1

Hydrozoan jellyfish blooms and interactions with

Scottish salmon aquaculture:

prediction, prevention and mitigation

by

Anna H. Kintner

4-month Report, 27 January 2012

Submitted in partial requirement for fulfillment of the degree of Doctor of Philosophy in Marine Biology

Phialella quadrata medusa (Hydrozoa, Leptomedusae. )

Photo by Otto Larink, courtesy of Scottish Association for Marine Sciences.

5mm

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TABLE OF CONTENTS

I. Introduction

II. Jellyfish and Scottish industry: current state of knowledge and future outlook

III. Life cycle of Phialella quadrata

IV. When and why do hydrozoan blooms occur?

V. Forecasting a cnidarian bloom: recent investigations and projected strategies

VI. During a bloom: nematocysts, venom and infection

A. Predatory ecology of the cnidarian: the venom system

B. Bacterial infection and symbiosis

VII. Conclusion and proposed investigative avenues

VIII. References

IX. Appendix: Intended procedures for data collection and analysis

Objective 1: Identification of environmental factors governing the presence of P. quadrata

medusa

(a) Historical data

(b) Laboratory culture

(c) Field studies

Objective 2: Venom ecology of P. quadrata: cnidome, prey analysis, and basic venom

characterization

Objective 3: Defining the nature of P. quadrata symbiosis with the bacterium Tenacibaculum

maritimum

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I. Introduction

Phialella quadrata (Subclass Leptomedusae, Order Leptothecata, Family Phialellidae) is a colonial

hydrozoan species endemic to the British isles, with widespread distribution including inshore waters off

northern Europe, Morocco, Japan, New Zealand, and Chile (Russell 1953, Cornelius 1955). In Scotland, it

has produced two recorded large-scale blooms at salmon aquaculture facilities, in 1984 and 2008 (Bruno

& Ellis 1985, Ferguson et al. 2010). Both blooms resulted in up to 90% mortality in caged fish, recently

forcing the closure of a farm (Ferguson et al. 2010). A subsequent study showed that the medusae were

causing damage to the fish through a combination of envenoming and transmission of bacterial disease,

marking a first known instance of this phenomenon occurring in pelagic cnidarians (Ferguson et al.

2010). The present state of knowledge of hydrozoan blooms precludes informed prediction of blooms

such as these, leaving aquaculture facilities open to similar incidents.

The goals of this review will be to examine the physical and biological interactions which may have an

effect on the appearance of P. quadrata medusae in the plankton, and to consider its predatory ecology

and symbiosis with T. maritimum. This information will be used to develop data-gathering procedures

aimed at developing sustainable ways of mitigating the species’ potential effect on the aquaculture

industry, and contribute to the field of zooplankton seasonal ecology and an emerging pool of research

into cnidarian-prokaryote symbiosis.

II. Jellyfish and Scottish industry: current state of knowledge and future outlook

Impacts of jellyfish blooms and individual medusae on various coastal industries have been covered

previously in numerous review papers (e.g. Seaton 1989, J. Purcell et al. 2007, Richardson et al. 2009,

Nickell et al. 2010), but nonetheless bear brief reiteration. In Scotland, marine industries are vulnerable

to cnidarian blooms by way of stock losses in aquaculture, interference with coastal power generation,

and competitive or predatory interactions with commercial finfish species; while the latter two are

worth considering, the main focus of my research will be toward the ecological factors affecting Scottish

salmon production.

The British Isles are host to two classes of cnidarian that produce pelagic medusae. The Scyphozoa

include 13 endemic species, which tend to be larger and more easily identified than the Hydrozoa, which

include about 90 species (Russell 1953, Nickell et al. 2010). Members of both classes have been involved

in fish kills at aquaculture facilities. The first reported damage to aquaculture took place in 1984, with a

Phialella quadrata bloom that caused mass mortality of salmon smolts in Shetland in 1984 (Bruno & Ellis

1985). Since then, various species of scyphozoan and hydrozoan medusae including Cyanea capillata

and Aurelia aurita (Seaton 1989, Baxter et al. 2011, Mitchell & Rodger 2011), Solmaris corona (Tørud &

Håstein 2008), and Pelagia noctiluca (Doyle et al. 2008) have also been implicated, and other jellyfish-

related fish kills have been reported without identification of the species involved (McKibben & Hay

2002). Mechanisms for mortality may include direct results of jellyfish envenoming via stinging, clogging

of net pens resulting in localized anoxia, and spreading of disease (Doyle et al. 2008, Tørud & Håstein

