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Biochemical Systematics andEcology, Vol. 12, No. 4, pp. 43,6-440,1984. 0305-1978184 $3.00+0.00 Printed in Great Britain. PergamonPressLtd. Variation of Individual Electromorphs in Microtuspennsylvanicus and Peromyscus leucopus TERRANCE DAVIN, RAYMOND P. MORGAN II and GEORGE A. FELDHAMER Appalachian Environmental Laboratory, Center for Environmental and Estuerine Studies, University of Maryland, Frostburg State College Campus - Gunter Hall, Frostburg, Maryland 21532, U.S.A. Key Word Index - Peromyscus leucopus; whita-footed mouse; Microtus pennsyivanicus; meadow vole; eiectrophoresis; albumin; tmnsferrin. Abstract - Blood samples were taken from individual meadow voles (Microtus pennsylvanicus) and white-footed mice (Peromyscus leucopus) every three weeks. Two serum proteins, albumin and transferdn, were examined using acrylemide eiectrophoresis and densitometry. In individuals of both species, electrophoretic mobility and banding pattern changed. Such changes have been documented in other proteins, and may reflect changes in the intamei or external environment of the indivkluals. Individual changes in serum proteins may seriously affect their interpretation, when used as genetic markers in population studies. Inl~ducl~on Various hypotheses have been presented to explain population cycles in microtines. Food supply [1], predation [2, 3], stress [4], behavior [5, 6 and others], and genetics [7, 8] have all been advanced as controlling factors. Over the past 20 years, much of the attention devoted to the study of population cycles has been on the hypothesis proposed by Chitty [9], in which changes in behavior are said to be caused by changes in the gene pool of the population. This hypothesis has promoted two broad types of population studies - behavioral and genetical. Field studies of behavior are somewhat difficult to conduct [10], so more studies have dealt with the genetics of popula- tions. • ~,o genetic loci, transferrin (Tf) and leucine aminopeptidase (LAP), have been investigated by several researchers [11-14]. In these studies, the frequencies of various alleles at both loci were examined in relation to population variables in- cluding density, mass of individuals at first capture, rate of survival, reproductive condition, rate of growth and age at sexual maturity. In all cases, individual genetic markers were assumed to remain constant. However, McGovern and Tracy [15] took blood samples from individual M. ochrogaster over time to determine if Tf and LAP remained constant within individuals. Of 41 (Received 10 December 1983) animals trapped and subjected to various environ- mental conditions, 26 (63.4%) showed changes in at least one of the two loci. Using the meadow vole (Microtus pennsyl- vanicus) and the white-footed mouse (Peromyscus leucopus), immediately after trapping and then during an extended period of holding [16], we studied Tf and albumin to determine if any changes in electrophoretic mobility would take place over time. Results StatisticalAnalyses We analysed primarily albumin and transferrin (see Experimental). Figure 1 shows an average densitometry graph, including albumin (peak B) and the two Tf peaks (peaks E and F- bend designations do not neces~rily correspond to electrophoretic designations of previous studies). The other peaks shown occurred in over 50% of the samples. These other peaks were not included in the statistical analyses, but were analysed for effects of heparin on serum proteins [17]. Two aspects of each peak were analysed: (a) the amount of protein in each band, represented by the height of each peak, and (b) the migration distance of each bend, equal to the distance from the beginning of the graph to where the highest point of the peak occurs. In order to analyse the graphs statistically, in- ternal standards were used to factor out variation 435

Variation of individual electromorphs in Microtus pennsylvanicus and Peromyscus leucopus

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Biochemical Systematics and Ecology, Vol. 12, No. 4, pp. 43,6-440, 1984. 0305-1978184 $3.00+0.00 Printed in Great Britain. Pergamon Press Ltd.

Variation of Individual Electromorphs in Microtus pennsylvanicus and Peromyscus leucopus

TERRANCE DAVIN, RAYMOND P. MORGAN II and GEORGE A. FELDHAMER Appalachian Environmental Laboratory, Center for Environmental and Estuerine Studies, University of Maryland,

Frostburg State College Campus - Gunter Hall, Frostburg, Maryland 21532, U.S.A.

