Pseudoceros indicus is an Indo-Pacific species of free-living marine flatworm (Platyhelminthes, Polycladida) named for its conspicuous margin of irregular blue or purple spots (fig 1). The dorsal surface ranges from cream to white, often with brighter white flecks  (Newman and Schupp, 2002). Pseudotentacles are present at the head end and are part of the ruffled margin. Common in the shallow waters of the sub-littoral zone, mudflats and mangroves, this flatworm is an active predator which feeds on various colonial ascidians. Like most polyclads, P. indicus is hermaphroditic and reproduces via sexual reproduction between two individuals (Newman and Schupp, 2002).

Individuals are dorso-ventrally flattened and elongate, often described as ‘leaf shaped’ (Newman and Schupp, 2002). Soft-bodied and relatively small, ranging from 10-40mm in length. Margin is ruffled and pinched at the head, forming two ear-like pseudotentacles (Bolaños et al., 2016). 

Dorsal surface white or cream, often with irregular white spots present in darker specimens. Margin composed of irregular blue or purple spots which vary in size, shape and spacing (Bolaños et al., 2016). The absence of a median stripe distinguishes P. indicus from many closely related species such as P. concinnus (Bolaños et al., 2016). The pattern of blue spots appears to be unique between individuals. During the regeneration experiment described below, the differences in the relative size, shape and spacing of the spots were able to be used to distinguish between different individuals and to match photos of the same individuals across the 5-week experimental period. In some instances, the shape of the cerebral eyespot was also considered. Figure 2 provides an example of the way in which the spots were used to uniquely identify each animal. 

Little is known about the ecology of P. indicus, though it has been recorded in a number of habitats, ranging from mudflats to reef crests and the sublittoral zone (Newman and Schupp, 2002; Bolaños et al., 2016). Marine turbellarians are active predators and thus the abundance of ascidian prey Eudistoma toealensis and the yellowish, colonial ascidian, E. viride drive the abundance of P. indicus (Newman and Schupp, 2002). Newman and Schupp (2002) observed that the secondary metabolites incorporated into the flatworm corresponded to the species of ascidian on which the worm had been collected. This suggests either specialised, species specific feeding or fast incorporation of secondary metabolites with short retention time (Schupp et al., 2002). It is hypothesised that these metabolites may act as a chemical defence against reef fish predators (Newman and Schupp, 2002). 

Reproduction & Development 

Polyclads are all hermaphroditic, with each individual consisting of both female and male reproductive systems. Unlike many other animals, flatworms do not have discrete gonads, and thus male and female reproductive organs are scattered throughout the body. While self-fertilisation is possible, it is rare and mating almost always takes place between two individuals. Fertilisation of sperm and egg occurs internally, however the mechanisms of reproduction employed are species-specific and thus species are often distinguished by the intricacies of their reproductive systems. 

P. indicus exhibits complex reproductive behaviour, including distinct stages of insemination, oviposition and parental care of the eggs. Armed with a single, sharp stylet, mature worms battle to inseminate each other in a process called hypodermic insemination, or ‘penis-fencing’.  This penis is used to transfer sperm bundles into the dorsal of ventral tissue of the other individual which are then absorbed by the epidermis to reach the eggs scattered within the body (Fig 3). Deliberate motions are made by each animals as they attempt to inseminate the other while avoiding insemination themselves. This fight occurs as each individual attempts to minimise the cost of reproduction in favour of sperm donation. Sperm receipt incurs far greater energetic costs to the animal as it is followed by long periods of egg laying and intensive parental care of the eggs. The adults of P. indicus are observed to brood their eggs, covering the eggs with their body for the full developmental period, potentially improving the adhesion of the eggs to the substrate (Chim et al., 2015). 

P. indicus, like most other polyclads, lay multiple batches of benthic egg plates over several weeks, laying up 3,000 eggs across approximately 17 laying events (Chim et al., 2015). These batches of eggs are deposited adjacent to previous batches in an irregular pattern, laying later batches over older eggs, whether hatched or not. It is hypothesised that the staggered laying of eggs is an adaptive advantage, increasing survival rate of the embryos in unstable habitats susceptible to environmental fluctuations (Chim et al., 2015). Unlike many polyclads, P. indicus do not undergo direct development and eggs produce free-swimming Müller’s larvae which must metamorphose before reaching adult form (Chim et al., 2015). Larvae spend approximately 27 days in the plankton before settlement and metamorphosis occurs (Chim et al., 2015).  

Regeneration Experiment 

The regenerative abilities of freshwater flatworms have been of significant scientific interest for many years, particularly in stem cell research. Owing to their body tissue (parenchyma) being full of totipotent cells (neoblasts), planarians are capable of full body restoration from fragmented halves which many species exploit as a mode of asexual reproduction. This is because totipotent cells are able to be differentiated into any other type of cell to replace damaged tissues (Rink, 2013). Furthermore, the continuous replenishment of damaged cells allows for a unique digestive system function and daily self-renewal of tissues (Sheiman and Kreshchenko, 2015). Interestingly, memory trace is able to be retained in both fragments of a divided freshwater planarian where sufficient portions of the nervous system are preserved in each half (Shomrat and Levin, 2013).

