Hydrodynamics and sliding posture analysis of the Cambrian arthropod Ercaicunia multinodosa

Dynamic mechanical analysis offers the opportunity to explore the motility and feeding strategies of extinct organisms, but the prerequisite is to have an accurate recovery engineering model. As shown with micro-computed tomography (CT) scanning, fossils of the bivalved arthropod Ercaicunia multinodosa from the Cambrian (Series 2, Stage 3) Chengjiang biota of China have well-preserved three-dimensional (3D) morphological details with a certain degree of compression. Here, we propose a palaeontological restoration method using computational fluid dynamics (CFD) to analyse multiple hypothetical models based on the fossil information to obtain a reasonable restoration model. Furthermore, we carry out hydrodynamic experiments to verify the palaeontological restoration results. Our simulation and experimental results suggest that, a dorsally convex and ventrally straight body shape with the valves opening at an angle of approximately 120° works best for E. multinodosa to overcome resistance, and in the meantime obtain most lift while sliding in the water column. The combination of three-dimensional reconstruction, CFD simulation, and hydrodynamic experiments provides a useful method for restoring the morphologies of extinct animals and exploring their palaeoecology.     

In-Frame Deletions Allow Functional Characterization of Complex Cellulose Degradation Phenotypes in Cellvibrio japonicus

The depolymerization of the recalcitrant polysaccharides found in lignocellulose has become an area of intense interest due to the role of this process in global carbon cycling, human gut microbiome nutritional contributions, and bioenergy production. However, underdeveloped genetic tools have hampered study of bacterial lignocellulose degradation, especially outside model organisms. In this report, we describe an in-frame deletion strategy for the Gram-negative lignocellulose-degrading bacterium Cellvibrio japonicus. This method leverages optimized growth conditions for conjugation and sacB counterselection for the generation of markerless in-frame deletions. This method produces mutants in as few as 8 days and allows for the ability to make multiple gene deletions per strain. It is also possible to remove large sections of the genome, as shown in this report with the deletion of the nine-gene (9.4-kb) gsp operon in C. japonicus. We applied this system to study the complex phenotypes of cellulose degradation in C. japonicus. Our data indicated that a Δcel5B Δcel6A double mutant is crippled for cellulose utilization, more so than by either single mutation alone. Additionally, we deleted individual genes in the two-gene cbp2ED operon and showed that both genes contribute to cellulose degradation in C. japonicus. Overall, these described techniques substantially enhance the utility of C. japonicus as a model system to study lignocellulose degradation.     

Trace fossils from the Cambro-Ordovician cow head group,newfoundland, and their paleobathymetric implication

U-shaped burrows identified as the trace fossil Arenicolites occur in the Cambro-Ordovician Cow Head Group, a series of thin-bedded limestones interbedded with graptolitic shale and thick beds of limestone breccia and conglomerate. The lithology, limestone petrography, and trace fossils indicate deposition on a submarine slope of a slowly submerging carbonate platform. The limestone bed containing Arenicolites probably represents a period of slow deposition. The steeply inclined U-shaped burrows are assumed to have been formed by polychaete worms of the family Mochtyellidae which thrived in an argillaceous carbonate mud bottom of a carbonate platform slope, in depths exceeding 200 m. Although Arenicolites is believed to denote a shallow-water environment, its presence in the Cow Head extends the ecological niche of the Arenicolites-producing organisms into the outer shelf and continental slope. The domichnia of suspension feeders thus extends from shallow- to deep-water environments.     

Defining the Epigenetic Mechanism of Asymmetric Cell Division of Schizosaccharomyces japonicus Yeast

A key question in developmental biology addresses the mechanism of asymmetric cell division. Asymmetry is crucial for generating cellular diversity required for development in multicellular organisms. As one of the potential mechanisms, chromosomally borne epigenetic difference between sister cells that changes mating/cell type has been demonstrated only in the Schizosaccharomyces pombe fission yeast. For technical reasons, it is nearly impossible to determine the existence of such a mechanism operating during embryonic development of multicellular organisms. Our work addresses whether such an epigenetic mechanism causes asymmetric cell division in the recently sequenced fission yeast, S. japonicus (with 36% GC content), which is highly diverged from the well-studied S. pombe species (with 44% GC content). We find that the genomic location and DNA sequences of the mating-type loci of S. japonicus differ vastly from those of the S. pombe species. Remarkably however, similar to S. pombe, the S. japonicus cells switch cell/mating type after undergoing two consecutive cycles of asymmetric cell divisions: only one among four “granddaughter” cells switches. The DNA-strand–specific epigenetic imprint at the mating-type locus1 initiates the recombination event, which is required for cellular differentiation. Therefore the S. pombe and S. japonicus mating systems provide the first two examples in which the intrinsic chirality of double helical structure of DNA forms the primary determinant of asymmetric cell division. Our results show that this unique strand-specific imprinting/segregation epigenetic mechanism for asymmetric cell division is evolutionary conserved. Motivated by these findings, we speculate that DNA-strand–specific epigenetic mechanisms might have evolved to dictate asymmetric cell division in diploid, higher eukaryotes as well.     

