Interrelationships of Staphyliniform groups inferred from 18S and 28S rDNA sequences, with special emphasis on Hydrophiloidea (Coleoptera, Staphyliniformia)

The series Staphyliniformia is one of the mega‐diverse groups of Coleoptera, but the relationships among the main families are still poorly understood. In this paper we address the interrelationships of staphyliniform groups, with special emphasis on Hydrophiloidea and Hydraenidae, based on partial sequences of the ribosomal genes 18S rDNA and 28S rDNA. Sequence data were analysed with parsimony and Bayesian posterior probabilities, in an attempt to overcome the likely effect of some branches longer than the 95% cumulative probability of the estimated normal distribution of the path lengths of the species. The inter‐family relationships in the trees obtained with both methods were in general poorly supported, although most of the results based on the sequence data are in good agreement with morphological studies. In none of our analyses a close relationship between Hydraenidae and Hydrophiloidea was supported, contrary to the traditional view but in agreement with recent morphological investigations. Hydraenidae form a clade with Ptiliidae and Scydmaenidae in the tree obtained with Bayesian probabilities, but are placed as basal group of Staphyliniformia (with Silphidae as subordinate group) in the parsimony tree. Based on the analysed data with a limited set of outgroups Scarabaeoidea are nested within Staphyliniformia. However, this needs further support. Hydrophiloidea s.str., Sphaeridiinae, Histeroidea (Histeridae + Sphaeritidae), and all staphylinoid families included are confirmed as monophyletic, with the exception of Hydraenidae in the parsimony tree. Spercheidae are not a basal group within Hydrophiloidea, as has been previously suggested, but included in a polytomy with other Hydrophilidae in the Bayesian analyses, or its sistergroup (with the inclusion of Epimetopidae) in the parsimony tree. Helophorus is placed at the base of Hydrophiloidea in the parsimony tree. The monophyly of Hydrophiloidea s.l. (including the histeroid families) and Staphylinoidea could not be confirmed by the analysed data. Some results, such as a placement of Silphidae as subordinate group of Hydraenidae (parsimony tree), or a sistergroup relationship between Ptiliidae and Scydmaenidae, appear unlikely from a morphological point of view.     

Highly specialized Cretaceous beetle parasitoids (Ripiphoridae) identified with optimized visualization of microstructures

Extremely miniaturized longipedes insects (body length c. 0.3 mm) embedded in two pieces of Cretaceous amber from Myanmar are described and interpreted. Using inverted fluorescence and light microscopy for detailed analysis of microstructures, the inclusions were identified as primary larvae of the beetle family Ripiphoridae, subfamily Ripidiinae. While the structure of thoracic and abdominal segments including appendages corresponds well with the groundplan known in recent members of Ripidiinae, a curved prosternal ridge with prominent spines (each c. 5 μm), the reduced condition of stemmata and antennae and the lack of sharp mandibles are unique features within the entire family, apparently apomorphies of the longipedes larvae. A sinuate prosternal edge with a dense row of spines (prosternoctenidium) might be homologous with ‘head ctenidia’ in some previously described miniaturized conicocephalate larvae, but further investigation is needed. The morphological differences between the head of longipedes larvae and extant Ripidiinae are interpreted as adaptations to different groups of hosts and life strategies. Palaeoethology of the longipedes larvae is briefly discussed. In addition, the systematic placement of conicocephalate larvae from Canadian, Myanmar and Russian Cretaceous ambers, already interpreted by various authors as primary instars within Coleopterida (assigned to either Strepsiptera or to the coleopteran Tenebrionoidea: Ripiphoridae), is discussed.     