These remarkable capabilities make planarians popular research subjects for stem cell studies in higher organisms and a plethora of studies into the cellular sources and molecular processes of regeneration have been conducted. Additionally, the regulatory factors of morphogenesis and asexual reproduction as well as the proliferation of neoblasts are under investigation (Rink, 2013). Despite this, many mechanisms of planarian regeneration are poorly understood and long-term in vivo cultures of planarian stem-cells have not yet been successful (Sheiman and Kreshchenko, 2015). 

In contrast, little research has been done into the regenerative capacities of marine polyclads and reports of asexual reproduction in marine species are scarce. I therefore conducted a small experiment over a 5-week period investigating regeneration in P. indicus. This experiment aimed to determine if complete regeneration of two new individuals from one worm cut in half was possible, as is common in freshwater species. 

Methods

I collected 6 adult Pseudoceros indicus specimens from Burleigh Heads, Queensland which were held in a tank at the University of Queensland for the duration of the experiment at 25-26˚C. Once a week for five weeks, I removed the specimens from the tank and observed under a dissecting microscope in a petri dish containing filtered sea water. In the first week, each worm was observed and photographed under the microscope individually before being cut in half. I made a transverse cut was half-way down the organism, dividing the worm into an anterior (‘head’) and posterior (‘tail’) end. The fragments were housed in a mesh box sunk under a rock in the aquarium. I grouped the head and tail fragments of each individual in the same compartment, but kept each individual separate in order to track differences in growth between individuals. 

Observations and Results

Following the cut, the anterior end continued to swim around the dish in the same manner as previously, while the posterior end did not move following amputation.  After one week, the cut margin showed clear signs of healing, with blue spots beginning to reappear on one specimen. Distinct difference in the behaviour of the two types of fragments were observes, with the posterior ends curled into balls and showing little signs of life compared to the swimming head ends.  By the end of the second week, blue spots had appeared on the cut margin of all the individuals, on both anterior and posterior fragments. The posterior fragments began to exhibit directionality in their movement but no signs of eyespots or pseudotentacles were observed. Like the first week, they remained curled up in balls unless disturbed. 3 weeks following the amputation, most of the anterior fragments appeared almost entirely whole, with only slight irregularities in the margin where the cut was made. The posterior ends were considerably more active than the previous week, occasionally swimming and adhering to the substrate. Rather than growing outwards as with the anterior fragments, the posterior fragments folded in on themselves and the two halves of the margin sealed together, with the exterior edges of the margin standing erect at times, bearing some resemblance to pseudotentacles. No eyespots were observed. Figure 4 illustrates the growth of the worms over the five week period. 

While no head was grown on the posterior (tail) fragments, this may be due to a number of factors. Firstly, P. indicus may not be capable of full regeneration of a head where the brain is cut off as was likely the case here. Secondly, the posterior fragments did not possess a mouth or pharynx, and thus may have been unable to meet the energy requirements for regeneration. Finally, more time may simple have been required in order to observe the growth of a head.

An unexpected observation was made where one individual appeared to become damaged during the experimental process. This animal, pictured in figure 5, appears to have grown a third pseudotentacle. This irregularity in the margin was not observed to move and contract with the rest of the margin, but rather with the movements of the pseudotentacles. Errors in regeneration occurring in response to damage to marine polyclads has been recorded a number of times, including growth of a second tail (Newman et al., 2003). 

The general anatomy of Pseudoceros indicus is illustrated in figure 6. Flatworms are relatively simple, unsegmented worms with bilateral symmetry. They are acoelomates, meaning they have no cavities within their body. As a result, their organs are scattered roughly throughout the tissue (Newman et al., 2003). 

Generally, polyclads have two main discernible features: their eyes and pseudotentacles. P. indicus possesses a dorsal cerebral eyespot consisting of approximately 40 ocelli clustered in a horseshoe shaped arrangement. Ocelli are simple eyes lacking any lens composed of photosensitive cells and a concave cup which allow flatworms to sense the direction of light (Newman et al., 2003). Structures resembling the ventral or pseudotentacular eyespots in other Pseudoceros species were observed on all six P. indicus specimens from Burleigh Heads (fig 7), however more research is required to determine the exact nature of these spots as there is no record of ventral eyespots in P. indicus.

Polyclads possess a large bundle of nerve cells below the pseudotentacles which acts a brain (Newman et al., 2003). The specific structure of these nerve bundles differ between taxa and Pseudoceros are generally classified as having a small, single-lobed brain (Quiroga, 2008). These brains are relatively complex and integrate all the activity of the worm into a central nervous system (Quiroga, 2008). Polyclads also possess a well-developed muscular system, with muscles throughout the body wall and pharynx (Rawlinson, 2010; Bolaños et al., 2016).