2008, Mitchell et al. 2011). Outside of Scotland, but near enough to be of concern, the same list of

cnidarian species plus others such as Apolemia uvaria and Muggaiea atlantica have caused mortality of

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caged salmon in Norway and Ireland (Tørud & Håstein 2008). It has been suggested that worldwide

jellyfish populations, and associated deleterious events, may be on the rise (Purcell et al. 2007,

Richardson et al. 2009). Shifts toward cnidarian-dominated food webs, decreased finfish landings, and

numerous blooms which interfere with power stations, have received increasing attention in the last

decade (Lynam et al. 2005, Purcell et al. 2007, Richardson et al. 2009, Miller 2011). An increasing body

of evidence suggests that larger and more frequent jellyfish blooms are to be expected in the future,

due to factors such as climate change and overfishing (Richardson et al. 2009). It is reasonable to

suspect that P. quadrata and other British cnidarian species will follow suit.

As stated previously, Phialella quadrata blooms have been implicated in two major incidents of salmon

mortality in Scottish aquaculture. Both blooms occurred in Shetland, though concerns regarding the

presence of the medusae throughout the salmon-producing areas of Scotland are mentioned in various

reviews (e.g. Seaton 1989, Nickell et al. 2010). In the 2008 bloom, 90% loss of salmon stock took place

over the course of about eight weeks, with fish eventually dying of respiratory failure as a result of

severe gill lesions (Bruno & Ellis 1985, Ferguson et al. 2010). Jellyfish were present for most of this time,

though their first appearance was not marked; it is approximated that they were present for at least six

of the eight weeks in question. It was found that the P. quadrata medusae appeared to be transmitting

the infectious bacterial species Tenacibaculum maritimum, probably accounting for at least some of the

damage to gill tissue (Ferguson et al. 2010). The causative strain of T. maritimum was matched to a

strain found living on the mouth parts of the medusae (Ferguson et al. 2010), raising a number of

pertinent questions regarding the aetiology of infection and the symbiosis between P. quadrata and T.

maritimum. These questions will be further considered in Part VI.

III. Life cycle of Phialella quadrata

In examining the ecological context of hydromedusan blooms, it is important to understand the life cycle

features that contribute to bloom appearance. Hydrozoans undergo an alternation between a colonial

benthic stage and a freeswimming medusa stage. Gametes from pelagic medusae are externally

fertilised and zygotes develop into a motile planula larva, which eventually settles onto a substrate;

initial reproduction takes place via asexual budding into a colony of polyps connected by hydrorhizae

(Boero 1987). These are specialized either as feeding hydranths, or as medusa-producing gonangia.

Colonies are dioecious and release only female or male medusae. Once in the water column, medusae

mature and release gametes. The timing of this life cycle varies across taxa, but tentative estimates of

time to maturity in P. quadrata are approximately 15-30 days (based on P. fragilis, a former conspecific)

(Boero, 1987). Total lifespan in the plankton may range up to 70 days, though medusae typically begin

to deteriorate once gamete release has occurred (Boero 1987).

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a

b

c

d

e

f

g

h

ji

k

l

m

n

Figure 1. Representative life cycle of Phialella fragilis (formerly conspecific with P. quadrata). Dioecious hydroid

colonies consist of interconnected sessile polyps, of two distinct types: reproductive gonangia and feeding

hydranths. Gonangia (a) produce the pelagic, sexually reproducing medusae, which develop within the protective

gonotheca (b) before being released into the plankton. Colonies grow asexually, forming both lateral creeping

hydrorhizae (c) and branching stalks (d). Hydranth polyps are specialized for feeding only, and do not produce

medusae (e: retracted hydranths; f: extended, actively feeding hydranth). After release from the gonangia, juvenile

medusae emerge with 4 tentacles at about 0.6mm in bell diameter (g). After 5-7 days, the medusae will reach

about 2mm bell diameter with 8 tentacles (h). After about 12 days, medusae become fully mature, with a

maximum bell size of 8-10mm in P. fragilis and up to 16mm reported in P. quadrata (i), and up to 16 tentacles.

Gametes are released and fertilized externally, usually between 12-30 days after medusa release (j). Zygotes (k)

develop in the plankton into ciliated planula larvae (l). After this several hours to several days, planulae attach by

the anterior pole to an appropriate substrate and cells collapse around this point, eventually re-developing into the

tissues of the primary polyp of a new colony (m), which increases in size by asexual budding (n). Drawn by A.

Kintner, from Boero (1987).

