the behavioural ecology of trophic...
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THE BEHAVIOURAL ECOLOGY OF
TROPHIC EGG-LAYING
Jennifer C. Perry B.Sc. (Hon.) Environmental Biology, University of Alberta, 2001
Thesis submitted in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
In the Department
of Biological Sciences
O Jennifer C. Perry 2004
SIMON FRASER UNIVERSITY
July 2004
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ABSTRACT
Across diverse taxa, animals produce infertile trophic eggs that are consumed by
offspring. The objective of this thesis research was to establish a behavioural ecology framework
for the study of trophic eggs; to model their evolution; and to investigate their adaptive function,
if any, in the ladybird beetle Harmonia axyridis.
Current trophic egg hypotheses suggest two adaptive functions: provisioning offspring or
reducing parent-offspring conflict over sibling cannibalism. On the other hand, trophic eggs may
simply represent infertility - they may not be an adaptation at all, a distinction given insufficient
attention in the literature. I consider trophic egg laying in the context of sibling cannibalism
behaviour. A frequency-independent analysis of sibling cannibalism suggests that there is a
critical value of increase in offspring survival above which cannibalism benefits both parents and
offspring; then mothers should adopt tactics (e.g., trophic egg laying) to facilitate cannibalism.
However, the frequency-independent approach does not incorporate game interactions. I used a
genetic algorithm approach to model the co-evolution of trophic eggs, sibling cannibalism, and
hatching synchrony. Results suggest that, when game interactions occur or when infertile eggs are
present, cannibalistic tendencies increase dramatically, as does the maternal response to limit
cannibalism. Furthermore, trophic egg laying and hatch synchrony appear to be viable tactics for
facilitating egg-eating.
We tested trophic egg function in H. axyridis, predicting that mothers should lay fewer
trophic eggs in food-rich environments and more in low food environments where offspring gain
a larger relative benefit from eating an egg. As predicted, ladybirds produced 64% more trophic
eggs when provided with information that they were in a low vs. high food environment (P =
0.0093). This result demonstrates that ladybirds produce trophic eggs to increase the chance that
offspring survive starvation. In a second observation set, I tested whether the spatial distribution
and oviposition sequence pattern of trophic eggs is over- or under-dispersed compared to random.
There was no indication of a non-random distribution.
In conclusion, this research is of interest in behavioural ecology because it shows that an
unusual parenting strategy, killing off some offspring to benefit others, can be adaptive.
Acknowledgements First and foremost, I thank the members of my supervising committee - Dr. Bernie
Roitberg, Dr. Bernie Crespi and Dr. Felix Breden - for their carefully considered advice as I
prepared for and conducted this thesis research. 1 particularly appreciate the guidance and
mentorship of my senior supervisor, Bernie Roitberg, throughout this project.
I am grateful to Dr. Carl Schwarz, who provided extremely useful statistical help, and
Jabus Tyerman, who wrote the randomisation test program described in Chapter 4.
The manuscripts were improved by thoughtful and appreciated comments from Geoff
Allen, Jay Biernaskie, Felix Breden, Kelly Campbell, Bernie Crespi, Brian Ma, Jason Peterson,
Bernie Roitberg, Maxence Salomon, and Jabus Tyerman.
I am obliged to laboratory assistants Andy Capadouca, Nick Charrette, Beth Ann Nyboer,
Jennifer O'Neil, Eva Poon, and Andy Yang, for their assistance.
Members of the Behavioural Ecology Research Group, the Entomological Society of
British Columbia, and the DOGG Journal Club provided helpful advice as I planned and
conducted experiments.
The research was supported by the National Science and Engineering Research Council
(NSERC) in graduate support and grants to Bernie Roitberg. I also acknowledge graduate support
from the Department.
Finally, I must express my appreciation for the Roitberg lab group. Frequent discussions
with Roitbergers improved all aspects of this work, and lab interactions made this research
process tremendously enjoyable.
Table of Contents . . Approval ................................................................................................................................... 11
... Abstract ................................................................................................................................. nl
Acknowledgements ........................................................................................................................ iv
Table of Contents ........................................................................................................................... v .. List of Figures ............................................................................................................................... vu
List of Tables ................................................................................................................................. i x
Chapter 1
Chapter 2
Chapter 3
Overview of the Thesis .......................................................................................... 1 Overview .................................................................................................................... 1 Literature Cited .......................................................................................................... 3
The Behavioural Ecology of Trophic Egg Laying: Hypotheses and Evidence ................................................................................................................... 4
...................................................................................................................... Abstract 4 Introduction ................................................................................................................ 4 Parent and offspring interests: inclusive fitness approach ......................................... 5 Two sets of trophic egg hypotheses ........................................................................... 7
The parent-offspring conflict reduction hypothesis ........................................... 8 Cooperation hypothesis .......................................................................................... 9
What evidence is required to distinguish hypotheses? ............................................. 10 Evidence from natural history and behavioural observations .............................. 11 Evidence from behavioural and life history experiments ..................................... 11 Comparative analyses ........................................................................................... 13
Challenges for future research ................................................................................. 14 .............................................................................................................. Conclusions 15
Literature cited ......................................................................................................... 17
Co-Evolution of Maternal and Offspring Effectors of Sibling Cannibalism ........................................................................................................... 31
Abstract .................................................................................................................... 31 Introduction .............................................................................................................. 31 Model ....................................................................................................................... 33
Biological system .......................................................................................... 33 Inclusive fitness argument .................................................................................... 34 Genetic algorithm-based simulations: the basic model ........................................ 35 Computer experiments ......................................................................................... 36
Results and discussion ............................................................................................. 38 Relative benefit of egg eating .............................................................................. 38 Trait co-evolution ................................................................................................. 40 Non-evolved infertility ......................................................................................... 41 Time extension ..................................................................................................... 42
Conclusions .............................................................................................................. 43
3.5
3.6
Chapter 4
4.1 4.2 4.3
4.4
4.5
4.6
4.7 4.8
Chapter 5 5.1 5.2 5.3
5.4 5.5
Trophic egg laying ............................................................................................... 45 . . Future directions ................................................................................................... 46
Appendix .................................................................................................................. 47 Details of genetic algorithm and simulations ....................................................... 47
Literature cited ......................................................................................................... 49
Ladybird Mothers Mitigate Offspring Starvation Risk by Laying Trophic Eggs .......................................................................................................... 71 Abstract .................................................................................................................... 71 Introduction ........................................................................................................... 71 Methods I: Food level experiments .......................................................................... 74
Study system and experimental animals .............................................................. 74 Food level experiments ........................................................................................ 74 Statistical analyses ............................................................................................... 76
Results I: Food level experiments ............................................................................ 77 Internal cue experiment ........................................................................................ 77
....................................................................................... External cue experiment 79 Methods 11: Non-random patterns of infertile egg production ................................. 80
Spatial patterns ..................................................................................................... 80 Oviposition patterns ............................................................................................. 80
Results 11: Non random patterns of infertile egg production ................................... 81 Spatial patterns ..................................................................................................... 81
............................................................................................. Oviposition patterns 81 ................................................................................................................ Discussion 81
......................................................................................................... Literature cited 85
Summary of the Thesis and Suggestions for Future Work ............................ 101 ............................................................................................................ Introduction 101
Thesis summary ..................................................................................................... 101 Suggestions for future work ................................................................................... 102
............................................................... Approaches to answering question (1) 103 A suggested approach for question (2) ............................................................... 105 A suggested approach for question (3) ............................................................... 105
Conclusions ............................................................................................................ 105 ....................................................................................................... Literature cited 107
APPENDIX I ............................................................................................................................... 108 I . 1 Frequency of sibling cannibalism in Harmonia axyridis ....................................... 108
Methods .............................................................................................................. 108 ......................................................................................... Results and summary 109
1.2 Does egg viability or hatching time depend on egg mass? .................................... 109 .............................................................................................................. Methods 109
Results and summary ......................................................................................... 110 1.3 Trophic egg production in the laboratory and field ................................................ 111
Methods .............................................................................................................. 111 Results and summary ......................................................................................... 112
