Plasmodial Stage Research Articles

Ternary structure of Plasmodium vivaxN-myristoyltransferase with myristoyl-CoA and inhibitor IMP-0001173.

Plasmodium vivax is a major cause of malaria, which poses an increased health burden on approximately one third of the world's population due to climate change. Primaquine, the preferred treatment for P. vivax malaria, is contraindicated in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, a common genetic cause of hemolytic anemia, that affects ∼2.5% of the world's population and ∼8% of the population in areas of the world where P. vivax malaria is endemic. The Seattle Structural Genomics Center for Infectious Disease (SSGCID) conducted a structure-function analysis of P.vivax N-myristoyltransferase (PvNMT) as part of efforts to develop alternative malaria drugs. PvNMT catalyzes the attachment of myristate to the N-terminal glycine of many proteins, and this critical post-translational modification is required for the survival of P. vivax. The first step is the formation of aPvNMT-myristoyl-CoA binary complex that can bind to peptides. Understanding how inhibitors prevent protein binding will facilitate the development of PvNMT as a viable drug target. NMTs are secreted in all life stages of malarial parasites, making them attractive targets, unlike current antimalarials that are only effective during the plasmodial erythrocytic stages. The 2.3 Å resolution crystal structure of the ternary complex of PvNMT with myristoyl-CoA and a novel inhibitor is reported. One asymmetric unit contains two monomers. The structure reveals notable differences between the PvNMT and human enzymes and similarities to other plasmodial NMTs that can be exploited to develop new antimalarials.

Killing of Plasmodium Sporozoites by Basic Amphipathic α-Helical Fusion Peptides.

Membranolytic molecules constitute the first line of innate immune defense against pathogenic microorganisms. Plasmodium sporozoites are potentially exposed to these cytotoxic molecules in the hemolymph and salivary glands of mosquitoes, as well as in the skin, blood, and liver of the mammalian host. Here, we show that sporozoites are resistant to bacteriolytic concentration of cecropin B, a cationic amphipathic antimicrobial insect peptide. Intriguingly, anti-tumoral cell-penetrating peptides derived from the anti-apoptotic protein AAC11 killed P. berghei and P. falciparum sporozoites. Using dynamic imaging, we demonstrated that the most cytotoxic peptide, called RT39, did not significantly inhibit the sporozoite motility until the occurrence of a fast permeabilization of the parasite membrane by the peptide. Concomitantly, the cytosolic fluorescent protein constitutively expressed by sporozoites leaked from the treated parasite body while To-Pro 3 and FITC-labeled RT39 internalized, respectively, binding to the nucleic acids and membranes of sporozoites. This led to an increase in the parasite granularity as assessed by flow cytometry. Most permeabilization events started at the parasite's posterior end, resulting in the appearance of a fluorescent dot in the anterior part of sporozoites. Understanding and exploiting the susceptibility of sporozoites and other plasmodial stages to membranolytic molecules might foster strategies to eliminate the parasite and block its transmission.

RNA-seq analysis of parasitism by Intoshia linei (Orthonectida) reveals protein effectors of defence, communication, feeding and growth.

Orthonectida is a group of multicellular endoparasites of a wide range of marine invertebrates. Their parasitic stage is a multinuclear shapeless plasmodium infiltrating host tissues. The development of the following worm-like sexual generation takes place within the cytoplasm of the plasmodium. The existence of the plasmodial stage and the development of a sexual stage within the plasmodium are unique features to Bilateria. However, the molecular mechanisms that maintain this peculiar organism, and hence enable parasitism in orthonectids, are unknown. Here, we present the first-ever RNA-seq analysis of the plasmodium, aimed at the identification and characterization of the plasmodium-specific protein-coding genes and corresponding hypothetical proteins that distinguish the parasitic plasmodium stage from the sexual stage of the orthonectid Intoshia linei Giard, 1877,parasite of nemertean Lineus ruber Müller, 1774.We discovered 119 plasmodium-specific proteins, 82 of which have inferred functions based on known domains. Thirty-five of the detected proteins are orphans, at least part of which may reflect the unique evolutionary adaptations of orthonectids to parasitism. Some of the identified proteins are known effector molecules of other endoparasites suggesting convergence. Our data indicate that the plasmodium-specific proteins might be involved in the plasmodium defense against the host, host-parasite communication, feeding and nutrient uptake, growth within the host, and support of the sexual stage development. These molecular processes in orthonectids have not been described before, and the particular protein effectors remained unknown until now.

