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Soil moisture and temperature interact to affect growth, survivorship, fecundity, and fitness in the earthworm Eisenia fetida
Camp. B&hem. Physiol. Vol. 114A,No. 4, pp. 319-326, Copyright 0 1996 Elsevier Science Inc.
ISSN 0300-9629/96/$15.00 PI1 SO300-9629(96)00017-5
1996
ELSEVIER
Soil Moisture and Temperature Interact to Affect Growth, Survivorship, Fecundity, and Fitness in the Earthworm Eisenia fetida M. Lance Presley, Tom C. McElroy, and Walter _I. Diehl DEPARTMENT OF BIOLOGICALSCIENCES,MISSISSIPPISTATE UNIVERSITY, P.O. DRAWER GY, MISSISSIPPISTATE, MS 39762, USA
ABSTRACT.
Life history and fitness characters of the earthworm Eisenin fetida were measured for all combina-
tions of three soil moistures growth early in ontogeny,
(2,3,4
ml HzO/g peat moss) and four soil temperatures
fecundity
(cocoons
per week per earthworm),
(15,20,
25, 28°C).
and fitness (fecundity
Maximal
X survivorship)
occurred in high moistures and moderate temperatures. After reproductive maturity, maximal growth and survivorship occurred in moderate/high moistures and low temperatures. Soil moisture and temperature interacted to affect all life history and fitness characters such that high moisture often alleviated the adverse effects of high temperature
to some extent.
Because more variation also a good predictor studies examining
Growth
early in ontogeny
in fitness was contributed
was a good predictor
by fecundity
of fitness. This supports the use of early ontogenetic
the effects of individual genotypic
KEY WORDS. Soil moisture,
temperature,
variability.
growth,
of fecundity
than survivorship,
but not survivorship.
early ontogenetic
growth was
growth as a surrogate
of fitness in
COMP BIOCHEMPHYSIOL114A;4:319-326,
survivorship,
fecundity,
fitness,
life history,
1996.
earthworm,
Eisenia fetida
INTRODUCTION Soil temperature and moisture are important environmental factors affecting the physiology and ecology of earthworms (17). Upper thermal limits for survival are variable among lumbricid species, ranging from 2533°C (18,31). Below 25”C, greater temperature promotes greater rates of hatching, development, and individual growth (4,14) at a cost of reduced cocoon viability and output (27). The effects of soil moisture are more complex.
Below a
critical soil moisture, there is an adverse osmotic effect (desiccation) on earthworms (10). This critical soil moisture depends on the physical properties of the soil (3) and is species-specific (17). Desiccation tolerance is also speciesspecific (10). Above this critical point, tissue is fully hy drated, but soil moisture still has important, nonosmotic effects on earthworms. Moderately low soil moisture depresses aerobic metabolism (8), whole organismal growth rate (28) and fecundity (21). High soil moisture depresses growth rate and fecundity (21). Intermediate moisture levels (e.g., 70% water in cow manure for Eisenia fetida) are considered optimal. Above the critical point for desiccation, the effects of
Addwss reprint requests to: W.J. Diehl, Department of Biological Sciences, Mississippi State University, P.O. Drawer GY, Mississippi State, MS 39762. Received 27 September 1995; revised 7 January 1996; accepted 18 January 1996.
soil moisture are postulated to be largely respiratory (29) because of both the difference in oxygen diffusion rate in water vs. air (5) and the effect of soil moisture on cutaneous oxygen uptake (17). Despite the importance of both temperature and moisture to earthworms, little is known of the effects of these factors varying together on life history and fitness of earthworms especially late in ontogeny. Kaplan et al. (15) showed that early in ontogeny, E. fetida gained maximal weight when raised at 28°C and 3 ml Hz0 per g dry manure. Weight gain declined with lesser temperature and with lesser or greater moisture than 3 ml/g. One could not determine from these data whether there was an interaction between temperature and moisture affecting growth or whether these environmental effects would be similar for growth measured at other times in ontogeny or for other life history/fitness characters. Growth early in ontogeny has been used as a surrogate of fitness in previous studies reporting correlates of m&locus heterozygosity lations
have
in the earthworm E. fetida (2,6). Such correbeen
reported
commonly
in many species
(19,32) even though the specific relationship between growth and fitness may be unknown. Variation in a physiological or fitness phenotype derives from variation in the environment, the genotype, or both. We report the results of an experiment that makes use of variation in the environment (soil temperature, moisture) to produce variation in life history and fitness characters in order to examine rela-
M. L. Presley et al.
320
tionships among them. We test the hypotheses that responses of various life history and fitness characters to a variable environment are similar and that early life history characters (e.g., growth) are good predictors of adult fitness (e.g., fecundity, survivorship).
MATERIALS Earthworms
AND
METHODS
were raised in all combinations
of three soil
moistures (2, 3, 4 ml HlO/g dry peat moss) and four soil temperatures (15, 20, 25, 28OC). Treatments were initiated with 200 newly hatched earthworms weighing about 20 mg each (about 3-weeks old), except for the 25°C treatments which were initiated with 100 earthworms. Dead earthworms were replaced with other newly hatched earthworms in some treatments to maintain sample sizes for other experiments. Newly hatched earthworms were placed separately into 25ml plastic vials. The growth medium was prepared by adding an appropriate volume of aged tap water to dry peat moss containing
calcium carbonate
(5% w/w) to main-
tain a soil pH of 7. Vials were covered with aluminum foil and plastic caps with a small hole to permit some air flow while limiting moisture loss. Vials were kept in temperature-controlled incubators (15, 20, 25’C) or on the laboratory benchtop for the duration of the experiment. Although the air temperature of the laboratory fluctuated between 26 and 30°C diurnally (because the air conditioning was
0
10
20
30
40
50
60
TIME (weeks)
FIG. 1. Typical life history characteristics of Eisenia fetida throughout ontogeny. (A) Fresh weight, (B) survivorship, and (C) total number of cocoons. Data are taken from the ZO”C, 3 ml/g treatment (initial N = 210).
(e.g., 2-week growth rates, time to clitellum development, etc.) were analyzed by two-way ANOVA with PROC GLM
turned off at night), the temperature of the growth medium in the benchtop treatments maintained 28 + 1°C. None
of SAS for Windows@ (23). Frequency data that were obtained for each treatment (e.g., survivorship, fitness) were analyzed by log-linear contingency tests with the BIOM-
of the moisture levels was likely to have affected the tissue hydration of these earthworms (8). Earthworms were fed a
PC package of computer programs of Sokal and Rohlf (26). Where environmental effects were significant (0 < 0.05),
cornmeal-based ration (7) ad l&turn weekly. The growth medium was replaced every two weeks.
results were reported as second-order response surface diagrams (1). Coefficients were obtained with Proc GLM of SAS for Windows (23). Figures were drawn with PSI-Plot’
Survivorship,
fresh weight and number of cocoons
were
recorded every 2 weeks. Earthworms were rinsed in aged tap water, blotted dry and weighed to the nearest 0.1 mg. The week in which the clitellum developed (indicating reproductive maturity)
was recorded. Earthworms
are hermaph-
(Poly Software (Software
International)
Publishing
and Harvard Graphics@ 3.0
Corporation).
