The influence of year, laying date, egg fertility and incubation, individual hen, hen age and mass and clutch size on maternal immunoglobulin Y concentration in captive Steller's and spectacled eider egg yolk

Developmental and Comparative Immunology 52 (2015) 10–16

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The influence of year, laying date, egg fertility and incubation, individual hen, hen age and mass and clutch size on maternal immunoglobulin Y concentration in captive Steller’s and spectacled eider egg yolk Katrina L. Counihan a,*, John M. Maniscalco a, Maryann Bozza b, Jill M. Hendon c,1, Tuula E. Hollmén a,d a

Alaska SeaLife Center, PO Box 1329, Seward, AK 99664, USA Oregon State University Hatfield Marine Science Center, 2030 Marine Science Dr., Newport, OR 97365, USA Department of Biological Sciences, The University of Southern Mississippi, 118 College Drive, Box #5018, Hattiesburg, MS 39406, USA d School of Fisheries and Ocean Sciences, University of Alaska Fairbanks, PO Box 1329, Seward, AK 99664, USA b c

A R T I C L E

I N F O

Article history: Received 16 January 2015 Revised 28 March 2015 Accepted 12 April 2015 Available online 16 April 2015 Keywords: Steller’s eider Spectacled eider Immunoglobulin Y Maternal transfer Egg yolk Passive immunity

A B S T R A C T

Steller’s eiders and spectacled eiders are sea duck species whose populations have declined significantly and infectious diseases could influence offspring survival. Therefore, the maternal transfer of immunoglobulin Y (IgY) into yolk was investigated in captive Steller’s and spectacled eiders during the 2007–2013 breeding seasons. This project had two objectives: establish baseline IgY levels in Steller’s and spectacled eider yolk under controlled captive conditions and evaluate the effect of year, laying date, egg fertility, egg incubation duration, individual hen, hen age and mass, and laying order to determine which variables influenced IgY levels. Average IgY concentrations were 0.03–0.48 mg ml−1 in Steller’s eider yolk and 0.10–0.51 mg ml−1 in spectacled eider yolk. The year and individual hen influenced IgY concentration in Steller’s and spectacled eider yolk. The laying date was negatively correlated with egg IgY levels for most Steller’s eider hens, but laying order was positively correlated with egg IgY concentration for spectacled eiders. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Steller’s eiders (Polysticta stelleri) and spectacled eiders (Somateria fischeri) breed in coastal regions of western and northern Alaska, but both species have experienced significant population declines (Quakenbush et al., 2004; Stehn et al., 1993). The Alaska breeding population of Steller’s eiders was listed as threatened under the Endangered Species Act in 1997 due to reductions in their numbers and nesting habitat (Federal Register, 1997). Spectacled eiders were listed as threatened in 1993 because of population decline, especially in western Alaska (Federal Register, 1993). The reason for the precipitous decline in the eider populations is unknown, although predation, climate change, contaminants and disease may be factors (U.S. Fish and Wildlife Service, 1996, 2002). Brood survival can be

* Corresponding author. Alaska SeaLife Center, PO Box 1329, Seward, AK 99664, USA. Tel.: +1 907 224 6336; fax: +1 907 224 6371. E-mail address: [email protected] (K.L. Counihan). 1 Current address: Gulf Coast Research Laboratory, The University of Southern Mississippi, 703 East Beach Drive, Ocean Springs, MS 39564, USA. http://dx.doi.org/10.1016/j.dci.2015.04.005 0145-305X/© 2015 Elsevier Ltd. All rights reserved.

