Allometric Engineering: A Causal Analysis of Natural Selection on Offspring Size

Barry Sinervo; Paul Doughty; Raymond B. Huey; Kelly Zamudio

Science, New Series, Vol. 258, No. 5090. (Dec. 18, 1992), pp. 1927-1930.

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 Allometric Engineering: A Causal Analysis of
during any of these censuses were assumed
to have died (1 7). We analyzed patterns of
Natural Selection on Offspring Size
hatchling survival for first-clutch hatchlings
separately from later clutches because
Barry Sinervo, Paul Doughty, Raymond B. Huey, Kelly Zamudio hatchlings from later clutches must have
competed not only with other later clutch
Techniques of offspring size manipulation, "allometric engineering," were used in com- -
hatchlings but also with first clutch hatch-
bination with studies of natural selection to elucidate the causal relation between egg size lings, which were larger by virtue of their
and offspring survival of lizards. The results experimentally validate premises underlying age.
"
theories of optimal egg size: fecundity selection favoring the production of large clutches Lack's first prediction, that selection on
of small eggs was balanced by survival selection favoring large offspring. However, large egg size should be optimizing, was found in
hatchlings did not always have the highest survival, contrary to most theoretical expec- seven of eight cases (Fig. 1, except in first
tations. Optimizing selection on offspring size per se was the most common pattern. clutch Los Bafios 1990). Contrary to theo-
Moreover, matches between average and optimal egg size were qualitative, not quanti- ry, however, optimizing selection did not
tative, perhaps reflecting known functional constraints on the production of large eggs. usually result from a simple interaction
between conflicting directional-selection
components. The fecundity component (me
curve) was invariablv directional and fa-
T h e effect of egg and clutch size on fitness in natural populations of a lizard. vored females that prbduced large clutches
has been a central focus in evolutionary We collected eggs from reproductive of small eggs, whereas the offspring survival
ecology (1) since the work of Lack (2, 3). side-blotched lizards (Utu stansburiunu), a component (1, curve) was strictly direction-
For organisms with extended ~ a r e n t a lcare small iguanid lizard (3 to 10 g) that matures al and favored the largest egg size in only
(for example, birds and mammals), Lack in 1 year, at two localities in the inner two of eight cases [later clutch Los Bafios
predicted that natural selection favors the Coast Range of California, along Del Puer- (LB) and Del Puerto (DP) in 19891. Sur-
clutch (or litter) size that produces the most to Canyon Road (Stanislaus County, Cali- vival selection was significantly optimizing
surviving offspring (2). He tested this hy- fornia) and near Los Bafios Grandes along in three cases (LB first clutch 1989, LB and
pothesis by manipulating clutch size, and Billy Wright Road (Merced County, Cali- DP later clutches 1990), optimizing but
this elegant protocol has become the stan- fornia) (9). Females typically lay small eggs nonsignificant in two cases (DB first clutch
dard in avian studies. For organisms with- (mean of 0.39 g) on the first clutch and 1989 and 1990; however, if data from the 2
out parental care (for example, most am- larger eggs (0.43 g) on later clutches (5, 6, years are pooled, significant optimizing se-
phibians, reptiles, fish, and insects), Lack 10). Lack's hypothesis would be supported lection is detected. P < 0.05). , , and unre-
(3) realized that natural selection should if (i) females that lay intermediate-sized lated to size in one case (LB first clutch in
instead favor a compromise (optimizing se- eggs leave the most surviving offspring (op- 1990; however, see below).
