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Bait-subsidized diets and their effects on ovigerous North American (Homarus americanus)

Jason S. Goldstein & Jeffrey D. Shields

Aquaculture International Journal of the European Society

ISSN 0967-6120

Aquacult Int DOI 10.1007/s10499-018-0285-8

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Aquacult Int https://doi.org/10.1007/s10499-018-0285-8

Bait-subsidized diets and their effects on ovigerous North American lobsters (Homarus americanus)

Jason S. Goldstein1,2 & Jeffrey D. Shields3

Received: 9 February 2018 /Accepted: 3 July 2018 # Springer Nature Switzerland AG 2018

Abstract Ovigerous American lobsters (Homarus americanus) display a protracted period of ovary maturation and maternal care when incubating their eggs, potentially influencing offspring fitness. Lobsters consume a wide range of food items; however, trap bait may comprise a larger proportion of their in some fished areas compared to non-fished areas, and the long-term consequences of a bait-based diet remain largely unexplored in lobsters. We tested the hypothesis that disproportionate amounts of bait in the diets of pre-ovigerous females affect the quality of their ovaries and eggs. We held pre-ovigerous lobsters (n =29) over a period of ~ 300 days (range = 270–378) and fed them diets of bait, natural prey items (, , urchin, macroalgae), or a combination of both diet types. Nutritional status, measured as biweekly blood indices and total glucose levels, suggest differences between lobsters fed a natural or combination diet and lobsters fed a bait-based diet (ANOVA; P < 0.05). We found that bait diets contained more protein (58.5%) and lipids (31.6%) compared to natural diets (34.5 and 13.2%, respectively) and lipid levels in ovaries and eggs significantly correlated with each other for all treatments (r = 0.76, n =15,P = 0.028). Histo- pathological analysis indicates that ovaries contained more variable maturation in starved lobsters or those fed with bait, with some showing delayed gonad maturation. Results suggest that a varied diet promotes the overall fitness of ovigerous lobsters and the associated reserves that are used for ovarian development and subsequent oocyte formation.

Keywords Maternal effects . Ovigerous lobsters . Egg lipids . Atlantic herring

* Jason S. Goldstein [email protected]

1 Wells National Estuarine Research Reserve, The Maine Coastal Ecology Center, 342 Laudholm Farm Road, Wells, ME 04090, USA 2 Department of Biological Sciences and School of Marine Sciences and Engineering, University of New Hampshire, 46 College Road, Durham, NH 03824, USA 3 Department of Aquatic Health Sciences, Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point, VA 23062, USA Author's personal copy

