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Rhodope placozophagus (Heterobranchia) a new species of turbellarian-like Gastropoda that preys on placozoans
Zoologischer Anzeiger 270 (2017) 43–48
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Research Paper
Rhodope placozophagus (Heterobranchia) a new species of turbellarian-like Gastropoda that preys on placozoans Rodrigo Cuervo-González Laboratorio de Evolución y Embriología, Facultad de Ciencias Biológico y Agropecuarias, Universidad Veracruzana, Carretera Tuxpan-Tampico, Km 7.5, C.P. 92860, Tuxpan, Ver., Mexico
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Article history: Received 29 April 2017 Received in revised form 18 September 2017 Accepted 26 September 2017 Available online 28 September 2017 Keywords: Rhodope Placozoa Predator Gulf of Mexico Spermatophore
a b s t r a c t Rhodope placozophagus nov. spec. from the western Gulf of Mexico that actively preys on placozoans is described. This white-translucent rhodopemorph was discovered on glass slides introduced in marine aquariums as part of an assemblage of small organisms including the vorticellids, ciliates, acoela, heliozoans and high populations of placozoans, which correlate with an abundance of this worm-like mollusk. When R. placozophagus comes into contact with placozoans, it immediately everts the anterior portion of the buccal bulb and sucks large portions of the disc-shape organism. The new species is likely to be a specialized predator since it was never observed chasing other microorganisms and especially because it can be reared for several generations while feeding exclusively on placozoans. It is known that the lipid granules called shiny spheres, located in the dorsal epithelium of Trichoplax, contain venoms and toxins that serve as an anti-predator defense. These properties can therefore be transmitted, at least transitorily, to a predator. Apart from one report of Riedl (1959) of Rhodope veranii preying on Trichoplax, no other predator-prey association has been described for placozoans. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction The Rhodope genus comprises five nominal species of small turbellarian-like Gastropoda, although around ten unnamed species have been reported in locations including the Mediterranean, Galapagos, Guam and the Norwegian coast (Brenzinger et al., 2011; Jörger et al., 2014). These records suggest that Rhodope live worldwide in tropical and subtropical oceans. The anatomy of rhodopemorpha is characterized by the absence of many of the distinguishing features of Mollusca, such as radula, anterior tentacles, foot, gills, mantle and heart, and they present other autapomorphies such as a very simplified excretory system (Brenzinger et al., 2013). These peculiarities have puzzled taxonomists since the description of the first species Rhodope veranii Kölliker, 1847, and they have been alternately described as turbellarian (Schultze, 1853) or as nudibranch (Haszprunar, 1997; Marcus and Marcus, 1952). Rhodope species are generally distinguished according to the number and color of transverse and longitudinal bands, which can be purple, red or orange, although Rhodope marcusi SalviniPlawen, 1991, and some other undescribed species are uniformly white (Salvini-Plawen, 1991). Another distinguishing characteristic
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is the presence of spicules below the epidermis that differ in length, roughness and number according to species. It is supposed that this endoskeleton gives corporal support and provides mechanical protection, which is valuable for interstitial life (Rieger and Sterrer, 1975). Rhodope species, together with the most worm-like mollusk Helminthope psammobionta Salvini-Plawen, 1991, present a series of body simplifications and regressive characters that has been called “meiofaunal syndrome” (e.g. loss of body appendages, extensive body ciliation, presences of caudal adhesive gland, sub-epidermal spicules etc), which is developed by several mesopsammic taxa (Brenzinger et al., 2013). These anatomical reductions and autapomorphies make Rhodopemorpha prone to the effects of homoplasy and problematic phylogenetic placement. However, recent molecular analysis finally resolved the ambiguous taxonomic place of Rhodopemorpha (Wilson et al., 2017), resulting in a monophyletic basal heterobranch taxa, sister of the shelled Murchisonellidae group that, together with the Rhodopemorpha, forms a new clade termed Allomorpha. Trichoplax adhaerens Schultze, 1883, is the only described species of the phylum Placozoa (Schulze, 1883); however, molecular studies have suggested the existence of several haplotypes living in tropical and subtropical waters around the world (Voigt et al., 2004). Because of their small size and slow-moving and benthic lifestyle, placozoans should be vulnerable to predators such
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Fig. 1. Rhodope placozophagus n. sp. (A) General view of in vivo adult hermaphrodite. (B) cup-shaped eyes with lens. (C) Statocysts above cerebral ganglion. (D) Cerebropleural ganglion, posterior spherical visceral ganglion and principal paired nerves. (E) Drop-shaped testes at posterior right side. (F) Corkscrew-head spermatozoa characteristic of heterobranch (Healy, 1993). (G) Egg masses with gelatinous capsule. (H) 4-cell stage embryos with characteristic D-quadrant cleavage of Spiralia. (I) 4-day-old embryos with direct development. (J) Juvenile organisms hatching after 6 days. (K) Detail of middle section of excretory organ. (L) Close-up showing the antero-ventral mouth. (M) The specialized buccal bulb characteristic of rhodopemorpha. (N) Everted anterior portion of buccal bulb during feeding. ag, adhesive gland; bb, buccal bulb; cg, cerebral ganglion; dg, digestive gland; l, lens; o, oocyte; on, olfactory nerves; pn, pedal nerves; t, testes; tx, Trichoplax; vg, visceral ganglion; vn, visceral nerves.
