Autofocusing MALDI mass spectrometry imaging of tissue sections and 3D chemical topography of nonflat surfaces.

We describe an atmospheric pressure matrix-assisted laser desorption-ionization mass spectrometry imaging system that uses long-distance laser triangulation on a micrometer scale to simultaneously obtain topographic and molecular information from 3D surfaces. We studied the topographic distribution of compounds on irregular 3D surfaces of plants and parasites, and we imaged nonplanar tissue sections with high lateral resolution, thereby eliminating height-related signal artifacts.

Functional ultrastructure and cytochemistry of vitellogenesis and mature vitellocytes of the digenean Cainocreadium labracis (Dujardin, 1845), parasite of Dicentrarchus labrax (L., 1758).

Vitellogenesis and vitellocytes of Cainocreadium labracis were studied by transmission electron microscopy (TEM) and TEM cytochemistry. Four developmental stages were distinguished during vitellogenesis: (I) stem cell of high nucleo-cytoplasmic ratio; (II) early differentiation with chief activity focused on the beginning of protein synthesis and shell globule formation; (III) advanced differentiation with rapid intensification of protein synthesis, progressive fusion of single shell globules into large globule clusters, and formation of unsaturated lipid droplets surrounded by ß-glycogen particles; and (IV) mature vitellocyte. Early vitellogenesis with vitellocyte maturation consists of: (1) increase in cell volume; (2) increased development of large, parallel cisternae of GER with production of proteinaceous granules; (3) development of small Golgi complexes that package granules; and (4) within vacuoles, progressive enlargement of proteinaceous granules into shell globule clusters formed during vitellogenesis. Three types of inclusions accumulate in large amounts in mature vitelline cells: (1) shell globule clusters, important component in the formation of egg shell; (2) numerous unsaturated lipid droplets. Though fewer, there are also diphasic droplets consisting of saturated and unsaturated lipids in the same droplet, and (3) a relatively small amount of ß-glycogen particles, usually surround a few groups of lipid droplets. The ß-glycogen and lipid droplets are nutritive reserves for embryogenesis. General pattern and functional ultrastructure of vitellogenesis greatly resemble those observed in some lower cestodes, such as bothriocephalideans and diphyllobothrideans. Variations and differences in the amount of lipids and of glycogen during vitellogenesis in lower cestodes and other trematodes are compared and discussed.

Methods for quantifying eggs and oviposition rate of Dawestrema cycloancistrium (Monogenea: Dactylogyridae), monogenean parasite of Arapaima gigas (Teleostei: Osteoglossidae).

Dawestrema cycloancistrium is the main ectoparasite causing mortality in fingerlings of pirarucu (Arapaima gigas) in Amazonian aquaculture. Very little is known about the D. cycloancistrium reproductive index and appropriate methods of collecting eggs for investigation. This study aimed to determine the oviposition rate of D. cycloancistrium. To achieve this aim, two egg quantification methods were tested: the estimative method (ME) and the total counting method (MT). Compared with the MT, the ME overestimated the number of eggs counted, which were 2943.5 ± 2840.6 and 1041.5 ± 533 eggs, and the oviposition rate, which was 80.1 ± 58.7 and 31.4 ± 16.4 eggs/parasite/day, for ME and MT, respectively. These results show that for studies quantifying D. cycloancistrium eggs, the total eggs in the sample must be counted, as the estimates made using subsamples are not representative. Using the MT, the oviposition rate for D. cycloancistrium was determined to be 31.4 ± 16.4 eggs per adult parasite per day. The present study demonstrates the egg production capacity of a monogenean species parasite of A. gigas, providing basic biological data for D. cycloancistrium.

Threading: A novel insilico indagation method for genetic characterization of some diplostomoid metacercariae (Digenea:Diplostomidae Poirier, 1886).

