Small steps or giant leaps for male-killers? Phylogenetic constraints to male-killer host shifts

Background  

Arthropods are infected by a wide diversity of maternally transmitted microbes. Some of these manipulate host reproduction to facilitate population invasion and persistence. Such parasites transmit vertically on an ecological timescale, but rare horizontal transmission events have permitted colonisation of new species. Here we report the first systematic investigation into the influence of the phylogenetic distance between arthropod species on the potential for reproductive parasite interspecific transfer.     

Replication of a Nuclear Polyhedrosis Virus in a Continuous Cell Culture of Spodoptera frugiperda: Purification, Assay of Infectivity, and Growth Characteristics of the Virus

Nonoccluded virus, polyhedra, and occluded virus were purified from a continuous cell culture of Spodopera frugiperda infected with nuclear polyhedrosis virus. The optimal temperature for the replication and lateral transmission of infectivity for the nuclear polyhedrosis viruses (NPV) in cell culture was 27 C. End-point dilution and plaque assay procedures for the measurement of infectivity are described and compared. Dose-response data demonstrated that a single particle could initiate an infection, and the validity of the relationship of 0.7 PFU per mean tissue culture infective dose (TCID(5 0)) further substantiated the accuracy of these infectivity assays. Particle-infectious unit calculations gave a ratio of 62 to 310 nonoccluded virus particles TCID(5 0). Growth cycle and lateral transmission experiments indicated that infectious material was released from cells 12 h postinfection (p.i.) and approached a maximal titer 4 days p.i. The number of polyhedra, nonoccluded virions, and TCID(5 0) produced per cell was also presented. Typical yields of NPV produced per liter flask suggested that insect cell culture systems represent a feasible means by which the replication of these viruses could be investigated.     

Improved gold chloride staining method for anatomical analysis of sensory nerve endings in the shoulder capsule and labrum as examples of loose and dense fibrous tissues

Consistency in gold chloride staining is essential for anatomical analysis of sensory nerve endings. The gold chloride stain for this purpose has been modified by many investigators, but often yields inconsistent staining, which makes it difficult to differentiate structures and to determine nerve ending distribution in large tissue samples. We introduce additional steps and major changes to the modified Gairns’ protocol. We controlled the temperature and mixing rate during tissue staining to achieve consistent staining and complete solution penetration. We subjected samples to sucrose dehydration to improve cutting efficiency. We then exposed samples to a solution containing lemon juice, formic acid and paraformaldehyde to produce optimal tissue transparency with minimal tissue deformity. We extended the time for gold chloride impregnation 1.5 fold. Gold chloride was reduced in the labrum using 25% formic acid in water for 18 h and in the capsule using 25% formic acid in citrate phosphate buffer for 2 h. Citrate binds gold nanoparticles, which minimizes aggregation in the tissue. We stored samples in fresh ultrapure water at 4° C to slow reduction and to maintain color contrast in the tissue. Tissue samples were embedded in Tissue Tek and sectioned at 80 and 100 μm instead of using glycerin and teasing the tissue apart as in Gairns’ modified gold chloride method. We attached sections directly to gelatin subbed slides after sectioning with a cryostat. The slides then were processed and coverslipped with Permount. Staining consistency was demonstrated throughout the tissue sections and neural structures were clearly identifiable.     

Catalytic core structure of the trans-acting HDV ribozyme is subtly influenced by sequence variation outside the core

The human pathogenic hepatitis delta virus (HDV) employs a unique self-cleaving catalytic RNA motif, the HDV ribozyme, during double-rolling circle replication. Fluorescence spectroscopy, circular dichroism, terbium(III) footprinting, and X-ray crystallography of precursor and product forms have revealed that a conformational change accompanies catalysis. In addition, fluorescence resonance energy transfer (FRET) has previously been used on a trans-acting HDV ribozyme to demonstrate surprisingly significant catalytic and global conformational effects of substrate analogues with varying 5' sequences, which reside as dangling overhangs outside the catalytic core. Here, we use the fluorescent guanine analogue 2-aminopurine (AP) in nucleotide position 76, immediately downstream of the catalytically involved C75, to monitor the relative structural effects of these substrate analogues on the ribozyme's trefoil turn of the catalytic core. Steady-state and time-resolved AP fluorescence spectroscopies show that the binding of each substrate analogue induces a unique local conformation with a specific AP76 stacking equilibrium. Binding of the 3' product results in a relative increase in AP fluorescence, suggesting that AP76 becomes more unstacked upon catalysis. These local conformational changes are kinetically concomitant with global conformational changes monitored by FRET. Finally, the rate constant of the local conformational change upon 3' product binding is fast and independent of 3' product concentration yet Mg2+ dependent. Our results demonstrate that the trefoil turn of the HDV ribozyme catalytic core is in a state of dynamic equilibrium not captured by static crystal structures and is highly sensitive to the identity of the 5' sequence and Mg2+ ions.