Gelatinous zooplankton and Scottish
salmon aquaculture: three investigations
into hydrozoan ecology
by
Anna H. Kintner
Clytia hemisphaerica (Hydrozoa) medusa collected from a
salmon farm in Lochaber, Scotland. Photo by the author.
9-month Report, 27 July 2012
Submitted in partial requirement for fulfillment of the degree of Doctor of Philosophy in Marine Biology
5mm
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Introduction
Blooms of jellyfish represent a major source of mortality, morbidity and expense to the salmon
aquaculture industry in Scotland. At present, very little information is available to apply toward avoiding
or relieving the effects of such blooms, and the industry remains at risk. The goals of my project for the
degree of Doctor of Philosophy in Marine Biology are to examine the physical and ecological factors
which may affect the occurrence of medusae at Scottish salmon aquaculture, particularly hydrozoan
species such as Phialella quadrata. This 9 month report gives three experiments toward this end taking
place over the summer season of 2012, plus additional sampling and long-range aims.
Contents of the 9 month review
I.
Investigation 1: Settlement preferences of medusozoan larvae in proximity to Scottish salmon
aquaculture facilities (page 3)
II. Investigation 2: Microbial endosymbiosis in Scottish jellyfish medusae: potential endosymbiotes
and fish pathogens (page 8)
-Pilot investigations completed to date
III. Investigation 3: Prospective seasonal monitoring of medusozoa at Scottish salmon aquaculture
facilities (page 12)
IV. Research timeline and other objectives (page 14)
-Co-objective:
-Establishment of a Phialella quadrata culture
-collection of P. quadrata medusae for venom ecology studies
-Timeline for future research
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Investigation 1: Settlement preferences of medusozoan larvae
in proximity to Scottish salmon aquaculture facilities
Introduction
Medusozoan zooplankton of the classes Scyphozoa and Hydrozoa are known sources of morbidity and
mortality in Scottish salmon aquaculture, particularly between late June and late September (e.g. Bruno
and Ellis 1985, Seaton 1989, McKibben and Hay 2002, Doyle et al. 2008, Tørud and Håstein 2008, Baxter
et al. 2011, Ferguson et al. 2011, Mitchell et al. 2011, Mitchell and Rodger 2011). The seasonal presence
of jellyfish around salmon aquaculture pens can cause anything from chronic lamellar thickening and
inflammation of the gills, leading to generally poorer condition and slower growth (e.g. Baxter et al.
2011), to outright fish kills during major jellyfish blooms, with mortality sometimes reaching 70% or
more (e.g. Ferguson et al. 2011).
With the notable exception of some pelagic scyphozoan species such as Pelagia noctiluca, jellyfish
medusae are produced asexually by a sessile, polypoid life stage; the medusae themselves mature in the
plankton and release gametes, which after fertilization settle out as larvae to develop into new polyps.
In the Class Hydrozoa, these polyps are colonial, with colonies developing structural complexity and
some differentiation into feeding polyps (hydranths) and reproductive polyps (gonangia). Within-colony
reproduction is asexual, with new polyps budding from others. In the Class Scyphozoa, polyps are
individual and reproduce asexually by strobilation (transverse fission of the body structure). Each
medusozoan species may have a different set of environmental stimuli that trigger the development and
release of medusae (e.g. Arai 1992). Nonetheless, it is obvious that a large benthic population can
potentiate greater production of medusae (Hoover and Purcell 2009). This may be of particular concern
in Scottish, Irish and Norwegian aquaculture. In the Northeast Atlantic region, salmon farming facilities
are usually sited within semi-protected sea lochs and fjords, where hydrographic regimes may vary from
frequently flushed zones with strong oceanic influences to areas with higher residence time, slower
currents, and fewer opportunities for produced medusae to be carried out into a larger zooplankton
milieu (Gillibrand and Turrell 1997). In areas of restricted flow, benthic polyp populations could have a
particularly deleterious effect, as most medusae produced would tend to be retained locally.
In considering possible ways to moderate the effects of jellyfish, then, it should be considered that
human-made structures may be artificially inflating the population of benthic polyps, by providing
habitat for larval settlement and colonial growth. Several species of scyphozoans (including Cyanea
capillata and Aurelia aurita, both of which have been shown to have deleterious effects in aquaculture)
have previously been shown to prefer artificial materials such as concrete, polystyrene, wood, plastic
and glass to natural materials such as shell and macroalgae (Holst and Jarms 2007). In addition, the
placement of artificial structures in muddy or sandy, soft-benthos environments can provide hard
surfaces for larvae to settle where they would normally be unable to do so, thus expanding the range of
the species into previously unavailable areas, such near the heads of estuarine bodies of water (Holst
and Jarms 2010). Hoover and Purcell (2009) showed that Aurelia labiata polyp populations expanded
quickly when presented with new dock building materials, as measured both by field-based
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experimental plates and in laboratory settlement experiments. The population growth capacity for the
polyp phase of medusozoans is augmented by the fact that they are able to survive in a wide range of
dissolved oxygen and salinity regimes, and thus take advantage of conditions where other fouling
organisms might be unable to survive and compete for substrate space (e.g. Holst and Jarms 2010, Ishii
and Katsukoshi 2010).