The protein encoding zone of Mitochondrial DNA region (inherited from single lineage) seems most suitable and effective for taxonomic, systematic, ecological, evolutionary, DNA barcoding, cryptic species and population studies, exploiting nucleotide/amino acid datasets (1D/2D/3D conformational level). Nowadays, expeditious computerized methods are in trend for analyzing genetic material to demonstrate variations at various levels of protein structures. Structural proteomics have implemented here for genetic identification, differentiation and relationship of species from information rich data of mt COI gene of the family Diplostomidae with inclusion of molecular tools. Various aspects have been utilized herein for re-validation and infallible discrimination of Trematode diplostomoid metacercariae (Tetracotyle lucknowensis Pandey, 1971; T. xenentodoni Chakrabarti, 1970; T. fausti Rai and Pande, 1969; T. muscularius Chakrabarti, 1970 and Diplostomulum minutum Pandey, 1968), the infective stage in the life cycle, causing severe damage to fish host, whose adults are found mainly in fish eating birds and mammals.

Resilin-like protein in the clamp sclerites of the gill monogenean Diplozoon paradoxum Nordmann, 1832.

Resilin is a soft and elastic protein, which is found in many exoskeletal structures of arthropods. Proteins with similar chemical properties have been described for other invertebrates including monogenean fish parasites. However, for the latter taxon no clear microscopic evidence for a resilin-like protein has been shown so far. Here, we present the results of detailed microscopic analyses of the clamp sclerites (attachment devices) of the monogenean Diplozoon paradoxum. Toluidine blue, which is known to stain resilin, selectively stained the material in the clamp sclerites. In addition, when exposed to UV light, this material exhibited an intense blue autofluorescence. The emission spectrum of this autofluorescence has its maximum at 424 nm and is nearly identical to emission spectra of blue autofluorescences observed in 2 well-studied structures containing high proportions of resilin in the exoskeleton of the locust Schistocerca gregaria. The results strongly indicate that the sclerite material of D. paradoxum contains a resilin-like protein. The presence of such a protein likely enhances the attachment efficiency of the clamp sclerites and increases their lifetime.

The neuromuscular system in continuously swimming cercariae from Belarus. II Echinostomata, Gymnocephala and Amphistomata.

The neuromuscular system in cercariae of Moliniella anceps, Echinostoma revolutum, Cathaemasia hians, Psilochasmus oxyurus, Sphaeridiotrema globulus, Paramphistomum cervi and Diplodiscus subclavatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR sensory receptors were investigated. The general patterns of musculature, 5-HT- and FMRFamide-IR neuronal elements in the 12 species studied here and in paper I are similar to those observed in other cercariae and reflect the morphology of the groups. The musculature of the tail shows variations which are related to the different strategies of host finding. In the Echinostomatoidea and Paramphistomoidea, the striated musculature of the tail is well developed compared to that in the Xiphidiocercariae. Specialized muscle fibres were found in S. globulus, which are able to change the shape of the tail. Nine of the species studied have seven paired 5-HT-IR neurons in the body, and two species have eight. No correlation between the body size and the number of 5-HT-IR neurons was observed. However, the size of the neurons followed the body size. The number of 5-HT-IR neurons in the brain ganglia increased from the primitive to the advanced forms. The number of FMRFamide-IR transverse commissures in the body correlates with the size of the cercariae. Regardless of the differences in the second intermediate host, the distribution of α-tubulin-IR sensory receptors shows a high degree of conformity in all species except in P. cervi, which encysts on plants.

Molecular characterization of pouched amphistome parasites (Trematoda: Gastrothylacidae) using ribosomal ITS2 sequence and secondary structures.

Members of the family Gastrothylacidae (Trematoda: Digenea: Paramphistomata) are parasitic in ruminants throughout Africa and Asia. In north-east India, five species of pouched amphistomes, namely Fischoederius cobboldi, F. elongatus, Gastrothylax crumenifer, Carmyerius spatiosus and Velasquezotrema tripurensis, belonging to this family have been reported so far. In the present study, the molecular phylogeny of these five gastrothylacid species is derived using the second internal transcribed spacer (ITS2) sequence and secondary structure analyses. ITS2 sequence analysis was carried out to see the occurrence of interspecific variations among the species. Phylogenetic analyses were performed for primary sequence data alone as well as the combined sequence-structure information using neighbour-joining and Bayesian approaches. The sequence analysis revealed that there exist considerable interspecific variations among the various gastrothylacid fluke species. In contrast, the inferred secondary structures for the five species using minimum free energy modelling showed structural identities, in conformity with the core four-helix domain structure that has been recently identified as common to almost all eukaryotic taxa. The phylogenetic tree reconstructed using combined sequence-structure data showed a better resolution, as compared to the one using sequence data alone, with the gastrothylacid species forming a monophyletic group that is well separated from members of the other family, Paramphistomidae, of the amphistomid flukes group. The study provides the molecular characterization based on primary sequence data of the rDNA ITS2 region of the gastrothylacid amphistome flukes. Results also demonstrate the phylogenetic utility of the ITS2 sequence-secondary structure data for inferences at higher taxonomic levels.

