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The influence of soil moisture and temperature on the survival, aestivation, growth and development of juvenile Aporrectodea tuberculata (Eisen) (Lumbricidae)
Pedobiologia 45, 121–133 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/pedo
The influence of soil moisture and temperature on the survival, aestivation, growth and development of juvenile Aporrectodea tuberculata (Eisen) (Lumbricidae) Linda A. Wever1, Timothy J. Lysyk2 and M. Jill Clapperton2* Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta, Canada, T1K 3M4. [email protected] 2 Agriculture and Agri-food Canada, Lethbridge Research Centre, P.O. Box 3000, Lethbridge, Alberta, Canada, T1J 4B1. [email protected] 1
Submitted: 30. June 2000 Accepted: 23. August 2000
Summary Soil moisture and temperature are the primary factors determining earthworm survival and growth. Few studies, however, have examined the combined effects of soil moisture and temperature on earthworm physiology and ecology. The objective of this study was to evaluate the influence of soil moisture and temperature on the survival, growth and sexual development of an endogeic species, Aporrectodea tuberculata. Juvenile earthworms were grown at all possible combinations of four soil moistures (10, 15, 20 and 25 %, dry weight basis) and temperatures (5, 10, 15 and 20 °C). Logistic regression was used to determine the relationships between the proportion of surviving individuals with soil moisture and temperature, and time. Stepwise regression analysis was used to determine the various possible relationships between temperature, moisture and time on earthworm growth. Soil moisture accounted for 48 % of the variation in earthworm survival with the lowest survival associated with 10 % soil moistures. The interaction terms, time × temperature × moisture, and time × temperature, accounted for 63 % of the variation in earthworm growth. The greatest increase in earthworm weight was in soil incubated with 25 % moisture at 15 and 20 °C. There was also an obvious relationship between survival and growth linked to the interaction *E-mail corresponding author: [email protected]
0031–4056/01/45/02–121 $ 15.00/0
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between soil temperature and moisture. After 10 wk the only post-clitellate individuals were observed in the 25 % and 20 °C treatment. Clitellate earthworms weighed 1.5 times more than individuals having only genital tumescences (GT). The results of the study showed that the effects of soil moisture on earthworm growth and survival are modified by soil temperature. Key words: Earthworms, juveniles, growth, survival, soil temperature, soil moisture, growth modeling, Aporrectodea tuberculata, aestivation
Introduction Soil moisture is a major factor limiting earthworm survival, growth and reproduction (Lee 1985). Temperature and moisture influence embryonic development, sexual maturation, reproductive success and longevity (Böström & Lofs 1996; Butt 1997; Butt et al. 1992; Daniel 1992; Frenot 1992; Hallatt et al. 1992; Holmstrup et al. 1996; Kretzschmar & Bruchou 1991; Muyima et al. 1994; Reinecke et al. 1992; Uvarov 1995; Viljoen & Reinecke 1992). Most laboratory experiments did not focus on endogeic or geophagus species that often dominate in agroecosystems (Auerswald et al. 1996; Baker et al. 1999; Clapperton 1999; Clapperton et al. 1997). A few studies have addressed how soil moisture and temperature interact to influence earthworm growth and development (Presley et al. 1996; Whalen & Parmelee 1999), however, only a limited range of temperatures and moistures were used in these studies and most do not apply to semi-arid agriculture. There is a critical requirement for such ecophysiological studies if we are to adequately explain observed seasonal variation in earthworm biomass, numbers and age structure between treatments in field studies (Garnsey 1994). The objective of this study was to examine the combined influence of soil moisture and temperature on survival, growth and development of Aporrectodea tuberculata, a species common to the agricultural fields in the Canadian prairies (Clapperton 1999). We used juvenile earthworms in the experiment to ensure that changes in live weight would reflect growth and not be confounded by the demands of reproduction (Daniel et al. 1996) and as early growth can be used to predict fitness and fecundity (Presley et al. 1996).
Materials and Methods Dark Brown Chernozemic Lethbridge Loam (Typic Borroll) soil was sieved to 2mm to ensure homogeneity. Juvenile A. tuberculata were collected from an agricultural field site in mid-October. All juvenile earthworms were sorted according to similar size and temporarily stored in field soil at 15 °C. Four different soil moistures (10, 15, 20 and 25 % water content on a dry mass basis) and temperatures (5, 10, 15 and 20 °C) were combined for a total of sixteen treatments with twenty replicates of each treatment. Each replicate consisted of one hundred grams of air-dried soil, 3 g of ground hard red spring wheat grain, an amount of tap water appropriate to the desired soil moisture, and one juvenile A. tuberculata placed in a 125 ml plastic container with a perforated lid. Earthworms were rinsed in tap water, carefully blotted and weighed prior to being added to the container.
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The treatments were then incubated in total darkness in constant temperature chambers for a total of 10 weeks. Each week, the contents of the containers were hand-sorted, the earthworm was observed for presence of sexual characteristics such as genital tumescences (GT), tubercula pubertatis (TP) and clitellum development, rinsed, blotted and weighed. The containers were also watered to weight in order to maintain the soil moisture. The proportion of surviving earthworms for a given week was determined by dividing the number of surviving earthworms by the total number of earthworms in each treatment (n = 20). A logistic regression was used to model the probability of earthworm survival as a function of soil moisture, temperature and time and any possible two or three way interactions. A binary variable was created for each observation where Y = 1 if the earthworm was alive or Y = 0 if the earthworm was otherwise dead or missing. The proportion of aestivating earthworms was also calculated for each week of the experiment and logistic regression used to examine the relationships between the probability of aestivation and soil moisture, temperature, time and any possible two or three way interactions. The terms time2 and time3 were included to account for curvilinearity in the relationship with time. A binary variable was created where Y = 1 if the earthworm was aestivating and 0 if not. All earthworms that were colourless, inactive and wound into a tight ball were considered to be aestivating. Data for growth was presented for only the first six weeks of the study, to when the first post-clitellate earthworm was observed. During the experiment it was found that a small number of the containers had A. trapezoides (n = 5). It was decided to include these individuals as part of the data set as they responded similarly to the treatments and were approximately the same size. In a previous experiment, Wever & Clapperton (1996) found that A. trapezoides had optimum growth at 25 % soil moisture at 15 to 20 °C. For modeling purposes, relative worm weight was calculated weekly for each worm as ri = weighti/weight0 where i = week the observation was made. Relative worm weights > 1 indicate that earthworms had increased weight, while relative weights < 1 indicate the worms had lost weight. Since all relative weights at week = 0 were 1, these were not included in the analyses. The relative weights were transformed to log(ri) to stabilize the variances. Stepwise multiple regression was used to determine the relationship between log(ri) and time (weeks), time2, temperature, moisture, and all possible two-way and three-way interactions between temperature, moisture and time. The term time2 was included to account for curvilinearity in the relationship with time. Variables were retained in the model if they contributed significantly (P < 0.05) to a reduction in error sums of squares of a model containing variables entered previously. The mean weight of earthworms with various sexual characteristics was calculated by pooling all of the treatments. The individual weights of earthworms with GT and/or TP observed over the course of the study were batched and an average weight and standard deviation were calculated.
