Category Archives: genetics

Connecting the Fraser salmon virus dots

Are the Fraser chinook that southern resident killer whales love to eat already infected by the Infectious Salmon Anemia virus (ISAV) just detected in 2 Fraser sockeye smolts?  Could this virus — not salmon leukemia — be what caused the the mortality-related genomic signature in Fraser sockeye reported earlier this year?

Remember that DFO scientist Miller-Saunders told Scientific American last spring that “there is some indication that the signature may be in Chinook and coho” salmon, too.  To what data was she referring, I wonder?  Was it derived from out-migrating smolts or returning adults, wild or hatchery fish?  Was she referring to Fraser Chinook and coho, or some other stocks?

In contemplating how ISAV may affect the Northeast Pacific ecosystem, the Final Recovery Plan for U.S. Atlantic Salmon (Gulf of Maine DSP, 2005) is truly frightening reading.  The section on ISAV (appended below) suggests: that the virus can kill 3-50% of each production cycle and can infect non-Atlantic salmon (coho salmon in Chilean pens), as well as non-salmonids like rainbow trout (cultured) and gadids (potentially our pollack and cod species!); that rainbow and brown trout can be asymptomatic vectors; and that wild Atlantic salmon have been infected.  The plan also notes that “sea lice have been shown to retain the ISA virus after feeding on infected salmon.”  That’s pretty troubling when juxtaposed with recent research on lice infestation of wild B.C. salmon

The outlook for the Salish Sea ecosystem (and particularly it’s endangered salmon stocks) looks even dimmer after perusing an article about experimental infection of herring with ISAV.  The take home message (from the abstract): “It is concluded that the ISA virus is able to propagate in herring and that the herring may be an asymptomatic carrier of the virus.”

It’s going to be fascinating (and probably depressing) to see whether a pandemic develops.  If it does, the long-term outlook for southern resident killer whales may be bleak, especially if DFO fails to act at least as quickly and rigorously with the salmon farming industry as the U.S. agencies did when attempting to control the initial outbreaks in Maine.

Excerpt from the Final Recovery Plan for U.S. Atlantic Salmon (Gulf of Maine DSP, 2005) starting on page 1-60 —

ISA is a contagious and untreatable viral disease that affects a fish’s kidneys and circulatory system with a variable mortality rate from 3% to more than 50% in one production cycle (USDA APHIS 2001). Atlantic salmon infected with clinical ISA are anemic, typically lethargic, swim near the surface and fail to swim upright. Experimental studies have demonstrated that the virus is transmissible through mucous, feces and blood of infected/diseased fishes (Nylund et al., 1994). This results in cultured fishes being particularly susceptible to exposure to ISAV by infected cagemates. Studies in Norway indicate that penned salmon populations held within five kilometers (km) of each other or the discharge of slaughter wastes are at greatest risk of contracting ISA (Jarp and Karlsen, 1997). There is no evidence that the virus spreads vertically (from parents to offspring) although poor disinfection of fertilized eggs may allow for external transfer of the virus. Poor culture practices in fish hatcheries and net-pens in an Atlantic salmon watershed could increase the risk of a wild population’s exposure to disease.

ISA is the most significant known disease threat to the DPS. The threat of ISA to the recovery of the DPS is both direct, through infection of wild fish, and indirect by compromising hatchery supplementation of the DPS. The infection of emigrating smolts or adults passing near infected net-pens may cause mortality. This risk is greatest in those rivers whose approaches are nearest the highest concentration of net-pens, specifically the Dennys, East Machias and Machias. Other DPS river populations may also be at risk if they migrate through areas where aquaculture facilities are concentrated.

ISA has the potential to compromise CBNFH and the GLNFH if ISA-infected fish are inadvertently brought into one of these facilities. For example, an ISA-infected salmon brought into CBNFH for broodstock purposes could potentially infect other fish at the facility. In fact in 2001, a Penobscot sea run salmon brought to CBNFH for use as broodstock initially tested positive for ISA. Subsequent tests were negative and no additional fish were found to be infected. Outbreaks of ISA in freshwater hatcheries have not been reported from major salmon producing countries that have experienced ISA outbreaks. Still the potential for juveniles that have never entered salt water to be carriers of the virus is currently unknown.

ISA has already had an impact on Atlantic salmon recovery efforts. An adult stocking experiment (see page 4-69) was not fully optimized due to ISA concerns. These concerns resulted in more than 50% of the net-pen reared broodstock being destroyed. This decision was made because fish health experts felt the close proximity of these fish to fish infected with the ISA virus (ISAV) in commercial aquaculture pens was a substantial risk to wild populations. This concern was later affirmed by the outbreak of ISA in marine pens in the Cobscook Bay region (see page 1-82).