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Introduction

Ovigerous (egg-bearing) American lobsters (Homarus americanus) typically show a protracted period of ovary maturation and maternal care, carrying their eggs externally on their pleopods in excess of 9 months (Bumpus 1891; Talbot and Helluy 1995). Such maternal effects (e.g., brooding behavior, nutrient provisioning), defined broadly as Bnongenetic traits that are acquired from the mother,^ elicit potentially significant impacts on the ecological and evolutionary life histories of marine organisms (reviewed in Bernardo 1996), including lobsters. Within marine populations, environmental stressors (e.g., salinity, temperature, pol- lution, ocean acidification) along with nutritional state can negatively affect egg size, fecun- dity, and offspring fitness (reviewed in Marshall and Keough 2008). In particular, maternal nutrition can have pronounced Bcarry over effects^ on offspring fitness as is the case in a variety of including beetles, worms, bryozoans, , and , among others (Qian and Chia 1991; Bernardo 1996;Fox2000;Marshalland Keough 2004; Sato and Suzuki 2010;Carrascoetal.2016; Peters-Didier and Sewell 2017), although there are exceptions to this (e.g., Lewis and Choat 1993). Based on these complex relationships, theoretical models that build on the linkages between maternal size and offspring fitness incorporate the influence of maternal nutritional state in addition to other factors including environmental changes and fishing pressure (Marshall and Keough 2008;Brander 2010;Molandetal.2010). American lobsters are fished intensively throughout their range in U.S. and Canadian waters, and the relies heavily on baited traps (ASMFC 2015). The coastal \Maine fishery is estimated to fish in excess of 650,000 traps per season (Maine DMR 2017), resulting in almost 70% of Atlantic herring (Clupea harengus) landings (70,000–75,000 metric tons) being re-introduced as bait into coastal benthic habitats (Salia et al. 2002; Grabowski et al. 2010). Consequently, this volume of herring biomass has been assumed to subsidize lobster growth and may contribute to increases in lobster biomass in some areas (Salia et al. 2002; Grabowski et al. 2009). Although the mechanisms in how lobster biomass has increased in the Gulf of Maine over the last decade are debated, factors that may shape and influence this pattern include (1) conservation measures in commercial lobster traps, creating a dispropor- tionate number of sub-legal lobsters that enter traps, feed, and escape only to repeat the cycle (Karnofsky and Price 1989;Juryetal.2001;Watsonetal.2009); (2) changes in trophic level dynamics, i.e., declines in large predatory fishes that release lobsters from risk (Steneck and Wilson 2011; Wahle et al. 2013); (3) continued long-term management practices such as the vigilant protection of egg-bearing females and size restrictions (Miller 1995); and (4) changing environmental conditions (e.g., temperature, climate change) (Drinkwater et al. 1996;Oviatt2004; Boudreau and Worm 2010) that may influence large-scale changes in ocean climate and fishery dynamics over time (Nye 2010; Pinsky and Fogarty 2012; Mills et al. 2013;Boudreauetal.2015). Trap bait may include a large proportion (30–55%) of the diet of a lobster, and this could substantially impact overall lobster health (Myers and Tlusty 2009). A recent survey of bait use by Nova Scotian lobstermen estimated an average of 860 g (1.9 lbs) of bait was used each time a trap was set, translating to over 5216 kg (11,500 lb) of bait/year/lobsterman (Harnish and Willison 2009). With such large volumes of bait being used in some areas, the ecological and economic implications of bait subsidies are of interest to both scientists and industry (Thunberg 2007). Nonetheless, the biological impacts resulting from a disproportionate amount of bait input into the lobster fishery are largely uninvestigated. For example, lobster Author's personal copy

Aquacult Int trap bait could operate as a potential nutritional stressor increasing the susceptibility of lobsters to a number of aquatic diseases (Sindermann 1990;Tlustyetal.2000, 2008; Myers and Tlusty 2009) or there may be little impact (see Bethoney et al. 2011). This is the hypothesis we wanted to test with ovigerous H. americanus. Lobster bait in the Gulf of Maine consists predominantly of salted or fresh herring or (Scomber scombrus) that, if not included among a more diverse diet, is depleted of certain nutritional constituents (e.g., amino acids, minerals; Gendron et al. 2001). The nutri- tional requirements of adult lobsters have yet to be fully resolved (reviewed in Conklin 1995; but see Hanson 2009); however, the best growth results from larval and juvenile studies have been obtained from a varied diet that includes algal matter, bivalves, and other (Conklin 1995;Tlustyetal.2005a, b). In the absence of fishing, lobsters forage among a wide spectrum of marine organisms that include , mollusks, echinoderms, , and macroalgae. Lobsters are also known to temporally shift their diet (Elner and Campbell 1987; Conklin 1995) and are considered keystone predators, capable of driving the trophic dynamics in many benthic communities (Mann and Breen 1972; Jones and Shulman 2008). In particular, ovigerous lobsters can undertake long seasonal movements, allowing them to encounter a variety of benthic habitats as well as fishing areas of varying trap densities (Cooper and Uzmann 1980; Campbell and Stasko 1985; Cowan et al. 2007; Goldstein and Watson 2015a). Taken together, such patterns of behavior, seasonal movements, and overall activity potentially affect the types and quantities of food items that lobsters may encounter. The prevalence of bait and natural foraging items for lobsters depends heavily on the locations they occupy, trap densities being fished, and the distribution and abundance of lobsters in an area (Jury et al. 2001; Geraldi et al. 2009; Watson et al. 2009; Grabowski et al. 2010). Laboratory studies using juvenile lobsters have shown that a diet composed primarily of fish can cause phenotypic changes in shell color, but it remains unknown if an exclusive fish-based diet induces deleterious consequences on maturation, egg production, or shell integrity in adult animals (Tlusty et al. 2008). There is some evidence to suggest negative, short-term consequences associated with lobsters consuming bait. Prince et al. (1995)for example observed a reduction in the overall incidence of shell disease in a lobster pound fed with an artificial diet compared to one that used salted racks. Gendron et al. (2001) reported that a varied natural diet, augmented by rock crab (Cancer spp.) rations, over a 3- week period had favorable effects on ovary condition throughout the maturation process. Using stable isotope ratio analysis, Grabowski et al. (2010) showed that bait-subsidized lobsters grew faster than those animals in seasonally closed fishing areas. However, the long-term consequences of lobsters subsidized primarily on bait, particularly the effects on ovary and egg condition in mature adult lobsters, remain unexplored (Bethoney et al. 2011). Pre-ovigerous lobsters provide an interesting and viable model for testing bait-subsidized effects since they also frequent lobster traps, possibly more often during periods of seasonal movements (Herrick 1895;Campbell1986; Goldstein and Watson 2015a). If these animals feed primarily on bait from traps, nutritionally mediated maternal effects could result in carry- over effects on ovarian condition, leading to changes in egg quality (e.g., low lipid content or poor quality lipids). Sexually mature females forage most of the year prior to egg extrusion to build energy reserves in preparation for ovarian maturation and oviposition (Waddy and Aiken 1992). It is not unreasonable to assume then that readily obtained food items such as trap bait and naturally abundant prey items would influence ovarian development. Newly extruded eggs are high in lipid content, as a result of maternal provisioning during the maturation process and the sequestering of yolk reserves during the process of vitellogenesis (Sasaki et al. 1986; Author's personal copy

