DOI: 10.5061/dryad.1vhhmgqt0
title: No evidence for increased fitness of offspring from multigenerational effects of parental size or natal carcass size in the burying beetle Nicrophorus marginatus

description: Multigenerational effects (often called maternal effects) are components
 of the offspring phenotype that result from the parental phenotype and the
 parental environment as opposed to heritable genetic effects.
 Multigenerational effects are widespread in nature and are often studied
 because of their potentially important effects on offspring traits.
 Although multigenerational effects are commonly observed, few studies have
 addressed whether they affect offspring fitness. In this study we assess
 the effect of potential multigenerational effects of parental body size
 and natal carcass size on lifetime fitness in the burying beetle,
 Nicrophorus marginatus (Coleoptera; Silphidae). Lifespan, total number of
 offspring, and number of offspring in the first reproductive bout were not
 significantly related to parental body size or natal carcass size.
 However, current carcass size used for reproduction was a significant
 predictor for lifetime number of offspring and number of offspring in the
 first brood. We find no evidence that multigenerational effects from
 larger parents or larger natal carcasses contribute to increased fitness
 of offspring.
description: <b>Source of
 burying beetles</b> To generate the
 laboratory-bred population to use for the experiment, we captured
 adult<i> N. marginatus</i> at the Utah Wetland Preserve near
 Goshen, Utah, USA (this is public land, and no permits were required to
 trap or collect beetles), in August 2014 using pitfall traps baited with
 aged chicken. We created 41 independent genetic lines from wild-caught
 pairs by providing a 40g mouse carcass for each pair and allowing them to
 breed. We designated the offspring from the wild-caught pairs as the first
 parental generation. After eclosion and emergence of this first
 laboratory-bred generation, we placed individuals in small plastic
 containers (15.6 x 11.6 x 6.7 cm), fed them <i>ad libitum</i>
 raw chicken liver twice weekly, and maintained them on a 14:10 h
 light:dark cycle at 21<b>°</b>C<b> </b>until they
 were used in experiments. We designated the date of eclosion as the first
 day of life for all beetles used in the experiments. <b>Experimental
 design</b> The purpose of this
 experiment was to determine the effect on lifetime fitness of potential
 multigenerational effects of parental body size and natal carcass size in
 <i>N. marginatus</i>. We measured three fitness response
 variables: lifespan, total number of offspring, and number of offspring
 produced in the first brood. Number of offspring in the first brood was
 used in addition to lifetime number of offspring because number in the
 first brood may or may not follow total lifetime numbers [39], and in the
 natural environment, number of reproductive bouts may be fewer than that
 observed in laboratory experiments. Thus, number in the first brood may
 represent natural conditions better than total lifetime number of
 offspring in a laboratory environment. To generate potential
 multigenerational effects from parental body size and natal carcass size,
 we allowed large and small beetles to reproduce on large or small
 carcasses (first parental generation). To assess fitness effects of
 multigenerational effects, we then allowed female offspring from this
 first parental generation to reproduce throughout their lifetime. We then
 measured the three fitness responses for all females of this second
 generation and compared them among treatment combinations. To determine what
 sizes constituted large or small beetles for assignment in the first
 parental generation, we used the distribution of pronotum widths from
 wild-caught beetles. We assigned beetles from the first parental
 generation with pronotum widths &gt; 1 standard deviation above and
 &lt; 1 standard deviation below the mean (derived from wild-caught
 population) as large and small, respectively. In the wild-caught
 population, the mean pronotum width of females was 6.67mm, with a standard
 deviation of 0.78mm (N = 50). The mean pronotum width of males was 6.87mm,
 with a standard deviation of 0.72mm (N = 50). Thus, the size range of
 large and small female beetles that we used in this experiment for the
 first parental generation was 7.44mm – 8.22mm and 5.11mm – 5.89mm,
 respectively, and the corresponding size range for large and small males
 was 7.60mm – 8.32mm and 5.42mm – 6.15mm, respectively. For this first part
 of the experiment, we used a fully crossed factorial design.  There were
 four parental size treatments - large male with large female, large male
 with small female, small male with large female, and small male with small
 female.  Each parental size treatment was crossed with both small (20g)
 and large (40g) carcass sizes, for a total of eight treatment
 combinations. We chose 20g and 40g carcass sizes based on a previous study
 that tested multiple carcass sizes from 5g to 50 g. The 20g and 40g sizes
 were both within the range of carcass sizes whereon <i>N.
 marginatus</i> experienced equally high reproductive success [69].
