Studying Annelida Body Regeneration Under Environmental Stress in Diopatra neapolitana

Abstract

The polychaete Diopatra neapolitana is a cosmopolitan annelid that can robustly regenerate both its anterior and posterior body part depending on the position of the amputation. Previous studies demonstrated that body regeneration represents a sensitive and unspecific response to environmental stresses, including contaminants and climate alterations.

The posterior body regeneration of D. neapolitana is thus a suitable, ecological and relevant biomarker in ecotoxicological and ecological risk assessment assays. Here we describe the amputation process, the monitoring of the regeneration process of the polychaete D. neapolitana and the quantification of the impact of environmental stresses on its regenerative capacity.

1 Introduction

Annelids are known for their efficient wound healing and their capacity to regenerate both anterior and posterior segments after loss by injury [1, 2]. This regenerative ability varies significantly within the phylum, and some species can regenerate an entire individual from a single segment while others are much more limited [3]. Previous works demonstrated that species of the genus Diopatra could regenerate anterior and posterior segments and prostomial structures [3,4,5,6]. This mechanism performs a critical role in survivorship after tissue loss due to sublethal predation and harvesting [5, 7]. Additionally, it can also aid in recovery from injuries due to physical alterations [8].

Several studies demonstrated that exposure to environmental stressors such as contaminants or abiotic alterations reduced the regenerative capacity of polychaetes [9,10,11,12,13,14,15,16,17], with organisms regenerating slower and usually fewer chaetigers (segments that have chaetae). Nusetti et al. [9] observed that the polychaete Eurythoe complanata exposed to crankcase oil took longer to regenerate a new region and regenerated fewer segments. Exposure to micro- and nanoplastics reduced the capacity of Perinereis aibuhitensis and Hediste diversicolor to regenerate their posterior ends [14, 15]. Diopatra neapolitana exposed to several contaminants, such as metals, pharmaceuticals, carbon nanotubes, and environmental enrichment presented a delay in posterior segments regeneration, taking longer to achieve complete regeneration, and regenerated fewer segments [10,11,12,13, 16, 18]. Moreover, exposure to abiotic alterations, including pH variations and salinity changes, also reduced the regenerative ability of D. neapolitana [17].

Although the majority of toxicity studies with polychaetes have been conducted using the species H. diversicolor [14, 19,20,21,22,23], most of those regarding the use of the regenerative ability as a biomarker were carried out with the species D. neapolitana due to this process being well documented for Diopatra species (e.g., [3, 5, 24]). Additionally, this species represents a wide spatial distribution, being reported in intertidal and shallow subtidal habitats, namely, in the Red Sea and Indian Ocean [25], Mediterranean Sea [25,26,28], and the Atlantic Ocean [6, 28,29,31]. Furthermore, Diopatra species play an important ecological and economic role. Their tubes stabilize the sediments, increasing their structural complexity and thus their biodiversity, by supplying refugia from disturbance and predation [32] and facilitating the settlement and the attachment of some algal species [33]. Moreover, this species is commonly harvested to be sold as fish bait [31, 34, 35]. Altogether, these studies suggest that the regenerative capacity of polychaetes is a suitable biomarker in ecotoxicological and ecological risk assessment assays since it is sensitive to environmental stressors, including organic and inorganic contaminants and climate alterations.

The mechanisms behind this sensitive yet unspecific response to environmental stresses in Diopatra remain to be elucidated. Some authors suggested that the delay in regenerative capacity could be related to exposure to oxidative stress [10, 11, 14, 17] since free radicals may damage the biochemical and cellular functions that underlie the regenerative process. Moreover, Soneja et al. [36] reported that oxidative stress prolonged chronic wound inflammation as it stimulates cells of the immune system. Delayed regeneration may impact the sexual reproduction of individuals, as organisms will canalize their reserves toward tissue regeneration rather than producing gametes [5,6,7]. Also, the delay of organisms in starting gamete production compromises population maintenance, with consequences for communities and ecosystems [8].

Consequently, due to this species’ ecological importance, understanding the interplay between environmental stresses and regenerative capacity is particularly pertinent since delays in regenerative capacity may negatively impact population and ecosystem function.

