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Reproductive allocation in Nicrophorus maliger and Nicrophorus guttula

Citation

Belk, Mark; Meyers, Peter; Creighton, J. Curtis (2022), Reproductive allocation in Nicrophorus maliger and Nicrophorus guttula, Dryad, Dataset, https://doi.org/10.5061/dryad.rv15dv48t

Abstract

Abstract: The cost of reproduction hypothesis suggests that allocation to current reproduction constrains future reproduction. How organisms accrue reproductive costs and allocate energy across their lifetime may differ among species adapted to different resource types. We test this by comparing lifetime reproductive output, patterns of reproductive allocation, and senescence between two species of burying beetles, Nicrophorus marginatus and N. guttula, that differ in body size, across a range of carcass sizes. These two species of burying beetles maximized lifetime reproductive output on somewhat different–sized resources. The larger N. marginatus did better on large and medium carcasses while the smaller N. guttula did best on small and medium carcasses. For both species, reproduction is costly and reproduction on larger carcasses reduced lifespan more than reproduction on smaller carcasses. Carcass size also affected lifetime reproductive strategies. Each species’ parental investment patterns were consistent with terminal investment on carcasses on which they performed best (optimal carcass sizes). However, they exhibited reproductive restraint on carcass sizes on which they did not perform as well. Reproductive senescence occurred largely in response to carcass size. For both species, reproduction on larger carcasses resulted in more rapid senescence. These data suggest that whether organisms exhibit terminal investment or reproductive restraint may depend on type and amount of resources for reproduction.

Methods

2.1. Burying Beetle Natural History

Burying beetles locate small vertebrate carcasses and use them as food resources for themselves and their offspring. Males and females compete with individuals of the same sex until a single pair, typically those with the largest body sizes, dominate the carcass [37,38]. Similar to within species competitive dynamics, larger species will displace smaller species when they co–occur on a carcass [10,31]. The winning pair of beetles buries the carcass under the soil, removes the feathers or hair, shapes the carcass into a ball, and coats it with oral and anal secretions that help prevent microbial growth. During carcass preparation, the female lays eggs in the soil, and larvae usually hatch on or after the fourth day. Parents adjust brood size through filial cannibalism, regurgitate food to newly hatched larvae, and provide defense of the carcass and larvae from conspecifics. Parental care continues until larvae disperse into the soil to pupate [31].

2.2. Experimental Design

We collected both N. marginatus and N. guttula at Goshen Ponds (39° 57.476’N, 111° 51.426’W) and Utah Lake Wetland Preserve (40° 6.933’N, 111° 47.589'W) in central Utah during June 2011 and July 2012 using pitfall traps baited with aged chicken. We transported beetles back to Brigham Young University and established laboratory populations for each species by breeding wild–caught pairs on a 30 g carcass. We kept newly eclosed offspring in small plastic containers (11.3 cm L x 7.6 cm W x 5.7 cm H), provided them ad libidum raw chicken liver, and maintained them on a 14L:10D cycle (a natural photoperiod for the summer breeding season at the source location). Beetles used in this experiment were F1, F2, and F3 individuals, and all crosses used different family lines to ensure no inbreeding occurred.

At 28 days (± 1 day) from eclosion, we randomly assigned females from both species to one of six carcass size treatments and one nonreproductive treatment (12 replicates for each treatment for a total of 84 females of each species). In six of the treatments, we allowed females to reproduce throughout their lifetime on one of six carcass sizes (5 g, 10 g, 20 g, 30 g, 40 g, or 50 g, ±1.0 g lab mouse carcasses; this range in size covers the range of carcass sizes available in the natural environment). For each reproductive bout, we placed each female with a virgin male (at least 21 days old, to ensure sexual maturity) on a carcass of their assigned size in a plastic container (20.3 cm L x 15.2 cm W x 9.8 cm H) filled with approximately 4.5 cm (depth) of commercially purchased topsoil and allowed them to reproduce. After 48 h, we removed males from the carcass to isolate the reproductive investment patterns of females (males were present only during the beginning of the carcass preparation phase). At the end of each reproductive attempt (defined as the point when all larvae dispersed from the carcass into the soil), we removed the females, placed them each in a small, individual container, and provided them with a moistened paper towel for water and chicken liver ad libidum. After 48 h, we placed females on a new carcass (of the same size as their previous reproductive attempt) with a virgin male and allowed them to produce offspring. This cycle was repeated until the female died. To assess effects of reproduction on lifespan, our seventh treatment was a “non–reproducing” treatment where females were fed on chicken liver (0.5 g to 1 g twice per week), but were not allowed to breed throughout their life.