lection) between the quantity and quality timizing selection) ; (ii) the optimum egg The discrepancy between the expected
(size) of offspring. His prediction was based size is small in the first clutch of the season pattern of strictly directional survival selec-
on two premises: (i) egg size is inversely but larger in later clutches; and (iii) the tion that favors large egg size and observed
related to clutch size (egg number), but (ii) observed seasonal shifts in average egg size patterns reflects a complicating effect of
offspring quality and hence offspring surviv- in these clutches coincides with shifts in season and hatchling sex. At LB, for exam-
al is positively related to egg size. Optirniz- the optimum. ple, directional selection consistently fa-
ine" selection results from the conflict be- Optimizing selection can be difficult to vored large female hatchlings (Fig. 2, A
tween fecundity selection (presumably fa- detect because the narrow size range of and 8). Nevertheless, size selection on
voring many small eggs) and survival selec- individuals in natural populations (1 1-1 3) males was comnlex. In three of four cases.
tion (presumably favoring a few large results in a low statistical power. Allometric size selection on males to 1 month of age
eggs). A t evolutionary equilibrium the engineering techniques (4, 5, 8) permitted included a significant optimizing compo-
average egg size should match the opti- us to decrease hatchling size by 1.3 SDs and nent that favored intermediate-sized hatch-
mum. The first premise is usually support- increase hatchling size by 1.6 SDs (14). lings in addition to a significant directional
ed in descriptive studies (4) and experi- Moreover, by experimentally separating size component that favored large hatchlings.
mental tests (5). However, with the ex- from other correlated traits (4, 5, 8), we In the fourth case, size selection was direc-
ception of a few descriptive studies (6, 7), could verify the causal links between size tional but favored small male hatchlines. "
the second premise has not been tested by and fitness (1 1-1 3). The sex of the hatchling is important be-
direct manipulation of offspring size anal- Newborn hatchlings were weighed (g), cause aggression (including cannibalism) by
ogous to manipulations of clutch size. marked, and released (May through Au- adult males, as well as dispersal away from
Therefore, we set out to test the second of gust) within 3 days on the site in small adult male territories, depends on hatchling
Lack's premises. We combined new tech- groups in close proximity to female home sex (16).
niques of offspring size manipulation (4, ranges. A total of 1668 control, miniatur- Lack's hypothesis also predicts that the
5 ) , "allornetric engineering" ( a ) , with a ized, and gigantized hatchlings were re- average egg size should track a seasonal shift
field study of natural selection to deter- leased at the two study sites over two (if one occurs) in the optimal egg size. The
mine the causal relation between egg size -- renroductive seasons (1 ~,4 ) . We recorded prediction always holds qualitatively. Both
and offspring quality as indexed by survival offspring survival to 1 month and to matu- average and optimal egg sizes increased
rity (15). We used a cubic spline algorithm . - 1).
seasonallv in three of four cases (Fig. ,,
B. S i n e ~ oand K. Zamudio, Department of Integrative (12) to determine the shape of the fitness and neither shifted in the remaining case
Biology, University of California, Berkeley, CA 94720,
and Department 6 f Zoology NJ-15, university of Wash- function relating offspring survival and off- (DP 1990) (18). Quantitatively, however,
ington,'Seattle WA 9819% spring size. Significance tests for directional average egg size did not shift nearly as much
P. Doughty, Department of Ethology, University of
and optimizing survival selection (16) were (-11% increase) as did optimal egg size
Tennessee, Knoxville, TN 3791 6.

