A significant respiratory role for the lungs is often dismissed for both species (Guimond and Hutchison 1976; Lenfant and Johansen 1967; Shield and Bentley 1973) because they can survive prolonged sub- mergence, because the lungs are simple sacs in compari- son to the highly septate lungs of Amphiuma and Siren, and especially because the pulmonary contribution to resting gas exchange is small (at 25 ~ C, <8% for 02 and <3% for CO2, Guimond and Hutchison 1972, 1973).

The limited aerial respiration of Cryptobranchus, paired with its lack of gills, may be responsible for its habitat being restricted to permanent streams and small rivers in the drainage systems of the Appalachian and Ozark mountains. The 02 tensions in these habitats are normally near air saturation and maximal temperatures are usually <25 ~ C (Beffa 1976; Brown 1985; Nickerson and Mays 1973). In the case of Necturus, the gills have been presumed to allow it to occupy hypoxic waters in spite of its poor lungs, which may explain its much wider range (Conant 1975). However, although some species of Necturus are reputed to occupy habitats that would be expected to become occasionally hypoxic (Har- ris 1959), we are not aware of any field data concerning the PO2 of the microhabitat of Neeturus.

In spite of the poor vascular iza t ion and lack of septa- t ion in the lungs of bo th species, the lungs are relatively large ( G u i m o n d and Hutch i son 1976), and they are ven- ti lated regularly (albeit it at low frequencies) at higher temperatures , as indicated by our observat ions and the presence of a measurab le p u l m o n a r y O2 up take (Bouti- lier et al. 1980; G u i m o n d and Hutch i son 1976; Miller and Hutch i son 1979). This suggests tha t they are serving some respira tory funct ion. Miller and Hutch i son (1979), for example, found that while a lmost all O2 up take in Necturus maculosus was aquat ic at 5 and 15 ~ C, that dur ing periods of spon taneous aerobic activity over 50% of the 02 uptake was pu lmonary . In addi t ion , mos t of the studies of gas exchange in bo th species have been done using aerated water, so the responses to hypoxia are no t well known. In studies of aquat ic hypoxia in Necturus (Miller and Hutch i son 1979; Shield and Bent- ley 1973), confl ict ing results have been ob ta ined regard- ing their abil i ty to regulate the rate of O2 consumpt ion .

Here we repor t that bo th of these sa lamanders can survive at least 5-11 days in near ly anoxic water at 20 ~ C by ut i l izing p u l m o n a r y respirat ion, showing that the lungs serve an i m p o r t a n t respi ra tory func t ion dur ing pe- riods of low water PO2. We also demons t ra te tha t bo th species are metabol ic O2 regulators when respir ing aquatical ly, a l though the critical 02 tens ion of Crypto- branchus is over twice that of Necturus.

Material and methods

Animals. Hellbenders (Cryptobranehus alleganiensis alleganiensis) were collected from Cypress Creek in northwestern Alabama and from the Hiwasee River in northeastern Georgia. Both sites are part of the Tennessee River drainage and are near the southern limits of the range of hellbenders (Conant 1975). Mudpuppies (Nee- turus maculosus maculosus) were purchased from Kons Scientific Co., and were collected in Wisconsin. The salamanders were kept in flowing well water that was adjusted to circa pH 7 by passage through marble chips; they were maintained at 20 + 2 ~ C with a 12L-12D photoperiod centered on 1400 h. The animals were fed mealworms, earthworms, and recently killed minnows, except that no animal was fed within a week of use in an experiment.

Oxygen consumption measurements. Oxygen consumption (VO2) of submerged animals was measured continuously using a flow- through respirometer similar to that described by Ultsch et al. (1981). Flow rate was set so the difference in PO 2 between incurrent and excurrent water was 10-20 mmHg in the range of metabolic O2 regulation. Corrections were made for electrode drift and for changes in barometric pressure during the experiment; in practice, the latter correction is usually so small that it could be ignored. When exit PO2 is constant (i.e. the system is in a steady state), the ~/O2 can be readily calculated according to the Fick principle. It is also possible to calculate the VO2 in a non-steady state, such as occurs when the input PO2 is changed and the chamber water is in a washout phase. The derivation of appropriate formulae have been discussed by Ultsch and Anderson (1988) and Steffensen (1989). Here we calculated the equilibrium exit PO2 (PO2eq) that would be reached in an eventual steady state from the PO2's of an interval during the preceding washout phase according to Eq. 1 :