1.4 Male-killing bacteria and the proportion of trophic eggs laid ................................ 113 Methods .............................................................................................................. 113 Results and summary ......................................................................................... 114
1.5 Literature cited ...................................................................................................... 115
List of Figures
Figure 2-1 The inclusive fitness interests in sibling cannibalism of a parent and her cannibalistic offspring; r is the coefficient of relatedness between siblings. Parent-offspring conflict or cooperation depends on the survival benefits of cannibalism, relative to survival of non-cannibalistic offspring. In the range from 0-r%, neither parents nor offspring benefit from sibling cannibalism. ............... 22
Figure 3-1 The average number of offspring cannibalized per clutch with various combinations of cannibalism delay and hatching synchrony values. Cannibalism delay alleles correspond to the number of time units offspring wait before eating an egg. Phenotypes for hatching synchrony alleles are shown in Table 3.1. The shaded area indicates combinations of traits that yield no sibling cannibalism. The number of cannibalized offspring increases diagonally to a maximum of eight (in a clutch of 16), as indicated by the arrow. ........................................................................................................................... 5 1
Figure 3-2 The evolution of cannibalism delay, hatching asynchrony and trophic egg laying, when other genes were prevented from evolving (Table 3.2). Least square means are from a one-way ANOVA with the factor 'relative benefit of egg eating', which was a significant factor for all three genes; P < 0.0001 in each test. Means with different letters are statistically different. The bars represent standard error. The y-axis is reduced for the trophic egg laying gene because average allele value never exceeded 8 ............................................................ 53
Figure 3-3 Evolution of the cannibalism delay gene over 1000 generations, for different levels of relative benefit of egg-eating, when the evolution of other traits was prevented (Table 3.2). Results from one representative simulation are shown for each level of relative benefit. Mean gene value was taken for the parental population every tenth generation. With the random generation of individuals in generation zero, initial average gene values were approximately 7.5 ...................... 55
Figure 3-4 Effect of infertility on the evolution of three traits. For each figure, least squares mean gene values are given for different levels of relative benefit of egg eating, given by the different lines, in the absence and presence of infertile eggs. (a) Cannibalism delay. The mean difference in gene value in the presence and absence of infertile eggs was 6.7 (4.8, 8.5) at 10% relative benefit and 3.8 (1.9, 5.6) at 25% relative benefit. The difference was not significant for any other level of benefits. For clarity, levels above 75% are not shown. (b) Hatching synchrony. The presencelabsence of infertile eggs affected the mean difference in gene values at 10% benefits (2.0 (0.9, 3.2)). The difference was not significant at any other level of relative benefit. For clarity, not all of the tested levels are shown. (c) Trophic egg-laying. Infertile eggs significantly decreased trophic egg production at the following levels of relative benefit, with the mean difference in gene value given: 0.9 (0.5, 1.3) at 75%; 0.8 (0.4, 1.2) at 90%; 1.1 (0.7, 1.5) at 100%; 0.8 (0.4, 1.2) at 110%; 0.9 (0.5, 1.3) at 125%; 0.9 (0.5, 1.3) at 150%. The y-axis range is reduced to show
vii
differences between the curves. For clarity, not all levels of relative benefit are shown. .................................................................................................................... 57
Figure 3-5 Trait evolution when other traits are constrained. Levels of relative benefit of egg eating are given by the different lines. (a) The evolution of cannibalism delay in offspring when maternal traits affecting cannibalism are free to evolve or constrained (set 1, Table 3.2). Least squares mean gene values are obtained from an ANOVA with the factors relative benefit, presence of constraints, and the interaction term. Constraining traits lead to delayed cannibalism; see discussion in text. Significant increases in delay times occurred at relative benefit levels of 25% (mean difference 6.6 time units (4.5, 8.7)) and 50% (2.2 time units (0.1,4.3)). The differences were not significant at other levels of relative benefit. (b) The effect of constraints on cannibalism delay and trophic egg laying on the evolution of hatching synchrony (set 2, Table 3.2). The difference in gene value when other traits were constrained, or free to evolve, was significant at 0% relative benefit (mean difference 5.2 (1.6, 8.8)) and 150% relative benefit (mean difference 3.8 (0.2, 7.4)). (c) The effect of constrained traits (set 3, Table 3.2) on the evolution of trophic egg laying. The mean difference between the trophic egg gene when traits were constrained, or free to evolve, was 0.7 (0.1, 1.4) at 75% relative benefit; 1.6 (0.9,2.2) at 125%; and 3.2 (2.6, 3.9) at 150%. Note that the y-axis scale for this trait differs from that in graphs of the other genes. ........................................ 61
Figure 4-1 External cue experimental chamber. Ladybirds were placed in the upper compartment with filter paper as an oviposition surface, a water source, and a small mass of pea aphids. The bottom compartment contained a bean leaf pair, inserted through Parafilm@ into a vial of water, and the treatment: either no or many pea aphids. ................................................................................................ 89
Figure 4-2 Proportion of trophic eggs produced when females were given information that there was low or high food availability through (a) the internal cue of food consumption or (b) the external cue of prey scent; see text for details. The mean proportion was smaller under the low food treatment in the internal cue but not the external cue experiment (Table 4.2). Horizontal lines represent means. Whiskers indicate the range of observations. .................................................. 9 1
Figure 1-1 Proportion of egg mass lost during the development of H. axyridis egg types. Trophic eggs lose significantly less mass than viable eggs (estimated difference and 95% CI: 0.025 (0.009,0.042)). Inviable eggs, where an embryo develops but does not emerge, lost an intermediate amount of mass. The initial mass of eggs was taken 12 hours afier oviposition; final mass was taken in the 24 hours before hatching. ................................................................................. 116
Figure 1-2 Hatching time in relation to egg mass within egg batch 60-1 5. Hatching time is expressed relative to the first hatching egg in the batch, which has a hatch time of zero. The initial mass was taken within 12 hours following oviposition. Heavier eggs tended to hatch sooner than lighter eggs, though clearly the trend has exceptions. ................................................................................ 1 18
... Vl l l
List of Tables
Table 2-1 Taxa that produce trophic eggs, grouped by the behavioural evidence that trophic egg laying is adaptive ...................................................................................... 24
Table 2-2 Hypotheses regarding the function of trophic eggs and predictions of ecological factors or species traits that should be correlated with trophic egg laying. Hypotheses and predictions are grouped by whether they consider trophic eggs a tactic for reducing sibling cannibalism or provisioning offspring. ..................................................................................................................... 2 7
Table 2-3 Evidence required to distinguish among hypotheses of trophic egg laying: whether trophic eggs are produced by non-evolved infertility or by maternal adaptation (constraint vs. adaptive hypotheses); and, if adaptive, whether trophic eggs function to reduce parent-offspring conflict over sibling cannibalism or to provision offspring (conflict reduction vs. provisioning hypotheses). Lines of evidence are grouped by how they are obtained: through observation (natural history), behavioural studies, or comparative analyses. Evidence may be 'consistent' (if it is not predicted by and does disprove a hypothesis), 'necessary but not sufficient' (if the evidence is not found, the hypothesis cannot be true, but finding the evidence does not mean the hypothesis is correct), or strongly or weakly supportive or falsifying. If investigators cannot obtain falsifying or strongly supportive evidence, the next best option is to collect several lines of weakly falsifying or supporting evidence. The framework is developed for the scenario of one trophic egg- eating or cannibalising event, but the qualitative predictions are general to the case where more than one egg or sibling is eaten. ....................................................... 29
Table 3-1 The range in hatching synchrony phenotypes, in terms of number of offspring hatching in each time period for each allele, that were tested in simulations. Mothers with each non-binary allele value produce clutches with the temporal hatching pattern indicated in the table. ........................................................................ 65
Table 3-2 Conditions for each set of simulations of the evolution of sibling cannibalism and maternal traits that influence cannibalism: the range of relative benefits that cannibal offspring receive; whether infertile eggs are present or absent; whether cannibalism is time-limited; and the presence of constraints, if any, on the evolution of any of the tested traits ................................................................... 67
Table 3-3 The effect of the relative benefit of egg eating, infertile eggs, and a time extension (whereby offspring could wait for viable siblings to hatch before eating eggs) on the evolution of the three genes .......................................................... 69
Table 4-1 Comparison of protocols for the internal and external cue experiments ...................... 93
Table 4-2 Effect of food level on proportion of trophic eggs and three measures of hatching synchrony (see text) when females were given information about food conditions through feeding (internal cue) or other senses (external cue). Sample sizes were: in the internal cue experiment, 7 1 for the proportion of
trophic eggs and 55 for hatching synchrony; for the external cue experiment, 38 for the proportion of trophic eggs and 17 for hatching synchrony .......................... 95
Table 4-3 Randomization tests for the spatial distribution of trophic eggs within egg batches produced by different females. P values were generated from simulations of possible distributions, given the spatial arrangement of each batch; values less than 0.025 indicate a clumped distribution while values greater than 0.975 indicate a uniform distribution. ...................................................... 97
Table 4-4 Runs tests (Zar 1999) of the temporal distribution of infertile eggs within oviposition sequences of different females .................................................................. 99
Table I- 1 The evidence, across studies, that female ladybirds adjust trophic egg production depending on male-killing infection status. The expected proportion hatch is a weighted mean proportion of hatching eggs from uninfected lines, based on data reported in each study. The observed proportion is also a weighted mean. The null hypothesis is that the observed hatching proportion does not differ from half of the hatching proportion in uninfected lines. If females can control the production of trophic eggs, infected females may benefit from reducing trophic egg production because hatching larvae will have dead male eggs to eat. When the lower limit of the 95% CI of the observed proportion exceeds the expected proportion, the evidence is taken to support the alternate hypothesis. ............................................... 120
Table 1-2 Evidence that females control trophic egg production from studies in which male-killing (MK) infected ladybirds were cured with antibiotic. The expected proportions for MK-infected lines before antibiotic treatment are calculated as half of the proportion of hatching eggs after antibiotic treatment. If the observed proportion is greater than the expected proportion, females appear to reduce trophic egg production when infected. For lines that were uninfected before treatment, the expected hatching proportion is the proportion observed following antibiotic treatment. If the observed proportion is greater than the expected, the antibiotic causes some reduction in trophic egg production regardless of MK infection status. .................................................... 122
Chapter 1 Overview of the Thesis
1.1 Overview
In some species, mothers produce non-developing trophic eggs that are consumed by other
offspring (Crespi 1992). This trophic egg-laying behaviour - noted across diverse taxa - has
rarely been studied in an evolutionary ecology context. In any given species, trophic eggs may
function to reduce parent-offspring conflict over sibling cannibalism or to reduce offspring
starvation risk by providing an easy meal for neonates. Trophic egg-laying, then, is one of several
parental tactics that could accomplish these functions, such as parental manipulation of clutch
size, egg size, or hatching synchrony.