RESULTS
roditic and although E. fetida usually reproduces by outcrossing (9), it is reportedly capable of limited selffertilization also (11). Thus cocoon production by single individuals was assumed to be proportional to cocoon production by outcrossing even though no offspring hatched from
Figure 1 shows a typical set of relationships among fresh weight (FW), survivorship and cocoon production in Eisenia fetida until constant weight was attained. The growth curve was sigmoidal and survivorship declined lin-
these cocoons
early with time. The clitellum
in any treatment
during the entire experi-
ment. Each treatment was maintained until the earthworms reached constant fresh weight or until survivorship declined below 50%. For simplicity, data were analyzed for four stages in ontogeny: newly hatched stage (3-5 weeks post-hatching = weeks O-2 of the experiment), juvenile stage (9-11 weeks post-hatching, before clitellum development in most treatments = weeks 6-8 of the experiment), early adult stage (15-17 weeks post-hatching, after clitellum development in most treatments = weeks 12-14 of the experiment ), and adult stage (sexually mature earthworms at maximum weight). Data that were obtained for each individual
developed
on about week
13; cocoon production began after week 15 and continued throughout the experiment. The timing of clitellum development and cocoon production was highly dependent on the environment (see below). During ontogeny an increase in the number of earthworms producing cocoons offset a decrease in the number of cocoons produced per earthworm (data not shown). Thus total cocoon production in the whole population (about 9.5 new cocoons per week) neither increased nor decreased during ontogeny. The great variability in cocoon production during ontogeny was probably caused by reduced fecundity associated with selfing.
Effect
of Environment
on Fitness
321
of Eisenia
TABLE 1. Two-way ANOVA tables analyzing the effects of moisture and temperature on fresh weight (FW) and 2-week growth rates of 5, 1l-, and 1‘I-week-old E&n& fhida
Variable S-Week
Model
FW
ll-Week
FW
17-Week
FW
3-5 Week growth rate
9-11 Week growth rate
15-17 Week growth rate
Moisture Temperature Moisture X Error Moisture Temperature Moisture X Error Moisture Temperature Moisture X Error Moisture Temperature Moisture X Error Moisture Temperature Moisture X Error Moisture Temperature Moisture X Error
df
temperature
temperature
temperature
temperature
temperature
temperature
F
2 372.02 3 75.42 6 38.52 2307 2 37.98 3 5.82 6 6.03 1768 2 344.45 2 60.11 3 51.95 1110 2 3 6 2309 2 3 6 1768 2 2 3 1107
P
15
315.76 122.83 21.15
8.57 62.10 17.26
0.0002
testing
the effects of moisture and temperature on fresh weights (FW) and two-week growth rates (d FW) of 3/5-, 9/l I- and 15/17 -week-old Eisenia fetida. Because sample sizes changed over time because of mortality, a repeated-measures ANOVA
was not used to test the effects of moisture and
temperature on growth during ontogeny. However, the probability of a Type I error among growth analyses was less than 0.01 according to a sequential Bonferroni test (22). There were significant interactive effects of soil moisture and temperature (p < 0.0001) on both fresh weight and two-week growth rates at each stage of ontogeny. The effects of soil moisture and temperature on these variables are depicted in Fig. 2. Early in ontogeny, the greatest growth rates occurred in high moistures (4 ml/g) and moderate temperatures (20,ZS”C). The least growth rates occurred in low moistures (2 ml/g) and low temperatures (15’C) as well as in low moistures (2 ml/g) and high temperatures (28“C).
(15’C). The least growth rates occurred in low moistures (2 ml/g) and high temperatures (28°C). This shift in the of greatest
growth
coincided
with
of sexual
maturity
in earthworms
(see
15
20
25
20
25
15
20
25
15
20
25
(“C)
FIG. 2. Effect of moisture and temperature on dy ontogenetic growth of ELFenia fetkh(A-C) Change in freshweight (g) per week between (A) 3-5 weeks, (B) 9-11 weeks, and (C) 15-17 weeks, respectively. (D-F) Absdute f+eshweight (g) of (D) 5., (E) lie, and (F) 17-week-old earthworms, respectively. Values are given for each contour.
that
the temperature/moisture
interaction
apparent late in ontogeny. Table 2 shows results of two*way ANOVAs
was more testing the
effects of moisture and temperature on maximum fresh weight (FW,,,), weeks to FW,,, and overall growth rate (FW,,/weeks
to FW,,,)
in E. fetida. Three
treatments
(2O”C, 4 ml/g; 28”C, 3 and 4 ml/g) were terminated because of high mortality before FW,,, was attained and were not included in these analyses. There were significant
interac-
tive effects of soil moisture and temperature (p < 0.0001) weeks to FW,,, and overall growth rate. The efon W,,,, fects of soil moisture
and temperature
on these variables
TABLE 2. Two-way ANOVA
tables dyzing the effects of moisture and temperature on maximum fresh weight and overall growth rate (FW_/ (Ew,,), weeks to m,,, weeks to FW,,_ ) in Eisenia fetida Variable
Fwmx
Weeks
to
Fw,,,
the develop-
Also by this time in ontogeny, growth rate had become more dependent on temperature than on moisture. Generally the effects of moisture and temperature on fresh weight (Fig. 2) were similar to those effects on growth rate with the excepment
25
TEMPERATURE
Later in ontogeny, the greatest growth rates occurred in moderate/high moistures (3, 4 ml/g) and low temperatures
environment
20
<0.0001
820.22 197.30 72.70
1 shows the results of two-way ANOVAs
15
tion
Table
25
below).
FW,/week
Model Moisture Temperature Moisture X temperature Error Moisture Temperature Moisture X temperature Error Moisture Temperature Moisture X temperature Error
df 2 3 3 838 2 3 3 838 2 3 3 838
F
P
1342.45 1519.90 34.77
3998.48 1336.38 19.30
<0.0001
567.59 249.59 65.90
M. L. Presley et al.
322
15
20
25
15
20
25
TEMPERATURE
15
20
25
(“C)
weeks to FW,,, and (C) overall growth rate (FW,,,Jweeks to Fw,,). Values are given for each contour.
are depicted in Fig. 3. The greatest FW,,, was attained by earthworms in high moistures (4 ml/g) and low temperatures (15’C).
It took up to five times longer for these earthworms to reach FW,,, than earthworms in other environments. Considering FW,,, and time to FW,,, together, earthworms in moderate temperatures (ZO’C) and high moistures (4 ml/g) reached the greatest FW,,, in the shortest time (i.e., they had the greatest overall growth rate). Earthworms in low moistures (2 ml/g) and high temperatures (28’C) had the least overall growth rate. Table 3 shows the results of a log-linear contingency
test
on the effects of moisture and temperature on the frequency of survivorship of adult earthworms. Only those paths testing the dependence of survivorship on moisture and/or temperature are reported; other paths are biologically
irrelevant
because moisture and temperature levels were fixed (26). The tests for conditional independence are analogous to tests of the interaction of main effects in an ANOVA. The test for complete independence of survivorship is somewhat analogous to tests of main effects in an ANOVA. The effect of moisture on survivorship depended on temperature and the effect of temperature on survivorship depended on moisture. Because both conditional independence tests (interaction effects)
were significant,
further consideration
of the
complete independence of survivorship from moisture and temperature was inappropriate (26). The effects of soil moisture and temperature
on survivorship of adult earthworms
TABLE 3. Three-way log&near contingency test (G,,,,) analyzing the effects of moisture (M) and temperature (T) on survivor-ship (S) in Eiseniafetida (The null hypothesis is independence) Variable
Survivorship
Model
Conditional Independence S x M from T S x TfromM Independence of S from M&T
df
G
P
8
113.88 307.56 333.33
CO.001
9 11
20
15
FIG. 3. Effect of moisture and temperature on final growth of Eiseniahida. (A) Maximum fresh weight (FW,,, g), (B)
25
TEMPERATURE (“C) FIG. 4. Effect of moisture and temperature on survivor-ship (%) of Eiseniafetida. Values are given for each contour.
are depicted
in Fig. 4. The greatest survivorship occurred
in moderate
temperatures
(ZOOC) regardless of moisture.
Generally survivorship was high in a wide range of moistures and temperatures. The least survivorship occurred in high moistures (4 ml/g) and high temperatures
(28’C).