POze q - P02(2)--PO2(D q-PO2(1) (Eq. 1) 1 -- e - v(a t/v)

where POz(1) is the exit PO2 at the beginning, and PO2(=) at the end, of the time interval A t; V is the flow rate through the respir-

251

ometer, and V is the water volume of the chamber and associated tubing and mixing pump. POzeq can then be substituted for the exit PO2 in the Fick equation to calculate VO2, and the PO2 at which this VO2 occurred can be approximated as the average of PO2(1) and PO2~2). An inherent assumption is that the "~Oz is con- stant throughout the interval A t. This assumption can be checked by comparing the calculated VO2 during the non-steady state with that found during the ensuing steady state. Over the range of ambi- ent PO2 where VO2 is regulated, we rejected any values that were more than 10% above the steady state value achieved after wash- out, on the assumption that the animal was active during the inter- val and that the calculated VOz therefore did not represent a mea- sure of the standard metabolic rate (SMR), which was the rate in which we were interested. However, once the ambient PO2 dur- ing the progressive hypoxia approach we used fell below the critical O2 tension (Pc, see below), then VO2 does decrease throughout the fall in POz associated with the time interval A t, and the data points can not be rejected by such a criterion. In practice, this did not appear to present a problem, and all data below the Pc were used.

Determination of Pc- Given VO2 as a function of ambient PO2, a variety of methods have been used to determine Po, ranging from fits of data by eye to a number of statistical approaches. We used the method of Yeager and Ultsch (1989), which basically involves assuming that the data can be satisfactorily fit by two regression lines whose point of intersection is an approximation of the Pc- The method utilizes a program that calculates the two best-fit regression lines and their intersection. It is imperative that these lines and their intersection be plotted on a graph of the data to verify visually that the intersection is a good approximation of the Pc; if it is not, then an alternative "midpoint approximation" is used (see Yeager and Ultsch 1989, for a discussion of both ap- proximations of Pc and a BASIC program for analysis of data). Here the Pc approximation derived from the intersection of the two best-fit regression lines was satisfactory in 15 of 21 cases.

Ecological metabolic 02 regulation and conformity. In order to pro- vide a reasonable range of PO2 over which to calculate regression lines above the Pc, we measured VO2 at a PO2 as high as near 200 mmHg. We also calculated Pc using only the {zO2 data for POz'S <156 mmHg (air-saturation) in order to describe the rela- tion between the two in the likely ecological range of PO2. For comparisons of SMR, we use the VO2 shown at 156 mmHg.

Tolerance of severe water hypoxia. The efficacy of the lungs as gas exchangers when the water PO2 was critically low was assessed by lowering the PO2 while allowing the animals access to air. One to three salamanders were placed in the respiration chamber and the input PO2 was lowered to <2 mmHg by using Nz as the gas source in the water equilibration system. The chamber was slightly inclined with two 1.2 cm holes in the lid left open to the atmo- sphere. This maneuver created an air pocket of 300400 ml con- nected to the atmosphere by the holes. Most animals quickly learned to position their heads below the open surface. Water POz usually remained near 9-10 mmHg (range 4.6-17.2), a level well below the Pc of both species, and one that would be lethal to submerged animals in < 24 h. Survival was noted daily.

In a separate set of experiments, surfacing frequency in recircu- lating hypoxic water was noted periodically for 3 hellbenders ob- served over 0.5 h intervals for a total of 5 h each, and for 3 mud- puppies observed over 0.5 and 1.0 h intervals for a total of 10 h each. As we did not want to inhibit surfacing by the animals, we did not take any measures to prevent diffusion of 02 from the air into the water. The entire water surface was open to the atmosphere, resulting in an equilibrium water P O 2 that ranged from 22 to 30 mmHg, which nevertheless represents a relatively severe hypoxia (about 16% of air-saturation). To eliminate influ- ences of body size, animals of similar body weight were used.