Trace fossils from lower Cambrian Hongjingshao Formation,Yunnan, China: Taxonomy,palaeoecology, palaeoenvironment

The Hongjingshao Formation (Cambrian Series 2, Stage 3) yields abundant, complex trace fossils that have not been systematically investigated up till now. Our detailed ichnological study of the traces from this formation in Malong area, Yunnan, China, recognizes four groups: (1) simple horizontal or sub-horizontal burrows; (2) complex branched burrow systems; (3) arthropod trails and; (4) simple vertical burrows belonging to the Cruziana ichnofacies. A trophic web is reconstructed for the community in Hongjingshao Formation based on both trace and body fossils. The ichnological and sedimentological features of the formation indicate an intertidal setting. The trace fossil assemblage demonstrates that Early Cambrian organisms were able to colonize very shallow marine environments, which further supports the landward expansion of the Early Cambrian ecosystems.     

Identification of the pathogens associated with skin ulceration and peristome tumescence in cultured sea cucumbers Apostichopus japonicus (Selenka)

The aquaculture of sea cucumbers Apostichopus japonicus (Selenka) has developed rapidly in China in recent years, but is increasingly affected by diseases such as skin ulceration and peristome tumescence. Previous studies on the pathogens causing these diseases focused largely on bacterial causes. In December 2008, we isolated four dominant bacterial species from lesions present in A. japonicus with the aforementioned diseases, from a farm in Yangkou (Qingdao, China). With two of these bacterial species, experimental infection of healthy A. japonicus resulted in the same disease symptoms that occurred in naturally infected A. japonicus. These two species were identified as Pseudoalteromonas sp. and Pseudoalteromonas tetraodonis. The early symptoms of infection for these bacterial species were ulcer spots on the dorsal skin and abdominal parapodia, followed by an increase in the number of ulcer spots or their merging into larger spots. Additionally, we isolated a spherical virus 100-250 nm in diameter and with a bilayer capsule, from A. japonicus with another disease from four different farms. By experimental infection with crude extracts of the virus, healthy laboratory-acclimatized A. japonicus developed the same symptoms as in natural infected cases. The early symptoms of viral infection comprised a decrease in tentacle activity, decay of dorsal papillate podia, peristome tumescence and abdominal ulceration. Our study demonstrates that the bacteria and virus were both responsible for skin ulceration and peristome tumescence in A. japonicus, but resulted in different early disease symptoms.     

Vein density of angiosperms as a paleoclimate proxy: a case study using fossil leaves of Zelkova and Machilus

Using vein density to study the relationship between plants and environment is an important topic for paleoclimate and paleoenvironment studies. In this paper, the vein density of fossil plants Zelkova ungeri Kovats, Machilus tiantaiensis Ding et Sun and their NLRs was analyzed using the one-dimensional analysis and two-dimensional analysis approaches. The mechanism of vein density responses to the environment variation was also discussed. Further, the differences of the vein density between the fossil and extant leaves were compared. The results indicated that the vein density of Z. schneideriana Hand.-Mazz. and M. pingii Cheng ex Yang increased closely with the increase of precipitation but not closely related with the variation of temperature. The vein density of Z. ungeri and M. tiantaiensis was respectively higher than that of their NLRs. Thus we deduced that the precipitation in eastern Zhejiang Province during the late Miocene was higher than that of nowadays and the paleoclimate was more humid at that time.     