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Talbot and Helluy 1995). If pre-ovigerous lobsters consume disproportionate amounts of low- quality bait in their diets, their health could potentially be altered at many levels (e.g., decreased disease resistance, altered maturation schedules, egg quality), and this could trans- late into variable egg quality and larval survival. Nutritional provisioning in lobsters from mother to offspring may have direct carry-over effects manifested at any level of their life- history as has been documented in other (e.g., urchins, Bertram and Strathmann 1998). The impetus for this study originated from a series of previous studies involving egg development, seasonal movements, and mating dynamics of ovigerous lobsters (Johnson et al. 2011;Pughetal.2013; Goldstein et al. 2014; Goldstein and Watson 2015a, b). In light of the potential disparity in quality between natural food sources and trap bait, the goal of this study was to determine the potential nutritional effects that bait-subsidized diets may have on lobster health by focusing on ovigerous lobsters and their ovary tissue and egg quality. Our objective was to evaluate the hypothesis that pre-ovigerous females that consume dispropor- tionate amounts of bait may compromise their ovary condition and egg quality (i.e., egg reserves). To address this, pre-ovigerous lobsters were collected and held in long-term, natural conditions while being fed diets of herring bait, natural pretty items, or a combination of the two.

Materials and methods

Animal collection and assessment

Pre-ovigerous female lobsters were collected by fishermen holding scientific collection permits in southern coastal Gulf of Maine waters in spring 2009. All lobsters were held onboard boats in live wells with running ambient seawater and transported to the University of New Hampshire’s Coastal Marine Laboratory in New Castle, New Hampshire, USA, where all subsequent work was conducted. For this study, 29 lobsters (size range = 85– 96 mm carapace length, CL, mean = 94 ± 2.2 SE) were measured using digital calipers to 1.0 mm (Mitutoyo IP 65; Mitutoyo Corp., ). There were no significant differences in the sizes of lobsters used between treatment groups (one-way ANOVA; F3,28 = 0.62, P = 0.62). The reproductive histories of all female lobsters were assessed in the laboratory as they were used in a previous mating study (duration ~ 30 days) and fed a combination of fresh and frozen fish, , and . In these cases, animals were visually confirmed by video (Pugh 2014) as having mated and were sampled noninvasively for the presence of a small amount of fresh plug from individual spermatophores (see Goldstein et al. 2014). At the start of the study (July 2009), all lobsters were tagged with small circular laminated disc tags (diameter = 2.0 cm; Floy Tag and Mfg., Inc. Seattle, WA) fastened to the base of their claw and held isolated in floating totes (81.3 cm × 50.8 cm × 38 cm, L × W × H, Fig. 1) subjected to ambient seawater flow (16.3 ± 1.6 °C; mean ± SE) and light in an outside impoundment until they extruded their egg clutches.

