Role of diurnal rhythm of oxygen consumption in emergence from soil at night after heavy rain by earthworms

Role of diurnal rhythm of oxygen consumption

in emergence from soil at night after heavy rain by earthworms

Shu-Chun Chuang

1

and Jiun Hong Chen

1,2,a

1

Institute of Zoology, National Taiwan University, Taipei, Taiwan

2

Department of Life Science, National Taiwan University, Taipei, Taiwan

Abstract. Two species of earthworms were used to unravel why some earthworm species crawl out of the soil at night after heavy rain. Specimens of Amynthas gracilis, which show this behavior, were found to have poor tolerance to water immersion and a diurnal rhythm of oxygen consumption, using more oxygen at night than during the day. The other species, Pontoscolex corethrurus,survived longer under water and was never observed to crawl out of the soil after heavy rain; its oxygen consumption was not only lower than that of A. gracilis but also lacked a diurnal rhythm. Accordingly, we suggest that earthworms have at least two types of physical strategies to deal with water immersion and attendant oxygen depletion of the soil. The first is represented by A. gracilis; they crawl out of the waterlogged soil, espe-cially at night when their oxygen consumption increases. The other strategy, shown by P. corethrurus,allows the earthworms to survive at a lower concentration of oxygen due to lower consumption; these worms can therefore remain longer in oxygen-poor conditions, and never crawl out of the soil after heavy rain.

Additional key words:rain, earthworm crawling, oxygen consumption

Darwin (1881) observed that, after heavy rain, many dead earthworms could be found lying on the soil sur-face. He considered that these earthworms were sick and that the heavy rain had hastened their death. Lan-kester (1921) suggested that earthworms were found dead on the soil surface because of lack of oxygen in the groundwater, implying that earthworms might drown when submerged in water. However, some earthworm species can remain alive in water for several days; as oxygen diffuses through their moist skin, they should survive under water for at least some time, as suggested by Nagano (1934). Nagano further suggested that lack of oxygen could be a primary cause of earthworm death while under water. However, this does not explain why some earthworm species crawl out of the soil after heavy rain, and do so only at night.

In Taiwan, some species of earthworm, including Amynthas gracilis KINBERG 1867, Amynthas

asp-ergillum, Amynthas robustus,and Metaphire schmardae, can be observed dispersed on the soil surface after heavy rain (Tsai 1964). Because some earthworm spe-cies have been demonstrated experimentally to have a

diurnal rhythm of oxygen consumption, with a high rate of consumption at night and a low rate during the day (Ralph 1957; Chuang et al. 2004), we suspected that the reason for earthworms leaving the soil may be their rate of oxygen consumption and tolerance of wa-ter submersion. On the other hand, because earth-worms live in the soil, their physical condition and crawling behavior may be affected by chemicals enter-ing solution, such as acid (soil pH) or heavy metals (Chen & Liu 2006), in flooded burrows (Gupta et al. 1999; Spurgeon et al. 2004). To examine this phenom-enon, two common earthworm species in Taiwan were chosen: A. gracilis was selected as the positive model, as its members are known to leave the soil during the night after heavy rain, while Pontoscolex corethrurus MU¨LLER

1856 was chosen as the control model species, as its members are never observed to leave the soil after rain.

Methods

Earthworms

Specimens of Amynthas gracilis (Megascolecidae) were collected from several fields in Taiwan. Pontos-colex corethrurus(Glossoscolecidae), formerly an ex-otic species but now widespread throughout Taiwan

Journal compilation r 2008, The American Microscopical Society, Inc. DOI: 10.1111/j.1744-7410.2007.00117.x

a

Author for correspondence. E-mail: chenjh@ntu.edu.tw

(Tsai et al. 2000), was collected in a park at National Taiwan University. The earthworms were raised in a photoperiod-controlled incubator under a 12:12 h light:dark regime. The temperature was maintained at 251C and the humidity of the soil at B70–80%. Husks of rice and green bean sprouts were routinely mixed into the soil as the nutrient source. In order to exclude any effects of body weight, only mature earth-worms weighing 0.6–0.8 g were used in this study.

Crawling behavior tests

The soil components used in this study were mod-ified from a 1984 technical report of the Organization for Economic Co-operation and Development (OECD). Four hundred grams of soil (70% loam and 30% sandy soil), with a humidity of 70% and a pH of 7.0 (soil moisture and pH meter; Soil Tester, Takemura Electric Works, Taipei, Taiwan), were packed into a circular container (radi-us 5 10.5 cm, height 5 5 cm). In order to mimic the mineral components of soil dissolved in rainwater, artificial spring water (ASW) (Pierce et al. 1984) was used. When testing the effects of water alone (pH 7.0), the ASW either just covered or was 1 cm above the soil surface. In the acidic-condition tests, the earthworms were kept in soil moistened, but not cov-ered, with ASW at different pH values and 70% hu-midity. In the heavy-metal test, earthworms were maintained in moist soil (pH 7.0, 70% humidity) containing cadmium (0, 3, 9.48, or 30 mg Cd g 1dry soil). Each experiment was repeated on 20 different earthworms. These tests were performed at 251C on a 12 h light dark 1 cycle. The tests were started at noon, and the number of earthworms that crawled out of the soil was recorded every hour. If the earth-worms did not crawl out of the soil, their survival was checked after 72 h.