For each treatment, we weighed females and measured their pronotum width at 28 days of age (±1 day), and when the female died we recorded her lifespan. For the six reproducing treatments, we weighed females before and after each reproductive attempt. We monitored each female and her brood daily to determine brood size and timing of larval dispersal. If, after 7 days, no offspring had appeared on the carcass, we designated the brood as a failure, and removed the female, gave her food, and isolated her for 48 h, then allowed her to breed again on a fresh carcass with a new virgin male. We recorded the initial and final number of offspring and mass of offspring as they dispersed into the soil for each reproductive attempt.

2.3. Statistical Analyses

2.3.1. Analysis of Optimal Carcass Size

Our first goal in the experiment was to determine optimal carcass sizes for each species. We defined “optimal” as the carcass size that resulted in the greatest number of offspring produced over a lifetime. To determine optimal carcass size, we used a general linear model to examine the effects of carcass size and species on lifetime number of offspring (GLM procedure; SAS 9.3 SAS Institute, Cary, NC, USA). In the model, carcass size and species were main effects, and we included an interaction between carcass size and species, and standardized female body size (pronotum width) as a covariate. As noted above, body size varies between the two species. We were interested in effects of body size within each species, and we did not want to confound differences in body size between species with within species variation, so we standardized body size across species by creating a z–score centered on the mean of each species. The response variable, lifetime total number of offspring, was log transformed to meet assumptions for the parametric model. One N. guttula female from the 20 g carcass size never reproduced and was removed from all analyses.

Total mass of offspring over the lifetime (i.e., total number of offspring multiplied by mean offspring mass per brood) is sometimes used to represent evolutionary fitness. We analyzed patterns of total mass of offspring over a lifetime using the same model as that used for total number of offspring. Results were consistent with results obtained from total number of offspring, so we present only total number of offspring in this paper.

2.3.2. Analysis of Patterns of Reproductive Allocation

Our second goal was to determine if reproductive allocation and resulting senescence followed a pattern of terminal investment or reproductive restraint. We used four response variables to characterize contrasting patterns of reproductive allocation as follows: lifespan, lifetime number of reproductive bouts, mass change of females through time, and proportion of offspring culled through time. Terminal investment would be characterized by shorter lifespans, fewer reproductive bouts, negative or neutral mass gain, and fewer offspring culled at older ages. In contrast, reproductive restraint would be characterized by longer lifespans, more reproductive bouts, positive mass gain, and more offspring culled at older ages.

To test for differences in lifespan, we used a general linear model (GLM procedure; SAS 9.3 SAS Institute, Cary, NC, USA) and for differences in number of reproductive bouts we used a generalized linear model (GenMod procedure; SAS 9.3 SAS Institute, Cary, NC, USA). For each model, carcass size and species were main effects, and we included an interaction between carcass size and species, and standardized female body size (pronotum width) as a covariate. As noted above, body size varies between the two species. We were interested in effects of body size within each species and we did not want to confound differences in body size between species with within species variation, so we standardized body size across species by creating a z–score centered on the mean of each species. The non–reproducing treatment was only included in the model for lifespan. Data for lifespan met the assumptions of the parametric model and was not transformed. For the number of successful reproductive bouts, we assumed a Poisson distribution and used a log–link function.