Table 4 shows the effects of moisture and temperature on factors reflecting the fecundity of earthworms. There were significant interactive effects of soil moisture and temperature on the frequency of earthworms with a clitellum (threeway log-linear contingency test, p < O.OOl), weeks to clitellum development
(two-way ANOVA,
p < 0.0001)
and co-
coons produced per surviving earthworm per week (two-way ANOVA, p < 0.0007). Because a large number of earthworms did not produce
cocoons,
the latter variable
was
coded and normalized by a square root of (x + 0.5 ) transformation prior to statistical analysis. Using data from only those earthworms that produced cocoons would have been desirable to reduce variance in fecundity, but it was necessary to use data from all earthworms whether they produced cocoons or not in order to include those treatments where no cocoons were produced in the analysis. The effects of soil moisture and temperature on fecundity are depicted in Fig. 5. The greatest percentage of earthworms that developed a clitellum during the course of the experiment occurred in high moistures (4 ml/g) and low temperatures (15°C); the least percentage of earthworms with a clitellum occurred in high temperatures (28°C) and low moistures (2 ml/g). In contrast, the clitellum developed most rapidly in high temperatures (28°C) and high moistures (4 ml/g) and least rapidly in low moistures (2 ml/g) and low temperatures (15°C; data not shown). The disparity between the percent of earthworms with a clitellum (53% overall) and the percent of earthworms producing cocoons (8% overall) suggests that the latter underestimates the true fecundity of the species, likely because of selfing. We assumed that selfing depressed cocoon production to the same extent in all treatments and that cocoon production as measured was a valid index of reproductive output. Reproductive output (co-
Effect of Environment on Fitness of Eiseniu
TABLE 4. Effects of moisture (M)
323
and temperature
(T) on fecundity
Variable
Model
Weeks to clitellum development
per earthworm
df
Conditional Independence F X MfromT F X TfromM Independence of F from M & T Moisture Temperature Moisture X temperature
Frequency of earthworms with clitellum
Cocoons
(F) in Eiseniafetida
Error Moisture Temperature Moisture X temperature Error
per week
For G
8 9 11 2 ; 1304 2 3 6 1308
R
377.11 1366.48 1505.80 59.69 16.41 12.73
10.52 5.32 3.94
<0.0001
0.0012 0.0007
was used to analyze frequency of earthworms with a clitellum. The null hypothesis is independence. A A three-way log-linear contingency test (G,,,,,,) two-way ANOVA table was used to analyze weeks to clitellum development and cocoons produced per earthworm per week. Data were transformed by the square root of (x + 0.5).
per week) apparently reflected a bal-
were considered to be fit, whereas 0% of the earthworms in
ance between the percent of earthworms attaining reproductive maturity and the time to reproductive maturity. Thus the greatest number of cocoons produced per earth-
the 2 ml/g, 28°C treatment were considered to be fit. For convenience and conservatism, frequency analyses were conducted on a sample size set at 100 earthworms per treat-
worm per week occurred
ment. The effect of moisture on relative fitness depended on temperature and the effect of temperature on relative
coons per earthworm
in high moistures
(4 ml/g) and
moderate temperatures (20, 25%). The least cocoon production occurred in low moistures (2 ml/g) and low temperatures ( lS°C)
as well as in low moistures (2 ml/g) and high
fitness frequency depended on moisture. The effects of soil moisture and temperature on relative fitness frequencies are
temperatures (28’C). Table 5 shows the results of a log-linear contingency test on the effects of moisture and temperature on the relative
depicted in Fig. 6. The greatest relative fitness occurred in
fitness of earthworms (- relative frequency of fit earthworms). A fitness index was computed as the product of survivorship and reproductive output for each treatment.
(2 ml/g) and low temperatures (15°C) as well as in low moistures (2 ml/g) and high temperatures (28’C). Because of the large number of statistical tests presented, a sequential Bonferroni test (22) was performed simulta-
The relative fitness of earthworms in each treatment was computed as a percent of the maximum fitness index among treatments. In effect this corrected for the reduced levels of fecundity because of selfing. Thus for relative fitness analyses, 100% of the earthworms in the 4 ml/g, 25°C treatment
high moistures (4 ml/g) and moderate temperatures (20, 25’C). The least relative fitness occurred in low moistures
neously on all statistics reported in Tables l-5
to determine
the probability of Type I errors. This is an extremely conservative analysis because Type I errors were already controlled within each of the ANOVAs. Nevertheless the probability of a Type I error occurring anywhere among these analyses of moisture and temperature effects was less than 0.01.
A
B TABLE 5. Three-way lwlinear contingency test (GWilli_) analyzing the effects of moisture (M) and temperature (T) on relative fitness (RF) in Ebenia fetida Variable Relative fitness
-15
20
25
15
TEMPERATURE
20
25
(“C)
FIG. 5. Effect of moisture and temperature on fecundity of Ehenia fetida(A) Percent of individuals developing a clitelhun and (B) cocoons produced per earthworm per week. Values are given for each contour.
Model
df
G
p
Conditional independence RF X M from T RF X T from M Independence of RF from M&T
8 9 11
404.33 373.27 586.60
Relative fitness for each treatment was computed as the number of cocoons per earthworm per week X survivorship and was expressed as a percent of the maximum fitness among treatments based on a sample size of 100 earthworms (see text). The null hypothesis is independence.
M. L. Presley et al.
324
ment: high moisture and moderate temperature.
Although
cocoon production was the best index of fecundity available, it could not take into account differences in the number of viable embryos within each cocoon which likely occurred in an environment-dependent manner. Also populations at high temperatures attained reproductive maturity before populations in other environments and thus may be able to produce more generations
per unit time.
Over time this would increase the fecundity of high-temper-
15
20
25
ature populations beyond that which was evident from cocoon production in one generation alone. The environmental pattern of fitness was more similar
TEMPERATURE (“C) FIG.6. Effect of moisture and temperatureon relativefitness of Eisenia bib. Relative fitness for each treatment was computed as the number of cocoons per earthworm per week x survivorshipand was expressed as a percent of the maximum fitness among treatments (see text). Values are given for each contour.
to that of fecundity than survivorship even though both variables were mathmatically equivalent in computing the fitness index. This occurred because the coefficient of variation in fecundity
among treatments
was almost five times
greater than that of survivorship. Thus fecundity contributed more to the variation in fitness than did survivorship. Fitness was maximal in high moisture and moderate temperature and declined as moisture decreased and as temperature
DISCUSSION The combination
of soil moisture and temperature
produc-
ing maximal growth, expressed as absolute fresh weight or change in fresh weight, changed during ontogeny. Although the most favorable soil moisture level for growth was always high (4 ml/g), the most favorable temperature shifted from moderate-high (ZO-2S°C) to low ( ISaC) by the time of reproductive maturity and remained that way until maxima1 fresh weight was attained. This shift may rep-
increased or decreased. The same limitations on the interpretation of the fecundity results also apply to the interpretation of fitness. In particular the fitness of high-temperature populations may have been underestimated by the inability to account for the greater number of generations that may occur in those environments. Although the effects of soil moisture and temperature on growth and fitness of E. fetida measured here were reason-
resent catch-up growth in low-temperature earthworms as high-temperature earthworms directed resources to repro-
ably consistent with previous reports, some conspicuous differences exist. Our study confirmed reports that rates of fecundity and growth early in ontogeny varied with
duction
temperature below the upper thermal limit (4,14,27).
rather
than
somatic
growth.