Results Survival of juvenile earthworms ranged from 0 to 100 % (Fig. 1). At 20(C, the survival curves for different soil moistures were well separated with the 25 % treatment having the highest rate of survival followed by 20 and 15%. The lowest rate of survival was associated with a soil moisture content of 10 % regardless of the incubation temperature. The logistic regression model model P(surv = 1) = 1/(1exp(β0+β1Z+β2XZ+β3XY)) where X = time in weeks, Y = temperature (°C), and Z = % moisture indicated that identified variables accounted for 54 % of the variation observed in earthworm survival (Table 1). The variables that significantly influenced the proportion of earthworms surviving were soil moisture, moisture × time and temperature × time with soil moisture accounting for 47.9 % of the variation in P(surv). The soil moisture by time in-
Fig. 1. A comparison of the observed and model survival of juvenile A. tuberculata incubated at 5, 10, 15 and 20 °C and 10, 15, 20 and 25 % soil moisture content over a 10 wk period. Model r2 = 0.544, n = 20
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Table 1. Results of logistic regression analysis for the effects of time, temperature and moisture content on earthworm survival Parameter Estimate SE t (β=0) model r2 β0 -6.7351 0.2972 -22.6 β1Z -0.6669 0.0286 23.3 0.479 β2X*Z -0.0135 0.0023 - 5.8 0.536 β3X*Y -0.0096 0.0018 - 5.4 0.544 The logistic regression equation is P (surv=1)=1/(1exp(β0+β1Z+β2XZ+β3XY)) where X = time in weeks, Y= temperature (C), and Z = % moisture teraction accounted for 5.7 %, and the time × soil temperature term accounted for an additional 0.8 % of the variation in survival. The observed and modeled proportion of surviving earthworms is shown in Figure 1. In general, proportion of the surviving earthworms decreased with time, the probability of survival increased with moisture content, and only slightly decreased with increasing temperature. The rate of decreasing earthworm survival increased with increasing soil temperature and decreasing soil moisture (Fig. 1). Aestivating earthworms were observed in every treatment with the exception of the three treatments that had survival rates (15 % after the first week. These were the 10 % soil moisture treatments at 5, 10 and 15(C (Fig. 2). The basic form of the aestivation model was P (aest = 1) =1-1/(1+exp(β0+ β1XZ+β2X2+β3Z2+β4X3+β5X+β6YZ+ β7Y+β8Z)) where X=time in weeks, Y=temperature (°C), and Z = % moisture. The model accounted for only 12 % of the variation in the number of individual earthworms aestivating (Table 2). The variables which significantly influenced the proportion of aestivating earthworms were time, temperature, temperature × moisture, moisture, moisture2, moisture × time. In general, the number of aestivating earthworms increased as soil moisture increased up to 20 % after which there was a decrease in the number of aestivating individuals. Earthworms reared at 10, 15 and 20 % soil moisture were more likely to aestivate in the first five weeks whereas earthworm at 25 % soil moisture are more likely to aestivate in the tenth week. As temperature increased, the proportion of aestivating earthworms peaked during weeks 3 and 4 as temperature increased. The model also predicted a delay in aestivation if soil moisture was 20 % or greater. Earthworms grew best at a soil moisture content of 25% and incubation temperatures of 15 and 20 °C (Fig. 3). However, juveniles reared in soil at 20 % and 5 °C grew equally as well as those in soil at 25 %. The model developed to explain this relation ship was ln(ri) = β0+β1X-β2X2+β3Y+β4Z-β5XY+β6XYZ where X= time in weeks, Y= temperature (°C), and Z = % moisture. When the soil moisture temperature interaction term was added the model significantly (F = 405.8; df = 6, 1144; P < 0.0001) accounted for 68 % of the variation in the relative weights of individual earthworms. The variables that significantly influenced the relative weights of individual earthworms were time, time2, temperature, moisture, time × temperature, and time × temperature × moisture (Table 3). The two interaction terms, time × temperature × moisture, and time × temperature, accounted for 63 % of the variation in ln(ri). These also had the highest partial r2, indicating they accounted for a larger proportion of the va-
Fig. 2. A comparison of the observed and modeled aestivation of surviving juvenile A. tuberculata incubated at 5, 10, 15 and 20 °C and 10, 15, 20 and 25 % soil moisture content over 10 wk Model r2 = 0.118
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Table 2. Results of logistic regression analysis for the effects of time, temperature and moisture content on earthworm aestivation Parameter Estimate SE t (β=0) model r2 β0 -9.2016 1.6373 -5.6199 β1X*Z -0.0558 0.0071 -7.8454 0.015 β2X2 -0.4934 0.0549 -8.9895 0.026 β3Z2 -0.0176 0.0041 -4.2913 0.057 β4X3 -0.0271 0.0033 -8.2878 0.074 β5X -1.5059 0.2716 -5.5447 0.102 β6Y*Z -0.0132 0.0027 -4.9678 0.104 β7Y -0.2490 0.0535 -4.6555 0.111 β8Z -0.5497 0.1586 -3.4663 0.118 The logistic regression equation is P(aest=1)=1-1/(1+exp(β0+β1XZ+β2X2+β3Z2+ β4X3+β5X+β6YZ+ β7Y+β8Z)) where X = time in weeks, Y= temperature (°C), and Z = % moisture
riation in ln(ri) given that all other parameters were in the model. The main effects, time, temperature, and moisture contributed an additional 2.5% to the variation already accounted for by the interaction terms, with individually accounted for 1-9% given all other variables were in the model. The change in relative weights of the earthworms with respect to time, temperature and moisture were examined from the partial derivatives (Fig. 3). The partial derivative with respect to moisture was δln(ri)/δZ = β4+β6XY and showed that the rate of gain generally increased with moisture content, and the increases were greater as temperature and time increased. The partial derivative with respect to temperature was δln(ri)/δY = β5X+β6XZ showing that the rate of gain with respect to temperature was positive, but decreased as time increased, and increased as both moisture and time increased. The partial derivative with respect to time was δln(ri)/dX = β1-2β2X-β5Y+ β6YZ, and again the rate of change of earthworm weights declined with time. HoweTable 3. Results of stepwise regression analysis for the effects of time, temperature and moisture content on relative weight of earthworms Parameter Estimate SE t (β = 0) model r2 β0 -1.0039 0.0881 -11.4 β6X*Y*Z -0.0013 0.0001 -17.7 0.1331 β5 X*Y -0.0299 0.0018 -16.9 0.6303 β4 % moisture (Z) -0.0345 0.0033 -10.4 0.6609 β3 C (Y) -0.0250 0.0035 - 7.2 0.6720 β1 Time (X) -0.1580 0.0326 - 4.8 0.6774 β2 Time2 (X2) -0.0159 0.0049 - 3.3 0.6840 Model is ln(ri)= β0+β1X-β2X2+β3Y+β4Z-β5XY+β6XYZ where X = Y = temperature (°C), and Z = % moisture
r2 (β1/βn) 0.2154 0.1996 0.0857 0.0428 0.0201 0.0093 time in weeks,
Fig. 3. A comparison of the observed and modeled earthworm growth rate of juvenile A. tuberculata incubated at 5, 10, 15 and 20 °C and 10, 15, 20 and 25 % soil moisture content over 6 wk. Model r2 = 0.684, n = 20.
