A histopathological-biochemical health and condition assessment of farmed blue mussels in a changing Gulf of Maine

Final report for ONE17-306

Project Type: Partnership
Funds awarded in 2017: $14,233.00
Projected End Date: 04/15/2019
Grant Recipient: University of New England
Region: Northeast
State: Maine
Project Leader:
Adam St.Gelais
University of New England
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Project Information

Summary:

This study employed a conservation physiology approach to assess mussel health, condition and resilience. Aquaculture of blue mussels, Mytilus edulis, is gaining popularity in the Gulf of Maine at a time when naturally occurring mussels are declining and environmental conditions are rapidly changing. Histology and lipid/fatty acid analysis were used to assess reproduction, energy investment and pathology. Mussels were collected twice a month for two years in Casco Bay, Maine to determine energy investment in somatic vs reproductive processes, assess the timing of gemetogenesis and spawing, as well as the farm population level pathology with a focus on common and emerging pathologies. Results show seasonality in gamatogenesis, as expected, but also demonstrate a clear inverse relationship between gonadal and somatic storage tissues. That is, Mussels are likely least physiologically resilient around times of spawning, which coincides with high summer temperatures and likely food limitation. Quantification of gametogenesis suggests mussels spawned twice in 2017 and only once in 2018. Energy investment, in terms of reproduction and storage, differed significantly between the two study years, indicating that reproduction and correspondingly, investment in non-reproductive longer term storage tissues is variable across seasons, and likely tied to environmental cues and modulation. Wet weight of fatty acids such as DHA and EPA corresponded to pre-spawning periods, when gonad tissue was most abundant. Fatty acid profile analysis shows distinct differences with season when sampled, indicating seasonally dependent dietary shifts.

An important finding for the farmer is that traditional condition index (a ratio of meat yield to shell size) is an inadequate metric for crop health. Mussels in Casco Bay are least resilient during late spring/early summer, during and following their main spawning event. This is when their storage tissues are at the lowest levels throughout the year. These energy stores are what the mussel is going to draw from in times of stress and low food availability. Low levels of storage tissue were most easily viewed in histological sections, not via CI.

While the overall presence of pathologies and parasites was low, oocyte atresia (the pre-spawning death and degradation of oocytes within mussel tissue) was prevalent throughout, indicating likely presence of as yet undetermined, but sustained environmental stressors.  Northward range expansion of pathogens typical of southern populations of blue mussels is also a concern. Also concerning, this study documents the northernmost presence of the subtropical trematode Proctoeces maculatus in the Northwest Atlantic. While occurance was low, this species is suptropical in nature and increasing prevlaence with increasing temperatures should be of concern to mussel growers in the region.

Project Objectives:

OUR GOAL WAS: To assess the intra-seasonal health and condition of farmed mussels in Casco Bay, Maine to provide valuable information to farmers on when their crop is the most healthy, stressed, and nutritious.

OUR QUESTION WAS: How do fatty acids, storage tissue abundance, reproduction and pathology vary in farmed blue mussels in Casco Bay, Maine both temporally and spatially?

OUR OBJECTIVE WAS: Through a partnership with Bang’s Island Mussel Farm in Casco Bay to use a combination of environmental monitoring, histopathology (a cost effective tool used to assess health and condition of mussels) and fatty acid analysis (measure of energy storage vs. expenditure due to metabolic maintenance) to determine how storage tissue quantity and quality (Adipogranular and Vesicular Connective Tissues coupled with fatty acid quantity and profile) as well as the severity and presence of pathological indicators vary seasonally across farm sites with different environmental conditions and within the same site.

This will serve as an important  baseline for monitoring farmed mussel populations in the future allow for the development of best management practices to mitigate product loss either through limited handling during periods during stress, optimization of stocking densities, raft spacing.

Introduction:

Globally, more than 3 billion people rely on fish and shellfish for at least 15% of their dietary protein (Rowley et al. 2014). By 2030, over 40 million metric tons of seafood are needed to sustain current consumption if predicted population growth is correct (Costa-Pierce 2010). Shellfish aquaculture is a growing industry, and a necessary solution to sustainably feed this ever-increasing human population (Byron et al. 2011a, Byron et al. 2011b, Rowley et al. 2014, Filgueira et al. 2016). Selected species of mussels are farmed intensely in some regions of the world to help meet these demands.

The US imports more bivalves than any other country (Dumbauld et al. 2009) as almost all mussels are grown outside of the US; a combined 90% being grown in Europe (France, Italy, Netherlands, and Spain) and China. Within North America, 50% of farmed mussels come from Prince Edward Island (Sunila et al. 2004). The US produces less than 1% of the world’s farmed mussels (Dumbauld et al. 2009). In general, most of the United States’ mussel production comes from the Gulf of Maine, sourced from wild beds. Less than 20% of mussels harvested from the state of Maine were cultured mussels (Sorte et al. 2016). Mussels in Maine are cultured via bottom leases (Maloy 2001) or, the focus of this study, Spanish raft culture (Maloy 2001, Sunila et al. 2004, Murray et al. 2007).

 A threat to mussel aquaculture, particularly blue mussel aquaculture in the Gulf of Maine is climate change. Blue mussels in the Gulf of Maine have been in decline for nearly 40 years (1972 – 2007) which has many repercussions in local waters. Without wild mussels in the Gulf of Maine, there is lowered capacity for mussel aquaculture (Sorte et al. 2013, Sorte et al. 2016) since cultured systems are dependent on wild seed (Camacho et al. 1991, Dankers & Zuidema 1995, Jeffs et al. 1999, Inglis et al. 2000). Possible contributing factors to this decline include an increase in wild mussel harvest (NOAA National Marine Fisheries Service 2016), an increase in hurricanes (Carrington 2002), predation by invasive green crabs (Carcinus maenas) (Leonard et al. 1999) and changes in trophic structure due to overfishing (Harris & Tyrrell 2001). Perhaps the most shocking factor is that sea surface temperatures in the Northeast US are increasing three times faster than the rest of the world. Specifically, in the Gulf of Maine, sea surface temperatures are increasing faster than 99% of the world’s ocean waters (Pershing et al. 2015, Markowitz et al. 2016). Lesser (2016) suggests that a decline in mussels in the Gulf of Maine may be a result of increasing water temperatures with additional stress coming from other changes including ocean acidification (Lesser 2016).

Mussels are poikilotherms therefore temperature is an important factor in determining their growth and activity levels (Incze et al. 1980, Filgueira et al. 2016). Historically in North America, M. edulis ranged from the Arctic to the coast of North Carolina. Yet over the past 50 years, the range of M. edulis has been shifting poleward as summer air and water temperatures increase (Jones et al. 2010). Multiple studies state that the optimal water temperature for M. edulis falls within 10-20°C (Coulthard 1929, Bayne et al. 1973, Widdows 1973a, Widdows 1973b, Incze et al. 1980). Water temperatures of 25°C can result in a decrease in filtration rate (Schulte 1975, Filgueira et al. 2016) while 27°C is the lethal temperature (Read & Cummings 1967, Incze et al. 1980, LeBlanc et al. 2010, Filgueira et al. 2016). Temperatures below the lethal level can still be detrimental to mussel health and condition; especially when combined with other stressors (Incze et al. 1980, Sorte et al. 2016). Warming is likely to continue, as will range shifts for M. edulis (Jones et al. 2010).

This study takes a conservation physiology approach to assess mussel health, condition and resilience. When faced with an issue like declining mussel populations, conservation physiology pushes research beyond an organism-presence/absence approach and gets to the “cause and effect” of why a species is disappearing (Wikelski & Cooke 2006, Cooke et al. 2013). For example, Sorte et al. (2016) discovered a decrease in blue mussel abundance in the Gulf of Maine using historical data, quadrat sampling and transect surveys, but could only speculate on the cause of this decline (Sorte et al. 2016). Concepts of physiology can be used to describe what is happening at the tissue and cellular level of the mussels to identify specific times when mussels are most vulnerable and least resilient. These pieces can be combined with environmental data in an effort to inform future decisions concerning both farmed and wild mussels (Ricklefs & Wikelski 2002, Wikelski & Cooke 2006).