R B Huey, Department of Zoology NJ-15, University

based on jackknife estimates of t values (-50% for cases with a shifting optimum).
of Wash~ngton,Seattle, WA 98195.
(13). Hatchlings that were not recaptured Moreover, in these cases, the average egg
SCIENCE * VOL. 258 18 DECEMBER 1992
 Los Bahos 1989 Del Puerto 1989 LOSBafios 1990 Del Puerto 1990
First clutch First clutch First clutch First clutch
10

.. . 2
A

0.0 0
-.-.. 02 0
0.4 0.6 0.8 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 02 0.4 06 08 U)

.-
C
.n
P
0
5 Later clutches Later clutches Later clutches Later clutches *
C
08 6
08 6 03 8

6
06 4 02

04 2 01 02

/ m e x le
02

iL 0 00 0 00 0 00
02 04 06 08 02 04 06 08 02 04 06 08 02 04 06 08

Fig. 1. Each panel plots empirically estimated linear regressions of
fecundity (me) on egg mass and cubic splines (12) of survlval probability
(I, for 1 month after release) on egg mass for hatchling llzards from two
localities, for clutches one versus later clutches and for 2 years. In all
cases me curves were significant, negative, and always linear These
relations indicate that fecundity selection was directional and favored
females laying small eggs (22). In contrast, I, curves varied ~nsignifi-
cance and direction, and some curves contained a significant quadratic
term, indicative of optimizing selection (22). Also plotted are the regres-
sions of me x I, versus egg size (product of a linear regression versus a
cubic spline, shaded lines), which provide an estimate of fitness (equal to
the number of survlvlng young as a function of egg size). Detection of
selection was facllltated by experimentally decreasing (white portion of
histogram) and increasing (solid portion of histogram) egg size relative to
natural var~ationin egg size (shaded portion of histogram). Mean egg size
for natural variation (solid triangles) and optimum egg size (open trian-
gles) are indicated. The optlmum egg size is the reproductive strategy
that leaves the most surviving offspring (21)

size in the first clutch was not as small as the
optimum egg size, and the average egg size
in later clutches was often not as large as
the optimum. The population at DP, which
shifted egg size in 1989 but not in 1990,
suggests that seasonal changes in egg size
were facultative. This observation, as well
as the significant between-year variation in
average egg size observed between 1989 (a
nondrought year) and 1990 (a drought
year), indicate that egg size showed pro-
nounced plasticity (18).
Known functional and physiological
constraints may explain why females do not
primarily track the seasonal shifts in opti-
mum egg size. Females with experimentally
decreased clutch size, and thus with en-
larged eggs (as above), often become egg-
bound or produce eggs that burst at ovipo-
sition (5). A pronounced increase in the
probability of such reproductive difficulties
occurs at egg sizes >0.48 g (9,well below
many of the later clutch (seasonal) optima
detected in our studies. Females that be-
come egg-bound would ultimately die in
natural populations, and thus the produc-
tion of excessively large eggs required to
match the optimum egg size on later clutch-
es would result in a cost of reproduction
that decreases future reproductive success of
females (19).
Offspring size manipulation has two ad-
vantages over correlational studies. Experi-
mental manipulations verify causal links be-
tween egg size and fitness (4, 8, 11-13).
These techniques also facilitate the detec-
tion of selection (12) by increasing the
representation of large and small individuals
in the population and thus experimentally
enhancing the natural variance in offspring
size by -54% (Fig. 1). These advantages can
be shown by a comparison of results from an
analysis of selection on offspring size that
used only natural variation in egg size with
analvses that used both natural and ex~eri-
menially enhanced variation. The patterns
of natural selection detected with the re-
stricted sample of natural variation agreed
with analyses based on natural and experi-
mentally enhanced variation in all cases,
and there were no pathological artifacts aris-
ing from our manipulations (20).
The best quantitative comparison using
only the data for natural variation compared
to the full range of natural and experimen-
tally enhanced variation is provided by se-
lection on female hatchlings. For example,
survival selection on female hatchlings at 1
month of age appeared to be fairly stable and
directional (for example, linear), although
the relative strength of selection changed on
first versus later clutches (Fig. 2A). In this
instance, the size dependence of survival [P
coefficient, which measures the intensity of
directional selection (1 ~.6)1 that was measured
with both natural and experimentally en-
hanced variation in hatchling female surviv-
al (p = 0.180, P < 0.0001) was very similar
to the intensity of selection that was mea-
sured with the restricted data set comprised
of natural variation ( p = 0.196, P < 0.01).
Thus. the enhanced survival of large " female
hatchlings is causally related to greater ma-
ternal provisioning and enhanced egg size.
For male hatchlings, analyses based on
experimentally enhanced variation indicat-
ed that survival was usually the best for
intermediate-sized hatchlings: smallest and
largest hatchlings typically had lower rates
of survival at 1 month of age. Using a
s a m ~ l erestricted to the natural variation in
male hatchling size (both populations and
years pooled; n = 240), we detected mar-
ginally significant optimizing selection on
the first clutch (P = 0.06) that favored
intermediate-sized hatchlings. Thus, the
survival decrement associated with the