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Table 4. Effect of temperature on the sexual development of juvenile A. tuberculata grown at 25 % soil moisture Temperature (°C)
No. of individuals No. of individuals No. of post-clitellate with GT and/or TP with a clitellum individuals 5 0 0 0 10 1 0 0 15 11 3 0 20 7 4 3 No clitellate individuals were present at any soil moisture below 25 % regardless of incubation temperature at the end of the ten week study
ver, changes in weight gains over time were dependent on temperature and moisture (Table 4). The coefficient for temperature alone indicated that changes in rate of weight gain were reduced at higher temperatures, the moisture by temperature interaction indicated that changes in rate of gain with respect to time were dependent on a combination of temperature and moisture, and increased as both temperature and moisture increased. During the course of the study some of the earthworms developed sexual characteristics. However, no clitellate individuals were observed at soil moistures lower than 25 % at any temperature and only one treatment (25 % and 20 °C) had post-clitellate individuals. The mean live mass at which GT and/or TP became consistently observable was 833 ± 209 mg (n=32). Clitellum development followed when earthworms reached 1250 ± 227 mg (n=8) while post-clitellate individuals weighed slightly less at 1235 ± 191 mg (n=3).
Discussion The results of our study indicate that the activity of earthworms is governed by the temperature of their surroundings, in agreement with other experiments (Daniel 1992; Viljoen & Reinecke 1992; Frenot 1992; Daniel et al. 1996). Earthworms gained more weight as the incubation temperature increased. A. tuberculata juveniles reared at 10 °C and 25 % soil moisture grew much slower than those at 20 °C and the same moisture content (Fig. 3). Whalen & Parmelee (1999) showed that the instant growth rate of A. tuberculata at 10 °C and 30 % moisture (w/w) was 10.8 ± 1.4 × 10-3 d-1 and doubled to 17.0 ± 1.6 × 10-3 d-1 at 18 °C. Soil moisture content is considered to be the primary factor limiting survival of earthworms (Doube & Styan 1996; Hallatt et al. 1992; Kretzschmar & Bruchou 1991; Lee 1985; Satchell 1967). The survival model showed that 54 % of the variation in the survival data could be explained by moisture (47 % variation), time by moisture and time by temperature interaction (Table 1). The decline in survival with time is likely to be a function of the handling of the earthworms or aging. Including a soil moisture and temperature interaction into the model did not significantly reduce the error sum of squares suggesting that this interaction was not influencing earthworm survival. However, as the temperature increased above 5 °C to
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10 and 20 °C earthworm survival at 20 % moisture declined (Fig. 1). At 20 °C survival was reduced in all treatments except for earthworms maintained at 25 % moisture (Fig. 1). Temperature has been reported to affect mitochondrial activity in earthworms (Arillo & Melodia 1991; Uvarov 1995). Earthworms subjected to constant temperature decreased in body weight, and had increased rates of reproduction and mortality (Uvarov 1995). Whereas, earthworms subjected to fluctuating temperatures had increased rates of survival but did not reproduce (Uvarov 1995). However, the response of earthworms to a change in temperature depends on the thermal history of the earthworms before experiments are initiated (Arillo & Melodia 1991). The earthworms used in our experiments were collected in October under cooler soil temperatures (8–12 °C), with adequate moisture (20–25%) the earthworms survived at all temperatures (Fig. 1) and gained weight (Fig. 3). Furthermore, it is likely that the decreasing survival and weight gain for earthworms at 10, 15 and to a lesser extent 20 % moisture in the 15 and 20 °C treatments resulted from inadequate moisture when the rate of earthworm metabolism was rapidly increasing (Arillo & Melodia 1991; Uvarov 1995). The data for earthworm aestivation in our experiments reinforced the link between survival and growth rate with respect to a temperature and moisture interaction. The proportion of aestivating earthworms increased with increasing temperature particularly at the two lowest soil moistures (10 and 15%) (Fig. 2) which increased the rate of survival of the earthworms in the 10% moisture and 20 °C soil temperature treatment (Fig. 1). These results likely reflect the effects of the thermal history of the earthworms used in our study (Arillo & Melodia 1991). Earthworms kept in higher soil moistures (20–25 %) at the two lowest soil temperatures responded in a field-like manner in that they gained some weight (Fig. 3) before entering torpor after 6 weeks at 5 °C and 8–10 weeks at 10 °C (Fig. 2). These results would be expected for earthworms collected from the field at (8–12 °C) given the results of other studies (Arillo & Melodia 1991; Uvarov 1995). However, the effects of handling on the earthworms may also have influenced the number of aestivating earthworms as the study progressed (Fig. 2). Our results reveal that temperature and moisture were interacting to influence the growth of juvenile A. tuberculata (Table 3). The growth rate of earthworms reared at 5 °C at 20 and 25 % moisture was similar (Fig. 3). After 4wk at 10 °C the growth rate for earthworms at 20 % moisture began to decline, the growth rate for the earthworms at 25 % moisture increased, and others remained the same (Fig. 3). This was mirrored in