Transitional Range of Temperature, Pivotal Temperatures and Thermosensitive Stages for Sex Determination in Reptiles

Transitional range of temperature, pivotal temperatures and thermosensitive stages for sex determination in reptiles

N. Mrosovsky1, C. Pieau2 1 Departments of Zoologyand Psychology,University of Toronto, Ontario, Canada M5S 1A1. 2 Institut Jacques Monod, CNRS et Université Paris 7, 2 placeJussieu, 75251 Paris Cedex 05, France.

Abstract. The function relating phenotypicsex ratio to incubation temperature in reptiles can vary in a number of ways in addition to simpledifferences in the temperatures giving 50% of each sex. This paper offersterminology and definitionsfor describingthese relationships.These definitionsaccomodate interactions between genetic and environmentaleffects on sexual differentiation,and variability within popula- tions. The paper also discussesthe concept of a sensitive stage/periodwithin incubation during which temperature can affect the direction of sexual differentiation.Thermosensitive period has previouslybeen assessedin a variety of ways. A suggestionfor a more generalway of definingthermosensitive stage/period is made.

The effects of temperature on sexual differentiation of the gonads in reptiles is a topic of interest not only to those studying mechanisms of sexual determination but also to evolutionary theorists, ecologists, wildlife managers and conservationists. Perhaps as a result of these differing approaches, and also of the recency of this area as a research field, terminology is sometimes used loosely or in different ways. In this paper we attempt to define some commonly used terms and to discuss their usefulness and limitations in the context of interactions between environmental and genetic factors determining sexual phenotype.

Transitional range of temperature and pivotal temperatures

Operational definitions and alternative terms

In many species of reptiles, the incubation of eggs at some temperatures yields 100% phenotypic males, whereas 100% phenotypic females are obtained at other temperatures (reviewed by Bull, 1980, 1983; Pieau, 1985; Raynaud and Pieau, 1985; Deeming and Ferguson, 1988). Both sexes may be obtained only in a range of temperatures, often narrow, between these male- and female-producing temperatures. Within this range, the "pivotal temperature" has been defined as that temperature 170

Figure 1. Pivotal temperatures derived (vertical dotted lines) from relationshipsbetween sex ratio and incubation temperature: a) case with one pivotal temperature, b) case with two pivotal temperatures. during incubation at constant temperature which gives 50 % individuals of each sexual phenotype (Mrosovsky and Yntema, 1980; Yntema and Mrosovsky, 1982; fig. 1a). This definition is entirely operational. It simply provides a term for describing one aspect of the results when constant temperature incubation experiments are performed. Other terms have also been used, for example threshold temperature" (Bull, 1980) and "critical temperature" (Pieau, 1976). The term "pivotal" is preferred because it is more vivid and because "threshold" is widely used in other contexts. The term "critical temperature" has priority, but may lead to confusion because "critical" is sometimes applied to the period or interval during which egg temperature affects sexual differentiation. Therefore the term "pivotal", already quite widely used, is retained. In some species, such as Chelydra serpentina (Yntema, 1976), two pivotal temperatures have been found (fig. 1b). In experiments with eggs incubated in a number of different incubators, each at a constant temperature, it often happens that none of the chosen temperatures produces exactly 50% of each sex. In these cases the pivotal value should be obtained by inter- polating between the values of the sex ratio on each side of the 50 % level. This pro- cedure is different from that used for Caretta caretta by Limpus et al. (1985). They fitted a curve to all the data points and from this curve they calculated the temperature that would produce 50% females (the SDT 50)' An example illustrates how the two methods can come up with different values (fig. 2). At 29° C only 40% of the hatchlings sexed from Heron Island clutch # 2 were female (Limpus et al., 1985). A 50% value, the pivotal, would therefore occur at a temperature higher than 29.0° C. Curve fitting, however, gave a SDT50-value of 28.9° C. This was obtained because even at temperatures as low as 27.0° C, production of females did not fall to zero.