Statistics. Differences between variables for the two species were tested for by a t-test. The significance of the slopes of individual

252

TaMe 1. Summary of oxygen uptake data for submerged Cryptobranchus alleganiensis and Necturus macu- losus acclimated to and tested at 20 ~ C. Pc, critical 02 tension; "~O2 (sat), standard rate of 02 consumption in air-saturated water (PO2= 156 mmHg); m (conformity)=A{rO2/AP02 in the zone of metabolic 02 conformity; m (regulation)= AVCOz/AP02 in the zone of metabolic O2 regulation. Data are 2_+ SE (range)

Weight n Pc g o 2 (sat) m (conformity) m (regulation) (g) (mmHg) (gl O2/g'h)

Cryptobranchus alleganiensis 395 _ 83 11 90.0 _+ 7.5 19.51 +_ 1.37 ~0.197 + 0.022 b0.007_+ 0.006 (69--796) (57.5--136.5) (12.99--26.73) (0 .094-0.320) (--0.0240.015) Necturus rnaculosus 124 _+ 8.6 I 0 c 39.7 _ 3.1 19.96 _ 0.99 a.c 0.429 + 0.051 b 0.008 + 0.005 (72-170) (27.1-62.5) (14.70-25.92) (0 .199-0 .673) (--0.012-0.037)

a significantly greater than zero b not significantly different from zero

significantly different from Cryptobranchus

regression lines used to determine Pc's was determined from a Stu- dent's t-table using the t-statistics generated by the data analysis program (Yeager and Ultsch 1989). The mean slope of ~'Oz on POz regressions in the ranges of regulation and conformity was tested for being significantly different from zero by calculating the confidence intervals. All significance levels were set at 95%.

Results

Standard metabolic rates (SMR) and critical 02 tensions (Pc). There was no difference in the SMR's of the 2 spe- cies (Table 1), even when an adjustment was made for the larger size of the hellbenders (see below). The Pc of the hellbenders was much higher than that of the mudpuppies. Because of these 2 results, the rate at which an animal could increase its Oz consumption with an increase in PO/(AVO2/APO2) in the zone of metabolic O2 conformity (e.g. at PO2's below Pc) for Necturus was over twice that of Cryptobranchus (Table 1). When the ambient PO2 was increased beyond the Pc, the mean AVOz/APO2 was not significantly different from zero for either species, indicating metabolic 02 regulation. However, when the individual data sets are considered, AVOz/AP02 above Pc was significantly different from zero for 3 of 10 Necturus and 4 of 11 Cryptobranchus (examples include Figs. 1 B, C and 3 A-C), being positive in all cases (perhaps due to slight increases in physically dissolved O2 in the blood), but always much less than in the zone of metabolic 02 regulation (Figs. 1-3).

As mentioned in the methods section, the Pc could be satisfactorily approximated in 15 of 21 cases by fitting 2 regression lines to the data and taking their intersec- tion as the Pc; examples are given in Figs. 1 C -F for Necturus and Fig. 3 for Cryptobranchus. In the remain- ing 6 cases, we used an approximation described by Yeager and Ultsch (1989) to determine Po (e.g. Figs. 1 A - B, and 2).

Ecological 0 2 conformation and regulation. If the elimi- nation of ~rO2 data at POz's > 156 mmHg resulted in a single regression line fitting the data with a coefficient of determination (r 2) _>0.90, then we accepted that the animal was an ecological 02 conformer, in spite of the

fact that it could regulate g o 2 a t PO2's above air-satura- tion. By this definition, 2 of 11 Cryptobranchus were eco- logical 02 conformers (e.g. Fig. 3 B), and 2 more were very close with r2=0.88 (e.g. Fig. 2C), a reflection of the high Pc of hellbenders. No Necturus were ecological O2 conformers, as all had a Pc well below the PO2 of air-saturated water (Table 1), and were therefore able to regulate aquatic O2 uptake over a fairly wide range of naturally occurring 02 tensions.

Tolerance of severe hypoxia. Six hellbenders survived se- vere water hypoxia (9-10 mmHg, see above) in the respi- ration chambers for the entire 5 11 day observation pe- riod when there was an air pocket from which to breathe (Table 2). They were removed when it was evident that severe water hypoxia was not an immediate threat; how long they could have survived was not determined. They spent most of their time near the air pocket, did not struggle, often floated, and recovered without incident. Similar results were obtained for mudpuppies, which oc- casionally moved their gills back and forth rapidly, but also often held them retracted against the body. Mud- puppies did not float. When Necturus were returned to aerated water, the gills were maximally protruded and perfused, and were waved vigorously.