Diet treatments

Lobsters were divided randomly among four diet treatment groups: (1) lobsters (n =5)that received no food items (control); (2) lobsters that received a mixed fish (bait) diet (n =8) Author's personal copy

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Fig. 1 Lobster diet treatment setup. Animals were kept in floating totes (dimensions—81.3 cm × 50.8 cm × 38 cm) and held at ambient light and temperature in an outside impoundment subject to natural flowing seawater consisting of equal amounts of frozen herring (Clupea harengus) and American shad ( sapidissima); (3) lobsters fed a natural diet (n = 8) consisting of fresh, local prey items including fish (same as bait diet), rock crab (Cancer spp.), blue mussel (Mytilus edulis), green (Strongylocentrotus droebachiensis), and a mix of red, green, and brown macroalgae (Ulva lactuca, Laminaria agardhii, Chondrus crispus, Rhodymenia palmata)all found in the surrounding benthic community; and (4) lobsters fed a mixed diet (n =8) composed of a ~ 50/50 combination of bait and natural feed items. Fish was purchased in bulk 18 kg frozen flat trays from a single source (Little Bay Lobster Co., Newington, NH). All natural prey items were collected locally on a periodic basis by SCUBA divers and held in separate holding totes from the lobsters. We attempted to weigh out equal portions of natural prey items to obtain relatively comparable proportions of each item. Lobsters were fed to satiation twice a week between May and November and once a week during the rest of the year. Uneaten food items were removed prior to each new feeding event, and totes were cleaned periodically. Prior to starting any of the treatments, all lobsters were starved for a total of 2 weeks to ensure that they were all in the same relative condition at the start of the experiment.

Nutritional status

The nutritional status of each lobster was monitored by collecting a small sample of hemo- lymph on a biweekly basis, using methods similar in other lobster studies (Leavitt and Bayer 1977; Oliver and MacDiarmid 2001; Ozbay and Riley 2002; Wang and Mcgaw 2014;Gutzler and Butler 2017). A total of ~ 30 μL of hemolymph was collected from the base (sinus) of the fifth walking leg of each using a 3.0 mL 27-gauge syringe. Samples were placed on an analog clinical protein refractometer (model CLX-1, resolution = ± 0.2; VEE GEE Scientific, Author's personal copy

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Inc. Kirkland, WA) and a blood refractive index (BRI) was recorded (g/100 mL). The refractometer was calibrated with deionized water prior to each use and bovine serum albumin (Sigma-Aldrich A7284, St. Louis, MO) was used to create a standardized curve by which raw data values could be compared. On three occasions (start, middle, and end of study), larger samples (1.0 mL) of whole lobster hemolymph were drawn and frozen at − 80 °C for total glucose analysis. Total glucose was quantified from total blood serum using a standardized colorimetric kit for in vitro analysis of glucose (kit no. 439-90901; Wako Chemicals USA Inc., Richmond, VA). Both hemolymph and total glucose values were averaged across all individuals for each of the four treatments and compared using a repeated-measures ANOVA using JMP v. 9.0.3 statistical software package (SAS Institute, Cary, NC). BRI values were square-root transformed and analyzed by one-way ANOVA. A single-step multiple comparison test (Tukey’s honestly significant difference test, Tukey’s HSD) was used to test potential differences between treatments. All means are reported ± SEM.