Both the benthic and pelagic phases are important to understand in predicting medusa blooms. While it

is the medusa life stage that causes damage to industry, it is the colonial hydroid stage that gives rise to

medusae, necessitating an investigation of its reproductive dynamics. First, the proportion of feeding

hydranths to reproductive gonangia is important: new polyps in a colony will become specialized as one

or the other, but not both. A useful line of inquiry would be the factors influencing colonial growth and

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the means by which polyps specialize, providing information toward estimating the potential magnitude

of a bloom produced by a hydroid colony. Second, P. quadrata blooms are evidently seasonal, with the

greatest number of medusae occurring during the summer months (Russell 1953, Bruno & Ellis 1985,

Ferguson et al. 2010), but there is a paucity of data supporting speculation as to what physical or

biological triggers lead to medusa production and release by the hydroid colony. An accurate forecast of

medusa appearance based on environmental conditions would provide the obvious benefit of enabling

aquaculturists to make spatial and temporal adjustments to their production timelines in order to avoid

blooms. Third, once released, the medusae remain in the plankton for a mean duration and may even

overlap with newly released generations of medusae; an accurate estimation of the lifespan in the

plankton would be useful in gauging the length of time a bloom could pose a threat to industry.

Procedures for gathering and analyzing data toward these ends are laid out in Part IX.

IV. When and why do hydrozoan blooms occur?

As previously described, the appearance and magnitude of Phialella quadrata blooms will be dependent

on hydroid colonial capacity to produce medusae (i.e. number of gonangia) and a stimulus or stimuli to

actually produce them. A wide variety of physical and biological conditions conducive to medusa

production and colony growth are cited for other hydrozoan species, including light (Brinckmann-Voss

1985, regarding Sarsia princeps; Costello 1988, regarding Cladonema californicum), temperature

(Werner 1958, regarding Rathkea octopunctata, and Werner 1961 and 1962 regarding Bougainvillea

superciliaris, in Arai 1992; Widmer 2004, regarding Mitrocoma cellularia), salinity (Goy 1973, regarding

Scolionema suvaensis), food availability (Roosen-Runge 1970, regarding Clytia gregarium; Miglietta et al.

2008 regarding numerous hydrozoan taxa), lunar phases (Elmhirst 1925, regarding Obelia geniculata;

Goy 1973, regarding Scolionema suvaensis), and various interactions amongst the above (Edwards 1978

& 1983, regarding Sarsia occulta, S. tubulosa, and S. cliffordi; Ma & Purcell 2005, regarding Moerisia

lyonsi). M. lyonsi in particular showed increased gonangium ratios amongst the colonial polyps and a

resulting increase of medusa release in response to rising temperatures, by as much as 25% per 1° C rise

in temperature (Purcell 2005). Alternatively, some species release medusae with regularity without

apparent regard to particular physical stimuli (Kubota 2008, regarding Eugymnanthea sp.). In some

cases, these conditions are met as a constant; that is, a particular species’ colony held at 11° Celsius will

produce medusae regularly and continuously and will do so significantly less at over 15° Celsius (Widmer

2004, regarding Mitrocoma cellularia); other conditions, such as a peak in prey abundance, may lead to

similarly temporary peaks in medusa production (e.g. Arai 1987, regarding Sarsia cliffordi). This may

represent an ecological situation in which resources are sufficiently flush that the hydroid colony can

invest resources in sexual reproduction. In other cases, the stimulus for medusa production may be

hormetic; that is to say, a short-term stressor such as rapidly dropping salinity, a damaging rise in

temperature, or chemical toxicity appearing in the water column may cause a benthic colony to produce

motile medusae which can escape unfavourable conditions, as well as increase diversity and population

fitness through genetic recombination (e.g. Stebbing 1981, Stebbing 2002, Widmer 2004).

Many of these conditions will be environmentally dependent, but the bulk of published works regarding

wild medusa populations in response to environmental flux has focused on larger scyphozoan medusae

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rather than the Hydrozoa. However, scyphozoan species studied have shown marked correlation

between climatological and oceanographic effects. For example, the Scyphozoan species including

Cyanea capillata, C. lamarckii, and Aurelia aurita have been found to have medusa populations

correlating with the North Atlantic Oscillation, with positive NAO phases leading to cooler water in the

North Sea and greater numbers of medusae (Lynam et al. 2004). Work is now underway to further

define these effects on scyphozoan polyp strobilation and metamorphosis rates in laboratory cultures of

these species (Widmer 2011). Furthermore, a number of coastal scyphozoan species have shown

increased medusa population in response to eutrophication, in particular from nitrogen and phosphorus

runoff, which tends to favour nutrient pathways toward low-energy species such as jellyfish rather than

higher-energy, more complex food webs (Greve & Parsons 1977, Purcell et al. 2007). This phenomenon

may also have an effect on P. quadrata colonies, particularly in sea loch environments subject to

nutrient inputs from agricultural runoff as well as aquacultural waste.