as nemerteans, polychaetes and platyhelminthes etc; however, only one publication exists to date describing a predator of Trichoplax: Rhodope veranii (Riedl, 1959). Some authors have reported that sabellid tubeworms reject Trichoplax as food and that gastropods and flatworms recoil upon contact (Pearse and Voigt, 2007). I have seen that potential predators like acoela, small snails or copepods abruptly turned and moved in another direction upon contact. In laboratory experiments, Trichoplax given as food to polyps of the hydroid Podocoryna carnea were found to induced paralysis or death; however, pellets of tissue without the shiny spheres did not exhibit this noxious effect, indicating that the lipid granules that Trichoplax carry in the upper epithelium work as an anti-predator defense mechanism (Jackson and Buss, 2009). The Trichoplax genome has several proteins known to be present in venoms, including blockers of acetylcholine and ryanodine receptors, metalloproteinases and phospholipase A2, as well as neurotoxins and cytolycins (Jackson and Buss, 2009). R. placozophagus has therefore developed strategies to manage its noxious prey and the toxic molecular cocktail contained in their upper shiny spheres. The present contribution constitutes the first record of a new species of the Rhodope genus from the Gulf of Mexico and includes a description of sexual intercourse in adult hermaphrodites and insemination via spermatophore. I suggest that, in addition to Rhodope veranii, other species, such as Rhodope marcusi, Rhodope
roskoi Haszprunar, 2005, Rhodope rousei Breizinger, 2011, and Helminthope psammobionta, may also prey on placozoans.
2. Materials and methods The first specimens of R. placozophagus nov. spec. were found in October 2015 in sea water aquariums where placozoans flourish. Thereafter, they were collected in situ in the Tuxpan Reef by shaking coralline gravel in plastic bags and collecting the sediments. This reef has an emerged platform with a surface area of 1.4 km2 localized at 12 km from the coastline (21.016667 N 97.186389 W). It belongs to the Lobos-Tuxpan reef system in the Western Gulf of Mexico. For the experiments, organisms were maintained in 24well culture plates with 2.5 ml of 0.22 m filtered sea water and fed with placozoans ad libitum. Placozoans were harvested from glass slides maintained in aquariums that harbored macro algae such as Caulerpa sertularioides, Caulerpa cupressoides and Halimeda opuntia obtained from the same reef. Every week, a partial change of the water was conducted, replacing 15% of the total volume with filtered and UV sterilized water obtained from the same reef. Some specimens were observed in vivo as whole squash-mounts, others were fixed in 5% formalin saturated with calcium carbonate then transferred to 70% ethanol. Acetic acid was used at 10% to evaluate the calcareous composition of spicules. Amputations were
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performed with tungsten needles, cutting the posterior third of the body of adult slugs. Specimens were photographed and recorded on video with a cell phone camera of 8 megapixels and a Moticam 2500 camera of 5 megapixels adapted to a compound microscope Leica DMLS. No specific permissions were required to obtain these samples because of the low environmental impact of the collecting methods employed and the fact that these organisms are not listed under CITES and have no commercial value. 3. Results Heterobranchia sensu Haszprunar, 1985 Murchisonelloidea Casey, 1904 Rhodopidae von Ihering, 1876 Rhodope Kolliker, 1847 Rhodope placozophagus nov. sp. Zoobank registration: urn:lsid:zoobank.org:pub:FEAA9EC5E36D-4CAD-8D7C-48D9D3A4A8A6 3.1. Type material Holotype, adult hermaphrodite collected on October 2, 2015. The adult holotype with three juveniles mounted on a glass slide with 1:1 glycerol-ethanol 70% plus 0.1% NaN3 is deposited in the National Collection of Mollusks of Universidad Nacional Autónoma de México, under catalog number CNMO 6559. 3.2. Etymology The species is named from Greek plakos, flat; zoion, animal and Latin phagus, eater (referring to its diet of placozoans). 3.3. Diagnosis Distinguishable from other Rhodope species by its whitish translucent body lacking orange, purple or red bands. Length about 2000 m and maximum width 350 m. Similar to R. marcusi in length and white color, but clearly different in terms of the size and form of the calcareous spicules. Spicules about 45–70 m in length, loosely dispersed over the entire body. Two cup-shaped eyes with lens and two statocysts above the cerebral region. Short and flexible cilia distributed homogeneously over whole-body surface. Tubular excretory organ at right side of the body with ciliary flame cells. Genital opening, nephropore and anus at the anterior right body side. 3.4. Description Anterior end slender and rounded, middle body slightly broader and posterior body slender and rounded with ventral adhesive gland (Fig. 1A). The body of a live hermaphrodite adult is 2000 m in length and 350 m in width. Large paired cerebropleural ganglion and above a pair of well-developed cup-shaped eyes and two spherical statocysts slightly larger than the eyes (Fig. 2B, C). Paired oral nerves, posterior spherical visceral ganglion, and at least one pair of thick pedal nerves and paired thick visceral nerves (Fig. 1D). Drop-shaped testes situated distally (Fig. 1A, E), corkscrew head spermatozoa of about 85 m length (Fig. 1F). Yolk-rich eggs at sides of digestive gland (Fig. 1A). Direct development of six to nine embryos per egg mass (Fig. 1G–I). After 6 days, juveniles of around 500 m length hatched (Fig. 1J). Tubular excretory organ at right side with ‘warts’ and ciliary flame (Fig. 1K). Antero-ventral mouth (Fig. 1L) and broad buccal bulb (Fig. 1M) adapted to suck its prey with strong peristaltic movements of the buccal bulb and coordinated contraction of the sac-like digestive gland (Fig. 1N).
Fig. 2. Characteristic spicules of Rhodope placozophagus. (A) Overall distribution of bow-shaped spicules. (B) Detailed view of slender and thick spicules. (C) in vivo squash-mount view of Hoechst stained nucleus lodged in the middle notch. (D) Spicules straighten and extend longitudinally when the body is lengthened. (E) New developing spicules in juveniles appear as needles. (F) Regenerating caudal region at ninth day after amputation. Arrowheads indicate the amputation plane. (G) Regenerating spicules are similarly to developing spicules. (H) Images taken during the first 15 s of fixed squash-mount Rhodope showing the dissolution of spicules with acetic acid.