The goals of this thesis were four-fold:
To organize hypotheses about the ecology and evolution of trophic egg-laying into a
behavioural ecology framework;
To model the co-evolution of maternal and offspring behaviours related to trophic egg-
laying;
To investigate the adaptive function of trophic eggs in the ladybird Harmonia axyridis;
To test for the use of a functionally equivalent tactic, hatching synchrony, in the same
species.
This chapter summarizes the contents of the thesis. It does not provide a general
introduction to trophic eggs; the literature review in Chapter 2 serves that function. Chapters 2, 3
and 4 were written as independent manuscripts. Chapter 5 suggests questions that should be
addressed in future work. Appendix I presents additional results obtained during this thesis
research.
In Chapter 2, I organize the hypotheses for the function of trophic egg into a framework for
their study. I identify two categories of hypotheses, grouped by the suggested adaptive function of
trophic eggs: to provision offspring or to reduce parent-offspring conflict over sibling
cannibalism. Another research objective should be to determine whether trophic eggs have an
adaptive function at all or simply represent infertility. I review the evidence necessary to
distinguish among the hypotheses, concluding that much evidence hitherto collected lacks power
to distinguish hypotheses, and suggest approaches for future work.
Chapter 3 investigates predicted patterns of co-evolution for three maternal and offspring
traits: trophic egg production, hatching synchrony, and sibling cannibalism. I use stochastic
simulations with a genetic algorithm component. Genetic algorithm-based models are a useful
approach for studying complex, multi-trait systems with game theoretic aspects (Forrest 1993). I
compare the results from the co-evolving parent-offspring game to the predictions of a frequency-
independent inclusive fitness analysis based on Eickwort (1973). I also analyze the effect of
relative versus absolute starvation risk, the presence of infertile eggs, and time limitations on
cannibalism behaviour.
The objective of the laboratory experiments with H. anyridis, presented in Chapter 4, was
to determine whether trophic eggs are adaptively produced. I tested for phenotypic plasticity in
trophic egg production, with the prediction that females would increase the proportion of trophic
eggs per clutch in low food conditions where offspring starvation risk was greater. Because
females might benefit from a uniform distribution of trophic eggs within a clutch (if it is then
more likely that offspring consume trophic eggs instead of viable siblings), I assessed whether the
distribution of trophic eggs within clutches and oviposition sequences differed from random. I
also tested whether females manipulated hatching synchrony to adjust the opportunity for
cannibalism among offspring. Results demonstrated that the infertile eggs of H. axyridis are
adaptively produced to increase the chance that offspring survive starvation.
The final chapter suggests questions that should be addressed in future trophic egg
research.
1.2 Literature Cited
Crespi, B. J. 1992. Cannibalism and trophic eggs in subsocial and eusocial insects. Pages 176-213 in M. A. Elgar, and B. J. Crespi, eds. Cannibalism: ecology and evolution among diverse taxa. Oxford University Press, Oxford.
Eickwort, K. R. 1973. Cannibalism and kin selection in Labidomera clivicollis (Coleoptera: Chrysomelidae). American Naturalist 107:452-453.
Forrest, S. 1993. Genetic algorithms: principles of natural selection applied to computation. Science 261 :872-878.
Chapter 2 The Behavioural Ecology of Trophic Egg Laying:
Hypotheses and Evidence
2.1 Abstract
Trophic eggs are non-hatching eggs that are consumed by offspring. They are produced in
diverse taxa with a wide variety of parental care systems. Mothers may lay trophic eggs to reduce
cannibalism among offspring or to provision offspring. In this review, we draw attention to two
sets of trophic egg hypotheses: whether trophic eggs represent unavoidable infertility or a
maternal adaptation; and, if an adaptation, whether they are a tactic for the reduction of sibling
cannibalism or starvation risk. We review an inclusive fitness model to illustrate that the
hypotheses are mutually exclusive in any given system, though each may explain trophic egg
laying in some animals. We outline the types of evidence useful in distinguishing the hypotheses
and highlight challenges for future research on trophic egg laying behaviour. Because trophic
eggs are related to parental care, parent-offspring conflict, inclusive fitness, and sibling rivalry,
they have the potential to become a useful research topic in behavioural ecology with further
study.
2.2 Introduction
In considering sibling interactions across taxa, an interesting pattern becomes apparent: not
infrequently, individuals make meals of their siblings - thereby eliminating some of their closest
relatives. In a specialized behaviour related to such sibling consumption, mothers provide
offspring with trophic eggs to eat (Table 2.1). Crespi (1992) provided the first adaptive synthesis
of trophic egg laying behaviour, defining trophic eggs as a maternal adaptation of "ovarian-
derived structures or fluid, homologous to fertile eggs, that cannot develop into viable offspring
and are normally eaten" (p. 176). Thus, trophic eggs are not simply infertile or malformed eggs
produced under some constraint - for example, sperm limitation or gamete incompatibility - even
if those eggs are consumed by offspring and even if egg-eating offspring derive great benefit.
Furthermore, the definition illustrates a central question for trophic egg research: it is necessary to
demonstrate the maternal control of trophic egg production, in order to distinguish infertile and
trophic eggs.
A wide variety of invertebrate and vertebrate taxa produce trophic eggs (Table 2.1). They
occur in animals with diverse parental care systems, ranging from no care to matriphagy (wherein
mothers are devoured by offspring), and may occur with or without additional cannibalism of
viable siblings. Table 2.1 creates two categories of trophic egg-laying animals. The first
comprises animals with limited parental care in which trophic eggs are morphologically indistinct
from viable eggs. In these systems, the crucial question is whether a maternal adaptation exists, or
whether offspring simply cannibalize infertile eggs without help from their mother. In the second
group, trophic eggs are morphologically different from other eggs, seemingly specialized to
offspring needs, or they occur along with extensive parental care behaviours. Here it seems
apparent that trophic eggs are adaptively produced, because mothers appear to make a special
effort in their production or distribution to offspring (but see discussion under 'Evidence').
Researchers can therefore proceed directly to determining the adaptive function of trophic eggs.
This review explores functional hypotheses for the production of trophic eggs in non-
eusocial taxa. Eusocial insects are excluded despite the extensive literature from these animals
(reviewed in Crespi 1992; see also Gobin et al. 1998; Masuko 2003). For example, a search for
the keyword 'trophic eggs' in biological studies from 1969-2004 returned 54 articles, 45 of these
in eusocial insects. They are not considered here because there are unique selective forces driving
the evolution of trophic eggs in eusocial taxa (see Crespi 1992), making their function and
evolution a separate topic.
Our discussion begins with an inclusive fitness model of mother and offspring interests in
egg cannibalism. We use the model to categorize hypotheses of the function of trophic egg
laying. Next, we comment on the evidence required to distinguish the adaptive vs. constraint and
conflict vs. cooperation hypotheses, and discuss noteworthy examples of current empirical
approaches. The review concludes with challenges for future studies of trophic eggs and
offspring.