  Resilience is a system’s capacity to recover from or adapt to stress or a disturbing force (Cropp & Gabric 2002, Thrush et al. 2008, Dynesius et al. 2009, Marsden & Maclaren 2010). Individual mussels makeup this study’s ‘system’ and the more storage tissue a mussel has in its mantle, hypothetically the better it can cope with stress. For this study, stressors most commonly take the shape of high water temperatures, low food availability and the presence of pathogens. The effects of stress on an organism can be articulated through physiological resilience. Sokolova et al. (2012) outlines the concept that organisms have a pool of energy from which they can draw from. In an ideal, low stress environment an organism may be able to invest equal amounts of energy from this pool into different bins such as reproduction, storage, growth and maintenance. However, if the organism finds itself in a stressful environment, such as warmer water temperatures, more energy may be required for overall body maintenance and less energy is put into other bins (Sokolova et al. 2012). We can also look at reproductive resilience, defined by a population’s ability to reproduce successfully, ensuring population stability in the long-term, in the face of disturbances (Lowerre-Barbieri et al. 2015, Biggs et al. 2018).

Extensive knowledge on mussel health from a cellular, tissue and physiological level is needed in order to properly assess resilience and identify current threats. This information is critical in areas such as the Gulf of Maine that are experiencing elevated climate change effects. In order to take a conservation physiology approach to mussel health and resilience, this study employs histology and lipid/fatty acid analysis to assess reproduction, energy investment, diet and pathology; all key components in determining resilience capacity of a mussel. Knowing when mussels are reproductively active and where they are investing energy can reveal their overall resilience to stress and pathogens. Changes in diet throughout the year may also enhance this understanding.   

In order to explore these aspects of mussel health, this study combined a suite of methods. Histology provided a way to study the mussels and their tissues at a cellular level. Histological processing led to the creation of a library of thousands of slides. When the mussels were in slide-form, further analysis was made in terms of mussel reproduction, energy investment and pathology. Staging was chosen to assess the reproductive cycle; staging is a ‘classic’ method that has been used for decades (Chipperfield 1953, Seed 1969, Duinker et al. 2008) that can reveal patterns in the reproductive cycle and gonad development. Energy investment was assessed using a novel image analysis technique that also made use of histological slides created for this study. Reproductive tissue and two types of storage tissue are easily visible in mussel mantles and each stain a distinct color. This allowed for easy separation during image analysis. Histological slides were also used to identify pathogens and stress responses present in the mussel tissue. Each of these components studied through histology have the potential to provide answers to questions concerning mussel health and resiliency. Lipid and fatty analysis were used to provide insight into mussel diet, and how it differs throughout the year. Mussel resiliency and condition can be inferred based on what types of fatty acids and lipids are present in their tissues. Additionally, Condition Index (CI) data were taken on all mussels sampled. CI is a commonly used metric in industry and research settings, but the information it conveys may be misconstrued. Efforts were made to determine how best to use CI in the context of mussel resiliency and physiological condition.

The use of general condition indices (CI) have been established and assessed for M. edulis and used in research and commercial practices for over 50 years. Davenport & Chen (1987) conducted a comparison of seven different methods of calculating CI, and suggest the following as the best assay, one not affected by prior freezing of samples:

CI is an accessible and inexpensive tool for assessing condition of mussels but does not provide insight to the cellular, pathological, and biochemical condition of the organism.

Several studies have employed histology as a tool to assess the pathology, reproduction and condition of mussels. Bignell et al. (2008) histologically assessed extensively 29 different specific pathological health parameters, as well as the presence and abundance of adipogranular cells (ADG) within vesicular connective tissues (VCT) of wild populations of M. edulis and Mytilus galloprovincialis in the UK for example. Duinker et al. (2008) similarly used an assessment of ADG and VCT in an assessment of health, condition and spawning in mussels in Norway.  ADG and VCT are the primary storage tissues for the mussel; VCT is associated with storage of glycogen and ADG with lipid and proteins (Mathieu & Lubet 1993).  Thus, ADG, VCT and reproductive status are all valuable indicators of energy organismal investment and potential resiliency in the face of stressors.

Especially informative to our proposal is a study by Sunila et al. (2004) in which the reproduction and pathology of M. edulis on an experimental mussel farm in Long Island Sound, Connecticut, USA was conducted. Long Island Sound (LIS) is considered the a southern extent for the species and prior to this study the pathology, reproduction and aquaculture potential for M. edulis in the LIS region was unknown. Results of the study found that despite exceptionally high growth rates, condition was greatly affected by high prevalence of the diagenetic trematode parasite Proctoeces maculatus, which is considered typically a tropical to sub-tropical species. The GoM is currently considered one of the fastest warming portions of the oceans and as warming continues there is high probability of sub-tropical parasites, akin to Proctoeces maculatus moving into the GoM. However there is little if any available information on the pathology and condition of farmed mussels in the GoM.

In response to a mass mortality event on mussel farms in Casco bay in 2016, the UNE Ocean Food Systems Group conducted a limited case study led by Center for Excellence in the Marine Sciences (UNE-CEMS) Research Scientist Adam St. Gelais, on M. edulis histopathology of farmed mussels impacted by the event. This has allowed the research team to perfect the methodology for conducting histology in this species. Moreover, preliminary results suggest the mussels affected in the mortality event were extremely gravid, but appeared to lack both VCT and ADG storage tissues (figure 1) suggesting a low resiliency to stress recovery due to inadequate energy stores at a time of year (late summer) when food is in limited supply.

Economic and environmental sustainability of Maine working waterfronts has declined as capture fisheries that support them have consolidated or collapsed. Adoption of sustainable aquaculture has become critical to the diversification of the working waterfront in Maine. Emerging as a foundational aquaculture species in Maine are rope-grown farmed blue mussels (Mytilus Edulis). Considered among the highest quality mussels on the market they command a premium yielding market prices upwards of $8.00/lb with a seemingly insatiable market demand.

Any yet, the blue mussel in the Gulf of Maine (GoM) is struggling in the face of climate change. The GoM is rapidly warming (Pershing et al., 2015), producing physiological disruptions (Lesser, 2016) and bringing with it acidic waters, invasive predators, competitor species. The result is an alarming decline in wild intertidal populations (Sorte et al, 2016.) Furthermore, in summer 2016 an unexplained mortality event of farmed mussels led to a loss of ~$60,000 worth of mussel stock on one farm in Casco Bay, Maine.

Despite this, there has been no assessment of the condition, pathology, or parasitology of farmed blue mussels in Maine. We propose an intra-seasonal health and condition assessment through combined histopathological and biochemical analysis of pathology, reproduction and fatty acids in mussels farmed in Casco bay, Maine.

Results will inform farmers of when their crop is healthy, stressed, and nutritious, and hopefully lead to the development of best management practices to mitigate product loss, and serve as an important pathological baseline for monitoring farmed mussel populations as the GoM and climate continue to change.

Maine rope-grown farmed blue mussels (Mytilus Edulis) have garnered a reputation as some of the highest quality mussels on the market commanding a premium farm-gate value and yielding market prices upwards of $8.00/lb. The industry is currently incapable of filling market demand and is poised for sustainable expansion. However, in the face of climate change the GoM is among the most rapidly warming portions of our global oceans (Pershing et al., 2015), making farming blue mussels in the Gulf of Maine not without its challenges and risks. In fact, mussels appear to be already struggling in the GoM; a recent alarming study reported on the severe long term decline in this species within the intertidal zone along the entire 3,500 mile coast of Maine (Sorte et al., 2016) and evidence suggests severe sub-lethal impacts and metabolic depression in response to climate change stressors as the organismal level (Lesser, 2016). In fact, farmers in the mid-coast Maine region during the 2016 growing season reported spontaneous mass mortality of market size product following spawning out of synch with normal reproductive cycles. This reproduction-mortality coupled phenomenon is indicative of severe stress in sessile marine invertebrates and has been documented previously in farmed mussels (Myrand et al., 2000). Despite these alarming events, there is paucity of data pertaining to the condition, pathology and parasitology of mussels in Maine. A histopathological and biochemical baseline for the region is needed in order to effectively monitor for changes in the health of farmed mussels in Maine in the face of a changing climate and a warming GoM. 