Key Word Index - Peromyscus leucopus; whita-footed mouse; Microtus pennsyivanicus; meadow vole; eiectrophoresis; albumin; tmnsferrin.

Abstract - Blood samples were taken from individual meadow voles (Microtus pennsylvanicus) and white-footed mice (Peromyscus leucopus) every three weeks. Two serum proteins, albumin and transferdn, were examined using acrylemide eiectrophoresis and densitometry. In individuals of both species, electrophoretic mobility and banding pattern changed. Such changes have been documented in other proteins, and may reflect changes in the intamei or external environment of the indivkluals. Individual changes in serum proteins may seriously affect their interpretation, when used as genetic markers in population studies.

Inl~ducl~on Various hypotheses have been presented to explain population cycles in microtines. Food supply [1], predation [2, 3], stress [4], behavior [5, 6 and others], and genetics [7, 8] have all been advanced as controlling factors. Over the past 20 years, much of the attention devoted to the study of population cycles has been on the hypothesis proposed by Chitty [9], in which changes in behavior are said to be caused by changes in the gene pool of the population. This hypothesis has promoted two broad types of population studies - behavioral and genetical. Field studies of behavior are somewhat difficult to conduct [10], so more studies have dealt with the genetics of popula- tions.

• ~,o genetic loci, transferrin (Tf) and leucine aminopeptidase (LAP), have been investigated by several researchers [11-14]. In these studies, the frequencies of various alleles at both loci were examined in relation to population variables in- cluding density, mass of individuals at first capture, rate of survival, reproductive condition, rate of growth and age at sexual maturity. In all cases, individual genetic markers were assumed to remain constant. However, McGovern and Tracy [15] took blood samples from individual M. ochrogaster over time to determine if Tf and LAP remained constant within individuals. Of 41

(Received 10 December 1983)

animals trapped and subjected to various environ- mental conditions, 26 (63.4%) showed changes in at least one of the two loci.

Using the meadow vole (Microtus pennsyl- vanicus) and the white-footed mouse (Peromyscus leucopus), immediately after trapping and then during an extended period of holding [16], we studied Tf and albumin to determine if any changes in electrophoretic mobility would take place over time.

Results Statistical Analyses We analysed primarily albumin and transferrin (see Experimental). Figure 1 shows an average densitometry graph, including albumin (peak B) and the two Tf peaks (peaks E and F - bend designations do not neces~rily correspond to electrophoretic designations of previous studies). The other peaks shown occurred in over 50% of the samples. These other peaks were not included in the statistical analyses, but were analysed for effects of heparin on serum proteins [17]. Two aspects of each peak were analysed: (a) the amount of protein in each band, represented by the height of each peak, and (b) the migration distance of each bend, equal to the distance from the beginning of the graph to where the highest point of the peak occurs.

In order to analyse the graphs statistically, in- ternal standards were used to factor out variation

435

436 TERRANCE DAVIN, RAYMOND P. MORGAN II AND GEORGE A. FELDHAMER

1 4 0 0

1 2 0 0 L

.~ K B

.~ 8 0 0

"~ 60C Q. o0/

2O0

1 0 6 0 = ' ' ' ~ ' ' 8oo 6oo 4oo 2~o' 6 M o b i l i t y

FIG. 1. AVERAGE DENSlTOMETRY GRAPH SHOWING PROTEIN PEAKS THAT OCCURRED IN OVER 50% OF THE SAMPLES (A = albumin, E, F = transfen'in).

in staining time, destaining time, and length of electrophoretic run. The standards used to measure mobility were peaks A and L (Fig. 1), since those peaks occurred in all of the samples. Peak A was set as the 0 point, and peak L was set equal to 1000. All other mobilities were then measured relative to these standards. Thus, differences due to electrophoresis run time were eliminated. To eliminate the varying affects of staining using fast green [18] and destaining, a similar procedure was followed. Peak A was measured, and the height arbitrarily set equal to 1000. All other peaks were then measured relative to this peak.