Any of these factors may play a role in P. quadrata’s appearance in Scottish waters. The summer timing

of its abundance suggests that seasonally-governed environmental factors such as temperature, salinity

and light may affect medusa production; however, since high-population density blooms of the medusae

are infrequent, it can also be inferred that these conditions are not met constantly or even often.

Therefore, none of the physical or biological factors listed above can yet be conclusively ruled out.

Again, approaches for investigating the life cycle dynamics of P. quadrata are laid out in Part IX.

V. Forecasting a cnidarian bloom: recent investigations and projected strategies

Effective prediction of a harmful jellyfish bloom is an obvious objective for marine industry, but requires

a far greater data bank than is currently available. Until the past decade, jellyfish have been of little

economic interest, so there have been few historical surveys on which to base estimates of population

flux. Several recent strategies for gathering data on gelatinous zooplankton occurrence in Scotland have

been tested and implemented, with varying degrees of success. First, in a 2008 Crown Estate-funded

survey of jellyfish impacts, aquaculture facility managers were provided with a standardized

questionnaire regarding frequency, severity, and causative species via the Scottish Salmon Producers

Organisation (SSPO). Of 257 potential respondents, only 9 responded (Nickell et al. 2010). One

aquaculture syndicate in Shetland cited insurance concerns as a reason for withholding their data

records. Obviously n = 9 is insufficient for meaningful data analysis, though it has been suggested that

an increased timescale for completing the questionnaire might lead to an increased rate of response

from other companies (Nickell et al. 2010). Remote sensing has also been investigated as a cnidarian

research tool (Lynam et al. 2005, Nickell et al. 2010). Acoustic detection of jellyfish is possible for large

medusae, though has not yet been put into broader-scale use as would be required for long-term

population dynamic study, and is not yet applicable to smaller medusae such as P. quadrata. Aerial

monitoring via spotter plane was trialled and found to be cost-prohibitive due to a combination of poor

aerial visibility of problem species, and poor average weather conditions in the west of Scotland (Nickell

et al., 2010). Remote sensing of blooms via satellite has been investigated but is restricted, again, by

weather conditions and optical acuity (Nickell et al. 2010). Both aeroplane and satellite monitoring carry

limitation when it comes to collecting data on P. quadrata appearance, for the basic reasons that the

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medusae are small (under 15mm) and transparent (Russell 1953). Furthermore, bloom prediction by

remote-sensing of jellyfish is effective only in detecting blooms that develop elsewhere and are carried

by current flow; this might apply, for example, to the 2007 bloom of Pelagia noctiluca that moved in

toward Northern Ireland and western Scotland, which could easily be sighted from the air before

arriving inshore (Doyle et al. 2008). In the case of P. quadrata, both blooms discussed in the literature

appear to have developed in the immediate vicinity of the salmon farms (Bruno and Ellis 1985, Seaton

1989, Ferguson et al. 2010); if this is accurate, aerial flyover or acoustic sampling data would be unable

to provide any advance warning at all to afflicted farms.

Prediction of Phialella quadrata occurrence, therefore, might ideally be based on oceanographic and

ecological factors leading to the mass release of medusae from the hydroid colony. This approach is

contingent on gathering sufficient data to correlate bloom occurrence with environmental events. As

very little data on P. quadrata is now publicly available, one approach to this might be combing through

fish farm managers’ own logbooks to correlate dates of blooms with confirmed oceanographic and

weather data. These correlations could be confirmed with an ongoing liaison with farms to collect data

when and where new blooms occur. Early forays into this area have revealed that other species may co-

occur during lower-level blooms, expanding the potential data available on medusa presence (Hope

pers. obs. 2011). Additional information can come from life cycle data as discussed in Part III; any clues

gleaned from historical P. quadrata appearances can be tested on laboratory cultures in order to

evaluate their contributions to bloom production, as laid out in Part IX.

VI. During a bloom: nematocysts, venom and infection

As previously discussed, the 2008 Phialella quadrata bloom yielded information that fish were being

affected in a two-part process: first, through envenoming by jellyfish nematocysts; and second, through

infection by T. maritimum, transmitted from the jellyfish (Ferguson et al. 2010). These two separate

means of pathology require separate consideration.