First characterization of the parasite Haplosporidium costale in France and development of a real-time PCR assay for its rapid detection in the Pacific oyster, Crassostrea gigas.

The Pacific cupped oyster Crassostrea gigas is one of the most 'globalized' marine invertebrates and its production is predominant in many parts of the world including Europe. However, it is threatened by mortality events associated with pathogenic microorganisms such as the virus OsHV-1 and the bacteria Vibrio aestuarianus. C. gigas is also a host for protozoan parasites including haplosporidians. In contrast with Haplosporidium nelsoni previously detected in Europe, H. costale was considered exotic although its presence in French oysters was suggested in the 1980s based on ultrastructural examination. Here, a combination of light and transmission electron microscopy, PCR and sequencing allowed characterizing the presence of the parasite in the context of low mortality events which occurred in 2019 in France. Histological observation revealed the presence of uninucleated, plasmodial and spore stages within the connective tissues of some oysters. Ultrastructural features were similar to H. costale ones in particular the presence of axe-shaped haplosporosomes in spore cytoplasms. Three fragments of the genome including partial small subunit rRNA gene, the ITS-1, 5.8S and ITS-2 array and part of the actin gene were successfully sequenced and grouped with H. costale homologous sequences. This is the first time that the presence of H. costale was confirmed in C. gigas in France. Furthermore, a TaqMan real-time PCR assay was developed and validated [DSe=92.6% (78.2-99.8) and DSp=95.5% (92.3-98.6)] to enable the rapid and specific detection of the parasite. The application of the PCR assay on archived samples revealed that the parasite has been present in French oyster populations at least since 2008. Considering the little information available on this parasite, the newly developed TaqMan assay will be very helpful to investigate the temporal and geographic distribution and the life cycle of the parasite in France and more generally in C. gigas geographic range.

Molecular diagnostic based on 18S rDNA and supplemental taxonomic data of the cnidarian coelozoic Ceratomyxa (Cnidaria, Myxosporea) and comments on the intraspecific morphological variation

Ceratomyxa amazonensis is a cnidarian myxosporean originally described with strongly arcuate crescent-shaped myxospores, absence of vegetative stages and infecting Symphysodon discus, an important Amazonian ornamental fish in the aquarium industry. As part of a long-term investigation concerning myxosporeans that infect discus fish Symphysodon spp. from different rivers of the Amazon Basin, thirty specimens of S. discus collected from Unini River were examined. Plasmodial vegetative stages therefrom were found freely floating in the bile of gall bladders from eighteen fish. Mature myxospores were slightly crescent-shaped, measuring 4.72 ± 0.1 (4.52–4.81) μm in length, 24.2 ± 0.4 (23.9–25.3) μm in thickness with polar capsules 2.31 ± 0.1 (2.29–2.33) μm in length and 2.15 ± 0.1 (2.13–2.17) μm in width. Strong morphological differences were observed between the newly isolated myxospores obtained and the previously described C. amazonensis; however, molecular assessment, based on 18S rDNA, revealed a high similarity (99.91%), with only a single nucleotide base change. This study provides new data, expanding the original description of the species with a discussion on differences in myxospore-morphology in the context of intraspecific morphological plasticity.

Morphological and molecular characterization of Ceratomyxa xanthopteri n. sp. (Myxosporea: Ceratomyxidae) from the marine ornamental fish Acanthurus xanthopterus Valenciennes 1835 (Acanthuridae) off Vizhinjam coast, Kerala.