highly variable in eiders, and understanding factors affecting duckling survival is important (Flint et al., 2006; Safine, 2013). Maternal transfer of antibodies via yolk is important for the health and survival of ducklings. Ducklings hatch with a functional innate immune system, but their adaptive immune system is still developing (Davison et al., 2008). Newly hatched ducklings depend on maternal immunoglobulin Y (IgY), which is the primary antibody in serum and the principal antibody involved in defense against systemic infections (Davison et al., 2008; Lundqvist et al., 2006). The passive immunity provided by the mother to the hatchling protects the offspring against disease agents present in the environment, and can be especially important in dense breeding populations where disease transmission may be enhanced (Addison et al., 2009; Garnier et al., 2012). Maternal IgY will form complexes with antigens to protect the duckling and alleviate pressure on the duckling’s developing immune system (Addison et al., 2009). Besides providing protection against pathogens, maternal antibodies can have additional benefits for the duckling. The presence of maternal IgY can stimulate B cell development and augment or modulate responses to antigens that will result in optimization of future reactions to the same antigen (Fink et al., 2008; Lemke and Lange, 1999). Idiotype alterations could also arise that modify the specificity of

K.L. Counihan et al./Developmental and Comparative Immunology 52 (2015) 10–16

immunoglobulins for antigens or even suppress particular antibody types (Lemke and Lange, 1999). The availability of maternal antibodies to defend the hatchling against parasites can decrease demands on its growth and development by reducing the need for an innate immune response which allows the duckling to focus resources on musculature and feather development (Grindstaff, 2008). Maternal IgY is transferred into yolk through the ovarian follicle from the serum of the hen and the quantity transferred is proportional to IgY concentrations in hen serum (Hamal et al., 2006; Kowalczyk et al., 1985). The IgY is transported to the circulation of the developing embryo across the yolk sac (Hamal et al., 2006; Kowalczyk et al., 1985). Immunoglobulin Y is the last antibody to be synthesized by newly hatched birds and generally begins around 2 weeks post-hatch in chickens and 3 weeks post-hatch in mallards (Hamal et al., 2006; Liu and Higgins, 1990; Mast and Goddeeris, 1999). Mallard ducklings have the highest concentration of maternal IgY in their serum after hatching and levels decline to their lowest point 2 weeks post-hatch (Liu and Higgins, 1990). The bursa of Fabricius fully matures when the hatchling is 5–7 weeks old and chickens are capable of the full range of B lymphocyte and antibody production at this point, but mallards do not synthesize adult levels of IgY until they are 10 weeks old (Davison et al., 2008; Liu and Higgins, 1990). The quantity of IgY a hen allocates into her eggs depends on several factors. First, the hen is only capable of transferring antibodies that are present in her serum (Kowalczyk et al., 1985). Hens have been shown to transfer specific antibodies into yolk due to vaccination (Al-Natour et al., 2004; Rollier et al., 2000) or because of an active infection prior to egg laying (Buechler et al., 2002; Gasparini et al., 2001). Therefore, differences in immunological experience among females could be reflected in the yolks of their eggs. The age of the female could also influence IgY deposition in yolk because humoral immunosenescence can occur in older females resulting in decreased antibody availability for disease resistance and distribution into yolk (Barua et al., 1998a, 1998b; Cichon et al., 2003; Haussmann et al., 2005). Environmental factors can impact yolk IgY concentrations, also. Higher breeding densities of birds have been linked to increased IgY concentrations potentially due to elevated pathogen prevalence (Muller et al., 2004), while a sufficient food supply results in lower IgY concentrations (Blount et al., 2002; Gasparini et al., 2007). Egg production can be biologically demanding, so the amount of IgY transferred into yolk is likely influenced by individual and environmental factors that vary each breeding season (Lochmiller and Deerenberg, 2000). The distribution of maternal antibodies within a clutch may also vary. Black-headed gulls provide the first laid egg with the most IgY (Groothuis et al., 2006), while collared flycatchers allocate the most IgY to the last egg in the clutch (Hargitai et al., 2006). Both species are asynchronously hatching species and earlier laid eggs usually have a higher chance of survival (Groothuis et al., 2006; Hargitai et al., 2006). Providing the first laid eggs with higher concentrations of IgY furthers their advantage, while distributing more IgY to later laid eggs may be an attempt to help them survive (Groothuis et al., 2006; Hargitai et al., 2006). The level of IgY provided by Steller’s and spectacled eider hens could be important for duckling survival in the event of disease exposure. Therefore, the maternal investment of IgY in yolk was investigated using samples collected from eggs laid by captive Steller’s and spectacled eiders during the breeding seasons of 2007– 2013. This project had two objectives: establish baseline IgY levels in Steller’s and spectacled eider egg yolk under controlled captive conditions and evaluate the effect of year, laying date, egg fertility, egg incubation duration, individual hen, hen age, hen mass, and laying order to determine which variables influenced IgY levels. The goal of this project was to characterize maternal transfer of IgY in Steller’s and spectacled eiders.