Careful selection of materials which minimize the artificial habitat provided to colonizing polyps could
cut back on the magnitude and range of medusa populations present at aquaculture facilities. This
study seeks to examine the settlement preferences of Scottish medusozoan species, so as to make
appropriate recommendations for this approach.
Methods
Two study sites were generously provided by Marine Harvest on the Scottish mainland and four sites
were provided by Hjaltland Seafarms/Grieg Seafoods in Shetland. These sites were selected in order to
cover a greater area of oceanographic influence than would be available on the mainland or on Shetland
alone.
Mainland sites:
Linnhe (N: 56°43’32”, W: 5°14’33”), a Marine Harvest site near the Ardgour side of the Corran Ferry in
Loch Linnhe, south of Fort William. This site has been subjectively described as “relatively jellyfish-free”.
As Loch Linnhe is a mainland sea loch with the Hebridean islands to the west acting as barriers to direct
North Atlantic wind and wave-driven currents, the predominant inflow of oceanic waters comes from
the north-flowing Scottish Coastal Current, which carries northeastern Atlantic waters from south of the
Hebrides along the Minch and inshore areas and delineates the northern limit of a number of algal and
invertebrate species (Hill et al. 1997, Connor and Little 1998). Local freshwater inputs occur from
adjoining lochs and tributary rivers, giving a salinity range of ______________. Southwesterly winds
prevail in this area, and since Loch Linnhe is in a southwest-northeast orientation with high surrounding
topography funneling wind along the length of the loch, it is subject to wind-driven surface currents and
wave action (Salama et al. 2011). These plus local tidal eddies will determine dispersal of hydromedusae
and other plankters.
Invasion Bay (N: 56° 40’52”, W:5°36’28”), a Marine Harvest site in Loch Sunart along the Ardgour
peninsula. This site is described as being subject to frequent blooms of scyphozoan medusae,
particularly Aurelia aurita and Cyanea capillata. Loch Sunart is divided into a series of basins by seven
shallowing sills throughout the loch; Invasion Bay is situated in the uppermost basin near to the head of
the loch (Edwards and Sharples 1986). While the proximate sill rises to a depth of 8m, strong internal
tidally-generated currents promote high-volume water exchanges in the upper layers of this basin (Black
2005), suggesting that large scypho medusae may be frequently swept in by this water movement and
retained by shore topography and benthic features. As with Linnhe, Atlantic water inputs to Invasion
Bay come from a Scottish Coastal Current source, and the loch is subject to riverine freshwater inputs.
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Figure 1: Map of mainland study sites
Shetland sites:
North of Papa (N: 60°7’57”, W: 1°16’57”), a site jointly operated by Hjaltland Seafarms and the North
Atlantic Fisheries College in Scalloway, Shetland on the southwest side of the Shetland main island.
Salmon cages are situated in 30-35m of water in a large, southwest facing bay with a number of small
islands utilized for sheep grazing (UKHO Chart 3283). Freshwater inputs to the area are limited to
rainfall with a mean of 1300-1700 mm annually (National Meteorological Library and Archive 2011) and
salinity fluctuates very little (Hope 2008). The bay is shared with several other salmon and mussel
growers’ sites within one mile of the study site, managed by Hjaltland Seafarms and Scottish Seafarms.
The primary water mass influence comes from the Shelf Edge Current through the North-West
Approaches, which maintains a flow of warm, saline, nutrient-rich water from the eastern Atlantic to the
west of the British Isles toward the Norwegian Sea (Maravelias and Reid 1997, Turrell et al. 1996.). This
current passes directly to the west of the Shetland Islands and continues north via the Faroe-Shetland
Channel (Turrell et al. 1996), with Atlantic water from this current communicating with the North Sea
via the relatively shallow and largely wind-driven Fair Isle Current to the south and the far northern East
Shetland Atlantic Inflow to the north of the Isle of Unst (Turrell et al. 1996, Napier 2006). Locally,
dispersal of plankton in the upper water column is affected by wind-driven surface currents; an easterly
or northeasterly wind would tend to lead to point-source dispersal of plankton (including
hydromedusae, scyphomedusae, and ephyrae) to the west, and eventual carriage by tidal currents to
other sites to the north and south. Westerlies would tend to cause aggregations of planktonic larvae in
the bay, particularly in small rocky inlets or geos. Since the prevailing winds in all of Shetland are
southwesterly, some (albeit limited) subpopulation development of larval-dispersed invertebrates such
as velvet crabs has been observed to occur (e.g. Henderson et al. 2005), though this has not been
investigated fully, nor applied to neritic gelatinous zooplankton.