Variation in pivotals temperatures

Pivotal temperature cannot be determined for a single egg. Pivotal is therefore a term applicable only to a number of eggs. This number might comprise a single clutch. It 171

Figure 2. Sex ratios of samplesfrom a clutch of loggerheadeggs as a function of incubation temperatures (crosses).Heavy dashed line showsthe SDT50as used by Limpus et al. (1985);this is based on a curve fitted to all the data points (crosses).Lighter verticaldashed line showsthe pivotal temperature as described in the text; this is derivedby interpolationbetween the twodata pointson either side of the 50% femalelevel. should not be assumed that pivotal temperatures for individual clutches necessarily provide a good estimate of pivotals for a larger sample. For example, the pivotal temperature in Emys orbicularis has been estimated to be 28.5° C (Pieau, 1976). How- ever, incubation of different clutches at 28.5 + 0.2° C showed that, although most clutches produced both males and females (in various proportions), some gave 100 % females and others 100% males (Zaborski et al., 1988 and unpublished results). A clear demonstration of inter-clutch variability in pivotal temperature comes from an experiment in which eggs from clutches of two different loggerhead turtles were placed in alternate positions within the same incubators, thus controlling for any spatial variation of ambient temperature within the incubators. Pivotal temperature differed by > 1°C between these two clutches (fig. 3).

Figure 3. Sex ratios in samplesfrom two clutchesof'loggerhead turtles incubatedsimultaneously in the same set of constant temperature incubators. Vertical dashed lines indicate pivotaltemperatures for each clutch (adapted from Mrosovsky,1988).

Possible causes of variations in pivotal temperatures: sexual genotypes

The main source of variation in pivotal temperatures, as defined operationally, appears to be genetic, although environmental sex determination (ESD) and genotypic sex determination (GSD) have sometimes been treated as if they were alternative 172 mechanisms that did not operate simultaneously (Bull, 1980). Work on salamanders has shown that genetic sex, as indicated by enzyme markers and heteromorphism of lampbrush sex chromosomes, operates at temperatures of 20° C giving 1:11 sex ratios. At 30-32° C sex ratio biases occur. That genetic sex is overridden at higher temperatures is evident from experiments in which larvae from such sexually reversed animals are reared at 20° C. The sex ratio of the offspring can be accurately predicted by Mendelian principles (reviewed by Dournon et al., 1990). For example, in Pleurodeles poireti, the mating of phenotypically reversed neo-females (ZZ) with standard males (ZZ) results in all male progeny if the larvae are reared at 20° C. Such demonstrations show that temperature can interact with genotypic sex determination and modify sexual differentiation of the gonads. That both genetic and epigenetic factors can operate simultaneously may be inferred from experiments with freshwater turtles, Emys orbicularis (Zaborski et al., 1988). When eggs are incubated at or close to the estimated pivotal temperature (28.5 5 0.2° C), blood cells type H-Y negative in phenotypic males whereas they type H-Y positive in most phenotypic females, indicating that at this temperature sexual phenotype generally conforms with expres- sion of H-Y antigen and therefore with sexual genotype (H-Y- males ZZ; H-Y + females ZW; Zaborski et al., 1988). It appears from this last finding that, in the range of temperature giving both sexes, the response is different according to the sexual genotype of individuals. Thus, in Emys orbicularis, the ZZ, ZW and WW individuals would have somewhat different pivotal temperatures (fig. 4). Therefore, if one tested a clutch of eggs that happened to be mostly genotype females, it would have a different pivotal temperature from one that happened to be mostly genotypic males.

