Morphology and phylogenetic position of two novel marine gregarines (Apicomplexa,Eugregarinorida) from the intestines of North‐eastern Pacific ascidians

Eugregarine apicomplexans parasitize marine, freshwater and terrestrial invertebrates, and have lifecycles involving trophozoites (feeding stages) with complex morphologies and behaviour. The genus Lankesteria refers to marine aseptate eugregarines that parasitize ascidians. We described the surface ultrastructure of two new gregarine species, L. chelyosomae sp. n. and L. cystodytae sp. n. that inhabit the intestines of Chelyosoma columbianum and Cystodytes lobatus, respectively, collected from the North‐eastern Pacific Ocean. Apart from inhabiting different hosts and major differences in the cell size of L. chelyosomae sp. n. (mean length 182 µm) and L. cystodytae sp. n. (mean length 70 µm), the morphology of both gregarine species was quite similar. The trophozoites ranged from elliptoid to obdeltoid in shape and were brownish in colour. The nucleus was situated at the anterior end of the cell just behind a pointed mucron. A dense array of epicytic knobs was present over the entire surface of trophozoites in both species, and longitudinal epicytical folds were only weakly developed. We also sequenced the small subunit rDNA from the gregarines collected from both hosts, which supported the establishment of two new Lankesteria species. Phylogenetic analyses of the new DNA sequences and those derived from other alveolates, demonstrated that both new species clustered in a strongly supported clade consisting of other Lankesteria species, Lecudina species, and some environmental sequences. These morphological and molecular phylogenetic data suggested that improved knowledge of gregarine diversity could lead to the recognition of more than one distinct clade (genus) of gregarines within ascidian hosts.     

EARLY EVOLUTIONARY HISTORY OF DINOFLAGELLATES AND APICOMPLEXANS (ALVEOLATA) AS INFERRED FROM HSP90 AND ACTIN PHYLOGENIES1

Three extremely diverse groups of unicellular eukaryotes comprise the Alveolata: ciliates, dinoflagellates, and apicomplexans. The vast phenotypic distances between the three groups along with the enigmatic distribution of plastids and the economic and medical importance of several representative species (e.g. Plasmodium, Toxoplasma, Perkinsus, and Pfiesteria) have stimulated a great deal of speculation on the early evolutionary history of alveolates. A robust phylogenetic framework for alveolate diversity will provide the context necessary for understanding the basic biological properties of the group and for developing appropriate strategies for management. We addressed the earliest stages of alveolate evolution by sequencing heat shock protein 90 (hsp90) genes from several ciliates, apicomplexans, and dinoflagellates, including key species thought to represent early diverging lineages: Oxyrrhis marina, Perkinsus marinus, Cryptosporidium parvum, and the eugregarine Monocystis agilis. Moreover, by sequencing the actin gene from Monocystis, we were able to examine the sister relationship between gregarines and cryptosporidians with a three‐protein concatenated data set (hsp90, actin, and β‐tubulin). Phylogenetic analyses of the hsp90 data set provided a robust topology for alveolate relationships: Alveolates were monophyletic and apicomplexans and dinoflagellates formed sister groups to the exclusion of ciliates. Oxyrrhis formed the earliest diverging sister lineage to the “core” dinoflagellates, and Perkinsus formed the earliest diverging sister lineage to the Oxyrrhis–dinoflagellate clade. This topology was strongly supported inall analyses and by a unique indel shared by Oxyrrhis and dinoflagellates. A sister relationship between Cryptosporidium and Monocystis was weakly supported by the hsp90 data set but strongly supported by the three‐protein concatenated data set.     

