Supercooling ability in two populations of the land snail Helix pomatia (Gastropoda: Helicidae) and ice-nucleating activity of gut bacteria

The land snail Helix pomatia (Gastropoda: Helicidae) is widely distributed in Northern and Central Europe where it may experience subzero temperatures during winter months. Its supercooling ability was studied in two populations of H. pomatia. One population originated from Southern Sweden (Gotaland) and the other from Central France (Auvergne). In the experimental design, they were acclimated, over 2 weeks, to artificial winter conditions (hibernation, T=5 degrees C). The Swedish snails showed a rather limited supercooling ability (temperature of crystallization, T(c)=-6.4+/-0.8 degrees C), significantly greater, however, than the supercooling capacity of the population from France (T(c)=-4.6+/-1.4 degrees C). In artificial spring conditions (3 months of hibernation followed by a progressive acclimation, over 2 weeks, to activity at T=20 degrees C), both populations exhibited a similar high T(c) (-2.0+/-1.0 degrees C). The lower T(c) of hibernating Swedish snails could be due to a greater loss of body water, accompanied by a higher concentration of solutes in the hemolymph. In both populations, the variation in hemolymph osmolality measured between hibernating (250-270 mOsm kg(-1)) and active (165-215 mOsm kg(-1)) snails may be explained by the variation in body water mass and did not suggest the production of colligative cryoprotectants. Moreover, the three bacterial strains, Buttiauxella sp., Kluyvera sp., and Tatumella sp. (Enterobacteriaceae) which were isolated from fed snails, but absent in starved snails, did not show any ice-nucleating activity at temperatures higher than -9 degrees C. Only the strain Kluyvera sp. initiated nucleation at -9 degrees C. This strain, therefore, is a weak, also termed a Type III or Class C ice-nucleating active bacterium, but with no influence on the supercooling ability of individual snails. In summary, fluctuations in body water mass of hibernating snail populations, triggering changes in osmolyte concentration, rather than the presence of endogenous ice-nucleating-active bacteria, accounts for fluctuations in their T(c).     

Flavonoids,benzoic acids and cinnamic acids isolated from shoots and roots of Italian rye grass (Lolium multiflorum Lam.) with and without endophyte association and arbuscular mycorrhizal fungus

Phenolic compounds present in Lolium multiflorum Lam. were isolated and characterized. Significant differences in their distribution were found, in shoots and roots of plants with (Lm+) and without (Lm−) endophyte association, grown with (Mic+) and without (Mic−) mycorrhizal fungi Glomus intraradices, indicating a systemic effect of these microorganisms on the phenolics metabolism of L. multiflorum.     

Sarcocystis neurona: molecular characterization of enolase domain I region and a comparison to other protozoa

Sarcocystis neurona causes protozoal myeloencephalitis and has the ability to infect a wide host range in contrast to other Sarcocystis species. In the current study, five S. neurona isolates from a variety of sources, three Sarcocystis falcatula, one Sarcocystis dasypi/S. neurona-like isolate, and one Besnoitia darlingi isolate were used to compare the enolase 2 gene segment containing the domain I region to previously sequenced enolase genes from Neospora caninum, Neospora hughesi, Toxoplasma gondii, Plasmodium falciparum, and Trypanosoma cruzi; enolase 2 segment containing domain I region is highly conserved amongst these parasites of veterinary and medical importance. Immunohistochemistry results indicates reactivity of T. gondii enolase 1 and 2 antibodies to S. neurona merozoites and metrocytes, but no reactivity of anti-enolase 1 to the S. neurona bradyzoite stage despite reactivity to T. gondii bradyzoites, suggesting expression differences between organisms.     