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Hamar Sound (N: 60°23’24”, W: 1°25’7”) a Hjaltland Seafarms site in the northwest of the Shetland main
island. Salmon cages here are situated in a western-exposed voe (fjord) 10-18m in depth (UKHO Chart
3281). Large-scale current and dispersal influences here are similar to those at North of Papa, though
this site is sheltered from the west by high sea-cliffs. The surrounding geology, shallower depth, and
directional exposure also contributes to lighter wave action due to diminished fetch and shelter from
incoming swell. Freshwater input and salinity and temperature fluctuations are within the same
seasonal range as North of Papa (Donaghy 2007).
Setterness (N: 60°23’24”, W: 1°7’24”), a Hjaltland Seafarms site on the northeast mainland, in
Boatsroom Voe in the Sound of Yell. This is a northeast-exposed site south of the island of Yell, with
salmon cages placed in 16-20m of water (UKHO Chart 3282). As with Hamar Sound and North of Papa,
limited temperature and salinity fluctuations occur. Prevailing water movement and planktonic
dispersal here takes place as a result of two factors: first, a shallow tidal race occurs in the Sound of Yell,
where relatively shallow water results in fast-moving tidal currents of up to 1.4 knots alternating
between southeasterly and northwesterly in direction (UKHO 1986). This race may have an effect on
dispersal of gelatinous zooplankton larvae and medusae. Second, the site is exposed to mixed North Sea
and Atlantic water masses via the East Shetland Atlantic Inflow flowing south from (Maravelias and Reid
1997). However, point-source dispersal by these currents is countered by the prevailing southwesterly
winds. Dispersal of settling zooplankton to and from this site may be complex and largely determined
by timing and volume of tidal inflows.
Wadbister (N: 60°15’45”, W:1°11’49”), a Hjaltland Seafarms site on the southeast mainland. Cages here
are in 20-22m of water, sheltered somewhat from prevailing southwesterly winds by a low western
headland (chart 3283). Water influxes are dominated by North Sea water masses (Maravelias and Reid);
some East Shetland Atlantic Inflow will be of consideration, but the bulk of this current lies well to the
east of this region of Shetland (Napier 2006). Of the four Shetland sites, this site has the least exposure
to North Atlantic inflows, and is the most sheltered from wind-driven wave action.
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Figure 2: Map of Shetland study sites.
Three replicate experimental arrays consisting of four different settlement plates will be placed at each
site. Arrays consist of a translucent polyethylene plastic box enclosed at top and bottom and on two
sides, with a front and back left open (Figure 1). The box is fitted with four 15 cm x 15 cm removable
settlement plates in a “bookshelf” design and held in place by a vertical plastic pin. Each plate is of a
different material: the uppermost plate is of high-density polystyrene foam, the second of stainless
steel, the third of opaque polyethylene plastic, and the fourth natural slate, with 5 cm vertical space
between each shelf.
Figure 3: Settlement array with four plates of varying material.
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Each array was hung at 4 m depth from floating pontoons at each study site. Checks on the arrays will
be carried out on a three-weekly basis. Arrays will be pulled up and removed to a bucket of seawater,
and the shelves removed one by one, photographed top and bottom, and percent cover of fouling of
each species calculated (including notable biofilms). Using a dissecting microscope, settled organisms
will be identified visually, and percent cover of medusozoan polyp species calculated. Historical data
shows reliable presence in the plankton of Leptomedusan species such as Clytia hemisphaerica and
Phialella quadrata until late September (Shucksmith unpublished data); in order to encompass the
whole of settlement opportunities by larval hydrozoans, this study will end in the first week of October.
Polyps will be removed by scraping with a razor and weighed to calculate wet biomass of each species.
Total percent cover, and species percent cover, will be separately compared using a generalised linear
model, with site and settlement material as variables.
Anticipated advisory outcomes
Results of this study will be used to develop information to help diminish or avoid deleterious effects on
salmon farms. An understanding of the settlement preferences of medusozoan larvae would afford a
near-term opportunity to make useful recommendations for aquaculturists regarding building materials
and possible ways to manage locally-blooming populations. Data collection will be complete by late
September 2012, and analysis completed in the following months.
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Marina, 56(2-3), pp.99-108.
Black, K. (coordinator), 2005. Ecological effects of sea lice medicines in Scottish sea lochs: Final report 9 February 2005. Scottish
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toxin, Cyanea capillata]. Bulletin of the European Association of Fish Pathologists, v.5, pp. 64-65
Baxter, E.J., Rodger, H.D., McAllen, R., and Doyle T.K., 2011. Gill disorders in marine-farmed salmon: investigating the role of
hydrozoan jellyfish. Aquaculture Environment Interactions, 1(3):245-257
Baxter, E., Sturt, M., Ruane, N., Doyle, T., McAllen, R., Harman, L., & Rodger, H., 2011. Gill damage to Atlantic salmon (Salmo
salar) caused by the common jellyfish (Aurelia aurita) under experimental challenge. PloS one, 6(4), p.18-29.