After the establishment of peak and mobility standards, the albumin and Tf's were measured. Comparisons were made between the heparinized and non-heparinized samples for both species using Student's t-test [19]. Data for P. leucopus were analysed using a two-factor analysis of variance with replictation, with sex as the fixed factor and time as the random factor. Data for the M. pennsylvanicus were analysed using a one- factor analysis of variance with replication. Because data from only one female were available for analysis, sex was not included in the analysis, only differences due to time. Because only one factor (time) was analysed for data on M. penn- sylvanicus, interactions were not tested. Equal samples of P. leucopus were analysed (four males and four females), while four males and one fe- male M. pennsylvanicus were tested. Both Tf alleles (bands E and F, Fig. 1) for both species had several zeros in the sets of data, so a log

transformation [ ) ( '= log (X+ I ) ] was used [19]. In both P. leucopus and M. pennsylvanicus, the level of significance was P <0.05. If differences were found between the sample periods, Duncan's multiple-range test was used to determine where the differences occurred [20].

Peromyscus leucopus Heparinized vs. non-heparinized capillary

tubes. Fourteen P. leucopus were bled using both types of capillary tubes. The average mobility of the proteins was not affected by the presence of the heparin. The peak height of the proteins, usually affected by heparin [17], also was not in- fluenced by heparin.

While heparin did not affect the average mobility or peak height of the proteins, it did affect the results from individual animals. All proteins (B-K) were examined for changes. In 24 cases, various proteins were present in one of the treatments (either heparinized or non-heparinized), but com- pletely missing in the other treatment. Proteins D, F, H and K were lost primarily from heparinized samples, while proteins C, E, G and I were lost from the non-heparinized samples (Table 1). There was no apparent pattern in loss or retention of bands.

Differences due to sex. A two factor analysis of variance with replication was used to test for differences in mobility or peak height of albumin

TABLE 1. PROTEIN BANDS FROM PEROMYSCUS LEUCOPUS AND MICROTUS PENNSYLVANICUS LOST FROM PAIRED SAMPLES DUE TO TREATMENT

Treatment

Heparinized Non-heparinized capillary tube capillary tube

Peromyscus leucopus No. 2 I

10 F, K 21 K,H E,G,I 22 B,K J 28 D,K C 34 K B 35 F E 37 F E,E

Microtus pennsylvanicus No. 42 45 45

*For bands see Fig. 1.

VARIATION OF INDIVIDUAL ELECTROMORPHS IN MICROTUS PENNSYL VANICUS AND PEROMYSCUS LEUCOPUS 437

TABLE 2. DIFFERENCES BETWEEN SAMPLE PERIODS FOR MEAN MOBILITY AND PEAK HEIGHT OF ALBUMIN FROM PEROMYSCUS LEUCOPUS (N=8)

Date Average Date Average sample taken mobility* sample taken peak height*

Group 1 27 Aug 1981 42 27 Aug 1981 447 Group 2 15 May 1981 48 15 May 1981 604 Group 3 30 Apr 1981 53 5 Jun 1981 668 Group 4 26 Jun 1981 66 30 Apr 1981 754 Group 5 17 Ju11981 69 26 Jun 1981 1" 844

5 Jun 1981 70 17 Sep 1981 861 9 Oct 1981 872

Group 6 6 Aug 1981 82 17 Ju11981 1" 893 17 Sep 1981 85 9 Oct 1981 87 6 Aug 1981 896

*Average mobilitisa and peak heights are relative to an internal standard and have no units. t" Duncan's Multiple-Range test could not clsafly differentiate between the average peak heights in groups 5 and 6, end

Are somewhat arbitrary. the two groups

or the Tf's. For P. leucopus, there were no differ- encas in either mobility or peak height of the proteins due to sex.

