Biologia 65/2: 289—293, 2010 Section Zoology DOI: 10.2478/s11756-010-0019-5

Temperature dependence and ontogenetic changes of metabolic rate of an endemic earthworm Dendrobaena mrazeki Vladimír Šustr & Václav Pižl Biology Centre Academy of Sciences of the Czech Republic, v.v.i., Institute of Soil Biology , Na Sádkách 7, CZ-37005 České Budějovice, Czech Republic; e-mail: [email protected]

Abstract: Ontogenetic changes and temperature dependency of respiration rate were studied in Dendrobaena mrazeki, an earthworm species inhabiting relatively warm and dry habitats in Central Europe. D. mrazeki showed respiration rate lower than in other earthworm species, < 70 µl O2 g−1 h−1 , within the temperature range of 5–35 ◦C. The difference of respiration rate between juveniles and adults was insignificant at 20 ◦C. The response of oxygen consumption to sudden temperature changes was compared with the temperature dependence of respiratory activity in animals pre-acclimated to temperature of measurement. No significant impact of acclimation on the temperature response of oxygen consumption was found. The body mass-adjusted respiration rate increased slowly with increasing temperature from 5 to 25 ◦C (Q10 from 1.2 to 1.7) independently on acclimation history of earthworms. Oxygen consumption decreased above 25 ◦C up to upper lethal limit (about 35 ◦C). Temperature dependence of metabolic rate is smaller than in other earthworm species. The relationships between low metabolic sensitivity to temperature, slow locomotion and reactivity to touching as observed in this species are discussed. Key words: earthworm; Lumbricidae; Dendrobaena; oxygen consumption; acclimation; metabolic rate; temperature

Introduction Dendrobaena mrazeki (Černosvitov, 1935) is an endemic earthworm species recorded from several localities in the Czech Republic and the westernmost part of Slovakia. The habitat selection of the species is atypical. In contrast to most of central European earthworms, it prefers steppe and forest-steppe ecosystems or even pine forests on sandy soils (Zajonc 1980; Pižl 2002) where it may reach remarkable density. The localities belong climatologically to the warmest and driest areas of the former Czechoslovakia, and show one or more irregular periods of soil drying-out during the course of vegetation season. Dendrobaena mrazeki is considered as a xerotolerant earthworm; however, the data on its ecophysiology needed for life form classification and explanation of its adaptations to relatively warm and dry environment are scarce. This species shows some features typical for endogeic species, some features of epigeic ones. It is larger than the related epigeic Dendrobaena octaedra (Savigny, 1826), with the fresh body mass about 0.6 g. D. mrazeki showed low relative water content and small relative amount of intestine content, similarly to epigeic D. octaedra (Šustr & Pižl 2009). The intestine of the species contained mineral soil as well as organic particles, the pH of the digestive tract and profile of digestive enzymes did not differ from those of other earthworms (Šustr & Pižl 2007). Values of melting and supercooling points of body fluid were simic 2010 Institute of Zoology, Slovak Academy of Sciences


lar to those of D. octaedra. Quiescence of adult individuals was observed in a laboratory culture at 15% soil moisture (Šustr & Pižl 2007). D. mrazeki creates mostly vertical burrows, which corresponds with an epiendogeic or anecic mode of life (Lee 1985). Body massspecific oxygen consumption rate of D. mrazeki measured at 15 ◦C was found extremely low in comparison with other earthworm species even after the adjustment for body water content and gut content (Šustr & Pižl 2009). However, the knowledge of temperature dependence and developmental changes of metabolism of D. mrazeki is absent. A number of studies about temperature dependence of metabolism in earthworms have been published in the last 60 years (Knoz 1957; Gromadska 1962; Phillipson & Bolton 1976; Uvarov 1998; Uvarov & Scheu 2004a, b) providing some data for comparison. In general, the metabolic rate of earthworms increases with temperature with Q10 about 2 (Lee 1985). The Q10 3.76 was measured for D. octaedra in the temperature range 2–10 ◦C using calorimetry (Calderon et al. 2009). The decrease of Q10 in some intervals of temperature was interpreted several times as metabolic adaptation based on metabolic rate “regulation” inside limits of “optimal” temperature (Uvarov 1998; Uvarov & Scheu 2004b). The upper lethal temperature limits of about 25–37 ◦C were observed for central European earthworms (Dunger 1980) or may be deduced from respirometric data (Knoz 1957).