This chapter presents a detailed protocol to study the impacts of environmental stressors in the posterior regenerative capacity in field-collected organisms of the polychaete D. neapolitana .

2 Materials

All reagents should be prepared with sterile reverse osmosis water and stored at room temperature (RT) unless otherwise stated.

• 1. 20 × 15 × 40 cm (W × L × H) glass aquariums (see Note 1).

• 2. Sediments: clean medium or fine sand with low organic matter content. Collect from nonpolluted organisms’ sampling site (see Note 2).

• 3. Artificial seawater (ASW): 30 g/L commercial synthetic sea salt (e.g., Tropic Marin Sea Salt) (see Note 3). Prepare at least 1 day before use.

• 4. Aeration system.

• 5. Acclimated culture room: photoperiod (12 h light:12 h dark), controlled temperature, constant aeration.

• 6. Diopatra food: collect shellfish in a clean site, cut in 2 mm³ cubes, store until needed at −20 °C (see Note 4).

• 7. Contaminated ASW (e.g., 0 to 0.25 μg/L of arsenic).

• 8. Contaminated sediments (e.g., 0 to 9 mg/kg of lead).

• 9. Anesthetizing solution: 4% (w/v) MgCl₂·6H₂O in ASW.

• 10. Imaging setup: stereomicroscope with a camera attached and a ruler for measuring organisms.

3 Methods

3.1 Collection of Organisms and Acclimation

• 1. Setup the acclimated culture room to the desired conditions (see Note 5).

• 2. Fill each aquaria to be used with 3 L of sediment (see Note 6).

• 3. Add 9 L of ASW.

• 4. Add aeration to the aquaria.

• 5. Choose a sampling site where D. neapolitana can be found (see Note 7).

• 6. Identify a tube containing a specimen (Fig. 1a, see Note 8).

• 7. Pitch a shovel 10 cm away from the tube, with an inclination of about 45° and deep up to 30 cm (Fig. 1b).

• 8. Expose the tube by digging the shovel.

• 9. Transfer Diopatra neapolitana inside their tube into a transport bucket (see Note 9).

• 10. Repeat steps 6 to 9 until sufficient specimens are collected.

• 11. Repeat steps 5 to 10 until all the target sampling sites have been explored.

• 12. Transfer the animals to the lab (see Note 10).

• 13. Fill a beaker with 2 L ASW.

• 14. Pick a Diopatra tube using a pair of tweezers.

• 15. Flush the anterior end of the tube to force the specimen out of its tube into the beaker using ASW (see Note 11).

• 16. Transfer organisms with more than 60 chaetigers into the prepared aquaria but discard regenerating specimens (Fig. 2a, b, see Note 12).

• 17. Repeat steps 14 to 16 until all tubes are processed.

• 18. Repeat steps 13 to 17 until all transfer buckets are emptied of their animals.

• 19. Wait 24 h.

• 20. Discard animals that have not rebuilt a new tube.

• 21. Discard unhealthy animals (see Note 13).

• 22. Wait 24 h.

• 23. Repeat steps 21 and 22 for 5 more days.

• 24. Renew water of every aquarium.

• 25. Place a piece of Diopatra food near the entrance of each tube as such as organisms can detect it.

• 26. Wait 2 h for the animals to consume the food.

• 27. Remove the food that is not consumed.

• 28. Repeat steps 21 and 22 for 2 more days (see Note 14).

• 29. Repeat steps 25 to 28 two more times.

• 30. Renew water of every aquarium.

• 31. Discard animals that have not healed their damaged posterior part (see Note 15).

• 32. Repeat steps 25 to 30 to maintain the culture of Diopatra (see Note 16).