In aquaria open to the air with a moderately severe water hypoxia (22-30 mmHg), hellbenders air-breathed about 6 x more than mudpuppies of a similar size (Ta- ble 3). I-Iellbenders spent much more time at the surface, often floating for up to 10 rain, and occasionally swam vigorously at the surface as if attempting to escape. Mudpuppies were generally inactive, and were not seen to float.

Discussion

Rates of 0 2 cosumption. The VO2's reported here are for animals that have no access to air, and therefore could have been somewhat depressed relative to those of the same animals breathing both air and water (Ultsch 1976b), which means that the rates can not be unequivo- cally compared to those found by other workers. How-

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Fig. 1. Examples of the standard rate of 02 consumption (SMR) of submerged Necturus maculosus at 20 ~ C as a function of ambient oxygen tension. W (weight, g); Pc (critical 02 tension, mmHg); SMR calculated as rate in air-saturated water (S, 156 mmHg); m, rate of increase in ~'O~ with increasing PO2 in the zone of metabolic 02 conformity. Po determined as the intersection of two best-fit regression lines (C F), and occasionally as an apparent discontin- uity between the two best-fit regression lines (A B, see text)

ever, as the aerial components of the "s 2 of bimodally respiring Cryptobranchus (Guimond and Hutchison 1973) and Necturus (Guimond and Hutchison 1972) are small, metabolic rates reported by these authors can be qualitatively compared to those found by us. Using Qlo'S from these authors, and assuming that s is a function of W ~176 among salamanders (Gatten et al. 1990), our data can be compared to that of Guimond and Hutchison (1976). We found the VO2's for our sub- merged hellbenders and mudpuppies were 27% lower and 5% higher, respectively, than those of the bimodally respiring slamanders studied by Guimond and Hutch- ison (1976). Neither of these differences is great, consid- ering the differences in techniques and the approxima- tions used in the calculations.

Among amphibians, salamanders have a lower rate

of 0 2 consumption than anurans (Feder 1976) by about a third (Gatten et al. 1990). However, among salaman- ders, neither this study nor those of Guimond and Hutchison (1972, 1973, 1976) found the VO2 ofmudpup- pies or hellbenders to be especially low. Whether the 27% reduction of VO2 below the rough 'predicted ' value described above is real is equivocal. Such a depression can be viewed as adaptive for an animal with a relatively limited gas exchange capability, if it were real. But if our data for Cryptobranchus is scaled to the same body size as for our Necturus using a regression equation gen- erated from our Cryptobranchus data CQO2/W~~- 46.36 W -~ pl O2/g 'h and g, weight range 69-796 g), then a submerged hellbender of 124 g is predicted to have a VO2 7% higher than a mudpuppy, which is to say that there is no difference in the SMR of similarly sized animals of the the two species. Because of this observation and the above comments, it does not appear that hellbenders have an SMR that is below that of mudpuppies, and perhaps not even below that of sala- manders in general, although the intraspecific data on large species is sparse. Therefore the lack of gills and the relatively infrequent use of lungs under resting condi- tions do not seem to inhibit the SMR of hellbenders, cutaneous respiration being sufficient to maintain a nor- mal SMR because of the skin folds, dorsoventral flatten-

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255

Table 2. Tolerance of Necturus maculosus and Cryptobranchus alle- ganiensis to hypoxic water (9-10 mmHg) at 20 ~ C with access to air. All animals were removed alive (and subsequently recovered) at the end of an experiment. Data are ~_+ SE (range)

Species n Weight Minimal survival (g) (days)

Necturus 5 88 _+ 11 6.4 -+ 1.2 maculosus (60-123) (5-11 ) Cryptobranchus 6 89 _+ 15 6.5 _+ 0.9 alleganiensis (44-152) (5 11)

Table 3. Surfacing frequency of similarly-sized individuals of Cryp- tobranchus alleganiensis and Necturus maculosus in hypoxic water (PO2 = 22-30 mmHg) at 20 ~ C. Data are i _ SE (range)

Species Number of Weight (g) Surfacing observations (n = 3) frequency

(surfacings/h)

Cryptobranchus 30 83_+12 47.13_+4.48 alleganiensis (67-107) (4-88) Necturus 33 85 _+ 17 * 7.79 _+ 0.75 maculosus (60-118) (2-16)

* Significantly different from Cryptobranchus

ing, and the fact that the capillaries reach into the epider- mis (Noble 1925).