Difference due to time. Albumin showed statis- tically significant differences over time for both mean mobility (F=3.06, P<0.005), and peak height (F=3.01, P<0.005). Differences in the mean mobility and peak height of the albumin bands and the six groups of peak heights and mobilitias established by Duncan's muitiple-range tests are given in Table 2. No temporal pattern was evident for either the mobility or peak height figures.

The first Tf allele (E) showed no changes over time for mean mobility or peak height of the pro- tein bands. However, the second Tf allele exhib- ited significant differences in both mean mobility

(F=2.85, P<0.025) and peak heights (F=2.93, P<0.025) (Table 3). Again, no temporal pattern was evident for either the mobility or peak heights.

Microtus pennsylvanicu$ Hepatinized vs. non-hepa~nized. Four M. penn- sylvanicus were sampled using both heparinized and non-heparinized capillary tubes. Neither average mobility nor peak height were changed by treating the samples with heparin. However, as with the P. leucopus, individual animals did exhibit changes. Two proteins (H, I) were lost from the non-heparinized samples, but appeared in the corresponding heparinized samples. One protein (K) was absent from the heparinized samples, but was present in the non-heparinized samples (Table 1 ).

TABLE 3. DIFFERENCES BETWEEN SAMPLE PERIODS FOR MEAN MOBILITY AND PEAK HEIGHT OF THE SECOND TRANSFERRIN ALLELE (BAND F) FROM PEROMYSCUS LEUCOPUS (N= 8)

Date Average Date Average sample taken mobility* sample taken peak height*

Group 1 5Jun 1981 65 5Jun 1981 70 Group 2 30 Apr 1981 132 30 Apr 1981 208

26Jun 1981 237 27 Aug 1981 296

Group 3 26 Jun 1981 195 15 May 1981 416 27 Aug 1981 198

Group 4 15 May 1981 321 17 Sep 1981 506 6 Aug 1981 331 6 Aug 1981 523

17 Sep 1981 360 17 Jul 1981 598 Group 5 17 Ju11981 461 9 Oct 1981 858

9 Oct 1981 485

"Average mobilities and peak heights are relative to an internal standard and have no units.

438 TERRENCE DAVIN, RAYMOND P. MORGAN 11 AND GEORGE A. FELDHAMER

TABLE 4. DIFFERENCES BETWEEN SAMPLE PERIODS FOR THE MEAN PEAK HEIGHT OF THE FIRST TRANSFERRIN ALLELE (BAND E} FROM MICROTUS PENNSYL VANICUS (N= 4)

Average Date peak height*

Group 1 17 July 1981 506 Group 2 6 August 1981 579 Group 3 17 September 1981 795

*Average peak heights are relative to an internal standard and have no units.

Differences due to time. Only the first Tf (E) allele showed significant differences over time in the mean peak height values (F= 6.72, P< 0.01 ). Peak heights of this Tf band increased from lowest to. highest through time (Table 4). The other proteins showed no significant differences over time for either mobility or peak height.

Individual Banding Differences for Both Species During the course of the study, various protein bands disappeared and reappeared within indivi- duals. The most important of these changes occurred in the Tf bands E and F. Because bands E and F are alleles of the same protein, changes in these bands were considered together. Changes

1 0 0 0 I

500 I

0

500

0

5OO

0

500

0

5OO

0

50O

1000

./%

750 500

M o b i l i t y

9 Oct

i t

1 7 Sep

6 A u g

i i

17 Jul

b - .~

26 June

5 J u n e

i J

3 0 A p r

2;0 6

FIG. 2. TRANSFERRIN REGIONS OF DENSITOMETRY GRAPHS OF P. LEUCOPUS ~ 35 SHOWING CHANGES IN MOBILITY AND PEAK HEIGHT THAT OCCURRED OVER SAMPLING PERIOD.