However
because
How-
earthworms in high moisture and low temperature attained the greatest maximal fresh weight among earthworms of all
ever in the present study, maximal fresh weight and percent of earthworms reaching reproductive maturity varied in-
treatments,
versely with temperature. Earthworms became acclimated to low temperature sufficiently well that physiological rates later in ontogeny exceeded those of high-temperature earth-
it is also likely that this shift represents an onto-
genetic shift in environmental tolerance. The effects of soil moisture and temperature on survivorship were different from those on growth. Maximal survivorship occurred at moderate temperature (20°C) and moderate moisture (3 ml/g). The pattern did not change throughout ontogeny (data not shown) and survivorship was generally more dependent on temperature than on soil moisture. The different ontogenetic patterns of growth response vs. survivorship response to the environment
suggest
that two different mechanisms of physiological acclimation were operating. The effects of soif moisture and temperature on fecundity were more complex. For those earthworms that reached reproductive maturity, the time to maturity occurred most rapidly at high moisture and high temperature. However more earthworms reached maturity at low temperature than at high temperature. Both of these factors influence (but not exclusively) the number of cocoons produced in different environments. Nevertheless cocoon production was maximal in what would seem to be an intermediate environ-
worms. Our study failed to confirm that maximal
growth
and fecundity occurred in a moderate soil moisture as opposed to high or low moisture, contrary to that reported by Reinecke and Venter (2 1). Th’ 1s d‘rscrepancy is likely caused by the different physical properties of the soils used between the studies (peat moss vs. manure). Peat moss retains water to a greater extent than manure (pers. obs.) so that for the same volumetric water content, less water is available to earthworms in peat moss than in manure. Treatments with moisture content greater than 4 ml HLO/g peat moss (80% moisture) would have likely been required to depress growth and fitness in E. fetida. Finally, previous studies (15) have not identified the subtle (but highly significant) interactions between temperature and moisture that affect growth, fecundity and survivorship of E. fetida. Growth rate, fecundity and fitness were more sensitive to temperature at low moisture than at high moisture. On the other hand, survivorship and maximal fresh weight were more sensitive
Effect of Environment
to temperature
on Fitness of
Eiseniu
325
at high moisture than at Iow moisture. Either
contrary to the work of Hawkins er al. (12,13)
and Koehn
way, appropriate soil moisture levels may ameliorate the ef-
and Bayne ( 16). In short, growth measured anytime in on-
fects of suboptimal temperature on earthworm physiology. Early in ontogeny, growth (FW, change in FW) was a
togeny has the potential to contribute some information about the htness (fecundity or survivorship) of the individ-
good predictor of fecundity (cocoons per week per earthworm) but not survivorship based on visual comparisons
ual. Growth measured prior to reproductive the greatest potential in this regard.
among response surface diagrams. Early growth was a good predictor of overall fitness since more of the variation in fitness could be attributed to survivorship.
to variation
After reproductive
change in FW, FW,,,)
in fecundity
than
maturity, growth (FW,
was a marginal predictor of survivor-
ship to the extent that both greater growth and survivorship occurred in lower temperature earthworms. The relationship between early ontogenetic growth and fecundity is predicted by the balanced energy equation (30) where somatic growth and reproduction represent competing pathways for energy use. Typically the energy use between pathways is segregated ontogenetically as seen here. This relationship should be independent of environmental variability so long as reproduction
is not postponed. This was confirmed in the
present study where early growth (d EW, weeks 3-5) was also correlated with fecundity (total no. cocoons of fecund earthworms) within the constant 20°C 3 ml/g treatment (r = 0.340; df = 33; p < 0.05). The marginal relationship between growth of mature earthworms
and survivorship
is
not as well grounded in bioenergetic theory (20). It may reflect the outcome of an optimai strategy between early growth and fitness cost (i.e., mortality) (24) extended over a variable environment that produces the same artifacts as comparisons among species (25). As such, a relationship between mature growth and survivorship would be dependent on the presence of environmental variability. This was also confirmed in the present study because mature growth (d Fw, weeks 15-17) was not correlated with survivorship (weeks to death) within the constant 20°C 3 ml/g treatment (r = 0.055; df = 190; NS). We sought to test two hypotheses:
(a) patterns
history and fitness responses to environmental
of life
variability
would be similar to each other, and (b) growth early in ontogeny would be a good predictor of adult fitness. The first hypothesis was not supported. Responses of growth, fecundity, survivorship and ftmess to the environment differed from each other to various degrees. Responses of growth to the environment changed with ontogeny. The second hypothesis was supported. The environmental response of growth early in ontogeny
was very similar to that of fecun-
dity and fitness. This supports the use of early ontogenetic growth as a surrogate of fitness in studies examining the effects of individual multilocus heterozygosity in earthworms (6,2). Because we used variation in the environment to produce variation in fitness, it is possible that the associations reported here would not hold for genetically-induced variation in fitness. If this were confirmed, it would imply that the genetic effect on fitness operates through different bioenergetic mechanisms than the environmental effect,
maturity has
We thank Melinda Chow, Rebecca Brasfeiti, Warren Cox, April Heinsch, Gzrie Loundensluger, Tara Mann, Yonya Nabors and Theresa Pouchfurassistance in dam colkctim and earthwum husbandry. This research was suppurredby NSF grant DEB-9221094 to WID.
References 1. Alderdice, D.F. Factor combinations responses of marine poikilotherms to environmental factors acting in concert. In: Kinne, 0. (ed). Marine ecology, vol. 1, Part 3. London: WileyInterscience; 1972:1659-1722. 2. Audo, M.C.; Diehl, W.J. Effect of quantity and quality of environmental stress on multilocus heterozygosity-growth relationships in Eisenia fetida(Annelida: Oligochaeta). Heredity 75:98-105;1995. 3. Buckman, H.O.; Brady, N.C. The nature and properties of soils, 7th ed. London: The Macmillan Co.; 1969. 4. Butt, K.R. The effects of temperature on the intensive production of Lumbricus terresrris (Oligochaeta: Lumbricidae). Pedobiologia 35:257-2641991. 5. Cameron, J.N. Principles of physiological measurement. Orlando: Academic Press; 1986. 6. Diehl, W.J. Genetics of carbohydrate metabolism and growth in Eisenia foetida (Oligcchaeta: Lumbricidae). Heredity 61: 379-387;1988. 7. Diehl, W.J. Apparent differences in kinetic properties among isozymes in Eisenia foetida (Oligochaeta: Lumbricidae). Comp. Biochem. Physiol. 968:343-348;1990. 8. Diehl, W.J.; Williams, D.L. Interactive effects of soil moisture and food on growth and aerobic metabolism in Eisenia fetida (Oligochaeta). Comp. Biochem. Physiol. 102A:179-184; 1992. 9. Gates, G.E. Contributions to a revision of the earthworm family Lumbricidae. XXII. The genus Eisenia in North America. Megadrilogica 3:131-147;1978. 10. Grant, W.C. Studies on moisture relationships in earthworms. Ecology 36:400-407;1955. 11. Hartenstein, R.; Neuhauser, E.F.; Easton, E.G. Growth and fecundity of Fz Eisenia foetida derived from FiS, both reared in isolation from birth. Megadrilogica 3:185-187;1980. 12. Hawkins, A.J.S.; Bayne, B.L.; Day, A.J. Protein turnover, physiological energetics and heterozygosity in the blue mussel, My&s edulils: the basis of variable age-specific growth. Proc. R. Sot. Lond. B 229:161-176;1986. 13. Hawkins, A.J.S.; Bayne, B.L.; Day, A.J.; Worrall, CM. Genotype-dependent interrelations between energy metabolism, protein metabolism and fitness. In: Ryland, J.S.; Tyler, P.A. (eds). Reproduction, Genetics and Distributions of Marine Organisms. Fredensborg, Denmark: Olsen & Olsen; 1989: 283-292. 14. Holmstrup, M.; Ostergaard, I.K.; Nielsen, A.; Hansen, B.T. The relationship between temperature and cocoon incubation time for some lumbricid earthworm species. Pedobiologia 35: 179-184;1991. 15. Kaplan, D.L.; Hartenstein, R.; Neuhauser, E.F.; Malecki, M.R. Physiccchemical requirements in the environment of the earthworm Eisenia foe&. Soil Biol. B&hem. 12:347-352;1980.
