The 5′ Terminal Trailer Region of Vesicular Stomatitis Virus Contains a Position-Dependent cis-Acting Signal for Assembly of RNA into Infectious Particles

The cis-acting genomic RNA requirements for the assembly of vesicular stomatitis virus (VSV) ribonucleocapsids into infectious particles were investigated. Using a biological assay based on particle infectivity, we demonstrated that subgenomic replicons that contained all four possible combinations of the natural genomic termini, the 3′ leader (Le) and 5′ trailer (Tr) regions, were replication competent; however, a 3′ copyback replicon (3′CB), containing the natural 3′ terminus but having the 5′ Tr replaced by a sequence complementary to the 3′ Le for 46 nucleotides, was unable to assemble infectious particles, despite efficient replication. When a copy of Tr was inserted 51 nucleotides from the 5′ end of 3′CB, infectious particles were produced. However, analysis of the replication products of these particles showed that the 51 nucleotides which corresponded to the Le complement sequences at the 5′ terminus were removed during RNA replication, thus restoring the wild-type 5′ Tr to the exact 5′ terminus. These data showed that a cis-acting signal was necessary for assembly of VSV RNAs into infectious particles and that this signal was supplied by Tr when located at the 5′ end. The regions within Tr required for assembly were analyzed by a series of deletions and exchanges for Le complement sequences, which demonstrated that the 5′ terminal 29 nucleotides of Tr allowed assembly of infectious particles but that the 5′ terminal 22 nucleotides functioned poorly. Deletions in Tr also altered the balance between negative- and positive-strand genomic RNA and affected levels of replication. RNAs that retained fewer than 45 but at least 22 nucleotides of the 5′ terminus could replicate but were impaired in RNA replication, and RNAs that retained only 14 nucleotides of the 5′ terminus were severely reduced in ability to replicate. These data define the VSV Tr as a position-dependent, cis-acting element for the assembly of RNAs into infectious particles, and they delineate RNA sequences that are essential for negative-strand RNA synthesis. These observations are consistent with, and offer an explanation for, the absence of 3′ copyback defective interfering particles in nature.     

Optimal foraging and gut constraints: reconciling two schools of thought

To ecologists, factors such as a forager's encounter rate with prey and its own susceptibility to predation are dominant determinants of foraging. In contrast, digestive physiologists consider foraging to be determined by factors like rates of digestion and absorption of ingested food. We reconcile these views in a model combining encounter rate, external handling, and internal handling of food in the gut. With internal food handling, two food properties become important; energy: external handling time (e/h) and energy: bulk (e/b). We show that internal handling is only one of a suite of rate limiting factors. The gut never reaches full capacity, indicating that spare capacity may be intrinsic to gut structure. Regardless of gut fullness, a food of sufficiently high e/b will always be harvested. Two isolegs in the state space of resource abundances determine diet selectivity. These isolegs, which we call the Mitchell and Pulliam isolegs, divide the state space into regions in which 1) the forager's optimal strategy is opportunism; 2) the forager is always selective on the food with the greater e/h and partially selective on the second food; 3) the forager is always selective on the food with the greater e/h and ignores the second food. The development and analysis of the isolegs thus reconcile the heretofore disparate perspectives of the ecological and the physiological frameworks of foraging.     

A new family of H3 receptor antagonists based on the natural product Conessine

A new family of Histamine H(3) receptor antagonists (5a-t) has been prepared based on the structure of the natural product Conessine, a known H(3) antagonist. Several members of the new series are highly potent and selective binders of rat and human H(3) receptors and display inverse agonism at the human H(3) receptor. Compound 5n exhibited promising rat pharmacokinetic properties and demonstrated functional antagonism of the H(3) receptor in an in-vivo pharmacological model.     

Fire and flood: Soil‐stored seed bank and germination ecology in the endangered Carrington Falls Grevillea (Grevillea rivularis,Proteaceae)

Abstract Seed germination is dependent on the interaction between the dormancy state of a seed and the presence of favourable environmental conditions. Thus, the spectacular pulse of seedling recruitment in many Australian vegetation communities following disturbances such as fire can be attributed to changes in microsite conditions and/or the dormancy‐breaking effect of the disturbance on accumulated seed banks. Grevillea rivularis is a threatened species endemic to the area immediately above Carrington Falls in the NSW Southern Highlands. Most of the population is confined to the riparian vegetation zone in woodland and heath, and is therefore subject to periodic disturbance from fire and flood. For this species, a pulse of seedling recruitment has been recorded after fire, flood and mechanical soil disturbance. The aims of this study were to examine the density and vertical distribution of the soil‐stored seed bank and to investigate the role of heat and scarification as cues for germination of fresh and soil‐stored seed. There was a large seed bank under the canopies of established individuals (194 ± 73 seeds m^?2) and most seeds were found in the 0–2 cm and leaf‐litter layers of the soil profile. The germination response of soil‐stored and fresh seed was examined using a hierarchical series of laboratory experiments. Seeds of G. rivularis showed marked dormancy polymorphism. Thirty‐six percent of soil‐stored seed germinated without treatment, whereas no untreated fresh seeds germinated. Scarification or heating caused significant germination of dormant soil‐stored seed, but only scarification resulted in germination of dormant fresh seeds. These results highlight important differences in the dormancy state of soil‐stored and fresh seed. Thus, being a riparian species in a fire‐prone environment, the dormancy mechanisms in seeds of G. rivularis suit this species to disturbance by both fire and flood.     

