Nancy Ostiguy

RNA Viruses in Hymenopteran Pollinators: Evidence of Inter-Taxa Virus Transmission via Pollen and Potential Impact on Non-Apis Hymenopteran Species

Although overall pollinator populations have declined over the last couple of decades, the honey bee (Apis mellifera) malady, colony collapse disorder (CCD), has caused major concern in the agricultural community. Among honey bee pathogens, RNA viruses are emerging as a serious threat and are suspected as major contributors to CCD. Recent detection of these viral species in bumble bees suggests a possible wider environmental spread of these viruses with potential broader impact. It is therefore vital to study the ecology and epidemiology of these viruses in the hymenopteran pollinator community as a whole. We studied the viral distribution in honey bees, in their pollen loads, and in other non-Apis hymenopteran pollinators collected from flowering plants in Pennsylvania, New York, and Illinois in the United States. Viruses in the samples were detected using reverse transcriptase-PCR and confirmed by sequencing. For the first time, we report the molecular detection of picorna-like RNA viruses (deformed wing virus, sacbrood virus and black queen cell virus) in pollen pellets collected directly from forager bees. Pollen pellets from several uninfected forager bees were detected with virus, indicating that pollen itself may harbor viruses. The viruses in the pollen and honey stored in the hive were demonstrated to be infective, with the queen becoming infected and laying infected eggs after these virus-contaminated foods were given to virus-free colonies. These viruses were detected in eleven other non-Apis hymenopteran species, ranging from many solitary bees to bumble bees and wasps. This finding further expands the viral host range and implies a possible deeper impact on the health of our ecosystem. Phylogenetic analyses support that these viruses are disseminating freely among the pollinators via the flower pollen itself. Notably, in cases where honey bee apiaries affected by CCD harbored honey bees with Israeli Acute Paralysis virus (IAPV), nearby non-Apis hymenopteran pollinators also had IAPV, while those near apiaries without IAPV did not. In containment greenhouse experiments, IAPV moved from infected honey bees to bumble bees and from infected bumble bees to honey bees within a week, demonstrating that the viruses could be transmitted from one species to another. This study adds to our present understanding of virus epidemiology and may help explain bee disease patterns and pollinator population decline in general.

Cross-species transmission of honey bee viruses in associated arthropods.

There are a number of RNA virus pathogens that represent a serious threat to the health of managed honey bees (Apis mellifera). That some of these viruses are also found in the broader pollinator community suggests the wider environmental spread of these viruses, with the potential for a broader impact on ecosystems. Studies on the ecology and evolution of these viruses in the arthropod community as a whole may therefore provide important insights into these potential impacts. We examined managed A. mellifera colonies, nearby non-Apis hymenopteran pollinators, and other associated arthropods for the presence of five commonly occurring picorna-like RNA viruses of honey bees - black queen cell virus, deformed wing virus, Israeli acute paralysis virus, Kashmir bee virus and sacbrood virus. Notably, we observed their presence in several arthropod species. Additionally, detection of negative-strand RNA using strand-specific RT-PCR assays for deformed wing virus and Israeli acute paralysis virus suggests active replication of deformed wing virus in at least six non-Apis species and active replication of Israeli acute paralysis virus in one non-Apis species. Phylogenetic analysis of deformed wing virus also revealed that this virus is freely disseminating across the species sampled in this study. In sum, our study indicates that these viruses are not specific to the pollinator community and that other arthropod species have the potential to be involved in disease transmission in pollinator populations.

Plant-mediated alteration of the peritrophic matrix and baculovirus infection in lepidopteran larvae.

The peritrophic matrix (PM) lines the midgut of most insects, providing protection to the midgut epithelial cells while permitting passage of nutrients and water. Herein, we provide evidence that plant-mediated alteration of the PM contributes to the well-documented inhibition of fatal infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) of Heliothis virescens F. larvae fed cotton foliage. We examined the impact of the PM on pathogenesis using a viral construct expressing a reporter gene (AcMNPV-hsp70/lacZ) orally inoculated into larvae with either intact PMs or PMs disrupted by Trichoplusia ni granulovirus occlusion bodies containing enhancin, known to degrade insect intestinal mucin. Larvae possessing disrupted PMs displayed infection foci (lacZ signaling) earlier than those with intact PMs. We then examined PMs from larvae fed artificial diet or plant foliage using electron microscopy; foliage-fed larvae had significantly thicker PMs than diet-fed larvae. Moreover, mean PM width was inversely related to both the proportion of larvae with lacZ signaling at 18h post-inoculation and the final percentage mortality from virus. Thus, feeding on foliage altered PM structure, and these foliage-mediated changes reduced baculoviral efficacy. These data indicate that the PM is an important factor determining the success of an ingested pathogen in foliage-fed lepidopteran larvae.