 We completed six replicates of each of the eight treatment combinations
 resulting in an initial sample size of 48 pairs for the first parental
 generation. We began each
 first-generation experimental replicate by randomly choosing a genetically
 unrelated pair of beetles that fit into one of the parental size
 treatments. We placed the pair in a small brood container and randomly
 assigned them either a 20g (± 1.0g) or a 40g (± 2.0g) mouse carcass. We
 checked the brood containers daily for larvae, and after larvae arrived on
 the carcass, we removed the lid of the small brood container and placed
 the small container in an abandonment chamber (see [70] for details). We
 checked abandonment chambers daily for leaving adults or dispersal of
 larvae, and when the carcass was consumed and larvae dispersed into the
 soil, we removed and weighed the remaining parent(s). The larvae from each
 brood reached eclosion 4-5 weeks after dispersal. We weighed each
 newly-eclosed adult offspring, placed them in an individual container, and
 fed them <i>ad libitum</i> chicken liver until they reached
 sexual maturity.We used results from this first-generation experiment to
 test for effects of body size of parents (male and female separately) and
 carcass size on reproductive output and offspring traits[65]. In the
 first-generation experiment female body size generated significant effects
 on offspring body size, but male size had no effect on offspring traits
 and there were no significant interactions between male and female body
 size [65]. For this reason, we included parental size treatments as four
 independent treatments in analysis of the second-generation experiment
 (this paper) rather than as a factorial. This reduced complexity of the
 second-generation analysis by eliminating some two-way and three-way, and
 all four-way interactions. For additional information on the first
 parental generation methods and results from the experiment, see Smith and
 Belk [65]. For the second
 parental generation, we used the offspring from the first parental
 experiment described above to determine how the size of parental
 <i>N. marginatus </i>beetles and the size of carcass that they
 reproduced on affected lifetime fitness of their offspring. For the second
 parental generation, we randomly chose two female offspring from each of
 the first parental replicates for the experiment. These two females (from
 replicates of the eight first generation treatment combinations) were
 randomly assigned either large (40g) or small (20g) carcasses for
 reproduction, for a total of sixteen treatment combinations in the second
 experiment. We started six replicates for each of the sixteen treatments
 for a total of 96 replicates. Five experimental pairs failed to reproduce
 (one each for five of the treatment combinations), so the realized sample
 size was 91 pairs. To determine fitness
 of second-generation beetles, sexually mature females (age &gt; 21
 days) were paired with a randomly selected, sexually mature, genetically
 unrelated male, and the pair was placed in a plastic container (14 × 13 ×
 17cm) filled with 10cm of moist soil and given either a 20g (±1.0g) or a
 40g (± 2.0g) mouse carcass (based on the random assignment described
 above). The containers were kept in an environmental chamber at 21°C on a
 14:10 h light:dark cycle. We checked for larvae daily, and after larvae
 arrived on the carcass, we removed the male from the container so that
 only the female cared for the offspring. After the larvae dispersed into
 the soil to pupate, we removed the female and placed her in a small
 container with <i>ad libitum</i> chicken liver for two days,
 and then set her up to mate again with a new, randomly selected,
 genetically unrelated, virgin male on the same size carcass (20g or 40g).
 This cycle continued for her entire life. In the second-generation
 experiment, number of successful reproductive bouts ranged from 1 to 5
 with a mean of 2.9 bouts. We measured lifespan as the number of days the
 female survived after eclosion. After the offspring from each brood of
 this second-generation experiment eclosed as adults, we recorded the sex,
 weight, and pronotum width for each beetle. We summed the number of
 offspring from each brood to determine a female’s lifetime number of
 offspring. <b>Analysis</b> To determine if parental size or natal carcass size influences fitness of offspring we used a mixed model analysis of covariance (ANCOVA) approach (SAS, Proc MIXED). We used three response variables to represent fitness of females from the second parental generation: i.e., total lifetime number of offspring, total number of offspring in the first brood, and lifespan.  Predictor variables for each of the three models were first parental generation body size (four levels), natal carcass size (two levels), current carcass size (two levels), and pronotum size of second parental generation focal female (covariate). Initially we included all two-way and the three-way interactions of main effects in the model. However, interaction terms were not significant in any of the models, so, we report results from a reduced model that included only one, two-way interaction – natal carcass size by current carcass size. This interaction was of interest because of the significant effect of current carcass size on fitness measures, and the possibility of a multigenerational effect that natal carcass size might provide some “priming” for efficiency of use of a similar carcass size. Because we used two individuals from each of the first-generation broods to breed for the second generation, we used first generation parental brood ID as a random effect to account for the relatedness of individuals from the same brood. We used raw data for the analysis, and inspection of residual plots showed no departure from assumptions for the parametric model.
description: Data are arranged in columns. Column 1 is parental brood ID,
 column 2 is offspring female ID, column 3 is the parental size treatment,
 column 4 is the natal carcass size treatment, column 5 is the current
 carcass size treatment, column 6 is the pronotal width of the female
 offspring, column 7 is the number of successful reproductive attempts,
 column 8 is the lifetime number of offspring, column 9 is the total
 offspring mass, column 10 is the lifespan, and column 11 is the number of
 offspring in the first brood. Key to treatment
 designations Parental size treatment: 1=large male
 large female, 2=large male small female, 3=small male large female,
 4=small male small female Natal carcass size treatment:
 1=20g natal carcass, 2=40g natal carcass Current
 carcass size treatment: 1=20g natal carcass, 2=40g natal carcass