Overall, our results show that H. carunculata fireworms can regenerate amputated anterior segments and organs, including fully functional sensory structures, mouth opening, and pharynx. Laboratory experiments showed that regeneration occurred at both 14°C and 22°C, but they were significantly slower down at 14°C. Complete regeneration during the experimental period was observed only at 22°C; this experimental evidence is consistent with the thermophilic nature of H. carunculata, native of the southernmost (and warmest) coastal areas of the Mediterranean Sea.
During the regenerative process at 22 ºC some malformations were also observed in some individuals. Commonly, the morphology of the head of H. carunculata includes median caruncle followed by a median antenna, two pairs of eyes, two antennae, and two palps. Malformations can occur at the level of median antenna, but also in the development of the branchial filaments, which were observed to collapse after 8 weeks, eventually resembling a small knob, before re-acquiring their typical morphological features by the time that the regenerative process is completed. By investigating the phenotypic plasticity of H.carunculata specimens collected from different locations worldwide, it was suggested the depletion of dissolved oxygen may locally drive an epigenetic adaptive response, with the development of a higher number of gill filaments (Ahrens et al. 2013).
Although the initial regenerative patterns were similar across the different experimental conditions (14°C vs 22°C, SW vs LW), the regeneration process started but did not complete at 14°C (all experimental worms died by 24 wpa). A difference of water temperature of 8°C between the two experimental conditions (22 vs 14°C) suddenly produced a shift of ten weeks at the start of the regenerative blastema formation. It has to be considered that the metabolic rates of H. carunculata appear strongly temperature-dependent (Bosch-Belmar et al. 2024). The regeneration rate was observed to be Temperature dependent also in other polychaete species, such as Sabella spallanzanii (Gmelin, 1791), Sabella pavonia (Savigny, 1822) and Diopatra neapolitana Delle Chiaje, 1841 (Berrill 1931; Licciano et al. 2012; Pires et al. 2015), all showing an enhanced regeneration at warmer temperatures.
Small-size worms (~ 4 g) showed a faster regeneration than large-sized worms (~ 25 g). A comparable result was obtained by Planques et al. (2019) on Platynereis dumerilii (Audouin and Milne Edwards, 1833) posterior regeneration. The concurrent bidirectional regeneration at the same time in the same fragment was also observed, as most occurs in planarians (Saló and Baguña 2002). In the small-sized worms, the number of regenerated segments posterior to the new prostomium was lower than the amputated segments occurring in the original specimens. On average, the number of regenerated segments was half the number of the amputated segments, i.e., 3 regenerated segments were formed following a 6-segment amputation, and about 5–6 regenerated segments were formed following a 10-segment amputation.
Among annelids, several species exhibit the ability to regenerate the anterior end, with the extent of this capability varying according to the location of amputation (i.e., the number of removed segments) along the anterior-posterior axis (Kostyuchenko and Kozin 2021). In some species, a reduction of the regenerative potential is observed when more segments are removed (Nengwen et al. 2011). Taking into consideration the limitations of our experiment - only to two different amputation sites (6th or 10th segment posterior to prostomium) - preliminary experimental evidence suggests H. carunculata maintains a proportional segment regeneration potential along the body axis, at least to the 10th segment posterior to the prostomium.
As the regeneration process in annelids is determined mainly by epimorphosis (Bely 2014), i.e., the involvement of a proliferative blastema of undifferentiated stem cells, an equally distributed regeneration potential along the main body axis might be secured by an evenly proportionate distribution of stem cells from the anterior to the posterior body ends. However, this hypothesis needs to be addressed by more specific investigations.