in the Tf bands occurred in 3 of 10 individual M. pennsyIvanicus, and in 11 of 13 P. leucopus. The most dramatic of the changes occurred in one of the P. ieucopus. Animal ~f 35 was a female, and was kept in captivity from 30 April to 9 October 1981. During that time, seven blood samples were analysed for changes. Of the seven samples taken, none of the Tf regions remained the same (Fig. 2) for two consecutive sample periods. This animal exhibited bands E and F, and at least one, and possibly two additional bands in the Tf region. Both of the new bands had slower mobilities than band F, with the one definite band occurring closest to band F. This new band occurred in two of the seven samples (17 September and 9 October). The other new band occurred as a shoulder on the first new band, and only occurred in the sample from 9 October. Of six possible changes that could have occurred, six took place. In the other individuals in which changes took place in the transferrin region, the number of changes was quite variable (Table 5). Changes occurred in all of the bands except A and L, which were constant in all of the samples. No animal that was sampled more than once had an overall banding pattern that remained constant.

Discussion The Chit'b/hypothesis of population regulation is based on behavioral changes that involve the aggressiveness of individuals resulting from rapid changes in the genetic composition of the popu- lation [9]. In studies that look for genetic changes in populations, attempts are made to correlate allelic frequencies with certain phases of the population cycle. It has always been assumed that the mobility of marker alleles are fixed within individual animals. We found that such alleles are not fixed. If the mobility of an allele is variable, it may be mistaken for another allele. Our results indicate that in some cases there are significant differences in the mobility of alleles over time. In P. leucopus, mobility of the albumin and the second Tf allele (band F) changed over time. In M. pennsylvanicus, there was no difference over time in the mobility of the two proteins examined. However, the fact that no differences occurred may be due to the small number (3) of time periods examined. While the samples from the P. leucopus spanned seven months (April to October), the samples from the M. pennsylvanicus

VARIATION OF INDIVIDUAL ELECTROMORPHS IN MICROTUS PENNSYL VANICUS AND PEROMYSCUS LEUCOPUS 439

TABLE 5. CHANGES IN TRANSFERRIN ELECTROMORPHS OF INDIVIDUAL PEROMYSCUS LEUCOPUS AND MICROTUS PENNSYL- VANICUS DURING APPROXIMATE 3-WEEK INTERVALS

Microtus Date Peromyscus leucopus No. pennsylvanicus No.

sample taken 8 10 21 22 28 34 35 37 42 46

4 Nov 1980 F F E F 11 Dec 1980 F F F F E 30 Dec 1980 E E F E E 22 Jan 1981 E E E E E 12 Feb 1981 F ElF ElF E E 6 Mar 1981 E E E E E

28 Mar 1981 E E ElF E E 23 Apr 1981 E E E E E 15 May 1981 ElF ElF F E E 5 Jun 1981 E E F E E

28Jun 1981 E E/F E E E 17 Ju11981 F F E/F E E/F 6 Aug 1981 E F E/F E E/F

27 Aug 1981 ElF F E E 17 Sap 1981 F F E/F F E 9 Oct 1981 E F F F F

F F F E F E F E E E F F F F E E/F F

F E F I F F FI/F II F

E E/F F E/F F E/F F F

E

Single lette~ denote e homozygotal double letters a heterozygote. Individuals which had no changes in Tf or for which sampling periods are minimal are not included. Blank spaces in columns occur when samples were not taken.

covered only three months (July to September). If a population were examined at several monthly intervals for differences in alleiic frequencies at these changing proteins, possibly a significant difference may have been noticed, when in fact the same alleles would have been present. In some individuals even more drastic changes took place, with a possibility of four alleles being repre- sented in one animal over a period of six months.

Changes in peak height, while not as critical as changes in mobility, can affect the observed presence or absence of a protein. Peak height on a densitometry graph is equivalent to protein density on an acrylamide gel. If the density is low enough, the band may not be visible to the naked eye, and it may be scored as absent. There is evidence that the amount of protein in blood serum can change over time. In several rodent species, the amount of serum proteins changed regularly throughout the year in species that hibernate [21].