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2. Materials and methods 2.1. Study species Captive adult Steller’s and spectacled eiders were housed outdoors separately at the Alaska SeaLife Center (ASLC) in specially designed enclosures. The frame work of the habitat was aluminum and supported fiberglass pools that were 7′ × 6′ and 2′ deep. The decking was level with the edge of the pool and was perforated fiberglass, covered with 3M Nomad matting. Waste water drained through these substrates into an aluminum catchment drain and was piped to the ASLC waste water sump where it was sanitized. The eiders were kept in larger flocks during the non-breeding season with access to salt water pools. During the breeding season, breeding pairs were separated from the main flock and provided access to fresh water pools. Salt or fresh water was supplied to each of the pools, flowed constantly and skimmed off the surface. The habitat sides were enclosed with nylon mesh with holes of 1″ square and the roof panels were nylon gillnet with holes of 3″ square. Visual barriers were placed between flocks or breeding pairs. Birds were fed a diet of Mazuri (Purina, St. Louis, MO USA) sea duck pellets. These pellets were dispensed in automatic feeders which have a paddle that the birds push on when they want food. Their diet was supplemented with krill (Euphasia pacifica), mussels (Mytilus trossulus), silversides (Menidia menidia) and clams (Spisula solidissima), all of which comprised less than 5% of their total diet. Fresh drinking water was provided when the pools contained salt water. Pools were cleaned and disinfected every week and pen surfaces were disinfected every 2 weeks. Pen surfaces were hosed off as needed. The feeding areas were cleaned daily and the automatic feeders disinfected monthly. The eiders were weighed monthly except during the breeding season (June–July) to prevent disruption of nesting. Eggs were collected from the hens after laying, marked to note laying order, and incubated at least 5 days to assess fertility. Eggs were cut around the mid-section and the yolk and albumen were separated. The yolk was homogenized and stored at −20 °C until IgY extraction. Yolk samples were obtained from 180 Steller’s and 223 spectacled eider eggs collected during the breeding seasons of 2007–2013. 2.2. IgY extraction from egg yolk Proteins were extracted from the yolk by mixing 0.5 ml PBS with 0.5 ml yolk and adding 2.0 ml chloroform. The solution was vortexed for 10 minutes and incubated at room temperature for 1 hour. The mixture was centrifuged at 1400 rpm for 35 minutes and the supernatant removed and stored at −20 °C until analysis in the IgY assay. 2.3. IgY assay Immunoglobulin Y levels were measured in the egg yolk extracts using an enzyme-linked immunosorbent assay. Flat-bottom 96-well plates were coated with 10 μg ml−1 rabbit anti-chicken IgY capture antibody (Sigma-Aldrich, St. Louis, MO, USA) in carbonate– bicarbonate coating buffer. The plates were washed 4 times with 0.25 ml of wash buffer (PBS with 0.05% Tween) using a plate washer and then 0.05 ml of yolk extract, standard or blank (PBS) was added to the plate. Dilutions of chicken IgY were used as the standard. The plates were incubated at 37 °C for 1 hour and then washed 4 times with wash buffer. Alkaline phosphatase labeled rabbit anti-chicken IgY (Sigma-Aldrich) was diluted 1:2000 and 0.05 ml added to each well. The plates were incubated at 37 °C for 1 hour then washed 4 times with wash buffer. Each well had 0.05 ml of p-nitrophenyl phosphate disodium salt (PNPP, Thermo Scientific, Rockford, IL, USA) substrate added to it and the plate was incubated for 30 minutes

