Jessica Jurzenski

Developing an in vivo toxicity assay for RNAi risk assessment in honey bees, Apis mellifera L.

Maize plants expressing dsRNA for the management of Diabrotica virgifera virgifera are likely to be commercially available by the end of this decade. Honey bees, Apis mellifera, can potentially be exposed to pollen from transformed maize expressing dsRNA. Consequently, evaluation of the biological impacts of RNAi in honey bees is a fundamental component for ecological risk assessment. The insecticidal activity of a known lethal dsRNA target for D. v. virgifera, the vATPase subunit A, was evaluated in larval and adult honey bees. Activity of both D. v. virgifera (Dvv)- and A. mellifera (Am)-specific dsRNA was tested by dietary exposure to dsRNA. Larval development, survival, adult eclosion, adult life span and relative gene expression were evaluated. The results of these tests indicated that Dvv vATPase-A dsRNA has limited effects on larval and adult honey bee survival. Importantly, no effects were observed upon exposure of Am vATPase-A dsRNA suggesting that the lack of response involves factors other than sequence specificity. The results from this study provide guidance for future RNAi risk analyses and for the development of a risk assessment framework that incorporates similar hazard assessments.

Identifying priority conservation areas for the American burying beetle, Nicrophorus americanus (Coleoptera: Silphidae), a habitat generalist

Conservation efforts leading to the recovery of the federally endangered American burying beetle (ABB), Nicrophorus americanus Olivier, have been challenging because of the unknown causes of its decline, difficulty in establishing habitat requirements, and unclear population distribution across the species’ range. Extant populations of this widespread generalist species occur in broadly separated regions of North America with varying habitat characteristics. A habitat suitability model for ABB in the Nebraska Sandhills was developed over the course of 3 years resulting in a final cross-validated spatial model. The succession of models from 2009 to 2011 indicated that most of the predictive variables stayed constant, but biased sampling and extrapolation areas affected classifier values differently. Variables associated with ABB occurrence were loamy sand, wetland and precipitation. Five variables, loam soil, agriculture, woodland, the average maximum temperature, and urban development, were associated with ABB absence. The 2011 cross-validated model produced an AUC value of 0.82 and provided areas designated as highly likely to support ABBs. By limiting the model extent to the Sandhills ecoregion and using threshold-dependent classifiers, the final habitat suitability model could be an important resource for wildlife managers engaged in the recovery of this habitat generalist.

Evaluation of Marking Techniques, Mark Retention, and Mortality Due to Marking of Burying Beetles (Coleoptera: Silphidae)

ABSTRACT The American burying beetle, Nicrophorus americanus Olivier, was listed as an endangered species in 1989 by the U.S. Fish and Wildlife Service. The recovery plan outlines the need for self-sustaining populations with at least 500 individuals, which requires mark and recapture sampling methods for measurement. To estimate populations, a marking technique must be used that will stay intact during the study and not adversely affect the beetles behavior. Four marking techniques (bee tags, enamel paint, elytron-clipping, and elytron-cauterizing) have been used in studies involving the marking of N. americanus studies. In our study, we used two surrogate species, Nicrophorus marginatus F. and Nicrophorus orbicollis Say, to test these different marking techniques. There were no significant differences in mortality rates among the four techniques for mature or teneral beetles. Bee tags had a significantly higher retention rate than paint in day-to-day activities and during reproductive cycles. In a field study of N. americanus where bee tags and elytral cauterizing were used, bee tags had a loss rate of 19.7%. A combination of permanent marks and bee tags will allow researchers to perform more accurate population estimates as well as track individual beetles to gain knowledge of their ecology and biology.

RNAi induced knockdown of a cadherin-like protein (EF531715) does not affect toxicity of Cry34/35Ab1 or Cry3Aa to Diabrotica virgifera virgifera larvae (Coleoptera: Chrysomelidae).