A. Predatory ecology of the Cnidaria: the venom system

All cnidarian classes at some point in the life cycle possess cnidocyte cells containing stinging organelles

called cnidocysts. Cnidocysts can be divided into three groups: spirocysts, which are exclusive to the

Anthozoa; ptychocysts, which are exclusive to the cerianthid anemones; and nematocysts, found across

the phylum (Östman 2000). While spirocysts and ptychocysts are monotypic within their classification,

nematocysts are diverse in type, placement on the body, structure, and predatory function (Hessinger &

Lenhoff 1988, Östman 2000, Underwood and Seymour 2007). However, all are engaged in some form of

delivery of venom and/or prey capture (Hessinger & Lenhoff 1988). Broadly speaking, nematocysts

consist of a hollow capsule with a contained, inverted tubule which everts rapidly when stimulated by

prey or self-defense to do so (Hessinger & Lenhoff 1988, Östman 2000). The predatory purpose of these

tubules falls into two categories: those which entangle or stick to prey, and those which deliver venom

(Hessinger & Lenhoff 1988). These were formerly termed astomocnidae and stomocnidae, in reference

to a closed or open tubule tip that would allow for a flow of capsule contents (Weill 1934), but more

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recent scanning electron microscopy images have found that nematocyst discharge often causes rupture

of astomocnid tubules, and the terminology has been discarded (Östman 2000). However, for the

purposes of discussing cnidarian predatory ecology, nematocysts can be viewed as penetrant or non-

penetrant. Non-penetrants serve to tether or stick to microstructures on the outside of prey such as

setae or exoskeletal plates, while penetrant nematocysts pierce prey integument either for venom

delivery or for further tethering (Carrette et al. 2002, Hessinger & Lenhoff 1988).

The total complement of nematocysts (cnidome) and physical arrangement on the tentacles and bell

varies taxonomically and ontogenetically, as does the toxin complement of contained venom (Carrette

et al. 2002, Gravier-Bonnet 1987, Hessinger & Lenhoff 1988, Yanagihara et al. 2002, Underwood &

Seymour 2007); therefore, differentiation between and within these types can help to discern the

predatory habits, prey preferences, and ontogeny of the cnidarian in question (Carrette et al. 2002). In

the context of Phialella quadrata ecology, a detailed knowledge of the hydroid and medusary cnidomes

could contribute to estimates of prey capture capabilities and colonial growth potential, in turn aiding in

predictions of bloom occurrence and magnitude. For example, if the P. quadrata hydroid cnidome is

one best adapted to a specific type of zooplankton, it may follow that the local population of the same

may correspond to medusa bloom magnitude in a given season.

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Figure 2. Nematocyst organelles consist primarily of an enclosed capsule and a coiled, inverted tubule (a);

appropriate stimuli will cause the tubule to evert rapidly (b), either stinging or entangling prey. (Nematocysts from

Carukia barnesi, 1000x. Photograph J. Seymour 2002). Bands of nematocysts on Chiropsella bronzei (fm.

Chiropsalmus sp.) tentacle shown at 100x (c) and 400x (d) show a diversity of types, including penetrant envenoming

nematocysts and non-penetrant tethering and adhering nematocysts. (Tentacle photographs of Chiropsella bronzei

by A. Kintner 2005). Nematocyst types may also vary according to ontogeny and placement on the body (e), with the

proportion of nematocyst types on Carukia barnesi tentacles differing from those on the bell (Underwood and

Seymour 2007) (Photograph by J. Seymour 2002.).

Meanwhile, there is virtually no information regarding the dynamics of P. quadrata venom and its

contributions to fish kills during a bloom event. Ferguson et al. (2010) cite a variety of cnidarian venom

mechanisms, but these run a very wide pharmacological and ecological gamut. Reported action of

various hydrozoan and scyphozoan venoms has included neurotoxic pathways, type-specific muscle

tissue signaling inhibition, enzymatic attack of epitheliar tissues, various cytolytic pathways, and

induction of pain-producing hormones such as histamines and serotonin (e.g.Tamkun & Hessinger 1981,

Hessinger & Lenhoff 1988, Mustafa et al. 1995, Torres-Ramos & Aguilar 2003). Most of these studies

show little species overlap of the molecular pathways leading to harmful outcomes, underlining the

unique nature of each species’ venom regardless of taxonomic proximity. Further complications have

been encountered as cnidarian venoms are notoriously difficult to study, contributing to an ongoing

debate into preparation, preservation and purification of what can be considered genuine, ecologically

relevant venom from the nematocysts (Bloom et al. 1998, Carrette & Seymour 2004, Kintner 2005).

While a detailed investigation of venom mechanisms and valid extraction techniques is outside the

b

a

d e

c

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scope of this review, this list must demonstrate the futility of generalizing the actions of P. quadrata

venom without at least a cursory investigation of its own particular actions.