To test for differences in mass change and proportion brood culled within lifetimes, we used a generalized linear mixed model (GLMM; GLIMMIX procedure; SAS 9.3 SAS Institute, Cary, NC, USA). Mass change was measured as mass of female at the end of the reproductive bout minus mass of the female at the beginning of the reproductive bout. Mass loss would be observed as a negative number and mass gain would be positive. Mass change is a continuous variable, and raw data met assumptions for a parametric model, so no transformations were used. Proportion brood culled was measured as number of offspring culled relative to initial brood size, so we assumed a binomial distribution with a logit–link function. We used a repeated measures design to analyze patterns of allocation through time. Species, carcass size, and age (indexed by reproductive bout) were predictor variables (i.e., main effects). We used standardized female body size as a covariate and included all two–way and three–way interactions among main effects. A single N. guttula female never reproduced and thus we removed her from all analyses. Because we had multiple measures of the same individual through time, individual ID was used as a random effect in the model.

2.3.3. Analysis of Patterns of Reproductive Senescence

To assess patterns of reproductive senescence, we used three response variables as follows: initial brood size, final brood size, and offspring body mass at dispersal. An increased rate of senescence would be characterized by a negative slope of initial offspring number and final offspring number with increasing age; whereas, a decreased rate of senescence or delayed senescence would be characterized by a zero slope of initial offspring number and final offspring number with increasing age. Because both the female and the offspring feed exclusively on the carcass during brood development, fewer offspring should result in larger offspring body size. Thus, senescence would result in fewer but larger offspring in older individuals; whereas, delayed senescence would result in about the same number and size of offspring with increasing age.

To test for differences in initial brood size, final brood size, and individual offspring mass within lifetimes we used generalized linear mixed models (GLMM; GLIMMIX procedure; SAS 9.3 SAS Institute, Cary, NC, USA). We ran separate models for each of the three response variables. Initial brood size was the number of larvae that first appeared on the carcass before culling had occurred. Final brood size was the number of larvae that dispersed into the soil and represents the brood size after culling has occurred. For each trait we used a repeated measures design to analyze patterns of senescence through time. Species, carcass size, and reproductive bout (age) were predictor variables (i.e., main effects). We used standardized female body size as a covariate and included all two–way and three–way interactions. For both initial and final brood sizes, we assumed a Poisson distribution and used a log–link function. Offspring mass was a continuous variable, so we used a log–link function. Because we had multiple measures of the same individual through time, individual ID was used as a random effect in the model. A single N. guttula female never reproduced and thus we removed her from all analyses. In addition, we removed two bouts from a single female from the analysis for mean offspring mass because in each bout, only two offspring were produced and they were abnormally small (1/5 the size of any other offspring).

Usage Notes

Two datasets:

first is the "lifetime" dataset, "." is missing data

Variables (columns) are as follows:

species (1=N. marginatus 2=N. guttula)

ID - individual ID number

carcass size (0=5g, 1=10g, 2=20g, 3=30g, 4=40g, 5=50g, and 7= non-reproductive)

lifespan (in days)

size (pronotum width of female)

sbouts = successful bouts 

tbouts = total bouts

totoffmass = total offspring mass

numoffspring = number of offspring

 

Second data set is "within", "." is missing data

Variables (columns) are as follows:

species (1=N. marginatus 2=N. guttula)

carcass size (0=5g, 1=10g, 2=20g, 3=30g, 4=40g, 5=50g)

ID - individual ID number

age - reproductive bout

cmass - actual carcass mass in g

pw - pronotum width 

startmass - mass of female at beginning of reproductive bout

endmass - mass of female at end of reproductive bout

startbrood - initial number of offspring in brood 

finalbrood - final number of offspring in brood 

moffsize - mean offspring size

totoffmass - total offspring mass per brood

masschange - mass change of female during bout

efficiency - brood mass divided by carcass mass