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at room temperature. Color development was stopped by adding 0.05 ml of 2N NaOH to each well and the plate was read on a plate reader at 405 nm. The IgY concentration in the samples was determined using a standard curve developed based on the pure chicken IgY. The use of anti-chicken IgY to detect eider IgY was validated by Frank (2004). The cross-reactivity of anti-chicken IgY with eider IgY was tested in Grabar Williams, Ouchterlony and Western blot assays, and the anti-chicken IgY demonstrated binding to both chicken and eider IgY in all of the validation experiments. The anti-chicken IgY was also validated in ELISAs with both chicken and eider IgY. A commercially available anti-duck IgY did not bind to eider IgY (Frank, 2004). 2.4. Statistics A number of predictor variables were tested for potential effects on IgY concentrations for each species using an information theoretic approach (Anderson, 2010). Akaike’s Information Criterion (AIC; Akaike, 1973) adjusted for small sample bias (AICc; Hurvich and Tsai, 1989) was compared among multiple additive models simultaneously with a null intercept model to determine the parameter structures that best fit the data, i.e., models with the lowest AICc values. To minimize the chances of overfitting the data, we took a 2-stage approach to the data analysis. That is, we divided the predictor variables into 2 categories for 2 stages of data analysis. Variables for which we were uncertain had any biological relevance to IgY levels were separated from those that were suspected as biologically relevant into Categories 1 and 2, respectively. Category 1 included year, laying date, fertility of the egg, and number of days the egg was incubated. Category 1 variables were first compared in individual models along with a null intercept model to assess their potential relevance. Variables that were modeled within 2 AICc points of the null model were considered unimportant and removed from further analysis. The others were retained for further testing with Category 2 variables which included an individual effect (hen ID), age of the eider, estimated mass at first laying date, and number of the egg in the clutch. Hen mass was not always recorded immediately prior to laying but ranged from 4 to 58 d for Steller’s eiders and from 0 to 76 d for spectacled eiders before first laying date. Therefore, mass was estimated by regressing measured mass on number of days prior to first egg laid for each species then adding the residual for each hen to the y-intercept. Some variable pairs were not tested in the same models together to minimize autocorrelation. Those pairs not tested together included hen age and year, hen ID and mass, and egg number and laying date. Finally, since spectacled eiders were all the same age and their first year of laying was the same for all individuals, their first year of laying was tested against all other years for its relative effect on IgY concentration. 3. Results Immunoglobulin Y was measured in the yolk of 180 eggs from 7 Steller’s eider hens and 223 eggs from 5 spectacled eider hens during the years of 2007–2013. Overall, average IgY content of Steller’s eider yolk ranged from 0.03 to 0.48 mg ml−1 and IgY in spectacled eider yolk ranged from 0.10 to 0.51 mg ml−1. Seventy-four of the 180 Steller’s eider eggs were fertile and 39 of the fertile eggs were incubated past 7 days before the egg was dissected. There were 120 fertile spectacled eider eggs of the 223 sampled and only 6 fertile eggs were incubated past 7 days. Number of days that the egg was incubated and the fertility status of the egg were found to have no influence on model selection during the first stage of data analysis for either species. Therefore, those 2 variables were not included in the second stage of data analysis. However, year and laying date

Table 1 Parameter structure and AICc statistics for second stage modeling of (a) spectacled eider and (b) Steller’s eider IgY in eggs. Model structure

Param.