A new species of Ceratomyxa infecting the gallbladder of the marine ornamental fish Acanthurus xanthopterus collected from the Vizhinjam coast of Kerala is described. The parasite exhibited a prevalence of 100%. Mature spores recovered from the gallbladder were slightly crescentic with rounded lateral extremities and possessed convex anterior and slightly concave to straight posterior margins. Spore valves two, equal, joined by a straight and prominent suture. Myxospores measured 5.5 ± 0.6 μm in length and 15.9 ± 2.3 μm in thickness. Polar capsules two, equal, spherical, positioned anteriorly on either sides of the suture, 2.3 ± 0.2 μm long and 2.2 ± 0.2 μm wide. Polar filament with four to five coils, 21.2 ± 0.6 μm when extruded. Posterior angle 173.6 ± 5.2°. Early sporogonic stages and monosporic, disporic, and multisporic plasmodial stages were spherical to irregular in shape, with or without filopodia. Histopathologic analysis revealed that spores and developing stages were attached to the gallbladder wall as well as found free in the lumen. Morphologic and morphometric comparison of the present parasite with known species of Ceratomyxa indicated significant differences. In molecular and phylogenetic analyses, the present myxosporean revealed high divergence with related forms and occupied an independent position within the Ceratomyxa clade with high nodal support. Considering the morphological, morphometric, molecular, and phylogenetic dissimilarities with the previously described species of Ceratomyxa and the differences in host and geographic locations, the present species of myxosporean is treated as new and is named Ceratomyxa xanthopteri n. sp.

A novel myxozoan parasite, Ellipsomyxa boleophthalmi sp. nov. (Myxozoa: Ceratomyxidae) in the brackishwater fish, Boleophthalmus dussumieri Valenciennes, 1837 (Perciformes: Gobiidae) from India.

A novel myxozoan parasite is identified and described from mudskipper, Boleophthalmus dussumieri, collected from a brackishwater ecosystem in Maharashtra, India. Ellipsomyxa boleophthalmi sp. nov. was found in the gallbladder of 58 of 60 fish examined (96.7%). The parasite formed disporous plasmodia that varied in size and shape, and the thin-walled, ellipsoidal and elongated myxospores measured 9.0-10.7 × 6.0-7.8 μm. The two, spherical polar capsules measured 2.7 μm in diameter and enclosed 3-4 coils of polar tubules. Histological observations of infected gallbladder revealed the attachment of disporous plasmodial stages of the parasite to the gallbladder wall with fine pseudopodia. Under the scanning electron microscope (SEM), the myxospores showed a distinct central sutural line and two distinct depressions on the opposite sides at the openings of polar capsules. SEM also revealed the engulfment of microvilli of gallbladder wall by pseudopodia of the plasmodial stages. Analysis of the partial fragment of the SSU rDNA region (1386 bp) showed less than 98% sequence similarity with the other reported Ellipsomyxa spp. In the phylogenetic tree, the present species formed as a distinct subclade within the major clade of Ellipsomyxa spp. The unique morphological and morphometric features of the myxospore, together with the molecular analysis, allowed us to conclude that the present myxozoan is a new species and is named Ellipsomyxa boleophthalmi sp. nov., after the generic name of the host. This is the first report on the occurrence of the genus Ellipsomyxa in B. dussumieri.

Transmission-blocking compound candidates against Plasmodium vivax using P. berghei as an initial screening.

BACKGROUND Different strategies for improvement of malaria control and elimination are based on the blockage of malaria parasite transmission to the mosquito vector. These strategies include the drugs that target the plasmodial sexual stages in humans and the early developmental stages inside mosquitoes.OBJECTIVES Here we tested Malaria Box compounds in order to evaluate their activity against male and female gametocytes in Plasmodium berghei, mosquito infection in P. vivax and ookinete formation in both species.METHODS/FINDINGS The membrane feeding assay and the development of ookinetes by a 24 h ex vivo culture and the ookinete yield per 1000 erythrocytes were used to test transmission-blocking potential of the Malaria Box compounds in P. vivax. For P. berghei we used flow cytometry to evaluate male and female gametocyte time course and fluorescence microscopy to check the ookinete development. The two species used in this study showed similar results concerning the compounds’ activity against gametocytes and ookinetes, which were different from those in P. falciparum. In addition, from the eight Malaria Box compounds tested in both species, compounds MMV665830, MMV665878 and MMV665941 were selected as a hit compounds due the high inhibition observed.CONCLUSION Our results showed that P. berghei is suitable as an initial screening system to test compounds against P. vivax.