(Map of region studied and full citations are attached in Research section below.)

Cooperators

Click linked name(s) to expand
  • Matthew Moretti
  • Carrie Byron, PhD (Researcher)
  • Connor Jones (Researcher)

Research

Materials and methods:

References and figures

Sampling and Study Sites

Mussels were sampled on a bi-monthly basis from farm sites located in Casco Bay, Maine (Fig. 1). Mussels were taken from the surface (less than a meter deep), either off the actual ropes or from the black plastic floats supporting the rafts. Samples were placed into mesh bags and stored on ice for immediate transportation to the lab. This study covers samples taken between February 2017 and October 2018. For each sampling day, at least 10 mussels were processed for histology and six were processed for lipid and fatty acid analysis. Mussels were cleaned of fouling organisms and general measurements were taken for each animal: total weight (g), shell length (mm), shell width (mm), shell depth (mm). Length measurements were taken with a pair of manual calipers. All samples were between 25mm and 85mm in shell length. Samples were then shucked, and the meat was removed from the shell. A sex estimate was recorded along with the following measurements: wet meat weight (g), shell weight (g).

 

Condition Index

Condition Index (CI) was calculated according to Davenport & Chen (1987).

CI = (wet meat weight (g)/wet shell weight (g)) * 100

This method was chosen because the required metrics were already being collected for mussels being processed for histology and lipid/fatty acid analysis.

 

Environmental Data

Data were collected on the two environmental parameters identified as being most influential for mussel reproduction; food availability and water temperature (Chipperfield 1953, Newell et al. 1982, Maloy 2001). Chlorophyll concentration was collected by Friends of Casco Bay, on an hourly basis, using a YSI optical sensor at a monitoring station in Casco Bay, ME (43.7519896, -70.1392618). These data represent relative abundance. Chlorophyll data is used as a proxy for phytoplankton, a main source of food for mussels. Figure 2 illustrates the average monthly water temperature (°C) and average monthly chlorophyll concentration (mg/L) during the sampling period of this study. Water temperature was also collected by Friends of Casco Bay, on an hourly basis, between the surface and a depth of 5 meters.

 

Histology

After being shucked from their shells, samples were cut transversely along the dorsoventral axis. The anterior half of the organism was placed in a labeled 20 mL vial filled with a 10% zinc-buffered formalin (z-fix) and filtered seawater solution. The tissues remained in the fixative for at least 24 hours and were subsequently rinsed three times with 70% ethanol and placed into labeled jumbo cassettes. Cassettes were stored in 70% ethanol until the mussels could be cleared in a tissue processor (submerged in increasing concentrations of alcohol, then xylene washes, then paraffin infiltration) at the University of New England (UNE) Histology & Imaging Core. After processing, mussels were embedded, cut side down, in paraffin wax blocks. Blocks were sectioned on a Leica Microtome producing 5µm thick transverse sections. A total of 5 slides (10 sections) were made from each block/individual mussel. Slides were separated by 1mm (50 turns of the microtome set at 20mm) and two sections within each width were included on each slide. Slides air dried overnight before baking at 60°C for at least two hours. Slides were then de-paraffinized and stained with Hematoxylin and Eosin using a Leica auto-stainer and cover-slipped by hand.

Reproduction

The acini/follicles of each mussel were staged using a guide created with the help of Michele Condon, based on Duinker et al. 2008 and Chipperfield 1953. Two slides were chosen out of the five generated for each mussel. These slides were usually the second and fourth or the third and fifth in the sequence. The first slide was generally avoided because these sections did not always include the entire organism. Analyzing every other slide ensured that there was about 2mm of space between each section. From each slide, 10 acini/follicles were staged from the left side of the mantle and 10 were staged from the right side. This resulted in 20 acini being staged per slide, and 40 total acini being staged per individual mussel. Staging scale is as follows. Also see Figure 3 for micrograph examples of each stage.

 

Stage Classification

            For a visual representation of each stage, see Figure 3.

Stage 0 – no gametes or gametes are too undeveloped to determine a sex, acini are empty or not even present, cannot determine sex

Females

            Stage 1 – Immature stage, sex determined, acini are small, oocytes present, SOME of the oocytes are starting to develop a visible nucleus (light purple circle), ALL eggs attached to acini wall

            Stage 2 – Developing stage, acini have grown in size, MOST oocytes have visible nucleus (light purple within dark purple), MOST eggs are attached to acini wall, some oocytes are detached and fully mature

            Stage 3 – Mature/Ripe stage, acini are large and take up most of the mantle area, ALL oocytes contain visible nucleus, many oocytes are detached from acini wall

            Stage 4 – Spawning stage, similar to Stage 3, fewer oocytes present within each acini, more interstitial space *(for visualizing the data in this study, stage 3 and stage 4 follicle counts were combined for females into what is labeled as ‘Stage 3’, follicles given a Stage 5 tag are simply called ‘Stage 4’)

            Stage 5 – Post-spawning stage, acini are almost completely empty, few mature oocytes still present

            *for the results, discussion and figures Stage 4 acini were combined with Stage 3 acini to create a ‘mature and beginning to spawn’ stage. Stage 5 acini were renamed ‘Stage 4’, but all the acini are still in a post-spawning stage. This in an effort to have equal numbers of stages for females and males.

 

Males

            Stage 1 – Immature stage, sex determined, no spermatozoa, acini are small

            Stage 2 – Developing stage, acini have increased in size, majority of acini filled with immature sperm (larger cells at margin of acini), spermatozoa present at acini center, pink sperm tails visible at acini center

            Stage 3 – Mature/Ripe stage, acini take up most of the mantle area, spermatozoa outnumber immature sperm cells

            Stage 4 – Post-spawning stage, acini are mostly empty, some mature and immature sperm still present along acini margins 

 

Energy Investment

Using the same two slides used for staging, four pictures were taken of the mantle tissue (e.g., one from the left mantle of slide #2, one from the right side of the mantle of slide #2, repeat for slide #4) at 100x magnification. Pictures (.jpg files) were uploaded into ImageJ or FIJI. If necessary, the border of the mantle was traced with the ‘polygon tool’ and the outside was cleared. The total number of pixels for the mantle in frame was determined. Then, using ‘Image > Adjust > Color Threshold’ the total number of pixels for gonad tissue (purple), vesicular connective tissue (VCT) (white), and adipogranular tissue (ADG) (pink) were determined based on their color difference. For each individual mussel, there were four measurements for total mantle, gonad, VCT, and ADG. These numbers were summed for each mussel, to create an aggregate pixel count for total mantle, gonad, VCT, and ADG. Ratios of gonad, VCT and ADG to total mantle were calculated and averaged either by month or by individual sampling date.

 

Parasite/Pathological Factor Survey

            Of the five slides produced from each mussel sampled, two were chosen for the pathology survey. The first slide was generally avoided because these sections did not always include the entire organism. Slides were chosen based on which slides were most representative, containing all tissue types and structures to be assessed via this study. Adjacent slides were not selected, ensuring that there was about 2mm of space between each section. An initial, rapid review of the first 1-2 months of slides revealed the most common pathologies that became the basis for this pathological survey. These included gill ciliates, digestive gland atrophy (DGA), oocyte atresia and hemocyte filled mantle follicles (Fig. 4) and these are the factors that this study focuses on. Identification was aided by the “Histopathological Atlas: Marine Bivalve Molluscs” (Darriba 2017). Presence/prevalence and intensity of these factors were recorded on a monthly basis using the following equations from Apeti et al. (2014):

Prevalence = ((Σ hosts with parasite or pathology) / (number of hosts analyzed)) x 100

Intensity = (Σ number of occurrences of pathology) / (number of hosts with pathology)

 

Counts of gill ciliates and hemocyte filled mantle follicles were made to inform intensity calculations. The total number of ciliates from each slide were summed to create the: number of occurrences of pathology. This was because there was a low chance that ciliates would be counted twice with 2mm between the two slides. Hemocyte filled mantle follicles however had a high chance of being counted twice, so only the slide with the highest number of hemocyte filled follicles was used to inform the: number of occurrences of pathology. Oocyte atresia and DGA were graded on a 0 – 4 point scale based on their presence in the target tissue (digestive system for DGA, mantle for oocyte atresia):

0: no presence of condition in target tissue

1: condition is present in up to 25% of target tissue

2: condition is present in 25-50% of target tissue

3: condition is present in 50-75% of target tissue

4: condition is present in over 75% of target tissue

Intensity values for oocyte atresia and DGA are therefore between 0 and 4. Intensity in general is only looking at infected individuals. Trematode prevalence was also recorded (Fig. 4), in an effort to determine if Proctoeces maculatus could be found in Casco Bay.