Comparative morphology of the euglenid pellicle. II. Diversity of strip substructure

The morphological diversity associated with the strip substructure of the euglenid pellicle was examined, and after identifying characters and states, we outlined hypotheses about their evolution. We have attempted to standardize terms necessary for analytical comparisons of strips by providing a glossary and comparing published synonyms. Most of the substructural diversity found in euglenids is demonstrated with 13 representative taxa. Strips are generally composed of two subcomponents: frames and projections. Frames support the basic shape of strips and many can be described as either S-shaped, plateau-shaped, M-shaped, or A-shaped. Projections branch laterally from the frames, are usually periodic, and can be described as thread-like structures, an indented plate, tooth-like structures, and plate-like structures. The ancestral state included strips that were few in number, flat, and fused. The strips became S-shaped and disjoined in the lineage leading to most euglenid taxa. These strips became secondarily flattened and fused in one lineage. In some lineages of phototrophs, the strips became increasingly robust. Two strips of different morphology formed the repeating pellicular unit or doublet in four taxa. These doublets evolved convergently at least three times and may provide insights into developmental patterns of the cytoskeleton.     

A screen for mutations in human homologues of mice exencephaly genes Tfap2alpha and Msx2 in patients with neural tube defects

BACKGROUND: Very little is known about the identity of genetic factors involved in the complex etiology of nonsyndromic neural tube defects (NTD). Potential susceptibility genes have emerged from the vast number of mutant mouse strains displaying NTD. Reasonable candidates are the human homologues of mice exencephaly genes Tfap2alpha and Msx2, which are expressed in the developing neural tube. METHODS: A single-strand conformation analysis (SSCA) mutation screen of the coding sequences of TFAP2alpha and MSX2 was performed for 204 nonsyndromic NTD patients including cases of anencephaly (n = 10), encephalocele (n = 8), and spina bifida aperta, SBA (n = 183). A selected number of SBA patients was additionally tested for specific mutations in MTHFD, FRalpha, and PAX1 already shown to be related to NTD. RESULTS: Two TFAP2alpha point mutations in individual SBA patients were silent on the amino acid level (C308C, T396T). On nucleic acid level, these mutations change evolutionary conserved codons and thus may influence mRNA processing and translation efficiency. One SBA patient displayed an exonic 9-bp deletion in MSX2 leading to a shortened and possibly less functional protein. None of these mutations was found in 222 controls. Seven polymorphisms detected in TFAP2alpha and MSX2 were equally distributed in patients and controls. Patients with combined heterozygosity of an exonic MSX2 and an intronic TFAP2alpha polymorphism were at a slightly increased risk of NTD (OR 1.71; 95% CI 0.57-5.39). CONCLUSIONS: Although several new genetic variants were found in TFAP2 and MSX2, no statistically significant association was found between NTD cases and the new alleles or their combinations. Further studies are necessary to finally decide if these gene variants may have acted as susceptibility factors in our individual cases.     

Amotin and amoenin,two sesquiterpenes of the picrotoxane group from Dendrobium amoenum

Two new sesquiterpenes, amotin and amoenin, of the picrotoxane group were isolated from Dendrobium amoenum. The constitution of amoenin was evident from spectral investigations and by its conversion into α-dihydro-picrotoxinin on oxidation with oxygen in the presence of platinum. The constitution of amotin was shown by its spectral properties and the conversion of aduncin into amotin by hydrogenolysis of its epoxide ring. That the configuration at C-4 in aduncin and amotin is R was supported by circular dichroism measurements on some derivatives of picrotoxinin and aduncin.     

Molecular Phylogeny of Marine Gregarine Parasites (Apicomplexa) from Tube‐forming Polychaetes (Sabellariidae,Cirratulidae, and Serpulidae), Including Descriptions of Two New Species of Selenidium