Deformed wing virus in western honey bees (Apis mellifera) from Atlantic Canada and the first description of an overtly-infected emerging queen

Deformed wing virus (DWV) in western honey bees (Apis mellifera) often remains asymptomatic in workers and drones, and symptoms have never been described from queens. However, intense infections linked to parasitism by the mite Varroa destructor can cause worker wing deformity and death within 67 h of emergence. Ten workers (eight with deformed wings and two with normal wings) and three drones (two with deformed wings and one with normal wings) from two colonies infected with V. destructor from Nova Scotia, Canada, and two newly-emerged queens (one with deformed wings and one with normal wings) from two colonies infected with V. destructor from Prince Edward Island, Canada, were genetically analyzed for DWV. We detected DWV in all workers and drones, regardless of wing morphology, but only in the deformed-winged queen. This is the first report of DWV from Atlantic Canada and the first detection of a symptomatic queen with DWV from anywhere.

Sunflower as a Potential Trap Crop of Halyomorpha halys (Hemiptera: Pentatomidae) in Pepper Fields

ABSTRACT The brown marmorated stink bug, Halyomorpha halys (Stål), feeds on a variety of fruits and vegetables, and is an economically important invasive hemipteran pest. Trap cropping of H. halys was examined at the Pennsylvania State University Southeast Agriculture Research and Extension Center (SEAREC) in Lancaster Co., PA, from 2012 to 2013, with sunflowers used as a trap crop to protect bell pepper. H. halys were observed frequently on sunflowers planted surrounding the pepper field, and in both years of this experiment significantly more H. halys were observed in sunflowers than peppers. Both adults and nymphs were observed with equal frequency, with higher numbers of both observed in September. A 2:1 ratio of females to males was observed throughout both years. While sunflowers were attractive to H. halys, no difference in fruit damage was observed in peppers surrounded by the sunflower trap crop versus those peppers surrounded by peppers. While sunflowers present an interesting potential trap crop for H. halys, future research is needed to clarify the feasibility of this crop protection technique.

Honey bee Apis mellifera parasites in the absence of Nosema ceranae fungi and Varroa destructor mites

Few areas of the world have western honey bee (Apis mellifera) colonies that are free of invasive parasites Nosema ceranae (fungi) and Varroa destructor (mites). Particularly detrimental is V. destructor; in addition to feeding on host haemolymph, these mites are important vectors of several viruses that are further implicated as contributors to honey bee mortality around the world. Thus, the biogeography and attendant consequences of viral communities in the absence of V. destructor are of significant interest. The island of Newfoundland, Province of Newfoundland and Labrador, Canada, is free of V. destructor; the absence of N. ceranae has not been confirmed. Of 55 Newfoundland colonies inspected visually for their strength and six signs of disease, only K-wing had prevalence above 5% (40/55 colonies = 72.7%). Similar to an earlier study, screenings again confirmed the absence of V. destructor, small hive beetles Aethina tumida (Murray), tracheal mites Acarapis woodi (Rennie), and Tropilaelaps spp. ectoparasitic mites. Of a subset of 23 colonies screened molecularly for viruses, none had Israeli acute paralysis virus, Kashmir bee virus, or sacbrood virus. Sixteen of 23 colonies (70.0%) were positive for black queen cell virus, and 21 (91.3%) had some evidence for deformed wing virus. No N. ceranae was detected in molecular screens of 55 colonies, although it is possible extremely low intensity infections exist; the more familiar N. apis was found in 53 colonies (96.4%). Under these conditions, K-wing was associated (positively) with colony strength; however, viruses and N. apis were not. Furthermore, black queen cell virus was positively and negatively associated with K-wing and deformed wing virus, respectively. Newfoundland honey bee colonies are thus free of several invasive parasites that plague operations in other parts of the world, and they provide a unique research arena to study independent pathology of the parasites that are present.