Brown, J. 2005. Hydrographic Survey Report: Boatsroom Voe. Prepared for Hjaltland Seafarms Ltd. Shetland Seafood Quality
Control, Port Arthur, Scalloway.
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Doyle, T., De Haas, H., Cotton, D., Dorschel, B., Cummins, V., Houghton, J., Davenport, J., and Hays, G., 2008. Widespread
occurrence of the jellyfish Pelagia noctiluca in Irish coastal and shelf waters. Journal of Plankton Research, 30(8), pp.963-968.
Edwards, A., and Sharples, F. 1986.
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for farmed salmon (Salmo salar). Journal of veterinary diagnostic investigation: official publication of the American Association
of Veterinary Laboratory Diagnosticians, Inc. 22(3), pp.376-82.
Gillibrand, P.A., and Turrell, W.R. 1997. The use of simple models in the regulation of the impact of fish farms on water quality
in Scottish sea lochs. Aquaculture 159: 1-2, pp. 33-46.
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of velvet crabs (Necora puber) in Shetland. North Atlantic Fisheries College Development Note 17.
Hill, A.E., Horsburgh, K.J., Garvine, R.W., Gillibrand, P.A., Slesser, G., Turrell, W.R., Adams, R.D. 1997. Observations of a densitydriven recirculation of the Scottish Coastal Current in the Minch. Estuarine, Coastal and Shelf Science 45, pp. 473-484.
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German Bight, North Sea. Marine Biology 151: 3 pp. 863-871.
Holst, S., and Jarms, G. 2010. Effects of low salinity on settlement and strobilation of Scyphozoa (Cnidaria): Is the lion’s mane
Cyanea capillata (L.) able to reproduce in the brackish Baltic Sea? Hydrobiologia 645, pp. 43-68.
Hoover, R.A., and Purcell, J.E. 2009. Substrate preferences of scyphozoan Aurelia labiata polyps among common dock-building
materials. Hydrobiologia 616, pp. 259-267.
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Control, Port Arthur, Scalloway.
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Tokyo Bay. Journal of Oceanography 66, pp. 329-336.
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abundance using generalized additive models. Marine Ecology Progress Series 147, pp. 1-9.
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samples in Loch Shieldaig , Western Scotland in relation to local salmon farm production cycles . International Council for the
Exploration of the Sea, Copenhagen.
Mitchell, S.O., Baxter, E. J., and Rodger, H.D., 2011. Gill pathology in farmed salmon associated with the jellyfish Aurelia aurita.
The Veterinary Record 169 (3) pp.1-3.
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Napier, I.R. 2006. A preliminary analysis of CPR and other environmental data for the waters around Shetland. University of
Highlands and Islands Staff Sabbatical Project, NAFC Marine Centre/UHI Millenium Institute.
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19th International Congress on Modelling and Simulation, Perth, Australia 12-16 December 2011.
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Shucksmith, R. unpublished. Seasonal changes in the abundance and community composition of gelatinous zooplankton in
Sandsound, Shetland, northern Scotland. Data collected through North Atlantic Fisheries College, Port Arthur, Scalloway,
Shetland.
Tørud, B. and Håstein, T., 2008. Skin lesions in fish: causes and solutions. Acta Veterinaria Scandinavica, 50 (Suppl 1), p.S7.
Turrell, W.R., Slesser, G., Payne, R., Adams, D., Gillibrand, P.A. 1996. Hydrography of the East Shetland Basin in relation to
decadal North Sea variability. ICES Journal of Marine Science 53, pp. 899-916.
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Hydrographic Office, Taunton.
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Office, Taunton.
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Investigation 2: Microbial flora in Scottish jellyfish medusa:
potential endosymbiotes and fish pathogens
Introduction
A 2008 fish kill event at a Scottish salmon farm in the Shetland Islands was found to be the result of a
bloom by the jellyfish Phialella quadrata, a member of class Hydrozoa. Jellyfish blooms are well known
sources of morbidity and mortality at fish farms via two principal mechanisms: envenoming fish via
stinging nematocysts on the tentacles (e.g. Seaton 1989) or by causing localized anoxic conditions during
particularly high-density blooms (e.g. Doyle et al. 2008). In this particular bloom event, a possible third
mechanism was added: the bacterium Tenacibaculum maritimum was found to be colonizing both the
gills of affected fish and the mouth parts of the jellyfish themselves, suggesting that the medusae may
act as vectors for the bacteria. If this is the case, this would be the first time that jellyfish have been
found to be playing such a role (Ferguson et al. 2010).