the survival curves at the same incubation temperatures, which showed similar survival rates for 15, 20 and 25 % moisture at 5 °C, and a slight decline in survival at 20 % moisture at 10 °C (Fig. 1). At 15 °C there was a further, more rapid decline in growth for all treatments which can also be clearly seen in the divergence between the survival curves for earthworms at all soil moisture levels (Fig. 1). The survival of earthworms incubated at 20 °C clearly differed depending on the soil moisture (Fig. 1). This was also the only incubation temperature where, after 6wk, there were still some earthworms at 10 % moisture, and a rapid and steady decline in the growth of earthworms at 15 % moisture. It appeared that low temperatures modified the effects of inadequate soil moisture. At low temperatures, water loss from respiration, and excretion are minimized due to lower metabolic rates (Edwards & Lofty 1972) and soil moisture is less limiting to growth. Conversely, our results showed that as the soil temperature and earthworm metabolic rate increase there is a greater requirement for water. Our model showed that 67 % of the variation in the growth rate data could be explained by a temperature moisture interaction (Table 3). This is consistent with the
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literature for other earthworm species that showed that water loss could be readily replenished under high soil moisture conditions as pF decreases with increasing temperature despite a doubling of the metabolic rate of earthworms for every 10 degree rise in temperature (Lee 1985). This makes it easier for earthworms to extract water from the soil to regulate body water content and drive cell processes of division and growth and consequently, more energy can be allocated to growth and development. High soil moistures also allow for evaporative cooling (Satchell 1967). Kretzschmar and Bruchou (1991) suggested that water availability and not soil water content influenced earthworm growth and that the threshold for the temperature-moisture interaction would differ according to soil texture (Doube & Styan 1996; Kretzschmar & Bruchou, 1991). Together with our study, it is clear that the effects of soil moisture on earth worm growth and survival should be examined with respect to the modifying effects of temperature. The environmental conditions which enhance earthworm growth also promote sexual maturity so it is not surprising that the only clitellate individuals in our study were associated with the high soil moisture and temperature treatments. Muyima et al. (1994) reported that substrate moisture profoundly influenced the growth rate and clitellum development of juvenile Dendrobaena veneta. The literature also suggests that temperature dramatically influences sexual development of other earthworm species such as Allolobophora chlorotica, Dendrobaena veneta, Dendodrilus rubidus (Butt 1997; Muyima et al. 1994; Frenot 1992). In our study the sexual maturity of A. tuberculata appeared to be influenced by soil moisture and temperature. Tubercula pubertatis and/or genital tumescences were observed at a mean live weight of 833 ± 209 mg. As expected, clitellate individuals weighed significantly more (1251 ± 227 mg) than non-clitellate A. tuberculata, and the earthworms continued to increase in weight until they become post-clitellate. Published weights for the closely related species Aporrectodea trapezoides collected from the field were much lower: 498 ± 34 mg with TP and 953 ± 47 mg with clitellum (McCredie et al. 1992). In the same study, aestivating individuals collected and reared in the laboratory until the clitellum was fully developed weighed nearly 1400 mg. This demonstrates the need for caution when comparing field and laboratory studies. In the semi arid agricultural ecosystems of the Canadian prairies A. tuberculata is most often found below 10 cm soil depth and more abundant under no tillage (Clapperton 1999). It is well known that the spring soil temperature is lower and the soil moisture content higher under reduced and no tillage systems compared with conventional tillage. Despite the lower temperature under no tillage, studies consistently report more earthworms (Parmelee et al. 1990; Nuutinen 1992; Francis & Knight 1993) and increased species diversity (Clapperton et al. 1999) in reduced and no tillage treatments. Interestingly, the literature suggests that a lower soil temperature is required for hatchling survival (Viljoen et al. 1992). Uvarov (1995) found that temperatures fluctuating between 5 and 25 °C resulted in increased survival but reduced fertility, and weight gain. Earthworms become abundant in semi-arid agriculture at a soil temperature between 5 and 10 °C in spring on the Canadian prairies (Clapperton 1999) under 18 to 25 % soil moisture content, and begin aestivating before soil moisture content is below 15% and soil temperatures are above 20 °C (Clapperton 1999). The results of our study are consistent with the behaviour of A. tuberculata in the field under semi-arid agriculture demonstrating that an interaction between soil moisture and temperature limit the survival, growth, and maturity this earthworm species.
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Acknowledgements We are very grateful to J. Unrau, F. Dunstan, D. Kanashiro, and N. Lee for their technical assistance, Geoff Baker (CSIRO, Division of Entomology, Canberra Australia) for improving the manuscript, and Canada-Alberta Environmentally Sustainable Agriculture (CAESA) Agreement for partially funding this study.