Statistical tools to interpret risks that arise from rare events in host specificity testing

Host specificity testing to predict host range is one of the key steps to predicting the risk a biological control agent will present to non-target organisms in the new environment. When host specificity testing data contain discrepancies, or unacceptable levels of uncertainty, it can be difficult for decision-makers to adequately address this uncertainty. To better understand the uncertainty in host specificity testing, we used a range of statistical tools to examine a data set associated with the leaf weevil Cleopus japonicus (Curculionidae), a biological control agent for the weed Buddleja davidii (Buddlejaceae) in New Zealand. Significant uncertainty arose during the early stages of host specificity testing when one C. japonicus larva reared to pupation on a culturally important native plant. Further trials were conducted to evaluate the suitability of C. japonicus as a biological control agent, and despite the uncertainty, C. japonicus was released in New Zealand in 2006, and has since established populations at each release site. However, the possibility of larvae completing their life cycle on the native plant initiated this evaluation of the statistics associated with testing biological control agents. We present results from analyses of the C. japonicus survival data using confidence intervals, equivalence testing, power analyses and survival curves to highlight the appropriateness of each of these tools for interpreting host specificity tests in biological control.     

Determination of paleoseasonality of fossil brachiopods using shell spiral deviations and chemical proxies

Brachiopods have been extensively used in paleoclimatic and paleoecological reconstructions, but their utility would greatly increase if paleoseasonality information could be obtained from their shells. Determining seasonal seawater temperature variations from fossil brachiopods requires knowledge of specimen ontogenetic ages, which is difficult to determine compared to other organisms secreting a shell by accretion. In this study, the combination of the spiral deviation methodology and chemical proxies is tested for determining specimen ontogenetic ages and paleoseasonality using two species of fossil brachiopods, Laqueus rubellus and Terebratula terebratula, of Pleistocene and Late Miocene age, respectively. Spiral deviations were obtained for Laqueus and Terebratula using an R program developed for modern taxa, and well-preserved shells were analyzed using oxygen isotopes and Mg/Ca ratios as chemical proxies for past seawater temperature. Results reveal that locations of spiral deviations on shells of L. rubellus displayed a strong direct relationship with Mg concentrations, and resulting Mg/Ca-derived paleotemperatures were seasonal. Conversely, specimens of T. terebratula did not show a consistently strong relationship between Mg concentrations and spiral deviations, although resulting paleotemperatures agreed with those from previous studies. Overall, the results from this study indicate that the spiral deviation methodology combined with chemical proxies presents great potential for utility in past seasonal seawater temperature reconstructions in pristinely preserved, biconvex fossil brachiopods.     

Ichnology of deep‐sea fan overbank deposits of the Ganei slates (Eocene,Switzerland)— a classical flysch trace fossil locality studied first by Oswald Heer

Ganei (Switzerland) is a classical locality for trace fossils. At this site, Heer (1877) described a large number of trace fossils, several of which were new taxa. The trace fossils occur in thin‐bedded turbidites in which the basal divisions of the Bouma sequence are typically absent; the turbidites are assigned to the Ganei Slates and are Eocene in age. They are interpreted to have been deposited in an overbank environment within an upper to middle fan area distal to a channel. Two trace‐fossil associations occur: the first (I) is characterized by bulldozing organisms producing biodeformational structures, Scolica, and Nereites irregularis; the second (II) association shows a distinct tiering pattern with near‐surface graphoglyptids and a mixed layer with simple tubes such as cf. Palaeophycus and Planolites, plus patterned tubes such as Nereites cirrinalis, and Chondrites. Deeper turbidite layers were colonized by Chondrites and Gyro‐phyllites. All trace fossils show a normal size spectrum compared to previously studied trace‐fossil associations, so the degree of oxygenation probably did not influence the composition of either trace‐fossil association. Seafloor sediment was probably soft and did not affect the trace‐fossil associations. Sedimentation rate and event frequency did not change and are estimated to have been in a range of 5–10 cm/1000 years and 2–5 events per 1000 years, respectively. The composition of trace‐fossil associations I and II is therefore interpreted to have been controlled by the benthic food content being higher for trace‐fossil association I than for II.     