We refer to non-evolved infertile eggs produced due to constraint as infertile eggs.
However, in many instances we refer to consumed eggs as trophic eggs for cases in which their
adaptive nature has not been demonstrated, acknowledging here that until the relevant evidence is
collected their 'trophic' status is unknown. As a shorthand, we refer to offspring feeding on
trophic eggs as cannibalism.
2.3 Parent and offspring interests: inclusive fitness approach
Trophic egg production is necessarily tied to the offspring behaviour of eating eggs. A
useful first step, then, is to consider when egg-eating - equivalently, sibling cannibalism - is
adaptive from mother and offspring perspectives. Eickwort (1 973) outlined the inclusive fitness
conditions under which offspring should eat siblings. Summarizing the argument (see also
Chapter 3), allow p to be the probability of survival of an offspring that does not consume an egg.
An egg-eating offspring receives an increase in survival, p', through reduced starvation risk, and
has a total chance of survival (p + p'). By re-stating Hamilton's Rule (Hamilton 1964) for the
spread of a selfish act (Mock and Parker 1997), sibling cannibalism is favoured when the relative
benefit to the cannibal exceeds the cost to the victim, weighted by relatedness - that is, when p' >
pr, which can be restated as
(P'JP) > r (1)
For an individual to benefit from eating a full sibling (r = OS), that individual must
increase its probability of survival by 50% or greater. Note the constraint that (p + p') 51;
combining the inequalities and solving for p yields the condition that, for offspring to benefit
from sibling cannibalism, p < l/(r+l) - else cannibalism cannot increase survival by more than
(r)%. Note also that it is the relative benefit of egg eating that matters, not the absolute increase in
survival.
The argument is easily extended to find a similar condition for parental benefit from sibling
cannibalism. Intuitively, parents benefit from sibling cannibalism when they gain more offspring
from the increased survival of cannibalism than they lose as victims (see O'Connor's (1978)
model of brood reduction). With pairwise sibling interactions (i.e., one egg or sibling per
cannibal), this occurs when
P' > P (2)
that is, when the cannibal's total survival (p + p') is at least doubled compared to a non-
cannibal offspring. The relatedness term does not occur in (2) because mothers are equally related
to cannibal and victim offspring.
Combining (1) and (2) reveals predicted conditions of agreement and conflict between
parents and offspring over sibling cannibalism (Fig. 2.1). At a relative benefit of egg-eating of
less than intra-clutch relatedness, neither offspring nor parents derive benefit from cannibalism. A
relative benefit between r and one represents conflicting interests, and parents should try to limit
cannibalism. When the relative benefit exceeds one, parents should cooperate with offspring and
adopt behaviours that facilitate cannibalism. With the pairwise nature of the modeled interaction,
the implication is that when sibling cannibalism is favoured, mothers prefer that half their
offspring consume the others!
This inclusive fitness approach can be compared with two related sets of models. The first
considers the evolution of clutch size in invertebrates in which offspring engage in sibling
cannibalism (e.g., Godfray 1987; Parker and Mock 1987; Mock and Parker 1997). These models
use different assumptions than the inclusive fitness approach (i.e., they assume declining per
capita offspring fitness with increasing clutch size, that cannibals benefit from reduced
competition rather than direct nutritional gain, and that offspring do not kill as many siblings as
opportunity permits). Furthermore, they consider a different maternal oviposition decision -
number of offspring per clutch - and it is not yet clear how trophic egg laying and clutch size
decisions interact. A second set of related models is the brood reduction models developed since
O'Connor (1 978; see Godfray and Harper 1990; Forbes 1993; Mock and Parker 1997). They
identify several important factors for maternal brood reduction decisions: how resources are
divided after siblicidal brood reduction (whether siblicidal offspring benefit from reduced
competition) and whether victims are random or pre-selected 'runts' (which affects how easily
sibling cannibalism genes can invade). While factors like these can change the predicted optimal
sibling cannibalism behaviour, the above analysis is valid as a basic model and its assumptions
(of pairwise sibling interactions; that the benefit of siblicide accrues only to the cannibal; random
selection of victims; flat clutch size-per capita fitness curve) may be adjusted to develop the
approach further.
2.4 Two sets of trophic egg hypotheses
We draw attention to two questions that must be addressed in any adaptive investigation of
trophic egg laying behaviour: is a maternal adaptation present and if so, what is the adaptive
function? We consider an adaptation to be a phenotype modified by selection to serve some
function; adaptations are detectable as the phenotype that yields the highest fitness from a set of
actual and possible alternatives in a specified environment (Reeve and Sherman 1993), and this
often illuminates the function of that phenotype (Thornhill 1990).
The first distinction is one of 'adaptation versus constraint', with the constraint hypothesis
suggesting that offspring consume infertile or damaged eggs, produced because egg development
and oviposition are imperfect processes. The offspring behaviour of eating these structures may
be an adaptation - and indeed, that is the assumption - but in refemng to the infertile eggs as
trophic eggs, we imply maternal control of their production. It is therefore necessary to eliminate
the hypothesis of non-adaptation (alternatively phrased, the hypothesis of offspring adaptation
alone). We return to this point in the section 'Evidence'.
If trophic egg laying is an adaptation, we can organize hypotheses about the function of
trophic eggs using the above dichotomy of parent-offspring cooperation and conflict. We
elucidate these hypotheses in the following section, then discuss predictions derived from them
(summarized in Table 2.2). We then consider evidence that may be useful in distinguishing the
predictions. A point to stress is that the cooperation and conflict reduction are not competing
hypotheses. While they are mutually exclusive for any given animal (because the relative benefits
of egg eating are either greater than or less than the benefit threshold for parents, Fig. 2. l), and
we might be interested in distinguishing them for a given animal, both are likely true for some
taxa.
The parent-offspring conflict reduction hypothesis
Crespi (1992) posited an important addition to Alexander's (1974) hypothesis that trophic
eggs are an adaptive maternal behaviour: in some systems, parents may use some offspring as
food for others, not as a provisioning tactic per se but to reduce the cannibalism of viable siblings
when it is against a mother's interests (Fig. 2.1). When mothers benefit from preventing
cannibalism among offspring, a possible tactic would be to provide offspring with trophic eggs
that provide sufficient energy to potential cannibals such that they avoid eating siblings (Crespi
1992). A reduction in cannibalism might occur even without an offspring preference for trophic
over viable eggs, if the presence of trophic eggs makes it less likely that cannibals will happen
upon, and consume, viable siblings.
A necessary condition for the hypothesis is that trophic eggs are less costly to produce than
viable eggs (Crespi 1992). If trophic eggs are equally or more costly to produce than viable eggs,
producing trophic eggs is equivalent to a mother eliminating a viable offspring instead of a
cannibal sibling, since she could have used the resources to create a viable offspring. Moreover,
trophic eggs may have to be substantially less costly to make their production worthwhile for all
but very large clutch sizes. And if trophic eggs are less costly - and thus, presumably, contain
fewer complex lipids and proteins - offspring are likely to prefer viable to trophic eggs, since
viable eggs would provide greater survival benefit (and, presumably, RBE exceeds the benefit
threshold for offspring, if mothers are producing trophic eggs). It may be difficult for mothers to
strike a balance between making a cost-effective trophic structure and making a nutritious enough
structure that offspring relinquish eating their siblings. Modelling is needed in this area.
Crespi (1992) made two predictions for systems in which trophic eggs function to reduce
conflict. Though not major points within Crespi's review of trophic eggs, they represent the only
evolutionary discussion of this hypothesis in the literature and, thus, are noteworthy here.
(1) Trophic eggs will occur more frequently in monogamous species with high within-
clutch relatedness, that is, when offspring benefit less from eating siblings. It may then be easier
for mothers to 'persuade' offspring to avoid eating viable siblings. The opposite pattern might
also occur - trophic egg laying being more common in polygamous than monogamous species -
because with lower relatedness between siblings, the range of benefits from egg eating for which
parents and offspring disagree is greater (Fig. 2.1). Parent-offspring conflict over sibling
cannibalism should, then, be more extensive, and conflict reduction may be a greater selective
pressure on mothers than in monogamous species.
(2) If trophic egg production is at an optimum for both mothers and offspring, there will be
no cannibalism of viable siblings. Co-evolutionary models of conflict reduction via trophic egg-
laying are required to predict whether a dual optimum would be evolutionarily stable.