The birds and the bees: pollinator behaviour and variation in the mating system of the rare shrub Grevillea macleayana

Background and Aims

In Australia, honey-bees have invaded systems that evolved without social insect pollinators, where many plants are adapted to vertebrate pollination. Behavioural differences between pollinators are likely to influence mating patterns, but few studies have examined this empirically in long-lived, woody, perennials. It was shown previously that outcrossing rates in Grevillea macleayana vary among populations. Here tests were conducted to determine whether the behaviour of birds and honey-bees differed between a population previously found to be highly outcrossed and two inbreeding populations.

Methods

Visit frequencies and movement patterns of the visitors to inflorescences at three sites over two seasons were compared. A caging experiment was used to test the effects of excluding birds on pollen removal from newly opened flowers and on pollen deposition on stigmas that had been washed clean.

Key Results

Honey-bees were the most frequent visitors overall, but honeyeaters were more frequent visitors in the population previously found to have a high outcrossing rate than they were in either of the other populations. More visits by honeyeaters were from distant plants. Pollen removal did not vary greatly among sites, and was not affected by bird exclusion; however, more pollen was deposited on the stigmas of cleaned pollen presenters in the population previously observed to be highly outcrossing than in the other two. This high level of pollen deposition was reduced by experimental bird exclusion.

Conclusions

Honey-bees were the most frequent visitors, by an order of magnitude, and excluding vertebrates revealed that bees were removing most of the pollen but deposited fewer pollen grains on stigmas. Birds were more frequent visitors at the site previously found to be outcrossing than the other two sites, and they moved further between plants and visited fewer inflorescences on a plant during a foraging bout than bees did. These characteristics of bird visits to G. macleayana would be sufficient to produce significant variation in outcrossing rates among sites.Key words: Grevillea macleayana, Apis mellifera, honey-bees, honeyeaters, pollinator behaviour, pollen removal, pollen deposition, outcrossing rate     

A comparison of Aedes vigilax larval population densities and associated vegetation categories in a coastal wetland,Northern Territory,Australia

Darwin's northern suburbs border an extensive coastal reed and upper mangrove wetland recognized as an important larval habitat for Aedes vigilax (Skuse), the northern salt marsh mosquito, an established vector for Ross River and Barmah Forest viruses and an appreciable pest species. We sought to identify the most important vegetation categories associated with Ae. vigilax breeding to maximize the efficiency of mosquito control efforts. Using a generalized linear model with negative binominal distribution and log link, this study compares larval densities, determined by focused dipping, between 13 discernable vegetation categories. The incidence rate ratios (RR) generated can be used to compare the magnitude of larval densities for each vegetation category, compared with the reference category. Aedes vigilax larval densities were almost ten times greater in artificial drainage areas (RR=9.82), followed by tide‐affected reticulate (Sporobolus/Xerochloa) areas (RR=8.15), then Schoenoplectus/mangroves (RR=2.29), compared with the reference vegetation category “lower mangroves.” Furthermore, larval densities were highest in May, due to tidal inundation, for drainage areas and tide‐affected reticulates (RR=12.2, 11.7, respectively) compared with March, the reference month. Thus, to maximize the efficiency of aerial salt marsh mosquito control operations in this wetland, larval control is best accomplished by concentrating on drains, Schoenoplectus/mangroves, and tide‐affected reticulate areas, commencing early after the wet season. These results should apply to other areas of salt marsh mosquito breeding across northern Australia.     

Vector competence of Australian Culex gelidus Theobald (Diptera: Culicidae) for endemic and exotic arboviruses

The recent recognition of established populations of the mosquito, Culex gelidus Theobald, in Australia has raised concerns about local transmission of arboviruses. The vector competence of a mainland population of Cx. gelidus was investigated for two local alphaviruses, Ross River (RRV) and Barmah Forest (BFV) viruses, and three flaviviruses, Japanese encephalitis (JEV), Kunjin (KUNV) and Murray Valley encephalitis (MVEV) viruses. Colonised mosquitoes were exposed to virus via blood-soaked pledgets and transmission was tested using a capillary-tube method. The important Australian vectors, Aedes vigilax (Skuse) and Culex annulirostris Skuse, were used as internal controls for the alphaviruses and flaviviruses, respectively. Overall, Cx. gelidus was a more efficient vector of flaviviruses than alphaviruses. Culex gelidus was refractory to infection with BFV, and nearly 25% transmitted RRV, which was comparable to Ae. vigilax . Culex gelidus was susceptible to all three flaviviruses, with transmission rates of 96%, 95% and 41% for JEV, KUNV and MVEV, respectively. JEV transmission rates in Cx. annulirostris were unexpectedly low and this was possibly due to differences in susceptibility to JEV genotypes I and II. Considering the high susceptibility to the flaviviruses demonstrated here, and the natural infections with RRV and JEV that have been detected from northern Australian populations, the establishment of the exotic mosquito, Cx. gelidus , in Australia is potentially a significant public health concern.