Description of Idiomarina insulisalsae sp. nov., isolated from the soil of a sea salt evaporation pond,proposal to transfer the species of the genus Pseudidiomarina to the genus Idiomarina and emended description of the genus Idiomarina

A halophilic, aerobic Gram-negative bacterium, designated strain CVS-6^T, was isolated from a sea salt evaporation pond on the Island of Sal in the Cape Verde Archipelago. Phylogenetic analysis of the 16S rRNA gene sequence revealed a clear affiliation of the organism with members of the family Idiomarinaceae. Sequence similarities between CVS-6^T and the type strains of the species of the genera Pseudidiomarina and Idiomarina ranged from 93.7% to 96.9%. The major isoprenoid quinone was ubiquinone 8 (Q-8). The major cellular fatty acids were 15:0 iso (21.8%), 17:0 iso (12.5%), 17:1 iso ω9c (10.7%), and 16:1 ω7c (10.6%). The DNA G+C content was 51.6 mol%. The species represented by strain CVS-6^T could be distinguished from the species of the genera Pseudidiomarina and Idiomarina; however, it was not possible to distinguish both genera from each other using the phenotypic or chemotaxonomic characteristics examined. Consequently, we propose that the species classified in the genus Pseudidiomarina should be transferred to the genus Idiomarina. We also propose that, on the basis of physiological and biochemical characteristics, strain CVS-6^T (=LMG 23123=CIP 108836) represents a new species which we name Idiomarina insulisalsae.     

Use of ribosomal DNA sequence data to characterize and detect a neogregarine pathogen of Solenopsis invicta (Hymenoptera: Formicidae)

A neogregarine parasite of the red imported fire ant, Solenopsis invicta, was discovered recently in Florida and tentatively placed in the Mattesia genus based on morphological characterization. S. invicta infected with this Mattesia species exhibited a characteristic yellowing of the cuticle which was designated Mattesia "yellow-head disease" (YHD). The 18S rRNA gene sequence from Mattesia YHD was elucidated and compared with the neogregarine pathogens, Mattesia geminata and Ophriocystis elektroscirrha. The sequence data support the previous conclusion that Mattesia YHD is a new species that infects S. invicta. Furthermore, high sequence identity between Mattesia YHD, M. geminata (95.7%), and O. elektroscirrha (86.2%) correctly place the YHD organism in the Mattesia genus and Neogregarinorida order. Oligonucleotide primer pairs were designed to unique areas of the 18S rRNA genes of Mattesia YHD and S. invicta. Multiplex PCR resulted in sensitive and specific detection of Mattesia YHD infection of S. invicta.     

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 26.87 h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3 weeks post inoculation. The highest level of spore production was at 4 weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34 ± 0.9 × 10⁶ spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.     

Liang Bua Homo floresiensis mandibles and mandibular teeth: a contribution to the comparative morphology of a new hominin species

In 2004, a new hominin species, Homo floresiensis, was described from Late Pleistocene cave deposits at Liang Bua, Flores. H. floresiensis was remarkable for its small body-size, endocranial volume in the chimpanzee range, limb proportions and skeletal robusticity similar to Pliocene Australopithecus, and a skeletal morphology with a distinctive combination of symplesiomorphic, derived, and unique traits. Critics of H. floresiensis as a novel species have argued that the Pleistocene skeletons from Liang Bua either fall within the range of living Australomelanesians, exhibit the attributes of growth disorders found in modern humans, or a combination of both. Here we describe the morphology of the LB1, LB2, and LB6 mandibles and mandibular teeth from Liang Bua. Morphological and metrical comparisons of the mandibles demonstrate that they share a distinctive suite of traits that place them outside both the H. sapiens and H. erectus ranges of variation. While having the derived molar size of later Homo, the symphyseal, corpus, ramus, and premolar morphologies share similarities with both Australopithecus and early Homo. When the mandibles are considered with the existing evidence for cranial and postcranial anatomy, limb proportions, and the functional anatomy of the wrist and shoulder, they are in many respects closer to African early Homo or Australopithecus than to later Homo. Taken together, this evidence suggests that the ancestors of H. floresiensis left Africa before the evolution of H. erectus, as defined by the Dmanisi and East African evidence.     