Food is taken up via the mouth into a complex, branched pharynx which can crush, suck or engulf prey (Newman et al., 2003). Platyhelminthes have no anus, and excretion occurs via the protonephridial system which also assists in water balance (Newman et al., 2003). They do not possess any circulatory or respiratory system, and thus rely on diffusion for the movement of molecules in and out of their bodies (Newman et al., 2003). 

As mentioned above, polyclads are hermaphroditic, possessing both male and female reproductive systems. These organs are not discrete as in larger animals, but rather, scattered throughout the body. The ventral male pore occurs directly below the mouth, followed by a posterior female pore (Newman et al., 2003). The male reproductive system consists of the male pore, holding a single penis, as well as vas deferens. The female pore consists of a vagina which does not accept the penis but acts only as the passage for eggs during egg laying (Chim et al., 2015). Around the vagina are numerous cement glands which secrete an adhesive substance allowing the eggs to adhere to the substrate (Newman et al., 2003).

The specimens collected for observation for the purposes of this webpage were found in the sub-tidal zone at Burleigh Heads, Queensland.  Records of P. indicus sightings demonstrate a wide Indo-Pacific distribution, with data collected in Moreton Bay (Queensland), Stradbroke Island (Queensland), Singapore, Micronesia, Lizard Island (Queensland), South Africa, Mayotte, Réunion Island and Lakshadweep (India).

Pseudoceros contains most brightly coloured individuals which are most easily distinguished from other species by markings (Newman and Cannon, 1995). In many species, specific patterns and colours may be signals of toxicity, acting as deterrents to protect individuals from predation by reef fishes (Newman et al., 2003). Alternatively, where the worm is not toxic, it may co-evolve with another species, mimicking its markings in order to avoid predation.  Mimicry of nudibranchs by marine polyclads has been reported many times, though the exact dynamics of this mechanism are not always known (Tan, 2015). As it is unlikely that its bright colours are of any use in camouflage, it is possible that the blue margin around P. indicus acts as a warning signal or deterrent against predation. Schupp et al. (2002) suggested that P. indicus incorporates toxic compounds from its prey which may render the worm unpalatable to reef fish. If this is the case, the blue markings may be indicative of this acquired toxicity, though much more research is required. 

Little is known about the conservation status of marine polyclads as they are relatively understudied with a cryptic nature. However, due to the permeable nature of their body plan which allows for water flow and gas exchange, it is likely that like most marine life, they will be susceptible to strong environmental fluctuations (Newman et al., 2003). 

Bidgrain, P. 2017. Flatworm forum from South-West Indian Ocean. Retrieved from http://seaslugs.free.fr/flatworm/planaire/a_miniature.htm

Bolaños, M., B. Q. Gan, and R. S. L. Ong. 2016. First records of pseudocerotid flatworms (Platyhelminthes- Polycladida- Cotylea) from Singapore- A taxonomic report with remarks on colour variation. Raffles Bulletin of Zoology (Supplement No. 34):130-169. 

Chim, C. K., R. S. L. Ong, and B. Q. Gan. 2015. Penis fencing, spawning, parental care and embryonic development in the cotylean flatworm Pseudoceros indicus (Platyhelminthes- Polycladida- Pseudocerotidae) from Singapore. Raffles Bulletin of Zoology 31:60-67. 

Newman, L. J., and L. R. G. Cannon. 1995. Colour pattern variation in the tropical flatworm, Pseudoceros (Platyhelminthes: Polycladida), with descriptions of three new species Raffles Bulletin of Zoology 43(2):435-446. 

Newman, L. J., L. R. G. Cannon, and A. Flowers. 2003. Marine flatworms : the world of polyclads

Marine flatworms the world of polyclads. Melbourne : CSIRO Publishing.

Newman, L. J., and P. Schupp. 2002. A new species of pseudocerotid flatworm (Platyhelminthes, Polycladida) from the Indo-Pacific. Micronesia 34(2):177-184. 

Quiroga, S. 2008. Systematics and taxonomy of polyclad flatworms with a special emphasis on the morphology of the nervous system. In: M. K. Litvaitis (ed.). ProQuest Dissertations Publishing.

Rawlinson, K. A. 2010. Embryonic and post-embryonic development of the polyclad flatworm Maritigrella crozieri; implications for the evolution of spiralian life history traits. Frontiers in Zoology 7(1):12. (journal article) doi: 10.1186/1742-9994-7-12

Rink, J. C. 2013. Stem cell systems and regeneration in planaria. Development Genes and Evolution 223(1-2):67-84. doi: 10.1007/s00427-012-0426-4

Schupp, P., P. Proksch, and V. Wray. 2002. Further New Staurosporine Derivatives from the Ascidian Eudistoma toealensis and Its Predatory Flatworm Pseudoceros sp. Journal of Natural Products 65(3):295-298. doi: 10.1021/np010259a

Sheiman, I. M., and N. D. Kreshchenko. 2015. Regeneration of planarians: Experimental object. Russian Journal of Developmental Biology 46(1):1-9. doi: 10.1134/s1062360415010075

Tan, Y. K. 2015. Apparent mimicry of marine flatworm and nudibranch. Singapore Biodiversity Records:85-86.