Critical 0 2 tensions and regulation of 02 consumption. We found Necturus maeulosus to be capable of regulation of VO2 over a range of PO2 extending down to approxi- mately 30% of normoxic values, in contrast to Shield and Bentley (1973), who found ~rO 2 to increase linearly with P O 2 up to 150 mmHg at 20 ~ C and therefore con- cluded that mudpuppies were metabolic O2 conformers. As these investigators reported metabolic rates at 150 mmHg about twice those we and Guimond and Hutchison (1972) found, the conflicting conclusions may be the result of activity in their animals. Miller and Hutchison (1979), using mudpuppies at 5 and 15 ~ C also found that that Necturus could regulate ~'O2 down to at least 40 mmHg.

The Pc of several fish species, as determined in a similar system at 20 ~ C, is near 20-30 mmHg (Ott et al. 1980; Ultsch et al. 1981). Although there was trend in the salamanders toward an increase in Pc with body size, it was not significant for either species (P = 0.08 for Nec- turus and 0.15 for Cryptobranchus). At 40 mmHg, the Po of Necturus is somewhat higher, but considering the much more efficacious respiratory system of fishes, the ability of mudpuppies to tolerate hypoxia is rather im- pressive. The role of the external gills is evident, as the mean P~ for 4 hellbenders that were of similar size (69- 132 g) to the mudpuppies was 75 mmHg. In addition, the gills take up twice as much 02 as does the skin at 5-25 ~ C, and can account for up to 61% of total VO2 in animals breathing both air and water at 15 and 25 ~ C (Guimond and Hutchison 1972).

Responses and adaptations to aquatic hypoxia. Necturus is unarguably more adapted to hypoxia than Crypto- branchus, but mudpuppies may not be more tolerant of hypoxia than most other water-breathing animals such as fishes, as evidenced by the higher Pc of mudpup- pies. This means that they can not be assumed to be capable of inhabiting environments that become severely hypoxic unless they can gain access to the surface. In portions of the range where ice cover may occur for extended periods during overwintering, this would ex- clude this species from shallow ponds subject to anoxic winterkill, and in fact the most typical habitat for mud- puppies in northern areas is large lakes.

In more southern areas, there are several physiologi- cal adaptations that could allow mudpuppies to inhabit relatively hypoxic waters, although we stress that envi- ronmental measurements of the degree of hypoxia in habitats of mudpuppies are lacking. Mudpuppies have the highest blood 02 affinity yet found among salaman- ders (Pso = 10.6 mmHg at 20 ~ C, Weber et al. 1985). The low Pso is not only important because of its obvious value in enabling the animal to remove 02 from hypoxic water, but also because hypoxic water is often hyper- carbic (Ultsch 1973b, 1976b; Wakeman and Ultsch 1975). Necturus blood has a large Bohr shift (Weber et al. 1975), which could inhibit Oz loading from hyp- oxic-hypercarbic waters if the degree of hypoxia was large. The extremely low Pso can be viewed as adaptive to this Bohr shift, allowing a mudpuppy in hypoxic water to maintain an adequate saturation of cutaneous and gill efferent blood with a minimum of surfacing in spite of any hypercarbia, which could save energy and reduce the risk of predation. The survival for days when in water of very low PO2 (Table 2) when the ani- mals have access to air indicates that the lungs can be of crucial importance during periods of extreme hypoxia. Finally, the high value for AVO2/APO 2 at 02 tensions below the Pc would enable mudpuppies to pay back an 02 debt or recycle lactate to glucose (Miller and Hutch- ison 1979) with a much smaller increase in ambient P O 2 than would be the case for hellbenders.