Fig. 1

(a) Tube of Diopatra neapolitana at sediment surface and (b) shovel with the inclination that should be used to catch D. neapolitana specimens

Fig. 2

Diopatra neapolitana anterior end ventral view (a) and dorsal view (b); (c) D. neapolitana specimen regenerating the posterior end, (d) D. neapolitana with posterior end regenerated. The newly regenerated chaetigers have a lighter color, being possible to observe the blood vessels through the body wall. 10—chaetiger 10, 60—chaetiger 60, P—Prostomium, Br—Branchiae, Pa—parapode, R—width of the regenerated chaetiger; NR—width of the not regenerated chaetiger (chaetiger 60); RS—specimen with posterior end fully regenerated

3.2 Regeneration Assay

Experiments should be carried out with acclimatized specimens of similar size. The impacts of environmental stresses are tested by exposing the regenerating organisms to contaminated sediments and/or contaminated water.

• 1. Follow steps 1 to 4 in Subheading 3.1 to prepare the aquariums for each condition that will be tested and for the controls (see Note 17).

• 2. Follow steps 13 to 15 in Subheading 3.1 to remove the specimens from their tubes.

• 3. Transfer an animal to a petri dish filled with 100 mL of anesthetizing solution.

• 4. Wait 15 min for the animal to anesthetize (see Note 18).

• 5. Transfer the dish under a stereomicroscope.

• 6. Measure the width of the tenth chaetiger (without parapodia) using the ruler (Fig. 2b, see Notes 19 and 20).

• 7. Amputate the anesthetized organism at the 60th chaetiger (see Note 21) (Fig. 2b) with a scalpel.

• 8. Transfer the animal into a beaker filled with 100 mL ASW.

• 9. Wait 20 min for the animal to “wake up” and start to swim in ASW.

• 10. Select organisms with similar sizes for the regeneration assay (see Note 20).

• 11. Place the amputated specimen in the experiment aquarium.

• 12. Repeat steps 3 to 11 to measure and amputate at least nine individuals per condition.

• 13. Follow steps 25 to 30 in Subheading 3.1 to feed the regenerating animals.

• 14. Renew the water of each tank with the corresponding culture condition.

• 15. Repeat steps 3 to 5 to anesthetize an animal.

• 16. Measure the width of the regenerated body part (R), the width of the last nonregenerated segment (NR) (Fig. 2c) and count the number of regenerating segments (RS), identified by the lighter color and/or the narrower width compared to the rest of the body (Figs. 2c and 3a–f, see Note 22).

• 17. Return the anesthetized animal to its experiment aquarium.

• 18. Repeat steps 15 to 17 to measure all animals.

• 19. Repeat steps 14 to 18 once a week until complete regeneration (see Notes 23 and 24).

• 20. Quantify the regenerative capacity of each condition through three parameters: the percentage of body regenerated (R/NR, Figs. 2c and 3a–f), the number of segments regenerated (RS), and the time needed to achieve complete regeneration (i.e., when R = NR, Fig. 2d).

Fig. 3

Different levels of posterior regeneration of Diopatra neapolitana exposed to sediments contaminated with lead (0.0, 3.0, 9.0 mg/kg). Photographic record of the regeneration process 14 (left column) and 28 days (right column) after amputation at control (a and b), 3.0 mg/kg (c and d), and 9.0 mg/kg (e and f)

4 Notes

• 1. Keep animals at a maximum density of 50 individuals/m². For example, this 20 × 15 cm aquarium can contain 14 organisms. Vary the size of the aquaria based on the number of organisms that will be used in each experiment.

• 2. If possible, use the sediments of the same sampling site where organisms will be collected. If these sediments do not have good characteristics, use sediment from another clean site. Alternatively, use commercial sand.

• 3. Salinity and pH should be adapted to parameters recorded in the sampling site. Alternatively, 0.22 μm filter-sterilized seawater (FSW) collected from the sampling site can be used.

• 4. Cockles and mussels are Diopatra’s preferred shellfish. Make sure to collect in a clean site. Alternatively, use commercial fish food (46% protein, 11% lipids) as feed. If commercial fish food is adopted, feed each organism with about 10 mg.

• 5. Organisms should be maintained at a constant temperature, salinity and pH similar to those measured in the collection site. In our case, experiments are usually carried out at a temperature between 17–20 °C, salinity between 28 and 30, and pH 7.8.

