Introduction

Earthworms are well recognized as soil engineers that significantly affect soil ecosystems by enhancing the soil macro and micronutrients as well as the soil structure1,2,3. Amongst the reported megascolid terrestrial earthworm, Pontodrilus is the only genus that is found globally distributed on sandy beaches and coastal areas. At present, two species of the littoral earthworm genus Pontodrilus have been recorded from the seashore of Thailand and Peninsular Malaysia namely P. litoralis (Grube, 1855) and P. longissimus Seesamut and Panha, 2018. Habitat preferences for P. litoralis and P. longissimus are obviously different with P. litoralis being widely distributed throughout tropical and sub-tropical sandy beaches around the world, while P. longissimus is only found at specific localities. Moreover, P. litoralis can be found in a wide range of habitats, such as sandy beaches, estuaries, mangrove swamps, and the lakes with a very low salinity4,5,6,7, and different types of soil texture. In contrast, P. longissimus can only be found in fine and muddy sand at the ecotone between marine and terrestrial-freshwater aquatic linked habitats6,8.

Variation in salinity is a major environmental major influence on the distribution of organisms in estuaries9,10,11. Previous studies reported that increments in the salinity had harmful effects on the growth, mortality, and reproduction of earthworms, while sodium chloride shows negative effects on the earthworm’s life cycle12,13. As mentioned above, the littoral earthworm genus Pontodrilus are euryhaline and are often exposed to salinity fluctuations that may range between 1 and 33 ppt6,7. In addition, the specific euryhalinity character of Pontodrilus has been reported in some detail14,15, and is believed to be an important tool for the littoral earthworm to regulate the osmotic concentration of their body fluid in order to survive and adapt to that extreme environment.

Earthworms in general can tolerate limited salinity, but some earthworms, like Eisenia fetida16, are highly salt-tolerant. In this research, the salinity tolerance of P. litoralis and P. longissimus, which both occur in saline habitats but in different microhabitats6,17, was evaluated based upon the following. To verify the distribution pattern of P. litoralis and P. longissimus, it is necessary to further identify the main driving factor behind this habitat selection process. The present study was, therefore, dedicated to identifying their salinity preferences in determining the successful adaptation of these two Pontodrilus species with respect to their recorded habitat. Experimentally, we observed the survival rate, weight change, behavior, and osmolality of P. litoralis and P. longissimus when subjected to different salinity concentrations over both a short- and a relatively long-term exposure (0–96 h and 28 d, respectively). We evaluated two hypotheses (i) a higher salinity will negatively affect the survival and weight of Pontodrilus, and (ii) that, since P. litoralis is a cosmopolitan earthworm that occurs naturally in the sand surface areas where sharp salinity gradients are frequent along with a high salinity exposure, it will be able to survive wider salinity ranges and higher salinity concentration than P. longissimus, a species that inhabits a deeper level in the sand and preferentially occurs in estuaries.

Results

Field sampling and observation

A total of 15 habitats of Pontodrilus were observed, and the salinity level was recorded in the field (Fig. 1, Supplementary Table 1, Supplementary Fig. 1). Pontodrilus litoralis was found in various habitat types for example under trash or leaf litter on the sandy beach, in mangrove swamp, sanitary sewer emptying to a sandy beach, and estuaries. These sites included both undisturbed and disturbed areas. However, P. longissimus mostly occurred in estuaries. We observed six localities where P. litoralis was found co-existing with P. longissimus in estuary habitats. At these locations, P. litoralis was found under debris or trash in the topsoil while P. longissimus was found deeper than P. litoralis; at a depth of 10–50 cm. The highest salinity recorded in a P. litoralis habitat was 33 ppt, whereas it was 21 ppt for sites with P. longissimus.

Figure 1
figure 1

Map of the sampling locations where Pontodrilus species were found in this study. Circle and star symbols represent localities for P. litoralis and P. longissimus, respectively, while square symbols are the localities where both Pontodrilus species were found. (Abbr. are mentioned in Supplementary Table 1).

Full size image

Long-term (28 d) exposure to different salinity concentrations

Data on the survival rate and weight change of littoral earthworms exposed for 28 d to different concentrations of salinity are presented in Fig. 2. Pontodrilus litoralis was able to survive a wide range of salinities (0–50 ppt) with the highest survival rate observed at ≤ 30 ppt. The survival rate then decreased as the salinity increased above 30 ppt, falling to 97.5% and 87.5% at 40ppt and 50 ppt, respectively. In contrast, the survival of P. longissimus was more strongly affected by changes in salinity with the highest survival rate recorded at 10ppt (ca. 97%), being lower at 0 ppt (ca. 56%), and decreasing at salinities above 10 ppt to ca. 76% at 20 ppt and 0% at 30–50 ppt. Relatively, P. litoralis have a higher survival rate than P. longissimus and is better adapted for survival over a wider salinity range.