Cooperation hypothesis
Alexander (1974) suggested that in some taxa, parents use some offspring as a food source
for others. With the conceptual model of Figure 2. I , mothers benefit from sibling cannibalism
when the relative benefit of egg-eating is greater than one. A solution is to neglect to fertilize
some eggs, effectively making them into meals for developed offspring. Under the cooperation
hypothesis, trophic eggs may be as costly as viable eggs, or potentially more costly if they are
specialized to the needs of cannibal offspring. We also refer to the cooperation hypothesis as the
'maternal provisioning hypothesis'; although trophic eggs provision offspring under the conflict
reduction hypothesis as well, in that case provisioning is the proximate, not the ultimate, selective
pressure.
Several extensions of the cooperation hypothesis have been made (Table 2.2).
Trophic eggs and ecological variables
Two hypotheses relate trophic eggs to features of the environment. The first is the widely
cited icebox effect (a concept from Alexander, 1974; dubbed the icebox hypothesis by Polis
198 I), which predicts that trophic egg laying should occur frequently in taxa that experience high
food availability during offspring production, but low food conditions during offspring
provisioning or foraging (Table 2.2). Then trophic offspring are a stable food storage solution for
the core brood. A second pair of hypotheses links the production of trophic eggs to poor food
environments. When the mean starvation risk experienced by offspring is high, but parents have
limited information about future resources, mothers should produce trophic eggs as a bet-hedging
strategy (Osawa 1992; Mock and Parker 1997; Dixon 2000). If the production of trophic eggs is
facultative, then we can also expect to see trophic eggs used in predictable environments: parents
should adjust the proportion of trophic eggs to match the resource conditions offspring will
experience (Chapter 4; Frechette and Coderre 2000; Osawa 2003).
Trophic eggs and species characteristics
Three hypotheses comment on how trophic eggs should be related to other characteristics
of species (Table 2.2).
1) Constraints on other tactics. Because trophic egg production may be called an extreme
provisioning strategy, it may be more likely to evolve when other possible provisioning tactics
are constrained (Alexander 1974; Polis 198 1 ; Polis 1984; Crespi 1992). For example, parents
might provision offspring by creating larger eggs (Polis 198 1 ; Polis 1984; Crespi 1992; Mock and
Parker 1997). Such a strategy would reduce the loss of energy associated with digestion of trophic
eggs (Dixon 2000). However, studies in a variety of taxa suggest that egg size is often fixed (e.g.,
Dixon and Guo 1993; Honek 1993; Christians 2002) - constrained, perhaps, by ovariole size,
which is constrained by body size and the tradeoff between ovariole size and number. There
would then be an advantage to trophic egg laying if it is adjustable between environments.
2) Protein limitation. Because siblings are proteinaceous food items (Polis 1984), trophic
eggs may provide a greater relative benefit if the diet is protein limited, as in herbivorous animals
and the ladybird and lacewing predators of aphids (e.g., Cohen and Brummett 1997; Specty et al.
1999). While carnivorous animals are also protein-limited when prey are not accessible, this
hypothesis states that species with an especially protein limited diet are more likely to evolve
trophic egg provisioning tactics.
3) Differential costs of foraging. Mothers may provide trophic eggs when it is easier
(Nakahira 1994; Kim and Roland 2000) or less risky (McKaye 1986) for mothers to forage than it
is for offspring. Mothers might forage before offspring require food (and store resources in their
bodies to produce trophic eggs later) or during offspring feeding (and produce trophic eggs if, for
example, it is difficult for offspring to consume cumbersome prey items, e.g. Masuko 2003).
However, it is likely that parents generally have lower foraging costs than neonate offspring, so
this hypothesis may not have much power to predict which species trophic eggs will occur in.
2.5 What evidence is required to distinguish hypotheses?
In Table 2.3, we summarize the evidence that may be useful in distinguishing ( I ) trophic
eggs from non-evolved infertility (the adaptive vs. constraint hypotheses), and (2) conflict vs.
cooperation hypotheses. We now turn to justifying our conclusions on the value of the evidence.
Evidence from natural history and behavioural observations
For some animals, the behavioural or life history context in which trophic eggs occur
suggests that they are produced by adaptation: it indicates a level of complexity that is not
expected by chance alone (Williams 1966). For example, in some species trophic eggs are clearly
different from other eggs in shape or size, implying that they are specialized to offspring needs
(Table 2.1, 2.3). A maternal adaptation is also implied when mothers repeatedly bring trophic
eggs to offspring; for example, in some tropical frogs, tadpoles live in small closed water bodies
and mothers return multiple times to lay trophic eggs, apparently the only food source for
offspring (Table 2.1). If a vast number of infertile eggs are provided (e.g., tens of thousands in
sand sharks, Duellman and Trueb 1986; Dopazo and Alberch 1994), adaptation is also implied
because a clearly complex maternal phenotype exists. However, mothers may deliver infertile
eggs to offspring; then the adaptation is the act of bringing the infertile material to offspring
rather thanproducing a trophic egg. This may occur if, for example, the level of non-evolved
infertility is very high; e.g., in the coronated treefrog Anotheca spinosa, 90% of apparently
fertilized eggs do not develop (Jungfer 1996; Table 2.1). It also seems reasonable to surmise that
trophic eggs are an adaptation when they occur with other extreme offspring provisioning
behaviours (e.g., matriphagy in some spiders, Table 2.1); however, it is still possible that the
adaptation is offspring consumption of infertile eggs and not maternal production of trophic eggs.
Behavioural observations may also contribute to distinguishing the conflict reduction vs.
cooperation adaptive hypotheses. If trophic eggs occur with other complex provisioning
behaviours (such as extended parental care, repeated visits to offspring, or matriphagy), or if
other cannibalism-influencing tactics are constrained, there is weak support for the provisioning
hypothesis. If cannibalism of viable siblings is frequently noted, there may be weak evidence
against the conflict-reduction hypothesis (Crespi 1992; but see comments under discussion of that
hypothesis above).
Evidence from behavioural and life history experiments
Four lines of evidence may be collected from trophic egg behavioural studies (Table 2.3):
the increased survival offspring experience from eating eggs; whether the proportion of trophic
eggs changes with starvation risk; maternal investment into trophic eggs (relative to viable eggs);
and the spatial distribution (random or non-random) of trophic eggs within clutches.
We separate the evidence that offspring benefit from eating eggs at all from quantitative
estimates of that benefit to call attention to the need for quantification in order to draw inferences
(Table 2.3). If benefits are quantified, one or the other adaptive hypotheses (of parent-offspring
conflict or cooperation) may be eliminated. However, falsifying one adaptive hypothesis does not
imply that the other adaptive hypothesis is unequivocally supported, because the observed level
of benefits might occur by coincidence. That is, a measurement of positive benefits does not show
that mothers use any tactic to facilitate egg eating or cannibalism (a fact not always recognized in
empirical studies; e g , Osawa 1992; Dixon 2000; Kudo and Nakahira 2001; Osawa 2002; Osawa
2003). Obtaining relevant quantitative estimates of the relative benefit of egg eating may be
challenging: the conditions under which trophic egg laying evolved may differ from average
conditions today, and offspring starvation risk is likely to vary temporally and spatially. Still, if,
for example, relative benefits of egg-eating are less than one in a large number of studies under a
wide range of conditions, it seems reasonable to conclude that reducing sibling cannibalism was a
selective factor in the evolution of trophic eggs in the study animal. Furthermore, if inferences are
to be drawn about the adaptive nature of consuming potentially viable siblings from an
offspring's perspective, relatedness within clutches must be estimated (to use equation (1)).
A demonstration that mothers adjust the proportion of trophic eggs to match offspring
starvation risk would be strong evidence for the cooperation hypothesis (Chapter 4; Frechette and
Coderre 2000; Osawa 2003). However, sibling cannibalism might be more likely in low food
environments; then mothers might increase the proportion of trophic eggs as a conflict reduction
strategy. This concern arises only if offspring assess food levels before eating siblings (e.g.,
Nakahira 1994; Evans et al. 1995); in many animals offspring have no information about
resources before sibling cannibalism (e.g., in many insects, Dickinson 1992; Desbuquois et al.
2000; Frechette and Coderre 2000). A second experimental difficulty is to separate the effects of
information and physiology - to give females information that they their offspring will face a
poor food environment without worsening their physiological state (Chapter 4). Some studies
have found no difference in the proportion of infertile eggs produced in natural habitats that
differed in offspring starvation risk (Dickinson 1992; Baur and Baur 1998) or in different
laboratory conditions (Frechette and Coderre 2000). However, in Chapter 4 I found that the
ladybird beetle Harmonia axyridis increased the proportion of infertile eggs produced in high
starvation risk conditions; I attempted to control for the confound between nutrition and
information.