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Kretzschmar, A., Bruchou, C. (1991) Weight response to the soil water potential of the earth worm Aporrectodea longa. Biology and Fertility of Soils 12, 209–212. Lee, K. E. (1985) Earthworms: their ecology and relationship with soils and land use. Academic Press, Sydney, Australia. McCredie, T. A., Parker, C. A., Abbott, I. (1992) Population dynamics of the earthworm Aporrectodea trapezoides (Annelida:Lumbricidae) in Western Australia pasture soil. Biology and Fertility of Soils 12, 285–289. Muyima, N. Y. O., Reinecke, A. J., Viljoen-Reinecke, S. A. (1994) Moisture requirements of Dendrobaena veneta (Oligochaeta), a candidate for vermicomposting. Soil Biology and Biochemistry 26, 973–976. Nuutinen, V. (1992) Earthworm community response to tillage and residue management on different soil types in southern Finland. Soil Tillage Research 23, 221–239. Parmelee, R.W., Beare, M.H., Cheng, W., Hendrix, P.F., Rider, S.J., Crossley, Jr. D.A., Coleman, D.C. (1990) Earthworms and enchytraeids in conventional and zero-tillage agroecosystems: a biocide approach to assess their role in organic matter breakdown. Biology and Fertility of Soils 10, 1–10. Presley, M. L., McElroy, T. C., Diehl, W. J. (1996) Soil moisture and temperature interact to affect growth, survivorship, fecundity, and fitness in the earthworm Eisenia fetida. Comparative Biochemistry and Physiology 114A, 319–326. Reinecke, A.J., Viljoen, S.A., Saayman, R. J. (1992) The suitability of Eudrilus eugeniae, Perionyx excavatus and Eisenia fetida (Oligochaeta) for vermicomposting in southern Africa in terms of their temperature requirements. Soil Biology and Biochemistry 24, 1295–1307. Satchell, J. E. (1967) Lumbricidae. In: Burgess, A., Raw, F. (eds) Soil Biology, Academic Press, Sydney, Australia. Uvarov, A. (1995) Responses of an earthworm species to constant and diurnally fluctuating temperature regimes in laboratory microcosms. European Journal of Soil Biology 31, 111–118. Viljoen, S.A., Reinecke, A.J. (1992) The temperature requirements of the epigeic earthworm species Eudrilus eugeniae (Oligochaeta) – a laboratory study. Soil Biology and Biochemistry 24, 1345–1350. Viljoen, S.A., Reinecke, A.J., Hartman, L. (1992) The influence of temperature on the life-cycle of Dendrobaena veneta (Oligochaeta). Soil Biology and Biochemistry 24, 1341–1344. Wever, L., Clapperton, M. J. (1996) Effects of temperature and moisture on the growth of juvenile Aporrectodea trapezoides (Oligochaeta). Canadian Journal of Soil Science 76, 437–438. Whalen, J. K., Parmelee, R. W. (1999) Growth of Aporrectodea tuberculata (Eisen) and Lumbricus terrestris L. under laboratory and field conditions. Pedobiologia 43, 1–10.
The influence of soil moisture and temperature on the survival, aestivation, growth and development of juvenile Aporrectodea tuberculata (Eisen) (Lumbricidae) Linda A. Wever1, Timothy J. Lysyk2 and M. Jill Clapperton2* Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta, Canada, T1K 3M4. [email protected] 2 Agriculture and Agri-food Canada, Lethbridge Research Centre, P.O. Box 3000, Lethbridge, Alberta, Canada, T1J 4B1. [email protected] 1
Submitted: 30. June 2000 Accepted: 23. August 2000
Summary Soil moisture and temperature are the primary factors determining earthworm survival and growth. Few studies, however, have examined the combined effects of soil moisture and temperature on earthworm physiology and ecology. The objective of this study was to evaluate the influence of soil moisture and temperature on the survival, growth and sexual development of an endogeic species, Aporrectodea tuberculata. Juvenile earthworms were grown at all possible combinations of four soil moistures (10, 15, 20 and 25 %, dry weight basis) and temperatures (5, 10, 15 and 20 °C). Logistic regression was used to determine the relationships between the proportion of surviving individuals with soil moisture and temperature, and time. Stepwise regression analysis was used to determine the various possible relationships between temperature, moisture and time on earthworm growth. Soil moisture accounted for 48 % of the variation in earthworm survival with the lowest survival associated with 10 % soil moistures. The interaction terms, time × temperature × moisture, and time × temperature, accounted for 63 % of the variation in earthworm growth. The greatest increase in earthworm weight was in soil incubated with 25 % moisture at 15 and 20 °C. There was also an obvious relationship between survival and growth linked to the interaction *E-mail corresponding author: [email protected]
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between soil temperature and moisture. After 10 wk the only post-clitellate individuals were observed in the 25 % and 20 °C treatment. Clitellate earthworms weighed 1.5 times more than individuals having only genital tumescences (GT). The results of the study showed that the effects of soil moisture on earthworm growth and survival are modified by soil temperature. Key words: Earthworms, juveniles, growth, survival, soil temperature, soil moisture, growth modeling, Aporrectodea tuberculata, aestivation
Introduction Soil moisture is a major factor limiting earthworm survival, growth and reproduction (Lee 1985). Temperature and moisture influence embryonic development, sexual maturation, reproductive success and longevity (Böström & Lofs 1996; Butt 1997; Butt et al. 1992; Daniel 1992; Frenot 1992; Hallatt et al. 1992; Holmstrup et al. 1996; Kretzschmar & Bruchou 1991; Muyima et al. 1994; Reinecke et al. 1992; Uvarov 1995; Viljoen & Reinecke 1992). Most laboratory experiments did not focus on endogeic or geophagus species that often dominate in agroecosystems (Auerswald et al. 1996; Baker et al. 1999; Clapperton 1999; Clapperton et al. 1997). A few studies have addressed how soil moisture and temperature interact to influence earthworm growth and development (Presley et al. 1996; Whalen & Parmelee 1999), however, only a limited range of temperatures and moistures were used in these studies and most do not apply to semi-arid agriculture. There is a critical requirement for such ecophysiological studies if we are to adequately explain observed seasonal variation in earthworm biomass, numbers and age structure between treatments in field studies (Garnsey 1994). The objective of this study was to examine the combined influence of soil moisture and temperature on survival, growth and development of Aporrectodea tuberculata, a species common to the agricultural fields in the Canadian prairies (Clapperton 1999). We used juvenile earthworms in the experiment to ensure that changes in live weight would reflect growth and not be confounded by the demands of reproduction (Daniel et al. 1996) and as early growth can be used to predict fitness and fecundity (Presley et al. 1996).
Materials and Methods Dark Brown Chernozemic Lethbridge Loam (Typic Borroll) soil was sieved to 2mm to ensure homogeneity. Juvenile A. tuberculata were collected from an agricultural field site in mid-October. All juvenile earthworms were sorted according to similar size and temporarily stored in field soil at 15 °C. Four different soil moistures (10, 15, 20 and 25 % water content on a dry mass basis) and temperatures (5, 10, 15 and 20 °C) were combined for a total of sixteen treatments with twenty replicates of each treatment. Each replicate consisted of one hundred grams of air-dried soil, 3 g of ground hard red spring wheat grain, an amount of tap water appropriate to the desired soil moisture, and one juvenile A. tuberculata placed in a 125 ml plastic container with a perforated lid. Earthworms were rinsed in tap water, carefully blotted and weighed prior to being added to the container.