AICc

(a) YR + ID + EggNo YR2 + ID + EggNo YR + ID Age + ID + EggNo YR2 + ID Age + ID + LDate YR YR2 Age + Mass + EggNo Age + Mass Age + Mass + LDate Mass + EggNo ID EggNo Mass + LDate LDate Mass Null model

12 7 11 7 6 7 7 2 4 3 4 3 5 2 3 2 2 1

580.40 580.98 590.81 591.10 593.32 600.28 601.31 603.26 607.18 611.78 613.25 621.61 621.80 625.04 625.50 627.48 628.35 632.16

(b) YR + ID + LDate Age + ID + LDate YR + ID + EggNo Age + ID Age + ID + EggNo ID YR + Mass + LDate Age + Mass + LDate Age + LDate LDate Mass + LDate YR YR + Mass YR + Mass + EggNo Age + Mass Age + Mass + EggNo Null model Mass EggNo Mass + EggNo

14 9 14 8 9 7 9 4 3 2 3 7 8 9 3 4 1 2 2 3

448.86 454.29 470.21 480.10 482.25 491.33 560.91 563.68 563.83 568.75 570.02 570.46 571.79 573.76 575.33 577.26 582.27 583.41 583.93 585.08

ΔAICc

AICc weight

Log L

0.00 0.58 10.41 10.70 12.92 19.88 20.92 22.96 26.78 31.38 32.85 41.21 41.41 44.65 45.10 47.08 47.96 51.77

0.57 0.49 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

276.30 282.14 282.64 287.20 289.39 291.79 292.31 298.62 298.45 301.79 301.48 306.71 304.70 309.47 308.65 310.68 311.12 314.05

0.00 5.43 21.35 31.24 33.38 42.47 112.05 114.82 114.96 119.89 121.16 121.60 122.93 124.89 126.47 124.89 133.41 134.55 135.07 136.22

0.94 0.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

207.97 216.50 218.64 230.52 230.47 237.25 269.81 276.67 277.80 281.31 280.90 276.81 276.37 276.23 283.55 276.23 289.10 288.64 288.90 288.43

Abbreviations: YR = year of laying, YR2 = first year compared to every other year combined, ID = individual hen, EggNo = egg number in clutch, LDate = laying date of egg.

were influential in stage 1 model fitting for both species and retained for the second stage of analysis. Age and mass of the hen were not favored among models that best fit the data and so had little correlation with IgY levels in yolk for both species. IgY concentrations were more strongly influenced by an individual effect of hen and variation between years for both species (Tables 1 and 2). Egg number in the clutch was also influential in model selection for spectacled eider eggs; whereas Steller’s eiders had a stronger influence of laying date in model selection related to IgY concentrations. Eggs that were laid later in the spectacled eider hen’s clutch generally had higher levels of IgY than early eggs (Figs. 1 and 2). On the other hand, IgY levels decreased with laying date among Steller’s eider eggs (Figs. 3 and 4). 4. Discussion Several factors were tested in an AICc modeling framework to determine which provided the most explanatory value for IgY concentrations in Steller’s and spectacled eider yolk. The number of days the egg was incubated and fertility status were not correlated with IgY concentrations. In studies conducted with chickens, embryonic absorption of IgY begins around day 7, but the majority of maternal antibody uptake occurs 3 days prior to hatch (Kowalczyk et al., 1985). The majority of the eggs in this study were incubated 5 days, long

K.L. Counihan et al./Developmental and Comparative Immunology 52 (2015) 10–16

Table 2 Parameter estimates from models that best fit the data for (a) spectacled eider and (b) Steller’s eider IgY in eggs. Note: for each set of parameters, the year 2007 and one individual hen were defined by the intercept. Coefficients

Estimate

Std error

t-Value

(a) Intercept YR2008 YR2009 YR2010 YR2011 YR2012 YR2013 ID10015 ID10018 ID10019 ID10024 EggNo.