Figure 2
figure 2

Pontodrilus litoralis and P. longissimus after a 28-day exposure to different salinity concentrations. The percentage survival rates are shown as lines while the changes in the fresh weight are shown as bars. Data are shown as the mean ± SD, derived from four replications.

Full size image

The weight change of P. litoralis was significantly affected by the salinity of the test medium, an increment in weight was observed when P. litoralis was exposed to a salinity of less than 40 ppt (highest increase as the salinity increased above 10 ppt to 40 ppt), but with a marked weight loss at 50 ppt. In contrast, P. longissimus exhibited a high weight loss at a low salinity of 0–20 ppt and was unable to survive at salinities above 20 ppt. These results clearly indicated that P. litoralis is better adapted than P. longissimus to changes in salinity, which would likely account for its wide distribution in different environments.

Short-term (0–96 h) exposure to different salinity concentrations

Pontodrilus litoralis exhibited the same weight gain pattern when exposed to a salinity of less than 30 ppt, where a significant weight gain was clearly observed over the first 12 h of exposure (Fig. 3) followed by a sharp decrease in the weight gain and ending in a constant weight after nearly 96 h. A marked weight loss was observed when P. litoralis was exposed to a salinity of 40 and 50 ppt. For P. longissimus, there was an increment in weight over the first 72 h of exposure to a salinity of 0 ppt and 10 ppt, but a prolonged exposure (96 h) resulted in a significant weight loss.

Figure 3
figure 3

Weight change (%) of adult P. litoralis and P. longissimus after 3, 6, 12, 24, 48 h, 72 h and 96 h of exposure to different salinity concentrations. Data are shown as the mean ± SD, derived from three replications. Means with a different letter are significantly different (ANOVA: Tukey’s test; P < 0.05).

Full size image

Pontodrilus litoralis and P. longissimus behaved differently when exposed to different salinity levels; not all individual presented the same behavior patterns initially but towards the end of the exposure period (96 h), all of them showed similar patterns in the introduced test medium. At a salinity of 0–30 ppt, P. litoralis immediately coiled and knotted (Fig. 4A) its posterior end. This response was maintained for up to 24 h of exposure and then they started to relax and loosen the knot by fully stretching their body (Fig. 4B) and maintained a calm state with slow movement until the end of the experiment. Increasing the medium’s salinity resulted in an increased body movement in P. litoralis with the most active was recorded at a salinity of 30 ppt. At 40 ppt, P. litoralis started to loosen its knot and stretch its body after 6 h of exposure, and the posterior end turned reddish (Fig. 4C), but they still moved a moderate amount until the end of the experiment. At 50 ppt, P. litoralis was inactive and became thinner at the posterior end (Fig. 4D). Besides these behavioral changes, the size changed significantly when P. litoralis was exposed to a high or a low salinity (Supplementary Fig. 2). In contrast, P. longissimus reacted differently compared to P. litoralis. At a salinity of 0–20 ppt, slow movement and coiled at posterior end was observed in P. longissimus. After 12 h of exposure, they started to relax and loosen the knot by fully stretching their body until the end of the experiment. At 30 ppt, P. longissimus coiled and knotted its posterior end from the beginning of exposure until towards 96 h of exposure. At high salinities (40–50 ppt), P. logissimus immediately coiled and knotted its body with rapid movement, after 3 h of exposure P. longissimus was inactive and the whole body of the earthworm turned reddish and became thinner with 100% mortality was observed in all replicates of treatment.

Figure 4
figure 4

Photographs showing Pontodrilus behavior when exposed to different salinities and time: (A) coiled and knotted [10 ppt after 3 h of exposure], (B) stretching [10 ppt after 48 h of exposure], (C) posterior end turns reddish [40 ppt after 6 h of exposure], (D) thinner in size at the posterior end [50 ppt after 24 h of exposure].