Survival rate in water

To mimic the environmental conditions of earth-worms in waterlogged soil, each earthworm was kept in a bottle (height 5 4.7 cm, diameter 5 2.0 cm, total volume 5 17 mL) filled with ASW. One earthworm was kept in a 90-mm-diameter plastic dish with moist #1 filter paper as a control. All treatments were per-formed in a 251C, photoperiod-controlled incubator. Some bottles containing earthworms were left open to the air, while others were sealed with polyvinyl-chloride (PVC) membrane and modeling clay to prevent air dissolving in the water during the exper-iment. Earthworms were classified as dead when they did not respond to a gentle prodding of their

pros-tomia with a sharp needle (OECD 1984), either directly (open bottles) or through the PVC mem-brane and clay (sealed bottles). If the earthworms were alive when tested, bottles were re-sealed and the experiment was continued. The residual oxygen con-centration of the water was measured when an earth-worm was classified as dead. Because the survival time in a Petri dish with wet filter paper of members of P. corethrurus was longer than those of A. gracilis, we ended the testing time at 96 and 72 h, respectively. However, in sealed bottles, we ended the test at 76 h for P. corethrurus, because the water was becoming muddy. Each treatment was repeated on 20 worms.

Oxygen consumption

Oxygen consumption was measured using a bio-logical oxygen meter (YSI5300, YSI 5301, Yellow Springs Instruments, Yellow Springs, OH, USA) as described in Chuang et al. (2004). Briefly, an earth-worm was placed in a water chamber filled with 12 mL of air-saturated water, and the oxygen concentration was measured every 15 min for 2 h (Clark-type oxygen electrode) at 251C. The oxygen concentration in a chamber with no earthworm was used as the negative control and was found to de-crease B5–7% at the beginning, but soon stabilized for the 2 h.

Air-saturated water at normal atmospheric pres-sure and various temperatures contains X mg O2/mL,

which was recorded using a digital oxygen meter (Lutron, Do 5510, Taipei, Taiwan), so that Y mLs contains X Y mg of O2. When an earthworm

weighing G g causes the oxygen concentration of the water to decline by Z% (the oxygen percentage in the chamber with a earthworm minus that with no earth-worm) in t min, the rate of oxygen consumption is (X Y  Z%)  t/60 mg h 1, and the oxygen con-sumption rate of a unit weight (g) of earthworm (mg h g 1) is [(X Y  Z%)  t/60]/G.

Rates of oxygen consumption by individuals of A. gracilis and P. corethrurus were determined over three periods, namely morning (04:00–06:00 hours), mid-day (11:00–13:00 hours), and night (19:00–21:00 hours); these periods were chosen on the basis of pilot studies. Each treatment was tested using six worms.

Statistical analysis

The effects of the different conditions on crawling out of the soil were tested using the w2 distribution. Two-way ANOVA and Duncan’s test were used to analyze differences in earthworm survival rates. The accumulated oxygen consumption of the two species

of earthworms during the different time periods was analyzed by a nested design and least-significant difference test. A probability of po0.05 was accept-ed as significant.

Results

Conditions affecting emergence

At a normal soil humidity of 70–80%, individuals of Amynthas gracilis always remained in the soil, but crawled out in r17 h when water reached the soil surface, or inr10 h when the surface was submerged to 1-cm depth (Table 1). In contrast, under acidic conditions or in the presence of cadmium, they did not crawl out of the soil (w2distribution, po0.001). None of the earthworms died in the water saturation or acidic-condition tests, and only 20% died when exposed to 0, 3, or 9.48 mg g 1 of cadmium. Apart from one individual, exposed to 30 mg g 1of cadmi-um, individuals of Pontoscolex corethrurus did not crawl out of the soil under any of the conditions test-ed (w2 distribution, p 5 0.8940.05 compared with 70% humidity, pH 7.0).