 

Lipid and Fatty Acid Analysis

Six mussels from each sampling event were shucked and the meat was placed in a lipid-cleaned glass vial along with 2mL of chloroform and topped off with 3 seconds of Nitrogen gas before being capped and sealed with Teflon tape. The tubes were stored in a -80°C freezer to prevent tissue degradation. Select samples were chosen from certain times of the year based on characteristics observed in the histology slides and energy investment analysis.

Samples were sent to Memorial University of Newfoundland where the lipid samples were extracted in accordance with Parrish (1999). Each mussel was homogenized in a 2:1 solution of ice-cold chloroform:methanol using a Polytron PCU-2-110 homogenizer (Brinkmann Instruments, Rexdale, Ontario, Canada). The ratio of cholorform:methanol:water was brought to 8:4:3 by adding chloroform extracted water. Samples were sonicated in an ice bath (4-10 minutes) and centrifuged at 5000 rpm (2-minutes). The bottom organic layer was extracted/removed via a double pipetting technique. This ensured that the aqueous layer was not included. Chloroform was added back to the extraction test tube and the process was repeated three additional times. The successfully extracted organic layers were combined in a lipid-cleaned vial and concentrated using a flash-evaporator (Buchler Instruments, Fort Lee, NJ).

Composition of lipid classes was determined using an Iatroscan Mark VI TLC-FID, silica coated Chromarods and a three-step development method (Parrish 1987). Lipid extracts were added to the Chromarods, 100% acetone was used to focus them to a narrow band. Hexane:diethyl ether:formic acid (99.95:1:00.05) was the first development system, the rods developed for 25-minutes, were removed from the system for 5-minutes then replaced again for 20-minutes. Hexane:diethyl ether:formic acid (79:20:1) was the second development system, in which the rods spent 40-minutes. The last development system consisted of two steps. Step one, 100% acetone for two, 15-minute periods. Step two, two 10-minute periods in chloroform:methanol:chloroform-extracted water (5:4:1). Rods were dried a constant humidity chamber before each solvent system. Rods were scanned after each development system in the Iatroscan and the data were collected using Peak Simple software (ver 3.67, SRI Inc). Chromarod calibration was completed using standards from Sigma Chemicals (Sigma Chemicals, St. Louis, MO).

Lipid extracts were transesterified using sulfuric acid and methanol for 1-hour at 100oC.  The fatty acid methyl esters (FAME) were analysed on a HP 6890 gas chromatograph flame ionization detection (GC FID) equipped with a 7683 autosampler. The GC column was a ZB wax+ (Phenomenex, USA). Columns were 30m long with an internal diameter of 0.32mm. The column temperature started at 65°C, this temperature was maintained for 30-seconds. The temperature increased to 195°C at a rate of 40°C/min, maintained for 15-minutes then increased to a final temperature of 220°C at a rate of 2°C/min. The final temperature was maintained for 45-seconds. Hydrogen was used as the carrier gas. It flowed at a rate of 2 ml/minute. The injector temperature began at 150°C and increased to a final temperature of 250°C at a rate of 120°C/minute. Temperature of the detector remained constant at 260°C. Peaks were identified using retention times from standards purchased from Supelco, 37 component FAME mix (Product number 47885-U), Bacterial acid methyl ester mix (product number 47080-U), PUFA 1 (product number 47033) and PUFA 3 (product number 47085-U).  Chromatograms were integrated using the Varian Galaxie Chromatography Data System, version 1.9.3.2. A quantitative standard purchased from Nu-Chek Prep, Inc (product number GLC490) was used to check the GC column after every 300 samples, or once a month, to ensure the areas returned were as expected.

For this study, select fatty acid biomarkers were used to identify what the mussels were feeding on (Table 1) (Kelly & Scheibling 2012, Parrish 2013).  

Statistical Analyses

            Nine linear regressions were created with variables of temperature, chlorophyll, ADG, VCT and gonad to determine their degree of correlation (Table 2). Model II theory with geometric means was utilized (RStudio, version 1.1.463), as neither the independent nor dependent variables were controlled (Laws & Archie 1981).  

A t-test (two tailed, equal variance, a=0.05, df=110) was used to determine degree of interannual variability in energy investment between 2017 and 2018, specifically during the months of April, May and June, both as individual months and with the three months combined for each year. Differences in means were recorded for each month as well as the aggregate period of April to May.

Principal coordinates analysis (PCO) was performed on all the fatty acid data (percent composition) collected. PCO (Fig. 5) was used to visualize the similarity between samples based on their fatty acid composition. Data were transformed via a square root transformation. SIMPER was used to assess similarity between and within groups, resemblance based on S17 Bray-Curtis similarity, with a cut off for low contributions at 70%. PCO was run on PRIMER 7 (version 7.0.13, from PRIMER-e, serial no. 4784, Quest Research Limited).  

 

 

Research results and discussion:

Environmental Data

            Environmental data is summarized in Table 3 as monthly averages. Chlorophyll concentration peaked in March for both years, but it was significantly higher in March of 2018 than March of 2017 (t-test, p<0.001). August was the hottest month for both years in terms of water temperature while January 2018 was the coldest month.

Condition Index (CI)

Condition Index peaked in April in both 2017 and 2018, closely mirroring the gonad peaks in both years. CI was lowest during the mid-summer months. After hitting a low point in the summer of 2017, CI began an upward trajectory until it peaked again in April 2018. After the trough of 2018, CI began to recover, then stayed relatively stable at roughly 100 for the rest of the year (Fig. 6).

Reproduction

A total of 463 mussels were staged for this study. Of these mussels, 228 were female and 235 were male. Three of these mussels showed characteristics of being hermaphrodites. In Figure 7, to keep the figures consistent between males and females, Stages 3 and 4 follicles were combined for female mussels, while Stage 5 follicles are referred to as Stage 4. For males and females, a Stage 3 label denotes follicles are full of gametes that are fully developed and ready to spawn. The highest percentage of Stage 3 female follicles in 2017 occurred in May in (95%). In 2018 the female Stage 3 peak occurred in June (93%) (Fig. 7). For males, highest percentages of Stage 3 follicles occurred September 2017 (73%), and June 2018 (97%) (Fig. 8). The lowest percentage of mature follicles occurred in November 2017 (0%) and October 2018 (14%) for females. This corresponded with high occurrences of Stage 1 developing follicles (Fig. 7). For males in 2017, February and March yielded 0% Stage 3 follicles. The lowest percentage occurred in 2018 during October (43%) (Fig. 8).

Energy Investment

A total of 434 mussels were analyzed. Of these, 213 were female, 214 were male and 7 were unable to be positively identified as either male or female due to lack of gonad tissue or hermaphroditic characteristics. For the purposes of this study, males, females and unidentifiable sexes were all combined as energy investment of the population as a whole is the focus, not whether there were any differences in investment between sexes. Area taken up by gonad, for males and females combined, peaked in April in 2017 (63%) and both May and June (77%) in 2018. Gonad tissue was lowest November 2017 (15%). Adipogranular tissue (ADG) was highest in November 2017 (36%) and lowest in May (3%) and June (1%), 2017 and 2018, respectively. Vesicular connective tissue (VCT) remained fairly constant throughout this timeseries and reached minimums in correspondence with maximum levels of gonad tissue (May 2017, April-July 2018) (Fig. 6).