1928 SCIENCE * VOL. 258 . 18 DECEMBER 1992

Fig. 2. (A) Empir~cally A 3. , The Natural Regulation of Animal Num-
Los Baiios 1989 Los B a b s 1990 bers (Clarendon, Oxford, 1954).
derived c u b ~ c spline
1 .o 4. B. Sinewo. Evolution 44 279 11990).
(12) of survival proba- 5. a n
\ ,

d P. Licht, Science252, 1300 (1991); J.

bil~ty (I, to 1 month) Exp. Zoo1 257, 252 (1991).
versus egg size for 6. G W. Ferguson and S. F. Fox, Evolution 38, 342
male and for female (1984).
hatchlings from LB, for 7. B C. Jayne and A. F. Bennett, ibid 44, 1204
clutches one versus lat- _n 0.4 (1990).
8. B Sinervo and R. B Huey, Science 248, 1106
er clutches and for 2 $ (1990). Allometrlc engineering provides an exper-
years. For females, sur- 5 0.2 mental assessment of the size effects on offspring
vival is generally posi- v, tralts that are detected in correlational analyses.
tively (and in most cas- 0.0 0.0 Offspring slze is manipulated in lizards by altering
es s~gnificantly)related 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 levels of yolk utilization in the egg (4) or yolk
Egg mass (g) provlsloning of the egg by the female (5)
to egg size; for males,
9 Near-term gravld female Uta stansburiana were
however, surv~valcan B Survival to maturity
Fitness collected from March to August 1989 and 1990.
be positively, negat~ve- All eggs were collected and Incubated under
1.o 2
ly, or even nonlinearly standardized condltlons (28"C, -200 kPa) (4).
related to egg size 3 m
.-h
Hatchling sue increased with egg size (4, 5).
(23). (B) Empirically .g 0.8 10. H A Nussbaum Oecologia 49, 8 (1981)
der~ved cubic spline o,6 = 11, J, A. Endler, Natural Selection in the Wild (Pr~nce-
ton Univ Press, Prlnceton N J 1986); M J Wade
(12) of surv~valproba- a m 1 and S Kalisz Evolution 44, 1947 (1990)
bility (I,) of offspring to g 0.4 .-> 12. D Schluter Evolution 42, 849 (1988).
maturity versus egg 13. T Mitchell-Olds and R. G. Shaw, ibid 41, 1149
slze for female (solid z> '5 (1987)
lines) and male (shad- '5 O.'
v,
14 To increase the frequency of small hatchlings, we
collected near-term females from our study s~tes
ed lines) offspr~ng(LB v, 0.0 o
and aspirated -18% of the yolk from half of the~r
0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8
1989 and 1990 freshly laid eggs (4. 5, 8) This manipulation
pooled). Selection on Egg mass (g) produced qualitatively normal hatchlings (4, 5, 8)
egg mass during the that were miniaturized by -18%. The remaining
first month of life persists to matur~ty,and selection was stronger on female offspring (24). Fitness eggs in these clutches served as sham-manipu-
lated (syringe inserted but no yolk removed),
(equal to the number of offspring surv~vingto maturity) was obtained by multiplying mecurves (see
full-sized controls. To increase the frequency of
Fig. 1) and I, curves. Opt~mumegg size (open tr~angles)on later clutches was larger than optimum large hatchlings, we anesthet~zed(w~thmetafane)
egg slze on the first clutch for both female and male offspring, and thus there was selection for a early vitellogenic females and surgically removed
seasonal shift In egg size (solid triangles). First clutch optima for both sexes bracketed observed the yolk from all but two or three of their follicles
egg size. However, later clutch optima were much higher than the observed egg size and effect~vely (4). In this way yolk normally allocated to the
beyond functional constraints on maximum egg slze (5) entire clutch was distributed to the two or three
remaining folllcles as the folllcles completed
growth This approach resulted in eggs -20%
larger than those from elther sham-manipulated or
smallest and largest male hatchlings that was a combination of directional and opti- unmanipulated females (5) Suwlval of offspr~ng
was detected in natural variation is causally mizing survival selection for egg size during from sham-manipulated (anesthesia and surgery
related to egg size. Moreover, this result the first month of life, not directional sur- but no foll~clesablated) and unmanipulated fe-
males was sim~lar.Sample slzes for each treat-