290 To obtain information about the thermal dependence of poikilotherm metabolic rate and understanding of physiological adaptive mechanisms, the complex measurements are needed with respect to several aspects. The capability of an organism to a physiological (non-genetic) adaptation, in the sense of Precht et al. (1973) may be assessed from the comparison between the shape of oxygen consumption/temperature curves of animals acclimated to none or to only one constant acclimation temperature (ET × AT design reflecting reaction on sudden temperature changes, ET = experimental temperature, AT = acclimation temperature) and of animals acclimated to each experimental temperature (ET = AT design). The combination of both approaches enabling the testing of adaptation ability was used rarely in earthworms (Phillipson & Bolton 1976). The ET × AT design was used by Knoz (1957) and Gromadska (1962). Uvarov (1998) and Uvarov & Scheu (2004a) focussed on the measurement of metabolic rate of animals acclimated to the experimental temperature including fluctuating temperature regimes. Respiration of D. octaedra taxonomically related to D. mrazeki was measured at constant and varying temperature regimes (Uvarov 1998). The metabolic rate of juveniles was found higher (Phillipson & Bolton 1976) as well as lower (Uvarov & Scheu 2004b) than that of adult earthworms. The aim of this study is to study the ontogenetic and thermal aspect of the metabolic adaptations of D. mrazeki. The aspect of desiccation tolerance must be a subject of further studies. The hypotheses tested in this study are derived from the low oxygen consumption of D. mrazeki discovered in a previous study (Šustr & Pižl 2009). The absence of noticeable differences of metabolic rate between juveniles and adults worms was hypothesized because the low oxygen consumption indicates the strategy of long ontogenesis broken by inactivity during unfavourable dry periods resulting in the low growth rate of juveniles. The low sensitivity of metabolic rate of the species to temperature was hypothesised in this species because it may reduce the energy loses by respiration during starvation in dry and warm periods. Moreover the high temperature sensitivity of metabolism would be advantageous rather for the species using the quick ontogenetic strategies exploiting short periods of favourable conditions to complete the individual development. The ability to make vertical burrows offers this species the possibility to migrate deep across the soil profile to avoid unfavourable temperatures on the soil surface (behavioural adaptation). The absence of acclimation to temperature and average resistance to high temperatures may be hypothesized because of the poor ability to adapt physiologically. Material and methods The site and earthworm sampling Dendrobaena mrazeki was collected by hand-sorting of

V. Šustr & V. Pižl soil and litter in the thermophilous oak forest Quercion pubescentis – petraeae located in the Milovice Hills, the eastern part of the Pálava Biosphere Reserve (Czech Republic, 48◦ 49 50 N, 16◦ 41 43 E) in spring 2003, 2004 and 2006. The soil of study site was Orthic Luvisol on tertiary sediments, humus form – mull. The climate of the area is dry and warm, with average annual temperature and precipitation of 9.6 ◦C and 337 mm, respectively. The average monthly soil surface temperatures (measured 0–5 cm above the ground) at the study site varied between −5 and 20 ◦C (5–20 ◦C in the vegetation season) in 2003 and 2004. The maximum diurnal temperature fluctuation (−1 to 23.4 ◦C) was measured on March 31, 2004. All experimental earthworms were kept in laboratory in plastic containers containing the substrate from the study site in thermostat at 12 ◦C and high moisture, in the dark, for about two weeks before the measurement. Respirometry Oxygen consumption of single animals was measured in round Warburg apparatus with 14 manometers with water bath (Glasswerke Stutzerbach, Germany) over 2-hour period at constant temperature; 30 minutes were allowed for thermostabilisation. Cylindrical 10 ml respirometric vessels were used supplied with 0.15 ml of 0.1 N KOH as CO2 absorbance agents placed in glass cylinder at the bottom of vessel. The cylinder was covered by plastic sieve to prevent contact of earthworms with the KOH solution. Worms were placed individually into vessels with 0.5 cm circle of filter paper moistened with distilled water to keep high moisture inside the vessel. The vessels were connected to manometers filled with Brodie’s manometric fluid and immersed in the water bath of the apparatus adjusted to the temperature of measurement. The oxygen consumption was measured in the fed worms to prevent the decrease of metabolism due to starvation. The procedure and measurement conditions were based on the methodology of manometric respirometry (Kleinzeller 1965; Sláma 1984), with the details of the procedure optimized during preliminary tests. The cumulative oxygen consumption was read four times in half hour intervals. The regression of oxygen consumption on time was calculated for every sample to verify the constancy of oxygen consumption during the time of measurement. Regression coefficients ranged from 0.8 to 1.0. Fresh mass (W) of animals was measured by weighing immediately after the respirometric measurements and oxygen consumption was expressed as body mass-specific metabolic rate (M/W in µl O2 g−1 h−1 ). Ontogenetic differences To obtain basic information about the changes of metabolic rate during the worm maturation the body mass-specific metabolic rate of five adult animals with formed clitellum and six juveniles was measured at constant temperature of 20 ◦C. Temperature changes of oxygen consumption The impact of temperature on oxygen consumption was studied using independent groups of worms measured at every temperature. The body mass differences among the groups may lead to increased variability of results, because of the dependency of body mass-specific metabolic rate (M/W ) on body mass (M/W = a · W b−1 ). Therefore respiratory activity was expressed as body mass-adjusted metabolic rate (AM = M/W b ). AM is identical to a coefficient in the equation M/W = a·W b−1 and is independent on the body mass. The linear regression of individual metabolic