ISA was first reported in Norway in 1984 (Thorud and Djupvik 1988). In more recent years, cases of the disease have been reported from eastern Canada (Mullins et al. 1998), Scotland (Rodger et al. 1998), the Faroe Islands (OIE 2000), and in Cobscook Bay, Maine (Bouchard et al. 2001). The virus has also been associated with disease in cultured coho salmon in Chile (Kibenge et al. 2001) and very recently has been detected in cultured rainbow trout in Ireland.

The ISA virus has been known to cause disease in cultured fishes, principally in Atlantic salmon, although other species may act as carriers of the virus without signs of the disease. Species other than Atlantic salmon can become infected with ISAV and must be considered in the epizootiology of outbreaks and management of ISA. In laboratory studies, brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) have been shown to be asymptomatic carriers of the ISA virus that can transmit the virus to salmon by co-habitation (Nylund and Jakobsen 1995; Nylund et al. 1995; Nylund et al. 1997). Escaped or caged rainbow trout may pose a threat to wild Atlantic salmon by serving as a reservoir of ISAV.

Recent studies in the United States and Canada indicate non-salmonids (i.e., gadids) can become infected with ISAV. Whether these species act as reservoirs in wild populations remains to be determined. Assays of non-salmonid fishes taken from pens containing ISA-diseased cultured Atlantic salmon resulted in isolation of virus from tissues of asymptomatic cod (MacLean et al. 2003).

Results of recent studies conducted in Scotland and Canada indicate that ISAV exists at a low level in wild salmonids. ISAV has been found in Atlantic salmon aquaculture escapees (Olivier 2002; Raynard et al. 2001). There has been one case of wild salmon exhibiting ISA in Canada, but these wild fish were confined in a trapping facility with infected salmon of aquaculture origin.

At the time of the listing of the DPS as endangered in December 2000 (65 FR 69459), some U.S. net-pen sites in Cobscook Bay, the location of Maine’s greatest concentration of salmon aquaculture pens, were within five km of Canada’s ISA positive sites, raising concerns about the potential for this disease to infect U.S. aquaculture and wild salmon stocks. Subsequent to the listing of the Gulf of Maine DPS of Atlantic salmon as endangered, the disease spread to U.S. aquaculture sites within Cobscook Bay. The first known case of ISA in Maine occurred in Cobscook Bay at a salmon aquaculture net-pen site. The infection probably occurred in 2000 and was confirmed in February 2001. By September 2001, 50% of the net-pen sites in Cobscook Bay were ISAV-infected or diseased.

In January 2002, in an effort to control a catastrophic outbreak of ISA in Cobscook Bay, the Maine Department of Marine Resources (DMR), with the assistance of the U.S. Department of Agriculture’s Animal and Plant Health Inspection Service (USDA/APHIS), ordered the destruction of an estimated 1.5 million cultured salmon in the Bay. The industry was required to remove all fish from the Bay and a fallowing period, between sixty and ninety days, was imposed for the entire Bay in an attempt to eradicate the disease. The industry was also required to remove, clean and disinfect all the associated net-pens, barges and equipment at all the farms. The January 2002 order followed the voluntary removal by the aquaculture industry of nearly one million ISA- infected or exposed fish. In March 2002, ISA was also detected in an aquaculture facility in Passamaquoddy Bay. In response, the DMR issued an eradication order for the approximately 140,000 fish at the site.
In response to the ISA outbreak in Cobscook Bay, Maine DMR implemented new fish health regulations. The new DMR rules include mandatory surveillance and reporting of all test results for ISAV in salmon culture facilities. Sites with confirmed presence of ISAV are automatically subject to a remedial action plan developed by the DMR in cooperation with the salmon growing industry. Under the new regulations, the movement of vessels and equipment is also restricted. Prior to the rule changes, surveillance was not mandatory and reporting was only required when a case of the disease was confirmed.

The new rules require monthly sampling for all active finfish facilities in Cobscook Bay and quarterly testing for aquaculture facilities elsewhere in Maine. Reporting of results is mandatory and reports are provided to DMR. The DMR can require monthly testing for finfish facilities outside of Cobscook Bay if a positive case of ISAV is detected. The new rules expand DMR’s authority to take action at not only infected facilities, but also those exposed to ISAV. The rules require DMR to consult with all relevant state and federal entities with expertise in ISA control to keep ISA from spreading and prevent further outbreaks.