Adult fertile hermaphrodites have about 190 sub-epidermal bow-shape spicules, while juveniles hatch with about 55 (Fig. 2A). Spicules 45–70 m in length and 3–7 m in width, hinged at the middle with a characteristic notch that bear a cell nucleus (Fig. 2B, C). Large spicules grow to twice the thickness of the slender spicules
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Fig. 3. Spermatophore mediated dermal insemination. (A) Bundles of spermatozoa in the genital terminal gland. (B) Everted distal portion of gonoduct leading to gonopore. (C) Extruded spermatophore, note the flagella of the spermatozoa directed to the exterior. (D) Spermatophore attached in the tail region (E) The sticky property of a spermatophore attached in the head. e, eye; s, spermatozoa; t, Trichoplax; tg, terminal gland.
(Fig. 2C), and are extended longitudinally when the body lengthens (Fig. 2D). In juveniles, de novo developing spicules can be seen growing as needles that gradually increase in thickness (Fig. 2E). Following amputation of posterior third of the body (Fig. 2F), adhesive gland regenerates within 72 h and new spicules are clearly seen growing de novo in the caudal blastema after nine days (Fig. 2G). In whole squash-mounts of fixed organisms, the spicules dissolved within seconds on addition of 10% acetic acid (Fig. 2H). 3.5. Reproduction In mature organisms, bundles of spermatozoa accumulate in the barrel shaped terminal gland (Fig. 3A). The terminal ring shaped gland is everted through the genital pore to extrude a spermatophore (Fig. 3B) that is composed of a sticky substance mixed with spermatozoa (Fig. 3C). Adult hermaphrodites engage in reproductive behavior mainly while feeding: with the snout, they touch the flanks of the partner and proceed to stick an spermatophore in the epidermis of the head, tail or flanks (Fig. 3D, E. and Video 1). The spermatophore is internalized with seconds and allosperms can be seen in the haemocoel space. On occasion, the individuals interact with the anterior right side of the body and momentarily mutually join their genital pores, however this behavior rarely occurs. Self-fertilization was evaluated by rearing individually ten newly-hatched R. placozophagus individuals on culture plates. All of these specimens grew and matured normally and after 18–20 days started to lay egg masses that developed into healthy juveniles. 3.6. Preying on placozoans Abundant fragmented and irregular shaped placozoans were concurrent with R. placozophagus of all stages and with egg masses on dozens of glass slides (Fig. 4A). I observed that R. placozophagus is a very active and fast-moving organism that, upon fortuitous contact with placozoans, immediately begins to suck the tissues
of its prey using strong peristaltic movements of the buccal bulb and digestive gland (Video 2). R. placozophagus does not suck the loose interior content of its prey, which consists mainly of fibrous cells (Buchholz and Ruthmann, 1995), but instead eats the complete tissues including the upper epithelium which is covered with shiny spheres (Fig. 4B). I captured several R. placozophagus and fed them with placozoans following overnight starvation. I observed that they pull and tear their prey and sometimes eat for up to 50 min (Fig. 4C–E). Moreover, it appears that the placozoans are unaware that they are being predated and do not detect the presence of the predator or make any attempt to escape. Since placozoans are able to regenerate their structures and regrow from small fragments, predation is not necessarily detrimental to them (Fig. 4F). Occasionally, fragmented placozoans becomes spherical (Thiemann and Ruthmann, 1991), which makes them unable to be caught by their predators (Video 3). I observed that newly hatched organisms do not accept placozoans but, after a few hours they eat avidly (Fig. 4D and Video 4). They duplicate in size after 6 days and at around 15 days reach 1800 m in length with large oocytes at sides of the digestive gland and complete their lifecycle in as little as 18 days when new egg masses appear adhered to the walls of the wells. Thereafter, they can lay egg masses every third day and, at least in vitro, attained a life span of around 60 days. In conclusion, this rhodopidae new species can self-fertilize, has a short life cycle and can be reared exclusively with placozoans for several generations. 4. Discussion Rhodopemorphs have been found in temperate and tropical waters in intertidal and subtidal sand habitats, as is the case for this new species, which inhabits the sand and coralline gravel zones of a platform reef with abundant seagrass, Caulerpa and Alimeda algae in the Western Gulf of Mexico, an ocean basin with a narrow connection to the Atlantic Ocean. The direct development of this species and low reproductive output (in this species less than 9
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Fig. 4. Predator-prey interaction. Glass slide with irregular and stretched (arrow) Trichoplax, inset shows small fragments normally formed after Rhodope feeding. (B) Shiny spheres become evident by phase contrast illumination and detail of upper epithelium. (C) Adult Rhodope pulling and feeding on Trichoplax. (D) Juveniles eating following overnight starvation. (E) Close up of everted anterior buccal bulb siphoning whole tissues of Trichoplax including shiny spheres (arrowheads). (F) New small well-formed Trichoplax formed from depredated adults.