Metabolic rate of Dendrobaena mrazeki 90

Metabolic rate [µl O2.g-1.h-1]

80

291 Statistical evaluation The ontogenetic difference in the body mass specific metabolic rate was tested using T -test. The impact of temperature on AM was tested using ANOVA. Arcus Tangent transformation of data was used to satisfy the presumption of homogeneity of variance. All the tests were executed using Statistica v6.0 software (StatSoft, Inc.). Q10 coefficients were calculated from the body massadjusted metabolic rate as:

average S.E.

70 60 50 40




30

Q10 =

20

(10/(T2 −T1 ))

where MAT1 and MAT2 are metabolic rates at the temperatures T 1 and T 2, respectively.

10 0

MAT2 MAT1

adults

juveniles

Fig. 1. Metabolic rate in adults and juveniles of Dendrobaena mrazeki at 20 ◦C.

rate (log M ) and logarithm of body mass (log W ) was calculated for every temperature of measurement in both experimental designs. The slope of the regression line represents the b coefficient from the relationship M = a · W b between individual metabolic rate (M ) and body mass (W ). The differences among b coefficients obtained from single experimental groups were tested by T -test to verify the homogeneity of b coefficient among all groups. The common b coefficient may be then calculated for all groups and used for the calculation of body mass-adjusted metabolic rate (AM = M/W b ). Two experimental designs were used to test the ability of the species under study to acclimate the metabolic rate to different temperatures: Reaction to sudden temperature changes (AT × ET design) The respiration rate of 11, 20, 22, 22, 22, 11 and 10 earthworms was measured at temperatures 5, 10, 15, 20, 25, 30 and 35 ◦C, respectively, following the protocol of measurement described above. The adult or sub-adult animals were chosen for the measurement. The design reflected metabolic reactions to sudden temperature changes because all animals had the same acclimation history (about two weeks at 12 ◦C) without previous acclimation of animals to temperature of measurements except the half an hour of thermostabilisation. Oxygen consumption was expressed as body mass-adjusted metabolic rate (AM). Animals acclimated to temperature of measurement (AT = ET design) Adult and sub-adult individuals of D. mrazeki were acclimated at the temperatures (5, 10, 15, 20 and 25 ◦C) in laboratory in plastic containers containing the substrate from the study site at high moisture, in the dark, for 25 days before the measurement. Oxygen consumption of these animals was measured after this acclimation period at the temperature identical to acclimation temperature. The 18, 22, 17, 9 and 7 specimens were measured at single temperatures respectively. The technique of the measurements was identical to that in the previous experiments. Oxygen consumption was expressed as body mass-adjusted metabolic rate (AM).