Ellipsomyxa ariusi sp. nov. (Myxosporea: Ceratomyxidae), a new myxosporean infecting the gallbladder of threadfin sea catfish Arius arius in India.

The present study describes a new species of myxosporean, Ellipsomyxa ariusi sp. nov., infecting the gallbladder of the threadfin sea catfish Arius arius (Hamilton, 1822). E. ariusi sp. nov. is characterized by bivalvular, ellipsoid or elongate-oval myxospores with smooth spore valves and a straight suture, arranged at an angle to the longitudinal spore axis. Mature myxospores measured 10.1 ± 0.8 µm in length, 6.8 ± 0.5 µm in width and 7.7 ± 0.7 µm in thickness. Polar capsules are equal in size and oval to pyriform in shape. They are positioned at an angle to the longitudinal myxospore axis and open in opposite directions. Polar capsules measured 2.8 ± 0.3 µm in length and 2.5 ± 0.4 µm in width; polar filaments formed 4-5 coils, and extended to 32.2 ± 2.1 µm in length. Monosporic and disporic plasmodial stages attached to the wall of gallbladder. Molecular analysis of the type specimen generated a 1703 bp partial SSU rDNA sequence (MN892546), which was identical to the isolates from 3 other locations. In phylogenetic analyses, genus Ellipsomyxa appeared monophyletic and E. ariusi sp. nov. occupied an independent position in maximum likelihood and Bayesian inference trees with high bootstrap values. The overall prevalence of infection was 54.8% and multiway ANOVA revealed that it varied significantly with location, year, season, sex and size of the fish host. Histopathological changes associated with E. ariusi sp. nov. infection included swelling, vacuolation and detachment of epithelial layer, reduced mucus production and altered consistency and colour of bile. Based on the morphologic, morphometric and molecular differences with known species of Ellipsomyxa, and considering differences in host and geographic locations, the present species is treated as new and the name Ellipsomyxa ariusi sp. nov. is proposed.

Haplosporosomes, sporoplasmosomes and their putative taxonomic relationships in rhizarians and myxozoans.

This paper reviews current knowledge of the structure, genesis, cytochemistry and putative functions of the haplosporosomes of haplosporidians (Urosporidium, Haplosporidium, Bonamia, Minchinia) and paramyxids (Paramyxa, Paramyxoides, Marteilia, Marteilioides, Paramarteilia), and the sporoplasmosomes of myxozoans (Myxozoa - Malacosporea, Myxosporea). In all 3 groups, these bodies occur in plasmodial trophic stages, disappear at the onset of sporogony, and reappear in the spore. Some haplosporidian haplosporosomes lack the internal membrane regarded as characteristic of these bodies and that phylum. Haplosporidian haplosporogenesis is through the Golgi (spherulosome in the spore), either to form haplosporosomes at the trans-Golgi network, or for the Golgi to produce formative bodies from which membranous vesicles bud, thus acquiring the external membrane. The former method also forms sporoplasmosomes in malacosporeans, while the latter is the common method of haplosporogenesis in paramyxids. Sporoplasmogenesis in myxosporeans is largely unknown. The haplosporosomes of Haplosporidium nelsoni and sporoplasmosomes of malacosporeans are similar in arraying themselves beneath the plasmodial plasma membrane with their internal membranes pointing to the exterior, possibly to secrete their contents to lyse host cells or repel haemocytes. It is concluded that these bodies are probably multifunctional within and between groups, their internal membranes separating different functional compartments, and their origin may be from common ancestors in the Neoproterozoic.