• 6. The height of the sediment in the aquarium should be about 10–12 cm. Diopatra neapolitana adults are very long, but organisms are not collected whole. Additionally, organisms construct their tubes with some inclination; thus, this height of sediment is suitable.

• 7. D. neapolitana are cosmopolitan tubiculous animals that live in muddy or muddy sand intertidal areas. They can be easily detected by the presence of their tubes on the sediment surface since they protrude a few millimeters above the surface of the sediment (Fig. 1a).

• 8. A tube with water is an inhabited tube. On the other hand, if the tube contains sediment inside, it is not inhabited by the polychaete .

• 9. Organisms are not collected entire, as they are very long; only the anterior end is usually collected. Avoid collecting organisms during the reproductive period (usually during summer months [27, 37]).

• 10. Animals can stand up to 2 h in transport buckets. The tubes could be complemented with macroalgae to maintain the organisms up to 6 h in the buckets.

• 11. It is important to remove the organisms from their tubes because their tubes usually have attached pieces of algae and other materials in the vicinity of the tube that will decompose and contaminate the clean sand. Organisms without tube will construct a new tube with the sand in a few hours.

• 12. Sampled specimens that show signs of undergoing regeneration will not be used in the experiment. They can be distinguished by the lighter color and/or the narrower regenerating chaetigers compared to the rest of the body (Fig. 2c).

• 13. In dead or dying organisms, the anterior end (antennae and some segments) usually remains outside the tube. To check their vitality, touch them in the portion located outside of the tube. If the polychaete runs into the tube, it means that it is alive. However, if it does not run into the tube, it means that it is dying. In this situation, remove the organism from the aquarium with its tube. Dead organisms should be immediately removed from the aquarium because they begin to decompose very quickly and contaminate water and sediment.

• 14. If needed, animals can be fed every 2 days.

• 15. Since organisms are very long, they cannot be harvested as a whole; consequently, it is necessary to ensure that their integrity is reestablished. This period is critical to understand if the organisms have already started to heal the posterior end. Healthy organisms should have at least healed the damaged part after 1 week. Do not use specimens that have not healed after these 2 weeks.

• 16. Diopatra can be maintained in culture for several months. Healthy organisms will heal and completely regenerate the posterior end in 2 months [38]. In this case, organisms should be changed for larger aquaria, with 20 cm of sediment (height).

• 17. We advise to prepare at least three aquariums per condition.

• 18. Organisms take about 15 to 20 min to become anesthetized. Do not maintain organisms anesthetized longer than 1 h because they may not recover.

• 19. The width of the tenth chaetiger is commonly used as the unit of size among Diopatra species as it is challenging to capture entire Diopatra animals [39, 40].

• 20. Organisms can differ by a margin of about 2 mm; for example, choose organisms with the width of the tenth chaetigers between 6 and 8 mm.

• 21. Studies conducted by Pires et al. [5] further revealed that, under laboratory conditions simulating environmental conditions, D. neapolitana specimens are able to regenerate the anterior body part only when organisms are amputated up to the 15th chaetiger, where the posterior end can regenerate the missing anterior part. Polychaetes, when amputated at chaetiger 3, 10 and 15 have a survival capacity of 87.5%, 75% and 50%, respectively and regenerated the anterior end. Individuals amputated around chaetiger 20 cannot regenerate and do not survive. D. neapolitana organisms amputated at chaetiger 25 and beyond only regenerated the posterior part. Individuals amputated between the 25th chaetiger, 40th and after branchial region (around 60th chaetiger) are able to regenerate and present a survival capacity ranging from 50%, 81.3% and 100%, respectively [5]. Considering these results, we suggest to amputate the animals at chaetiger 60 for all organisms to survive the procedure.

• 22. On the first week, the regenerated portion does not form individualized segments; therefore, it is not possible to count the chaetigers.

• 23. Full regeneration as evidenced by the same width between the older and the newly formed chaetigers (Fig. 2d) is observed for D. neapolitana organisms (not exposed to stressful conditions) between day 50 and 60 after amputation [38]. The regenerated portion appears lighter than the original segments (Fig. 2d).

• 24. Shorter experiments could be conducted for 28 days only, but regeneration speed will not be measurable.