demonstrates the enhanced statistical pow- vival selection, as is commonly believed ment are presented below by local~ty,year, and
er of experimentally enhanced variation. (22). Because we did not detect effects of egg clutch (number of mlniaturlzed hatchlings re-
Whereas the deleterious effects of large and size o n body size or fecundity of offspring at leased, number of controls, and number of gigan-
maturity, the effects of egg size on fitness tlzed, total number of these hatchlings recaptured
small hatchling size of male offspring were at 1 month) LB 1989 f~rstclutch (65, 72, 0, 72),
marginally significant with a pooled sample were mediated largely by the effects on off- later clutches (98, 117, 32: 85); 1990 flrst clutch
(n = 240) that was comprised of only spring survival at maturity (Fig. 2B) (23). (167, 182, 33, 230),.later clutches (64,91, 21, 68),
Moreover, the match between average and and DP 1989 first clutch (56, 51, 0: 40), later
natural variation, such effects were signifi-
clutches (104, 87, 25; 38), 1990 first clutch (150,
cant in most clutches and most years for the optimal egg size (Figs. 1 and 2) is only 144, 22, 142) later clutches (30, 42, 15, 31).
population at LB with relatively small sam- qualitative, not quantitative, perhaps re- 15. After the hatchlings were released, we staged
ple sizes (as small as n = 72) that were flecting a known functional constraint on several major recaptures on-slte (LB, rad~us-300
m; DP, radius -100 m) and in a large, surround-
comprised of natural and experimentally the maximum egg size females can produce. ing buffer zone (LB, extending an additional 250
induced variation (Fig. 2B). Thus, allomet- Our allometric engineering techniques, m; DP, extending 200 m) We recaptured hatch-
ric engineering provides a causal assessment which can be applied to a wide range of lings first during the summer, at about 4 weeks
(28 2 10 days) after they were in~tiallyreleased.
of selection on offspring size that greatly egg-laying organisms in addition to other We recaptured them again at maturity the follow-
enhances the detection of selection relative reptiles (4, 5, 8), could be used to extend ing spring (March to May)
to natural variation. the generality of our observations o n adap- 16. R. Lande and S. J Arnold, Evolution 37, 1210
tation and constraint in the evolution of (1983).
Our results clearly validate Lack's first
17. Thls assumption was probably valid because d ~ s -
premise (21, which is thought to govern the lizard reproductive patterns to other orga- persal rates into our buffer zone were very low (-5%
evolution of egg size: egg number is inversely nisms without parental care. of all recaptures) [P E. Doughty, thesis, Unlversityof
--
related to egg size in these and other lizards
(4, 5). However, our results question the
Tennessee, Knoxville (1991)l and capture efficiency
during the summer was high (89%) and indepen-
REFERENCESANDNOTES dent of hatchl~ngsize (P > 0 36)
universalitv of Lack's second uremise (2), on 18. Analvsis of covariance (ANCOVA, w ~ t hcovariate
which most evolutionary models ( 1 ) of off- 1. C. C Smith and S D. Fretwell Am. Nat. 108, 499 for female postlaying mass slgniflcant, P < 0.05)
(1974), G. C. Williams, ibid. 100, 687 (1966), of mean egg size revealed sign~f~cant differences
'pring are based. Large offspring do not Adaptation and Natural Selection (Princeton Univ in egg size between years (P < 0.001), popula-
invariably survive best: survival depends o n Press, Pr~nceton,NJ, 1966) H. M Wilbur, Am. tlons (P < 0.001) and first versus later clutches
offspring size, season, and even offspring sex. Nat. 111, 43 (1977) S. C Stearns, 0.Rev 5101. (0.0001),as well as significant population by year,
5 1 , 3 (1976);W. Y Brockelman, Am Nat 109,677 clutch by year (P 4 0 Ol), and populat~onby
The strength of selection on offspring size (1975) A McGinley, D, Ternme, A
clutch (P 4 0 05) lnteract~oneffects. The Interac-
also varies between years and between pop- Geber, ibid. 130, 370 (1987). tion between populat~onand clutch was not slg-
ulations (21): The most common pattern 2 D. Lack, Ibis 89, 302 (1947). nlficant (P 1 0 16). ANCOVA of egg size (popu-