Questions remain about the nature of the relationship between P. quadrata and T. maritimum, as well
as the proposed route for the development of tenacibaculosis in fish. While the above study (Ferguson
et al. 2010) clearly linked the two species in situ in affected fish gills during a bloom event, it did not
seek out T. maritimum from P. quadrata medusae not associated with the bloom, thus failing to rule out
the possibility that the medusae had acquired T. maritimum infection from the fish rather than vice
versa (Ferguson et al. 2010). Other researchers have suggested that T. maritimum may be part of the
“normal flora” in North Atlantic salmon gills, because congeners have been found infrequently in
Norwegian salmon farms (Steinum et al. 2009, Mitchell pers. comm. 2012). In this model, stings by P.
quadrata nematocysts cause primary insult to the gills, which then enables opportunistic and
pathogenic overgrowth by the T. maritimum bacteria already there (Mitchell, pers.comm. 2012). Until
P. quadrata medusae not associated with a harmful bloom can be sampled for the presence of T.
maritimum, this model remains a possibility. However, Delannoy et al. (2011) have found T. maritimum
colonizing the mouth parts of Pelagia noctiluca medusae captured far offshore and in no association
with aquaculture, lending credence to the idea that T. maritimum may be a symbiont of other jellyfish.
The possibility of microbial endosymbiosis, and pathogenic vectoring by medusae bears further
investigation from ecological and epidemiological standpoints alike. Examining the assemblage of
endogenous flora and the relationships these species have with host medusozoan species may shed light
on aspects of the jellyfish life cycle and predation strategies. An understanding of regional variation and
spread of bacterial species associated with jellyfish, plus a better awareness of which of these may be
pathogenic in salmon aquaculture, could help inform strategies for mitigating certain types of morbidity
and mortality. This study aims to gather data serving both of these purposes.
Methods
Two salmon aquaculture sites will be selected based on geographic location around the Shetland
mainland. Access to these sites has been granted by Hjaltland Seafarms. Bacteria will be sampled and
cultured from three different species of jellyfish:
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1) Phialella quadrata, the hydrozoan species initially found to be carrying T. maritimum (Ferguson
et al. 2010);
2) Aurelia aurita, a common and widely distributed scyphozoan species implicated in long-term gill
pathology in farmed salmon (e.g. Baxter et al. 2011, Mitchell et al. 2011);
3) Clytia hemisphaerica, a hydrozoan species also common and with cosmopolitan distribution, but
not presently known to cause disease in farmed salmon.
Eight individual medusae of each species will be collected at each site and held separately for 60
minutes in microbe-free, aerated seawater kept at 10°C, with water changed completely at 30 minutes;
this will ensure that any bacteria cultured from each are endogenous rather than from environmental
sources. In addition, water samples will also be collected from each site to use as negative controls.
After 60 minutes’ holding period has elapsed, the medusae will be removed from the seawater and
processed for bacterial flora. One medusa of each species will be placed in 4% buffered formalin and a
second placed in 3% buffered glutaraldehyde for later microscopy, if required. The six remaining A.
aurita medusae will be placed exumbrella side down, and the oral surfaces swabbed using a sterile
cotton swab (Copan Diagnostics, Brescia, Italy). One swab from each medusa will be transferred to
FMM (Flexibacter maritimus media, Conda Laboratories, Barcelona) broth, and a second to BHI (brainheart infusion) broth. Two sections of tentacle will be excised and placed in FMM and BHI broths and
macerated. Each remaining C. hemisphaerica and P. quadrata whole medusa will be halved and
macerated, and one half placed in FMM broth and one in BHI broth. Negative control samples of water
from each site will be filtered of particulate matter >2 µm in size and a loopful of water transferred to
vials of FMM and BHI broths. All broth samples will then be incubated at 25° C for 48 hours, then
treated with glycerol to a final concentration of 20% glycerol and stored at -20°C. Thawed broths will be
loop sampled and streaked to BHI or FMM agar plates, and incubated at 25°C for a further 48 hours.
Resulting bacterial colonies will be removed individually using a loop to fresh FMM and BHI agar plates
in order to establish isolated bacterial monocultures. Should settled polyps of these jellyfish species in
question be available, exemplars of these at each site will also be sampled as above.
After isolation, cultures will be identified using PCR amplification according to Cepeda et al. (2003) and
Ferguson et al. (2010), using 20F and 500R universal primers and a QIAquick PCR purification kit
(QIAGEN, Hilden, Germany). Nucleotide sequencing will be carried out by Eurofins MWG Operon
Laboratories in Ebersberg, Germany, and sequenced products will be compared to other 16s rRNA
sequences in the EMBL database by BLAST analysis. Second-round PCR and sequencing may be
conducted if necessary, using species-specific primers. Occurrence of specific bacterial presence on
jellyfish will be compared using a generalised linear model of the sample percentage of medusae
carrying each bacterial species, where site and jellyfish species are used as variates.
Anticipated outcomes
A better understanding of the risks to salmon from pathogens carried by medusae may be useful in
planning aspects of aquaculture, such helping to inform strategic use of antibiotics or to adding to the
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vaccinations given to smolts prior to release to pens. Insight into bacterial involvement into the jellyfish
life cycle could also contribute to bloom prediction, depending on the ecological roles played by the
microbial flora. Completion of this study is anticipated for late 2012, with PCR identification of cultures
and resulting statistical analyses being conducted after the summer sampling season.