Survival in water

The mean survival time of specimens of A. gracilis in water was 5.471.34 h in a sealed bottle and

13.476.95 h in an open bottle (Fig. 1). A control earthworm remained alive for 472 h (end of testing) in a humidity box. However, individuals of P. core-thrurussurvived longer than those of A. gracilis un-der the same conditions, surviving in water for 45.473.58 h in a sealed bottle, to the end of the test (96 h) in an open bottle, and for  96 h (end of test) in a humidity box. The differences between the spe-cies were significant (po0.001). The survival time for both species was longer in open bottles than in sealed bottles, the difference being highly significant (po0.001). Because there was no significant differ-ence (p 5 0.59240.05) among the weights of individ-ual worms or between the two species, the influence of body weight on oxygen consumption was not a factor in this study.

The ASW was saturated with air by vigorous stirring immediately before an earthworm was placed in the bottle. The oxygen concentration of the ASW in the bottles before an earthworm was added was 5.8670.4 mg mL 1

at 251C. When specimens of A. gracilis were classified as dead, the mean residual ox-ygen concentration in the ASW in sealed or open bot-tles was 1.87 or 1.5 mg mL 1, respectively (Fig. 2). However, individuals of P. corethrurus could survive until the mean oxygen concentration in a sealed or open bottle declined to 0.73 or 0.64 mg mL 1, respec-tively. The minimal oxygen concentration for survival of the two species differed significantly (po0.05).

Table 1. Results of crawling experiments.

Treatment Number crawling

out of soil (n 5 20)

Average time to crawl out of soil (h) Mortality in 3 d (%) Amynthas gracilis Pontoscolex corethrurus A. gracilis P. corethrurus A. gracilis P. corethrurus Water (pH 7) Humidity 70% 0 0 — — 0 0

Water to soil surface 20 0 1770.8 — 0 0

Water 1 cm higher than soil surface 20 0 1071.2 — 0 0 pH (humidity 70%) 4.0 0 0 — — 0 0 5.0 0 0 — — 0 0 6.0 0 0 — — 0 0 7.0 0 0 — — 0 0 8.0 0 0 — — 0 0 Cadmium (mg g 1) (humidity 70%, pH 7) 0 0 0 — — 0 0 3 0 0 — — 20 0 9.48 0 0 — — 20 0 30 0 1 — 26 0 20

Oxygen consumption versus time of day

For A. gracilis, the average oxygen consumption for the period 11:00–13:00 hours was lower than that for the periods 19:00–21:00 and 04:00–06:00 hours (Fig. 3), and therefore showed a diurnal rhythm, be-ing higher at night than in daytime. The cumulative oxygen consumption showed a significant difference between 11:00–13:00 hours and the other 2-h periods, while no difference was seen in oxygen consumption between the 19:00–21:00 and 04:00–06:00 periods (Fig. 3A, Table 2). In P. corethrurus, consumption did not differ significantly among time periods (Fig. 3B, Table 2). At 19:00–21:00 and 04:00–06:00 hours, the oxygen consumption in A. gracilis was signifi-cantly higher than in P. corethrurus (Fig. 3C).

Discussion

It is a common phenomenon to see earthworms crawling out of the soil during the night or morning

after heavy rain. In the past, researchers thought that earthworms could drown in water (Lankester 1921). However, our results and other reports (Nagano 1934; Edwards & Bohlen 1996) show that earth-worms can respire in water and live for at least sev-eral hours or even days. Thus, there must be another factor driving earthworms out of the soil after heavy rain.

Fig. 2. Oxygen remaining (mean7SD, n 5 20) in artificial spring water-containing bottles at 251C, measured immediately after the worm was found to be dead. Different letters indicate significant difference.

A

B

C

Fig 3. A. Cumulative oxygen consumption in Amynthas gracilis. Note the higher respiratory rate at night than in the daytime. B. Cumulative oxygen consumption in Pontoscolex corethrurus. Note the steady increase. C. Total oxygen consumption (mean7SD, n 5 6) in 2-h periods at 251C for A. gracilis and P. corethrurus. Different letters within time periods indicate significant differences between species.

Fig. 1. Survival times of submerged earthworms (mean7SD, n 5 20). Different letters and numbers indicate significant difference among treatments.

When soil is waterlogged, chemicals, such as heavy metals, might be put into solution. The heavy metal cadmium and acidic rain can affect some physical responses of earthworms, such as cocoon production or hatching (Gupta et al. 1999; Spurgeon et al. 2004). However, no significant influence of these factors on crawling behavior in Amynthas gracilis or Pontoscolex corethrurus was observed in this study, except that a few individuals died in the cadmium-containing soil. These results show that neither acidic rain nor cadmium is responsible for driving earthworms to escape the waterlogged soil.

Waterlogging significantly decreases the oxygen concentration in the soil (Drew 1983); water, with a lower oxygen concentration and diffusion rate, dis-places air in soil pores. Therefore, once waterlogged, the oxygen concentration in soil decreases rapidly (Barrett-Lennard et al. 1986).