2.163 0.792 1.012 0.459 1.028 0.829 1.363 −0.286 0.025 −0.520 −0.707 0.074

0.222 0.202 0.222 0.249 0.209 0.210 0.262 0.229 0.197 0.206 0.185 0.021

9.756 3.926 4.555 1.846 4.927 3.953 5.207 −1.247 0.125 −2.523 −3.818 0.021

(b) Intercept YR2008 YR2009 YR2010 YR2011 YR2012 YR2013 ID11885 ID1251 ID1961 ID2832 ID2834 ID8635 LDate

6.346 −0.136 0.788 0.398 0.107 0.822 0.802 1.718 1.106 0.715 1.401 1.485 3.604 −0.036

1.375 0.408 0.400 0.402 0.564 0.426 0.411 0.225 0.304 0.250 0.189 0.214 0.327 0.008

4.614 −0.332 1.969 0.990 0.189 1.929 1.952 7.630 3.631 2.864 7.404 6.943 11.005 −4.570

enough to determine fertility status, but not long enough to allow significant embryonic absorption to occur, which is likely why fertility and incubation duration were not correlated with IgY concentration. Immunosenescence can influence antibody transfer into yolk. The age of the Steller’s eider hen was not correlated with IgY concentration, but the Steller’s eiders only varied in age by 4 years. The ages of the seven females during the study period were: 3–5, 3–8, 2–8, 4–8, 4–7, 4–6, and 3–4 years old. Immunosenescence has been

Fig. 1. Box and whisker plots of the relationship between IgY (mg/ml) concentrations and egg number in clutch for all spectacled eiders combined.

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reported in birds, but Steller’s eiders are a long-lived species, so the females in this study were still relatively young and a 4 year age difference may not have been enough to detect differences between the females in the study (Cichon et al., 2003; Haussmann et al., 2005). Haussmann et al. (2005) noted that long-lived species appear to experience less immune function loss per year. The effect of the individual hen appeared to have a predominant influence on IgY concentration in both Steller’s and spectacled eider egg yolk in our study. The effect was most noticeable in Steller’s eiders, in which the hen with the lowest levels of yolk IgY had an average concentration of 0.1 mg ml−1 and the hen with the highest concentration of yolk IgY had an average of 0.39 mg ml−1. Neotropical passerines (Addison et al., 2009) and chickens (Kowalczyk et al., 1985) also had IgY levels that varied within a species similar to what we observed in our study. The IgY transferred into yolk is dependent on the mother’s acquired immunity status (Garnier et al., 2012). In chickens, it was determined that 10–20% of serum IgY is transported into the developing oocyte daily; however, serum IgY levels of individual chickens varied (Kowalczyk et al., 1985). The concentration of IgY in yolk has been found to be proportional to serum IgY of the mother (Hamal et al., 2006). Therefore, individual variations in IgY levels will lead to differences in IgY deposition in yolk. Hens from broiler flocks that had either no, medium or high levels of infectious bursal disease virus (IBDV) antibodies laid eggs with either no, medium or high levels of anti-IBDV antibodies in their yolk, respectively (Al-Natour et al., 2004). Because the eiders in our study were from a captive healthy flock, the primary source of individual variation in maternal IgY transfer may have been genetically determined as has been shown in chickens, pigeons and mammals (Grindstaff et al., 2003). The level of stress experienced by a female could be another factor influencing the amount of IgY an individual allocates to her eggs. Breeding can result in stressful social dynamics that impact females differently. Stress can elevate or depress immunoglobulin production depending on its severity (Hargitai et al., 2006; Morales et al., 2004). Pied flycatchers with increased stress levels, as measured by heat-shock protein levels, laid eggs with higher concentrations of IgY (Morales et al., 2004). Conversely, higher stress periods could repress the immune system. A study by Hargitai et al. (2006) found that high levels of stress led to lower amounts of IgY in eggs. However, the captive eider flocks were managed to keep stress to a minimum to maintain the health of the birds. Therefore, individual physiology is likely responsible for variations in IgY levels rather than stress within the flocks. The year during which the eggs were laid was correlated with the amount of IgY present in the yolk. Steller’s and spectacled eiders both had the highest yolk IgY concentrations in 2013, but there were generally no shared IgY concentration patterns for the other years. Gasparini et al. (2007) conducted a study with kittiwakes and determined that food supplemented birds transferred less IgY into eggs while non-supplemented birds transferred more IgY (Gasparini et al., 2007). The food supplemented birds may have anticipated good environmental conditions with abundant resources to provide their offspring while the non-supplemented kittiwakes transferred more IgY into their eggs to assist their offspring in potentially difficult environmental conditions (Gasparini et al., 2007). However, the eiders in our study had free access to food in all years of the study; therefore, if they anticipated favorable environmental conditions based on food availability they would have been under this assumption during all years of this study. A common environmental factor, such as weather, may have impacted IgY levels, but would require additional study. However, the annual fluctuations in IgY levels likely reflect annual individual variations in IgY deposition due to the lack of a shared pattern between species. Immunoglobulin Y concentration in Steller’s eider yolk declined with laying date in five of seven individuals in our study. This