Diet and egg analysis

Representative feed samples from each diet treatment (reference samples) were collected at three time intervals (the outset of the trials, mid-January, and June, day chosen randomly). A total of two samples per treatment per time period were collected and temporarily frozen at − 80 °C, before being freeze dried at − 40 °C for 24 h (Labconco Freeze Dryer 5, Kansas City,

MO). Samples for each diet treatment were combined (ntotal = 6 samples/treatment) to obtain enough sample (~ 15–20 g) for analysis and were ground down into a fine powder using an industrial-grade milling machine (Wiley Mill #4, 40-μm mesh screen; Thomas Scientific, Swedesboro, NJ) and stored in labeled glass storage vials. All samples were sent to the Forage Testing Laboratory (Ithaca, New York) for analysis. Total protein, lipid, ash, and calcium were analyzed using a Thermo IRIS Advantage HX or ICAP 6300 intrepid inductively coupled plasma radial spectrometer after microwave digestion (method 942.05; AOAC, 2006; Dairy One Cooperative, Inc.) and reported on a percent-dry-weight basis. Small samples of ovary tissue were extracted from each lobster ~ 1 month prior to egg extrusion (see Johnson et al. 2011 for methodological details). Briefly, a small square was cut into the lateral side of the carapace (behind the eye), and a section of ovarian tissue was removed with a pair of blunt-tipped forceps. The incision was sealed with the use of cyanoacrylate glue, gauze, and adhesive tape. Preliminary trials with a separate set of lobsters (CL = 84 ± 5.1, n = 20) demonstrated that all lobsters survived over a 4-month period. Upon egg extrusion, ~ 500 eggs/lobster were gently removed in clumps with forceps, washed with cold, sterile seawater, and placed in clean, labeled storage vials. Both egg and ovary samples were processed similarly to feed samples as described above.

Histolopathological analysis

Once egg extrusion was completed, each lobster was sacrificed so that tissue samples could be extracted and analyzed. Samples of ovary, hepatopancreas, claw muscle, shell cuticle, and midgut were removed, stored in individual tissue cassettes (Fisher Tissue Path cassettes IV), and preserved in a solution of 10% neutral buffered formalin. After 36 h, tissue samples were transferred into 70% ethanol and shipped to the Virginia Institute of Marine Science (VIMS, Gloucester Point, VA). All tissue samples were processed using paraffin histological Author's personal copy

Aquacult Int techniques and stained with Mayer’s hematoxylin and eosin as in Shields et al. (2012). Prepared slides were viewed and photographed using an Olympus BX51 compound micro- scope and a Nikon DXM1200 digital camera.

Results

Hemolymph indices

The nutritional status of individual ovigerous lobsters was assessed using the blood refractive index, or BRI. Animals in the natural diet treatment had the highest BRI (5.4 ± 0.80) followed by bait (4.9 ± 0.63), 50/50 (3.7 ± 0.72), and starved (3.1 ± 0.44) diets (Fig. 2). Post hoc follow-up tests (Tukey’s HSD) revealed differences in diets and the starved treatment differed significantly from all other treatments (P <0.001,Fig.2). With the exception of the starved treatment, all other treatments indicated an increasing trend (Fig. 2). The largest increase in nutritional condition over the course of the study was seen in lobsters in the natural diet treatment (+ 54%); conversely, the biggest decrease occurred in animals that were starved (− 79%).

Diet Average ± sem Range

naturala 5.4 ± 0.80 3.8 – 7.7

baita 4.9 ± 0.63 3.9 – 6.0

50/50a 3.7 ± 0.72 1.7 – 4.8

starvedb 3.1 ± 0.44 0.9 – 5.0 Fig. 2 Mean (± SEM) hemolymph values (blood refractive index, BRI) over time for lobsters held in one of four diet treatments. Values were averaged for all lobsters in each treatment (n =8,n = 5 starved). BRI values in the starved treatment were only measured until week 13, as there was 100% mortality thereafter. Treatments that share a superscript letter are not considered different (P < 0.001) Author's personal copy

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Glucose levels

The relationship between glucose values and absorbance (standard curve values) were fit by linear regression along with the absorbance data to obtain a linear effect and were significant (r2 = 0.99, P < 0.001; Fig. 3). Hemolymph glucose values changed significantly over time within (RMANOVA; F2,28 = 19.28, P = 0.0014) and between the four treatment groups (F3,4 = 7.80, P = 0.0379), including a significant interaction term (time*trt) (F5,28 =6.20,P =0.017). Total hemolymph glucose values (means ± SEM) for lobsters held in each of four diet treatments and sampled over three time periods are represented in Fig. 4.