Variation in the morphometrics of diplostomid metacercariae (Digenea: Trematoda) infecting the catfish, Clarias gariepinus in Tanzania.

Diplostomum species are economically important worldwide due to their metacercariae which parasitize the eyes of fish, in both natural and aquaculture systems. However, their striking morphological similarity, especially at the metacercarial stage, makes species separation difficult. Three closely related diplostomid metacercariae, namely Diplostomum mashonense (type 3), Tylodelphys sp. 1 and 2 (type 1 and 2, respectively), coexist in the cranial cavity of the catfish, Clarias gariepinus from Mindu dam, Lake Victoria, Msimbazi, Ruvu and Kilombero rivers. The morphometrics of these three species were analysed by discriminant function analysis to investigate the degree of variation among the populations from the five localities sampled. The first canonical functions for all visual examination plots accounted for over 50% of the between-group variability. The observed differences were mainly from measurements associated with length, indicating that these measurements are important in the description of population characteristics. Visual examination of the samples along the canonical functions showed a clear between-population differentiation. The overall random assignment of individuals into their original groups was high (97%). These extensive morphometric variations introduce doubt about the reliability of measurements in the determination of species in these trematodes, as they may lead to misidentifications.

Field preservation of monogenean parasites for molecular and morphological analyses.

In the present work we examined the efficacy of three different chemical solutions (EtOH 70%, DMSO-NaCl solution, and Longmire buffer) in field preservation of fish gills to be subsequently screened for monogenean specimens destined to morphological and molecular analyses. Degree of difficulty in collecting monogeneans from gills, morphological state of parasites, integrity of their DNA and reliability of sequence reading were observed and qualitatively compared to those of gills and parasites stored in 5% formalin and 99% ethanol. Data were collected over a period of 2 months. Storage in Longmire buffer resulted in dissociation of gills and parasites, while both DMSO and 70% ethanol provided a fine physical and molecular preservation of gills and monogeneans, allowing rapid collection of parasites from lamellae, and easy extraction, amplification and sequencing of parasitic DNA.

Histopathological effects of the intramolluscan stages of Zygocotyle lunata, Echinostoma trivolvis, and Ribeiroia ondatrae on Helisoma trivolvis and observations on keratin in the trematode larvae.

The histopathological effects of Zygocotyle lunata, Echinostoma trivolvis, and Ribeiroia ondatrae in naturally infected Helisoma trivolvis were studied in hematoxylin and eosin sections of infected digestive glands fixed in 10% neutral buffered formalin. The larval stages of all three trematodes damaged the snail digestive gland. Most notable histopathology included disrupted digestive gland tubules, lysed cells, compressed tubules, and edematous spaces between tubules. Considerable damage was done by rediae ingesting digestive cells. There was a detectable hemocytic response by H. trivolvis in response to the rediae and cercariae of Z. lunata. Histochemical studies on sectioned material stained with the Ayoub-Shklar method for keratin detected the presence of this protein in the rediae and cercariae of Z. lunata and R. ondatrae. The presence of keratin is probably related to its role in cercarial encystment.

A novel Kunitz protein with proposed dual function from Eudiplozoon nipponicum (Monogenea) impairs haemostasis and action of complement in vitro.

