Biology, Ecology, and Management of Brown Marmorate Stink Bug in Orchard Crops, Small Fruit, Grapes, egetables, and Ornamentals Our long-term goals for this project are to develop economically and environmentally sustainable pest management practices for the brown marmorated stink bug (BMSB), Halyomorpha halys (Stål), in specialty crops and to implement a coordinated, rapid delivery system to disseminate critical information generated from this project to specialty crop end-users. USDA-NIFA SCRI # 2011-51181-30937 Inside this Issue OBJECTIVES Objective 1. Establish biology and phenology of BMSB in specialty crops. Objective 2. Develop monitoring and management tools for BMSB. Objective 3. Establish effective management programs for BMSB in specialty crops. Objective 4. Integrate stakeholder input and Rapid transcriptome research findings Pheromone-Based Tools sequencing Pg. 1 Pg. 11 PROJECT DIRECTORS Tracy Leskey, USDA-ARS George Hamilton, Rutgers University Cerruti Hooks, University of Maryland Gregorz Krawczyk, Penn State University Classical biological Chris Bergh / Tom Kuhar, Virginia Tech Future Outreach Efforts Pg. 16 control Pg. 7 Carrie Koplinka-Loehr / Steve Young, Northeastern IPM Center Peter Shearer, Oregon State University Jim Walgenbach, North Carolina State Uni- versity Art Agnello, Cornell University Joanne Whalen, University of Delaware Jay Brunner / Elizabeth Beers, Washington State University Project Outputs Pg. 18 2014 Summary Report Year 3 Rapid transcriptome sequencing of an invasive pest, the brown marmorated stink bug, Halyomorpha halys by Leslie Pick and Julie Dunning- Hotopp Understanding the biology of the brown marmorated stink bug requires investigations at all levels, from population and behavioral measures to dissection of the genetic and genomic information that provides instructions for all developmental and physiological processes of the organism. In a study published this summer in BMC Genomics, investigators at the Institute for Genome Sciences at the University of Maryland School of Medicine and the University of Maryland Department of Entomology used state-of-the-art next generation sequencing methods to identify the sequences of all of the genes active in BMSB, the so-called transcriptome of BMSB. These researchers utilized new strategies to rapidly sequence the full transcriptome that allowed them to skip the time-consuming first step of breeding genetically identical individual animals in the laboratory, which is normally a first step in analysis of new genomes. Instead, the researchers were able to sequence and analyze all of the genetic variants that arose in their population of stink bugs, and to do so at all points in the insects’ life cycle, from the egg stage through late adulthood, including animals in diapause. This type of rapid sequencing provides a wealth of information about the biology of BMSB that is now available to the entire research community. In addition, it sets a precedent for an approach to sequencing that can provide a rapid response to other pest invasions similar to that already described for human disease epidemiology. The genome of each organism, present in each and every cell, is the information center that harbors the code for virtually every process carried out by the organism over its entire life cycle. This code is in the form of genes - DNA sequences with unique combinations of 4 different bases (A,T,C,G) in unique orders and of unique length. To be active, genes must be copied by cellular machinery into RNA that can then act on its own to execute cellular functions, or which, more often, is translated into proteins that function as enzymes, structural molecules, hormones, etc., responsible for everything from fertility, to embryonic development to homeostasis and aging. The differential expression of genes at different times during Figure 1. Life stages of BMSB starting with st nd rd th th development and in different cells of the animal eggs followed by 1 , 2 ,3 , 4 , and 5 instar is the key step in making cells and processes nymphs, and an adult in a counter-clockwise different from one another. For example, cells spiral outwards and from largest to smallest. The bar in the low left represents 1 cm. destined to become wing cells express a particular set of genes while cells destined to become parts of the brain express a different and unique complement of genes. Thus, This research project supports Objective 1. Establish biology and phenology of BMSB in specialty crops 2 2014 Summary Report Year 3 the key step required for a gene to be active in a cell or organism is the copying or transcription into RNA. Identifying all of the different RNA molecules in an organism tells us which genes are active in that organism. The goal of the study published in BMS Genomics was to - in one fell swoop - identify as many genes as possible that are transcribed by BMSB at any stage of the life cycle of BMSB. This approach would provide an overview of virtually all the genes that are active in this species. To