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The treatments were then incubated in total darkness in constant temperature chambers for a total of 10 weeks. Each week, the contents of the containers were hand-sorted, the earthworm was observed for presence of sexual characteristics such as genital tumescences (GT), tubercula pubertatis (TP) and clitellum development, rinsed, blotted and weighed. The containers were also watered to weight in order to maintain the soil moisture. The proportion of surviving earthworms for a given week was determined by dividing the number of surviving earthworms by the total number of earthworms in each treatment (n = 20). A logistic regression was used to model the probability of earthworm survival as a function of soil moisture, temperature and time and any possible two or three way interactions. A binary variable was created for each observation where Y = 1 if the earthworm was alive or Y = 0 if the earthworm was otherwise dead or missing. The proportion of aestivating earthworms was also calculated for each week of the experiment and logistic regression used to examine the relationships between the probability of aestivation and soil moisture, temperature, time and any possible two or three way interactions. The terms time2 and time3 were included to account for curvilinearity in the relationship with time. A binary variable was created where Y = 1 if the earthworm was aestivating and 0 if not. All earthworms that were colourless, inactive and wound into a tight ball were considered to be aestivating. Data for growth was presented for only the first six weeks of the study, to when the first post-clitellate earthworm was observed. During the experiment it was found that a small number of the containers had A. trapezoides (n = 5). It was decided to include these individuals as part of the data set as they responded similarly to the treatments and were approximately the same size. In a previous experiment, Wever & Clapperton (1996) found that A. trapezoides had optimum growth at 25 % soil moisture at 15 to 20 °C. For modeling purposes, relative worm weight was calculated weekly for each worm as ri = weighti/weight0 where i = week the observation was made. Relative worm weights > 1 indicate that earthworms had increased weight, while relative weights < 1 indicate the worms had lost weight. Since all relative weights at week = 0 were 1, these were not included in the analyses. The relative weights were transformed to log(ri) to stabilize the variances. Stepwise multiple regression was used to determine the relationship between log(ri) and time (weeks), time2, temperature, moisture, and all possible two-way and three-way interactions between temperature, moisture and time. The term time2 was included to account for curvilinearity in the relationship with time. Variables were retained in the model if they contributed significantly (P < 0.05) to a reduction in error sums of squares of a model containing variables entered previously. The mean weight of earthworms with various sexual characteristics was calculated by pooling all of the treatments. The individual weights of earthworms with GT and/or TP observed over the course of the study were batched and an average weight and standard deviation were calculated.
Results Survival of juvenile earthworms ranged from 0 to 100 % (Fig. 1). At 20(C, the survival curves for different soil moistures were well separated with the 25 % treatment having the highest rate of survival followed by 20 and 15%. The lowest rate of survival was associated with a soil moisture content of 10 % regardless of the incubation temperature. The logistic regression model model P(surv = 1) = 1/(1exp(β0+β1Z+β2XZ+β3XY)) where X = time in weeks, Y = temperature (°C), and Z = % moisture indicated that identified variables accounted for 54 % of the variation observed in earthworm survival (Table 1). The variables that significantly influenced the proportion of earthworms surviving were soil moisture, moisture × time and temperature × time with soil moisture accounting for 47.9 % of the variation in P(surv). The soil moisture by time in-
Fig. 1. A comparison of the observed and model survival of juvenile A. tuberculata incubated at 5, 10, 15 and 20 °C and 10, 15, 20 and 25 % soil moisture content over a 10 wk period. Model r2 = 0.544, n = 20
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Table 1. Results of logistic regression analysis for the effects of time, temperature and moisture content on earthworm survival Parameter Estimate SE t (β=0) model r2 β0 -6.7351 0.2972 -22.6 β1Z -0.6669 0.0286 23.3 0.479 β2X*Z -0.0135 0.0023 - 5.8 0.536 β3X*Y -0.0096 0.0018 - 5.4 0.544 The logistic regression equation is P (surv=1)=1/(1exp(β0+β1Z+β2XZ+β3XY)) where X = time in weeks, Y= temperature (C), and Z = % moisture teraction accounted for 5.7 %, and the time × soil temperature term accounted for an additional 0.8 % of the variation in survival. The observed and modeled proportion of surviving earthworms is shown in Figure 1. In general, proportion of the surviving earthworms decreased with time, the probability of survival increased with moisture content, and only slightly decreased with increasing temperature. The rate of decreasing earthworm survival increased with increasing soil temperature and decreasing soil moisture (Fig. 1). Aestivating earthworms were observed in every treatment with the exception of the three treatments that had survival rates (15 % after the first week. These were the 10 % soil moisture treatments at 5, 10 and 15(C (Fig. 2). The basic form of the aestivation model was P (aest = 1) =1-1/(1+exp(β0+ β1XZ+β2X2+β3Z2+β4X3+β5X+β6YZ+ β7Y+β8Z)) where X=time in weeks, Y=temperature (°C), and Z = % moisture. The model accounted for only 12 % of the variation in the number of individual earthworms aestivating (Table 2). The variables which significantly influenced the proportion of aestivating earthworms were time, temperature, temperature × moisture, moisture, moisture2, moisture × time. In general, the number of aestivating earthworms increased as soil moisture increased up to 20 % after which there was a decrease in the number of aestivating individuals. Earthworms reared at 10, 15 and 20 % soil moisture were more likely to aestivate in the first five weeks whereas earthworm at 25 % soil moisture are more likely to aestivate in the tenth week. As temperature increased, the proportion of aestivating earthworms peaked during weeks 3 and 4 as temperature increased. The model also predicted a delay in aestivation if soil moisture was 20 % or greater. Earthworms grew best at a soil moisture content of 25% and incubation temperatures of 15 and 20 °C (Fig. 3). However, juveniles reared in soil at 20 % and 5 °C grew equally as well as those in soil at 25 %. The model developed to explain this relation ship was ln(ri) = β0+β1X-β2X2+β3Y+β4Z-β5XY+β6XYZ where X= time in weeks, Y= temperature (°C), and Z = % moisture. When the soil moisture temperature interaction term was added the model significantly (F = 405.8; df = 6, 1144; P < 0.0001) accounted for 68 % of the variation in the relative weights of individual earthworms. The variables that significantly influenced the relative weights of individual earthworms were time, time2, temperature, moisture, time × temperature, and time × temperature × moisture (Table 3). The two interaction terms, time × temperature × moisture, and time × temperature, accounted for 63 % of the variation in ln(ri). These also had the highest partial r2, indicating they accounted for a larger proportion of the va-