Serine peptidases are involved in many physiological processes including digestion, haemostasis and complement cascade. Parasites regulate activities of host serine peptidases to their own benefit, employing various inhibitors, many of which belong to the Kunitz-type protein family. In this study, we confirmed the presence of potential anticoagulants in protein extracts of the haematophagous monogenean Eudiplozoon nipponicum which parasitizes the common carp. We then focused on a Kunitz protein (EnKT1) discovered in the E. nipponicum transcriptome, which structurally resembles textilinin-1, an antihemorrhagic snake venom factor from Pseudonaja textilis. The protein was recombinantly expressed, purified and biochemically characterised. The recombinant EnKT1 did inhibit in vitro activity of Factor Xa of the coagulation cascade, but exhibited a higher activity against plasmin and plasma kallikrein, which participate in fibrinolysis, production of kinins, and complement activation. Anti-coagulation properties of EnKT1 based on the inhibition of Factor Xa were confirmed by thromboelastography, but no effect on fibrinolysis was observed. Moreover, we discovered that EnKT1 significantly impairs the function of fish complement, possibly by inhibiting plasmin or Factor Xa which can act as a C3 and C5 convertase. We localised Enkt1 transcripts and protein within haematin digestive cells of the parasite by RNA in situ hybridisation and immunohistochemistry, respectively. Based on these results, we suggest that the secretory Kunitz protein of E. nipponicum has a dual function. In particular, it impairs both haemostasis and complement activation in vitro, and thus might facilitate digestion of a host's blood and protect a parasite's gastrodermis from damage by the complement. This study presents, to our knowledge, the first characterisation of a Kunitz protein from monogeneans and the first example of a parasite Kunitz inhibitor that impairs the function of the complement.

Lipid content and fatty acid composition of the monogenean Neobenedenia girellae and comparison between the parasite and host fish species.

Neobenedenia girellae, a capsalid monogenean, is a destructive fish parasite. We studied the lipid content and fatty acid composition of N. girellae and the skin and cutaneous mucus of a host fish, the amberjack Seriola dumerili (Carangidae). The lipid content of adult N. girellae was less than one-fourth that of both the skin and cutaneous mucus of its host. Adult N. girellae, S. dumerili skin and mucus had a relatively high weight-percentage of C16:0, C18:1(n-9), C18:0 and C22:6(n-3) fatty acids. When S. dumerili were fed a diet supplemented with [13C] fatty acids, [13C] fatty acids were detected in S. dumerili skin and adult N. girellae on S. dumerili, but no [13C] fatty acids were detected in the S. dumerili cutaneous mucus. In addition, the epidermis of S. dumerili, attached with N. girellae, was markedly thin. These results suggest that N. girellae feeds primarily on host epithelial cells. We then infected 2 host fishes, S. dumerili and the spotted halibut Verasper variegatus (Pleuronectidae; a host less susceptible to N. girellae infection), and compared the fatty acid composition of N. girellae with that of the skin and cutaneous mucus of the hosts. The fatty acid profiles from all samples were qualitatively and quantitatively similar. Thus, the fatty acid composition of the host may not contribute to the difference in susceptibility between S. dumerili and V. variegatus. These results may serve to develop new strategies for the control of N. girellae infections.

The use of fluorescent fatty acid analogs as labels in trematode experimental infections.

We examined the utility of fluorescent fatty acid analog dyes for labeling larval trematodes to use in experimental infections. Our goals were to identify two dyes that label larval trematodes belonging to the species Maritrema novaezealandensis and Coitocaecum parvum, determine if the dyes influence survival and infectivity of larval trematodes and/or host mortality, and if larval trematodes labeled with alternative dyes could be distinguished post-infection. The two dyes tested, BODIPY FL C(12) and BODIPY 558/568 C(12), successfully labeled all treated larval trematodes, did not influence cercariae survival or infectivity, and did not influence host mortality in either host-parasite system. All larval parasites were fluorescent and distinguishable after 5 days in amphipod intermediate hosts. In addition, larval Acanthoparyphium sp. were strongly fluorescent with both dyes after 5 weeks within cockle hosts. This method should be extremely useful for experimental studies using trematode-host systems as models for addressing a range of ecological and evolutionary questions.

Lipid profiles in Himasthla elongata and their intermediate hosts, Littorina littorea and Mytilus edulis.

In this research, the lipid profiles in rediae and free-living larvae (cercariae) of the trematodes Himasthla elongata were studied. It was shown that the lipid profiles of cercariae and rediae reflect the selective accumulation of membrane phospholipids and polyunsaturated fatty acids. In addition, infected tissues' lipid compositions in the intermediate hosts Littorina littorea (whole organism) and Mytilus edulis (foot) were studied. Modifications in the molluscs' lipid compositions were mainly caused by the parasites' metabolic requirements, as the parasites are unable to engage in de novo lipid biosynthesis, and thus, they utilise the host tissues' metabolites. The assumption that changes in the lipid unsaturation of infected intermediate hosts may affect their temperature resistance was discussed.

