Benjamin Miner

INFECTIOUSNESS OF SEA STAR Western Washington WASTING DISEASE University Shannon Point Marine Center FUNDING AND COLLABORATORS

 NSF . co-PI Ian Hewson  WA SeaGrant . Co-PI Melissa Miner  Western Washington University SEA STAR WASTING SYNDROME

 General description for a set of symptoms that are found in sea stars

 Typically, lesions appear in the ectoderm followed by decay of tissue surrounding the lesions, leading to eventual fragmentation of the body and death GEOGRAPHIC EXTENT

 Data from MARINe  Locations of die-offs . Vancouver area BC (fall 2013) . Seattle area WA (fall 2013) . Monterey Bay area, CA (fall 2013) . Southern CA (winter & spring 2014) . Oregon coast (spring 2014) . Salish sea (late spring 2014) . Sitka AK & Northern Vancouver Island (fall 2014) . BC, CA SPECIES AFFECTED

 Evidence of disease . Pycnopodia helianthoides (sunflower star) . Evasterias trochelii (mottled star) . Pisaster ochraceus (ochre star) . Pisaster brevispinus (giant pink star) . Solaster dawsoni (morning sun star) . Patiria miniata (bat star) . Solaster stimpsoni (striped sun star) . Orthasterias koehleri (rainbow star) . Pisaster giganteus (giant star) . Dermasterias imbricata (leather star) . Leptasterias spp (six-armed star) . Mediaster aequalis (vermilion star) . Henricia spp. . Linkia columbiae (fragile star) . Astropecten spp. (sand star) . Crossaster papposus (rose star) . Stylasterias forreri (velcro star) . Astrometis sertulifera (fragile rainbow star)  Appears unaffected . Pteraster spp. CAUSE OF THE DISEASE

 Evidence suggests that it is a virus.  There is also evidence that environmental factors are involved.  Star probably die of a secondary bacterial infection. QUESTIONS QUESTIONS

Can individuals with signs of the disease affect “healthy” conspecifics? .Is the viral load of the densovirus associated with wasting disease higher in “sick” individuals? Does temperature affect the progression of the disease? INFECTIOUSNESS EXPERIMENT DESIGN PYCNOPODIA HELIANTHOIDES

 Collection sites . “Sick” site: Oil docks, Mukilteo, WA on SCUBA . High proportion of stars with symptoms . “Healthy” site: Langley marina, Whidbey Island, WA on SCUBA  Treatments . Direct contact . Symptomatic star with asymptomatic star . Water contact . 3 liters of water from “sick” site . Control . Two asymptomatic stars  Replication . Each treatment was replicated with 3 aquaria  Aquaria . Held in a large room heated to 10C, SPMC . Water changed every third day . Each aquarium was filtered  Stars fed mussels INFECTIOUSNESS EXPERIMENT RESULTS PYCNOPODIA HELIANTHOIDES

Mortality . All symptomatic stars died within 3 days . All stars in Direct Contact tanks died within 15 days . Both stars in 1 Water aquarium died after 15 and 25 days . Both star in 1 Control aquarium died after 30 and 31 days METAGENOMICS

100000000  A densovirus was initially identified 10000000 Healthy by sequencing the viromes of 4 1000000 Diseased 100000 individuals 10000  1000 qPCR was used to measure the viral 100 load of the densovirus in 325 10 1 individuals Pycnopodia Pisaster Evasterias . qPCR Primers and probes (TaqMan ochraceus trocheli chemistry) designed around the VP4 region 1 0.9 of the SSWDZV genome HealthyPrev% 0.8 DiseasedPrev% 0.7 . Normalized to wet tissue weight 0.6 0.5  The densovirus was present in both 0.4 0.3 “healthy” and “diseased” individuals 0.2 0.1 0 Hewson et al. (in press) PNAS Pycnopodia Pisaster Evasterias ochraceus trocheli VIRAL LOAD

Viral loads from experimental stars were inconsistent with data from previous slide. Surviving stars were held for two weeks after an arm was collected for viral load analyses

