A single-step multiplex PCR assay for the detection of European Peristenus spp., parasitoids of Lygus spp.

Lygus Hahn (Hemiptera: Miridae) are serious pests of agricultural and greenhouse crops throughout North America. In Europe, bivoltine Peristenus Förster (Hymenoptera: Braconidae) species have a significant impact on Lygus populations. Release and establishment of European P. digoneutis Loan in Lygus lineolaris (Palisot de Beauvois) populations in northeastern USA has renewed interest in the intended liberation of European parasitoids for Lygus control in Canada. Accurate identification of natural enemies is the cornerstone of biological control but conventional methods for identifying Peristenus species and estimating parasitism rates rely on tedious and time-consuming dissection and rearing methods. The present study describes species-specific PCR primers for three species of Peristenus, and the use of a multiplex PCR assay to detect P. digoneutis and P. stygicus Loan eggs and larvae from Lygus rugulipennis Poppius nymphs. Results indicate that the primers amplify uniquely sized, species-specific PCR products for the three species and are capable of detecting single eggs in parasitized nymphs within 3 days post-parasitism. Using a multiplex PCR assay, the primers maintain specificity and sensitivity, and allow detection of each of the three species in a single reaction. Although molecular diagnostics have previously been used in the identification of parasitoids and estimation of parasitism rates, this is the first time a single-step multiplex PCR protocol has been described.     

The influence of consumer diversity and indirect facilitation on trophic level biomass and stability

The relationship between species diversity and the stability and production of trophic levels continues to receive intense scientific interest. Though facilitation is commonly cited as an essential underlying mechanism, few studies have provided evidence of the impact that indirect facilitation may have on diversity–ecosystem functioning relationships. In this laboratory study, we examined the effect of zooplankton species diversity on trophic structure (total algal and zooplankton biomass) and temporal stability of total zooplankton biomass. We utilized four species of pond zooplankton grown in either monoculture or in polyculture. When comparing responses in polycultures with responses averaged across monocultures, a positive effect of diversity on total zooplankton biomass was observed. This occurred as a result of positive facilitative effects among competing zooplankton. Daphnia pulex , a biomass dominant in monoculture, was negatively affected by the presence of interspecific competitors. In contrast, Diaphanosoma brachyurum , a species that performed poorly in monoculture, was strongly and positively affected by the presence of interspecific competitors, driving positive diversity effects on total zooplankton biomass. Positive temporal covariances among zooplankton were detected in several polyculture replicates, increasing temporal variability of total zooplankton biomass. However, this destabilizing effect was weak relative to effects of high biomass yields in polyculture which caused temporal biomass variability (as measured by the coefficient of variation) to be lower in polyculture relative to monocultures. Zooplankton diversity effects on total algal biomass were not detected. However, increased zooplankton diversity significantly altered the size structure of algae, increasing the relative abundance of large, grazer-resistant algae.     

The ones we left behind: Comparing plot sampling and floristic habitat sampling for estimating bryophyte diversity

An efficient method for estimating bryophyte diversity in forest stands must consider more than just the dominant forest mesohabitat. We compared two methodologies commonly used for estimating diversity in forest ecosystems. Floristic habitat sampling (FHS) utilizes stratification of all forest mesohabitats, which includes the natural diversity of microhabitats found within and stratifies a mosaic of mesohabitats (e.g. forest, streams, seeps, and cliffs) and microhabitats (e.g. rocks logs, etc.) that are often not considered in forest research projects that use plot sampling to estimate species diversity. In Canadian cedar hemlock forest, FHS methodology recorded more than twice as many bryophyte species as plot sampling (PS). A comparison of the dominant forest mesohabitat concluded that plot sampling was not as efficient as FHS in estimating bryophyte diversity and that plot sampling can result in different interpretations of species diversity. Rare species ordination of stands sampled using FHS showed strong clustering of sites with respect to biogeoclimatic zones and age since the last major disturbance (fire or logging) as compared with rare species ordinations from PS data, which showed no delineation of stands along temporal gradients. Plot sampling has many useful applications in ecology, but floristic habitat sampling is more efficient for quantifying overall bryophyte diversity. FHS provides an excellent way to record a comprehensive list of species.     

Strategic sperm allocation under parasitic sex-ratio distortion

Parasitic sex-ratio distorters are a major selective force in the evolution of host mating behaviour and mate choice. Here, we investigate sperm limitation in the amphipod Gammarus duebeni and the impact of the microsporidian sex-ratio distorter Nosema granulosis on sperm allocation strategies. We show that males become sperm limited after three consecutive matings and provide uninfected, high fecundity, females with more sperm than infected females. We show that sperm limitation leads to a decrease in female productivity. The outcome of sex-ratio distortion has been shown theoretically to be sensitive to the mating limits of males. Our results indicate that strategic sperm allocation under male rarity will have a greater impact on infected females and has the potential to regulate spread of parasitic feminisers in host populations.     