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K.L. Counihan et al./Developmental and Comparative Immunology 52 (2015) 10–16

Fig. 2. Least squares regression lines for each individual spectacled eider hen showing the relationship between IgY (mg/ml) concentrations and the egg number in clutch. *Significantly different levels of IgY between the first and last eggs in a clutch (P < 0.01).

relationship has been observed in black-headed gulls, and the authors speculated that maternal depletion may be responsible for the decrease in IgY concentration in the clutch (Groothuis et al., 2006). While 10–20% of serum IgY is transferred to the developing oocyte daily, 30–40% of serum IgY is also degraded due to metabolic turnover; therefore, some species may not have the immunological capacity to synthesize enough IgY for their own maintenance and egg production (Kowalczyk et al., 1985). Immunoglobulin Y increased within the clutch of spectacled eiders indicating they did not experience maternal depletion, but some Steller’s eiders may not have the same immunological capacity. The Steller’s hens also

laid larger clutches than the spectacled eiders which could have resulted in declining IgY at the end of the clutch. Immunoglobulin Y concentration was positively correlated with egg number in clutch for spectacled eiders. Collared flycatchers transfer more IgY into later laid eggs than earlier ones, but they hatch asynchronously with the last hatchling having a survival disadvantage to earlier ones, so increased IgY levels could benefit chicks later in the clutch (Hargitai et al., 2006). Spectacled eiders hatch synchronously, so increasing deposition of IgY into later eggs in the clutch would not provide the same advantage (Gill, 2003). Robertson and Cooke (1993) found that in common eider clutches of three to six eggs, the third and fourth eggs were most likely to hatch while the first egg was least likely to hatch. Spectacled eiders may allocate more immunological resources to later laid eggs if their hatching success in the wild is similar to common eiders. We found potential evidence for a different strategy in intraclutch IgY deposition between Steller’s and spectacled eiders. This study, to our knowledge, was the first one to investigate the maternal transfer of IgY into Steller’s and spectacled eider egg yolk. Using eggs collected from captive flocks during 2007–2013, the average baseline levels of IgY in yolk were determined for these species. The AICc modeling statistics provided evidence that year influenced IgY concentration in both species and that the individual hen had the most impact on IgY. The date the egg was laid was negatively correlated with IgY concentration in most Steller’s eider eggs, but the egg number in the clutch was positively correlated with IgY concentration in spectacled eider eggs indicating that these species may have different strategies for intraclutch IgY provisioning. Discovering more about passive immunity in threatened Steller’s and spectacled eiders may provide further insight into their population dynamics.

Acknowledgements

Fig. 3. Box and whisker plots of the relationship between IgY (mg/ml) concentrations and Julian date of egg laying for all Steller’s eiders combined.