achieve this, RNA was extracted from BMSB eggs, 1st instar nymphs, 2nd instar nymphs, 3rd instar nymphs, 4th instar nymphs, 5th instar nymphs, an active adult male, an active adult female, an adult male in diapause, and an adult female in diapause (Figure 1). The RNA was divided into two pools: pre-adult stages and adult stages in order to get a first glimpse at the differential expression of genes between developing animals and adults. From this RNA, strand-specific libraries were constructed and Illumina HiSeq was used to gather the sequences of these transcripts. Bioinformatic analysis, using the program Trinity, as well as a novel method that analyzed the strand-specificity of the data, led to the identification of 53,071 putative transcripts from 18,573 components (Figure 2). By integrating other data, such as comparison to known genes from other insects, this number was further narrowed to 13,211 representative transcripts (Table 1). For comparison, Drosophila Figure 2. Strand- specific Sequencing Method. An over- view of the steps in- volved in strand spe- cific sequencing. The final sequencing reads can be as- signed to a particular strand of DNA. The methods and steps undertaken in gener- ating the libraries are illustrated including how the sequences are loaded on the flow cell. melanogaster, the insect best-studied at the molecular level, is estimated to express ~ 14,000 genes (http://www.nature.com/scitable/topicpage/eukaryotic-genome- complexity-437), suggesting that this single sequencing approach likely identified the majority of the genes active in BMSB. To begin to determine the different functions carried out by the transcripts identified, their sequences were compared to genes from other insects and the transcripts were assigned to putative functional categories based upon the types of proteins they likely encode (Figure 3). Approximately 5,000 of the sequenced This research project supports Objective 1. Establish biology and phenology of BMSB in specialty crops 3 2014 Summary Report Year 3 transcripts could be assigned to functions based on this sequence comparison. They fall into the following major categories: information storage and processing, cellular processes and signaling, and metabolism. The top five sub-categories were general function prediction only (22.2%); DNA replication, recombination and repair (8.2%); posttranslational modification, protein turnover, and chaperones (7.9%); transcription (7.5%); and amino acid transport and metabolism (7.1%). Overall, this is very similar to the distribution of gene categories in other transcriptomes of eukaryotes, including insects. However, now that the BMSB-specific sequences are available for these genes, they can be used for monitoring and tracking pest populations and for designer gene targeting using approaches such as RNA interference (RNAi). Table 1. Summary of Assembly and Annotation Characteristic Before filtering After filtering Number of reads (both pools) 366,689,206 N/A Number of putative transcripts (both pools) 194,729 13,211 Average length (bp) 1,005 2,026 Standard deviation (bp) 1,474 1,592 Median length (bp) 439 1,649 Maximum length (bp) 27,655 24,046 Length >1000 bp 50,599 9,657 With a Uniref100 hit (e-value < 1e-10) 80,536 11,513 Matching unique Uniref100 proteins 37,160 9,993 With a NR hit (e-value < 1e-10) 80,262 11,497 Matching unique NR proteins 37,346 10,007 Number of Trinity components 123,175 13,211 Number of ORFs 89,684 13,210 >450 bp 61,569 11,141 With a function assigned 57,197 9,811 The mass-sequence information obtained is publically available and accessible to the scientific community for use by researchers in many different ways, dependent upon their individual research programs. Here we suggest several immediate applications that the transcriptome data will support: (1) Population tracking. The assembled transcriptome sequences will have heterogeneity that reflects some of the genetic heterogeneity of the US BMSB population. Such heterogeneity at the sequence level was identified in 11,462 different putative transcripts, 86.8% of the total number of putative transcripts identified. These polymorphisms can be used to examine the future spread of BMSB in the US, as well as for a retrospective tracking of current populations from the original invasion in Allentown. (2) Monitoring of insecticide resistance. Transcripts with functions related to pesticide detoxification were identified This research project supports Objective 1. Establish biology and phenology of BMSB in specialty crops 4 2014 Summary Report Year 3 in both the juvenile and adult RNA pools. Interestingly, there were 25 genes encoding proteins related to cytochrome P450 and glutathione S-transferase that were more abundant in adults. There were nine such proteins that were more abundant in the pre- adult RNA pool. Differential expression of these and other detoxification genes under field conditions may occur, providing an early warning signal for the emergence of pesticide-resistance in a population.