Ortholinea scatophagi (Myxosporea: Ortholineidae), a novel myxosporean infecting the spotted scat, Scatophagus argus (Linnaeus 1766) from southwest coast of India

A new species of myxosporean, Ortholinea scatophagi n. sp. infecting the urinary bladder of the spotted scat, Scatophagus argus (Linnaeus 1766) is described. O. scatophagi n. sp. is characterized by spherical myxospores with a slightly flattened anterior end and equal spore valves with extra sutural ridges on its surface; measured 7.34 ± 0.67 μm in length, 6.90 ± 0.71 μm in width and 6.48 ± 0.37 μm in thickness. Two polar capsules, equal, spherical to oval in shape, arranged diametrically opposite and measured 2.59 ± 0.42 μm in length and 2.24 ± 0.35 μm in width. Polar filaments, 21.84 ± 2.86 μm long, with four to five coils. Scanning electron microscopy revealed the presence of extra sutural ridges on spore surface. Pansporoblasts spherical to irregular in shape, measured 31.08 ± 2.67 μm in length and 13.88 ± 5.40 μm in width; Monosporic, disporic and polysporic plasmodial stages were observed; plasmodia spherical or irregular in shape with granular cytoplasm containing refractile granules. The species was compared with 23 existing nominal species of Ortholinea, based on morphology and morphometry. Molecular analysis resulted in a 1773 bp long SSU rDNA sequence (GenBank accession number MN 310514). In phylogenetic analyses the present parasite clustered with other members of Ortholinea, under the freshwater urinary clade. Considering the morphologic, morphometric and molecular differences with previously described species of Ortholinea, and differences in host and geographic locations, the present species is treated as new and the name Ortholinea scatophagi n. sp.is proposed.

The molecular machinery of translational control in malaria parasites.

Translational control regulates the levels of protein synthesized from its transcript and is key for the rapid adjustment of gene expression in response to environmental stimuli. The regulation of translation is of special importance for malaria parasites, which pass through a complex life cycle that includes various replication phases in the different organs of the human and mosquito hosts and a sexual reproduction phase in the mosquito midgut. In particular, the quiescent transmission stages rely on translational control to rapidly adapt to the new environment, once they switch over from the human to the mosquito and vice versa. Three control mechanisms are currently proposed in Plasmodium, (1) global regulation that acts on the translation initiation complex; (2) mRNA-specific regulation, involving cis control elements, mRNA-binding proteins and translational repressors; and (3) induced mRNA decay by the Ccr4-Not and the RNA exosome complex. The main molecules controlling translation are highly conserved in malaria parasites and an increasing number of studies shed light on the interwoven pathways leading to the up or downregulation of protein synthesis in the diverse plasmodial stages. We here highlight recent findings on translational control during life cycle progression of Plasmodium and discuss the molecules involved in regulating protein synthesis.

Transcript and protein expression analysis of proteases in the blood stages of Plasmodium falciparum

Proteases are crucial enzymes with varying roles in living organisms. In the malaria parasite Plasmodium falciparum, the role of proteases has been deciphered mainly in the asexual blood stages and shown to represent promising drug targets. However, little is known about their functions in the sexual blood stages, which are important for transmission of the disease from the human to the mosquito vector. Determination of their stage-specific expression during the malaria life-cycle is crucial for the effective design of multi-stage anti-malaria drugs aimed at eradicating the disease. In this study, we screened the P. falciparum genome database for putative proteases and determined the transcript and protein expression profiles of selected proteases in the plasmodial blood stages using semi-quantitative RT-PCR and indirect immunofluorescence assay. Database mining identified a total of 148 putative proteases, out of which 18 were demonstrated to be expressed in the blood stages on the transcript level; for 12 of these proteins synthesis was confirmed. While three of these proteases exhibit gametocyte-specific expression, two are restricted to the asexual blood stages and seven are found in both stages, making them interesting multi-stage drug targets.

The Life Cycle of Didymium laxifilum and Physarum album on Oat Agar Culture.