Fig. 2. A comparison of the observed and modeled aestivation of surviving juvenile A. tuberculata incubated at 5, 10, 15 and 20 °C and 10, 15, 20 and 25 % soil moisture content over 10 wk Model r2 = 0.118
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Table 2. Results of logistic regression analysis for the effects of time, temperature and moisture content on earthworm aestivation Parameter Estimate SE t (β=0) model r2 β0 -9.2016 1.6373 -5.6199 β1X*Z -0.0558 0.0071 -7.8454 0.015 β2X2 -0.4934 0.0549 -8.9895 0.026 β3Z2 -0.0176 0.0041 -4.2913 0.057 β4X3 -0.0271 0.0033 -8.2878 0.074 β5X -1.5059 0.2716 -5.5447 0.102 β6Y*Z -0.0132 0.0027 -4.9678 0.104 β7Y -0.2490 0.0535 -4.6555 0.111 β8Z -0.5497 0.1586 -3.4663 0.118 The logistic regression equation is P(aest=1)=1-1/(1+exp(β0+β1XZ+β2X2+β3Z2+ β4X3+β5X+β6YZ+ β7Y+β8Z)) where X = time in weeks, Y= temperature (°C), and Z = % moisture
riation in ln(ri) given that all other parameters were in the model. The main effects, time, temperature, and moisture contributed an additional 2.5% to the variation already accounted for by the interaction terms, with individually accounted for 1-9% given all other variables were in the model. The change in relative weights of the earthworms with respect to time, temperature and moisture were examined from the partial derivatives (Fig. 3). The partial derivative with respect to moisture was δln(ri)/δZ = β4+β6XY and showed that the rate of gain generally increased with moisture content, and the increases were greater as temperature and time increased. The partial derivative with respect to temperature was δln(ri)/δY = β5X+β6XZ showing that the rate of gain with respect to temperature was positive, but decreased as time increased, and increased as both moisture and time increased. The partial derivative with respect to time was δln(ri)/dX = β1-2β2X-β5Y+ β6YZ, and again the rate of change of earthworm weights declined with time. HoweTable 3. Results of stepwise regression analysis for the effects of time, temperature and moisture content on relative weight of earthworms Parameter Estimate SE t (β = 0) model r2 β0 -1.0039 0.0881 -11.4 β6X*Y*Z -0.0013 0.0001 -17.7 0.1331 β5 X*Y -0.0299 0.0018 -16.9 0.6303 β4 % moisture (Z) -0.0345 0.0033 -10.4 0.6609 β3 C (Y) -0.0250 0.0035 - 7.2 0.6720 β1 Time (X) -0.1580 0.0326 - 4.8 0.6774 β2 Time2 (X2) -0.0159 0.0049 - 3.3 0.6840 Model is ln(ri)= β0+β1X-β2X2+β3Y+β4Z-β5XY+β6XYZ where X = Y = temperature (°C), and Z = % moisture
r2 (β1/βn) 0.2154 0.1996 0.0857 0.0428 0.0201 0.0093 time in weeks,
Fig. 3. A comparison of the observed and modeled earthworm growth rate of juvenile A. tuberculata incubated at 5, 10, 15 and 20 °C and 10, 15, 20 and 25 % soil moisture content over 6 wk. Model r2 = 0.684, n = 20.
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Table 4. Effect of temperature on the sexual development of juvenile A. tuberculata grown at 25 % soil moisture Temperature (°C)
No. of individuals No. of individuals No. of post-clitellate with GT and/or TP with a clitellum individuals 5 0 0 0 10 1 0 0 15 11 3 0 20 7 4 3 No clitellate individuals were present at any soil moisture below 25 % regardless of incubation temperature at the end of the ten week study
ver, changes in weight gains over time were dependent on temperature and moisture (Table 4). The coefficient for temperature alone indicated that changes in rate of weight gain were reduced at higher temperatures, the moisture by temperature interaction indicated that changes in rate of gain with respect to time were dependent on a combination of temperature and moisture, and increased as both temperature and moisture increased. During the course of the study some of the earthworms developed sexual characteristics. However, no clitellate individuals were observed at soil moistures lower than 25 % at any temperature and only one treatment (25 % and 20 °C) had post-clitellate individuals. The mean live mass at which GT and/or TP became consistently observable was 833 ± 209 mg (n=32). Clitellum development followed when earthworms reached 1250 ± 227 mg (n=8) while post-clitellate individuals weighed slightly less at 1235 ± 191 mg (n=3).
Discussion The results of our study indicate that the activity of earthworms is governed by the temperature of their surroundings, in agreement with other experiments (Daniel 1992; Viljoen & Reinecke 1992; Frenot 1992; Daniel et al. 1996). Earthworms gained more weight as the incubation temperature increased. A. tuberculata juveniles reared at 10 °C and 25 % soil moisture grew much slower than those at 20 °C and the same moisture content (Fig. 3). Whalen & Parmelee (1999) showed that the instant growth rate of A. tuberculata at 10 °C and 30 % moisture (w/w) was 10.8 ± 1.4 × 10-3 d-1 and doubled to 17.0 ± 1.6 × 10-3 d-1 at 18 °C. Soil moisture content is considered to be the primary factor limiting survival of earthworms (Doube & Styan 1996; Hallatt et al. 1992; Kretzschmar & Bruchou 1991; Lee 1985; Satchell 1967). The survival model showed that 54 % of the variation in the survival data could be explained by moisture (47 % variation), time by moisture and time by temperature interaction (Table 1). The decline in survival with time is likely to be a function of the handling of the earthworms or aging. Including a soil moisture and temperature interaction into the model did not significantly reduce the error sum of squares suggesting that this interaction was not influencing earthworm survival. However, as the temperature increased above 5 °C to
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10 and 20 °C earthworm survival at 20 % moisture declined (Fig. 1). At 20 °C survival was reduced in all treatments except for earthworms maintained at 25 % moisture (Fig. 1). Temperature has been reported to affect mitochondrial activity in earthworms (Arillo & Melodia 1991; Uvarov 1995). Earthworms subjected to constant temperature decreased in body weight, and had increased rates of reproduction and mortality (Uvarov 1995). Whereas, earthworms subjected to fluctuating temperatures had increased rates of survival but did not reproduce (Uvarov 1995). However, the response of earthworms to a change in temperature depends on the thermal history of the earthworms before experiments are initiated (Arillo & Melodia 1991). The earthworms used in our experiments were collected in October under cooler soil temperatures (8–12 °C), with adequate moisture (20–25%) the earthworms survived at all temperatures (Fig. 1) and gained weight (Fig. 3). Furthermore, it is likely that the decreasing survival and weight gain for earthworms at 10, 15 and to a lesser extent 20 % moisture in the 15 and 20 °C treatments resulted from inadequate moisture when the rate of earthworm metabolism was rapidly increasing (Arillo & Melodia 1991; Uvarov 1995). The data for earthworm aestivation in our experiments reinforced the link between survival and growth rate with respect to a temperature and moisture interaction. The proportion of aestivating earthworms increased with increasing temperature particularly at the two lowest soil moistures (10 and 15%) (Fig. 2) which increased the rate of survival of the earthworms in the 10% moisture and 20 °C soil temperature treatment (Fig. 1). These results likely reflect the effects of the thermal history of the earthworms used in our study (Arillo & Melodia 1991). Earthworms kept in higher soil moistures (20–25 %) at the two lowest soil temperatures responded in a field-like manner in that they gained some weight (Fig. 3) before entering torpor after 6 weeks at 5 °C and 8–10 weeks at 10 °C (Fig. 2). These results would be expected for earthworms collected from the field at (8–12 °C) given the results of other studies (Arillo & Melodia 1991; Uvarov 1995). However, the effects of handling on the earthworms may also have influenced the number of aestivating earthworms as the study progressed (Fig. 2). Our results reveal that temperature and moisture were interacting to influence the growth of juvenile A. tuberculata (Table 3). The growth rate of earthworms reared at 5 °C at 20 and 25 % moisture was similar (Fig. 3). After 4wk at 10 °C the growth rate for earthworms at 20 % moisture began to decline, the growth rate for the earthworms at 25 % moisture increased, and others remained the same (Fig. 3). This was mirrored in the survival curves at the same incubation temperatures, which showed similar survival rates for 15, 20 and 25 % moisture at 5 °C, and a slight decline in survival at 20 % moisture at 10 °C (Fig. 1). At 15 °C there was a further, more rapid decline in growth for all treatments which can also be clearly seen in the divergence between the survival curves for earthworms at all soil moisture levels (Fig. 1). The survival of earthworms incubated at 20 °C clearly differed depending on the soil moisture (Fig. 1). This was also the only incubation temperature where, after 6wk, there were still some earthworms at 10 % moisture, and a rapid and steady decline in the growth of earthworms at 15 % moisture. It appeared that low temperatures modified the effects of inadequate soil moisture. At low temperatures, water loss from respiration, and excretion are minimized due to lower metabolic rates (Edwards & Lofty 1972) and soil moisture is less limiting to growth. Conversely, our results showed that as the soil temperature and earthworm metabolic rate increase there is a greater requirement for water. Our model showed that 67 % of the variation in the growth rate data could be explained by a temperature moisture interaction (Table 3). This is consistent with the
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literature for other earthworm species that showed that water loss could be readily replenished under high soil moisture conditions as pF decreases with increasing temperature despite a doubling of the metabolic rate of earthworms for every 10 degree rise in temperature (Lee 1985). This makes it easier for earthworms to extract water from the soil to regulate body water content and drive cell processes of division and growth and consequently, more energy can be allocated to growth and development. High soil moistures also allow for evaporative cooling (Satchell 1967). Kretzschmar and Bruchou (1991) suggested that water availability and not soil water content influenced earthworm growth and that the threshold for the temperature-moisture interaction would differ according to soil texture (Doube & Styan 1996; Kretzschmar & Bruchou, 1991). Together with our study, it is clear that the effects of soil moisture on earth worm growth and survival should be examined with respect to the modifying effects of temperature. The environmental conditions which enhance earthworm growth also promote sexual maturity so it is not surprising that the only clitellate individuals in our study were associated with the high soil moisture and temperature treatments. Muyima et al. (1994) reported that substrate moisture profoundly influenced the growth rate and clitellum development of juvenile Dendrobaena veneta. The literature also suggests that temperature dramatically influences sexual development of other earthworm species such as Allolobophora chlorotica, Dendrobaena veneta, Dendodrilus rubidus (Butt 1997; Muyima et al. 1994; Frenot 1992). In our study the sexual maturity of A. tuberculata appeared to be influenced by soil moisture and temperature. Tubercula pubertatis and/or genital tumescences were observed at a mean live weight of 833 ± 209 mg. As expected, clitellate individuals weighed significantly more (1251 ± 227 mg) than non-clitellate A. tuberculata, and the earthworms continued to increase in weight until they become post-clitellate. Published weights for the closely related species Aporrectodea trapezoides collected from the field were much lower: 498 ± 34 mg with TP and 953 ± 47 mg with clitellum (McCredie et al. 1992). In the same study, aestivating individuals collected and reared in the laboratory until the clitellum was fully developed weighed nearly 1400 mg. This demonstrates the need for caution when comparing field and laboratory studies. In the semi arid agricultural ecosystems of the Canadian prairies A. tuberculata is most often found below 10 cm soil depth and more abundant under no tillage (Clapperton 1999). It is well known that the spring soil temperature is lower and the soil moisture content higher under reduced and no tillage systems compared with conventional tillage. Despite the lower temperature under no tillage, studies consistently report more earthworms (Parmelee et al. 1990; Nuutinen 1992; Francis & Knight 1993) and increased species diversity (Clapperton et al. 1999) in reduced and no tillage treatments. Interestingly, the literature suggests that a lower soil temperature is required for hatchling survival (Viljoen et al. 1992). Uvarov (1995) found that temperatures fluctuating between 5 and 25 °C resulted in increased survival but reduced fertility, and weight gain. Earthworms become abundant in semi-arid agriculture at a soil temperature between 5 and 10 °C in spring on the Canadian prairies (Clapperton 1999) under 18 to 25 % soil moisture content, and begin aestivating before soil moisture content is below 15% and soil temperatures are above 20 °C (Clapperton 1999). The results of our study are consistent with the behaviour of A. tuberculata in the field under semi-arid agriculture demonstrating that an interaction between soil moisture and temperature limit the survival, growth, and maturity this earthworm species.
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Acknowledgements We are very grateful to J. Unrau, F. Dunstan, D. Kanashiro, and N. Lee for their technical assistance, Geoff Baker (CSIRO, Division of Entomology, Canberra Australia) for improving the manuscript, and Canada-Alberta Environmentally Sustainable Agriculture (CAESA) Agreement for partially funding this study.
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