Investigation of Chemical Rinses Suitable for Very Small Meat Plants To Reduce Pathogens on Beef Surfaces

Numerous antimicrobial interventions are capable of reducing the prevalence of harmful bacteria on raw meat products. There is a need to identify effective and inexpensive antimicrobial interventions that could, in practice, be used in very small meat plants because of limited financial, space, and labor resources. Eight antimicrobial compounds (acetic acid, citric acid, lactic acid, peroxyacetic acid, acidified sodium chlorite, chlorine dioxide, sodium hypochlorite, and aqueous ozone) were applied at various concentrations with small, hand-held spraying equipment, and bactericidal effectiveness was examined. Beef plate pieces were inoculated with fecal slurry containing a pathogen cocktail (Escherichia coli O157:H7, Salmonella Typhimurium, Campylobacter coli, and Campylobacter jejuni) and natural populations of aerobic plate counts, coliforms, and E. coli. Antimicrobial solutions were applied to beef surfaces via a portable, pressurized hand-held spray tank, and treated surfaces were subjected to appropriate methods for the enumeration and isolation of pathogens and hygiene indicators. Relative antimicrobial effectiveness was determined (from greatest to least): (i) organic acids, (ii) peroxyacetic acid, (iii) chlorinated compounds, and (iv) aqueous ozone. Using the equipment described, a 2% lactic acid rinse provided 3.5- to 6.4-log CFU/cm(2) reductions across all bacterial populations studied. Conversely, aqueous ozone yielded 0.02- to 2.9-log CFU/cm(2) reductions in pathogens and hygiene indicators, and did not differ significantly from a control tap water rinse (P = 0.055 to 0.731). This 2% lactic acid rinse will be subsequently combined with a previously described water wash to create a multistep antimicrobial intervention that will be examined under laboratory conditions and validated in very small meat plants.

Testing a combination of control tactics to manage Varroa destructor (Acari: Varroidae) population levels in honey bee (Hymenoptera: Apidae) colonies

Abstract Four combinations of strategies to reduce population growth of Varroa destructor Anderson and Trueman were tested in honey bee colonies located at two apiary sites. The strategies were combinations of mite tolerant queen stock, screen inserts, and T-02® strips (AI thymol). The effectiveness of the treatment combinations differed between the apiary sites. Site 1 apiary was in a cleared forest surrounded by secondary growth, while Site 2 was on the top of a hill, exposed to the wind. At Site 2, all but one colony survived the winter, while at Site 1, over-winter mortality of colonies was lowest in those colonies that combined hygienic queens and screen inserts. Mite populations at the end of the study did not differ among treatments or between sites. However, colony populations at Site 2 were significantly larger and overall colony survival was greater than at Site 1. The study demonstrates how the effectiveness of strategies to reduce the impact of Varroa on colony survival might ultimately depend upon the location of an apiary and how this affects the growth of colony populations.

Destruction of bacterial spores by phenomenally high efficiency non-contact ultrasonic transducers

Abstract. Conventional wisdom stipulates that high power ultrasound without direct or indirect transducer contact with the medium to be treated is not possible. This seemingly correct notion is based upon two major hurdles: inefficient transmission of ultrasound from the piezoelectric material into air/gases and exorbitant attenuation of ultrasound by gases. The latter is a natural phenomenon about which nothing can be done, and the former requires an un-conventional approach to transducer design. After many years of R& D in this area, we have finally succeeded in producing transducers that generate immense acoustic pressure in air in the frequency range of ∼50 kHz→10 MHz. By using these transducers without any contact with the material, we demonstrate destruction of 99.9% of dried bacterial spore samples of a close relative of anthrax, Bacillus thuringiensis. Following further refinement of the transducers and the mechanism of their excitation, we anticipate that non-contact ultrasound will have numerous applications including inactivation of agents of bioterrorism and sterilization of medical and surgical equipment, food materials, and air-duct systems of buildings, airplanes, space stations, and others.

Overwintered brood comb honey: colony exposure to pesticide residues

Summary To address beekeeper concerns about pesticide residues in overwintered honey, paired samples were obtained from the extracted supers and the brood chamber of the same colony. Only eight residues were detected: coumaphos, fluvalinate, boscalid, dimethoate, atrazine, bentazon, dichlorobenzene and thymol. Honey from extracted supers was significantly less likely to contain pesticide residues than honey from brood comb. Fluvalinate was detected only in overwintered brood comb honey, and coumaphos was found at significantly higher levels in the overwintered samples from the brood comb-honey super pairs. Pesticide residues in honey, while low in comparison to other substrates in the hive, contribute to the overall pesticide exposure of honey bees, with overwintered brood comb honey contributing more than honey stored in other locations in the hive.