Limitations to usefulness ofpivotal temperature values

Genetic contribution to the pivotal value, operationally defined, limits the usefulness of this measurement, but it does not make it useless. Of course, values derived from a small sample (e.g., a clutch) may not necessarily be a good reflection of values based on much larger samples. In fact, however, in a number of experiments, pivotals do not vary greatly between clutches. This is despite the fact that the types of incubators used in these experiments often do not hold temperatures constant with an accuracy more than + 0.5° C. Yntema (1976) gave data on 33 clutches of snapping turtles. ° The two pivotals, although not determined clutch by clutch, were clearly close to 21 C and 28° C for most of them. Mrosovsky (1988) found that pivotals of 6 clutches of loggerhead turtles were within about 2° C of each other. Samples of hatchlings of this species taken from 45 clutches in the field were more than 90 °?o female (Mrosovsky and Provancha, 1989). Wide variation in pivotal temperatures between individual clutches would have been unlikely to produce such a result since for much of the season the mean sand temperatures were only about 2° C above pivotals obtained in laboratory studies on a few clutches (Mrosovsky, 1988). Had the pivotals of some of 173

Figure 4. Transitional range of temperature (TRT) and pivotal temperatures (vertical dashed lines) for Emysorbicularis. Pl, P2 and P3 representthe pivotalsthat might be expectedfrom WW, ZW and ZZ samples, respectively.1m shows the limit belowwhich 100% of the embryosare masculinized;If showsthe limit above which 100% of the embryos are feminized.

the large sample of clutches from the field been 2° C lower, then more males should have been produced. In European pond turtles from Brenne, out of 53 clutches studied, pivotals below 28.25° C or above 29.25° C have not been found (Pieau, 1976 and unpublished results). Therefore specification of a pivotal value, even derived from a modest sample, can be a valuable guide both for experiments, or for wildlife manage- ° ment, when production of a particular sex is desired. In nature, however, even a 0.5° C difference in population values for pivotal temperatures could make considerable differences to the sex ratios produced. In attempting to correlate pivotal values with geographical distribution, or with other influences on the thermal environment, extrapolations from a few laboratory measurements of pivotals should only be made with caution, especially considering that temperatures within largish incubators often vary by 0.50 C. Smaller differences in pivotals may not be detectable with most pres- ent day technology, but acting cumulatively over many years could considerably influence demographic structure in the wild.

Other aspects of temperature-sex ratio relationships

Pivotal temperature describes only one aspect of curves relating incubation temperature to sex ratio: the point where the curve passes through the 1:11 sex ratio. So far the discussion has proceeded as if only this point varies (i.e., as diagrammed in fig. 4), without other changes in the shape of these curves. This is a simplification because there are numerous different possible curves relating temperature to sex ratio and numerous different ways in which these could vary. A number of these will now be considered, though of course it is recognized that further combinations are possible.

High and low temperature asymptotes and asymmetries

Figure 5a represents a situation where 100% phenotypic females are produced at high temperatures, but at low temperatures asymptotic values are not as extreme. Some- 174

Figure 5. a) showsdefinition of high and low temperatureasymptotes for valuesof sex ratios. b-d: various ways in which the shape of the function relating sex ratio and temperature may change without a change in pivotaltemperature (vertical dashed lines). In b) both high and low asymptotesdiffer from 100 and 0%. The horizontaldashed line showswhat might be expectedif GSD prevails. In c) only the low temperature asymptotehas changed. In d) both asymptotesremain unchanged, but the steepnessof the functiondiffers. thing approaching this type of curve sometimes occurs in sea turtles, with a few females occurring even at temperatures several ° C below the pivotal (Limpus et al., 1985; Mrosovsky, 1988). It has to be stressed again that incubators in laboratory experiments are seldom perfectly constant. Brief excursions from low to high temperature may be more important than the reverse type of change, because more developmental time occurs at high temperatures (see Pieau, 1982; Bull, 1985). There- fore, to conclude that there is a real asymmetry, females should be detected at temperatures sufficiently below the pivotal to be sure that excursions at the upper end of normal variations do not bring temperatures into the female zone. Both high and low temperature asymptotes may differ from 100 % female and 100 % male (fig. 5b). It may be speculated that these types of curves reveal strong genetic influences, since a perfectly flat sex ratio-temperature relationship is what prevails in GSD.