Selenidium is a genus of gregarine parasites that infect the intestines of marine invertebrates and have morphological, ecological, and motility traits inferred to reflect the early evolutionary history of apicomplexans. Because the overall diversity and phylogenetic position(s) of these species remain poorly understood, we performed a species discovery survey of Selenidium from tube‐forming polychaetes. This survey uncovered five different morphotypes of trophozoites (feeding stages) living within the intestines of three different polychaete hosts. We acquired small subunit (SSU) rDNA sequences from single‐cell (trophozoite) isolates, representing all five morphotypes that were also imaged with light and scanning electron microscopy. The combination of molecular, ecological, and morphological data provided evidence for four novel species of Selenidium, two of which were established in this study: Selenidium neosabellariae n. sp. and Selenidium sensimae n. sp. The trophozoites of these species differed from one another in the overall shape of the cell, the specific shape of the posterior end, the number and form of longitudinal striations, the presence/absence of transverse striations, and the position and shape of the nucleus. A fifth morphotype of Selenidium, isolated from the tube worm Dodecaceria concharum, was inferred to have been previously described as Selenidium cf. echinatum, based on general trophozoite morphology and host association. Phylogenetic analyses of the SSU rDNA sequences resulted in a robust clade of Selenidium species collected from tube‐forming polychaetes, consisting of the two new species, the two additional morphotypes, S. cf. echinatum, and four previously described species (Selenidium serpulae, Selenidium boccardiellae, Selenidium idanthyrsae, and Selenidium cf. mesnili). Genetic distances between the SSU rDNA sequences in this clade distinguished closely related and potential cryptic species of Selenidium that were otherwise very similar in trophozoite morphology.     

Species boundaries in gregarine apicomplexan parasites: a case study-comparison of morphometric and molecular variability in Lecudina cf. tuzetae (Eugregarinorida, Lecudinidae)

Trophozoites of gregarine apicomplexans are large feeding cells with diverse morphologies that have played a prominent role in gregarine systematics. The range of variability in trophozoite shapes and sizes can be very high even within a single species depending on developmental stages and host environmental conditions; this makes the delimitation of different species of gregarines based on morphological criteria alone very difficult. Accordingly, comparisons of morphological variability and molecular variability in gregarines are necessary to provide a pragmatic framework for establishing species boundaries within this diverse and poorly understood group of parasites. We investigated the morphological and molecular variability present in the gregarine Lecudina cf. tuzetae from the intestines of Nereis vexillosa (Polychaeta) collected in two different locations in Canada. Three distinct morphotypes of trophozoites were identified and the small subunit (SSU) rDNA was sequenced either from multicell isolates of the same morphotype or from single cells. The aim of this investigation was to determine whether the different morphotypes and localities reflected phylogenetic relatedness as inferred from the SSU rDNA sequence data. Phylogenetic analyses of the SSU rDNA demonstrated that the new sequences did not cluster according to morphotype or locality and instead were intermingled within a strongly supported clade. A comparison of 1,657 bp from 45 new sequences demonstrated divergences between 0% and 3.9%. These data suggest that it is necessary to acquire both morphological and molecular data in order to effectively delimit the "clouds" of variation associated with each gregarine species and to unambiguously reidentify these species in the future.     

Development of a new version of the Liverpool Malaria Model. II. Calibration and validation for West Africa

Mathematical models have been used to provide an explicit framework for understanding malaria transmission dynamics in human population for over 100 years. With the disease still thriving and threatening to be a major source of death and disability due to changed environmental and socio-economic conditions, it is necessary to make a critical assessment of the existing models, and study their evolution and efficacy in describing the host-parasite biology. In this article, starting from the basic Ross model, the key mathematical models and their underlying features, based on their specific contributions in the understanding of spread and transmission of malaria have been discussed. The first aim of this article is to develop, starting from the basic models, a hierarchical structure of a range of deterministic models of different levels of complexity. The second objective is to elaborate, using some of the representative mathematical models, the evolution of modelling strategies to describe malaria incidence by including the critical features of host-vector-parasite interactions. Emphasis is more on the evolution of the deterministic differential equation based epidemiological compartment models with a brief discussion on data based statistical models. In this comprehensive survey, the approach has been to summarize the modelling activity in this area so that it helps reach a wider range of researchers working on epidemiology, transmission, and other aspects of malaria. This may facilitate the mathematicians to further develop suitable models in this direction relevant to the present scenario, and help the biologists and public health personnel to adopt better understanding of the modelling strategies to control the disease