embryos per egg masses) indicates low dispersal abilities and suggests high endemism and localized speciation (Brenzinger et al., 2011). R. placozophagus is differentiated from other Rhodope species by the absence of orange, red or purple color. R. marcusi does have a white body but, despite being of similar size, differs in the form
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and size of the spicules. R. placozophagus has bow-shape spicules maximally 70 m in length and 3–7 m in width, with a characteristic middle notch, whereas R. marcusi (found in bay of Santos, Brazil) has more curved and larger C-shaped spicules of maximum length 130 m. The spicules drawn in the first description do not shown a notch and there is not a type specimen (Haszprunar and Hess, 2005; Marcus and Marcus, 1952). In addition, R. marcusi has two bundles of longer blue gland cells at the sides of the mouth and cyanophilous ventral glands (Marcus and Marcus, 1952). The only colored structure of R. placozophagus are the yellowish oocytes. It has been proposed that, in mesopsammic taxa, the endoskeleton provides body support, anchorage during locomotion and mechanical protection advantageous to insterstitial life; however, there is little knowledge regarding the intracellular origin, composition and growth of the spicules (Rieger and Sterrer, 1975). R. placozophagus has spicules with a cell nucleus just in the middle notch and are able to regrow new spicules in the regenerating blastema in the posterior third of the body. The phylum Mollusca has no species capable of regenerating the whole body; however, it is known that several groups can regenerate the foot, tentacles, mantle, head and even the shell (Bely et al., 2014). Spicules rapidly dissolve with acetic acid, indicating their calcareous nature and they constitute an endoskeleton that may be useful to provide strength to the body wall while siphoning the placozoans. Many hypothesis have been proposed for the origin of the skeleton in vertebrates and invertebrates: as a sensory enhancement structure, a barrier for osmosis, the result of calcium metabolism reorganization, a reservoir for bio-limiting elements or a structure for disposal waste by products (Donoghue and Sansom, 2002; Marin et al., 1996). These ideas may be useful to infer additional roles for the endoskeleton in Rhodope, as well as other taxa of meiofauna with endoskeletons. Riedl (1959) reported that mature specimens of R. veranii combined their anterior right side of the body and remain in this position for few seconds to put the sexual pores in contact in order to transfer sperm (Riedl, 1959). This author also supposed insemination by unidirectional transfer of spermatozoa. I occasionally observed this form of mutual intercourse in R. placozophagus, but I suggest that it may instead represent some kind of recognitiondomination interaction. Based on the aphallic nature of Rhodope rousei, it has recently been suggested that Rhodope uses dermal insemination via spermatophores and that sperm possibly penetrates by short-term lysis of a small stretch of the epidermis (Brenzinger et al., 2011). As shown here for R. placozophagus, dermal insemination via spermatophores certainly could be the reproduction type employed by rhodopemorpha. Predation is defined as the consumption of individuals or tissues belonging to species of the same or lower trophic levels (Elewa, 2007), as is the case with Trichoplax and R. placozophagus. The concomitant abundance of placozoans and R. placozophagus, together with the particular anatomy of the buccal apparatus and the toxic nature of its prey, suggests that this microslug, and perhaps all Rhodopidae (see below), are specialized predators, although whether they feed on other soft and toxic prey, such as polyps of cnidarians, has yet to be determined. Despite the diversity of microorganisms and predator-prey interactions occurring together with placozoans, only a single case of predation has been reported several decades ago (Riedl, 1959). The results shown here support the observations reported by Riedl about R. veranii: a creeping Rhodope reaches a Trichoplax, touches the feeding prey with the mouth edges, everts a small portion of buccal bulb, slightly crumples the body and suction begins accompanied by strong peristaltic movements of the buccal bulb and digestive gland. Considering that most reports relate observations of organisms such as small snails or rhabdocoel flatworms that avoided placozoans (Pearse and Voigt, 2007), it is surprising that no further characterization of this
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predatory activity followed this initial report, especially considering the kleptochemistry phenomenon of several opisthobranchs (Marín and Ros, 2004) and the resistance that R. veranii must have to the toxins of Trichoplax. Furthermore, it would be tempting to determine whether a specific and exclusive predation-prey relationship exists in the rhodopidae family with placozoans since R. roskoi, R. marcusi, R. rousei and Helminthope psammobionta have all been reported in locations where Trichoplax have been reported as: the north coast of France, the coast of Sao Paulo Brazil, the southeast coast of Australia and Bermuda (Brenzinger et al., 2011; Eitel et al., 2013; Haszprunar and Hess, 2005; Marcus and Marcus, 1952; Salvini-Plawen, 1991). Finally, this predator-prey association is particularly unusual: on the one hand, it seems that R. placozophagus cannot easily detect its prey; it needs to make physical contact in order to recognize its food. It is possible that, among the loss and simplification of organs and structures in Rhodopidae, there was a degeneration of the osphradium, an important chemosensory organ (Lindberg and Sigwart, 2015). On the other hand, Trichoplax cannot sense when they are being predated and, due to the fact that placozoans are very simple organisms that are able to reshape and regenerate their own structure from small fragments (Schwartz, 1984), predation can actually make a contribution to asexual reproduction; i.e., the resulting fragments will disperse and grow and eventually reproduce again by binary fission. Several organisms lose body parts by autotomy in order to preserve the individual, but this implies an energetic cost to regrow the lost structure. In the case of Trichoplax, the benefits may overcome the costs and, at the same time, due to the toxicity of their prey, R. placozophagus may in turn obtain protection against potential predators. Acknowledgements The author thanks Dra. Edna Naranjo García of the Biology Institute, UNAM, for helping with the type specimen. This work was supported by Grant No. 151757 from Consejo Nacional de Ciencia y Tecnología (CONACyT). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jcz.2017.09.005. References Bely, A.E., Zattara, E.E., Sikes, J.M., 2014. Regeneration in spiralians: evolutionary patterns and developmental processes. Int. J. Dev. Biol. 58, 623–634, http://dx. doi.org/10.1387/ijdb.140142ab. Brenzinger, B., Wilson, N.G., Schrödl, M., 2011. 3D microanatomy of a gastropod worm, Rhodope rousei n. sp. (Heterobranchia) from southern Australia. J. Molluscan Stud. 4, 375–387. Brenzinger, B., Haszprunar, G., Schrödl, M., 2013. At the limits of a successful body plan—3D microanatomy, histology and evolution of Helminthope (Mollusca: Heterobranchia: Rhodopemorpha), the most worm-like gastropod. Front. Zool. 10, 37.