Variations without changes in pivotal temperatures

From the considerations above it is clear that the relationships between temperature and sex ratio could vary without altering pivotal value. Figure 5c diagrams one 175 possibility. Whether such variation exists between clutches, and if so, what it might derive from, is unknown. Another type of variation without change in pivotal concerns the steepness of the curve (fig. 5d). The pivotals of loggerhead turtles (Mrosovsky, 1988) and of leather- back turtles (Rimblot et al., 1985; Rimblot-Baly et al., 1986-1987) are close, within about 1 ° C for those clutches tested so far. Yet the steepness of the curve is much greater in leatherbacks. The change from 100°?o male to 100?/o female occurs over < 1 ° C. In loggerheads it requires larger temperature changes to produce this effect (cf. fig. 3). It may be wondered whether flatter curves reflect a greater influence of genetic factors controlling sex determination.

Figure 6. Estimationof transitionalrange of temperature('rR'I'). Dashed vertical lines showestimate that might be obtainedwith moderatesample sizes. Solid vertical lines showhow estimate ofTRT might increase with large sample sizes. 1mand It define limits beyond which asymptotic temperatures are reached: at temperatures lower than 1mthere is no further increasein the % of males; at temperatures higher than 1, there is no further increase in the % of females. Shorter vertical dashed line (rnarked P) shows pivotal temperature.

Transition ranges of temperature

Because of the numerous ways in which sex ratio may vary as a function of temperature, specification of a pivotal temperature, though a natural beginning, and sometimes useful in describing a particular parameter of interest, is not adequate in itself. As seen above, different pivotals can be found for different clutches, probably mainly because of variations in the proportions of genotypic males and genotypic females in the samples. Therefore, delimitation of a transitional range of temperature (TRT) beween male- and female-producing temperatures is required to characterize a population. Ideally it should be based on experiments involving a large number of clutches, though it can be approximately determined with small samples. The TRT is bounded by two limits: the limit for maximum masculinization (100% individuals in most cases) and the limit for maximum feminization (1m and If, fig. 4 and fig. 6). There may be changes in the estimation of the transition range of temperature in that 176

studying larger samples might result in widening of this range (solid lines in fig. 6). Outside the 1m and If thresholds, asymptotic sex ratio values always occur - or have always occurred in all experiments to date.

Thermosensitive periods/stages

It has been found that temperature changes affect the sexual differentiation of the gonads only during a certain period in the incubation of reptile eggs. This period generally corresponds to the first steps oF histological differentiation of the gonads (Pieau and Dorizzi, 1981), somewhere at the end of the first third and beginning of the second third of embryonic life. To specify when the thermosensitive period is, and so discover with what embryological stages it is associated, two types of experiments have been performed. In the first type of experiment, eggs were shifted once, at different stages of embryonic development, from a male-producing temperature to a female-producing temperature (or vice versa) for the remainder of development. In the second type of experiment, eggs were submitted, for one to several days, to pulses of a masculinizing temperature on the background of a feminizing temperature (or vice versa). Sex ratio in the different experimental series was established at hatching or around hatching. On the basis of these experiments different authors used different criteria to delimit the thermosensitive period. Pieau and Dorizzi ( 1981 ) defined this period as the minimal duration of exposure at a male- or a female-producing temperature which results either in 100% phenotypic males or in 100% phenotypic females. Yntema and Mrosovsky (1982) did not use this criteria but based their thermosensitive window on the period during which temperature manipulations altered sex ratio but not necessarily to the 100% value. Bull and Vogt (1981) distinguished between a primary and a secondary temperature-sensitive period. The primary sensitive period included all stages in which sexual phenotype is irreversibly determined; regardless of temperatures experienced prior to this, sexual differentiation of gonads is controlled by temperature in this period. Incubation temperature prior to the primary period can also influence gonadal phenotype but not irreversibly. The secondary temperature-sensitive period corresponds to these prior stages. However, it is arguable whether these distinctions are of much use because the duration of the primary sensitive period depends on the particular temperature values used. For example, for female differentiation in snapping turtles, stages 14-19 are sensitive if 30° C is used as the female-producing temperature. However, if 20° C is used as the female-producing temperature, stages 14-16 are temperature sensitive; 26° C was used as the male-producing temperature in both cases (Yntema, 1979). Deeming and Ferguson (1988) argue against the concept of a thermosensitive period. They propose (but have not yet proved) that for alligators sufficient doses of some gene product are required to produce males whereas the default condition is to be female. The necessary dose may be reached rapidly if high temperatures prevail 177