The plasmodial slime molds is the largest group in the phylum Amoebozoa. Its life cycle includes the plasmodial trophic stage and the spore-bearing fruiting bodies. However, only a few species have their complete life cycle known in details so far. This study is the first reporting the morphogenesis of Didymium laxifilum and Physarum album. Spores, from field-collected sporangia, were incubated into hanging drop cultures for viewing germination and axenic oat agar plates for viewing plasmodial development and sporulation. The spores of D.laxifilum and P.album germinated by method of V-shape split and minute pore, respectively. The amoeboflagellates, released from spores, were observed in water film. The phaneroplasmodia of two species developed into a number of sporangia by subhypothallic type on oat agar culture. The main interspecific difference of morphogenesis was also discussed.

Mycosphere Essays 3. Myxomycete spore and amoeboflagellate biology: a review

The spore and amoeboflagellate stages of the heterothallic myxomycete life cycle are generally a uninucleate haploid alternative to the multinucleate diploid plasmodial stage. The spore is produced in a sporangium which develops from the plasmodium, and is a dispersal and resistant stage which has ornamentations that can be used in taxonomy. These small spores have thick pigmented walls and are generally wind dispersed and require water and other biological and physical conditions in order to germinate; this dispersal can occur over long distances and the germination conditions apparently vary between and within a species. The amoeboflagellate stage has three alternative phases: myxamoeba, swarm cell and cyst. The myxamoeba is a typical pleomorphic amoeboid cell that is the major phase, since it is the vegetative phase that ingests bacteria and yeasts, and divides mitotically to form a clonal population of cells. The myxamoeba of heterothallic strains can also, after reaching a certain cell density, become sexually competent and fuse to produce the plasmodial stage. The swarm cell phase develops from the myxamoebal stage when free water is present; it also feeds on bacteria but does not divide unless it reconverts to the myxamoebal phase. The cyst phase develops from the myxamoebal stage, when growth conditions become adverse, by the condensing of the cell and the formation of a resistant wall.

Plant hairy root cultures as plasmodium modulators of the slime mold emergent computing substrate Physarum polycephalum.

Roots of the medicinal plant Valeriana officinalis are well-studied for their various biological activities. We applied genetically transformed V. officinalis root biomass to exert control of Physarum polycephalum, an amoeba-based emergent computing substrate. The plasmodial stage of the P. polycephalum life cycle constitutes a single, multinucleate cell visible by unaided eye. The plasmodium modifies its network of oscillating protoplasm in response to spatial configurations of attractants and repellents, a behavior that is interpreted as biological computation. To program the computing behavior of P. polycephalum, a diverse and sustainable library of plasmodium modulators is required. Hairy roots produced by genetic transformation with Agrobacterium rhizogenes are a metabolically stable source of bioactive compounds. Adventitious roots were induced on in vitro V. officinalis plants following infection with A. rhizogenes. A single hairy root clone was selected for massive propagation and the biomass was characterized in P. polycephalum chemotaxis, maze-solving, and electrical activity assays. The Agrobacterium-derived roots of V. officinalis elicited a positive chemotactic response and augmented maze-solving behavior. In a simple plasmodium circuit, introduction of hairy root biomass stimulated the oscillation patterns of slime mold's surface electrical activity. We propose that manipulation of P. polycephalum with the plant root culture platform can be applied to the development of slime mold microfluidic devices as well as future models for engineering the plant rhizosphere.

Representation of shape mediated by environmental stimuli in Physarum polycephalum and a multi-agent model

The slime mould Physarum polycephalum is known to construct protoplasmic transport networks which approximate proximity graphs by foraging for nutrients during its plasmodial life cycle stage. In these networks, nodes are represented by nutrients and edges are represented by protoplasmic tubes. These networks have been shown to be efficient in terms of length and resilience of the overall network to random damage. However, relatively little research has been performed in the potential for Physarum transport networks to approximate the overall shape of a data-set. In this paper we distinguish between connectivity and shape of a planar point data-set and demonstrate, using scoping experiments with plasmodia of P. polycephalum and a multi-agent model of the organism, how we can generate representations of the external and internal shapes of a set of points. As with proximity graphs formed by P. polycephalum, the behaviour of the plasmodium (real and model) is mediated by environmental stimuli. We further explore potential morphological computation approaches with the multi-agent model, presenting methods which approximate the Convex Hull and the Concave Hull. We demonstrate how a growth parameter in the model can be used to transition between Convex and Concave Hulls. These results suggest novel mechanisms of morphological computation mediated by environmental stimuli.