Data collected to date: June 2012
Several species of jellyfish have now been sampled in order to establish appropriate techniques.
Preliminary investigations were conducted as follows.
Six blue lion’s mane jellyfish, Cyanea lamarckii, were collected from the beach at East Sands. Four were
sampled for presence of bacteria using several methods:
1) sterile cotton swab to the gut and streaking to plate of FMM (Flexibacter maritimus medium) agar
2) tentacle excision, maceration, and suspension in FMM broth
3) mouth part excision, maceration, and suspension in FMM broth.
Two smaller medusae were also sampled by whole jellyfish maceration and suspension in FMM broth.
All samples were incubated at 25°C for 48 hours. In addition, several gut-swab samples were collected
using preservative swabs and left on the bench at variable room temperature for the same time period,
in order to establish whether samples obtained in the field could be safely preserved without special
treatment.
Results: Two distinct bacterial colonies grew on each sample plate, interspersed heterogeneously.
Superficially, one measured 5 mm in diameter with gram-positive bacilli; the other measured 2 mm in
diameter with chain-linked gram-negative cocci. These were subsampled using a plastic loop to transfer
individual colonies to fresh plates of FMM agar, and further incubated for another 48 hours. Pure
cultures were then analysed using 16s PCR, QIAquick purification, and sequencing by Eurofins Operon
Laboratories as described above. The BLAST database was used to identify the two colonies as Vibrio
splendidus (or similar Vibrio sp.) and Pseudoalteromonas sp., both of which had been previously isolated
from jellyfish in unpublished results.
A second round of testing was conducted in order to establish negative-control water sampling, to
exclude the possibility of bacterial sources being exogenous to the medusae. This was done using 4
Eutinina indicans hydromedusae from East Sands macerated in FMM broth to develop fresh cultures of
bacteria, then taking loopfuls of water from the same vial and streaking it to a plate of FMM agar. This
resulted in visually-identical colonies of bacteria developing in the samples cultured from the medusae
as well as from the water, and PCR identification was not performed. In addition, owing to the methods
used to culture bacteria from water samples, we could not say with certainty that the medusae had not
contaminated the water rather than vice versa.
A third round of testing was then conducted in order to establish good practice for negatively controlling
for exogenous sources of bacteria in culture. This was done by thoroughly rinsing freshly sampled
Aurelia aurita scyphomedusae and Clytia hemisphaerica hydromedusae from Loch Sunart in sterile
saline before sampling, plus filtering sampled seawater through 2 µm pore size filter paper, in order to
remove large particulate contamination, as a negative control. This resulted in a perceived reduced
13
density of bacterial growth, though not its elimination; however, we were unable to obtain sterile filter
papers at this time of this trial, and therefore could not say with certainty whether the source of
cultured bacteria was the water or the filter paper itself. As this trial was conducted for the purpose of
investigating negative control methods, we did not subculture or identify bacterial samples collected
from Aurelia aurita or Clytia hemisphaerica medusae.
Conclusions for methodology:
-All bacterial sampling techniques were effective in obtaining cultures from jellyfish; this included
directly-plated samples as well as samples placed in broth and plated in second-round incubation.
-Nonsterile filtration was not 100% effective in negative controlling for bacterial contamination. Future
negative controls will be obtained using sterile filter paper.
-All jellyfish sampled will be kept alive, in oxygenated, temperature- and salinity-controlled water for at
least 60 minutes with one complete water change halfway through, and shaken gently to dislodge
bacteria which may be environmentally incidental rather than endogenously incorporated in order to
eliminate false-positive results.
References
Baxter, E., Sturt, M., Ruane, N., Doyle, T., McAllen, R., Harman, L., & Rodger, H., 2011. Gill damage to Atlantic salmon (Salmo
salar) caused by the common jellyfish (Aurelia aurita) under experimental challenge. PloS one, 6(4), p.18-29.
Cepeda ,C., García-Márquez , S., Santos, I., 2003. Detection of Flexibacter maritimus in fish tissue using nested PCR
amplification. J Fish Dis 26, p. 65–70
Delannoy, C.M.J., Houghton, J.D.R., Fleming, N.E.C., Ferguson, H.W., 2011. Mauve stingers (Pelagia noctiluca) as carriers of the
bacterial fish pathogen Tenacibaculum maritimum. Aquaculture 311(1-4), p. 255-257.
Doyle, T., De Haas, H., Cotton, D., Dorschel, B., Cummins, V., Houghton, J., Davenport, J., and Hays, G., 2008. Widespread
occurrence of the jellyfish Pelagia noctiluca in Irish coastal and shelf waters. Journal of Plankton Research, 30(8), pp.963-968.