Quantifying the relative cost of trophic eggs, a measurement that has not been conducted in
any study of trophic eggs, may help in distinguishing the hypotheses. An infertile egg produced
by constraint may fail to develop because it is unfertilized (and, then, should have equal cost as a
viable egg if sperm is not limiting) or because it receives too little yolk material. If there are few
infertile eggs, their reduced production cost is not sufficient evidence for an adaptive function.
However, accidental under-provisioning is probably infrequent, so if there are many of these
eggs, the constraint hypothesis is rejected and there is weak support for the conflict reduction
hypothesis. If trophic and viable eggs are equally costly to produce, conflict reduction cannot be
driving their production; they may be produced by constraint or to provision offspring. Finally,
the result that trophic eggs are more metabolically expensive to produce than viable eggs -that
they are specialized to offspring needs - can only mean that the cooperation hypothesis drives
their production in the system of study. An experimental challenge, however, is to determine
whether trophic eggs are, in total, more or less expensive to produce. For example, even if
evolved to reduce conflict over cannibalism, trophic eggs may secondarily become specialized to
offspring nutritional needs and may contain more proteins or lipids than other eggs. They might
still have lower total cost if they are smaller.
It may be advantageous to mothers to distribute trophic eggs uniformly within clutches, if
this maximizes the probability that cannibal offspring consume trophic rather than viable eggs
(Chapter 4). Thus, an over-dispersed spatial distribution of trophic eggs would indicate adaptive
production, without distinguishing between the adaptive hypotheses. A random spatial
distribution would not indicate non-adaptive production - it might simply mean that the
distribution of trophic eggs is not important for provisioning offspring or limiting sibling
cannibalism.
In general, experimenters should aim to demonstrate maternal control of trophic egg
production, because such a result provides strong support for the adaptive hypothesis. For
example, experiments in the white-eyed spider (Amaurobius ferox) show that females, after
laying a clutch of fertile eggs, produce a second clutch of trophic eggs if stimulated by the
presence of offspring; but if offspring are removed, the second clutch is also composed of fertile
eggs (Kim and Roland 2000; Kim and Horel2000). The result demonstrates that mothers are not
constrained to producing infertile eggs in this species.
Comparative analyses
Several comparative results may establish the adaptive nature of trophic egg production
(Table 2.2,2.3). For example, a straightforward test within any group of homogeneous animals
(e.g., the coccinellid beetles) would be to compare the percentage of infertility in species that lay
eggs in batches and that exhibit sibling cannibalism to the nearest relative species that does not. If
infertility is higher for species in which offspring consume the eggs, then some portion of that
infertility is due to an evolved response in mothers. These results would, furthermore, establish
cooperation as the functional explanation for trophic eggs - unless, as noted above for the
plasticity hypothesis, sibling cannibalism is also related to those predictive variables and mothers
lay trophic eggs to limit that cannibalism response. Researchers must take care that sibling
cannibalism tendencies are not a confounding factor in such studies. Another consideration is that
there are probably different selective forces driving the evolution of trophic eggs in different
groups. For this reason, it will be best to choose a homogenous group of trophic egg laying
animals for a comparative analysis (e.g., tropical frogs or aphidophagous insects; Table 2.1).
There have been no cross-taxa studies of trophic egg laying to date.
2.6 Challenges for future research
Future empirical studies on trophic egg laying animals should focus on determining
whether a) trophic eggs are a maternal adaptation and b) whether the functional explanation is an
attempt to reduce sibling cannibalism or an attempt to ensure that egg-eating among offspring
occurs. The above text highlights some empirical and theoretical challenges in collecting
evidence about the adaptive value of trophic eggs.
Beyond studies of particular species, there is a need to set trophic egg laying behaviour in
the context of other parental care, sibling rivalry, and provisioning theory. Three areas of research
will facilitate this process.
First, there is a great need for theoretical studies. Currently, models of sibling cannibalism
must be co-opted for trophic egg questions (but see Chapter 3). Theoretical questions include
implications of a decline in per capita offspring fitness with increasing clutch size on the
inclusive fitness model outlined here. It is also not clear how the cost of trophic eggs affect their
use in facilitating or limiting cannibalism: how cheap do trophic eggs have to be before they can
be used efficiently to reduce sibling cannibalism?
Second, work is needed in considering how trophic eggs may have originated and evolved.
Before a maternal adaptation of producing trophic eggs could arise, a necessary pre-adaptation is
that offspring consume material in or near the egg batch. Egg cannibalism by offspring might
arise to exploit (1) infertile or inviable eggs, or (2) viable siblings with delayed hatch (Crespi
1992).1n (I), mothers might have then been selected to increase the production of infertile eggs.
However, evolving intentional infertility may come with imperfect control of infertility, and may
be selected against because it would sometimes lead to the creation of more infertile eggs than
would be adaptive for mothers (in the same way that hosts of cuckoo brood parasites may accept
cuckoo eggs because rejection of parasite eggs comes with the risk of rejecting host eggs as well;
Lotem et a1 1995). However, it may also be possible for mothers to increase infertility without
frequent mistakes causing excessive levels of infertility. Furthermore, even if occasionally more
infertile eggs are made, the fitness cost of excess infertility may not be high, because if mothers
are creating infertile eggs it implies that the relative benefit to offspring exceeds 100%.
In (2), trophic egg laying might arise to enhance sibling cannibalism or to limit cannibalism
of viable offspring. Phylogenetic analyses within homogeneous groups in which trophic eggs may
presumably serve the same adaptive function (e.g., tropical frogs; see Weygoldt 1980) may help
uncover how trophic egg laying arises. It may be possible to map the relevant traits - presence of
infertile or inviable eggs, sibling cannibalism of eggs with delayed hatch, and trophic egg laying
- to a phylogenetic tree. If there is enough variation in the traits, it may be possible to determine
the sequence of origin of each.
Third, to understand the occurrence of trophic eggs across taxa, it will be necessary to
predict when trophic eggs should be employed rather than other alternative tactics that facilitate
or limit sibling cannibalism (such as clutch size, egg size, managing cannibalism of viable
offspring (see 'Cooperation'; reviewed by Crespi 1992), and parental care). Furthermore, we can
predict the use of particular tactics in species depending on environmental condition. If parents in
some taxa face environments that are homogeneous in starvation risk for offspring, then an
(apparently) inflexible trait such as egg size may be used, and we should expect egg size to be
negatively linked to starvation risk across taxa. On the other hand, many species will encounter
environments that vary in offspring starvation risk, either across clutches or across parents. If
variation in offspring starvation risk is unpredictable, we suggest that parents will often
manipulate clutch size to mitigate offspring starvation risk (e.g., to produce insurance offspring).
If risk is predictable, a solution is to use potentially plastic strategies, such as hatching synchrony
and trophic eggs, that can be adjusted between environments that differ in risk. Finally, we may
consider trophic eggs a subset of the larger adaptation of producing trophic offspring, including
viable eggs that could have hatched if not eaten and hatched offspring that are killed and
cannibalized.
2.7 Conclusions
Alexander (1974) proposed that trophic eggs are an example of parental manipulation of
offspring - that parents use some offspring as meals for others. Parents may do so to reduce
offspring starvation risk or to reduce cannibalism among offspring. These two adaptive
possibilities, parent-offspring cooperation or conflict, must be tested against the hypothesis that
trophic eggs are produced by unavoidable constraint. Distinguishing among the hypotheses may
be difficult in practice, because much evidence that is related to trophic egg laying and egg
consumption by offspring cannot falsify any hypotheses.
To establish that trophic egg production is an adaptation, researchers should focus on
collecting evidence that falsifies the constraint hypothesis (Table 2.3). Such evidence would
include observations of the morphology of trophic eggs or the associated maternal care, or the
plastic adjustment of trophic egg levels between environments that differ in starvation risk. To
determine the adaptive explanation for trophic egg laying, studies should concentrate on evidence
that can distinguish between the two right-most columns of Table 2.3. The strongest evidence
would be a reaction norm of trophic egg production along an offspring starvation risk gradient, a
demonstration that trophic eggs are more metabolically costly than normal eggs, or a comparative
study relating trophic eggs to predicted variables. In the absence of strong evidence, researchers
should attempt to collect several lines of equivocal evidence supporting one or the other
hypotheses, preferably with evidence falsifying the other. We emphasize the mutual exclusivity
of the conflict vs. cooperation hypotheses in any particular system, though both may explain
trophic eggs in some systems. Awareness of this incompatibility has not been apparent in the
literature (e.g., Nakahira 1994; Kim and Roland 2000; Kudo and Nakahira 2001).