Full size image

Osmolality of the coelomic fluid

Field collected P. litoralis earthworm (Fig. 5) exhibited a coelomic fluid osmolarity of 765 mOsm kg−1. When they were placed into 0 ppt (1–9 mOsm kg−1, mean = 4 mOsm kg−1) test medium, they regulated the water content of the body and so although the coelomic fluid osmolarity increased, it remained hypertonic (553 mOsm kg−1) to the medium. A similar trend was observed when the worms were introduced into 10 ppt salinity (233–376 mOsm kg−1, mean = 314 mOsm kg−1), where the coelomic fluid osmolarity increased to 653 mOsm kg−1. Further increasing in the medium’s salinity of 20 ppt (533–817 mOsm kg−1, mean = 641 mOsm kg−1) and 30 ppt (783–1198 mOsm kg−1, mean = 961 mOsm kg−1), the coelomic fluid of P. litoralis rises until it meets isotonicity (852 mOsm kg−1 and 1341 mOsm kg−1 respectively) at 96 h of exposure. Further increased the salinity of the test medium to 40 (1309–1825 mOsm kg−1, mean = 1465 mOsm kg−1) or 50 ppt (1557–2118 mOsm kg−1, mean = 1748 mOsm kg−1) resulted in the coelomic fluid osmolarity becoming isotonic after 48 h of exposure, but the worms started to die if immersed in either of these salinities for more than 48 h. Pontodrilus longissimus showed a similar trend when it was placed in a 0 ppt salinity test medium (5–10 mOsm kg−1, mean = 6 mOsm kg−1), it remained hypertonic (505 mOsm kg−1) to the medium (Fig. 6). When they were placed into medium with a salinity of 20 ppt (560–677 mOsm kg−1, mean = 608 mOsm kg−1) or 30 ppt (912–1030 mOsm kg−1, mean = 960 mOsm kg−1), the coelomic fluid of P. longissimus becoming isotonic after 48 h of exposure. However, at a salinity of 40 ppt (1315–1649 mOsm kg−1, mean = 1413 mOsm kg−1), P. longissimus was not able to survive for more than 48 h of exposure.

Figure 5
figure 5

The coelomic fluid osmolarity of P. litoralis after exposure to different salinity concentrations of: (A) 0 ppt [different scale of Y axis was used to plot the graph], (B) 10 ppt, (C) 20 ppt, (D) 30 ppt, (E) (40) ppt, and (F) 50 ppt. Horizontal constant dash lines represent field values.

Full size image
Figure 6
figure 6

The coelomic fluid osmolarity of P. longissimus after exposure to different salinity concentrations of (A) 0 ppt, (B) 10 ppt, (C) 20 ppt, (D) 30 ppt, and (E) 40 ppt. Horizontal constant dash lines represent field values.

Full size image

Discussion

The established cosmopolitan littoral earthworm Pontodrilus litoralis was found to live in various types of habitats whereas P. longissimus was found only in estuaries with muddy sand. Sympatry of these two Pontodrilus species occurred in some localities, where P. litoralis was repeatedly found in the sand surface, while P. longissimus was found at deeper levels below the surface than P. litoralis6,17. Pontodrilus litoralis has been reported as a bioluminescence earthworm and uses this to avoid predation by littoral predators because P. litoralis live on the sand surface17,18,19,20. We suggest that salinity is one of the key factors affecting the distribution and habitat preference of Pontodrilus in this region. Congeneric P. longissimus inhabited mostly estuaries with a lower salinity compared to P. litoralis.

The 28-d exposure to different salinity concentrations demonstrated a negative relationship between high salinity stress and P. longissimus survival, which only survived at low salinity concentrations and with a lower survival rate observed when compared to P. litoralis. We suggest that P. litoralis has a higher tolerance to salinity fluctuations than P. longissimus and that both species of littoral earthworms prefer to inhabit the ecotone between terrestrial and marine habitats. Our results support the current distribution patterns of these two littoral earthworm species, where P. litoralis is a cosmopolitan species and occurs in a very wide range of sub-temperate and tropical coastal ecosystems4,6,7,21,22,23,24 with dispersal mechanisms including transportation by wooden vessels5,25. Salinity fluctuation is a principal environmental factor affecting the distribution of marine and estuarine species26 and high euryhalinity may be a factor in its global distribution similar to the case of the marine intertidal gastropod Stramonita brasiliensis27. In contrast, a poor tolerance to salinity changes is a factor that limits the dispersal ability of P. longissimus, concordant with it only being reported in the coastline of Thailand and Peninsular Malaysia6,8.

The effects of salinity have also been reported in other earthworms, where it affected the quality of products in composting, limited the growth of earthworms, and reduced the weight and survival percentage of earthworms12,28,29,30. Here, the salinity was found to affect the weight change of both littoral earthworm species. The highest level of weight gain in P. litoralis was observed at 12 h in all salinity concentrations, which we suggest supports the notion that water enters by osmosis increasing the worm’s weight but this response becomes limited over time. The result showed that P. litoralis was able to resist salinity changes and match the salinity fluctuations in their natural microhabitat as previously reported in P. bermudensis (which is now considered a synonym of P. litoralis)14, with a salinity concentration of 20–30 ppt being the optimal range for P. litoralis.