The strongest relationships between tissue types were found for gonad and ADG tissue and gonad and VCT (r of -0.7, Table 2). There was a positive relationship between ADG and VCT (r of 0.5, Table 2.). There was no obvious relationship between any of the tissue types and any of the environmental parameters (Table 2).

Based on a t-test (two tailed, equal variance), there was significant interannual variability between the population level reproductive investment at or around the months of peak gravidity in 2017 vs 2018. The “peak gravidity” period is defined by the months of April, May and June, when gonad tissue is most present in the mantle. This period in 2018 saw significantly more gonad tissue than that of 2017 (p<0.001). Gonad tissue during this bulk period was 18.6% higher in 2018 than 2017. Inversely, storage tissues during the bulk reproductive period were significantly lower in 2018 than 2017 (p<0.001 for each type); ADG tissue was 3.4% lower in 2018, while VCT was 12.3% lower.  

Lipids

            Lipid classes were identified from 21 individual mussels. These mussels represent seven different months with three replicates per month: August 2017, November 2017, January 2018, March 2018, May 2018, July 2018 and August 2018. These months align with times of the year that represent pre and post spawning mussels and describe times when reproductive tissue was at a maximum, storage tissue was at a maximum or there was a combination of the two. Lipid classes identified were as follows: acetone mobile polar lipids, ethyl ketones, free fatty acids, hydrocarbons, phospholipids, sterols and triacylglycerols. The most abundant classes are graphed in Figure 9, along with total lipids. Lipids were reported as weights (mg/g of mussel meat wet weight) and percentages (percent of total lipids identified). Weights were not able to be calculated for January 2018 mussels. Total lipid values were calculated for each month using weight values. Triacylglycerols and Phospholipids were the most abundant classes throughout the timeseries. In terms of weight, triacylglycerols and phospholipids were highest in March and May of 2018 (Fig. 9). Phospholipids were consistently the most abundant lipid class, in terms of percentage of all lipids, throughout the timeseries. In July and August of 2018, they made up over 60% of the lipids identified in the samples.

 

Fatty Acids

            Fatty acid composition was determined for the same 21 mussels used for lipid analysis. Like lipids, fatty acids were reported as weights (mg/g of mussel meat wet weight) and percentages (percent of total fatty acids identified) and weights were not able to be calculated for January 2018 mussels. For all months, for both weight and percentage, diatom, dinoflagellate and detritus fatty acid biomarkers were the most abundant. In terms of weight, diatom biomarkers were highest in March and May of 2018. In terms of percentage, they were highest January, March and May of 2018 (Fig. 10). Peaks in both fatty acids and lipids occur after the 2018 spring bloom (Fig. 10) and around the time of highest investment in reproductive tissue (Fig. 9 & 11).  

Principal coordinates analysis (Fig. 5) reveals that samples are split into two groups based on their similarity in percent fatty acid composition. These groups can be categorized as samples from the winter and spring and samples from the summer and fall. 

Pathology

Pathological survey data are summarized in Table 4. The pathological survey revealed that these mussels had low levels of parasites, but comparatively high levels of stress induced conditions such as oocyte atresia, digestive gland atrophy and hemocyte filled mantle follicles. Oocyte atresia was present in the majority of female mussels for all but one month of this study. Atresia is not an abnormal process and atretic eggs are commonly found pre and post spawning in gonad tissues. The abundance of atretic eggs was not identified in this study but increased numbers of atretic eggs appeared to be occurring. Five cases of trematode infection were recorded in mussels from this sampling period; two from August 2017 and three from January 2018. Although intensity values for trematodes were not calculated from this study, it should be noted that one mussel, a male from January 2018, contained over 100 trematode sporocysts throughout its entire body. Due to its positioning within the mussel tissue, this species is likely Proctoeces maculatus (Canzonier 1972).

 

Research conclusions:

This study is a broad scale health assessment that covers mussel reproduction, energy investment and pathology through the lens of conservation physiology. It reveals seasonal and interannual variation of a population of farm-grown blue mussels (M. edulis) in Casco Bay, Maine and determines times of least resilience. Based on these data and previous studies, most aspects of mussel condition appear to be related to reproduction. In turn, reproduction is thought to be mediated by environmental conditions and cues (Chipperfield 1953, Newell et al. 1982, Maloy 2001, Suárez et al. 2005).

Condition Index

Condition Index is a measurement often used by farmers to inspect the quality of their crop in terms of meat yield (Davenport & Chen 1987). Throughout this time series, CI has closely followed the gonad curve (Fig. 6). When spawning occurs, meat yield decreases as material is being lost to the environment. CI does not take into account resiliency or energy reserves and may give a false impression on what is actually happening in the mussel physiologically. The highest monthly average condition indexes were reported during the spring and early summer. This is when the mantle is most full of reproductive material, and least full of storage tissue. Mussels yield the most meat at these times of the year, but they are poorly equipped to cope with stress, as most of their resources have been put towards reproduction (Incze et al. 1980). It may be inferred that high investment in reproduction does not necessarily equate to high reproductive or organismal resilience, because this cannot be sustained as a long-term strategy, especially if there are lacking levels of food. A more comprehensive metric could be used to determine resiliency, such as a ratio of storage tissue to gonad tissue (Fig. 12). When this ratio is high, mussels have more energy reserves. It tends to be highest when CI is lowest. When used together, CI and Storage:Gonad may be able to reveal the dynamics between reproductive investment and resilience. CI alone does not fit into the goals of a conservation physiology study, as CI on its own is not informative enough.

Reproduction

Staging reveals that throughout nearly the entire year, mussels contain mature follicles, with gametes that are ready to be spawned. From a physiological resilience standpoint, these mussels have always been able to put some energy towards reproduction. Stretches in time where no mature gametes are present in mantle follicles may indicate stress levels that warranted diverting energy away from reproduction (Sokolova et al. 2012). This observation may also be a direct result of mussels being reproductively oriented physiologically. Mussels may always have to put some percentage of energy towards reproduction regardless of season. This then leaves less energy for other bins.  

Through the use of an adapted staging scale, spawning times were estimated. The transition from Stage 3 follicles to Stage 4 follicles in both males and females, seen in Figure 7 and Figure 8, respectively, was used as an indicator of a spawning event. During May 2017, the majority of follicles staged (female: 95%, male: 67%) were Stage 3 (gametes are ripe, mature, ready to be spawned). During the following months, the percentage of Stage 3 follicles decreased as the percentage of Stage 4 (post spawning, empty acini) follicles increased. From this, a spawning event between May and July in 2017 can be inferred but not confirmed. It is not uncommon to see mussels spawn twice in one year in this part of the world (Chipperfield 1953, Seed 1969, Emmett et al. 1987) and a second spawn may have occurred between August and October of 2017. Mussels are synchronous spawners, so females and males should spawn around the same time to ensure successful external fertilization (Newell et al. 1982). In 2018, the onset of the spawning event seemed to have shifted later in the year, with the highest percentage of Stage 3 follicles in the mantle occurring in June 93% for females and 97% for males. Again, high investment into reproduction may not necessarily equate to reproductive resiliency, especially when food and energy reserves are lacking. These high levels of mature follicles continued into August. There also seemed to be a lack of a distinct, second spawning event in 2018. In 2017, the percentage of Stage 3 follicles recovered after spawning to levels close to the spring. In contrast to 2018, where only a single spawning event is suggested. The farmer had noted that they observed a spawning event on July 14th in 2016, a spawn in 2017 occurred earlier than in 2016, and the spawn in 2018 occurred later than in 2017 (correspondence with partner mussel farmers).