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16. Koehn, R.K.; Bayne, B.L. Towards a physiological and genetical understanding of the energetics of the stress response. Biol. J. Linn. Sot. 37:157-171;1989. 17. Lee, K.E. Earthworms Their Ecology and Relationships with Soils and Land Use. Sydney: Academic Press; 198533-55. 18. Miles, H.B. Heat-death in A~/o/obophoratemesnis (Sav.) forma hgu (Ude) and Eisenia foetida (Sav.). Nature 199:826; 1963. 19. Mitton, J.B.; Grant, M.C. Associations among protein heterozygosity, growth rate, and developmental homeostasis. Ann. Rev. Ecol. Syst. 15:479-499;1984. 20. Peters, R.H. The Ecological Implications of Body Size. Cambridge: Cambridge University Press; 1983:1188146. 21. Reinecke, A.J.; Venter, J.M. Moisture preferences, growth and reproduction of the compost worm Eisenia fetida (Oligochaeta). Biol. Fertil. Soils 3:135-141;1987. 22. Rice, W.R. Analyzing tables of statistical tests. Evolution. 43: 223-225;1989. 23. SAS. SAS/STA?TM User’s Guide, Release 6.03 Ed., Chapt. 20. Car-y: SAS Institute Inc.; 1988:549-640. 24. Sibley, R.; Calow, P.; Nichols, N. Are patterns of growth adaptive? J. Theoret. Biol. 112:553-574;1985.
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25. Sibley, R.M.; Calow, P. Growth and resource allocation. In: Calow, P. (ed). Evolutionary Physiological Ecology. Cambridge: Cambridge University Press; 1987:37-52. 26. Sokal, R.R.; Rohlf, F.J. Biometry, 2nd Ed. New York: W.H. Freeman and Co.; 1981:747-765. 27. Tsukamoto, J.; Watanabe, H. Influence of temperature on hatching and growth of Eisenia foetida (Oligochaeta, Lumbricidae). Pedobiologia 17:338-342;1977. 28. Viljoen, S.A.; Reinecke, A.J. Moisture and growth, maturation and cocoon production of Er&ilu.s eugeniae (Oligochaeta). Rev. Ecol. Biol. Sol 26:291-303;1989. 29. Williams, D.L.; Diehl, W.J. Interactive effects of soil moisture and food on glycolytic metabolism in Eisenia fetida (Oligochaeta). Comp. Biochem. Physiol. 102B:911-917;1992. 30. Winberg, G.G. Rate of metabolism and food requirements of fishes. Fish. Res. Bd. Can., Translation Ser. no. 194 [1960] 1956. 31. Wolf, A.V. Notes on the effect of heat in Lumbricus terrestris L. Ecology 19:346-348;1938. 32. Zouros, E.; Foltz, D.W. The use of allelic isozyme variation for the study of heterosis. Isozymes: Current Topics Biol. Med. Res. 13:1-59;1987.
ISSN 0300-9629/96/$15.00 PI1 SO300-9629(96)00017-5
1996
ELSEVIER
Soil Moisture and Temperature Interact to Affect Growth, Survivorship, Fecundity, and Fitness in the Earthworm Eisenia fetida M. Lance Presley, Tom C. McElroy, and Walter _I. Diehl DEPARTMENT OF BIOLOGICALSCIENCES,MISSISSIPPISTATE UNIVERSITY, P.O. DRAWER GY, MISSISSIPPISTATE, MS 39762, USA
ABSTRACT.
Life history and fitness characters of the earthworm Eisenin fetida were measured for all combina-
tions of three soil moistures growth early in ontogeny,
(2,3,4
ml HzO/g peat moss) and four soil temperatures
fecundity
(cocoons
per week per earthworm),
(15,20,
25, 28°C).
and fitness (fecundity
Maximal
X survivorship)
occurred in high moistures and moderate temperatures. After reproductive maturity, maximal growth and survivorship occurred in moderate/high moistures and low temperatures. Soil moisture and temperature interacted to affect all life history and fitness characters such that high moisture often alleviated the adverse effects of high temperature
to some extent.
Because more variation also a good predictor studies examining
Growth
early in ontogeny
in fitness was contributed
was a good predictor
by fecundity
of fitness. This supports the use of early ontogenetic
the effects of individual genotypic
KEY WORDS. Soil moisture,
temperature,
variability.
growth,
of fecundity
than survivorship,
but not survivorship.
early ontogenetic
growth was
growth as a surrogate
of fitness in
COMP BIOCHEMPHYSIOL114A;4:319-326,
survivorship,
fecundity,
fitness,
life history,
1996.
earthworm,
Eisenia fetida
INTRODUCTION Soil temperature and moisture are important environmental factors affecting the physiology and ecology of earthworms (17). Upper thermal limits for survival are variable among lumbricid species, ranging from 2533°C (18,31). Below 25”C, greater temperature promotes greater rates of hatching, development, and individual growth (4,14) at a cost of reduced cocoon viability and output (27). The effects of soil moisture are more complex.
Below a
critical soil moisture, there is an adverse osmotic effect (desiccation) on earthworms (10). This critical soil moisture depends on the physical properties of the soil (3) and is species-specific (17). Desiccation tolerance is also speciesspecific (10). Above this critical point, tissue is fully hy drated, but soil moisture still has important, nonosmotic effects on earthworms. Moderately low soil moisture depresses aerobic metabolism (8), whole organismal growth rate (28) and fecundity (21). High soil moisture depresses growth rate and fecundity (21). Intermediate moisture levels (e.g., 70% water in cow manure for Eisenia fetida) are considered optimal. Above the critical point for desiccation, the effects of
Addwss reprint requests to: W.J. Diehl, Department of Biological Sciences, Mississippi State University, P.O. Drawer GY, Mississippi State, MS 39762. Received 27 September 1995; revised 7 January 1996; accepted 18 January 1996.
soil moisture are postulated to be largely respiratory (29) because of both the difference in oxygen diffusion rate in water vs. air (5) and the effect of soil moisture on cutaneous oxygen uptake (17). Despite the importance of both temperature and moisture to earthworms, little is known of the effects of these factors varying together on life history and fitness of earthworms especially late in ontogeny. Kaplan et al. (15) showed that early in ontogeny, E. fetida gained maximal weight when raised at 28°C and 3 ml Hz0 per g dry manure. Weight gain declined with lesser temperature and with lesser or greater moisture than 3 ml/g. One could not determine from these data whether there was an interaction between temperature and moisture affecting growth or whether these environmental effects would be similar for growth measured at other times in ontogeny or for other life history/fitness characters. Growth early in ontogeny has been used as a surrogate of fitness in previous studies reporting correlates of m&locus heterozygosity lations
have
in the earthworm E. fetida (2,6). Such correbeen
reported
commonly
in many species
(19,32) even though the specific relationship between growth and fitness may be unknown. Variation in a physiological or fitness phenotype derives from variation in the environment, the genotype, or both. We report the results of an experiment that makes use of variation in the environment (soil temperature, moisture) to produce variation in life history and fitness characters in order to examine rela-
M. L. Presley et al.
320
tionships among them. We test the hypotheses that responses of various life history and fitness characters to a variable environment are similar and that early life history characters (e.g., growth) are good predictors of adult fitness (e.g., fecundity, survivorship).