SCIENCE VOL. 258 18 DECEMBER 1992 1929

 lations and years treated separately) revealed
that the seasonal change In egg slze was s ~ g n ~ f -
cant (P 0.05) for LB 1989, LB 1990, and DP
1989, but not for DP 1990 (P > 0 05)
19 D Rezn~ck,Oikos 44, 257 (1985)
20. The lower survival of enlarged hatchl~ngmales In
most years would suggest that surg~caleffects on
the female parent m~ghtcause a drop In offspr~ng
quallty even though size remains enlarged This is
unl~kelyfor the follow~ngreasons F~rst,we d ~ ndot
detect any effects of sham surgery (P > 0.561,
follicle ablation (P > 0.94) (51, or yolk removal
from eggs (P 0 47) (4) except for the compa-
rable effects that manipulations and natural varl-
ation have on egg slze and offsprlng survlval ( P 4
0.01) (ANCOVA with pooled data set). Second,
the pathology would have to be sex-dependent
(Fig. 2) the largest female hatchl~ngsalways
showed h ~ g hsurvival at the same tlme that the
largest male hatchllngs showed low surv~val.
Th~rd,on the f~rstclutch In 1989 for LB, we did not
release experimentally g~gantizedhatchl~ngs,a nd
thus the decreased surv~valof large hatchllngs
was a result of natural var~ation.Moreover, ~fwe
pool all results from both years and populat~onsto
estlmate selection on the first-clutch male hatch-
lings w ~ t honly the natural variat~on,we detected
marginally s~gnificantoptimizing select~on(P =
0.06, n = 240). Fourth, In the most extreme case
we observed direct~onalselectlon that favored
experimentally m~niaturizedmale hatchllngs (f~rst
clutch for LB 1990, Fig 2A), and in this case
s~gnificant directional select~on favor~ng small
males was also detected (P 4 0.01) with only the
natural range of varlatlon.
21 We tested (ANCOVA) for differences in I-month
offsprlng survlval as a function of egg size (cova-
r~ate)across clutches (f~rstversus later clutches),
between populations, and between years (as well
as higher order lnteract~onterms among these
three factors) We partitioned t h ~ sanalysls by sex
because female survlval curves were h e a r and
male surv~valcurves included a significant optimiz-
~ n cgomponent. For the female offspr~ng,we found
that most of the hlgher order interaction terms
involv~ngclutch (for example, flrst versus later
clutches) and the covar~ateegg slze were signifl-
cant or marginally significant, lncludlng clutch by
year (P < 0.05) clutch by population ( P 4 0.051,
clutch by year by egg slze (P = 0.061, clutch by
population by egg slze (P = 0.05) clutch by
population by year (P 4 0.01) , clutch by populat~on
by year by egg size (P < 0.05), as well as the term
for the covariate egg slze (P < 0 05). Thus, the
lntenslty of dlrectlonal selectlon on egg mass for
female offspring varles across first versus later
clutches, between populat~ons, and between
years. For the male offspr~ng,a s ~ g n ~ f ~ c a(egg
nt
mass)' term (optim~zingselection) was used as a
second covariate along w ~ t hthe covariate egg
mass (dlrect~onalselection). We found signlflcant
clutch (P 4 0.01), egg slze (P 4 0.01), and (egg
size)' (P 4 0 001) effects. The only interactlon term
that was s ~ g n ~ f ~ cwas
a n t clutch by egg size (P 4
0.01). Populat~on(P > 0.05) and year effects (P 1