On the systematic position of the family Gyrinidae (Coleoptera: Adephaga)

Various characters of adult and larval members of Adephaga and Cupedidae were analyzed, and suggest that Gyrinidae are the sister-group of the remaining Adephaga, and are not closely related to the remaining aquatic Adephaga. The aquatic families Noteridae, Amphizoidae, Hygrobiidae and Dytiscidae seem to form a well founded monophyletic unit. The following characters are considered as synapomorphies of Adephaga excluding Gyrinidae: bifurcate condition of the muscle (= M.) tentoriopraementalis inferior, reduction of hypopharynx, strongly developed prosternal process, reduction in size and specialized modification of the ventral sclerite of the mesothorax, strongly developed mesofurcal arms, a high mesopleural ridge, globular mesocoxae restricted to rotatory movements, invaginated sternum VIII (coxostemum), the strongly curved base of the median lobe of the aedeagus, which articulates with the parameres, the rotated position of the aedeagus in repose, fusion of the larval clypeolabrum with the frons and reduction of the larval lacinia. Mesal shifting of M. episterno-coxalis prothoracis, and the fusion of the apical portions of the malpighian tubules of either side are considered as synapomorphies of Adephaga excluding Rhysodidae and Gyrinidae. Lateral reduction of the meta “sternal” transverse ridge and the presence of the subcubital setal binding patch of the hind wing are considered as synapomorphic characters of Trachypachidae, Noteridae, Amphizoidae, Hygrobiidae and Dytiscidae. We postulate that the metacoxal fusion occurred independently in gyrmids and the common ancestor of Trachypachidae, Noteridae, Amphizoidae, Hygrobiidae and Dytiscidae. Consequently we consider this character state as another synapomorphy of Trachypachidae and Hydradephaga excluding Haliplidae and Gyrinidae. The following characters are considered as synapomorphies of Noteridae, Amphizoidae, Hygrobiidae and Dytiscidae: Loss of tactile setae on the head capsule, metafurcal origin on the intercoxal wall, expansion of the intercoxal wall, elongation of the subcubital setal binding patch, loss of Mm. furca-coxale anterior and posterior, reduction of the larval abdominal segments IX and X, and the shifting of the uropmphi onto the ventral side of segment VIII. Presence of M. tentorio-mandibularis and M. stipitopalpalis intemus are certainly primitive features of adult gyrinids but the distribution of these character states among most members of Adephaga is yet unclear. Chemical defence gland constituents point towards a very isolated position of Gyrinidae. The old age of the group, documented by a larva found in upper Permian deposits, may support the hypothesis of a sister-group relation-ship between Gyrinidae and the remainder of Adephaga.     

In vitro evolution of intrinsically bent DNA.

DNA fragments which are intrinsically bent or curved migrate anomalously during electrophoresis through polyacrylamide gels. Starting with an initial population of approximately 10(12) unique DNA sequences, DNA which exhibited the kind of anomalous mobility associated with DNA bending was selected and enriched using a variation of the SELEX procedure. After seven rounds of selection and amplification, the vast majority of the remaining population of DNA fragments migrated as bent DNA. Cloning and sequencing of 30 individual sequences from this population has yielded information regarding the relationship between DNA sequence and bending. Some of the previous conclusions on DNA bending have been confirmed while others have been modified, by the results presented here. In addition, the dinucleotide base step CA/TG, which had not been thought to be a major factor in DNA bending, appears to be important.     

Biodegradation Processes in a Laboratory-Scale Groundwater Contaminant Plume Assessed by Fluorescence Imaging and Microbial Analysis

Flow reactors containing quartz sand colonized with biofilm were set up as physical model aquifers to allow degrading plumes of acetate or phenol to be formed from a point source. A noninvasive fluorescent tracer technique was combined with chemical and biological sampling in order to quantify transport and biodegradation processes. Chemical analysis of samples showed a substantial decrease in carbon concentration between the injection and outflow resulting primarily from dilution but also from biodegradation. Two-dimensional imaging of the aqueous oxygen [O₂(aq)] concentration field quantified the depletion of O₂(aq) within the contaminant plume and provided evidence for microbial respiration associated with biodegradation of the carbon source. Combined microbiological, chemical, and O₂(aq) imaging data indicated that biodegradation was greatest at the plume fringe. DNA profiles of bacterial communities were assessed by temperature gradient gel electrophoresis, which revealed that diversity was limited and that community changes observed depended on the carbon source used. Spatial variation in activity within the plume could be quantitatively accounted for by the changes observed in active cell numbers rather than differences in community structure, the total biomass present, or the increased enzyme activity of individual cells. Numerical simulations and comparisons with the experimental data were used to test conceptual models of plume processes. Results demonstrated that plume behavior was best described by growth and decay of active biomass as a single functional group of organisms represented by active cell counts.     

DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency

Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K_d = 68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.