These data are often visualized as a timeseries with data pooled into months or days. If you arrange the data based on average water temperature from the week prior to the sampling day (Fig. 13), other patterns emerge. Looking specifically at the females from this study, their reproductive cycle, as it relates to water temperature can be broken down into three distinct periods. From -1.1°C – 9.1°C the mussels are in a developing period characterized by high levels of Stage 1 and 2 follicles, with Stage 3 follicles increasing in percentage as temperature increases. From 9.2°C – 12.3°C the mussels are mature, nearly 90% of the follicles from these sampling dates were filled with mature and ready to be spawned oocytes. At this point there was little presence of Stage 4 follicles, indicating that mussels had not begun large spawning efforts. The third period, spawning and redeveloping, occurs between 13°C – 19.2°C. This is when the highest percentage of post spawning stages are observed. Many lower stages are observed during these temperatures as well, indicating mussels that may be redeveloping after spawning in preparation for another spawning event. Chipperfield (1953) observed that mussel spawning in Britain aligned with sunny, rain-free weather when the water temperature reached 11-13°C (Chipperfield 1953), which aligns with the lower temperature range of this study’s spawning period.

Staging is a somewhat subjective process and many studies that employ it use modified scales based on the work of Chipperfield (1953) (Chipperfield 1953, Seed 1969, Duinker et al. 2008). This can make it difficult to compare staging data from different studies, and researchers within the same study can also have their own interpretations of a shared scale. A solution would be to create a standardized staging scale to be used for all M. edulis reproductive studies to facilitate comparison between studies in all areas where M. edulis grows or is farmed. Staging can lead to figures, such a percentage stacked bar plots (Fig. 7 & 8), that help to visualize the reproductive cycle and gametogenesis of a mussel over a given time period. However, these figures give little information on other aspects of mussel condition and resiliency, such as energy investment.   

Energy Investment

Image analysis of the mantle tissue may be a more refined and less subjective method compared to staging. The ImageJ color threshold analysis helps to corroborate potential spawning time estimates. Moreover, this assay can provide critical insight into other processes occurring within the mantle tissue, specifically all storage tissue allocation. Storage tissue is important to think about when it comes to mussel resilience in the face of environmental stress. In Figure 6 it is suggested that after the gonad line peaks, it declines because the mussels are losing gonad material as they spawn. It can be inferred that the main spawning events occurred following April in 2017 and June in 2018. The second spawning event of 2017 can be seen as starting after the small peak in September.

This methodology and Figure 6 also highlight the inverse relationship reproductive tissue has with storage tissue, adipogranular (ADG), and vesicular connective tissue (VCT). As the follicles are developing, they grow in size (Suárez et al. 2005), concurrently storage tissue cells shrink in size as nutrients from ADG and VCT are used for energy and structure in developing oocytes and sperm (Bayne et al. 1982, Gabbot 1983, Emmett et al. 1987, Pazos et al. 1997, Freites et al. 2003, Ojea et al. 2004, Narváez et al. 2008, Martínez-Pita et al. 2012). Out of all the variables, gonad tissue has the strongest relationship with the other tissues occupying the mantle (Table 2). This relationship is not evident if reproduction is only assessed through staging.

Figure 14 illustrates the relationship gonad tissue has with temperature. The highest levels of reproductive tissue in the mantle tend to fall between 5° C – 15° C. This is slightly broader than the temperature range observed for the ‘mature period’ in staging (9.2° C – 12.3° C). This reveals that just before the warmest part of the year, a mussel has invested the majority of its energy into reproduction, putting little towards storage and resilience.   

The energy requirements for gametogenesis and spawning can put organisms in a state where they are more susceptible to stress from temperature, parasites, pathogens and pollution (Li et al. 2007, Song et al. 2007, Petes et al. 2008, Li et al. 2009 a, b, c, Sokolova et al. 2012). Months where storage tissues are particularly low can be times of low resiliency for mussels. This is especially so when low storage tissue levels coincide with spawning events. Reproduction requires an input of materials from the mussel, which are then lost when gametes are released into the water (Emmett et al. 1987). Glycogen especially, stored in VCT, is used by animals during times of high environmental stress to maintain condition (Clements et al. 2018). Visual inspection of the mantles of mussels immediately after a spawning event reveals thin, watery, transparent tissue. To cope with the overall lack of material (storage and reproductive tissue) in the mantle after spawning, mussels will take in water to make up for lost volume (Chipperfield 1953). This was observed in specimens sampled during the summers of 2017 and 2018.  

One of the more interesting outcomes of this study was the reveal of the degree of interannual variability, most noticeable during the peak gravidity period. The reproductive period of 2018 saw significantly lower levels of both types of storage tissue (ADG and VCT). Glycogen, stored in VCT in mussels, is used by animals during times of stress to preserve normal condition, while lipids stored in ADG are used during times of low food availability (Clements et al. 2018). In oysters, glycogen storage levels can be used as an indicator of the animal’s resiliency (Dridi et al. 2007). Temperature was also significantly higher (t-test, p<0.001) during this period in 2018 than 2017. Clements et al. (2018) observed in a controlled laboratory study that mussels exposed to higher temperatures (22°C vs. 16°C) had lower levels of glycogen, lower CI and increased mortality. Although they did not see it, other studies have shown that an increase in temperature can also decrease protein and lipids in marine mollusk tissue (Valles-Regino et al. 2015, Tate et al. 2017). Reduced glycogen levels can negatively impact reproduction (Clements et al. 2018), which is perhaps why the mussels in question did not spawn a second time in 2018. Our CI did not reflect a decrease from 2017 to 2018 for the gravid season but was in fact higher in 2018. It is difficult to say what the 18.6% increase in energy allocated towards reproduction in 2018 was caused by, although the high concentration of chlorophyll in March 2018 may be an indication that there were high levels of food available to the mussels in the spring (Fig. 2). The mussels did show signs that are known to accompany temperature stress later in 2018. There was anecdotal evidence of a die-off and increased mortality in 2018, both on the farm and in the lab. During mid to late summer, closed shells were shucked to reveal an absence of meat.

A study of M. edulis in Maine’s Damariscotta River observed the death of mussels exposed to water at temperatures above 20°C. While mussels cannot physiologically acclimate well in water over 20°C, these mussels were not dying from increased temperatures alone. Peak mussel mortality always occurred after a phytoplankton decline. Mussels died due to low food availability during a time when they were facing stress due to high temperatures. Depending on where they were in the gametogenic cycle, they may have been lacking ample levels of storage tissue that they normally would have been able to draw from in times of stress and low food availability (Incze et al. 1980).

It has been suggested that mussels may have a pathway in place that determines whether the organism can provide energy to undergo a second spawning event or if it should store energy for the upcoming winter and the following spring (McKenzie 1986, Duinker et al. 2008). Based on some combination of conditions in 2017, the mussels were able to successfully spawn twice, and maintain some degree of storage tissue as they went into the winter. Mussels appeared to not have the capacity to spawn twice in 2018, evident by the low levels of storage tissue and the observed die-off. This variability reveals that not only is resiliency inconsistent throughout the year, it is also inconsistent between different years. Mussel health cannot be inferred from patterns observed in previous years. Yearly monitoring is required, at least until there is some understanding of the patterns that mussels in Casco Bay display.  

Lipids & Fatty Acids

Lipid and fatty acid data are informative on multiple fronts when it comes to mussel health and resiliency. Lipids in mussels are directly related to reproduction as they provide structure to gonad membranes and serve as an energy store that embryos will need during development (Gabbot 1983, Pazos et al. 1997, Freites et al. 2003, Ojea et al. 2004, Narváez et al. 2008, Martínez-Pita et al. 2012). A study by Martínez-Pita et al. (2012) saw lipids increase in the mantles of female mussels as their oocytes matured. Neutral lipids, such as triacylglycerols and polar lipids (phospholipids) accumulate in eggs. Triacylglycerols are used for energy and phospholipids are used for cell structures (Gadner & Riley 1972, Swift 1977, Soudant et al. 1996a, Pazos et al. 1997, Martínez-Pita et al. 2012). Although this study did not distinguish between males and females for lipid analysis, it is likely that the spike in triacylglycerols and phospholipids during March and May (Fig. 9) can be attributed to oocytes in the mantle in preparation for a spawning event.