Distribution of Tetrodotoxin in the New Zealand Clam, Paphies australis, Established Using Immunohistochemistry and Liquid Chromatography-Tandem Quadrupole Mass Spectrometry.

Tetrodotoxin (TTX) is one of the most potent neurotoxins known. It was originally thought to only occur in puffer fish but has now been identified in twelve different classes of freshwater and marine organisms, including bivalves. Despite being one of the world’s most studied biotoxins, its origin remains uncertain. There is contradictory evidence regarding the source of TTX and its pathway through food webs. To date, the distribution of TTX has not been examined in bivalves. In the present study, 48 Paphies australis, a TTX-containing clam species endemic to New Zealand, were collected. Thirty clams were dissected, and organs and tissues pooled into five categories (siphons, digestive gland, adductor muscles, and the ‘rest’) and analyzed for TTX using liquid chromatography-mass spectrometry (LC-MS). The micro-distribution of TTX was visualized in the remaining 18 individuals using an immunohistological technique incorporating a TTX-specific monoclonal antibody. The LC-MS analysis revealed that siphons contained the highest concentrations of TTX (mean 403.8 µg/kg). Immunohistochemistry analysis showed TTX in the outer cells of the siphons, but also in the digestive system, foot, and gill tissue. Observing TTX in organs involved in feeding provides initial evidence to support the hypothesis of an exogenous source in P. australis.

Identification and partial characterization of a novel serpin from Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea).

BACKGROUND: Serpins are a superfamily of serine peptidase inhibitors that participate in the regulation of many physiological and cell peptidase-mediated processes in all organisms (e.g. in blood clotting, complement activation, fibrinolysis, inflammation, and programmed cell death). It was postulated that in the blood-feeding members of the monogenean family Diplozoidae, serpins could play an important role in the prevention of thrombus formation, activation of complement, inflammation in the host, and/or in the endogenous regulation of protein degradation. RESULTS: In silico analysis showed that the DNA and primary protein structures of serpin from Eudiplozoon nipponicum (EnSerp1) are similar to other members of the serpin superfamily. The inhibitory potential of EnSerp1 on four physiologically-relevant serine peptidases (trypsin, factor Xa, kallikrein, and plasmin) was demonstrated and its presence in the worm's excretory-secretory products (ESPs) was confirmed. CONCLUSION: EnSerp1 influences the activity of peptidases that play a role in blood coagulation, fibrinolysis, and complement activation. This inhibitory potential, together with the serpin's presence in ESPs, suggests that it is likely involved in host-parasite interactions and could be one of the molecules involved in the control of feeding and prevention of inflammatory responses.

Differential temperature acclimatization responses in the membrane phospholipids of Posthodiplostomum minimum and its second intermediate host, Lepomis macrochirus.

The effects of temperature change on phospholipid content in metacercariae of Posthodiplostomum minimum and their second intermediate hosts, Lepomis macrochirus, were examined to gauge similarities in the homeoviscous adaptation of host and parasite membranes to environmental thermal change. Heart, liver, and muscle tissues from individual L. macrochirus responded to environmental temperature declines with a decrease in the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Increases in membrane PE concentration increase membrane fluidity, maintaining fish membrane function as environmental temperature declines. However, the metacercariae of P. minimum exhibit changes in cholesterol levels, total lipid levels, and lipid composition (PE/PC) that contrast the normal changes for homeoviscous membrane adaptation exhibited by their fish intermediate hosts. The parasites seem to rely on their hosts for homeoviscous adaptation within normal developmental temperature ranges, pooling both cholesterol and PE as energetic stores for development and ontological transitions signaled by elevated temperatures.

Partial characterization and isolation of 130 kDa antigenic protein of Dicrocoelium dendriticum adults.