Response of predatory snails to a novel prey following the geographic range expansion of an intertidal barnacle

During a geographic range expansion, the success of a species colonizing a new region may be influenced by how resident predators respond to this novel prey. The volcano barnacle Tetraclita rubescens has recently expanded its range > 300 km northward along the coast of California, USA. In the historic portion of its range, Tetraclita is preyed upon by a southern dogwhelk, Nucella emarginata. In newly colonized northern habitats, Tetraclita has joined an assemblage of mid-intertidal barnacles preyed upon by two species of northern dogwhelks (N. ostrina and N. canaliculata). Using a combination of field enclosures and laboratory experiments, we tested the impacts of northern whelks on volcano barnacles relative to other barnacle species. In the field, juvenile Tetraclita were rarely consumed. In laboratory trials with larger barnacles, Tetraclita were nearly immune to drilling by northern whelks, perhaps due to superior morphological defenses. In all experiments, prior encounters with Tetraclita did not appear to increase the frequency of predation on this barnacle; northern whelk species rarely preyed on Tetraclita even when collected from a site where this barnacle was abundant. In contrast, the southern whelk N. emarginata readily preyed on Tetraclita in laboratory trials, perhaps reflecting a longer shared evolutionary history between these species. Overall, our results suggest that the success of a population established beyond a species' historical range boundary may be influenced by the functional traits of that species in relation to the composition and evolutionary history of the surrounding community.     

Phylogeny of Phaeomollisia piceae gen. sp. nov.: a dark, septate, conifer-needle endophyte and its relationships to Phialocephala and Acephala

Dark, septate endophytes (DSE) were isolated from roots and needles of dwarf Picea abies and from roots of Vaccinium spp. growing on a permafrost site in the Jura Mountains in Switzerland. Two of the isolates sporulated after incubation for more than one year at 4 °C. One of them was a hitherto undescribed helotialean ascomycete Phaeomollisia piceae gen. sp. nov., the other was a new species of Phialocephala, P. glacialis sp. nov. Both species are closely related to DSE of the Phialocephala fortinii s. lat.-Acephala applanata species complex (PAC) as revealed by phylogenetic analyses of the ITS and 18S rDNA regions. Morphologically dissimilar fungi, such as Vibrissea and Loramyces species, are phylogenetically also closely linked to the new species and the PAC. Cadophora lagerbergii and C. (Phialophora) botulispora are moved to Phialocephala because Phialocephala dimorphospora and P. repens are the closest relatives. Several Mollisia species were closely related to the new species and the PAC according to ITS sequence comparisons. One DSE from needles of Abies alba and one from shoots of Castanea sativa formed Cystodendron anamorphs in culture. Their identical 18S sequences and almost identical ITS sequences indicated Mollisia species as closest relatives, suggesting that Mollisia species are highly euryoecious.     

Resistance to antiparasitic drugs: the role of molecular diagnosis

Chemotherapy is central to the control of many parasite infections of both medical and veterinary importance. However, control has been compromised by the emergence of drug resistance in several important parasite species. Such parasites cover a broad phylogenetic range and include protozoa, helminths and arthropods. In order to achieve effective parasite control in the future, the recognition and diagnosis of resistance will be crucial. This demand for early, accurate diagnosis of resistance to specific drugs in different parasite species can potentially be met by modern molecular techniques. This paper summarises the resistance status of a range of important parasites and reviews the available molecular techniques for resistance diagnosis. Opportunities for applying successes in some species to other species where resistance is less well understood are explored. The practical application of molecular techniques and the impact of the technology on improving parasite control are discussed.     

Molecular phylogeny of Phoma and allied anamorph genera: Towards a reclassification of the Phoma complex

The present generic concept of Phoma is broadly defined, with nine sections being recognised based on morphological characters. Teleomorph states of Phoma have been described in the genera Didymella, Leptosphaeria, Pleospora and Mycosphaerella, indicating that Phoma anamorphs represent a polyphyletic group. In an attempt to delineate generic boundaries, representative strains of the various Phoma sections and allied coelomycetous genera were included for study. Sequence data of the 18S nrDNA (SSU) and the 28S nrDNA (LSU) regions of 18 Phoma strains included were compared with those of representative strains of 39 allied anamorph genera, including Ascochyta, Coniothyrium, Deuterophoma, Microsphaeropsis, Pleurophoma, Pyrenochaeta, and 11 teleomorph genera. The type species of the Phoma sections Phoma, Phyllostictoides, Sclerophomella, Macrospora and Peyronellaea grouped in a subclade in the Pleosporales with the type species of Ascochyta and Microsphaeropsis. The new family Didymellaceae is proposed to accommodate these Phoma sections and related anamorph genera. The present study demonstrated that Phoma radicina, the type species of Phoma sect. Paraphoma and Phoma heteromorphospora, the type species of Phoma sect. Heterospora can be assigned to the Phaeosphaeriaceae and Leptosphaeriaceae respectively.     