Ferguson, H.W., Delannoy, C., Hay, S., Nicolson, J., Sutherland, D., & Crumlish, M., 2010. Jellyfish as vectors of bacterial disease
for farmed salmon (Salmo salar). Journal of veterinary diagnostic investigation: official publication of the American Association
of Veterinary Laboratory Diagnosticians, Inc. 22(3), pp.376-82.
Mitchell, S.O., Baxter, E. J., and Rodger, H.D., 2011. Gill pathology in farmed salmon associated with the jellyfish Aurelia aurita.
The Veterinary Record, pp.1-3.
Mitchell, S.O. pers. comm.. 29 March 2012. Gill Health Workshop, Dunstaffnage Marine Laboratory.
Seaton, D.D., 1989. Fish Kills by Planktonic Organisms. Aquaculture Information Series, no. 9.
Steinum, T., Sjåstad, K., Falk, K., Kvellestad, A., Colquhoun, D.J. 2009. An RT PCR-DGGE survey of gill-associated bacteria in
Norwegian seawater-reared Atlantic salmon suffering proliferative gill inflammation. Aquaculture 293(3-4), p. 172-179.
14
Investigation 3: Prospective seasonal monitoring of
medusozoan occurrence at Scottish salmon aquaculture facilities
Introduction
Salmon aquaculture relies on the use of pens placed in fjords or harbors, minimizing infrastructure,
energy use, and cost of rearing fish to marketable size. However, sea-caged fish are vulnerable to
environmental factors such as blooms of gelatinous zooplankton, which can cause considerable
morbidity as well as outright fish kills in some cases (e.g. Doyle et al. 2008, Ferguson et al. 2010). At
present, little information is available which might predict blooms in advance, preventing the
implementation of preparatory strategies and sometimes leading to economic losses in the millions of
pounds (e.g. Doyle et al. 2008); in addition, it is not clear what the long-term cost of sublethal exposure
of salmon to cnidarian zooplankton might be in terms of growth and health indices. No prospective
studies in Scotland have yet been made which would provide this information, and industry members
have, at best, poor retrospective records or none at all (Nickell et al. 2010). This investigation seeks to
collect information regarding the appearances of both classes of medusae at salmon aquaculture
facilities throughout the 2012 summer season, in order to begin developing a bank of data from which
patterns of zooplankton occurrence might be discerned.
Materials and methods
Four salmon aquaculture sites along the west coast of Scotland have been provided by Marine Harvest
Ltd: Linnhe, in Loch Linnhe on the Scottish mainland; Invasion Bay, in Loch Sunart on the mainland; and
sites off Greshornish and Portnalong on the Isle of Skye. Lochs Linnhe and Sunart have been previously
detailed in Investigation 1 of this report. Portnalong is sited within Loch Harport, a tributary of Loch
Bracadale at the Isle of Skye, and as with Linnhe and Invasion Bay, subject to similar water mass inflows
from the north-flowing Scottish Coastal Current. The bulk of Loch Bracadale is lacking in sills and is
largely unsheltered from southwesterly winds and waves generated over the Minch, but Loch Harport
itself is somewhat sheltered from direct southwesterly influences by the island of Oronsay, a narrowing
at the Ardtreck peninsula, and a shallowing slope within the fjord (Highland Council 2002). These
features also reduce flushing time of the loch, potentiating problems such as retention of medusan or
other planktonic blooms. Greshornish is sited within Loch Greshornish, a smaller fjord within Loch
Snizort at the Isle of Skye. Here, prevailing southwesterlies would tend to generate surface currents
which flow from the head to the mouth of the loch, reducing retention of plankters in the upper layers
of the water column. Loch Greshornish is sheltered from other wave action and surface currents
generated in greater Loch Snizort by the Greshornish and Lyndale Point peninsulas (Ordnance Survey
2012). Although the loch shallows from its mouth in Loch Snizort, no marked sill exists, further reducing
the potential for larval planktonic retention (UKHO 2011).
15
Figure 4: Map of study sites
Each site has been provided with a 0.5 m ring net plankton tow with a 270 µm mesh cod end attached to
a 5 m tow line, preservative materials and sample jars, and training in their use. On a weekly basis, and
at slack high tide, site staff will obtain three replicate vertical towed samples from 5 m depth at the
salmon cage pontoons. These will be preserved in 4% buffered formalin. Hydrozoan medusae and
scyphozoan juvenile medusae, or ephyrae, will be identified visually using a dissecting microscope and
counted, plus total volume of gelatinous zooplankton biomass measured. Large scyphozoan medusae,
identifiable without magnification, will be observed by 20 m transect walk along the seaward edge of
the salmon pontoons; again, species observed and counts will be recorded. Volume of water filtered in
each haul has been calculated as πr2l, where r = net radius and l = the vertical distance towed, and
gelatinous biomass and species counts will be expressed as units per cubic meter of water. In addition
to site, these data will be compared according to the following parameters: gill health score of salmon as
per Mitchell et al. (2011), primary productivity (as measured by satellite-derived chlorophyll data
provided to Marine Harvest), salinity and sea surface temperature, meteorological patterns, and
estimated flushing times of the surrounding sea lochs. Analyses of any correlations between these data
and medusozoan occurrences will be conducted using a generalised linear model.