Diet and nutritional analysis

Lobster diets were composed of very different bulk constituents in terms of proteins, lipids, and ash (Fig. 5). Herring bait contained higher amounts of protein (58.5%) and lipids (31.6%), compared with natural diets (34.5% and 13.2%, respectively). In addition, ash comprised > 50% of the constituents in the natural diet, and this included large amounts of calcium. The proportion of total lipids in the ovaries of lobsters were not different among treatments, with the exception of ovaries from starved lobsters (ANOVA; F3,28, P <0.05;Fig. 6). A similar trend was seen in lobster eggs, with those in the starved treatment having somewhat lower lipid levels (P = 0.081; Fig. 6). Lipid levels in the ovaries were well correlated with those in the eggs for all treatments (r =0.76,n =15,P = 0.028).

Histolopathological analysis

Several tissues were examined via histology in this study, but the ovary and hepatopancreas showed distinct changes associated with diet treatments. In the hepatopancreas and midgut region of lobsters that were starved as well as those animals in the bait treatment, the hepatopancreas had few observable reserve inclusion (RI) cells or the RI cells were not apparent (Figs. 7 and 8). There was a noticeable reduction in the spongy connective tissues supporting elements of the vascular system, and the fixed phagocytes were reduced in number.

0.45 0.40 0.35 0.30 0.25 0.20 0.15 Absorbance 0.10 y = 0.0025x + 0.021 R² = 0.9857 0.05 0.00 0 25 50 75 100 125 150 175 Glucose (mg/dL) Fig. 3 The relationship between glucose values and absorbance (standard curve values). Absorbance values were log transformed to obtain a linear effect. Bovine serum albumin was used as a protein standard Author's personal copy

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Fig. 4 Total hemolymph glucose values (mean ± SEM) for lobsters held in each of four diet treatments and sampled at three times: (1) initial (fall), (2) middle (winter), and (3) late (spring). Values were averaged for all lobsters in each treatment (n =8,n = 3 starved). Values in the starved treatment were unavailable in the spring (late period) due to 100% mortality. Significant differences (P < 0.05) between time periods within each treatment are indicated by an asterisk (*)

Within the hepatopancreatic tubules, the R-cells were highly vacuolated with nuclei condensed and smaller than lobsters that were fed. The F-cells were inactive or effete, barely noticeable within the tubules. B-cells and E-cells were present but they did not exhibit obvious changes, with the possible exception that the E-cells may have had a narrower band at the tip of the tubule than in fed animals. In starved animals, the area around the midgut had effete RI cells or they were not present (Fig. 8c). In animals fed a natural diet, the RI cells stained intensely with eosinophilic uptake; the cells were irregular, large, and the nuclei were subjacent to the center of the cell. In animals fed a fish (bait) diet, the RI cells showed less intense eosinophilic staining, were globular and ovoidal in shape while in ovarian tissue, some mobilization of yolk reserves was observed that was similar to the starved lobster treatment.

Discussion

This study sought to address the long-term effects of a bait-subsidized diet on the overall health of ovigerous lobster ovary and hepatopancreas condition in mature H. americanus.We

Fig. 5 Nutritional components for bait and natural diets expressed on a percent-dry-weight basis. The break- down of diet components was obtained from processed diet materials that were pooled from three separate time periods (ntotal = 6 samples/treatment, pooled over all three time periods) Author's personal copy

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Fig. 6 Comparison of total lipid (as %) for both egg and ovary samples from lobsters of all diet treatments. The correlation between egg and ovary values (r =0.76,n = 15) was significant (P = 0.028)