MATERIALS Earthworms
AND
METHODS
were raised in all combinations
of three soil
moistures (2, 3, 4 ml HlO/g dry peat moss) and four soil temperatures (15, 20, 25, 28OC). Treatments were initiated with 200 newly hatched earthworms weighing about 20 mg each (about 3-weeks old), except for the 25°C treatments which were initiated with 100 earthworms. Dead earthworms were replaced with other newly hatched earthworms in some treatments to maintain sample sizes for other experiments. Newly hatched earthworms were placed separately into 25ml plastic vials. The growth medium was prepared by adding an appropriate volume of aged tap water to dry peat moss containing
calcium carbonate
(5% w/w) to main-
tain a soil pH of 7. Vials were covered with aluminum foil and plastic caps with a small hole to permit some air flow while limiting moisture loss. Vials were kept in temperature-controlled incubators (15, 20, 25’C) or on the laboratory benchtop for the duration of the experiment. Although the air temperature of the laboratory fluctuated between 26 and 30°C diurnally (because the air conditioning was
0
10
20
30
40
50
60
TIME (weeks)
FIG. 1. Typical life history characteristics of Eisenia fetida throughout ontogeny. (A) Fresh weight, (B) survivorship, and (C) total number of cocoons. Data are taken from the ZO”C, 3 ml/g treatment (initial N = 210).
(e.g., 2-week growth rates, time to clitellum development, etc.) were analyzed by two-way ANOVA with PROC GLM
turned off at night), the temperature of the growth medium in the benchtop treatments maintained 28 + 1°C. None
of SAS for Windows@ (23). Frequency data that were obtained for each treatment (e.g., survivorship, fitness) were analyzed by log-linear contingency tests with the BIOM-
of the moisture levels was likely to have affected the tissue hydration of these earthworms (8). Earthworms were fed a
PC package of computer programs of Sokal and Rohlf (26). Where environmental effects were significant (0 < 0.05),
cornmeal-based ration (7) ad l&turn weekly. The growth medium was replaced every two weeks.
results were reported as second-order response surface diagrams (1). Coefficients were obtained with Proc GLM of SAS for Windows (23). Figures were drawn with PSI-Plot’
Survivorship,
fresh weight and number of cocoons
were
recorded every 2 weeks. Earthworms were rinsed in aged tap water, blotted dry and weighed to the nearest 0.1 mg. The week in which the clitellum developed (indicating reproductive maturity)
was recorded. Earthworms
are hermaph-
(Poly Software (Software
International)
Publishing
and Harvard Graphics@ 3.0
Corporation).
RESULTS
roditic and although E. fetida usually reproduces by outcrossing (9), it is reportedly capable of limited selffertilization also (11). Thus cocoon production by single individuals was assumed to be proportional to cocoon production by outcrossing even though no offspring hatched from
Figure 1 shows a typical set of relationships among fresh weight (FW), survivorship and cocoon production in Eisenia fetida until constant weight was attained. The growth curve was sigmoidal and survivorship declined lin-
these cocoons
early with time. The clitellum
in any treatment
during the entire experi-
ment. Each treatment was maintained until the earthworms reached constant fresh weight or until survivorship declined below 50%. For simplicity, data were analyzed for four stages in ontogeny: newly hatched stage (3-5 weeks post-hatching = weeks O-2 of the experiment), juvenile stage (9-11 weeks post-hatching, before clitellum development in most treatments = weeks 6-8 of the experiment), early adult stage (15-17 weeks post-hatching, after clitellum development in most treatments = weeks 12-14 of the experiment ), and adult stage (sexually mature earthworms at maximum weight). Data that were obtained for each individual
developed
on about week
13; cocoon production began after week 15 and continued throughout the experiment. The timing of clitellum development and cocoon production was highly dependent on the environment (see below). During ontogeny an increase in the number of earthworms producing cocoons offset a decrease in the number of cocoons produced per earthworm (data not shown). Thus total cocoon production in the whole population (about 9.5 new cocoons per week) neither increased nor decreased during ontogeny. The great variability in cocoon production during ontogeny was probably caused by reduced fecundity associated with selfing.
Effect
of Environment
on Fitness
321
of Eisenia
TABLE 1. Two-way ANOVA tables analyzing the effects of moisture and temperature on fresh weight (FW) and 2-week growth rates of 5, 1l-, and 1‘I-week-old E&n& fhida
Variable S-Week
Model
FW
ll-Week
FW
17-Week
FW
3-5 Week growth rate
9-11 Week growth rate
15-17 Week growth rate
Moisture Temperature Moisture X Error Moisture Temperature Moisture X Error Moisture Temperature Moisture X Error Moisture Temperature Moisture X Error Moisture Temperature Moisture X Error Moisture Temperature Moisture X Error
df
temperature
temperature
temperature
temperature
temperature
temperature
F
2 372.02 3 75.42 6 38.52 2307 2 37.98 3 5.82 6 6.03 1768 2 344.45 2 60.11 3 51.95 1110 2 3 6 2309 2 3 6 1768 2 2 3 1107
P
15
315.76 122.83 21.15
8.57 62.10 17.26
0.0002
testing
the effects of moisture and temperature on fresh weights (FW) and two-week growth rates (d FW) of 3/5-, 9/l I- and 15/17 -week-old Eisenia fetida. Because sample sizes changed over time because of mortality, a repeated-measures ANOVA
was not used to test the effects of moisture and
temperature on growth during ontogeny. However, the probability of a Type I error among growth analyses was less than 0.01 according to a sequential Bonferroni test (22). There were significant interactive effects of soil moisture and temperature (p < 0.0001) on both fresh weight and two-week growth rates at each stage of ontogeny. The effects of soil moisture and temperature on these variables are depicted in Fig. 2. Early in ontogeny, the greatest growth rates occurred in high moistures (4 ml/g) and moderate temperatures (20,ZS”C). The least growth rates occurred in low moistures (2 ml/g) and low temperatures (15’C) as well as in low moistures (2 ml/g) and high temperatures (28“C).
(15’C). The least growth rates occurred in low moistures (2 ml/g) and high temperatures (28°C). This shift in the of greatest
growth
coincided
with
of sexual
maturity
in earthworms
(see
15
20
25
20
25
15
20
25
15
20
25
(“C)
FIG. 2. Effect of moisture and temperature on dy ontogenetic growth of ELFenia fetkh(A-C) Change in freshweight (g) per week between (A) 3-5 weeks, (B) 9-11 weeks, and (C) 15-17 weeks, respectively. (D-F) Absdute f+eshweight (g) of (D) 5., (E) lie, and (F) 17-week-old earthworms, respectively. Values are given for each contour.
that
the temperature/moisture
interaction
apparent late in ontogeny. Table 2 shows results of two*way ANOVAs
was more testing the
effects of moisture and temperature on maximum fresh weight (FW,,,), weeks to FW,,, and overall growth rate (FW,,/weeks
to FW,,,)
in E. fetida. Three
treatments
(2O”C, 4 ml/g; 28”C, 3 and 4 ml/g) were terminated because of high mortality before FW,,, was attained and were not included in these analyses. There were significant
interac-
tive effects of soil moisture and temperature (p < 0.0001) weeks to FW,,, and overall growth rate. The efon W,,,, fects of soil moisture
and temperature
on these variables
TABLE 2. Two-way ANOVA
tables dyzing the effects of moisture and temperature on maximum fresh weight and overall growth rate (FW_/ (Ew,,), weeks to m,,, weeks to FW,,_ ) in Eisenia fetida Variable
Fwmx
Weeks
to
Fw,,,
the develop-
Also by this time in ontogeny, growth rate had become more dependent on temperature than on moisture. Generally the effects of moisture and temperature on fresh weight (Fig. 2) were similar to those effects on growth rate with the excepment
25
TEMPERATURE
Later in ontogeny, the greatest growth rates occurred in moderate/high moistures (3, 4 ml/g) and low temperatures
environment
20
<0.0001
820.22 197.30 72.70
1 shows the results of two-way ANOVAs
15
tion
Table
25
below).