Some oligochaetes, such as Alma emini (MICHAELSEN 1892), can subsist in waterlogged

swamps. The interstitial spaces in the soil are gener-ally anoxic and the soil is highly reducing. Members of A. emini have evolved a remarkable unique respi-ratory strategy; the posterior-dorsal part of the body is converted into a ‘‘lung,’’ across which gases are ex-changed at the soil surface or under water (Maina et al. 1998). Terrestrial earthworms do not possess this respiratory strategy; although earthworms can respire in water for a period, they do not survive well in oxygen-poor water (Nagano 1934).

In this study, we measured the oxygen consump-tion of earthworms from a limited reservoir. Com-monly, in oxygen-consumption studies, animals are maintained in a small vessel and bathed in flowing, oxygen-laden water for a period of time; after the test animal is acclimatized, the oxygen-saturated water

flow is stopped, and oxygen consumption is mea-sured. In this study, however, we wanted to mimic an unusual waterlogged environment. The limited oxy-gen reservoir of our test instrument, designed for measurement of oxygen consumption rate, appropri-ately fits the experimental design of this study where earthworms were exposed to waterlogged soil and consumed oxygen under a diffusion-limited condi-tion. Accordingly, the earthworms were not kept in a resting state. In A. gracilis, because of the limited reservoir, the oxygen consumption rate decreased during the experiment; total consumption reached a plateau. On the other hand, while the consumption rate in P. corethrurus slowed, the total consumption continued to increase during the experiment. Corre-spondingly, at the end of the experiments, the water in which A. gracilis was tested retained a higher oxygen concentration than that of P. corethrurus. Therefore, we inferred that members of P. core-thrurus can survive better than those of A. gracilis in water with a lower oxygen concentration, due to lower oxygen consumption or better oxygen uptake. Some earthworms have been known to respire an-aerobically for a period (Davis & Slater 1928; Lee 1985; Edwards & Bohlen 1996). In this study, the oxygen concentration in the water at the end of the experiment was found to be very low, and so it is possible that members of P. corethrurus can respire anaerobically under oxygen-poor conditions.

Members of A. gracilis crawled out of the soil 10– 17 h from noon, at night, matching the diurnal rhythm of their higher oxygen consumption. Most terrestrial earthworms are more active at night (Chuang et al. 2004). Predation pressure (Christensen & Mather 2001; Csermely 2003), especially from birds, might be a factor selecting against day emergence of earth-worms during daytime. Ultraviolet light exposure may Table 2. Comparison of the accumulated oxygen consumption at different time periods, measured every 15 min for 2 h, using a nested design and a least-significant difference test. Numbers represent p values; the asterisks indicate significant differences.

Pontoscolex corethrurus Amynthas gracilis

04:00–06:00 11:00–13:00 19:00–21:00 04:00–06:00 11:00–13:00 19:00–21:00 P. corethrurus 04:00–06:00



0.281 0.299 0.0001 0.8927 0.0001 11:00–13:00



0.967 0.0001 0.345 0.0001 19:00–21:00



0.0001 0.3663 0.0001 A. gracilis 04:00–06:00



0.0001 0.7193 11:00–13:00



0.0001 19:00–21:00



_po0.05._po0.01.

be another negative selection factor against daytime emergence (Merker & Bra¨gunig 1927), but the inten-sity of UV light is low on a rainy day and should not be strong enough to threaten the earthworms under waterlogged conditions due to heavy rain.

Other factors, such as sickness (Darwin 1881), mat-ing, feedmat-ing, and migration (Edwards & Bohlen 1996), might cause earthworms to emerge above ground. However, these factors were also excluded to explain our results for the following reasons. Firstly, the mat-ing or feedmat-ing behavior of earthworms differs from their crawling behavior after rain. A study of mating behavior showed that an earthworm stretches its an-terior section out onto the ground to make contact with another earthworm, leaving its posterior section in the soil (Nuutinen & Butt 1997). This resembles its feeding behavior. According to our observations over the last 10 years, earthworms that crawl out of water-logged soil never mate, even though they may be in close contact with one another. Secondly, the vast ma-jority of earthworms that left the soil appeared to be healthy, because they could be maintained in our lab-oratory for several months (data not shown), thus ex-cluding the possibility of illness.

In summary, our results support our hypothesis that the night-time emergence behavior of earthworms is mediated by their diurnal rhythm of oxygen consump-tion. Earthworms of some species, such as A. gracilis, have lower oxygen consumption during the daytime and can remain in waterlogged soil; at night, however, especially when waterlogging due to heavy rain reduces oxygen availability in the soil, greater activity and higher oxygen consumption drive the earthworms to crawl out of the soil in order to obtain more oxygen. In contrast, earthworms such as P. corethrurus, which have lower oxygen consumption with no diurnal rhythm, can tolerate reduced oxygen levels due to wa-terlogging and never emerge after heavy rain.

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