Surveys of Gynaecotyla squatarolae and Microphallus koreana (Digenea: Microphallidae) Metacercariae in Two Species of Estuarine Crabs in Western Coastal Areas, Korea

To figure out the geographical distribution of Gynaecotyla squatarolae and Microphallus koreana metacercariae in Korea, shore crabs of southwestern coastal areas were examined. Eight coastal areas in Inchon-si (A), Gyeonggi-do (B), Chungcheongnam-do (C, D, and E), Jeollabuk-do (F), and Jeollanam-do (G and H) were selected, and 2 kinds of crabs, Macrophthalmus dilatatus and/or Macrophthalmus japonicus, were caught. After transportation to the laboratory, 15 crabs per each group were grouped and ground in a mortar and pestle, and examined for microphallid metacercariae. In M. dilatatus, G. squatarolae metacercariae were recovered from 3 (C, E, and H) out of 6 regions, but M. koreana metacercariae were not recovered. In the case of M. japonicus, G. squatarolae metacercariae were recovered from 6 (B, D, E, F, G, and H) of 7 areas surveyed, and M. koreana matacercariae were detected from 5 regions (A, B, D, F, and H). These results indicate that the life cycle of G. squatarolae is maintained in the western coastal areas using M. dilatatus and M. japonicus as intermediate hosts, while that of M. koreana is maintained only using M. japonicus.     

General Trends in Trace Element Utilization Revealed by Comparative Genomic Analyses of Co, Cu, Mo, Ni, and Se

Trace elements are used by all organisms and provide proteins with unique coordination and catalytic and electron transfer properties. Although many trace element-containing proteins are well characterized, little is known about the general trends in trace element utilization. We carried out comparative genomic analyses of copper, molybdenum, nickel, cobalt (in the form of vitamin B₁₂), and selenium (in the form of selenocysteine) in 747 sequenced organisms at the following levels: (i) transporters and transport-related proteins, (ii) cofactor biosynthesis traits, and (iii) trace element-dependent proteins. Few organisms were found to utilize all five trace elements, whereas many symbionts, parasites, and yeasts used only one or none of these elements. Investigation of metalloproteomes and selenoproteomes revealed examples of increased utilization of proteins that use copper in land plants, cobalt in Dehalococcoides and Dictyostelium, and selenium in fish and algae, whereas nematodes were found to have great diversity of copper transporters. These analyses also characterized trace element metabolism in common model organisms and suggested new model organisms for experimental studies of individual trace elements. Mismatches in the occurrence of user proteins and corresponding transport systems revealed deficiencies in our understanding of trace element biology. Biological interactions among some trace elements were observed; however, such links were limited, and trace elements generally had unique utilization patterns. Finally, environmental factors, such as oxygen requirement and habitat, correlated with the utilization of certain trace elements. These data provide insights into the general features of utilization and evolution of trace elements in the three domains of life.     

Repellence of nectar-thieving ants by a physical barrier: Adaptive role of petal hairs on Menyanthes trifoliata (Menyanthaceae)

Some plant species possess petal hairs, the defensive functions of which remain untested. I hypothesized that the petal hairs of an emergent plant species, Menyanthes trifoliata (Menyanthaceae), have a function in deterring nectar-thieving ants (Lasius japonicus). Lasius japonicus individuals were observed to stop moving forward after touching hairs and had difficulty in maintaining their balance when walking on hairs. In order to confirm the effectiveness of the hairs quantitatively, I trimmed hairs with a nose hair trimmer and then observed the behavior of flower-visiting L. japonicus. The success rate of L. japonicus in entering the floral tubes was significantly lower in the presence of intact hairs than when there were trimmed hairs. Moreover, it took L. japonicus twice as long to enter floral tubes with intact hairs, even when they succeeded. These results suggest that hairs on M. trifoliata petals are effective as a physical barrier against L. japonicus by restricting the movements of the ants.     

Characterization of tyrosinase inhibitory constituents from the aerial parts of Humulus japonicus using LC-MS/MS coupled online assay

In the screening of natural products for the development as cosmetic ingredients, the EtOAc-soluble fraction of Humulus japonicus showed tyrosinase inhibitory activity. HPLC-MS/MS coupled online tyrosinase assay of EtOAc-soluble fraction of H. japonicus characterized the twenty-eight constituents including two unknown ones and their tyrosinase inhibitory activity. Fractionation of H. japonicus using various chromatographic techniques yielded thirty-eight compounds. The chemical structures of isolated compounds were identified by spectroscopic analysis. As characterized by HPLC-MS/MS analysis, we isolated twenty-four predicted compounds and further identified two unknown ones, named humulusides A (1) and B (2). Additional ten compounds were also identified by purification. Tyrosinase inhibitory activity of isolated compounds were evaluated, which was closely correlated with the results from HPLC-MS/MS coupled online tyrosinase assay. Consistent with predicted data, two major compounds, trans-N-coumaroyltyramine (14) and cis-N-coumaroyltyramine (15) showed tyrosinase inhibition with IC₅₀ values of 40.6 and 36.4?μM. Taken together, H. japonicus is suggested as whitening ingredient in cosmetic products. In addition, HPLC-MS/MS coupled tyrosinase assay is powerful tool for predicting active compounds with short time and limited amounts, although identification of new compounds and verification of predicted data are also needs to be demonstrated by further experiment.     