0.05) were not significant, nor were any other
h~gherorder interactlon terms
22. Postlaying body mass was used as a second
covariate for the calculations of the directional
selection coeff~c~ents on female fecundity me.
Significance levels based on jackknife est~mates
of t values (13) for the directional (p) and stabi-
lizing (y) coeffic~entson fecundity (me) and off-
spring survival (I,) (15) [where suggested by
cubic spllne analyses ( I Z ) ] are as follows: LB
1989, first clutch. 1, (P = 2.686, P 4 0 01, y =
2 . 4 9 5 , P < 0.05) m,(p = -0.618, P < 00001),
later clutches I , (P = 0.162 P = 0.05) me (p =
-0.397, P < 0.001); 1990, first clutch I , [p =
0 . 0 0 7 . P > 0 05, not s~gn~ficant (NS)]. me (p =
0 . 7 2 9 . P < 0 0001) later clutches I , (p = 1.I75,
P 4 0.05; y = 0 968, P < 0.061,m,(p = -0.556,
P < 0.05);and DP 1989, f~rstc lutch: 1, (p = 0.878,
P > 0 05, NS; y = 1 . 0 2 4 , P > 0.05, NS), me (P
= -0 544, P 4 0 0001), later clutches I , (P =
0 177, P < 0 Ol), me (p = 0 745, P < 0.001);
1990, f~rstclutch. 1, (p = 0 510, P > 0.05, NS, y = In thls case, select~onon egg slze differed sign~f-
-0 444, P 1 0 05, N S ) me (p = -0 383, P 4 lcantly depend~ngon offspr~ngsex (ANCOVA, P
0 051, later clutches I , (P = 2 161, P 4 0 05, y = < 0 0 1 ) and the probab~lityof surv~valwas high-
- 2 191, P 4 0 05), me (p = 0 . 4 0 4 , P 4 0.0001) est for largest females but smallest males.
The balance between fecund~tyand surv~valse- 24 The curves relatlng survlval probab~l~ty to matur~ty
lect~on(me x ),I y~eldedan optimum egg slze ~n are I~near,wh~chpermlts a test for dlfferences In
seven of eight comparisons The optlmum egg surv~valbetween the sexes by ANCOVA Selec-
slze IS the reproduct~vestrategy that lefi the most tlon on egg slze to maturlty was slgnlficant (cova-
surv~v~ng offsprlng In one case (LB 1990, first rlate egg mass was s~gnificant,P 4 0 01), and
clutch) there was no optlmum per s e and the there were significant sex differences in the
female that laid the smallest eggs In the popula- strength and d~rect~on of select~onon the survival
tion produced the most survlvlng offspr~ng(arbl- of male and female offspr~ng(sex by egg mass
trar~lydef~nedas the optimum) lnteractlon term was slgnlflcant, P 4 0 05)
23. Slgnlflcance levels based on jackknife estimates 25 We thank R Nichols, S Edwards, and L Carroll
of t values (13) for the direct~onal(p) and stabl- for assistance wlth collect~ngeggs and hatchling
l ~ z ~ n(y)
g coefflc~entsfor survlval select~on(15) recaptures, P. Llcht for research support (DCB-
[where suggested by cubic spline analyses (12)] 88.480221, and M Wake for arranging support
are as follows LB 1989, flrst clutch (females p = from the Department of Zoology, Univers~tyof
0.186, P = 0 05, males p = 3 536, P < 0.02, y = Cal~forn~a (UC), Berkeley Th~swork was support-
-3 361, P 4 0 0 3 ) second clutch (females p = ed by a National Sc~enceFoundation grant to
0.079, P > 0 05, NS, males p = 1 196, P = 0 06, R B.H. and B S. (BSR-8919600) B S, was sup-
y = 1 051, P = 0 09); and 1990, f~rstclutch ported by the Miller Institute for Basic Research ~n
(females p = 0 150, P 4 0.02, males p = Sc~ence,UC, Berkeley We also thank P Stadler