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Research Paper
Rhodope placozophagus (Heterobranchia) a new species of turbellarian-like Gastropoda that preys on placozoans Rodrigo Cuervo-González Laboratorio de Evolución y Embriología, Facultad de Ciencias Biológico y Agropecuarias, Universidad Veracruzana, Carretera Tuxpan-Tampico, Km 7.5, C.P. 92860, Tuxpan, Ver., Mexico
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Article history: Received 29 April 2017 Received in revised form 18 September 2017 Accepted 26 September 2017 Available online 28 September 2017 Keywords: Rhodope Placozoa Predator Gulf of Mexico Spermatophore
a b s t r a c t Rhodope placozophagus nov. spec. from the western Gulf of Mexico that actively preys on placozoans is described. This white-translucent rhodopemorph was discovered on glass slides introduced in marine aquariums as part of an assemblage of small organisms including the vorticellids, ciliates, acoela, heliozoans and high populations of placozoans, which correlate with an abundance of this worm-like mollusk. When R. placozophagus comes into contact with placozoans, it immediately everts the anterior portion of the buccal bulb and sucks large portions of the disc-shape organism. The new species is likely to be a specialized predator since it was never observed chasing other microorganisms and especially because it can be reared for several generations while feeding exclusively on placozoans. It is known that the lipid granules called shiny spheres, located in the dorsal epithelium of Trichoplax, contain venoms and toxins that serve as an anti-predator defense. These properties can therefore be transmitted, at least transitorily, to a predator. Apart from one report of Riedl (1959) of Rhodope veranii preying on Trichoplax, no other predator-prey association has been described for placozoans. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction The Rhodope genus comprises five nominal species of small turbellarian-like Gastropoda, although around ten unnamed species have been reported in locations including the Mediterranean, Galapagos, Guam and the Norwegian coast (Brenzinger et al., 2011; Jörger et al., 2014). These records suggest that Rhodope live worldwide in tropical and subtropical oceans. The anatomy of rhodopemorpha is characterized by the absence of many of the distinguishing features of Mollusca, such as radula, anterior tentacles, foot, gills, mantle and heart, and they present other autapomorphies such as a very simplified excretory system (Brenzinger et al., 2013). These peculiarities have puzzled taxonomists since the description of the first species Rhodope veranii Kölliker, 1847, and they have been alternately described as turbellarian (Schultze, 1853) or as nudibranch (Haszprunar, 1997; Marcus and Marcus, 1952). Rhodope species are generally distinguished according to the number and color of transverse and longitudinal bands, which can be purple, red or orange, although Rhodope marcusi SalviniPlawen, 1991, and some other undescribed species are uniformly white (Salvini-Plawen, 1991). Another distinguishing characteristic
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is the presence of spicules below the epidermis that differ in length, roughness and number according to species. It is supposed that this endoskeleton gives corporal support and provides mechanical protection, which is valuable for interstitial life (Rieger and Sterrer, 1975). Rhodope species, together with the most worm-like mollusk Helminthope psammobionta Salvini-Plawen, 1991, present a series of body simplifications and regressive characters that has been called “meiofaunal syndrome” (e.g. loss of body appendages, extensive body ciliation, presences of caudal adhesive gland, sub-epidermal spicules etc), which is developed by several mesopsammic taxa (Brenzinger et al., 2013). These anatomical reductions and autapomorphies make Rhodopemorpha prone to the effects of homoplasy and problematic phylogenetic placement. However, recent molecular analysis finally resolved the ambiguous taxonomic place of Rhodopemorpha (Wilson et al., 2017), resulting in a monophyletic basal heterobranch taxa, sister of the shelled Murchisonellidae group that, together with the Rhodopemorpha, forms a new clade termed Allomorpha. Trichoplax adhaerens Schultze, 1883, is the only described species of the phylum Placozoa (Schulze, 1883); however, molecular studies have suggested the existence of several haplotypes living in tropical and subtropical waters around the world (Voigt et al., 2004). Because of their small size and slow-moving and benthic lifestyle, placozoans should be vulnerable to predators such
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Fig. 1. Rhodope placozophagus n. sp. (A) General view of in vivo adult hermaphrodite. (B) cup-shaped eyes with lens. (C) Statocysts above cerebral ganglion. (D) Cerebropleural ganglion, posterior spherical visceral ganglion and principal paired nerves. (E) Drop-shaped testes at posterior right side. (F) Corkscrew-head spermatozoa characteristic of heterobranch (Healy, 1993). (G) Egg masses with gelatinous capsule. (H) 4-cell stage embryos with characteristic D-quadrant cleavage of Spiralia. (I) 4-day-old embryos with direct development. (J) Juvenile organisms hatching after 6 days. (K) Detail of middle section of excretory organ. (L) Close-up showing the antero-ventral mouth. (M) The specialized buccal bulb characteristic of rhodopemorpha. (N) Everted anterior portion of buccal bulb during feeding. ag, adhesive gland; bb, buccal bulb; cg, cerebral ganglion; dg, digestive gland; l, lens; o, oocyte; on, olfactory nerves; pn, pedal nerves; t, testes; tx, Trichoplax; vg, visceral ganglion; vn, visceral nerves.