The fatty acid composition of a mussel is also largely governed by reproduction (Pollero et al. 1979, Chu et al. 1990, Abad et al. 1995, Pazos et al. 1997, Narváez et al. 2008, Martínez-Pita et al. 2012). Dridi et al. (2007) noted that, in Crassostrea gigas, fatty acid levels were highest when gonads were ripest. Conversely, fatty acid levels were lowest during spawning (Dridi et al. 2007) (Fig. 11). Two of the more commonly detected fatty acids (by wet weight) in this study were the essential fatty acids, DHA (Docosahexaenoic acid, 22:6w3) and EPA (Eicosapentaenoic acid, 20:5w3). These biomarkers can be used to determine mussel diet. EPA is produced by diatoms, and DHA by dinoflagellates (Khan et al. 2006). DHA contributes to the structure and function of cell membranes used in female gametogenesis (Marty et al., 1992, Soudant et al. 1996a, 1996b, 1999, Pazos et al. 2003, Martínez-Pita et al. 2012), while EPA may be used during gametogenesis as a source of energy (Martínez-Pita et al. 2012). Blue mussels are acquiring these lipids and fatty acids from their diet, most notably from phytoplankton (Fig. 10). The reproductive cycle observed in blue mussels in Casco Bay relies on large quantities of phytoplankton to be present in the water column at roughly the same time every year. If environmental conditions continue to drastically change, phytoplankton blooms may shift or decline in magnitude (Li & Smayda 1998), negatively impacting mussel reproduction.

Principal Coordinates Analysis (Fig. 5) reveals a seasonal split in the fatty acid composition of mussels in Casco Bay. There are two groupings of mussel samples, marked by 80% similarity in fatty acid composition, that can roughly be categorized into summer and fall samples and winter and spring samples. This split may be caused by a number of factors including water temperature, food availability and timing of reproduction. EPA may be important in maintaining cell membrane fluidity in colder temperatures (Hall et al. 2002). EPA was more commonly seen in the winter and spring samples, when water temperature is generally colder, however DHA has also been suggested to maintain membrane fluidity in cold water (Dey et al. 1993; Buda et al. 1994; Tiku et al. 1996; Logue et al. 2000; Pernet et al. 2007). The split may be due in part to food available to mussels, again revealed by EPA and DHA. The winter and spring samples contain a higher percentage of EPA, which is produced by diatoms, a common component of spring blooms. While the summer and fall samples have a higher percentage of DHA, produced by dinoflagellates. Dinoflagellates are more commonly found in blooms later in the year. The samples taken in the winter and spring may represent pre-spawning mussels, while the summer and fall mussels may represent post-spawning conditions. The fatty acids found in the winter and spring samples may be of high importance when it comes to reproduction, either in terms of materials that go into gamete production or the energy they provide to fuel gametogenesis and spawning. Resiliency may depend not just on the amount of food available to the mussels, but the quality of food. Certain species of phytoplankton are providing these essential fatty acids required for normal function.

Mussels may selectively retain essential fatty acids such as DHA, EPA and 20:4w6 during times of the year when other, non-essential fatty acids are decreasing in the tissue. This can be a sign of stress as it indicates that the mussels are not getting enough essential fatty acids from their diet (Zhukova et al. 1992, Alkanani et al. 2007) The percentage of DHA in mussel tissue increased in 2018 between the months of May and August, the warmest part of the year. Non-methylene interrupted dienes (NIMDs) may also be signs of stress, these indicate that the mussel is attempting to produce compounds similar to essential fatty acids (Alkanani et al. 2007). The percentage of 22:2NIMDb? was greatest in August 2017, and July and August of 2018. In terms of wet weight (mg/g), 22:2NIMDb? was highest in May of 2018. These data may indicate that mussels may be under higher levels of stress during the summer, when water temperatures are high and food availability is low, and during times of high reproductive investment (May 2018).      

Pathology

Results of a pathological survey can reveal the effect low resilience has on mussel health. However, for this study, no one factor stood out as being a major contributor to poor mussel health, or recent blue mussel declines for that matter. Throughout this timeseries, low levels of parasites were detected in sampled mussels. The main parasites found, gill ciliates, are considered to be commensal organisms and only pose a threat when present at high levels and intensities in individual mussels (Darriba 2017). Stress responses however were found in a high number of mussels, and at times, high intensities (Table 4). Higher prevalence and intensities were seen in the summer months, correlating with when storage tissue is low in the mussels (Fig. 6), as are food levels in the environment (Fig. 2). For example, digestive gland atrophy, the thinning of the walls of the digestive tubule, was observed to be most prevalent and most intense during the summer and fall months of both sampling years (Table 4). Digestive gland atrophy can be caused by poor nutrition and environmental pollutants (Winstead 1995, Apeti et al. 2014). For this study, a liberal approach was taken when identifying tubules as thinned and atrophied; more so than a pathologist would be. And many of the tubules were on the outer edge of the digestive ceca, not spread throughout the entire digestive system. Therefore, the DGA in these mussels is not as intense or concerning as the values may suggest.

Oocyte atresia, the main pathological factor found in this study, is an issue of energy investment as much as it can be an indicator of environmental stress. Oocyte atresia is the breakdown of female gametes and is often seen in bivalves, yet it is poorly understood. It is a natural part of the female reproductive cycle, where unspawned eggs are resorbed, and their nutrients recycled (Suárez et al. 2005). Hemocyte filled mantle follicles were often seen in mussels experiencing atresia, a possible sign that the mussels were resorbing oocyte materials. Atresia can also be caused by external conditions that are not conducive to spawning, such as less than ideal water temperatures and low food availability (Suárez et al. 2005). Endocrine disruptor compounds (EDCs), like polycyclic aromatic hydrocarbons (PAHs), have also been seen to increase the incidence of oocyte atresia. Even though the nutrients in the oocytes may be reabsorbed after atresia, the mussel still invested energy into reproduction instead of growth, maintenance or storage during that season and the reproductive resilience of this population is reduced. The resulting reduction in fecundity and reproductive output may have downstream impacts on recruitment. Individuals may be able to survive after failing to spawn, but if atresia is prevalent enough, the population as a whole may suffer over time (Suárez et al. 2005, Ortiz-Zarragoitia & Cajaraville 2010). More traditional assessments such as CI or visual inspections of mussel mantles may suggest that mussels are gravid at specific times. Yet through a conservation physiology approach, the use of histology has revealed that some of these gravid females may not be spawning some, or in extreme cases, the majority of their eggs.  

Trematode sporocysts were identified in five mussels from this sampling period. Close attention was paid towards the species, Proctoeces maculatus, as it has not been previously detected in Maine or Casco Bay. Hemocyte filled mantle follicles were observed in four out of the five infected mussels in this study. P. maculatus however does not occur in gonadal tubules, and therefore does not cause hemocyte reactions in the mantle follicles. The trematodes seen in this study were within the sinusoids of the mussels, which is where P. maculatus is known to occur (Canzonier 1972) making it likely that P. maculatus is in fact present in these mussels. Prior to this study, the furthest north P. maculatus had been observed was Great Bay, New Hampshire in 2016 (Markowitz et al. 2016). This study marks a possible new northern extent for this species. Historically, the cold waters north of Cape Cod, Massachusetts acted as barrier preventing this trematode from spreading (Markowitz et al. 2016). Detection this far north represents climate change effects that have allowed some organisms to expand, while leaving others, like blue mussels, to experience elevated stress levels.

In general, trematodes, including P. maculatus, have complex life cycles involving multiple vertebrate and invertebrate hosts (Buck et al. 2005, Thieltges et al. 2008, Galaktionov et al. 2014, Markowitz et al. 2016). However, P. maculatus in its northern range has adapted to completing its entire life cycle in M. edulis (Stunkard & Uzmann 1959, Wardle 1980, Sunila et al. 2004, Markowitz et al. 2016). High intensity P. maculatus infections can be visible with the naked eye. Larvae are orange and high numbers will turn the entire mantle orange in color – this is known as orange sickness. Trematode infections can cause thinning of the mantle tissue, macroscopic abscesses, organ damage, and castration (Sunila et al. 2004, Markowitz et al. 2016). Trematodes also rob mussels of glycogen and triglycerides which results in slower growth. Ultimately, an infection of P. maculatus may kill its mussel host (Apeti et al. 2014, Markowitz et al. 2016). From an industry standpoint, trematodes lower the quality of the meat and can be harmful for human consumption. Infections may cause toxic metabolites, such as butyric and short chain fatty acids, to accumulate in mussel tissue. This is a result of enzymes secreted by the trematodes that break down neutral fats (Sunila et al. 2004). Trematodes have also been linked to serious intestinal illnesses in humans (Apeti et al. 2014).