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an important maize pest throughout most of the U.S. Corn Belt. Bacillus thuringiensis (Bt) insecticidal proteins including modified Cry3Aa and Cry34/35Ab1 have been expressed in transgenic maize to protect against WCR feeding damage. To date, there is limited information regarding the WCR midgut target sites for these proteins. In this study, we examined whether a cadherin-like gene from Diabrotica virgifera virgifera (DvvCad; GenBank accession # EF531715) associated with WCR larval midgut tissue is necessary for Cry3Aa or Cry34/35Ab1 toxicity. Experiments were designed to examine the sensitivity of WCR to trypsin activated Cry3Aa and Cry34/35Ab1 after oral feeding of the DvvCad dsRNA to knockdown gene expression. Quantitative real-time PCR confirmed that DvvCad mRNA transcript levels were reduced in larvae treated with cadherin dsRNA. Relative cadherin expression by immunoblot analysis and nano-liquid chromatography - mass spectrometry (nanoLC-MS) of WCR neonate brush border membrane vesicle (BBMV) preparations exposed to DvvCad dsRNA confirmed reduced cadherin expression when compared to BBMV from untreated larvae. However, the larval mortality and growth inhibition of WCR neonates exposed to cadherin dsRNA for two days followed by feeding exposure to either Cry3Aa or Cry34/35Ab1 for four days was not significantly different to that observed in insects exposed to either Cry3Aa or Cry34/35Ab1 alone. In combination, these results suggest that cadherin is unlikely to be involved in the toxicity of Cry3Aa or Cry34/35Ab1 to WCR.

Opossums and Leopard Frogs Consume the Federally Endangered American Burying Beetle (Coleoptera: Silphidae)

The American burying beetle (ABB), Nicrophorus americanus Olivier, has disappeared from more than 90% of its historic range and has been listed as federally endangered since 1989. Prior to its listing, relatively few studies existed documenting ABB ecology and interactions with other animals, including its potential vertebrate predators. Bedick et al. (1999) documented a predation event by a blue jay, Cyanocitta cristata L. Walker and Hoback (2006) presented circumstantial evidence of predation by a bat of a beetle that was equipped with a radio transmitter. Shrews that fall into pitfall traps occasionally eat American burying beetles (W. W. Hoback, personal observation). Further evidence of potential predation by shrews was found in an illustration of a masked shrew consuming an American burying beetle (Zim and Hoffmeister 1955). Herein, we document video surveillance of predation of American burying beetle by Virginia opossum, Didelphis virginiana (Kerr), and pronota and elytra of ABB in feces of the northern plains leopard frog, Lithobates pipiens Schreber. We conducted two studies in the northeastern region of Nebraska’s Sandhills utilizing camcorders to record carrion beetle activity at night on rat (Rattus norvegicus Berkenhout) carcasses. The rat carcasses (approximately 300 g) were secured and could not be buried or removed from the site; however, beetles and some other animals could freely enter or exit the bait stations. Similar stations were used in 2010, but cages were used to eliminate access by opossums. The cages kept opossums out, but did not deter northern plains leopard frogs. Over 230 hrs of video were captured in 2009 and over 500 hrs in 2010, providing novel ABB footage and the opportunity to make behavioral observations. American burying beetle mortality caused by an opossum was confirmed by a video recorded on the

Patterns of Gene Expression in Western Corn Rootworm (Diabrotica virgifera virgifera) Neonates, Challenged with Cry34Ab1, Cry35Ab1 and Cry34/35Ab1, Based on Next-Generation Sequencing

With Next Generation Sequencing technologies, high-throughput RNA sequencing (RNAseq) was conducted to examine gene expression in neonates of Diabrotica virgifera virgifera (LeConte) (Western Corn Rootworm, WCR) challenged with individual proteins of the binary Bacillus thuringiensis insecticidal proteins, Cry34Ab1 and Cry35Ab1, and the combination of Cry34/Cry35Ab1, which together are active against rootworm larvae. Integrated results of three different statistical comparisons identified 114 and 1300 differentially expressed transcripts (DETs) in the Cry34Ab1 and Cry34/35Ab1 treatment, respectively, as compared to the control. No DETs were identified in the Cry35Ab1 treatment. Putative Bt binding receptors previously identified in other insect species were not identified in DETs in this study. The majority of DETs (75% with Cry34Ab1 and 68.3% with Cry34/35Ab1 treatments) had no significant hits in the NCBI nr database. In addition, 92 DETs were shared between Cry34Ab1 and Cry34/35Ab1 treatments. Further analysis revealed that the most abundant DETs in both Cry34Ab1 and Cry34/35Ab1 treatments were associated with binding and catalytic activity. Results from this study confirmed the nature of these binary toxins against WCR larvae and provide a fundamental profile of expression pattern of genes in response to challenge of the Cry34/35Ab1 toxin, which may provide insight into potential resistance mechanisms.