Died? TEMPERATURE EXPERIMENT DESIGN PISASTER OCHRACEUS

 Collection site . Post Point, Bellingham WA at low tide . Large proportion of stars with symptoms  Treatments Low ~ 9°C . Low treatment, similar to winter . 2 symptomatic stars . High treatment, similar to summer . 2 symptomatic stars  Replication . High treatment was replicated with 3 aquaria . Low treatment was replicated with 5 aquaria High ~ 12°C  Aquaria . Held in 9C cold room, WWU . Water changed about once a week . Each aquarium with own filter  Stars fed mussels collected from Post Point TEMPERATURE EXPERIMENT RESULTS PISASTER OCHRACEUS

Temperature slowed, but did not stop, the progression of the disease

INFECTIOUSNESS EXPERIMENT PISASTER OCHRACEUS

 Collection site . Post Point, Bellingham WA at low tide . Low proportion of stars with symptoms . All stars were collected from this site  Treatments . Direct contact . Symptomatic star with 2 asymptomatic stars . Control . 3 asymptomatic stars  Replication . Each treatment was replicated with 5 aquaria  Aquaria . Held in 10C cold room, WWU . Water changed about once a week . Each aquarium with own filter  Stars fed mussels collected from Post Point RESULTS

 All stars survived for two months  No evidence of “healthy” stars getting “sick”  Several stars with symptoms showed signs of healing

 We have not observed any deaths at Post Point, until June 2014 when the population of adults nearly disappeared in several weeks Pycnopodia Pycnopodia Pisaster ochraceus Evasterias RNA Virome Healthy Adenoviridae Diseased Alphatetraviridae Alvernaviridae Arteriviridae Asfarviridae Bacilladnavirus Baculoviridae Bidnaviridae Bunyaviridae Caliciviridae Caulimoviridae Circoviridae Coronaviridae Dicistroviridae Flaviviridae Geminiviridae Herpesviridae Iflaviridae Iridoviridae Marnaviridae Marseilleviridae Megaviridae Mimiviridae Nanoviridae Nodaviridae Nimaviridae Orthomyxoviridae Ourmarivirus Papillomaviridae Paramyxoviridae Parvoviridae Partiriviridae Phycodnaviridae Picobirnaviridae Picornaviridae Poxviridae Reoviridae Retroviridae Rhabdoviridae Tombusvirus Totiviridae Bacteriophage Unclassified Virus Procine bocavirus HN2 KC473563.1

Colobocentrotus atrata densovirus  Viral metagenomics used Sea Star Wasting Disease Associated densovirus to survey DNA viruses from 100 28 sea stars and RNA Densovirus SC116 JN857346.1

viruses from 4 stars Ixodes scapularis densovirus 2411173040

100 Acyrthosiphon pisum densovirus 320447039  Viral communities Tripneustes gratilla densovirus 62 dominated by 98 Periplaneta fuliginosa densovirus 9633605 bacteriophage & viruses 54 67 Echinometra mathaei densovirus infecting other 97

microorganisms Junonia coenia densovirus 23334597

Porcine parvovirus IA469 JX896320.1 59 63  Parvoviruses targeted Acanthaster planci densovirus because only animal virus Simian parvovirus U26342 seen in diseased stars 850 Adeno-associated virus 3B 100

565 Adeno-associated virus 2

Penaeus monodon densovirus 100

Infectious hypodermal and hematopoiec virus 0.1 substitutions / site PYCNOPODIA HELIANTHOIDES EXPERIMENT 1

 Collection site . Oil docks, Mukilteo, WA on SCUBA . High proportion of stars with symptoms . All stars were collected from this site  Treatments . Direct contact . Symptomatic star with asymptomatic star . Control . Two asymptomatic stars  Replication . Each treatment was replicated with 5 aquaria  Aquaria . Held in a large room heated to 10C . Water changed about once every day . Each aquarium was bubbled, but NOT filtered  Stars fed mussels bought from store IS SSWDZV ACTIVELY REPLICATING?

 Gene transcription (qRT-PCR) of SSWDZV VP4 gene indicates active 10000000 1 replication - 1000000 Healthy Diseased  Replication in Evasterias and 100000 Pycnopodia only detected in 10000 diseased stars and not in healthy 1000

100

10 VP4 Transcripts [Genome Copy] [Genome Transcripts VP4 ND ND 1 Evasterias Pisaster ochraceus Pycnopodia