as nemerteans, polychaetes and platyhelminthes etc; however, only one publication exists to date describing a predator of Trichoplax: Rhodope veranii (Riedl, 1959). Some authors have reported that sabellid tubeworms reject Trichoplax as food and that gastropods and flatworms recoil upon contact (Pearse and Voigt, 2007). I have seen that potential predators like acoela, small snails or copepods abruptly turned and moved in another direction upon contact. In laboratory experiments, Trichoplax given as food to polyps of the hydroid Podocoryna carnea were found to induced paralysis or death; however, pellets of tissue without the shiny spheres did not exhibit this noxious effect, indicating that the lipid granules that Trichoplax carry in the upper epithelium work as an anti-predator defense mechanism (Jackson and Buss, 2009). The Trichoplax genome has several proteins known to be present in venoms, including blockers of acetylcholine and ryanodine receptors, metalloproteinases and phospholipase A2, as well as neurotoxins and cytolycins (Jackson and Buss, 2009). R. placozophagus has therefore developed strategies to manage its noxious prey and the toxic molecular cocktail contained in their upper shiny spheres. The present contribution constitutes the first record of a new species of the Rhodope genus from the Gulf of Mexico and includes a description of sexual intercourse in adult hermaphrodites and insemination via spermatophore. I suggest that, in addition to Rhodope veranii, other species, such as Rhodope marcusi, Rhodope
roskoi Haszprunar, 2005, Rhodope rousei Breizinger, 2011, and Helminthope psammobionta, may also prey on placozoans.
2. Materials and methods The first specimens of R. placozophagus nov. spec. were found in October 2015 in sea water aquariums where placozoans flourish. Thereafter, they were collected in situ in the Tuxpan Reef by shaking coralline gravel in plastic bags and collecting the sediments. This reef has an emerged platform with a surface area of 1.4 km2 localized at 12 km from the coastline (21.016667 N 97.186389 W). It belongs to the Lobos-Tuxpan reef system in the Western Gulf of Mexico. For the experiments, organisms were maintained in 24well culture plates with 2.5 ml of 0.22 m filtered sea water and fed with placozoans ad libitum. Placozoans were harvested from glass slides maintained in aquariums that harbored macro algae such as Caulerpa sertularioides, Caulerpa cupressoides and Halimeda opuntia obtained from the same reef. Every week, a partial change of the water was conducted, replacing 15% of the total volume with filtered and UV sterilized water obtained from the same reef. Some specimens were observed in vivo as whole squash-mounts, others were fixed in 5% formalin saturated with calcium carbonate then transferred to 70% ethanol. Acetic acid was used at 10% to evaluate the calcareous composition of spicules. Amputations were
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performed with tungsten needles, cutting the posterior third of the body of adult slugs. Specimens were photographed and recorded on video with a cell phone camera of 8 megapixels and a Moticam 2500 camera of 5 megapixels adapted to a compound microscope Leica DMLS. No specific permissions were required to obtain these samples because of the low environmental impact of the collecting methods employed and the fact that these organisms are not listed under CITES and have no commercial value. 3. Results Heterobranchia sensu Haszprunar, 1985 Murchisonelloidea Casey, 1904 Rhodopidae von Ihering, 1876 Rhodope Kolliker, 1847 Rhodope placozophagus nov. sp. Zoobank registration: urn:lsid:zoobank.org:pub:FEAA9EC5E36D-4CAD-8D7C-48D9D3A4A8A6 3.1. Type material Holotype, adult hermaphrodite collected on October 2, 2015. The adult holotype with three juveniles mounted on a glass slide with 1:1 glycerol-ethanol 70% plus 0.1% NaN3 is deposited in the National Collection of Mollusks of Universidad Nacional Autónoma de México, under catalog number CNMO 6559. 3.2. Etymology The species is named from Greek plakos, flat; zoion, animal and Latin phagus, eater (referring to its diet of placozoans). 3.3. Diagnosis Distinguishable from other Rhodope species by its whitish translucent body lacking orange, purple or red bands. Length about 2000 m and maximum width 350 m. Similar to R. marcusi in length and white color, but clearly different in terms of the size and form of the calcareous spicules. Spicules about 45–70 m in length, loosely dispersed over the entire body. Two cup-shaped eyes with lens and two statocysts above the cerebral region. Short and flexible cilia distributed homogeneously over whole-body surface. Tubular excretory organ at right side of the body with ciliary flame cells. Genital opening, nephropore and anus at the anterior right body side. 3.4. Description Anterior end slender and rounded, middle body slightly broader and posterior body slender and rounded with ventral adhesive gland (Fig. 1A). The body of a live hermaphrodite adult is 2000 m in length and 350 m in width. Large paired cerebropleural ganglion and above a pair of well-developed cup-shaped eyes and two spherical statocysts slightly larger than the eyes (Fig. 2B, C). Paired oral nerves, posterior spherical visceral ganglion, and at least one pair of thick pedal nerves and paired thick visceral nerves (Fig. 1D). Drop-shaped testes situated distally (Fig. 1A, E), corkscrew head spermatozoa of about 85 m length (Fig. 1F). Yolk-rich eggs at sides of digestive gland (Fig. 1A). Direct development of six to nine embryos per egg mass (Fig. 1G–I). After 6 days, juveniles of around 500 m length hatched (Fig. 1J). Tubular excretory organ at right side with ‘warts’ and ciliary flame (Fig. 1K). Antero-ventral mouth (Fig. 1L) and broad buccal bulb (Fig. 1M) adapted to suck its prey with strong peristaltic movements of the buccal bulb and coordinated contraction of the sac-like digestive gland (Fig. 1N).
Fig. 2. Characteristic spicules of Rhodope placozophagus. (A) Overall distribution of bow-shaped spicules. (B) Detailed view of slender and thick spicules. (C) in vivo squash-mount view of Hoechst stained nucleus lodged in the middle notch. (D) Spicules straighten and extend longitudinally when the body is lengthened. (E) New developing spicules in juveniles appear as needles. (F) Regenerating caudal region at ninth day after amputation. Arrowheads indicate the amputation plane. (G) Regenerating spicules are similarly to developing spicules. (H) Images taken during the first 15 s of fixed squash-mount Rhodope showing the dissolution of spicules with acetic acid.