Tetrapod Ichnofacies: A New Paradigm

We recognize three fundamental terms in ichnology: (1) ichnoassemblage, which is an assemblage of ichnofossils conceptually equivalent to an assemblage of body fossils; (2) ichnocoenosis, which is a trace fossil assemblage produced by a biological community that can be characterized by morphological criteria; and (3) ichnofacies, which refers to recurrent ichnocoenoses that represent a significant portion of Phanerozoic time. There are two different kinds of ichnofacies, ethoichnofacies (mostly invertebrate ichnofacies) and biotaxonichnofacies (mostly tetrapod ichnofacies). Nonmarine invertebrate ichnologists now recognize five archetypal ichnofacies (Mermia, Skolithos, Scoyenia, Coprinisphaera, Psilonichnus) to which we add the Octopodichnus ichnofacies. We propose a coherent and consistent classification and nomenclature for tetrapod ichnofacies. We name five archetypal vertebrate ichnofacies for nonmarine environments: Chelichnus, Grallator, Brontopodus, Batrachichnus and Characichnos ichnofacies.     

Origin and diversification of the clawed lobster genus Metanephrops (Crustacea: Decapoda: Nephropidae)

A phylogenetic analysis of all 17 extant species of the clawed lobster genus Metanephrops based on mitochondrial 12S rRNA, 16S rRNA and cytochrome c oxidase I, and nuclear histone H3 gene sequences supports the morphological groupings of two of the traditional groups of the genus (the binghami and japonicus groups) but refutes monophyly of the other two groups (the arafurensis and thomsoni groups). The results in general support a recent morphology-based cladistic analysis of this genus except that this study suggests M. neptunus to be a basal rather than a derived species as indicated in the morphological analysis. This species is genetically diverse over its geographical range. Moreover, the two color forms of M. thomsoni are genetically distinct, most likely representing different species. The molecular phylogeny and current distribution pattern of the extant species, together with the fossil record, suggest that the genus originated in the Antarctica in the Cretaceous, followed by diversification and dispersal along the continental shelf of different continents as a result of the vicariant events associated with the breakup of the Southern Temperate Gondwana since Late Cretaceous.     

Mycobacterium ulcerans Fails to Infect through Skin Abrasions in a Guinea Pig Infection Model: Implications for Transmission

Transmission of M. ulcerans, the etiological agent of Buruli ulcer, from the environment to humans remains an enigma despite decades of research. Major transmission hypotheses propose 1) that M. ulcerans is acquired through an insect bite or 2) that bacteria enter an existing wound through exposure to a contaminated environment. In studies reported here, a guinea pig infection model was developed to determine whether Buruli ulcer could be produced through passive inoculation of M. ulcerans onto a superficial abrasion. The choice of an abrasion model was based on the fact that most bacterial pathogens infecting the skin are able to infect an open lesion, and that abrasions are extremely common in children. Our studies show that after a 90d infection period, an ulcer was present at intra-dermal injection sites of all seven animals infected, whereas topical application of M. ulcerans failed to establish an infection. Mycobacterium ulcerans was cultured from all injection sites whereas infected abrasion sites healed and were culture negative. A 14d experiment was conducted to determine how long organisms persisted after inoculation. Mycobacterium ulcerans was isolated from abrasions at one hour and 24 hours post infection, but cultures from later time points were negative. Abrasion sites were qPCR positive up to seven days post infection, but negative at later timepoints. In contrast, M. ulcerans DNA was detected at intra-dermal injection sites throughout the study. M. ulcerans was cultured from injection sites at each time point. These results suggest that injection of M. ulcerans into the skin greatly facilitates infection and lends support for the role of an invertebrate vector or other route of entry such as a puncture wound or deep laceration where bacteria would be contained within the lesion. Infection through passive inoculation into an existing abrasion appears a less likely route of entry.