Genetic structure of cherry fruit fly (Rhagoletis cingulata) populations across managed,unmanaged, and natural habitats

The cherry fruit fly (CFF), Rhagoletis cingulata Loew (Diptera: Tephritidae: Trypetini), is endemic to eastern North America and Mexico, where its primary native host is black cherry [Prunus serotina Ehrh. (Rosaceae)]. Cherry fruit fly is also a major economic pest of the fruit of cultivated sweet (Prunus avium L.) and tart (Prunus cerasus L.) cherries. Adult CFF that attack wild black cherry and introduced, domesticated cherries in commercial and abandoned orchards are active at different times of the summer, potentially generating allochronic isolation that could genetically differentiate native from sweet and tart CFF populations. Here, we test for host‐related genetic differences among CFF populations in Michigan attacking cherries in managed, unmanaged, and native habitats by scoring flies for 10 microsatellite loci. Little evidence for genetic differentiation was found across the three habitats or between the northern and southern Michigan CFF populations surveyed in the study. Local gene flow between native black cherry, commercial, and abandoned orchards may therefore be sufficient to overcome seasonal differences in adult CFF activity and prevent differentiation for microsatellites not directly associated with (tightly linked to) genes affecting eclosion time. The results do not support the existence of host‐associated races in CFF and imply that flies attacking native, managed, and unmanaged cherries should be considered to represent a single population for pest management purposes.     

Isolation of high molecular weight DNA suitable for BAC library construction from woody perennial soft-fruit species

We have developed a novel nuclei extraction method that allows for the extraction of high molecular weight DNA from leaves of woody perennial soft-fruit species that contain high levels of carbohydrates and polyphenolics. The method utilizes a modified buffer system including 4% (w/v) polyvinylpyrrolidone (PVP)-10 and a combination of nylon filters and Percoll gradients to purify nuclei extracts prior to embedding in agarose plugs. The effectiveness of the method was demonstrated on leaves of red raspberry (Rubus idaeus) and blackcurrant (Ribes nigrum), two soft-fruit species that have shown to be recalcitrant to standard genomic DNA extraction methods. Extracted DNA was readily digested by restriction enzymes and, as shown for raspberry, suitable for bacterial artificial chromosome (BAC) library construction.     

Host refractoriness of the tobacco hornworm, Manduca sexta, to the braconid endoparasitoid Cotesia flavipes

Cotesia flavipes (Hymenoptera:Braconidae) is a gregarious endoparasitoid of several pyralid stemborer larvae of economic significance including the sugarcane borer, Diatraea saccharalis. In this study, the ability of this parasitoid to develop in a sphingid host, Manduca sexta, was tested. First, second, third, fourth, and even pharate fifth instar host tobacco hornworm larvae were readily parasitized by the female C. flavipes parasitoids but no wasp larvae hatched from the eggs in this refractory host. Instead, the parasitoid eggs were invariably encapsulated by the host's hemocytes and, ultimately, no parasitoids emerged from tobacco hornworm hosts. The first stages of encapsulation were evident at 2 h post-parasitization of the host M. sexta larvae, when the beginning stages of capsule formation were seen. The developmental fate of the host larvae with encapsulated parasitoids was variable. Most succumbed as abnormally small fifth instars or as post-wandering prepupal animals, while a few developed normally to the pupal stage. Dissection of all the larvae or pupae with encapsulated wasp eggs showed evidence of hemocytic encapsulation and melanization of the C. flavipes eggs. This report describes the association between C. flavipes and M. sexta, which appears to be an excellent model system for studying the physiological processes accompanying wasp egg encapsulation that result in death of the host as well as the parasitoid. Since the parasitoid egg never hatches, the system offers an excellent opportunity to identify and study the effects of parasitoid-injected polydnavirus and venom on host physiology.     

Australia Antigen and Liver Function Tests Following Infectious Hepatitis—A Study of 111 Patients in Quest of Aids in Blood Donor Screening

An epidemic of infectious hepatitis involving 99 patients and employees of a state mental hospital revealed Australia antigen Au(1) to be absent from the blood of all but one of the subjects when tested at six weeks, three months, nine months and 12 to 18 months after onset of jaundice. The single patient with Au(1) at 12 months had no enzyme abnormality to indicate residual liver disease.If Au(1) is the virus of hepatitis these data would support the concept that persistent or long standing viremia is not a feature of epidemic hepatitis. Moreover, results of this study suggest that the Au(1) test should not be used to establish the absence of a past history of hepatitis in blood donors. These data do not establish the value of the Au(1) test in blood donors with active viremia, but do suggest that of 111 patients with recent hepatitis 1 percent had persistent antigenemia and 4 percent probably had circulating antigen antibody complexes and constituted a potential risk to recipients of their blood. The degree of risk to recipients from transfused blood of post-hepatitis patients without demonstrable Au(1) cannot be assessed.     

Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague

The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-V(C424S) proteins were overexpressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2mg/g of cell paste of 95% pure, mono-disperse protein having < or =0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-V(C424S) were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible alhydrogel adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-V(C424S) monomer; cysteine-capped F1-V monomer; cysteine-capped F1-V multimer; and a F1-V standard reported previously. After a two-dose vaccination with 2 x 20 microg of F1-V, respectively, 100%, 80%, 80%, and 70% of injected mice survived a subcutaneous lethal plague challenge with 10(8) LD(50)Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection.     