Trophic eggs are rarely treated in a functional context, yet they have the potential to
illustrate and test basic behavioural ecological ideas. There is a great need for theoretical work
and cross-species phylogenetic analyses in this area. Hypotheses for trophic egg laying behaviour
relate to some of the most important topics in behavioural ecology: inclusive fitness, parent-
offspring conflict, sibling rivalry and parental care. They are also an interesting example of an
extreme and complex parental care behaviour - sacrificing potentially viable offspring to improve
the quality of other offspring. Because trophic eggs occur in animals of diverse ecologies and life
histories (Table 2. l), and because there are several alternative parental care tactics that may
accomplish the same end (discussed under 'Challenges'), investigators can both identify and test
predictions about the ecological factors related to trophic egg laying.
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Figure 2-1 The inclusive fitness interests in sibling cannibalism of a parent and her cannibalistic offspring; r is the coefficient of relatedness between siblings. Parent-offspring conflict or cooperation depends on the survival benefits of cannibalism, relative to survival of non- cannibalistic offspring. In the range from 0-r%, neither parents nor offspring benefit from sibling cannibalism.
Offspring . - cannibalizing
I siblings
0 - Ph r - 100% > 100% Paornts benefit
Relative increase in offs~ring survival through cannibalism cannibalism among offspring
Table 2-1 Taxa that produce trophic eggs, grouped by the behavioural evidence that trophic egg laying is adaptive
Tax
on
Can
niba
lism
of
viab
le s
iblin
gs?
Sour
ce
1. N
o cl
ear
evid
ence
that
trop
hic
eggs
are
ada
ptiv
e (n
o pa
rent
al c
are;
off
spri
ng e
at n
on-d
iffe
rent
iate
d in
fert
ile e
ggs)
M
ilkw
eed
leaf
bee
tle, L
abid
omer
a cl
ivic
ollis
G
reen
lace
win
g, C
hrys
oper
la r
ufil
abri
s C
omm
on h
ouse
spi
der,
Ach
aear
anea
te
pida
rior
um
Lan
d sn
ails
Pro
sobr
anch
gas
trop
ods
Lad
ybir
d be
etle
s, C
occi
nelli
dae
2 sp
ot la
dybi
rd, A
dalia
bip
unct
ata
Che
ilom
ense
luna
ta
1 1 sp
ot l
adyb
ird,
Coc
cine
lla
I I-p
unct
ata
7 sp
ot l
adyb
ird,
Coc
cine
lla
sept
empu
ncta
ta
Col
eom
egil
la m
acul
ata
leng
i M
ulti-
spot
ted
Asi
an la
dybi
rd, H
arm
onia
axy
ridi
s
Lioa
dali
a fl
avom
acul
ata
Pro
pyle
a 14
-pun
ctat
a
2. B
ehav
iour
al e
vide
nce
that
trop
hic
eggs
are
ad
apti
ve
Bur
row
ing
cric
ket,
Anu
rogr
yllu
s m
utic
us
Ow
lfly
, Asc
alop
tynx
furc
iger
B
urro
wer
bug
, Adm
erus
tri
gutt
ulus
Spid
ers,
Ara
neae
W
hite
-eye
d sp
ider
, Am
auro
bius
fero
x
Aus
tral
ian
soci
al c
rab
spid
er, D
iaea
erg
andr
os
Yel
low
sac
spi
der,
Chi
raca
nthi
um in
clus
um
+ + + + + + + + + + + Uni
que
mor
phol
ogy?
+ + ? +
Pare
ntal
ca
re?
+***
+ +**
+**
+
Can
niba
lism
of
viab
le s
iblin
gs?
+
Dic
kins
on 1
992
Frec
hette
and
Cod
erre
200
0 V
aler
io 1
974;
Val
erio
197
7
Bau
r 19
90; D
esbu
quoi
s an
d M
adec
19
98; D
esbu
quoi
s et
al.
2000
B
aur
1992
(rev
iew
)
Ban
ks 1
956;
Dim
etry
197
1 B
row
n 19
72
Haw
kes
and
Mar
rine
r 19
27
Ban
ks 1
956
Pien
kow
ski
1965
C
hapt
er 4
; Kaw
ai 1
978;
Osa
wa
1989
; O
saw
a 19
91 ; O
saw
a 19
92
Bro
wn
1972
B
anks
195
6
Wes
t and
Ale
xand
er 1
963
Hen
ry 1
972
Nak
ahir
a 19
94; K
udo
and
Nak
ahir
a 20
0 1
Kim
and
Rol
and
2000
; Kim
and
Hor
el
2000
E
vans
et a
l. 19
95
Peck
and
Whi
tcom
b 19
70
Tax
on
Can
niba
lism
of
viab
le s
iblin
gs?
Sour
ce
Am
phib
ians
T
ropi
cal
frog
s D
endr
obat
idae
, Man
telli
dae
and
Lep
toda
ctyl
idae
-
+ C
rum
p 19
96 (r
evie
w);
Sum
mer
s an
d A
mos
199
7; H
eyin
g 20
01; G
ibso
n an
d B
uley
200
4 H
ylid
ae
+ +
+/-
* C
rum
p 19
96 (r
evie
w);
Jun
gfer
199
6;
Tho
mps
on 1
996
Rha
coph
orid
ae
+/-
* +
Was
sers
ug e
t al.
198 1
; Ued
a 19
86;
Kam
et a
l. 19
96; K
am e
t al.
2000
Sa
lam
ande
r, S
alam
andr
a at
ra
- ? - ?
Due
llman
and
Tru
eb 1
986;
Dop
azo
and
Alb
erch
199
4 O
ther
ver
tebr
ates
A
fric
an c
atfi
sh, B
agru
s rn
erid
iona
lis
- +
? M
cKay
e 19
86
- Sa
nd ti
ger
shar
k - ?
- ? +
spri
nger
194
8; W
ourm
s et
al.
198
8 * O
ccur
s in
som
e sp
ecie
s **
Mat
riph
agy
*** M
atri
phag
y af
ter
mot
her's
dea
th
Table 2-2 Hypotheses regarding the function of trophic eggs and predictions of ecological factors or species traits that should be correlated with trophic egg laying. Hypotheses and predictions are grouped by whether they consider trophic eggs a tactic for reducing sibling cannibalism or provisioning offspring.
Hyp
othe
sisl
pred
icti
on
Sour
ce
Tax
on r
efer
red
toa
1. P
aren
t-of
fspr
ing
conf
lict r
educ
tion:
C
resp
i 19
92; s
ee a
lso
Nak
ahir
a 19
94; M
ock
and
Adm
erus
trig
uttu
lus
(Nak
ahir
a);
mot
hers
pro
duce
trop
hic
eggs
to li
mit
or
prev
ent
cann
ibal
ism
am
ong
offs
prin
g T
roph
ic e
ggs
shou
ld b
e m
ore
com
mon
in
mon
ogam
ous
spec
ies
If tr
ophi
c eg
g pr
oduc
tion
is a
t an
optim
um
for
mot
hers
and
off
spri
ng, v
iabl
e of
fspr
ing
shou
ld n
ot b
e ea
ten
2. P
aren
ts u
se s
ome
offs
prin
g as
food
for
ot
hers
, 'pr
inci
ple
of p
aren
tal m
anip
ulat
ion'
%
Tro
phic
egg
s ar
e lik
ely
whe
n.. .