Results Ontogenetic differences Fresh body mass (W ) of adult earthworms was significantly higher than that of juveniles (T -test, P < 0.045, 0.801 ± 0.089 (SE) and 0.482 ± 0.095 g, respectively). Body mass-specific metabolic rate in juveniles was not significantly higher than the rate in adults (Fig. 1, T test, P > 0.70, 67.99 and 64.50 µl g−1 h−1 , respectively). Effect of temperature The regression coefficients between logarithm M and logarithm W fluctuated between −0.8 and 1.4 at different temperatures of both experimental designs, however neither any consistent trend of changes in b with increasing temperature nor the apparent difference in this coefficient due to the acclimation was observed. The pair testing of the differences among the coefficients in all possible subgroups did not reject the hypothesis of homogeneity of regression coefficients (T -test, P > 0.07 in all combinations of comparisons). The homogeneity enabled us to calculate common b from the regression of log M on log W across all temperatures and experimental designs. The value of the common b was 0.41 ± 0.09. The relationship between temperature and the bodymass adjusted metabolic rates (AM = M/W 0.41 ) was calculated to view temperature dependence with substantially suppressed influence of the body size differences. It means that the individual oxygen consumption of the worm was divided by its body mass raised to a power 0.41. The shape of the relationship between temperature and the body-mass adjusted metabolic rates of both acclimation variants is compared in Fig. 2. The respiration rates slowly increased with increasing temperature (maximum at 25 ◦C). The temperature Q10 coefficients were lower then 1.8 across the whole investigated temperature range in both acclimation variants (Table 1, Fig. 2). No clear difference in the shape of temperature response of metabolic rate is visible in the temperature range from 5 to 25 ◦C. The increase of oxygen consumption with temperature seems to be a little bit steeper in acclimated animals. However the ANOVA showed no significant im-

V. Šustr & V. Pižl

292 Table 1. The Q10 coefficients calculated from the relationship of oxygen consumption of Dendrobaena mrazeki adjusted for body mass (AM) on temperature using two different experimental designs (n.d. – not determined). Temperature interval ( ◦C) Design of measurement Acclimated Short response

5–15

10–20

15–25

20–30

25–35

1.42 1.55

1.70 1.55

1.23 1.50

n.d. 0.93

n.d. 0.75

70

-b

-1

AM [µ l.g .h ]

60 50 40 30 20 10 0 0

5

10 15 20 25 30 35 40

Temperature [°C] Fig. 2. The relation between temperature and respiration rate of Dendrobaena mrazeki adjusted for body mass (AM). Filled circles – reaction to short temperature changes, empty circles – animals acclimated to temperature of measurement. Error bars represent SE.

pact of the acclimation history on AM (P > 0.45). Similarly, the interaction between acclimation type and temperature was insignificant (P > 0.48). On the contrary, the influence of temperature on oxygen consumption was highly significant (P < 0.0001). In non-acclimated animals respiratory activity decreased with the increase of temperature starting from the interval between 25 and 30 ◦C. The exposition to 35 ◦C for two hours was lethal for 73% of the animals measured. No mortality was found during acclimation except for the group exposed to temperature 25 ◦C where 27% mortality was observed. Discussion Developmental differences in respiration rate were shown to be associated with growth and changing physiological condition in general (Phillipson & Bolton 1976). Phillipson & Bolton (1976) reported the higher annual average respiration rates in small immatures (<100 mg, not clitellate) and large immatures (>100 mg but not clitellate) than in adults of Aporrectodea rosea (Savigny, 1826) at 10 ◦C (78.56, 72.66 and 64.17 µl O2 g−1 h−1 , respectively). They concluded that there were no obvious and consistent differences among the three groups during the whole season, but the higher oxygen consumption of immatures was