The costs of autotomy and regeneration in animals: a review and framework for future research

Many organisms have the ability to shed an appendage (autotomy)to escape a predator or fouled molting event. Despite its immediateadvantage on survivorship, autotomy can have important consequencesfor locomotion, foraging, survivorship, and/or reproduction.Thus, regeneration is a way that animals alleviate some of thecosts associated with losing an appendage. Like autotomy, however,appendage regeneration can have important consequences for avariety of aspects of fitness; in a wide range of amphibians,reptiles, fishes, and arthropods, the allocation of resourcesto regenerate a lost appendage negatively affects somatic orreproductive growth. Previous research into the costs associatedwith regeneration has provided a strong framework to explorehow trade-offs associated with regeneration may have influencedits evolution. However, all research to date describing thecosts and benefits associated with autotomy and regenerationhave compared individuals autotomizing and regenerating an appendagewith individuals that have never lost an appendage. I suggestthat for studies of the evolutionary significance of regeneration,an alternative comparison is between individuals experiencingautotomy without regeneration and individuals experiencing autotomywith regeneration. Future work in this direction promises newinsights into the evolution of regenerative tendencies, as wellas how regeneration may be influencing animal form and function.     

Molecular phylogeny of the Arctoidea (Carnivora): effect of missing data on supertree and supermatrix analyses of multiple gene data sets

Phylogenetic relationships of 79 caniform carnivores were addressed based on four nuclear sequence-tagged sites (STS) and one nuclear exon, IRBP, using both supertree and supermatrix analyses. We recovered the three major arctoid lineages, Ursidae, Pinnipedia, and Musteloidea, as monophyletic, with Ursidae (bears) strongly supported as the basal arctoid lineage. Within Pinnipedia, Phocidae (true seals) were sister to the Otaroidea [Otariidae (fur seals and sea lions) and Odobenidae (walrus)]. Phocid subfamily and tribal designations were supported, but the otariid subfamily split between fur seals and sea lions was not. All family designations within Musteloidea were strongly supported: Mephitidae (skunks), Ailuridae (monotypic red panda), Mustelidae (weasels, badgers, otters), and Procyonidae (raccoons). A novel hypothesis for the position of the red panda was recovered, placing it as branching after Mephitidae and before Mustelidae+Procyonidae. Within Mustelidae, subfamily taxonomic changes are considered. This study represents the most comprehensive sampling to date of the Caniformia in a molecular study and contains the most complete molecular phylogeny for the Procyonidae. Our data set was also used in an empirical examination of the effect of missing data on both supertree and supermatrix analyses. Sequence for all genes in all taxa could not be obtained, so two variants of the data set with differing amounts of missing data were examined. The amount of missing data did not have a strong effect; instead, phylogenetic resolution was more dependent on the presence of sufficient informative characters. Supertree and supermatrix methods performed equivalently with incomplete data and were highly congruent; conflicts arose only in weakly supported areas, indicating that more informative characters are required to confidently resolve close species relationships.     

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.     

Leg regeneration stunts wing growth and hinders flight performance in a stick insect (Sipyloidea sipylus)

Major morphological structures are sometimes produced not once, but twice. For example, stick insects routinely shed legs to escape a predator or tangled moult, and these legs are subsequently re-grown. Here, I show that in Sipyloidea sipylus, re-growth of a leg during development causes adults to have disproportionately smaller wings and increases wing loading. These morphological consequences of leg regeneration led to significant reductions in several biologically relevant measures of individual flight performance. This previously unrecognized tradeoff between legs and wings reveals the integrated nature of phasmid phenotypes, and I propose how this tradeoff may have shaped phasmid evolution.     

Discovery of potent and orally active MTP inhibitors as potential anti-obesity agents

We have successfully identified a number of novel MTP inhibitors with single digit nanomolar potency. Analogues 10aq and 10dq demonstrated in vivo efficacy in a murine gut retention assay.     

Supervillin modulation of focal adhesions involving TRIP6/ZRP-1

Cell-substrate contacts, called focal adhesions (FAs), are dynamic in rapidly moving cells. We show that supervillin (SV)--a peripheral membrane protein that binds myosin II and F-actin in such cells--negatively regulates stress fibers, FAs, and cell-substrate adhesion. The major FA regulatory sequence within SV (SV342-571) binds to the LIM domains of two proteins in the zyxin family, thyroid receptor-interacting protein 6 (TRIP6) and lipoma-preferred partner (LPP), but not to zyxin itself. SV and TRIP6 colocalize within large FAs, where TRIP6 may help recruit SV. RNAi-mediated decreases in either protein increase cell adhesion to fibronectin. TRIP6 partially rescues SV effects on stress fibers and FAs, apparently by mislocating SV away from FAs. Thus, SV interactions with TRIP6 at FAs promote loss of FA structure and function. SV and TRIP6 binding partners suggest several specific mechanisms through which the SV-TRIP6 interaction may regulate FA maturation and/or disassembly.