The study focused on characterizing and isolating Dicrocoelium dendriticum antigens or their fractions that could be used for the immunological diagnosis of dicrocoeliosis. Somatic (SoAg) and excretory-secretory antigens (ESAg) were analyzed by SDS-PAGE and their specificity was evaluated by Western blot with homologous and heterologous sera. The antigens were partially purified by chromatographic techniques of gel-filtration (Sephacryl S-300) and ion exchange (Hitrap-DEAE-Sepharose). Western blot analysis using sera of ovine infected with D. dendriticum revealed eight main antigenic polypeptides ranging from 24 to 205 kDa for SoAg and seven for ESAg with apparent molecular mass in the range of 26-205 kDa. We detected a specific parasite protein with an approximate molecular weight of 130 kDa in SDS-PAGE gels, arranged as a 450 kDa tetramer in native conditions. It also showed strong immunoreactivity by Western blot against ovine sera experimentally infected with D. dendriticum. Gel filtration chromatography (Sephacryl S-300) also showed other specific proteins, one of about 24 kDa in SoAg and another of about 42 kDa in ESAg. The elution conditions of 450 kDa protein (130 kDa monomer) by DEAE chromatography were similar to those from the somatic antigen (pH 7.2, 0.1M NaCl, in 29-34 ml fractions) and from the excretion-secretion antigen (pH 8.0, 0.1M NaCl, in 29-35 ml fractions). The suitability of 130 kDa polypeptide for D. dendriticum infection diagnosis was confirmed by Western blot using a pool of sera as well as individual serum samples from experimentally infected sheep. The sequence of amino termini of 130 kDa polypeptide from both fractions was the same and identical to that reported for a peptide from D. dendriticum described as a globin. This sequence also revealed an appreciable similarity with the amino end of globins from some phylogenetically related worms.

Localisation, quantitation, and characterisation of neuropeptide F- and FMRFamide-immunoreactive peptides in turbellarians and a monogenean: a comparative study.

Over the past decade it has become clear that the nervous systems of platyhelminths are both complex and highly developed, particularly in peptidergic elements. The central position of an ancestral flatworm in the evolution of the Bilateria has placed a greater importance on the study of modern flatworms. Using antisera generated to the C-terminal region of platyhelminth neuropeptide F and the molluscan neuropeptide, FMRFamide, in immunocytochemistry at both light and ultrastructural levels, immunoreactivities have been localised within the nervous systems of three species of triclad turbellarians, Dugesia lugubris, Dendrocoelum lacteum, and Polycelis nigra, and one species of monogenean trematode, Diclidophora merlangi. Extensive immunostaining was obtained with both antisera throughout the central and peripheral nervous systems of all species studied, but intensity and abundance was significantly greater in the turbellarians. Indirect electron-immunogold labeling demonstrated that immunoreactivity to both neuropeptides was often colocalised in neurosecretory vesicles, although discrete populations of vesicles were also observed. Radioimmunoassay of extracts of all species confirmed that neuropeptide F immunoreactivity was consistently more abundant than FMRFamide immunoreactivity, and that the levels of both in the three turbellarians were several orders of magnitude greater than those found in the monogenean. Chromatographic analyses of turbellarian extracts revealed that neuropeptide F and FMRFamide immunoreactivities were attributable to different peptides. These data imply that the neuropeptidergic systems systems of turbellarians are considerably more extensive than those of monogeneans, and would suggest that a regression has occurred in the latter as a consequence of the adoption of a mere sedentary parasitic lifestyle.

Widespread distribution of histamine in the nervous system of a trematode flatworm.

In general, most flatworms contain very little histamine (HA) and their nervous systems often lack, or contain very few, histaminergic elements. However, preliminary studies in our laboratory have revealed that the frog lung parasite, Haplometra cylindracea (Trematoda: Digenea), contains HA in a very high concentration. For this reason, the present study was undertaken to study the localization and synthesis of HA in this worm by using immunocytochemistry and high-pressure liquid chromatography (HPLC). Essentially all parts of the nervous system of H. cylindracea showed HA-like immunoreactivity. The paired cerebral ganglia and nerves emanating from these, including the longitudinal nerve cords, were intensely immunoreactive. The musculature of the pharynx, oral and ventral suckers, and those of the reproductive organs were all innervated by HA-immunoreactive fibers. Fiber plexuses beneath the tegument and throughout the parenchyma also showed HA-like immunoreactivity. HPLC studies revealed one of the highest HA concentrations in the animal kingdom, 6.49 +/- 1.36 nmole/mg protein, in the worm. The frog lung and blood contained very low concentrations of HA and could be excluded as sources for HA, while an enzyme assay revealed that the worm produces HA by decarboxylation of histidine. Thus, it is likely that H. cylindracea uses HA as a neurotransmitter or modulator.