Fortunately, the last decade has seen a number of studies which broaden the scope of cnidarian venoms

investigation to include ecological rather than only pharmacological contexts, with many toxin pathways

being linked to prey preference and niche (Carrette et al. 2002, Torres-Ramos & Aguilar 2003, Kintner et

al. 2005). Many examples of neurotoxicity, for example, have been shown to operate most efficiently in

crustacean models and can be reproduced only poorly in vertebrate models (Torres-Ramos & Aguilar

2003), demonstrating prey-specific targeting of venom compounds. Cofamiliars with widely different

prey targets may exhibit a number of different venom toxin assemblages according to prey habit

(Carrette et al. 2002, Kintner et al. 2005). It is possible that venom toxin complement may, like the

cnidome, vary ontogenetically as prey preference and environmental conditions change.

Consideration of these dynamics may assist in developing mitigation strategies when P. quadrata

blooms occur. First, as with the cnidome, an understanding of venom ontogeny can help predict prey

ecology and resource availability for both pelagic medusae and benthic colonies. Second, a basic

understanding of toxicity may help in risk assessment of bloom magnitude—e.g., small or dispersed

blooms may be unable to deliver critical damage to fish, and can be calculated as such to avoid

unnecessary disruption of normal farm operation. Third, knowing the proportionate damage of venom

vs. bacterial infection would help to devise treatments for affected fish pens that minimize side effects

and environmental impact of chemical treatments. A procedure for investigating basic characteristics of

P. quadrata venom using cultured gill cells is given in Part IX.

B. Bacterial infection and symbiosis

The final aspect of P. quadrata bloom biology to consider is the role of Tenacibaculum maritimum

infection. A major component of fish morbidity is attributable to T. maritimum action in the gill tissues

(Ferguson et al. 2010). T. maritimum is a relatively well-known fish pathogen and is most often linked to

skin conditions, particularly in farmed fish, though tenacibaculosis of the gills is by no means rare

(Avendaño-Herrera et al. 2006). Occasionally the bacilli have been found living in fish dermal mucus,

suggesting the possibility of their being endemic there (Avendano-Herrera et al. 2005). Given this

situation, it has been suggested that the jellyfish may be picking up the bacteria from fish skins and

merely transferring them to gill tissue as the medusae are sucked in through the mouths of salmon;

however, tenacibaculosis in any form is virtually unheard of in Shetland salmon, including as a dermal

pathology (Sutherland pers. obs. 2012). This does not rule out the fish themselves as the origin of

bacteria, but certainly argues against the idea. Moreover, no other natural, non-pathogenic reservoirs

of T. maritimum are yet known (Avendaño-Herrera et al. 2006). Scanning electron microscopy of P.

quadrata medusae showed a heavy colonization of the bacteria on the mouth parts (Ferguson et al.,

2010). Therefore, the question of a long-term symbiosis between the two species should be considered.

Close microbial symbiosis in cnidarians is well known, particularly in the cases of shallow water hard

corals and various scyphozoans such as the Cassiopeia and Mastigias genera, all of which harbour

photosynthetic zooxanthellae. However, the P. quadrata bloom in Shetland marked the first recorded

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instance of a medusa symbiosis with a prokaryotic organism (Ferguson et al., 2010). A similar symbiosis

was found in the past year, with T. maritimum living on the mouth parts of Pelagia noctiluca medusae

which had had no contact with farmed fish (Ferguson et al., 2010). This lends credence to the possibility

that the relationship between medusa and bacteria is long term and not merely a case of pathogen

transference from skin to gills.

Figure 3. Microbial symbiosis is well-known in the cnidarian phylum, but until recently, only eukaryotic algal symbionts

have been found. These include the photosynthesizing zooxanthellae of two scyphozoan genera: the “upside-down”

jellyfish of the Cassiopeia genus (a) (Photograph T. Laman), the Mastigias sp. medusae best known from enclosed lakes

in Palau (b) (photograph T. Laman) and the perhaps best-known photosynthetic zooxanthellae of anthozoan hard corals

(c) (photograph B. Skerry). In the scyphozoans, nematocyst complement may be reduced or altered in the presence of

the zooxanthellae, with the jellyfish dependent on photosynthetically-derived energy.