visible from May till August (Fig. 4, in Phillipson & Bolton 1976). The metabolic cost of growth (Riisg˚ ard 1998) may be responsible for the increase of metabolic rate in intensively growing immature stages. In contrast, the mass specific respiration rates were higher in mature than in juvenile stages of Lumbricus rubellus Hoffmeister, 1843 at all constant temperature regimes in the range from 2 to 20 ◦C. The reproduction activity of mature L. rubellus was assumed to contribute to high respiratory activity measured (Uvarov & Scheu 2004b). Based on those sporadic data, the developmental changes of metabolic rate seem to be species specific. The similar metabolic intensity of growing juveniles and adults is in accordance with the proposed low developmental rate in D. mrazeki. The oxygen consumption-temperature curve of D. mrazeki characterised by Q10 values of 1.2–1.7 is very flat in comparison with most of other earthworms. In general, the oxygen consumption of earthworms increases with the temperature with Q10 about 2 (Lee 1985), but the variability of Q10 among species is extensive. Q10 coefficients of oxygen consumption of Eisenia fetida (Savigny, 1826) lies between 1.6 and 3.4, the low sensitivity of metabolism to temperature is restricted to a zone of relative temperature independency between 20 and 25 ◦C (Q10 = 0.9–1.1, Moment & Habermann 1979). Temperature response of epigeic species, the range of Q10 between 2.1–2.4 in D. octaedra (Uvarov 1989) or 2.3–3.9 in L. rubellus (Uvarov & Scheu 2004b), is more pronounced than that of endogeic earthworms (Q10 between 1.4 and 2.4 in all stages of A. rosea (Phillipson & Bolton 1976)), which may be a consequence of more active behaviour of epigeic species (Uvarov & Scheu 2004b). The low sensitivity of metabolism of D. mrazeki agrees with the proposed strategy of long development interrupted by irregular periods of soil desiccation. In this way the energy for the periods of restricted feeding activity may be saved without regard to temperature changes. The directly responsible physiological mechanisms remain unknown. The weak temperature response of D. mrazeki may be related to low locomotion activity of this species observed in both laboratory and field conditions, which should be tested. The oxygen consumption-temperature curve of D. mrazeki acclimated to the temperature of measurement is identical with that of non-acclimated earthworms. It corresponds to the acclimation type 4 (no-capacity adaptation) according to Precht et al. (1973) and indicates that the species is unable to adapt physiologically to different temperatures. The absence of an acclimation impact on the oxygen consumption-temperature relationship was previously reported for some other earthworms. Kirberger (1953) did not find translation or rotation of the Ql0 relation in differently acclimatised individuals of E. fetida. The temperature of acclimatisation did not give rise to any statistically significant differences in the relationship between oxygen consumption rate and experimental temperature for either adult or large immature of A. rosea (Phillipson & Bolton 1976). Moment & Habermann (1979) observed

Metabolic rate of Dendrobaena mrazeki an increase of oxygen consumption rate with increasing acclimation temperature in E. fetida. However, the increase was restricted to the temperatures 15–25 ◦C only. The absence of any clear difference between the two curves in D. mrazeki may be connected with the low locomotion activity of this species, and consequently less intensive locomotory escape reactions in animals exposed to sudden temperature changes. In conditions of the AT × ET measurements, the oxygen consumption decreased above 25 ◦C and most of earthworms did not survive the short exposition to 35 ◦C. In this earthworms cultured at 12 ◦C before the measurement, the lethal temperature may have been undervalued by thermal shock due to quick temperature change or overvalued due to short exposition to experimental temperature. About 25% mortality was noted during the acclimation at 25 ◦C for AT = ET measurements. The lethal limit of about 25 ◦C was reported for most of north and central European earthworms (Dunger 1980). It lies under 40 ◦C in earthworm E. fetida (Moment & Habermann 1979). The lethal temperature limit of D. mrazeki seems to be within the range reported for other earthworms and hardly indicates any physiological adaptation to warm conditions. However, the temperature resistance of earthworms acclimated to different temperatures must be tested before the final conclusion, because the temperature at which the maximum of the respirationtemperature curve was observed and possibly the upper lethal temperature as well may correlated with their acclimation level (Moment & Habermann 1979). It is probable that D. mrazeki is not extensively influenced by temperature fluctuation at the site owing to ecological adaptations connected with its epi-endogeic or anecic mode of life. The vertical migrations deeper to the soil may compensate the absence of any clear nongenetic physiological adaptation to temperature. The low metabolic rate poorly sensitive to temperature is probably species-specific genetically based adaptation to the slow ontogenetic strategy and may save the energy of organism for periods of inactivity forced by irregular dry weather periods. Acknowledgements The research was supported by the Grant Agency of the Czech Republic, project No. 206/03/0056, “Biology and ecophysiology of the endemic earthworm Dendrobaena mrazeki, and its effects on soil and soil organisms in a xerophilous oak forest”, and by the Research Plan of the ISB BC ASCR, No. AV0Z60660521.

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