Adult fertile hermaphrodites have about 190 sub-epidermal bow-shape spicules, while juveniles hatch with about 55 (Fig. 2A). Spicules 45–70 m in length and 3–7 m in width, hinged at the middle with a characteristic notch that bear a cell nucleus (Fig. 2B, C). Large spicules grow to twice the thickness of the slender spicules
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Fig. 3. Spermatophore mediated dermal insemination. (A) Bundles of spermatozoa in the genital terminal gland. (B) Everted distal portion of gonoduct leading to gonopore. (C) Extruded spermatophore, note the flagella of the spermatozoa directed to the exterior. (D) Spermatophore attached in the tail region (E) The sticky property of a spermatophore attached in the head. e, eye; s, spermatozoa; t, Trichoplax; tg, terminal gland.
(Fig. 2C), and are extended longitudinally when the body lengthens (Fig. 2D). In juveniles, de novo developing spicules can be seen growing as needles that gradually increase in thickness (Fig. 2E). Following amputation of posterior third of the body (Fig. 2F), adhesive gland regenerates within 72 h and new spicules are clearly seen growing de novo in the caudal blastema after nine days (Fig. 2G). In whole squash-mounts of fixed organisms, the spicules dissolved within seconds on addition of 10% acetic acid (Fig. 2H). 3.5. Reproduction In mature organisms, bundles of spermatozoa accumulate in the barrel shaped terminal gland (Fig. 3A). The terminal ring shaped gland is everted through the genital pore to extrude a spermatophore (Fig. 3B) that is composed of a sticky substance mixed with spermatozoa (Fig. 3C). Adult hermaphrodites engage in reproductive behavior mainly while feeding: with the snout, they touch the flanks of the partner and proceed to stick an spermatophore in the epidermis of the head, tail or flanks (Fig. 3D, E. and Video 1). The spermatophore is internalized with seconds and allosperms can be seen in the haemocoel space. On occasion, the individuals interact with the anterior right side of the body and momentarily mutually join their genital pores, however this behavior rarely occurs. Self-fertilization was evaluated by rearing individually ten newly-hatched R. placozophagus individuals on culture plates. All of these specimens grew and matured normally and after 18–20 days started to lay egg masses that developed into healthy juveniles. 3.6. Preying on placozoans Abundant fragmented and irregular shaped placozoans were concurrent with R. placozophagus of all stages and with egg masses on dozens of glass slides (Fig. 4A). I observed that R. placozophagus is a very active and fast-moving organism that, upon fortuitous contact with placozoans, immediately begins to suck the tissues
of its prey using strong peristaltic movements of the buccal bulb and digestive gland (Video 2). R. placozophagus does not suck the loose interior content of its prey, which consists mainly of fibrous cells (Buchholz and Ruthmann, 1995), but instead eats the complete tissues including the upper epithelium which is covered with shiny spheres (Fig. 4B). I captured several R. placozophagus and fed them with placozoans following overnight starvation. I observed that they pull and tear their prey and sometimes eat for up to 50 min (Fig. 4C–E). Moreover, it appears that the placozoans are unaware that they are being predated and do not detect the presence of the predator or make any attempt to escape. Since placozoans are able to regenerate their structures and regrow from small fragments, predation is not necessarily detrimental to them (Fig. 4F). Occasionally, fragmented placozoans becomes spherical (Thiemann and Ruthmann, 1991), which makes them unable to be caught by their predators (Video 3). I observed that newly hatched organisms do not accept placozoans but, after a few hours they eat avidly (Fig. 4D and Video 4). They duplicate in size after 6 days and at around 15 days reach 1800 m in length with large oocytes at sides of the digestive gland and complete their lifecycle in as little as 18 days when new egg masses appear adhered to the walls of the wells. Thereafter, they can lay egg masses every third day and, at least in vitro, attained a life span of around 60 days. In conclusion, this rhodopidae new species can self-fertilize, has a short life cycle and can be reared exclusively with placozoans for several generations. 4. Discussion Rhodopemorphs have been found in temperate and tropical waters in intertidal and subtidal sand habitats, as is the case for this new species, which inhabits the sand and coralline gravel zones of a platform reef with abundant seagrass, Caulerpa and Alimeda algae in the Western Gulf of Mexico, an ocean basin with a narrow connection to the Atlantic Ocean. The direct development of this species and low reproductive output (in this species less than 9
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Fig. 4. Predator-prey interaction. Glass slide with irregular and stretched (arrow) Trichoplax, inset shows small fragments normally formed after Rhodope feeding. (B) Shiny spheres become evident by phase contrast illumination and detail of upper epithelium. (C) Adult Rhodope pulling and feeding on Trichoplax. (D) Juveniles eating following overnight starvation. (E) Close up of everted anterior buccal bulb siphoning whole tissues of Trichoplax including shiny spheres (arrowheads). (F) New small well-formed Trichoplax formed from depredated adults.