In response to the ISA outbreaks, the Maine DMR, with assistance of the USDA/APHIS also implemented an ISA control and indemnity program for farm-raised salmon in the U.S. The funds provided by the USDA were used to help the State of Maine with epidemiology and surveillance, and to indemnify the industry for their losses due to ISA. Under the DMR rule, all salmon growers in Maine must participate in the program. The goal of this program is to control and contain the disease through rapid detection and depopulation of salmon that have been infected with or exposed to the ISA virus.

In Spring 2002, Maine DMR authorized the restocking of Cobscook Bay. The Bay had lain fallow since January 2002. This authorization followed USDA approval of the cleaning and disinfection of equipment and the fallowing period. Subsequent to approval, the aquaculture industry stocked 1.9 million smolts on seven farms in Cobscook Bay. The number of smolts stocked was 30% lower than the amount historically stocked in this area (DMR 2002). New husbandry standards have also been put in place as part of the ISA control program. These new standards are administered by DMR.

The ISA control program initially divided Cobscook Bay into two management areas, a southern and a northern zone. The southern zone was stocked in even years beginning in Spring 2002. The northern zone was stocked only in odd years, beginning in Spring 2003. Recently, USDA and Maine DMR have determined that the entire Cobscook Bay would be managed as a single area. DMR estimated that by there would be approximately 25% fewer fish in Cobscook Bay compared to previous levels. In addition, several conditions are required for each lot of smolts that are introduced into net-pens from freshwater hatcheries. All aquaculture facilities in Cobscook Bay are only permitted to raise a single-year class of fish. A minimum thirty-day fallowing period between production cycles is required. No more than 10% of the fish at a site may be carried over between production cycles and then only upon approval by DMR. This approval requires that no ISA is detected at the site during the production cycle, that general fish health is satisfactory, that fish are removed by September 1, and that there be a biweekly surveillance of the site by a fish health professional. Movement of fish between farms in the same zone requires a permit and verification that ISAV has not been detected at either site in the four weeks prior to movement. There will be no moving of fish between zones. In addition, farms, aquaculture vessels and processing plants are subject to routine third-party biosecurity audits. Despite these measures, additional cases of ISAV were detected at aquaculture sites in Cobscook Bay beginning in June 2003 and continuing in 2004.

The DMR’s bay management program was developed following an evaluation of other bay management and ISA control programs in Canada, Ireland, Scotland and Norway. These nations have developed control programs intended to prevent further outbreaks of the disease. The DMR plans to codify bay management husbandry standards in a rule and establish other bay management areas where finfish leases are located. Successful sea lice management and control is a necessary component of bay area management as sea lice have been shown to retain the ISA virus after feeding on infected salmon (Nylund et al. 1993).

During routine surveillance of all salmon culture sites in Maine, an apparently new strain of ISAV was detected in November 2003 at a site approximately 50 miles from Cobscook Bay. This was the first detection of ISAV at any site in Maine other than Cobscook Bay. The new strain did not cause disease in the cultured salmon and did not grow in the laboratory on various cell lines typically used in ISA isolation. Gene sequencing of this organism indicates it is more closely related to a Norwegian strain than the New Brunswick strain that has caused the mortalities in Cobscook Bay. Subsequently, this new strain has also been found in Cobscook Bay sites. Efforts are underway to sequence archived samples to determine the significance of the virus in the Cobscook Bay system.

One potential mode of disease transmission is through biological sampling conducted by various state and federal agencies in DPS rivers. The development and implementation of disinfection and biosecurity protocols reduces the risk of a pathogen being moved from one location to another (G. Russell Danner, IF&W fish pathologist, personal communication 2004). Disinfection and biosecurity protocols, where not already in place, should be developed and implemented for all research and sampling activities taking place in rivers within the DPS (see page 4-63).

Virus implicated in Fraser sockeye (and chinook?) mortality

The idea that a virus may play a part in the unpredictable Fraser river sockeye returns is (month) old news, but this article in Scientific American is the first to mention chinook that I’ve seen.  Perhaps the fate of the southern resident killer whales (who specialize on Fraser chinook in the summertime) is more connected than we thought to whatever marine factors govern the population dynamics of Fraser River sockeye?

“One of the most important findings of this study was the fact that salmon were already compromised before entering the river” on their journey home to spawn, she wrote. The scientists are currently studying juvenile salmon to see if the genomic signature is already present before they go out to the open ocean. Miller-Saunders also reports “there is some indication that the signature may be in Chinook and coho” salmon, too.