FW,/week
Model Moisture Temperature Moisture X temperature Error Moisture Temperature Moisture X temperature Error Moisture Temperature Moisture X temperature Error
df 2 3 3 838 2 3 3 838 2 3 3 838
F
P
1342.45 1519.90 34.77
3998.48 1336.38 19.30
<0.0001
567.59 249.59 65.90
M. L. Presley et al.
322
15
20
25
15
20
25
TEMPERATURE
15
20
25
(“C)
weeks to FW,,, and (C) overall growth rate (FW,,,Jweeks to Fw,,). Values are given for each contour.
are depicted in Fig. 3. The greatest FW,,, was attained by earthworms in high moistures (4 ml/g) and low temperatures (15’C).
It took up to five times longer for these earthworms to reach FW,,, than earthworms in other environments. Considering FW,,, and time to FW,,, together, earthworms in moderate temperatures (ZO’C) and high moistures (4 ml/g) reached the greatest FW,,, in the shortest time (i.e., they had the greatest overall growth rate). Earthworms in low moistures (2 ml/g) and high temperatures (28’C) had the least overall growth rate. Table 3 shows the results of a log-linear contingency
test
on the effects of moisture and temperature on the frequency of survivorship of adult earthworms. Only those paths testing the dependence of survivorship on moisture and/or temperature are reported; other paths are biologically
irrelevant
because moisture and temperature levels were fixed (26). The tests for conditional independence are analogous to tests of the interaction of main effects in an ANOVA. The test for complete independence of survivorship is somewhat analogous to tests of main effects in an ANOVA. The effect of moisture on survivorship depended on temperature and the effect of temperature on survivorship depended on moisture. Because both conditional independence tests (interaction effects)
were significant,
further consideration
of the
complete independence of survivorship from moisture and temperature was inappropriate (26). The effects of soil moisture and temperature
on survivorship of adult earthworms
TABLE 3. Three-way log&near contingency test (G,,,,) analyzing the effects of moisture (M) and temperature (T) on survivor-ship (S) in Eiseniafetida (The null hypothesis is independence) Variable
Survivorship
Model
Conditional Independence S x M from T S x TfromM Independence of S from M&T
df
G
P
8
113.88 307.56 333.33
CO.001
9 11
20
15
FIG. 3. Effect of moisture and temperature on final growth of Eiseniahida. (A) Maximum fresh weight (FW,,, g), (B)
25
TEMPERATURE (“C) FIG. 4. Effect of moisture and temperature on survivor-ship (%) of Eiseniafetida. Values are given for each contour.
are depicted
in Fig. 4. The greatest survivorship occurred
in moderate
temperatures
(ZOOC) regardless of moisture.
Generally survivorship was high in a wide range of moistures and temperatures. The least survivorship occurred in high moistures (4 ml/g) and high temperatures
(28’C).
Table 4 shows the effects of moisture and temperature on factors reflecting the fecundity of earthworms. There were significant interactive effects of soil moisture and temperature on the frequency of earthworms with a clitellum (threeway log-linear contingency test, p < O.OOl), weeks to clitellum development
(two-way ANOVA,
p < 0.0001)
and co-
coons produced per surviving earthworm per week (two-way ANOVA, p < 0.0007). Because a large number of earthworms did not produce
cocoons,
the latter variable
was
coded and normalized by a square root of (x + 0.5 ) transformation prior to statistical analysis. Using data from only those earthworms that produced cocoons would have been desirable to reduce variance in fecundity, but it was necessary to use data from all earthworms whether they produced cocoons or not in order to include those treatments where no cocoons were produced in the analysis. The effects of soil moisture and temperature on fecundity are depicted in Fig. 5. The greatest percentage of earthworms that developed a clitellum during the course of the experiment occurred in high moistures (4 ml/g) and low temperatures (15°C); the least percentage of earthworms with a clitellum occurred in high temperatures (28°C) and low moistures (2 ml/g). In contrast, the clitellum developed most rapidly in high temperatures (28°C) and high moistures (4 ml/g) and least rapidly in low moistures (2 ml/g) and low temperatures (15°C; data not shown). The disparity between the percent of earthworms with a clitellum (53% overall) and the percent of earthworms producing cocoons (8% overall) suggests that the latter underestimates the true fecundity of the species, likely because of selfing. We assumed that selfing depressed cocoon production to the same extent in all treatments and that cocoon production as measured was a valid index of reproductive output. Reproductive output (co-
Effect of Environment on Fitness of Eiseniu
TABLE 4. Effects of moisture (M)
323
and temperature
(T) on fecundity
Variable
Model
Weeks to clitellum development
per earthworm
df
Conditional Independence F X MfromT F X TfromM Independence of F from M & T Moisture Temperature Moisture X temperature
Frequency of earthworms with clitellum
Cocoons
(F) in Eiseniafetida
Error Moisture Temperature Moisture X temperature Error
per week
For G
8 9 11 2 ; 1304 2 3 6 1308
R
377.11 1366.48 1505.80 59.69 16.41 12.73
10.52 5.32 3.94
<0.0001
0.0012 0.0007
was used to analyze frequency of earthworms with a clitellum. The null hypothesis is independence. A A three-way log-linear contingency test (G,,,,,,) two-way ANOVA table was used to analyze weeks to clitellum development and cocoons produced per earthworm per week. Data were transformed by the square root of (x + 0.5).
per week) apparently reflected a bal-
were considered to be fit, whereas 0% of the earthworms in
ance between the percent of earthworms attaining reproductive maturity and the time to reproductive maturity. Thus the greatest number of cocoons produced per earth-
the 2 ml/g, 28°C treatment were considered to be fit. For convenience and conservatism, frequency analyses were conducted on a sample size set at 100 earthworms per treat-
worm per week occurred
ment. The effect of moisture on relative fitness depended on temperature and the effect of temperature on relative
coons per earthworm
in high moistures
(4 ml/g) and
moderate temperatures (20, 25%). The least cocoon production occurred in low moistures (2 ml/g) and low temperatures ( lS°C)
as well as in low moistures (2 ml/g) and high
fitness frequency depended on moisture. The effects of soil moisture and temperature on relative fitness frequencies are
temperatures (28’C). Table 5 shows the results of a log-linear contingency test on the effects of moisture and temperature on the relative
depicted in Fig. 6. The greatest relative fitness occurred in
fitness of earthworms (- relative frequency of fit earthworms). A fitness index was computed as the product of survivorship and reproductive output for each treatment.
(2 ml/g) and low temperatures (15°C) as well as in low moistures (2 ml/g) and high temperatures (28’C). Because of the large number of statistical tests presented, a sequential Bonferroni test (22) was performed simulta-
The relative fitness of earthworms in each treatment was computed as a percent of the maximum fitness index among treatments. In effect this corrected for the reduced levels of fecundity because of selfing. Thus for relative fitness analyses, 100% of the earthworms in the 4 ml/g, 25°C treatment
high moistures (4 ml/g) and moderate temperatures (20, 25’C). The least relative fitness occurred in low moistures
neously on all statistics reported in Tables l-5
to determine
the probability of Type I errors. This is an extremely conservative analysis because Type I errors were already controlled within each of the ANOVAs. Nevertheless the probability of a Type I error occurring anywhere among these analyses of moisture and temperature effects was less than 0.01.
A
B TABLE 5. Three-way lwlinear contingency test (GWilli_) analyzing the effects of moisture (M) and temperature (T) on relative fitness (RF) in Ebenia fetida Variable Relative fitness
-15
20
25
15
TEMPERATURE
20
25
(“C)
FIG. 5. Effect of moisture and temperature on fecundity of Ehenia fetida(A) Percent of individuals developing a clitelhun and (B) cocoons produced per earthworm per week. Values are given for each contour.