If medusae are living symbiotically with marine bacteria, this raises the question of the nature of the

symbiosis: parasitic, wherein the bacteria are pathogens of the medusae themselves; commensal,

wherein the bacteria are incidental to the survival and success of the medusae but benefit from a free

ride to whatever organisms the jellyfish may be preying on; or mutualistic, wherein the bacteria provide

some kind of selective advantage to the jellyfish and vice versa. This latter possibility might recall the

gut flora mutualism in higher animals, wherein endosymbiotic bacteria would provide digestive and

enhanced nutritional benefit to the medusae. Furthermore, if any form of symbiosis is long-term, it is

not known at what point in the hydrozoan life cycle the bacteria become involved, whether at the

colonial hydroid stage or exclusively during the pelagic medusa stage. Bacterial contributions (or

detriments) to P. quadrata reproductive fitness and survival and role in the various life stages should be

pertinent lines of inquiry into further study of bloom ecology. These investigations should dovetail with

other hydroid culture experiments already being undertaken, as described in Part IX.

VII. Conclusion and proposed investigative avenues

The present state of knowledge regarding Phialella quadrata’s involvement with the Scottish

aquaculture industry is inadequate to begin developing mitigation strategies. The primary goal of my

studies for the degree of Doctor of Philosophy in Marine Biology will be to provide information towards

these strategies and toward new questions in ecology raised by P. quadrata bloom events. First, P.

quadrata hydroid colony ecology will be examined, including the growth and polyp specialization

determinants. Environmental stimuli leading to the generation and release of medusae, and their

maturation and lifespan in the plankton will also be investigated. These lines of inquiry can be followed

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by a simultaneous approach with experimental laboratory culture of the hydroids, plus a collation of

historical data on the appearance and blooms of P. quadrata medusae. Second, the role of the venoms

system, including the cnidome and basic information on the venom itself, will be explored in order to

better inform the predator-prey relationship and predatory strategy. Finally, a characterization of the

relationship between P. quadrata and Tenacibaculum maritimum will be undertaken. These latter goals

can be accomplished in concert with hydroid colonial rearing, plus sufficient access to wild P. quadrata

medusae. These investigations will hopefully lead to an informed and effective approach in predicting

and treating future blooms of P. quadrata, and add to the body of knowledge of gelatinous zooplankton

ecology.

VIII. References

Arai, M.N., 1992. Active and Passive Factors Affecting Aggegations of Hydromedusae: A Review J. Bouillon et al., eds. Scientia

Marina, 56(2-3), pp.99-108.

Arai, M.N., 1987. Population ecology of the hydromedusae of Masset Inlet, British Columbia. In Modern Trends in the

Systematics, Ecology, and Evolution of Hydroids and Hydroimedusae. pp. 107-110.

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IX. Appendix: Intended procedures for data collection and analysis

This project will consider several aspects of Phialella quadrata biology, toward the stated goals of

prediction and mitigation of harmful blooms. Research objectives include:

1) Identifying environmental factors governing the presence of P. quadrata medusae, with a view

to using these to develop predictive protocols;

2) Examining P. quadrata venoms ecology and aetiology of gill injury at salmon farms, through

examination of the cnidome, prey analysis, and basic venom characterization;

3) Defining the nature of P. quadrata symbiosis with the bacterium Tenacibaculum maritimum.

Procedural plans for Objective 1: “Identification of environmental factors governing the presence of P.

quadrata medusae.” This will be accomplished jointly through (a) collection of historical data and (b)

laboratory culture of P. quadrata.

(a) Historical data will come from logged zooplankton appearances in aquaculture industry records.

To this end, I am coordinating with Rachel Hope of Hjaltland Seafarms in Lerwick, Shetland, and

Christopher Wallace of Marine Harvest Scotland, to contact and gather information from these

sources. Records of P. quadrata presence will be collated according to site, season, population

density (e.g. whether the animals are swarming in high numbers or are a low-level presence in a

zooplankton community), and oceanographic/meteorological data for a reasonable preceding

timeframe. This will result in a database of P. quadrata occurrence, hopefully dating from at

least the late 1990s and giving weekly or biweekly notes of the zooplankton community, though

this will undoubtedly vary from source to source. If sufficient data is available, retrospective

factorial analysis will be used to identify conditions contributing to swarm appearance. I will

aim to begin this aspect of research over the 2012 winter.

(b) Laboratory culture of P. quadrata will commence this summer. As many medusae as possible

will be collected, from beach sites near the Scottish Association for Marine Sciences in

Dunstaffnage, from pier sites in Vidlin Voe in Shetland, and (should a major bloom event occur)

from any affected aquaculture sites. The latter factor will depend on participation of the salmon

farming industry; as mentioned in part (a), both Hjaltland Sea Farms and Marine Harvest have

expressed interest in the project, and are helping to facilitate on-site contacts who will contact

me in the event of an obvious bloom and/or a fish kill with similar signs as occurred during the

2008 P. quadrata bloom in Shetland. Some captured medusae will be brought to research

facilities at SAMS and induced to spawn; others will be kept for use in Objective 2 (see below).

