Search

Non-parasitic flatworms are known to temporarily attach to the substrate by secreting a multicomponent bioadhesive to counteract water movements. However, to date, only species of two higher-level flatworm taxa (Macrostomorpha and Proseriata) have been investigated for their adhesive proteins. Remarkably, the surface-binding protein is not conserved between flatworm taxa. In this study, we sequenced and assembled a draft genome, as well as a transcriptome, and generated a tail-specific positional RNA sequencing dataset of the polyclad Theama mediterranea . This led to the identification of 15 candidate genes potentially involved in temporary adhesion. Using in situ hybridisation and RNA interference, we determined their expression and function. Of these 15 genes, 4 are homologues of adhesion-related genes found in other flatworms. With this work, we provide two novel key components on the flatworm temporary adhesion system. First, we identified a Kringle-domain-containing protein (Tmed-krg1), which was expressed exclusively in the anchor cell. This in silico predicted membrane-bound Tmed-krg1 could potentially bind to the cohesive protein, and a knockdown led to a non-adhesive phenotype. Secondly, a secreted tyrosinase (Tmed-tyr1) was identified, which might crosslink the adhesive proteins. Overall, our findings will contribute to the future development of reversible synthetic glues with desirable properties for medical and industrial applications.

Many free-living flatworms have evolved a temporary adhesion system, which allows them to quickly attach to and release from diverse substrates. In the marine Macrostomum lignano , the morphology of the adhesive system and the adhesion-related proteins have been characterised. However, little is known about how temporary adhesion is performed in other aquatic environments. Here, we performed a 3D reconstruction of the M. lignano adhesive organ and compared it to the morphology of five selected Macrostomum , representing two marine, one brackish, and two freshwater species. We compared the protein domains of the two adhesive proteins, as well as an anchor cell-specific intermediate filament. We analysed the gene expression of these proteins by in situ hybridisation and performed functional knockdowns with RNA interference. Remarkably, there are almost no differences in terms of morphology, protein regions, and gene expression based on marine, brackish, and freshwater habitats. This implies that glue components produced by macrostomids are conserved among species, and this set of two-component glue functions from low to high salinity. These findings could contribute to the development of novel reversible biomimetic glues that work in all wet environments and could have applications in drug delivery systems, tissue adhesives, or wound dressings.

Marine invertebrates are promising sources of novel bioactive secondary metabolites, and organisms like sponges, ascidians and nudibranchs are characterised by possessing potent defensive chemicals. Animals that possess chemical defences often advertise this fact with aposematic colouration that potential predators learn to avoid. One seemingly defenceless group that can present bright colouration patterns are flatworms of the order Polycladida. Although members of this group have typically been overlooked due to their solitary and benthic nature, recent studies have isolated the neurotoxin tetrodotoxin from these mesopredators. This review considers the potential of polyclads as potential sources of natural products and reviews what is known of the activity of the molecules found in these animals. Considering the ecology and diversity of polyclads, only a small number of species from both suborders of Polycladida, Acotylea and Cotylea have been investigated for natural products. As such, confirming assumptions as to which species are in any sense toxic or if the compounds they use are biosynthesised, accumulated from food or the product of symbiotic bacteria is difficult. However, further research into the group is suggested as these animals often display aposematic colouration and are known to prey on invertebrates rich in bioactive secondary metabolites.

Several polyclad flatworm species are known to contain high levels of tetrodotoxin (TTX), but currently TTX-bearing flatworms seem to be restricted to specific Planocera lineages belonging to the suborder Acotylea. During our ongoing study of flatworm toxins, high concentrations of TTXs were detected for the first time in the flatworm Prosthiostomum trilineatum , suborder Cotylea, from the coastal area of Hayama, Kanagawa, Japan. Toxin levels were investigated by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing that this species contains comparable concentrations of toxins as seen in planocerid flatworms such as Planocera multitentaculata . This finding indicated that there may be other species with significant levels of TTXs. The distribution of TTXs among other flatworm species is thus of great interest.