-0 136, P = 0 0 6 ) second clutch (females p = for permission to work on h ~ lsand All an~malcare
0 307, P < 0.01, males. P = 1 353, P < 0 01, y = and surgery were In full compl~ancew ~ t hthe UC,
1 . 2 0 1 , P < 0 05). When both responses were Berkeley, an~malcare and use comm~ttee.
linear (for example, first clutch of 1990) we could
test for slgnlficant dlfferences between the sexes 22 June 1992, accepted 20 October 1992
Components of Sterol Biosynthesis Assembled on
the Oxygen-Avid Hemoglobin of Ascaris
David R. Sherman, Brett Guinn, Monique M. Perdok,
Daniel E. Goldberg*
The parasitic nematode Ascaris infests a billion people worldwide. Much of its proliferative
success is due to prodigious egg production, up to lo6 sterol-replete eggs per day. Sterol
synthesis requires molecular oxygen for squalene epoxidation, yet oxygen is scarce in the
intestinalfolds the worms inhabit. Ascaris has an oxygen-avid hemoglobin inthe perienteric
fluid that bathes its reproductive organs. Purified hemoglobin contained tightly bound
squalene and functioned as an NADPH-dependent, ferrihemoprotein reductase. All com-
ponents of the squalene epoxidation reaction-squalene, oxygen, NADPH, and NADPH-
dependent reductase-are assembled on the hemoglobin. This molecule may thus func-
tion in sterol biosynthesis.
Ascariasis is an infection that pervades the aerobic in Ascaris, but a terminal cy-
world. The World Health Organization es- tochrome chain exists that uses oxygen as
timates that one billion people are infested available (7).
,-
with the etiologic parasitic helminth, As- How oxvgen is delivered to the cells
caris lumbricoides (1). Mortality rates for the remains unknown. Ascaris muscle has a
disease are estimated at 20,000 per year, myoglobin with high oxygen affinity (8-
due mostlv to biliarv and intestinal obstruc- 1I ) , and the perienteric fluid has an abun-
tions. Mdrbidity is 'somewhat higher, with
dant hemoglobin that binds oxygen 25,000
about one million cases per year exhibiting times more tightly than its mammalian
overt clinical manifestations (2, 3). Per- homolog. (The partial pressure of oxygen at
haps most devastating, ascariasis results in -
which the hemoglobin is half saturated is
decreased growth and development in mil- about 0.001 mm of mercury for Ascaris
lions of affected children (4, 5). Little is hemoglobin, and 25 mm of mercury for
known about the molecular metabolism of human hemoglobin.) (8, 10, 12, 13). The
Ascaris. Ascaris appears to be microaero- perienteric hemoglobin molecule is com-
~ h i l i c .consistent with its location in the prised of eight 40-kD subunits (14), but the
low-oxygen environment of the intestinal structural features that promote its robust
folds (6). Carbohydrate metabolism is an- oxveen affinitv are unknown. The function
,"
of this protein, which was detected spectro-
Department of Medicine and Department of Molecular
Microbiology, Wash~ngtonUniversity School of Medi- scopically by Keilin in 1925 (19) and char-
cine, and The Jew~shHospital of St LOUIS,St LOUIS, acterized with respect to oxygen affinity by
MO 63110. Davenport in 1949 (8), has also been a
*To whom correspondence should be addressed mystery. It binds oxygen too tightly to be
SCIENCE ' VOL. 258 18 DECEMBER 1992