Antigen-driven focal inflammatory death of malaria liver stages.

Multiple immunizations using live irradiated sporozoites, the infectious plasmodial stage delivered into the host skin during a mosquito bite, can elicit sterile immunity to malaria. CD8+ T cells seem to play an essential role in this protective immunity, since their depletion consistently abolishes sterilizing protection in several experimental models. So far, only a few parasite antigens are known to induce CD8+ T cell-dependent protection, but none of them can reach the levels of protection afforded by live attenuated parasites. Systematic attempts to identify novel antigens associated with this efficient cellular protection were so far unsuccessful. In addition, the precise mechanisms involved in the recognition and elimination of parasitized hepatocytes in vivo by CD8+ T cells still remain obscure. Recently, it has been shown that specific effector CD8+ T cells, after recognition of parasitized hepatocytes, recruit specific and non-specific activated CD8+ T cells to the site of infection, resulting in the formation of cellular clusters around and in the further elimination of intracellular parasites. The significance of this finding is discussed in the perspective of a general mechanism of antigen-dependent focalized inflammation and its consequences for the elimination of malaria liver stages.

Conducting polymer-coated Physarum polycephalum towards the synthesis of bio-hybrid electronic devices

This paper presents a generic method for the production of functionalized coatings on biological substrates. The specific method described involves the functionalization of the living plasmodial stage of Physarum polycephalum with the conducting organic polymer polypyrrole. The simple method involves localized treatment of tube sections with a solution of ferric chloride, followed by exposure to the vapour or a liquid solution of the pyrrole monomer. This technique enables the production of surface-coated conducting plasmodial tubes of certain lengths to be formed at specific points. Measurement of the electrical resistance of a 1 cm functionalized tube gave a value of 100 k. The use of this selective functionalization technique means that the majority of the growing plasmodium remains unfunctionalized and living; thus, a true hybrid device is formed. It can be seen how a range of functionalized polymers and materials whereby a chemical activator, for the formation of the product (or the pre-cursor) can be added to P. polycephalum (or other organisms) followed by reaction to form a hybrid material.

In vitro cultivation of Hematodinium sp. isolated from Atlantic snow crab, Chionoecetes opilio: partial characterization of late developmental stages.

Hematodinium is a parasitic dinoflagellate of numerous crustacean species, including the economically important Atlantic snow crab, Chionoecetes opilio. The parasite was cultured in vitro in modified Nephrops medium at 0 °C and a partial characterization of the life stages was accomplished using light and transmission electron microscopy (TEM). In haemolymph from heavily infected snow crabs two life stages were detected; amoeboid trophonts and sporonts. During in vitro cultivation, several Hematodinium sp. life stages were observed: trophonts, clump colonies, sporonts, arachnoid sporonts, sporoblasts and dinospores. Cultures initiated with sporonts progressed to motile dinospores; however, those initiated with amoeboid trophonts proliferated, but did not progress or formed schizont-like stages which were senescent artefacts. Plasmodial stages were associated with both trophonts and sporonts and could be differentiated by the presence of trichocysts on TEM. Macrodinospores were observed but not microdinospores; likely due to the low number of Hematodinium sp. cultures that progressed to the dinospore stage. No early life stages including motile filamentous trophonts or gorgonlocks were observed as previously noted in Hematodinium spp. from other crustacean hosts. All Hematodinium sp. life stages contained autofluorescent, membrane-bound electron dense granules that appeared to degranulate or be expelled from the cell during in vitro cultivation.