There is even more evidence that changes can occur in protein mobility. Hemoglobin changes occur between prenatal and adult deer mice (Peromyscus maniculatus) [22]. Tegelstrom [23] found that by treating liver esterases of the house mouse (Mus musculus) with various substances, the number of bends increased from 12 to 30. Changes in the number of bands of leucine

aminopeptidase in humans are associated with pregnancy and several blood disorders [24],

Transferrin has two branching chains, each terminating in a sialic acid residue [25]. It is a binding protein that can combine with two atoms of ferric iron (Fe 3.), and transport them to bone marrow and other storage organs [26]. The electrophoretic mobility of "If is related to sialic acid concentration, bicarbonate ion concentra- tion, neuraminidase concentration, and the number of iron atoms bound to each Tf molecule. Ih the presence of neuraminidase, sialic acid residues are removed from Tf in a stepwise fashion. As sialic acid residues are removed, there is a gradual slowing of the electrophoretic mobility of the Tf [26]. This decrease in mobility may be caused by either a change in the shape or charge of the molecule [26]. Neuraminidase is found in individuals with influenza [25] and in diphtheria toxin [27]. Any other bacteria that produce neuraminidese and were present in an organism could cause changes in the mobility of Tf.

The number of iron atoms bound to Tf can also change the charge and shape of the molecule [28]. Because it can bind two atoms of iron, there are three forms of Tf that can exist; iron free, one iron atom, or two iron atoms attached. Commer- cial starch used in electrophoresis contains suffi-

440 TERRANCE DAVIN, RAYMOND P. MORGAN II AND GEORGE A. FELDHAMER

cient iron to bind two atoms of iron to every Tf molecule [26]. Some types of bacteria are known to compete with Tf for iron in the blood [25, 29].

The third variable that may affect the electro- phoretic mobility of Tf is the concentration of bi- carbonate ion in the blood. For a Tf molecule to bind with the iron atoms, an anion must bind at the same time [30]. Bicarbonate ions form the most stable bonds with Tf, and help bind the iron atoms more strongly than any other anions [31]. While the concentration of bicarbonate ion is not as serious as changes in the other variables, in conjunction with the other variables, it could result in a change in the mobility of Tf within indi- viduals.

Experimental Trapping of live animals was conducted on three sites near Frostburg, Allegany County, Maryland (39040 ' N lat. 78o55 ° W long.). Nine M. pennsylvanicus and 19 P. leucopus were captured on 3-13 November 1980; two M. pennsyl- vanicus were captured on 23-27 March 1981; 10 M. pennsyl- vanicus and one P. leucopus were captured on 4-20 July 1981.

Blood samples were taken from the sub-orbital sinus using a non-heparinized capillary tube. They were centrifuged for 5 rain to separate serum from plasma. The serum component was frozen at - 70 ° until analysed.

After the initial blood sample was taken, animals were transferred to 21 cm x 21 cm x 24 cm holding cages. Food and water were provided ad libitum. Food consisted of a mixture of one part whole corn, and three parts 15% protein pallets. Once a week carrots were added to the diet [16].

Blood samples were taken from each animal every 3 weeks. Samples were taken in the mornings so that possible circadian influences would not affect the results. On occasion, two samples were taken from an animal on the same day. The first sample was drawn using a non- heparinized capillary tube, and the second sample was drawn using a heparinized capillary tube. Heparin may affect plasma proteins [17], and this section of the experiment was performed to assess the affects of heparin on proteins.

Electrophoretic procedure. Serum was analyzed via column-gel electrophoresis using polyacrylamide as the support medium. An 8% acryiamide gel was used in a Tris- glycine buffer system.

In the electrophoresis apparatus (Pharmacia GE-4), gels were covered with 3 I. of Tris-glycine buffer. A current of 2 mA/gel was then passed through the system for 20 min to remove any excess persulphate and by-products of polymer- ization from the gels [18]. A micro-syringe was then used to add a 5/JI sample of serum to each gel. A current of 5 mA/gel was then passed through the system for 1 ½ h. Fast green was used as the primary stain because of its use in densito- merry work [18]. If enough serum was available from a sample for more than one gel, the extra gels were stained with buffalo black. These gels were then compared with the gels stained in the fast green to ensure that both stains showed the same protein bands. Gels remained in the stain

for 6 -8 h, and then were destained. After destaining, gels were placed in the test tubes, covered with distilled water, and stored for later analysis.