Tetrodotoxin (TTX) is a potent neurotoxin that acts specifically on voltage-gated sodium channels on excitable membranes of muscle and nerve tissues. The biosynthetic process for TTX is unclear, although marine bacteria are generally thought to be the primary producers. The marine flatworm Planocera multitentaculata is a known TTX-bearing organism, and is suspected to be a TTX supplier to pufferfish. In this study, flatworm specimens were collected from an intertidal zone in Hayama, Kanagawa, Japan, the TTX content of the flatworm was measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and seasonal changes in TTX content were investigated. No significant difference in TTX concentration of the flatworm body was found between the spawning period and other periods. However, the TTX content in individual flatworms was significantly higher in the spawning period than at other times. The TTX content rose in association with an increase in the body weight of the flatworm.

The marine plathyhelminth Macrostomum lignano was recently isolated from Adriatic shore sediments where it experiences a wide variety of environmental challenges, ranging from hypoxia and reoxygenation, feeding on toxic algae, to exposure to anthropogenic contaminants. As multidrug resistance transporters constitute the first line of defense against toxins and toxicants we have studied the presence of such transporters in M. lignano in living animals by applying optical methods and pharmacological inhibitors that had been developed for mammalian cells. Application of the MDR1 inhibitor Verapamil or of the MRP1 inhibitors MK571 or Probenecid increased the intracellular fluorescence of the reporter dyes Fura-2 am, Calcein am, Fluo-3 am in the worms, but did not affect their staining with the dyes Rhodamine B, CMFDA or Ageladine A. The marine sponge alkaloid Ageladine A remained intracellularly trapped for several days in the worms, suggesting that it does not serve as substrate of multidrug resistance exporters. In addition, Ageladine A did not affect multidrug resistance-associated protein (MRP)-mediated dye export from M. lignano or the MRP1-mediated glutathione (GSH) export from cultured rat brain astrocytes. The data obtained demonstrate that life-imaging is a useful tool to address physiological drug export from intact marine transparent flatworms by using multiphoton scanning microscopy.

Even though tetrodotoxin (TTX) is a widespread toxin in marine and terrestrial organisms, very little is known about the biosynthetic pathway used to produce it. By describing chemical structures of natural analogs of TTX, we can start to identify some of the precursors that might be important for TTX biosynthesis. In the present study, an analog of TTX, 5,11-dideoxyTTX, was identified for the first time in natural sources, the ovary of the pufferfish and the pharynx of a flatworm (planocerid sp. 1), by comparison with totally synthesized (-)-5,11-dideoxyTTX, using high resolution ESI-LC-MS. Based on the presence of 5,11-dideoxyTTX together with a series of known deoxy analogs, 5,6, 11-trideoxyTTX, 6,11-dideoxyTTX, 11-deoxyTTX, and 5-deoxyTTX, in these animals, we predicted two routes of stepwise oxidation pathways in the late stages of biosynthesis of TTX. Furthermore, high resolution masses of the major fragment ions of TTX, 6,11-dideoxyTTX, and 5,6,11-trideoxyTTX were also measured, and their molecular formulas and structures were predicted to compare them with each other. Although both TTX and 5,6,11-trideoxyTTX give major fragment ions that are very close, m/z 162.0660 and 162.1020, respectively, they are distinguishable and predicted to be different molecular formulas. These data will be useful for identification of TTXs using high resolution LC-MS/MS.

The brominated pyrrole-imidazole Ageladine A was used for live imaging of the jellyfish (jellies) Nausithoe werneri, the sea anemone Metridium senile and the flatworm Macrostomum lignano. The fluorescence properties of Ageladine A allow for estimation of pH values in tissue and organs in living animals. The results showed that Nausithoe werneri had the most acidic areas in the tentacles and close to the mouth (pH 4-6.5), Metridium senile harbours aggregates of high acidity in the tentacles (pH 5) and in Macrostomum lignano, the rhabdoids, the gonads and areas close to the mouth were the most acidic with values down to pH 5.