Orca genetics talk by Phillip Moran

Using next generation sequencing to generate whole mitochondrial genomes for population genetics and phylogeography of cetaceans

Dr. Phillip Morin, Protected Resources Division, Southwest Fisheries Science Center

Abstract and bio

Live blog notes:

Hoelzel et al 2002 found extremely low genetic diversity in control region (1000 base pairs): only 13 haplotypes from 100 samples from global killer whales. LeDuc et al 2008 increased to 35 haplotypes in ~>180 samples, but still very little global structure in phylogenetic tree.

But there are good reasons to use whole mitochondrial genome (16.4 kilobase genome) broken into 2-3 overlapping products (4.8-9.4 kb). Next generation sequencing uses highly parallel sequencing of small (30-350bp) fragments, but generate 100 million to 10 billion copies very economically and quickly.

Gathered north pacific samples (only 5 offshore), including ENA (Eastern North Atlantic who differ most in tooth wear) type 1 and 2, offshore, resident, transient, unknown. Also had samples from Antarctic whales and by Andy Foote from N Atlantic whales. We used Baysian techniques and publicly available mitochondrial priors from a wide range of marine mammals and managed to date divergence in killer whales to ~700,000 years ago.

Killer whale mitogenetics show that transients diverged ~700ky ago. In comparison, residents and offshores diverged much more recently, ~175ky ago (e.g. conventional wisdom: beginning of the pliocene). Antarctic B/C diverged from each other 150ky ago, and from A/GoM 335ky. Nuances are: proximity of ENA (1/2) and a Hawaii whale to North Pacific residents/offhores hints of exchange through the Northwest passage; some Antarctic A individuals have a haplotype close to transients, suggesting there may be even more types of killer whales in Antarctica (Bob plans to find out).

De Queiroz, 2007: helps in defining of species/subspecies — a hot topic for killer whales

  • B/C Antarctic types have strong morphological, feeding behavior and prey, group size, and genetic differences.  Foote et al. 2010.
  • N Pac transients: should be distinct species, primarily due to genetic divergence, though they also differ in morphology, feeding behavior and prey, group size, acoustics, fatty acids, contaminants.
  • Resident/Offshores we tend to believe are different sub-species, or species awaiting more evidence.  We have especialluy little info about offshores (only 5 samples and minimal behavioral differences).
  • North Atlantic situation is undetermined.

So, we had this low world-wide diversity (even in microsatellites — why?).  With whole mitogenome, we have strong association of ecotypes and genotypes.  For species with low mtDNA sequence diversity or poor phylogenetics, these new techniques can be very useful!

Other species that could benefit:

  • Blue whales (taxonomy and population structure, using SNPs)
  • Fin whales (150 mitogenomes sequenced but not analyzed; clear need for analysis of whether N Pac and Atlantic are really the same species (likely a historic taxonomic mistake)
  • Sperm whales (even less diverse than KWs — globally about 30 haplotypes, but 90% of samples fall into 3 haplotypes)
  • Turtles (effectively dinosaurs — been around for millions of years w/only 7 species and handful of haplotypes; SNPs may help describe population structure of leatherback and green turtles that move around the globe and are currently hard to genotype to source location when caught in longline fisheries)

Mike Ford Q: have you estimated historic population sizes from your results?  We’ve only recently started those analyses and we’re overwhelmed with data.  A current Masters student is looking at rates of patterns of evolution in mitochondrial genome.  Hoping to fund a post-doc (or any other collaborators!) to look at historic population size.

Q: Did you differentiate between N Pacific residents: We had 1? southern resident and a couple from Russia, but no BC residents.

Q: What’s difference between ecotype and subspecies?  It’s a really tough call (demographically distinct, DPS, evolutionarily distinct…).  In my mind, a subspecies is one in which you have multiple lines of evidence (not necessarily including genetic) suggesting distinctive evolutionary trajectories.  There is likely gene flow in delphinids (some evidence from microsatellite data, but some is suspect inference).

Q: Is there an issue with nodes evolving at different rates?  Our MS student is working on that and has a manuscript in preparation, but we’re still confident in our times.

Q: What are the different potentials of mitochondrial, microsatellites, and SNPs as tools for understanding evolution?  I hate microsatellites because we don’t understand them, especially their mutation rates (overestimate gene flow and underestimate divergence time)!  They indicate divergence, but aren’t diverging linearly in time.  SNPs are so simple in comparison!