Fig. 7 a Cross-section of hepatopancreas tissue from a starved lobster (Lobster 01) showing few reserve inclusion cells in the spongy connective tissues and a reduction in the fixed phagocytes and spongy connective tissues around the tubules, and vacuolation of the R-cells (r) and inactive F-cells (arrows) within the tubules. b Hepatopancreas from a lobster fed a natural diet showing robust R-cells and F-cells within the tubules, and large, globular eosinophilic reserve inclusion cells (*) within the spongy connective tissues surrounding the tubules. Bar = 50 μm. c Hepatopan- creas from a bait-fed lobster (Lobster 4B) with a similar histological presentation as a starved lobster (same notation as in (a)). Bar = 50 μm. d Hepatopancreas near midgut of same bait-fed lobster at lower magnification showing accumulations of reserve inclusion cells (*) in the spongy connective tissues. Bar = 100 μm Author's personal copy

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Fig. 8 a Ovarian tissue sample for a lobster on a natural diet. Note the large but variable size of the individual ova. This is partly due to the section plane. Bar = 300 μm. b Ovary of a starved lobster (Lobster 01) showing several ova in early stages of development. Bar = 300 μm. c Connective tissue rind around the ovary in a starved lobster (01) showing mobilization of yolk resources (arrows) in the hemolymph. Bar = 50 μm. d Connective tissue rind around the ovary in a starved lobster (01) showing abnormal infiltration of granulocytes in the hemal sinus of the ovary. Bar = 50 μm. e Ovary from a bait-fed lobster (lobster 04) with ova in a synchronized developmental state and some mobilization of yolk reserves. Bar = 300 μm. f Ovary from a starved lobster (lobster 26) with similarly synchronized ova. Bar = 300 μm followed the health and egg quality in female lobsters over a period of ~ 300 days (range = 270–378) that were subjected to different diets. Our findings suggest that a varied diet is integral to the general health of ovigerous lobsters, and this was reflected in indicators of physiological state (BRI, glucose), lipid storage, and histological assessment of our Author's personal copy

Aquacult Int experimental animals. Although we did not see a significant difference in bait-fed lobsters from three of our other treatments, bait-fed lobsters tended to show some similar histological attributes to those of starved lobsters. Lobsters in many areas of the commercial fishery are often referred to as Bfarmed^ due to their propensity to enter traps, consume bait, and frequent other traps (Grabowski et al. 2010). In response to this, we attempted to quantify the potential effects of a bait diet on ovarian and ova development in lobsters. We did not follow ova through to fertilization, development, and eclosion, and therefore did not assess their success or failure in hatching. However, under this continuing scenario, we suspect that true dietary carry-over effects could manifest themselves with respect to larval competency and survivorship in lobsters consuming sub-optimal diets (Tlusty et al. 2008). In fact, other studies have shown such a link between maternal nutrition and the fecundity and hatching success in marine crustaceans, particularly with dietary lipids (Castell and Kean 1986). Herring bait has been suggested to augment growth rates in some lobsters (Saila et al. 2002; Grabowski et al. 2010); however, at least three factors may confound this effect, especially on a localized scale: changes in lobster density, variable fishing effort, and the availability and abundance of natural prey. In addition, both historic and recent analyses of in situ dynamics of lobsters in traps suggest that bait consumption is highly variable in lobsters, as they are often out-competed for bait by crabs and fish (Jury et al. 2001; Clark et al. 2015). Clearly, the nutritional aspects of bait consumption and its effects on different ontogenetic stages in lobsters (e.g., juveniles versus adults) should be examined in more detail. Given the differences in protein and lipid content of bait versus natural forage items, the effect of a bait diet should be investigated in the future for several biological processes (e.g., growth, maturation, and reproduction). Although lobsters are opportunistic foragers consuming prey over a variety of taxa (Conklin 1995; Sainte-Marie and Chabot 2002), there is some evidence indicating that they prefer rock crab ( and C. borealis; Boghen et al. 1982; Ojeda and Dearborn 1991; Gendron et al. 2001). Rock crab provides a high level of nutritional constituents (e.g., amino acids, proteins, minerals) to lobster diets compared to other fauna (Gendron et al. 2001).Lobstersareknowntousechemoreceptiontofindandpreyonrock crab, and this prey choice may contain mechanisms for stimulating their ingestion (Hirtle and Mann 1978). Because rock crabs are common decapods in many coastal communities (Palma et al. 1998; Wells et al. 2010), they comprise a large and readily available prey for lobsters. We incorporated rock crab into our natural diet treatment and our findings corroborate those of Gendron et al. (2001)thatCancer spp. are likely an integral dietary component as part of a healthy lobster diet. Although crabs are known to be an important dietary component for lobsters, especially to the growth and health of laboratory-cultured juvenile lobsters (Kean et al. 1985; Conklin 1995), the bait diet, although lacking dietary components found in the natural diet, was rich in lipids and contained the highest total lipid content in the hemolymph sera (Fig. 5). Lipids play a major role in egg development and are a vital source of metabolic reserves (Holland 1978). Lobster ovary sequesters the majority of lipids allocated for egg reserves, comprising over 30% of wet tissue weight (Castell and Covey 1976). For most crustaceans, lipids significantly affect ovarian development, fecundity, and hatchability of eggs (Amsler and George 1984;Sasakietal.1986). Lipid stores are important to several biochemical processes; for example, triglyceride reserves at the time of hatching can affect the diet of lobster larvae (Castell and Kean 1986;Sasakietal.1986; Sibert et al. 2004). Author's personal copy

