Elucidating the mechanism of pesticide resistance in a non-target amphipod, azteca: Genetic and Functional Genomic Approaches Helen Poynton, Donald Weston, Gary Wellborn, Michael Lydy, Bonnie Blalock, Padrig Tuck, Maria Sepulveda, John Colbourne University of Massachusetts, Boston; University of California, Berkeley; University of Oklahoma; Southern Illinois University; Purdue University; Indiana University

Abstract Voltage gated sodium channel (vgsc) Hyalella azteca transcriptomics While pesticide resistance has been heavily documented in target pest , few studies have The voltage gated sodium channel is the target site for pyrethroid pesticides Summary of Hyalella azteca transcriptome sequencing by 454 investigated the evolution of resistance in non-target . We have detected one of the first The VGSC is essential for nerve impulse transmission. When Total number raw sequencing reads 1,039,832 examples of pyrethroid pesticide resistance in a non-target , the amphipod Hyalella stimulated, the sodium channel opens and allows Na+ to flow into Total number of bases 205 Mb azteca. H. azteca is a freshwater epibenthic and the the cell, depolarizing the membrane and creating an action Average coverage of transcriptome 4X primary species used for freshwater sediment toxicity testing in the U.S. potential. When the action potential is reached, the VGSC Number of contigs 65,961 Because H. azteca is a which has diverged in North becomes inactivated. Repolarization occurs until the membrane Number of singletons 147,877 America over the past 11 million years, our initial identification of reaches the resting potential. Number of annotated sequences (with BLAST 15,830 pesticide tolerant H. azteca in California prompted us to explore the Pyrethroids bind to the VGSC matches, e-value <1E-4) genetic structure of the tolerant organisms. Our studies revealed inside the channel and inhibit the Number of sequences with assigned GO terms 5,167 three different species groups among the seven populations examined, Hyalella azteca inactivation of the sodium with high pesticide tolerance detected across different species groups. channel, and repolarization of Hyalella azteca oligo microarray (12 x135k format) was constructed to Due to its important role in the monitoring of sediment quality, the neuron. it was imperative to unravel the mechanism behind the Several critical amino acid uncover transcriptional responses to pyrethroids in resistant populations variable sensitivity to pesticides. To understand the origin of residues have been identified pesticide tolerance, we investigated whether tolerant within the sodium channel that Experimental Design exhibited target site resistance. We sequenced the voltage-gated A lab strain and resistant strain of H. azteca were are essential for stable Modeling study of critical residues exposed to their population specific NOEC (no sodium channel (vgsc) gene, whose gene product is the target of pyrethriod binding. Mutations in important in pyrethroid binding observable effect concentration) of cyfluthrin. Due pyrethroid toxicity, in several populations and identified two Role of VGSC in nerve impulse these amino acids are associated within the sodium channel. From: to potential hybridization differences to the single nucleotide mutations, which appear to be responsible for transmission. From: Davies, et al. (2007) with pyrethroid resistance in Usherwood et al. (2007) FEBS microarray between the two species, animals were reducing pyrethroid toxicity 100-1000-fold and providing IUBMB Life. 59:151-162. Letters. 581:5485–5492. only compared to their own species control. many insect pests. pesticide resistance. In addition, we performed a transcriptomic UC Berkeley - lab Grayson Creek study to determine how resistant animals responded to VGSC mutations are Sediment Quality Triad: H. azteca is used by pyrethroid exposure. Transcriptomic analysis suggests that government agencies as a key species in resistant H. azteca, eventually succumb to pyrethroid toxicity, at associated with pyrethroid bioassessments and the predominant test much higher concentrations compared with sensitive organisms, Unexposed 0.4 ng/L Unexposed 170 ng/L species for sediment toxicity. resistance in H. azteca. not through inhibition of the VGSC, but through oxidative stress. Cloning and sequencing of Domain II of the vgsc from laboratory and wild populations revealed two alleles While pyrethroid exposure in UCB containing point mutations that H. azteca affected genes involved in Pyrethroid sensitivity across populations Gene expression patterns in resistant and wild-type H. azteca change the amino acid sequence of neurological system processes, the genes affected in the GC population were related after exposure to pyrethroid pesticides. Differentially expressed While screening sediments for pesticide toxicity, several populations of H. the protein. These mutations change genes are organized based on Gene ontology (GO) terms to Methionine -918 to Leucine or to the oxidative stress response suggesting illustrate the major biological processes affected. Red represents azteca were found living in sediments with high pyrethroid contamination. Leucine -925 to Isoleucine and are an alternate mechanism of action. up-regulation, green represents down-regulation. only found in the populations with Seven populations from high pyrethroid tolerance. Studies in insects and in vitro experiments have Northern California were Conclusions Sacramento found these two residues to be (A) Four domain structure of the VGSC illustrating where M918 Watershed tested for pyrethroid critical for pyrethroid binding to the PG and L925 are found. (B) Nucleotide and amino acid sequences Sacramento toxicity. Genetic analysis sodium channel. of VGSC Domain II in different H. azteca populations. Similar resistant alleles identified across populations and in LL MO BR of the COI gene was two species groups suggests multiple origins and convergent San Francisco MS GC conducted to determine if San Francisco Bay San Jose San Joaquin River evolution. Watershed Watershed multiple populations represented different Pleasant Grove Creek – evolution in progress Tulare Evolution of pesticide resistance in non-target arthropods Watershed species groups. Four CH Over the past three years, H. azteca collected from PGC have gained such as H. azteca appears to occur rapidly and may be 40 miles species groups were increased tolerance to pyrethroid pesticides. found with varying levels common in exposed populations. Locations of H. azteca sampling sites. H. azteca were collected from seven locations in N. California where sediment pyrethroid concentrations were of pyrethroid tolerance known. Relative pyrethroid concentrations in sediments are indicated by text These results have important implications color: Non-detect, Low , Medium , High. Sediments with medium and high shown in the table below. concentrations of pyrethroids were toxic to the laboratory strain of H. azteca. for bioassessments that rely on arthropod species including H. azteca to determine Species Cyfluthrin 96-h Relative Collection Site Group LC50 (ng/L) Tolerance sediment quality. Lab cultures C UCB 4.8 (3.9-6.2) 1 Wild Populations A Laguna Lake (LL) 4.8 (3.7-5.8) 1 Cyfluthrin - pyrethroid B Blodgett Reservoir (BR) 1.3 (1.1-1.5) 1 Toxicity testing results for PGC H. azteca collected in 2010 Species Group and genotype distribution of H. azteca Acknowledgements pesticide Pleasant Grove Creek 11.8 (8.8-14.7) 3 (dotted line) and 2013 (solid line). collected in 2013 (left) and 2010 (right). (PG) Continuing Funding of genetic and D Morrison Creek (MO) 132 (63.5-174) 30  Toxicity testing demonstrated that H. azteca in the PGC population increased their pyrethroid support for Mosher Slough (MS) 211 (176-244) 50 tolerance by an order of magnitude over three years. transcriptomic studies: Grayson Creek (GC) >691 >175  Genetic analysis showed that two species groups co-exist at PGC. Hyalella azteca Chualar Creek (CH) 535 (403-650) 100  vgsc sequencing revealed the presence of the L925I resistance allele in Species Group B. This allele genome project Species Groups were identified as A, B, or D. Relative tolerance refers to the was not detectable in 2010, but has increased in frequency to about 30% in Group B. BOSTON level of tolerance compared to the lab culture and was determined by dividing  All organisms that survived a 125 ng/L pyrethroid exposure contained the resistance allele. the wild population LC50 by the lab culture LC50.