Vascular Streak Dieback of cacao in Southeast Asia and Melanesia: in planta detection of the pathogen and a new taxonomy

Vascular Streak Dieback (VSD) disease of cacao (Theobroma cacao) in Southeast Asia and Melanesia is caused by a basidiomycete (Ceratobasidiales) fungus Oncobasidium theobromae (syn. =Thanatephorus theobromae). The most characteristic symptoms of the disease are green-spotted leaf chlorosis or, commonly since about 2004, necrotic blotches, followed by senescence of leaves beginning on the second or third flush behind the shoot apex, and blackening of infected xylem in the vascular traces at the leaf scars resulting from the abscission of infected leaves. Eventually the shoot apex is killed and infected branches die. In susceptible cacao the fungus may grow through the xylem down into the main stem and kill a mature cacao tree. Infections in the stem of young plants prior to the formation of the first 3-4 lateral branches usually kill the plant. Basidiospores released from corticioid basidiomata developed on leaf scars or along cracks in the main vein of infected leaves infect young leaves. The pathogen commonly infects cacao but there are rare reports from avocado. As both crops are introduced to the region, the pathogen is suspected to occur asymptomatically in native vegetation. The pathogen is readily isolated but cultures cannot be maintained. In this study, DNA was extracted from pure cultures of O. theobromae obtained from infected cacao plants sampled from Indonesia. The internal transcribed spacer region (ITS), consisting of ITS1, 5.8S ribosomal RNA and ITS2, and a portion of nuclear large subunit (LSU) were sequenced. Phylogenetic analysis of ITS sequences placed O. theobromae sister to Ceratobasidium anastomosis groups AG-A, AG-Bo, and AG-K with high posterior probability. Therefore the new combination Ceratobasidium theobromae is proposed. A PCR-based protocol was developed to detect and identify C. theobromae in plant tissue of cacao enabling early detection of the pathogen in plants. A second species of Ceratobasidium, Ceratobasidium ramicola, identified through ITS sequence analysis, was isolated from VSD-affected cacao plants in Java, and is widespread in diseased cacao collected from Indonesia.     

Horizontal and vertical transmission of a Nosema sp. (Microsporidia) from Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

The gypsy moth, Lymantria dispar L. (Lepidoptera, Lymantriidae), a serious defoliator of deciduous trees, is an economically important pest when population densities are high. Outbreaking populations are, however, subject to some moderating influences in the form of entomopathogens, including several species of microsporidia. In this study, we conducted laboratory experiments to investigate the transmission of an unusual Nosema sp. isolated from L. dispar in Schweinfurt, Germany; this isolate infects only the silk glands and, to a lesser extent, Malpighian tubules of the larval host. The latent period ended between 8 and 15 days after oral inoculation and spores were continuously released in the feces of infected larvae until pupation. Exclusion of feces from the rearing cages resulted in a 58% decrease in horizontal transmission. The silk of only 2 of 25 infected larvae contained microsporidian spores. When larvae were exposed to silk that was artificially contaminated with Nosema sp., 5% became infected. No evidence was found for venereal or transovum (including transovarial) transmission of this parasite.     

Biogenesis of yeast mitochondrial cytochrome c: a unique relationship to the TOM machinery

The import of cytochrome c into the mitochondrial intermembrane space is not understood at a mechanistic level. While the precursor apocytochrome c can insert into protein-free lipid bilayers, the purified translocase of the outer membrane (TOM) complex supports the translocation of apocytochrome c into proteoliposomes. We report an in organello analysis of cytochrome c import into yeast mitochondria from wild-type cells and different mutants cells, each defective in one of the seven Tom proteins. The import of cytochrome c is not affected by removal of the receptor Tom20 or Tom70. Moreover, neither the transfer protein Tom5 nor the assembly factors Tom6 and Tom7 are needed for import of cytochrome c. When the general import pore (GIP)-protein Tom40 is blocked, the import of cytochrome c is moderately affected. Mitochondria lacking the central receptor and organizing protein Tom22 contain greatly reduced levels of cytochrome c. We conclude that up to two components of the TOM complex, Tom22 and possibly the GIP, are involved in the biogenesis of cytochrome c.     