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Most artificial lobster diets, especially bait diets, are devoid of carotenoids. However, these pigments play a key role in lobster reproduction (e.g., vitellin production, embryo develop- ment), as is the case in other marine crustaceans (Bordner et al. 1983;Conklin1995;Dalletal. 1995; Linan-Cabello et al. 2002). The natural forage diet used in this study included a variety of animal and plant constituents that contain components that are beneficial to the ovary and egg maturation process in some lobsters. Indeed, macroalgae are commonly found in lobster stomachs and are a viable nutritional component (Scarratt 1980; Elner and Campbell 1987; Hanson 2009). Herrick (1909) typically observed macroalgae in the stomachs of wild-caught lobsters and suggested that the mineral constituents in these species were beneficial to overall lobster health. In a laboratory-based formulated diet study, Gallagher et al. (1983) reported that optimal ratios of C/P in juvenile lobsters from algal species were also beneficial to adult lobsters. In this study, natural feed diets contained proportionately more inorganic materials (i.e., ash; Fig. 5) compared to other diet treatments and, as a result, may provide a variety of trace minerals for physiological function and overall health. There is a paucity of information on dietary requirements and nutritional deficiencies in adult lobsters, and most information relates to the production of juvenile lobsters from hatchery and aquaculture-related studies (Conklin 1995;Tlustyetal.2005a, b). More directed efforts investigating the nutrient requirements for adult lobsters and the foods they consume are paramount to further understanding the role of trap bait in augmenting lobster diets in intensive trap . Our results suggest that lobsters fed on an exclusive trap bait diet are compromised nutritionally. However, in the field, lobsters feed on a large variety of taxa including trap-subsidized bait. More research should be conducted on bait-subsidized lobster diets considering the increased input of bait into some areas of the lobster fishery, the variability and quality of herring bait (Melvin and Stephenson 2007), and the proportion of bait consumed by lobsters compared with natural foraging. Understanding these kinds of relationships would allow fisheries managers to address the impact of lobster trap bait on the health of this important fishery throughout its range.

Acknowledgments We thank Nancy Whitehouse (UNH, Dairy Research Center) who helped with the nutri- tional analyses as well as provided laboratory equipment for processing samples, and the encouragement and support from Prof. Win Watson. Thanks to several lobstermen from New Hampshire for their help in collecting live lobsters for this study. We appreciate the help from Tracy Pugh, Kate Masury, and Audra Chaput who all assisted in weekly feedings, sampling, and general maintenance throughout the study. Nathan Rennels and Noel Carlson of the UNH Coastal Marine Laboratory helped coordinate logistics and space.

Funding This research was supported from grants awarded to JSG from the UNH Marine Program and a Lerner-Gray Fund for Marine Research (American Museum of Natural History).

Compliance with ethical standards All lobsters were collected and held in accordance with NHFG permits MFD0920 and MFD1016 issued to the University of New Hampshire.

Conflict of interest The authors declare no conflict of interest.

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