Planula larvae will be placed in a culture dish and allowed to settle and form a stable attachment

to substrate for several days before regular feeding with Artemia nauplii commences.

Established colonies will be divided into separate culture plates for experimental investigation.

A pilot study examining effects of temperature and salinity using a two-way factorial

ANOVA design will be undertaken first in order to establish a baseline for medusa production.

Results from this study, plus information gleaned from the historical data culled above, will

contribute to design of a more comprehensive set of experimental comparisons using more

19

variables and examining both medusa production and colonial expansion (as measured by wet

biomass), using MANCOVA to analyse whether chosen variables are having a significant effect,

and whether colonies are allocating resources toward expansion, medusa production, or a

combination of the two. Variables to be tested will be selected based on Part (a), but may

include temperature, salinity, and food availability. Following this, the role of hormesis will be

investigated using new hydroid colonies held at in steady-state controlled conditions, followed

by a spike in a single factor (e.g. a rapid rise or fall in temperature). These will be analyzed using

single-factor ANOVA comparison to a negative control colony.

I will aim to complete the pilot study aspect of research, plus develop sufficient stock

cultures of P. quadrata hydroids for the second phase of experiments, over the summer of 2012

at SAMS. Follow-up experiments of more detailed environmental conditions will be conducted

at SOI using equipment already in use for similar procedures.

(c) Field surveys will be conducted in order to identify natural and manmade substrates hosting P.

quadrata colonies. This will be accomplished through collecting specimens from fouling

communities on aquaculture infrastructure. Hydroids obtained will be reared and identified; if

P. quadrata or its congeners are present, a field-settlement study examining preferred

substrate, preferred depth, and natural vs. artificial surfaces will be undertaken. Sampling may

commence in early spring of 2012 in Vidlin Voe, Shetland. Depending on outcomes,

experimental field settlement studies will likely take place in summer of 2013.

Procedural plans for Objective 2: “Venom ecology of P. quadrata: cnidome, prey analysis, and basic

venom characterization.”

P. quadrata venom will be collected from live medusae according to methods set out by Bloom

et al. (1998) and Carrette & Seymour (2002), involving tentacle decay and nematocyst

lyophilisation for storage, then extraction from rehydrated nematocysts when required. SDS-

PAGE gel electrophoresis will be used to obtain a profile of the number and molecular weights

of the proteins involved. Toxicity will be assessed using cultures of the established gill cell line

of RTgill-W1 rainbow trout lamellar tissue after Helmholz et al. (2010), using Sytox Green

immunofluorescence reagent to indicate cell death after treatment with venom doses. Results

will be analysed using linear regression as per Kintner et al. (2005). These studies will be

conducted during the winter of 2013, after a sufficient source of venom have been collected and

stored from source medusae. Facilities and supervision for these procedures have been made

available by Dr Val Smith at SOI.

Procedural plans for Objective 3: “Defining the nature of P. quadrata symbiosis with the bacterium

Tenacibaculum maritimum.”

The relationship between P. quadrata and T. maritimum will be investigated through several

avenues of inquiry. First, P. quadrata medusae taken from non-salmon farm sites, e.g. those not

involved in harmful blooms, will be sampled for the presence of T. maritimum in order to assess

20

the incidence of symbiosis outwith interaction with caged salmon. This will take place

simultaneously with medusa collection for culture (e.g. summers of 2012 and 2013). Cultured

and wild P. quadrata colonies, as well as medusae released during experiments, will also be

sampled. Any strains of T. maritimum found will be compared to strains found on fish skins

during medusa bloom events. These studies will be carried out at the Moredun Research

Institute in Edinburgh, in conjunction with Dr Christian Delannoy. Should T. maritimum be

found in conjunction with P. quadrata colonies, an attempt will be made to raise a sterile,

asymbiotic hydroid colony, in order to compare relative success of colonial growth and

reproductive success in the presence and absence of bacteria. Analysis of outcomes will be

carried out using one-way ANOVA. Should P. quadrata be found to acquire T. maritimum only

as hydromedusae, medusa success in the presence and absence of bacteria will be compared by

assessing time to gamete release and overall lifespan between T. maritimum-positive and T.

maritimum-negative medusae (to be compared using χ2 analysis). This approach will establish

whether T. maritimum inoculation is helpful, harmful, or neutral to P. quadrata in its various life

phases.