While the adaptations of mudpuppies to hypoxia suggest that they could inhabit hypoxic waters, it is clear that hellbenders are not well-adapted to hypoxia and that this may be why they are limited to habitats that are normally well-oxygenated. However, this does not mean that Cryptobranchus has no defenses against a moderate hypoxia. A mean Pc of 90 mmHg (Table 1) means that hellbenders can maintain their SMR at a 40% reduction from normoxic PO2, which in turn im- plies that they must have some control over 02 transport across the integument. One response is to rock the body from side to side in hypoxia, and the frequency of this rocking has been shown to increase as PO2 falls (Beffa 1976; Harlan and Wilkinson 1981). Presumably this be- havior enhances diffusion by reducing the water bound- ary layer (Burggren and Feder 1986) as well as by renew- ing the water near the skin in a manner similar to the waving of the gills of Necturus during hypoxia. Rocking in hypoxic or hypercarbic water raises blood PO2 by 4-7 mmHg (Boutilier and Toews 1981b; Harlan and

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Table 4. Transcutaneous 02 condutance (GO2) of Cryptobranchus alleganiensis as a function of temperature, calculated from cutane- ous routine "VO2 and water to blood PO2 gradient

Temp aCutaneous bTranscutaneous Cutaneous Qlo (o C) ~'O2 PO2 gradient GO2

(I.tl 02/g" h) (mmHg) (gl 02/ g. h- mmHg)

5 8 . 1 5 (158-17)=141 0.0578 - 15 18.57 (157-17)= 140 0.1326 2.29 25 28.52 (155-15)=140 0.2037 1.54

" from Guimond and Hutchison (1973) b from Moalli et al. (1981)

Wilkinson 1981). Since the animals are usually operating on the steep portions of their rather sigmoidal 02 disso- ciation curves (Boutilier and Toews 1981 a; McCutcheon and Hall 1937), these relatively small increases in the PO2 of the blood can be significant in terms of maintain- ing 02 saturation levels (Boutilier and Toews 198] b).

There also appears to some control in hellbenders over both O2 demand and the cutaneous conductance of 02 (GO2). As temperature is increased from 15 to 25 ~ C, Cryptobranchus has the lowest Qlo of the giant salamanders (1.48 vs. 1.96 for Amphiuma means, 2.19 for Siren lacertina, and 2.38 for Necturus maculosus, cal- culated from Guimond and Hutchison 1976). Cutaneous conductance has been argued to be a relatively noncon- trollable parameter because the movement of gas across the skin is primarily diffusion-limited (see review of Feder and Burggren 1985). In Cryptobranchus this was found to be the case for CO2, where increases in aquatic VCO2 were due to increases in the transcutaneous CO2 gradient associated with increases in acclimation temper- ature (Moalli et al. 1981), with the result that GCO2 was constant over a temperature range of 5-25 ~ How- ever, if the same approach used by these authors to cal- culate GCO2 is used to calculate GO2, then GO2 is found to increase substantially with temperature with no increase in the transcutaneous 02 gradient (Table 4). This could be the result of cutaneous capillary recruit- ment that increases the effective surface area for O2 dif- fusion (Burggren and Moalli 1984; Feder and Burggren 1985), an effect that may not be shown for CO2 because of the much higher diffusivity of this gas (i.e. perfusing additional cutaneous capillaries may not change the ef- fective surface area for diffusion of CO2). Presumably the same response could be used as PO2 falls at a given temperature, thereby partially explaining the ability of hellbenders to regulate VOz over a naturally occurring range of 60 mmHg.

Circulatory considerations and the role of the lungs. As mentioned, the value of the lungs of both species as gas exchange organs has been questioned, and they have often been stated to serve mainly a hydrostatic role. The latter role seems unlikely, particularly for Cryptobran- chus, which typically is a bottom dweller that inhabits fairly fast-flowing water. Animals in such habitats tend

to lose their buoyancy devices (e.g. darters, a group of fishes typically found in streams, have no gas bladder; a number of stream-dwelling salamanders have lost their lungs). The lungs of both mudpuppies and hellbenders are relatively large (Guimond and Hutchison 1976), and pulmonary respiration greatly extends survival at low water PO2 (Table 2). The lungs of both species should therefore be viewed as accessory respiratory structures that can be of importance during bouts of hypoxia that might occur, albeit rarely, during periods of drought and high temperatures. In addition, lungs may be impor- tant in the recovery from exercise, because of the limited aerobic scope of activity of both species (Boutilier et al. 1980; Miller and Hutchison 1979).