New bioassay method to assess the pathogenicity of Colombian strains of Metarhizium anisopliae (Metsch.) Sorokin and Paecilomyces sp. (Deuteromycotina: Hyphomycetes) against the subterranean burrower bug Cyrtomenus bergi Froeschner (Hemiptera: Cydnidae)

Using a new sand bioassay method, the pathogenicity of three Colombian strains of Metarhizium anisopliae (i.e., CIAT 224, 245, and 230) and one strain of Paecilomyces sp. (CIAT 308) were tested against fourth instars of the subterranean burrower bug Cyrtomenus bergi Froeschner (Hem.: Cydnidae) in the laboratory. The proposed bioassay simulates the habitat of C. bergi and at the same time enables the evaluation of a large number of insects in a short period of time and under controlled conditions. All tested fungal species/strains were pathogenic against C. bergi. However, only low levels of mortality were obtained, and never exceeded 55%. In the screening experiment, M. anisopliae strains CIAT 224 and CIAT 245 caused the highest levels of mortality. The use of the bioassay procedure described herein is discussed.     

Presence of a unique male-specific extension of C-terminus to the cytochrome c oxidase subunit II protein coded by the male-transmitted mitochondrial genome of Venustaconcha ellipsiformis (Bivalvia: Unionoidea)

Analyses of unionoidean bivalve male-transmitted (M) mtDNA genomes revealed an approximately 555 bp 3' coding extension to cox2. An antibody was generated against this predicted C-terminus extension to determine if the unique cox2 protein is expressed. Western blot and immunohistochemistry analyses demonstrated that the protein was predominantly expressed in testes. Weak expression was detected in other male tissues but the protein was not detected in female tissues. This is the first report documenting the expression of a cox2 protein with a long C-terminus in animals. Its universal presence in unionoidean bivalve testes suggests a functional significance for the protein.     

Sitobion avenae alatae infected by Pandora neoaphidis: their flight ability, post-flight colonization, and mycosis transmission to progeny colonies

Epizootics caused by the obligate Entomophthorales pathogen Pandora neoaphidis may result from more than one possible means of fungal dissemination among host aphids, but we hypothesize that wide dispersal of the fungus is most likely to be associated with the flight behavior of migratory alates. We tested this hypothesis in a simulation experiment by assessing the flight capability of Sitobion avenae alates infected with P. neoaphidis and the potential of their post-flight survival, colonization, and mycosis transmission to progeny. A total of 281 alates were inoculated with P. neoaphidis, individually flown for up to 5h and 9km in a computer-monitored flight mill system and then reared for 10 days on wheat seedlings. The infected alates were capable of surviving on average for 2.9 days (range 1-7 days) and leaving 4.6 nymphs prior to deaths. Transmission of fungal infection within progeny colonies occurred after the mother alates died from P. neoaphidis mycosis. The level of contagious infection among the nymphs reached up to 16.8% within 7 days but varied with the survival time of the infected mother alates after flight. Based on stepwise polynomial regression analysis, progeny colony size was highly correlated with the interactions of flight time with both post-flight survival time and the number of nymphs left per alate before death (r2 = 0.997). Progeny mortality on day 5 after colonization was inversely correlated with post-flight survival time (r2 = 0.949) whereas infection on day 7 was correlated with flight distance and an interaction of post-flight survival time with fecundity of the infected alates (r2 = 0.970). Progeny mortality observed on day 10 was merely correlated to mortality observed on day 5 (r2 = 0.946). These results indicate a successful transmission of alate-borne P. neoaphidis to progeny colonies and further support our hypothesis on the means of primary dispersal of aphid epizootics by migratory alates in a geographically wide range.