(in alligators high temperatures yield phenotypic males). If temperatures are not so high, then it takes longer. Deeming and Ferguson suggest that there is no unique developmental time when these events must occur. It remains true, nevertheless, that temperature shifts during the first few days or the last few days of embryonic development have no effects on sex ratio. Clearly there are periods not sensitive to temperature. The general point here is that the sensitive period obtained varies with the experimental procedures, and with the criteria employed. With extreme temperature manipulations, temperature can have effects at times at which lesser manipulations would be ineffective. Thus, in Emys orbicularis, 35° C is more feminizing than 30° C (Pieau, 1982). Given these complexities, there seem to be two options. The first is to recognize that the sensitive period does not exist. There are a number of sensitive periods for different procedures. These periods then can be specified only operationally with reference to the particular procedures and criteria employed. The second option is to use descriptions analogus to the transitional range of temperature (see above), but for the temporal dimension. Thus, a sensitive period could be defined by exclusion as the period between those times (or stages of development) when no temperature manipulation whatsoever has been found to have any influence on sex ratio. This leaves open the possibility that synthesis of some gene product, acting with a dose-effect (above a certain threshold) on gonadal differentiation, could be activated in different ways. Sufficient amounts could be reached either over a continuous production (in the case of incubation at constant temperature) or over a number of production bouts occurring at different times (in the case of temperature pulses). On this view there is a period by the end of which certain events must have happened to produce a particular sex, although within this period there is no critical time when these events must have occurred. We therefore suggest the term thermosensitive period (or thermosensitive stages) for that interval when variation of temperature (any kind of temperature variation) has been shown to have some significant effect (any kind of effect) on sex ratio. Concep- tually it may be better to think of this as the interval between the times/stages when temperature, no matter how much it is altered within the limits of viability of the eggs, cannot produce effects, or has not yet been shown to produce effects. With increasing sample size, some widening of the thermosensitive period is to be expected.

Concluding statements

We do not discuss here which of the phenomenological aspects of the relationships between temperature and sexual differentiation are the most important. This may depend on what questions one is primarily interested in answering and on the level of analysis. In any case, unambiguous description based on defined terminology could help in making comparisons between the results of different studies. We therefore suggest the following definitions. 178

Transitional range of temperature (TRT): that range(s) between male- and female- producing temperatures, within which both sexes may differentiate among individuals of a population. Masculinizing and feminizing limit temperatures (1m and I,): those temperatures delimiting the transitional range, below (or above) which masculinization or feminization are maximum (asymptotic). Pivotal temperature (P): that temperature within the transitional range giving 50?/0 of each sex in experiments in which eggs are incubated at constant temperatures. Pivotal temperatures can be determined for a number of clutches or for a particular clutch or for a sample from a clutch. Thermosensitiaeperiodlstages (TSP, TSS): that time span or developmental-stage span outside of which temperature manipulations do not exert any influence on sexual phenotype. We also suggest that more attention should be paid to the various different aspects of the relationship between temperature and sex ratio, in particular high and low temperature sex ratio asymptotes, width of transitional range of temperature, and the influence of sexual genotype on these variables, as well as their possible evolutionary significance.

Acknowlcdgements.We thank the Natural Sciencesand EngineeringCouncil of Canada for support. This paper, long in gestation, was induced by discussionswith colleaguesat the First World Congress of Herpetology,Canterbury, September 1989.

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