The regeneration rate can be affected by the individual stage of reproductive maturation, as the ability to regenerate missing parts appears reduced during the time of reproduction, because of the high energy investment for gametogenesis, impairing the ability of regeneration (Maginnis 2006). In regeneration experiments, a 100% mortality was observed in the sabellid Branchiomma luctuosum (Grube, 1870) during the spawning period (Licciano et al. 2012; 2015); similarly, a reduced rate of regeneration and survivorship was found in Myxicola infundibulum when worms were ripe (Prentiss et al., 2017). Conversely, H. carunculata seems not to follow this rule, since both small and large specimens can undergo gametogenesis and anterior regeneration at the same time. At the beginning of the experiment (November), histological analyses confirmed that the source population of experimental worms was in a spent phase; on the contrary, at the end of experiment the dissecti on of the worms maintained at the two different temperatures (14°C and 22°C) revealed that the gametogenic cycle was ongoing in both cases. In the field, H. carunculata reproduces in summer (Toso et al. 2020), with gametogenesis starting in May/June, and spawning occurring between July and September (80–100 days). The histological analysis of specimens maintained under laboratory conditions revealed the presence of different stages of gametogenesis, suggesting that this species can have multiple spawning events during the favorable season. The entire gametogenesis observed under laboratory conditions lasted 80–100 days, and all the worms at both temperatures (14°C and 22°C) showed the same patterns of gamete development after amputation and, therefore, there seems to be no energetic trade-off between sexual reproduction and regeneration in H. carunculata. However, although individuals reared at the low temperature (14°C) produced gametes, their oocytes were not as well differentiated as they were in the specimens maintained at 22°C. During the formation of gametes, a significant increase in lipid concentration (shifting from 98.7 ± 13.2 (SD) µg lipids/mg AFDW before amputation to 117.7 ± 13.4 (SD) µg lipids/mg AFDW after amputation) was observed in the SW during the regeneration time (12 weeks). The anabolic pathway of these molecules, reflecting a change in the metabolism of the animal, was around 16%. The same patterns in the concentration of lipids were detected in the worms of the control group, in which the lipids increased over the 12 weeks. Since these worms did not need to invest energy in the anterior regeneration, energies were employed for the production of gametes. As reported by Martinez (1991), the increase in lipid concentrations can be correlated to the production of gametes. Thus, these results seem to support the hypothesis that the high demand for energy needed for regeneration does not impair the production of gametes in H.carunculata.
Being the Amphinomidae one of the basal groups in the annelid phylogeny, our observations on H. carunculata corroborate the hypothesis by Zattara and Bely (2016), who considered anterior and posterior regeneration as an ancestral feature of Annelida and a key developmental innovation for the evolution of fission as a mode of asexual reproduction, particularly appropriate for rapid expansion of individuals and the consequent exploitation of seasonal resources, available for a brief period. Overall, asexual reproduction in the family Amphinomidae includes such ability. Nonetheless, throughout the present study and further investigation on H. carunculata demography, traces of in situ fission in H. carunculata (A. Toso, pers comm) were not detected. Conversely, laboratory-based, repeated mechanical stimulation (by forceps) on the same body segment of healthy worms, eventually led the worms to initiate and carry out fission. As mentioned, we observed an accidental, mechanical injury caused the rupture of a single specimen into four fragments, all starting bidirectional regeneration (Fig. 4). Altogether, these observations suggest the occurrence of an adaptive developmental toolkit in H. carunculata genome, granting fireworms the potential to face unfavourable ecological conditions and repair physical damage. It can be hypothesized htat the recruitment of bidirectional regenerative processes, as known in H. carunculata, may be involved but apparently not sufficient to drive the evolution of asexual reproduction patterns, such as the paratomic or architomic fission observed in our experimental manipulations. Future investigations will be required to determine whether the bidirectional regeneration ability is fully retained by H. carunculata in the field and whether the same regenerative molecular toolkit can be upregulated - in the absence of wounds and injuries - to carry out clonal reproduction by fission.
Notably, as reported by Celona and Comparetto (2010) and Tiralongo et al. (2023), fishermen frequently encounter a significant quantity of fireworms as accidental bycatch in their nets. Intentionally and unintentionally, they cut the worms into multiple fragments and discard them returing the fragments into the sea (A. Toso, pers comm). In light of the high regenerative potential of H.carunculata, this practice may inadvertently contribute to the proliferation and increase distribution of fireworms in the area, as observed in the harvesting of Diopatra aciculata Knox and Cameron, 1971 (Schoeman and Simon 2023).
In conclusion, H. carunculata seems to adapt to high stress levels such as a partial loss of its body, being able not only to recover from injuries through regeneration but also continue the reproductive cycle during a period in which is not able to feed, maintaining its fitness and being able to maintain constant its population. Moreover, the ability for anterior regeneration across different temperatures (without any apparent interference with sexual reproduction), along with the increasing abundance of fireworms observed during the last years (Toso et al. 2022), point out this species as a potential winner in the climate change scenario of higher seawater temperatures.