This project was funded by the U.S. Fish and Wildlife Service. We thank Rebekah Ziegman and Emily Ripley for assay assistance. We are also grateful to the husbandry staff for egg and data collection

K.L. Counihan et al./Developmental and Comparative Immunology 52 (2015) 10–16

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Fig. 4. Least squares regression lines for each individual Steller’s eider hen showing the relationship between IgY (mg/ml) concentrations and Julian laying date. *Significantly different levels of IgY between early and late laying dates (P < 0.01).

and the many lab staff, interns and volunteers who have helped with sample collection over the years. We would also like to thank Dr. Bobby Middlebrooks for his contribution to work on eider immunoglobulins. References Addison, B., Klasing, K., Robinson, W.D., Austin, S.H., Ricklefs, R.E., 2009. Ecological and life-history factors influencing the evolution of maternal antibody allocation: a phylogenetic comparison. Proc. R. Soc.B 276, 3979–3987. Akaike, H., 1973. Information theory and an extension of the maximum likelihood principal. In: Petrov, B., Csaki, F. (Eds.), The 2nd International Symposium on Information Theory. Akadeemiai Kiado, Budapest, Hungary. Al-Natour, M.Q., Ward, L.A., Saif, Y.M., Stewart-Brown, B., Keck, L.D., 2004. Effect of different levels of maternally derived antibodies on protection against infectious bursal disease virus. Avian Dis. 48, 177–182. Anderson, D., 2010. Model Based Inference in the Life Sciences: A Primer on Evidence. Springer, New York. Barua, A., Yoshimura, Y., Tamura, T., 1998a. Effects of ageing and oestrogen on the localization of immunoglobulin-containing cells in the chicken ovary. J. Reprod. Fertil. 114, 11–16. Barua, A., Yoshimura, Y., Tamura, T., 1998b. The effects of age and sex steroids on the macrophage population in the ovary of the chicken, Gallus domesticus. J. Reprod. Fertil. 114, 253–258. Blount, J., Surai, P., Nager, R., Houston, D., Moller, A.P., Trewby, M., et al., 2002. Carotenoids and egg quality in the lesser black-backed gull Larus fuscus: a supplemental feeding study of maternal effects. Proc. Biol. Sci. 269, 29–36. Buechler, K., Fitze, P., Gottstein, B., Jacot, A., Richner, H., 2002. Parasite-induced maternal response in a natural bird population. J. Anim. Ecol. 71, 247–252. Cichon, M., Sendecka, J., Gustafsson, L., 2003. Age-related decline in humoral immune function in Collared Flycatchers. J. Evol. Biol. 16, 1205–1210. Davison, F., Kaspers, B., Schat, K. (Eds.), 2008. Avian Immunology. Elsevier, San Diego. Federal Register, 1993. Final rule to list the spectacled eider as threatened. Fed. Regist. 58, 27474–27480. Federal Register, 1997. Endangered and threatened wildlife and plants; threatened status for the Alaska breeding population of the Steller’s eider. Fed. Regist. 62, 31748–31757. Fink, K., Zellweger, R., Weber, J., Manjarrez-Orduno, N., Holdener, M., Senn, B., et al., 2008. Long-term maternal imprinting of the specific B cell repertoire by maternal antibodies. Eur. J. Immunol. 38, 90–101. Flint, P.L., Morse, J.A., Grand, J.B., Moran, C.L., 2006. Correlated growth and survival of Spectacled Eider ducklings: evidence of habitat limitation? Condor 108, 901–911. Frank, J. 2004. Evaluation of immunoglobulin G (IgG) in the spectacled eider (Somateria fischeri) and development of monoclonal antibodies for use as a species specific reagent. Dissertation, University of Southern Mississippi, Hattiesburg. Garnier, R., Ramos, R., Staszewski, V., Militao, T., Lobato, E., Gonzalez-Solis, J., et al., 2012. Maternal antibody persistence: a neglected life-history trait with implications from albatross conservation to comparative immunology. Proc. Biol. Sci. 279, 2033–2041.

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