Anticipated advisory outcomes
Provision of advance notice for jellyfish blooms, and the mitigation of their effects, is the ultimate goal
for academia and industry alike. This investigation examines both locally-blooming species such as
Phialella quadrata (Ferguson et al. 2010) and pelagic species such Pelagia noctiluca which may be
carried in occasionally by current flow (Doyle et al. 2008). Information on both types of bloom will
represent an important first step in developing a “jellyfish early-warning system.” In addition, the
16
involvement of the aquaculture industry itself represents a means for expanding awareness and
potentially expertise outside of the academic realm, enabling sustainable and widespread data
collection at low cost. Intensive sampling will cease after late September 2012. Further sampling at
reduced frequency may continue through the winter, depending on the observed abundance of
medusae occurring after the summer season.
References
Doyle, T., De Haas, H., Cotton, D., Dorschel, B., Cummins, V., Houghton, J., Davenport, J., and Hays, G. 2008. Widespread
occurrence of the jellyfish Pelagia noctiluca in Irish coastal and shelf waters. Journal of Plankton Research, 30(8), pp.963-968.
Ferguson, H.W., Delannoy, C., Hay, S., Nicolson, J., Sutherland, D., & Crumlish, M. 2010. Jellyfish as vectors of bacterial disease
for farmed salmon (Salmo salar). Journal of veterinary diagnostic investigation : official publication of the American Association
of Veterinary Laboratory Diagnosticians, Inc. 22(3), pp.376-82.
The Highland Council 2002. Loch Bracadale Aquaculture Framework Plan. Produced by the Planning and Development Service,
The Highland Council.
Nickell, T., Davidson, K., Fox, C., Miller, P., Hays, G. 2010. Developing the capacity to monitor spatial and temporal distributions
of jellyfish in western Scottish waters. The Crown Estate, 70 pages, ISBN: 978-1-906410230.
Ordnance Survey 2012. Sheet 408, Explorer Map (1:25,000) Skye – Trotternish & The Storr.
United Kingdom Hydrographic Office 2011. Anchorages on the West Coast of Skye Sheet 2533. Admiralty Charts and
Publications. Hydrographic Office, Taunton.
17
Part 2: Objectives and timeline
Concurrent objectives
Two additional sampling activities will be conducted during the coming months. Greater detail on
procedural intent has been given previously in my 4-month report. In brief, a Phialella quadrata culture
will be established, through the capture and spawning of live medusae collected in Shetland. Resulting
planula larvae will be used to establish a captive hydroid colony for various investigations into life cycle
ecology, to be carried out over the winter of 2013 in St Andrews following husbandry techniques
developed by Chad Widmer in the Pelagic Ecology Research Group. A second collection of mature P.
quadrata medusae will also be undertaken, in order to amass sufficient numbers from which to extract
nematocyst venom. These will be used to investigate the predatory ecology of P. quadrata over 2013
and early 2014.
Summary timeline
June 18 – late September 2012 (end date to be defined as an 80% drop in observed medusa individuals
in plankton tows), Shetland Islands:
-Investigation 1 observations carried out
-Investigation 2 samples collected (projected August 2012)
-Investigation 3 samples collected
-Establishment of P. quadrata culture and collection of medusae for venom extraction
This residence at North Atlantic Fisheries College in Scalloway, Shetland is in partial fulfillment of
the conditions of the MASTS Prize Studentship, wherein students must spend several months in
residence at a MASTS institution separate from their host university. NAFC is part of the
University of Highlands and Islands.
September –December 2012, St. Andrews:
-Results analysis of Investigation 1
-Culturing, identification and analysis of bacteria isolated from medusae during Investigation 2
-Investigation 3 preliminary analysis conducted, plus ongoing infrequent samples collected and
analysed
-Cultivation of P. quadrata culture
January 2013 – May 2013, St. Andrews:
-Study of temperature, salinity and food availability on P. quadrata hydroid growth and
reproduction; these and other specific variables to test are contingent on correlational findings
observed in Investigation 3.
-Experimental venom extraction for hydromedusae developed.
-Role of bacteria in cultured P. quadrata investigated
May/June 2013 – September 2013:
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-Additional field studies into hydrozoan settlement preferences conducted as per results of
Investigation 1 (to include depth and potentially other substrates as variables)
-Study of environmental factors affecting medusa release expanded to include further variables
September – December 2013:
-Venom actions on cultured salmonid gill tissue cells investigated
-P. quadrata culture experiments continued as necessary
Key conferences upcoming
Fourth International Jellyfish Bloom Symposium: 5-7 June 2013, Hiroshima, Japan
The International Conference on Coelenterate Biology: 1-6 December 2013, Eilat, Israel
19