Understanding the fundamental knowledge of osmotic strategies in gaining salinity tolerance should provide insights into the evolutionary processes of physiological adaptation to saline waters and habitat selection strategies. Several studies have been reported on osmoregulation in earthworms28,31,32. Since, osmoregulation is very important in estuarine intertidal species, we investigated how the coelomic fluid osmolality of these two Pontodrilus increases with increasing medium salinity to evaluate how they the species handle salinity fluctuations. Changes in salinity concentrations will cause a change in the osmotic pressure of an organism through osmoregulation processes. The body fluid osmolarity changed in accord with the outside exposure medium. These findings are in agreement with the present hypothesis and confirm that P. litoralis and P. longissimus are osmoconformers with regard to salinity change33,34,35. In conclusion, the current study examined the effects of different salinities on the two species of earthworms in the genus Pontodrilus. A high salinity concentration resulted in a reduced weight and survival percentage of the cosmopolitan earthworm P. litoralis and was fatal to the endemic P. longissimus. The osmoregulation ability of these two different Pontodrilus species of littoral earthworms to changes in salinity appear to explain their distribution pattern and habitat preference throughout Thailand’s coastal areas, where P. litoralis has a higher tolerance to salinity fluctuations than P. longissimus corresponding with its greater ability to disperse throughout the world. Habitat patterns associated with salinity indicated that their natural habits are based upon the least osmolarity stressful environment. Theoretically, a specific adaptation is required, they either control their body fluid equivalent to the composition of the sea or actively conserve or shed excess salts.

Material and methods

Field sampling and observation

Littoral earthworm genus Pontodrilus were surveyed throughout the coastal areas of Thailand, both on the Gulf of Thailand (east coast) and the Andaman Sea (west coast) from January 2015 to January 2016. The sampling sites are shown in Fig. 1. During the field collection, geographic variation between the sampling sites was observed and photographed (type of biotope), with salinity of the sampling site were recorded.

Laboratory experiment

Test organism

Adult P. litoralis (weight 190.31 ± 30.21 mg) and P. longissimus (weight 532.49 ± 146.17 mg) with a well-developed clitellum were collected from Hat Bang Saen, Mueang Chonburi, Chonburi, Thailand and from Hat Pakmeng, Sikao, Trang, Thailand, respectively. Only active worms were acclimated to laboratory conditions. The earthworms were rinsed with distilled water and left for a day on wet paper, moistened with artificial seawater without soil to avoid their stomach contents.

Test substances and medium

The sand was transported back from the field to the laboratory and oven-dried for 24 h. Different salinities of the test medium (0, 10, 20, 30, 40 and 50 ppt) were prepared by adding artificial sea salt to distilled water, and the salinity was then adjusted accordingly using a refractometer (ATAGO, Master-S10M).

Experimental procedure

Long-term (28 d) salinity exposure

A similar working procedure was applied to the long-term exposure (28 d) where the earthworms were exposed to different salinities (0, 10, 20, 30, 40 and 50 ppt) in the presence of the sand as a substrate. Round plastic containers (internal diameter 170 mm, height 130 mm) were filled with 2 kg of sand. Each container had an inlet and an outlet to allow for running water and cleaning processes and was lined with a paper bag in order to prevent the worms from escaping out of the container. Ten earthworms were released into each container with four replicates of each treatment. The tests were conducted at ambient temperature (30 ± 2 °C) and a 16:8 h light:dark cycle (light intensity of 400–800 lx). Finely ground cow dung (30 g) was added each week to each container as food36. At the end of the exposure, the survival rate and weight change of each treatment were calculated.

Short-term (0–96 h) salinity exposure

The short-term exposure test was conducted in a round, flat-bottomed (internal diameter 48 mm, height 80 mm) container. Each test container was filled with 20 ml of the prepared medium with the designated salinity level (artificial seawater at 0, 10, 20, 30, 40 and 50 ppt) and then exposed for different duration (3, 6, 12, 24, 48, 72 and 96 h). Each treatment was performed with three replicates (one individual per replicate). The test units were closed with a perforated plastic film to prevent the earthworms from escaping. The experiment was carried out at ambient room temperature (25 ± 2 °C) with a 16:8 h light:dark illumination cycle (light intensity of 400–800 lx). The working medium was checked and changed every day in order to avoid salinity changes due to evaporation. The behavior of the earthworms was recorded using a Canon 650D camera. The earthworms were weighed after 0, 3, 6, 12, 24, 48, 72 and 96 h of exposure, and the change in weight was calculated. The earthworms were prepared for osmolality testing.

Data analysis

One-way ANOVA was used to evaluate differences among different treatments on the assessed parameters, while Tukey’s post hoc test was performed to test for the significance of any differences between the means of different treatments. All statistical analyses were performed using the Minitab 16 Statistical software and significance was accepted at P < 0.05 level.

Determination of the body (coelomic) fluid osmolarity

Earthworms from the short-term exposure experiment were used for osmolality determination. Their coelomic fluid (≥ 20 μL) was extracted from the coelomic cavity with the aid of a thin glass capillary. A Micro Osmometer (Fiske Model 210 Micro Osmometer) was used to determine the osmolality of each solution using freezing point depression and was calibrated using the manufacturer-supplied standards.