embryos per egg masses) indicates low dispersal abilities and suggests high endemism and localized speciation (Brenzinger et al., 2011). R. placozophagus is differentiated from other Rhodope species by the absence of orange, red or purple color. R. marcusi does have a white body but, despite being of similar size, differs in the form
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and size of the spicules. R. placozophagus has bow-shape spicules maximally 70 m in length and 3–7 m in width, with a characteristic middle notch, whereas R. marcusi (found in bay of Santos, Brazil) has more curved and larger C-shaped spicules of maximum length 130 m. The spicules drawn in the first description do not shown a notch and there is not a type specimen (Haszprunar and Hess, 2005; Marcus and Marcus, 1952). In addition, R. marcusi has two bundles of longer blue gland cells at the sides of the mouth and cyanophilous ventral glands (Marcus and Marcus, 1952). The only colored structure of R. placozophagus are the yellowish oocytes. It has been proposed that, in mesopsammic taxa, the endoskeleton provides body support, anchorage during locomotion and mechanical protection advantageous to insterstitial life; however, there is little knowledge regarding the intracellular origin, composition and growth of the spicules (Rieger and Sterrer, 1975). R. placozophagus has spicules with a cell nucleus just in the middle notch and are able to regrow new spicules in the regenerating blastema in the posterior third of the body. The phylum Mollusca has no species capable of regenerating the whole body; however, it is known that several groups can regenerate the foot, tentacles, mantle, head and even the shell (Bely et al., 2014). Spicules rapidly dissolve with acetic acid, indicating their calcareous nature and they constitute an endoskeleton that may be useful to provide strength to the body wall while siphoning the placozoans. Many hypothesis have been proposed for the origin of the skeleton in vertebrates and invertebrates: as a sensory enhancement structure, a barrier for osmosis, the result of calcium metabolism reorganization, a reservoir for bio-limiting elements or a structure for disposal waste by products (Donoghue and Sansom, 2002; Marin et al., 1996). These ideas may be useful to infer additional roles for the endoskeleton in Rhodope, as well as other taxa of meiofauna with endoskeletons. Riedl (1959) reported that mature specimens of R. veranii combined their anterior right side of the body and remain in this position for few seconds to put the sexual pores in contact in order to transfer sperm (Riedl, 1959). This author also supposed insemination by unidirectional transfer of spermatozoa. I occasionally observed this form of mutual intercourse in R. placozophagus, but I suggest that it may instead represent some kind of recognitiondomination interaction. Based on the aphallic nature of Rhodope rousei, it has recently been suggested that Rhodope uses dermal insemination via spermatophores and that sperm possibly penetrates by short-term lysis of a small stretch of the epidermis (Brenzinger et al., 2011). As shown here for R. placozophagus, dermal insemination via spermatophores certainly could be the reproduction type employed by rhodopemorpha. Predation is defined as the consumption of individuals or tissues belonging to species of the same or lower trophic levels (Elewa, 2007), as is the case with Trichoplax and R. placozophagus. The concomitant abundance of placozoans and R. placozophagus, together with the particular anatomy of the buccal apparatus and the toxic nature of its prey, suggests that this microslug, and perhaps all Rhodopidae (see below), are specialized predators, although whether they feed on other soft and toxic prey, such as polyps of cnidarians, has yet to be determined. Despite the diversity of microorganisms and predator-prey interactions occurring together with placozoans, only a single case of predation has been reported several decades ago (Riedl, 1959). The results shown here support the observations reported by Riedl about R. veranii: a creeping Rhodope reaches a Trichoplax, touches the feeding prey with the mouth edges, everts a small portion of buccal bulb, slightly crumples the body and suction begins accompanied by strong peristaltic movements of the buccal bulb and digestive gland. Considering that most reports relate observations of organisms such as small snails or rhabdocoel flatworms that avoided placozoans (Pearse and Voigt, 2007), it is surprising that no further characterization of this
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predatory activity followed this initial report, especially considering the kleptochemistry phenomenon of several opisthobranchs (Marín and Ros, 2004) and the resistance that R. veranii must have to the toxins of Trichoplax. Furthermore, it would be tempting to determine whether a specific and exclusive predation-prey relationship exists in the rhodopidae family with placozoans since R. roskoi, R. marcusi, R. rousei and Helminthope psammobionta have all been reported in locations where Trichoplax have been reported as: the north coast of France, the coast of Sao Paulo Brazil, the southeast coast of Australia and Bermuda (Brenzinger et al., 2011; Eitel et al., 2013; Haszprunar and Hess, 2005; Marcus and Marcus, 1952; Salvini-Plawen, 1991). Finally, this predator-prey association is particularly unusual: on the one hand, it seems that R. placozophagus cannot easily detect its prey; it needs to make physical contact in order to recognize its food. It is possible that, among the loss and simplification of organs and structures in Rhodopidae, there was a degeneration of the osphradium, an important chemosensory organ (Lindberg and Sigwart, 2015). On the other hand, Trichoplax cannot sense when they are being predated and, due to the fact that placozoans are very simple organisms that are able to reshape and regenerate their own structure from small fragments (Schwartz, 1984), predation can actually make a contribution to asexual reproduction; i.e., the resulting fragments will disperse and grow and eventually reproduce again by binary fission. Several organisms lose body parts by autotomy in order to preserve the individual, but this implies an energetic cost to regrow the lost structure. In the case of Trichoplax, the benefits may overcome the costs and, at the same time, due to the toxicity of their prey, R. placozophagus may in turn obtain protection against potential predators. Acknowledgements The author thanks Dra. Edna Naranjo García of the Biology Institute, UNAM, for helping with the type specimen. This work was supported by Grant No. 151757 from Consejo Nacional de Ciencia y Tecnología (CONACyT). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jcz.2017.09.005. References Bely, A.E., Zattara, E.E., Sikes, J.M., 2014. Regeneration in spiralians: evolutionary patterns and developmental processes. Int. J. Dev. Biol. 58, 623–634, http://dx. doi.org/10.1387/ijdb.140142ab. Brenzinger, B., Wilson, N.G., Schrödl, M., 2011. 3D microanatomy of a gastropod worm, Rhodope rousei n. sp. (Heterobranchia) from southern Australia. J. Molluscan Stud. 4, 375–387. Brenzinger, B., Haszprunar, G., Schrödl, M., 2013. At the limits of a successful body plan—3D microanatomy, histology and evolution of Helminthope (Mollusca: Heterobranchia: Rhodopemorpha), the most worm-like gastropod. Front. Zool. 10, 37.
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