Occurrence of bioluminescent and nonbioluminescent species in the littoral earthworm genus Pontodrilus

In this study, we confirmed that P. longissimus is nonbioluminescent, despite its close relationship to the luminous species P. litoralis (Supplementary Fig. S2)⁸. The presence of both luminous and nonluminous species in a single genus is likely widespread, but only a few examples have been confirmed; for example, the genera Vibrio and Photobacterium (marine bacteria)⁹, Epigonus (deep-sea fishes)¹⁰, Mycena (bonnet mushrooms)¹¹ and Eisenia (terrestrial earthworms)¹² have been reported to contain both luminous and nonluminous species. P. litoralis and P. longissimus can easily be collected at the same beach⁸ and reared in a laboratory; thus, they are suitable for studying the ecology and evolution of bioluminescence.

In vitro luciferin-luciferase cross-reaction tests of P. longissimus and P. litoralis confirmed that the luminescence ability of P. litoralis is due to the presence of multiple bioluminescent components in coelomic fluid, i.e., luciferin, luciferase and the light emitter. Cross-reaction tests have previously indicated that luminous earthworms in the genera Pontodrilus (Megascolecidae), Microscolex and Diplocardia (Acanthodrilidae) share the same basic bioluminescence mechanisms^5,7,13,14, despite their distant relationships to each other^15,16. It is expected that the ancestral state of Pontodrilus is nonbioluminescent because the nearest extant relatives of Pontodrilus belong to the genus Plutellus Perrier, 1873, and all members of this group are nonbioluminescent^6,17. These findings suggested that P. litoralis secondarily acquired bioluminescent properties through parallel evolution, similar to the case of bioluminescence in lampyrid and elaterid beetles¹⁸. We detected a clear difference in the protein composition of the secreted fluid between P. litoralis and P. longissimus (Supplementary Fig. S1). Luciferase and other bioluminescent components of luminous earthworms were not identified, and further comparative analyses of the protein bands, which appear only in the secreted fluid of luminous species, will be useful to understand the mechanisms of bioluminescence and its parallel evolution.

In Thailand, P. longissimus was found sympatrically with P. litoralis at the beaches along the coast, but the microhabitats of the two congeners are different; P. litoralis was collected on the beach surface (under trash or leaf litter on sandy beaches), whereas P. longissimus was found at a greater depth than P. litoralis, i.e., a depth of more than 10 cm, where trash and leaves are scarce⁸ (Fig. 4A–D). It has been hypothesized that the biological function of bioluminescence in Annelida, including P. litoralis, is to stun or divert attention as an anti-predator defense^{19,20,21,22,23,24,25}, but experiments and observations of the prey are limited. Sivinski & Forrest²⁵ reported that the luminescence of Microscolex phosphoreus deterred predation by the mole cricket Scapteriscus acletus under laboratory conditions, although the specimen was ultimately consumed. A British television program²⁶ presented by David Attenborough showed that the French luminous earthworm Avelona ligra glowed when attacked by the carabid beetle, but the beetle consumed the luminescent worm without any hesitation. We suggest that the absence of bioluminescence in P. longissimus may be associated with its presence in habitats with low predation pressure, whereas P. litoralis acquired a bioluminescent property during evolution that enabled it live on the surface of the beach, which is rich in nutrition and food sources^3,27 as well as potential predators.

Indeed, while we observed some burrowing bivalves, no potential predators were observed in the deep sand inhabited by P. longissimus. In contrast, various carnivorous invertebrates, such as earwigs, rove beetles and carabid beetles, were observed on the surface of beaches in Thailand and Japan, where P. litoralis live (Seesamut pers. obs.). We therefore performed a feeding experiment using maritime earwigs sympatrically distributed in a P. litoralis habitat. The maritime earwig Anisolabis maritima (Dermaptera, Anisolabididae) is a cosmopolitan species that can be found in Japan. It has well-developed compound eyes (Fig. 4E) and is considered a carnivorous animal that forages for prey at night^{28, 29}. A. maritima (body length ≤ 30 mm) was the predominant predator at the beach where P. litoralis was collected (Fig. 4F). Some rove beetles (Coleoptera, Staphylinidae) were found in the same habitat, but they seemed to be too small (< 10 mm) to consume P. litoralis, and during our laboratory observations, the rove beetle did not attack the worm. Thus, we think A. maritima is a major potential predator of P. litoralis at the beaches in Japan. Live P. litoralis and A. maritima were collected from the same beach on the same day, and we observed the predation behavior by the latter in a laboratory within a dark cage with beach sand spread on the bottom. Our observation of the predation of P. litoralis by earwigs (Supplementary Video 1) may provide important insight into the function of bioluminescence in P. litoralis. The earwigs immediately began aggressively attacking the worm with their mandibles and abdominal cerci, a pair of scissor-like pincers; the worm secreted luminescent mucus from its wounds (Supplementary Video 1), and it appeared that the retention of the glue-like luminescent mucus on the mouth and forelegs of the earwigs was unpleasant to them, since they attempted to remove the mucus by frequent grooming (Fig. 4E, Supplementary Video 2). Indeed, after aggressive attacks, the earwigs ultimately abandoned their attempts to consume the worm, and thus, the worm survived. To the best of our knowledge, this is the first observation of earthworm bioluminescence induced by predation under almost natural conditions. Based on these observations, we hypothesized that the luminous glue-like mucus of P. litoralis may function to deter and/or divert predation and that luminescence might even enhance the avoidance learning of the predator. Notably, the amount of mucus exuded following the same mechanical stimulation seemed to be much higher in P. litoralis than in P. longissimus. Nevertheless, we suppose that the global distribution of P. litoralis is a consequence of its adaptation to the beach surface (i.e., luminescence), which provides opportunities for dispersal by current, whereas P. longissimus is endemic to the coasts of the Thai-Malay peninsula^8,30 due to its inhabitation of deeper sand.

Based on microscopic observations, we confirmed that both species secrete coelomic cells following stimulation, but neither bioluminescence nor fluorescence was observed in P. longissimus. The presence and absence of fluorescence in a single genus of earthworm was also reported in the terrestrial genus Eisenia, and it has been suggested that the difference in fluorescent characteristics could be used as a “fluorescence fingerprint” to delineate these closely related species³¹. Therefore, the fluorescence fingerprint method is also applicable to Pontodrilus.

Littoral zones have rich species diversity of both macro- and microorganisms^32,33. They comprise a front of human pressure in marine ecology and are one of the most important zones for conservation^34,35; therefore, there is a need to understand littoral fauna. Earthworms typically have strong effects on soil ecosystems^36,37,38. Pontodrilus is a major “ecosystem engineer”³⁸ that inhabits littoral habitats. Thus, the identification of P. litoralis and P. longissimus is significant to the assessment of the littoral environment. These species are actually distinguishable by the internal morphology of the spermathecal diverticulum, but special skills and equipment are necessary for morphological analyses. In this study, we identified differences in the bioluminescent and fluorescent characteristics of P. litoralis and P. longissimus and demonstrated that the analysis of these differences provides an easy in situ methodology to identify these earthworms in marine ecological studies and for the conservation of littoral zones in Southeast Asia.

Source: Ecology - nature.com