Isolation of Pandemic Vibrio parahaemolyticus from UK Water and Shellfish Produce

Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium found commonly in temperate and warm estuarine waters worldwide. V. parahaemolyticus is considered an emerging bacterial pathogen in Europe and has been responsible for several recent seafood-associated outbreaks. During ad hoc testing of raw shellfish produce in May 2012, pandemic group (O3:K6) V. parahaemolyticus was isolated from Pacific oysters (Crassostrea gigas), harvested in Southern England. Follow-on testing of water and shellfish, encompassing a small number geographically diverse sites, also retrieved pandemic group isolates. These strains are amongst the most northerly pandemic strains described to date and represent the first instance of pandemic V. parahaemolyticus isolated in the UK, highlighting the expanding geographical distribution of these foodborne pathogens in the environment.     

Aboveground resource allocation in response to root herbivory as affected by the arbuscular mycorrhizal symbiosis

Aims

Arbuscular mycorrhizal (AM) fungi associate with the majority of terrestrial plants, influencing their growth, nutrient uptake and defence chemistry. Consequently, AM fungi can significantly impact plant-herbivore interactions, yet surprisingly few studies have investigated how AM fungi affect plant responses to root herbivores. This study aimed to investigate how AM fungi affect plant tolerance mechanisms to belowground herbivory.

Methods

We examined how AM fungi affect plant (Saccharum spp. hybrid) growth, nutrient dynamics and secondary chemistry (phenolics) in response to attack from a root-feeding insect (Dermolepida albohirtum).

Results

Root herbivory reduced root mass by almost 27%. In response, plants augmented investment in aboveground biomass by 25%, as well as increasing carbon concentrations. The AM fungi increased aboveground biomass, phosphorus and carbon. Meanwhile, root herbivory increased foliar phenolics by 31% in mycorrhizal plants, and increased arbuscular colonisation of roots by 75% overall. AM fungi also decreased herbivore performance, potentially via increasing root silicon concentrations.

Conclusions

Our results suggest that AM fungi may be able to augment plant tolerance to root herbivory via resource allocation aboveground and, at the same time, enhance plant root resistance by increasing root silicon. The ability of AM fungi to facilitate resource allocation aboveground in this way may be a more widespread strategy for plants to cope with belowground herbivory.

     

Evaluating gull diets: a comparison of conventional methods and stable isotope analysis

ABSTRACT Samples such as regurgitated pellets and food remains have traditionally been used in studies of bird diets, but these can produce biased estimates depending on the digestibility of different foods. Stable isotope analysis has been developed as a method for assessing bird diets that is not biased by digestibility. These two methods may provide complementary or conflicting information on diets of birds, but are rarely compared directly. We analyzed carbon and nitrogen stable isotope ratios of feathers of Glaucous Gull (Larus hyperboreus) chicks from eight breeding colonies in northern Alaska, and used a Bayesian mixing model to generate a probability distribution for the contribution of each food group to diets. We compared these model results with probability distributions from conventional diet samples (pellets and food remains) from the same colonies and time periods. Relative to the stable isotope estimates, conventional analysis often overestimated the contributions of birds and small mammals to gull diets and often underestimated the contributions of fish and zooplankton. Both methods gave similar estimates for the contributions of scavenged caribou, miscellaneous marine foods, and garbage to diets. Pellets and food remains therefore may be useful for assessing the importance of garbage relative to certain other foods in diets of gulls and similar birds, but are clearly inappropriate for estimating the potential impact of gulls on birds, small mammals, or fish. However, conventional samples provide more species‐level information than stable isotope analysis, so a combined approach would be most useful for diet analysis and assessing a predator's impact on particular prey groups.     

Standing geographic variation in eclosion time and the genomics of host race formation in Rhagoletis pomonella fruit flies

Taxa harboring high levels of standing variation may be more likely to adapt to rapid environmental shifts and experience ecological speciation. Here, we characterize geographic and host‐related differentiation for 10,241 single nucleotide polymorphisms in Rhagoletis pomonella fruit flies to infer whether standing genetic variation in adult eclosion time in the ancestral hawthorn (Crataegus spp.)‐infesting host race, as opposed to new mutations, contributed substantially to its recent shift to earlier fruiting apple (Malus domestica). Allele frequency differences associated with early vs. late eclosion time within each host race were significantly related to geographic genetic variation and host race differentiation across four sites, arrayed from north to south along a 430‐km transect, where the host races co‐occur in sympatry in the Midwest United States. Host fruiting phenology is clinal, with both apple and hawthorn trees fruiting earlier in the North and later in the South. Thus, we expected alleles associated with earlier eclosion to be at higher frequencies in northern populations. This pattern was observed in the hawthorn race across all four populations; however, allele frequency patterns in the apple race were more complex. Despite the generally earlier eclosion timing of apple flies and corresponding apple fruiting phenology, alleles on chromosomes 2 and 3 associated with earlier emergence were paradoxically at lower frequency in the apple than hawthorn host race across all four sympatric sites. However, loci on chromosome 1 did show higher frequencies of early eclosion‐associated alleles in the apple than hawthorn host race at the two southern sites, potentially accounting for their earlier eclosion phenotype. Thus, although extensive clinal genetic variation in the ancestral hawthorn race exists and contributed to the host shift to apple, further study is needed to resolve details of how this standing variation was selected to generate earlier eclosing apple fly populations in the North.