Densitometry procedure. Gels stained in fast green were analysed on a Beckman Model 25 Spectrophotometer. Light with a wavelength of 540 nm was passed through a slit with a width of 0.1 mm. One densitometry graph was produced for every blood sample taken.

A c k n o w l e d g e m e n t s - W e would like to thank Dr. Mike McGovem, Dr. Chrismarie Baxter and Dr. C. Alan Miller for helpful comments on the manuscript. Dr. Miller also aided substantially in statistical analyses. Ms. Fren Younger prepared the figures. This is Scientific Series No. 1510-AEL, Appalachian Environmental Laboratory, Center for Environ- mental and Estuarine Studies, University of Maryland.

References 1. Pitelka, F. A. (1958) Cold Spring Herb. Symp. Quanr.

Biol. 22" 237. 2. Elton, C. S. (1924) Br. J. Exp. Biol. 2, 119. 3. Pearson, O. P. (1971) J. Mammal. 52,41. 4. Christian, J. J. (1950) J. Mammal 31,247. 5. Krebs, C. J. (1964) Arctic Inst. N. Amer. Tech. Paper No.

15. 104 pp. 6. Christian, J. J. (1971) Biol. Reprod. 4,248. 7. Chitty, D. (1960) Can. J. Zool. 38,99. 8. Chitty, D. (1967) Proc. Ecol. Soc. Aust. 2, 51. 9. Krebs, C.J. (1978) Can. J. Zoo/. 56,2463.

10. Krebs, C. J. (1970) Eco/ogy§l, 34. 11. Tamarin, R. H. and Krebs, C. J. (1969) Evo/ution23, 183. 12. Gaines, M. S. and Krebs, C. J. (1971)Evo/udon 25, 702. 13. LeDuc, J. and Krebs, C. J. (1975) Can. J. Zoo/. 53, 1825. 14. Kohn, P. H. and Tamarin, R. H. (1978) Evo/u~'on32" 15. 15. McGovem, M. and Tracy, C. R. (1981) Oecologia 51,276. 16. Dietarich, R. A. and Preston, D. J. (1977) Lab. Anim.

Sci. 27, 494. 17. Jacques, L. B. (1978)Science298,526. 18. Gaal, O., Medgyesi, G. A. and Vereczykey, L. (1980)

John Wiley, England. Zar, J. H. (1974) Prentice-Hall, Englewood Cliffs, NJ. Walpole, R. E. and Myers, R. H. (1972) MacMillan, New York. Wenberg, G. M. and Holland, J. C. (1972) Comp. Biochem. Physiol. 462A, 989. Maybank, K. M. and Dawson, W. D. (1976) Biochem. Genet. 14, 389. Tegeistrom, H. (1975) Comp. Biochem. Physiol. 50B, 177. Scandalios, J. G. (1967) J. Hered. 58, 153. Gottshalk, A. (1966) Elsevier, Amsterdam. Putnam, F. W. (ed.) (1975) in The Plasma Proteins-- Structure, Function and Genetic Control Vol. 1, pp. 265- 315. Academic Press, New York. Poulik, M. D. (1961) Clin. Chem. Acta 6, 493. Root, W. S. and Bedin, N. I. (1974) Blood. Vol. V. Academic Press, New York. Kluger, M. and Rothenburg, B. A. (1978) Science 293, 374. Rogers, T. B., Borresen, T. and Feeney, R. E. (1978) Biochemistry 17, 1105. Rogers, T. B., Feeney, R. E. and Meeres, C. F. (1977) J. Biol. Chem. 252, 8108.

19. 20.

21.

22.

23. 24. 25. 26.

27. 28.

29.

30.

31.