Model
df
G
p
Conditional independence RF X M from T RF X T from M Independence of RF from M&T
8 9 11
404.33 373.27 586.60
Relative fitness for each treatment was computed as the number of cocoons per earthworm per week X survivorship and was expressed as a percent of the maximum fitness among treatments based on a sample size of 100 earthworms (see text). The null hypothesis is independence.
M. L. Presley et al.
324
ment: high moisture and moderate temperature.
Although
cocoon production was the best index of fecundity available, it could not take into account differences in the number of viable embryos within each cocoon which likely occurred in an environment-dependent manner. Also populations at high temperatures attained reproductive maturity before populations in other environments and thus may be able to produce more generations
per unit time.
Over time this would increase the fecundity of high-temper-
15
20
25
ature populations beyond that which was evident from cocoon production in one generation alone. The environmental pattern of fitness was more similar
TEMPERATURE (“C) FIG.6. Effect of moisture and temperatureon relativefitness of Eisenia bib. Relative fitness for each treatment was computed as the number of cocoons per earthworm per week x survivorshipand was expressed as a percent of the maximum fitness among treatments (see text). Values are given for each contour.
to that of fecundity than survivorship even though both variables were mathmatically equivalent in computing the fitness index. This occurred because the coefficient of variation in fecundity
among treatments
was almost five times
greater than that of survivorship. Thus fecundity contributed more to the variation in fitness than did survivorship. Fitness was maximal in high moisture and moderate temperature and declined as moisture decreased and as temperature
DISCUSSION The combination
of soil moisture and temperature
produc-
ing maximal growth, expressed as absolute fresh weight or change in fresh weight, changed during ontogeny. Although the most favorable soil moisture level for growth was always high (4 ml/g), the most favorable temperature shifted from moderate-high (ZO-2S°C) to low ( ISaC) by the time of reproductive maturity and remained that way until maxima1 fresh weight was attained. This shift may rep-
increased or decreased. The same limitations on the interpretation of the fecundity results also apply to the interpretation of fitness. In particular the fitness of high-temperature populations may have been underestimated by the inability to account for the greater number of generations that may occur in those environments. Although the effects of soil moisture and temperature on growth and fitness of E. fetida measured here were reason-
resent catch-up growth in low-temperature earthworms as high-temperature earthworms directed resources to repro-
ably consistent with previous reports, some conspicuous differences exist. Our study confirmed reports that rates of fecundity and growth early in ontogeny varied with
duction
temperature below the upper thermal limit (4,14,27).
rather
than
somatic
growth.
However
because
How-
earthworms in high moisture and low temperature attained the greatest maximal fresh weight among earthworms of all
ever in the present study, maximal fresh weight and percent of earthworms reaching reproductive maturity varied in-
treatments,
versely with temperature. Earthworms became acclimated to low temperature sufficiently well that physiological rates later in ontogeny exceeded those of high-temperature earth-
it is also likely that this shift represents an onto-
genetic shift in environmental tolerance. The effects of soil moisture and temperature on survivorship were different from those on growth. Maximal survivorship occurred at moderate temperature (20°C) and moderate moisture (3 ml/g). The pattern did not change throughout ontogeny (data not shown) and survivorship was generally more dependent on temperature than on soil moisture. The different ontogenetic patterns of growth response vs. survivorship response to the environment
suggest
that two different mechanisms of physiological acclimation were operating. The effects of soif moisture and temperature on fecundity were more complex. For those earthworms that reached reproductive maturity, the time to maturity occurred most rapidly at high moisture and high temperature. However more earthworms reached maturity at low temperature than at high temperature. Both of these factors influence (but not exclusively) the number of cocoons produced in different environments. Nevertheless cocoon production was maximal in what would seem to be an intermediate environ-
worms. Our study failed to confirm that maximal
growth
and fecundity occurred in a moderate soil moisture as opposed to high or low moisture, contrary to that reported by Reinecke and Venter (2 1). Th’ 1s d‘rscrepancy is likely caused by the different physical properties of the soils used between the studies (peat moss vs. manure). Peat moss retains water to a greater extent than manure (pers. obs.) so that for the same volumetric water content, less water is available to earthworms in peat moss than in manure. Treatments with moisture content greater than 4 ml HLO/g peat moss (80% moisture) would have likely been required to depress growth and fitness in E. fetida. Finally, previous studies (15) have not identified the subtle (but highly significant) interactions between temperature and moisture that affect growth, fecundity and survivorship of E. fetida. Growth rate, fecundity and fitness were more sensitive to temperature at low moisture than at high moisture. On the other hand, survivorship and maximal fresh weight were more sensitive
Effect of Environment
to temperature
on Fitness of
Eiseniu
325
at high moisture than at Iow moisture. Either
contrary to the work of Hawkins er al. (12,13)
and Koehn
way, appropriate soil moisture levels may ameliorate the ef-
and Bayne ( 16). In short, growth measured anytime in on-
fects of suboptimal temperature on earthworm physiology. Early in ontogeny, growth (FW, change in FW) was a
togeny has the potential to contribute some information about the htness (fecundity or survivorship) of the individ-
good predictor of fecundity (cocoons per week per earthworm) but not survivorship based on visual comparisons
ual. Growth measured prior to reproductive the greatest potential in this regard.
among response surface diagrams. Early growth was a good predictor of overall fitness since more of the variation in fitness could be attributed to survivorship.
to variation
After reproductive
change in FW, FW,,,)
in fecundity
than
maturity, growth (FW,
was a marginal predictor of survivor-
ship to the extent that both greater growth and survivorship occurred in lower temperature earthworms. The relationship between early ontogenetic growth and fecundity is predicted by the balanced energy equation (30) where somatic growth and reproduction represent competing pathways for energy use. Typically the energy use between pathways is segregated ontogenetically as seen here. This relationship should be independent of environmental variability so long as reproduction
is not postponed. This was confirmed in the
present study where early growth (d EW, weeks 3-5) was also correlated with fecundity (total no. cocoons of fecund earthworms) within the constant 20°C 3 ml/g treatment (r = 0.340; df = 33; p < 0.05). The marginal relationship between growth of mature earthworms
and survivorship
is
not as well grounded in bioenergetic theory (20). It may reflect the outcome of an optimai strategy between early growth and fitness cost (i.e., mortality) (24) extended over a variable environment that produces the same artifacts as comparisons among species (25). As such, a relationship between mature growth and survivorship would be dependent on the presence of environmental variability. This was also confirmed in the present study because mature growth (d Fw, weeks 15-17) was not correlated with survivorship (weeks to death) within the constant 20°C 3 ml/g treatment (r = 0.055; df = 190; NS). We sought to test two hypotheses:
(a) patterns
history and fitness responses to environmental
of life
variability
would be similar to each other, and (b) growth early in ontogeny would be a good predictor of adult fitness. The first hypothesis was not supported. Responses of growth, fecundity, survivorship and ftmess to the environment differed from each other to various degrees. Responses of growth to the environment changed with ontogeny. The second hypothesis was supported. The environmental response of growth early in ontogeny
was very similar to that of fecun-
dity and fitness. This supports the use of early ontogenetic growth as a surrogate of fitness in studies examining the effects of individual multilocus heterozygosity in earthworms (6,2). Because we used variation in the environment to produce variation in fitness, it is possible that the associations reported here would not hold for genetically-induced variation in fitness. If this were confirmed, it would imply that the genetic effect on fitness operates through different bioenergetic mechanisms than the environmental effect,
maturity has
We thank Melinda Chow, Rebecca Brasfeiti, Warren Cox, April Heinsch, Gzrie Loundensluger, Tara Mann, Yonya Nabors and Theresa Pouchfurassistance in dam colkctim and earthwum husbandry. This research was suppurredby NSF grant DEB-9221094 to WID.
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