CONCLUSIONS

Mussels in Casco Bay are least resilient during late spring/early summer, during and following their main spawning event. This is when their storage tissues are at the lowest levels throughout the year. These energy stores are what the mussel is going to draw from in times of stress and low food availability. Low levels of storage tissue were most easily viewed in histological sections, not via CI. CI does not equate to mussel resiliency, though CI is a good metric for when mussels are most gravid, as seen in the energy investment data (Fig. 6), this is when mussels have the least amount of storage tissue. CI alone cannot be the determinant factor when it comes to mussel health and resiliency.

Times of low resiliency may occur during times of the year when water temperatures are especially warm and when there is low food availability. Though this study did not see any strong correlation between water temperature and reproductive or storage tissue, the dataset in this particular study may be too limited to adequately derive temperature impacts. Temperature has been documented via many other studies as an important environmental mediator and cue. Temperatures may have influenced reproduction in the summer of 2018 (Fig. 2). Climate change effects may have led to these three events happening at roughly the same time. As water temperatures continue to warm, these events may occur together more frequently within a year or on a year-to-year basis. The detection of Proctoeces maculatus in Casco Bay blue mussels further emphasizes a rapidly changing environment that has allowed some species to thrive in new areas, while others are disappearing from their home ranges.

Studying a declining mussel population through the lens of conservation physiology provides a more complete picture of overall mussel health and resiliency. This is compared to more traditional studies that assess presence and absence while possibly overlooking the cause for species absence or decline.

Participation Summary
1 Farmer participating in research

Education & Outreach Activities and Participation Summary

8 Consultations
2 On-farm demonstrations
3 Published press articles, newsletters
13 Webinars / talks / presentations
20 Workshop field days

Participation Summary

2 Farmers
2 Number of agricultural educator or service providers reached through education and outreach activities
Education/outreach description:

Press (2018)

https://www.une.edu/news/2018/une-research-project-bangs-island-mussels-featured-nbc-boston

https://www.nbcboston.com/news/business/Maine-Mussel-Company-Teams-Up-With-University-of-New-England-for-Research-493087721.html

https://www.une.edu/news/2017/une-aquaculture-research-collaboration-bangs-island-mussels-featured-%E2%80%98lab-manager%E2%80%99-magazine

Presentations 

St. Gelais A., Jones C., Parker K., Condon M., Jane A. Histopathological and biochemical monitoring tools for enhancing an ecological approach to Mytilus edulis aquaculture. World Aquaculture Society, New Orleans, March, 2019.

Parker, K., Byron, C., St. Gelais, A. Histopathological Analysis of Parasites and Environmental Stress Responses of Farmed Blue Mussels (Mytilus edulis) In Casco Bay, Maine. New England Estuarine Research Society (NEERS), Portsmouth, NH, April 26, 2018. [Oral]

Jones, C., St. Gelais, A., Byron, C., Costa-Pierce, B., Smolowitz, R. ‘A histopathological health and condition assessment of farmed blue mussels (Mytilus edulis) in a changing Gulf of Maine’. American Fisheries Society (AFS), Atlantic City, NJ, August 2018. [Oral]

Parker, K., Condon, M., Jones, C., Byron, C., St. Gelais, A. ‘A histopathological health survey or farmed blue mussels (Mytilus edulis) in the Gulf of Maine’. Maine-North Atlantic & Arctic Education Consortium, Portland, ME, April 2018. [Poster]

Jones, C., Condon, M., Jane, A., Parker, K., St. Gelais, A., Byron, C. ‘A histopathological health survey of farmed blue mussels (Mytilus edulis) in the Gulf of Maine’. 2018 RARGOM Annual Science Meeting, Portland, ME, October 2018. [Poster]

Parker, K., Byron, C., St. Gelais, A. ‘Histopathological Analysis of Parasites and Environmental Stress Responses of Farmed Blue Mussels (Mytilus edulis) in Casco Bay, Maine’. University of New England College of Arts and Sciences 19th Annual Spring Research Symposium, Biddeford, ME, May 2018. [Oral]

Condon, M., Byron, C., St. Gelais, A. ‘Analysis of Reproduction and Energy Investment within a population of Farmed Blue Mussels (Mytilus edulis) in Casco Bay, Maine’. University of New England College of Arts and Sciences 19th Annual Spring Research Symposium, Biddeford, ME, May 2018. [Oral]

Condon, M., Parker, K., St. Gelais, A., Byron, C., Jones, C. ‘A Histopathological Health Survey of Farmed Blue Mussels (Mytilus edulis) in the Gulf of Maine’. SEANET 2018 All Hands Annual Meeting, Orono, ME, May 2018. [Poster]

Condon, M., Parker, K., St. Gelais, A., Byron, C., Jones, C. ‘A Histopathological Health Survey of Farmed Blue Mussels (Mytilus edulis) in the Gulf of Maine’. Milford Aquaculture Seminar, Shelton, CT, January 2018. [Poster]

Condon, M., St. Gelais, A., Byron, C., Jones, C. ‘Interannual Analysis of Reproduction and Energy Investment within a Population of Farmed Blue Mussels (Mytilus edulis)’. SEA Fellows Symposium at the Darling Marine Center, Walpole, ME, August 2018. [Poster]

Condon, M., St. Gelais, A., Byron, C., Jones, C. ‘Interannual Analysis of Reproduction and Energy Investment within a Population of Farmed Blue Mussels (Mytilus edulis)’. University of New England College of Arts and Sciences Annual Fall Research Symposium, Biddeford, ME, September 2018. [Poster]

Jane, A., Parker, K., Jones, C., St. Gelais, A., Byron, C. ‘Preliminary Assessment of Trematode Infection in Farmed Blue Mussels (Mytilus edulis) in Casco Bay, Maine’. University of New England College of Arts and Sciences 19th Annual Spring Research Symposium, Biddeford, ME, May 2018. [Poster].

Jane, A., Jones, C., St. Gelais, A., Byron, C., Parker, K. ‘An Assessment of Trematode Infection in Farmed Blue Mussels (Mytilus edulis) in Casco Bay, Maine’. Sustainble Ecological Aquaculture Network 2018 All Hands Annual Meeting, Orono, ME, May 2018. [Poster].

Jane, A., Jones, C., St. Gelais, A., Byron, C., Parker, K. ‘An Assessment of Trematode Infection in Farmed Blue Mussels (Mytilus edulis) in Casco Bay, Maine’. SEA Fellows Symposium at the Darling Marine Center, Walpole, ME, August 2018. [Poster].

Jane, A., Jones, C., St. Gelais, A., Byron, C., Parker, K. ‘An Assessment of Trematode Infection in Farmed Blue Mussels (Mytilus edulis) in Casco Bay, Maine’. University of New England College of Arts and Sciences Annual Fall Research Symposium, Biddeford, ME, September 2018. [Poster].

Learning Outcomes

2 Farmers reported changes in knowledge, attitudes, skills and/or awareness as a result of their participation
Key areas in which farmers reported changes in knowledge, attitude, skills and/or awareness:

Farmers have expressed the value of increased awareness of the physiological health and condition of their crop. An important finding for the farmer is that traditional condition index (a ratio of meat yield to shell size) is an inadequate metric for crop health. In fact, in the summer of 2018, our research team was able to give advance warning to the farmer that the conditions that precipitated significant crop loss in 2016 were again presenting themselves in summer 2018; namely, an over-investment in reproduction and low short and long term energy stores, coupled with a very hot and dry period of weather. 

Subsequently, the farmer reported crop moralities as hot, dry conditions persisted. The farmer was able to take the physiological information we provided and make more informed farm management decisions as to whether to harvest the crop prematurely to prevent loss of revenue, or to hold back on harvesting product to avoid handling and additional stress to the crop populations. 

Project Outcomes

3 Grants applied for that built upon this project
Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.