Since the lungs apparently can serve a respiratory function, this raises the possibility of adaptive shifts in perfusion of the various gas exchangers. Necturus macu- losus has a fenestrated and incomplete intraatrial sep- tum, no spiral valve in the conus arteriosus, a divided truncus arteriosus, and an incomplete intraventricular septum (Baker 1949; Putnam and Dunn 1978). Because the pulmonary artery arises from the radix aorta, there would seem to be no pathway for getting blood directly from the heart to the lungs without it first passing over the gills. This is a circulatory pattern apparently unique to Necturus (Romer and Parsons 1986), and one which would not be beneficial while the animal is breathing air when in severely hypoxic water, as 02 would be lost to the water via the gills. However, there are several possible solutions to this problem. Mudpuppy gills can be actively protruded or retracted, and when they are retracted the arborization of the filaments is not evident, which suggests that perfusion is also limited. Both Baker (1949) and Darnell (1949) have described vessels that might function as gill bypasses, and Baker (1949) sug- gested that lung bypass was also possible. Therefore it is likely that the mudpuppy is able to selectively shunt blood to the appropriate gas exchanger depending upon the PO2 of the water.

Cryptobranchus has a cardiac anatomy that, like Nec- turus, suggests that it is primarily a water breather (Put- nam and Parkerson 1985). There are no gills, but Baker (1949) found extensive branching of the pulmonary ar- tery to the stomach, raising the possibility of gastric respiration. We have mentioned that this animal fre- quently floats (tail down) at the surface in hypoxic water, but we could not tell if the frequent inspirations were resulting in any swallowing of air.

Ecological conclusions. The hypothesis that Cryptobran- chus is found only in larger streams and small rivers with a substantial and permanent flow rate because this salamander has a limited ability to take up Oz is sup- ported by our results. This limited ability is evident in the very high Pc and in the extended time required to recover from lactacidosis caused by exercise (Boutilier et al. 1980). Also important is that the mountain-stream habitat characteristically has a nil PCO2. The buffer ca- pacity of hellbender blood is not great (Boutilier and Toews 1981 a), although it is not especially low for ec- totherms. More importantly, the lungs are probably not

257

efficient enough to reduce the water to blood PCOz gra- dient during hypercarbia-induced hypercapnia, although this point is not certain since the experiments that have been done with hellbenders in hypercarbic water did not permit them access to CO2-free air (Boutilier and Toews 1981 b). During recovery from hypercapnia, be it gener- ated from exercise or exposure to hypercarbic water, lung ventilations increase markedly (Boutilier et al. 1980; Boutilier and Toews 1981 b), which suggests that pulmonary respiration plays some role in COz elimina- tion under extreme conditions. It is unlikely, however, that the lungs are as effective in this role as in other giant salamanders that typically inhabit hypercarbic waters (Heisler et al. 1982). In summary, hellbenders may be viewed as animals that are living at the limits of their gas exchange capabilities, which restricts them to relatively cool and flowing waters with high levels of dissolved Oz and nil levels of CO2. Under such condi- tions they can maintain rates of gas exchange compara- ble to other salamanders, but are restricted to a life style of minimal activity.

In contrast, Necturus is much more tolerant of hy- poxia because of its highly developed external gills, smaller size, and very low blood P5o- Whether it is also tolerant of the hypercarbia often found in hypoxic waters is uncertain. We have found that this species can tolerate 4% CO2 (unpublished data), but Stiffier et al. (1983) found 3% to be lethal; in addition the latter au- thors found that Necturus did not compensate blood pH after 24 h of a hypercarbia-induced (2% CO~) respi- ratory acidosis, while larval Ambystoma tigrinum com- pensate 26% when exposed to 3% CO2 in water. Wheth- er Necturus is thus limited to areas where hypoxia is not accompanied by hypercarbia, if in fact it inhabits hypoxic areas at all, remains to be investigated.

Acknowledgements. Robert Guthrie collected most of the hell- benders. Joesph Arceneaux, Keith Blackmon, David Carroll, Bu- lent Cinkilic, Scott Cochran, Phillip Craft, John Fischer, Charles Gehrdes, Kevin Johnson, Leonard Jones, Douglas Lewis, Abbott Martinson, Arnold Mayberry, Edward Purdue, Rosalind Moore, Deborah Stephens, Michael Sullivan, Shaun West, and Christopher Wright provided technical help. The figures were prepared by Dar- yl Harrison. The manuscript was prepared while GRU was on sabbatical at the Department of Zoology at the University of Flori- da, and we are thankful to that department for support and the use of its facilities, and particularly to John Anderson for supplying space in an already crowded environment. This research was par- tially supported by the Research Grants Committee of The Univer- sity of Alabama.

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