T
here
is a
bund
ant
food
dur
ing
offs
prin
g cr
eatio
n bu
t lim
ited
food
dur
ing
offs
prin
g pr
ovis
ioni
ng/f
orag
ing
(ice
box
hypo
thes
is)
Pare
nts
have
no
othe
r w
ay to
con
vert
re
sour
ces
to n
ouri
shm
ent
Egg
siz
e is
infl
exib
le
Off
spri
ng s
tarv
atio
n ri
sk i
s ge
nera
lly h
igh
Off
spri
ng s
tarv
atio
n ri
sk is
unp
redi
ctab
le a
t ov
ipos
itio
n R
esou
rce
leve
ls a
re p
redi
ctab
le; t
hen
mot
hers
sho
uld
adju
st p
rodu
ctio
n of
tr
ophi
c eg
gs a
ccor
ding
ly
Off
spri
ng d
iet i
s pr
otei
n lim
ited
It i
s ea
sier
or
less
risk
y fo
r m
othe
rs to
fin
d fo
od th
an f
or o
ffsp
ring
~a
rke
r 1997
; Kud
o 20
01
~ca
nth
oso
iati
dae
(Kud
o)
Cre
spi
1992
Cre
spi
1992
Cha
pter
s 3
,4; A
lexa
nder
197
4; S
tinso
n 19
79; P
olis
E
agle
s 19
81; G
odfr
ay 1
987;
Cre
spi
1992
; Osa
wa
1992
; D
ixon
; M
ock
1994
; Moc
k an
d ~
orb
es 19
95; P
fenn
ig 1
997;
D
ixon
200
0; F
rech
ette
and
Cod
erre
200
0
Ale
xand
er 1
974;
see
als
o M
ock
and
Forb
es 1
995;
M
ock
and
Park
er 1
997
Ale
xand
er 1
974;
Pol
is 1
981;
Pol
is 1
984;
Cre
spi
1992
Poli
s 19
8 1; P
olis
198
4; C
resp
i 19
92; M
ock
and
Park
er 1
997;
Dix
on 2
000
God
fray
198
7; C
resp
i 19
92; T
hom
pson
199
6 O
saw
a 19
92; M
ock
and
Park
er 1
997;
Dix
on 2
000
Cha
pter
4; F
rech
ette
and
Cod
erre
200
0; O
saw
a 20
03
McK
aye
1986
; Cre
spi
1992
; Nak
ahir
a 19
94; K
im a
nd
Rol
and
2000
; Mas
uko
2003
(Stin
son)
; Coc
cine
llida
e (O
saw
a;
Perr
y an
d R
oitb
erg)
; C
hrys
oper
la
rufi
labr
is (F
rech
ette
and
Cod
erre
)
Coc
cine
llida
e (D
ixon
)
Tro
pica
l fro
gs (
Tho
mps
on)
Coc
cine
llida
e (O
saw
a, D
ixon
)
C. r
ufila
bris
(Fr
eche
tte a
nd C
oder
re);
C
occi
nelli
dae
(Osa
wa,
Per
ry a
nd
Roi
tber
g)
Bag
rus
mer
idio
nalis
(M
cKay
e);
Am
auro
bius
fero
x (K
im a
nd R
olan
d);
Am
blvo
~on
e silv
estr
ii (M
asuk
o)
,1
" Lis
ted
for
auth
ors
who
refe
rred
to
part
icul
ar s
peci
es; m
any
auth
ors
mad
e ge
nera
l pre
dict
ions
Table 2-3 Evidence required to distinguish among hypotheses of trophic egg laying: whether trophic eggs are produced by non-evolved infertility or by maternal adaptation (constraint vs. adaptive hypotheses); and, if adaptive, whether trophic eggs function to reduce parent-offspring conflict over sibling cannibalism or to provision offspring (conflict reduction vs. provisioning hypotheses). Lines of evidence are grouped by how they are obtained: through observation (natural history), behavioural studies, or comparative analyses. Evidence may be 'consistent' (if it is not predicted by and does disprove a hypothesis), 'necessary but not sufficient' (if the evidence is not found, the hypothesis cannot be true, but finding the evidence does not mean the hypothesis is correct), or strongly or weakly supportive or falsifying. If investigators cannot obtain falsifying or strongly supportive evidence, the next best option is to collect several lines of weakly falsifying or supporting evidence. The framework is developed for the scenario of one trophic egg-eating or cannibalising event, but the qualitative predictions are general to the case where more than one egg or sibling is eaten.
Impl
icat
ions
for
.. .
Con
stra
int
Ada
ptiv
e hy
poth
esis
hy
poth
esis
E
vide
nce
Con
flic
t red
ucti
on
Pro
visi
onin
g 1.
Nat
ural
his
tory
T
roph
ic e
ggs
mor
phol
ogic
ally
dis
tinc
t M
othe
r bri
ngs
trop
hic
eggs
to o
ffsp
ring
O
ther
com
plex
pro
visi
onin
g be
havi
ours
C
anni
bali
sm o
f vi
able
sib
ling
s fre
quen
tly
occu
rs
Oth
er p
oten
tial
tact
ics
are
cons
trai
ned
2. B
ehav
iour
al s
tudi
es
Off
spri
ng b
enef
it f
rom
eat
ing
eggs
Rel
ativ
e be
nefi
t mos
t of
ten
0-10
0%
w
Rel
ativ
e be
nefi
t mos
t of
ten
> 1
00%
0
Dir
ecti
onal
Pla
stic
ity:
mot
hers
pro
duce
trop
hic
eggs
whe
n re
lati
ve
bene
fit>
100%
, and
pro
duce
few
er w
hen
rela
tive
bene
fit<
than
100
%
Infe
rtil
e eg
gs le
ss c
ostl
y to
pro
duce
than
via
ble
eggs
In
fert
ile
eggs
equ
ally
cos
tly to
pro
duce
In
fert
ile
eggs
mor
e co
stly
to p
rodu
ce
Ada
ptiv
e sp
atia
l dis
trib
utio
n of
infe
rtil
e eg
gs
3. C
ross
-tax
a co
mpa
riso
ns
Tro
phic
egg
layi
ng re
late
d to
tem
pora
l pat
tern
of r
esou
rce
abun
danc
e
Tro
phic
egg
s m
ore
com
mon
in p
rote
in li
mite
d ta
xa
Tro
phic
egg
s m
ore
com
mon
whe
n it
is e
asie
r or
less
risk
y fo
r mot
her
Fal
sifi
es
Con
sist
ent
Fals
ifie
s*
Con
sist
ent
Wea
kly
fals
ifie
s C
onsi
sten
t C
onsi
sten
t W
eakl
y fa
lsif
ies*
Con
sist
ent
Con
sist
ent
Con
sist
ent
Nec
essa
ry b
ut n
ot s
uffi
cien
t
Con
sist
ent
Con
sist
ent
Con
sist
ent
Fal
sifi
es
Fals
ifie
s F
alsi
fies
Wea
kly
fals
ifie
s W
eak
supp
ort
Con
sist
ent
Fal
sifi
es
Fal
sifi
es
Fal
sifi
es
Fals
ifie
s*
Con
sist
ent
Fals
ifie
s F
alsi
fies
, unl
ess
rela
ted
to
incr
easi
ng si
b ca
nnib
alis
m
Fals
ifie
s* *
Fal
sifi
es, u
nles
s re
late
d to
in
crea
sing
sib
cann
ibal
ism
Fa
lsif
ies
Fal
sifi
es, u
nles
s re
late
d to
Con
sist
ent
Con
sist
ent
Wea
k su
ppor
t C
onsi
sten
t
Wea
k su
ppor
t
Nec
essa
ry b
ut n
ot
suff
icie
nt
Fal
sifi
es
Con
sist
ent
Stro
ng s
uppo
rt
Con
sist
ent
Con
sist
ent
Stro
ng s
uppo
rt
Con
sist
ent
Str
ong
evid
ence
Stro
ng e
vide
nce
Stro
ng e
vide
nce
than
off
spri
ng to
fin
d fo
od
incr
easi
ng si
b ca
nnib
alis
m
* But
see
dis
cuss
ion
of th
is p
oint
in te
xt
** B
ut m
ay m
ean
offs
prin
g ad
apta
tion
is m
ore
impo
rtan
t in
thes
e ta
xa
Chapter 3 Co-Evolution of Maternal and Offspring Effectors
of Sibling Cannibalism
3.1 Abstract
Sibling rivalry and parent-offspring conflict games are generally modelled separately, yet
their interaction determines the co-evolved outcome for many games within family units. For
example, when sibling cannibalism occurs in clutches of more than two offspring, parent-
offspring conflict or cooperation may exist over the occurrence of cannibalism, and siblings may
compete over the opportunity to cannibalize. Using a game theoretic genetic algorithm approach,
we derive optimal offspring cannibalism behaviour and parental behaviours influencing
cannibalism (hatching synchrony and trophic egg production), under a range of values for the
relative benefit of cannibalism. The results are compared to the predictions of a frequency-
independent inclusive fitness analysis that does not incorporate the parent-offspring or between
sibling games. We also model the expected effect of relative versus absolute starvation risk, the
presence of infertile eggs, and time limitations on cannibalism behaviour. The addition of a game
component to the model led to a prediction of increased cannibalism, compared to the frequency-
independent prediction, as offspring competed to eat siblings. As cannibalism among offspring
increased, mothers increased behaviours to limit cannibalism. Absolute survival probability had
no influence on offspring or parent behaviours; as predicted by the analytical model, the relative
increase in survival from cannibalism was the important parameter. When infertile eggs were
present, offspring risked eating viable siblings in competition to eat infertile eggs, enhancing
parent-offspring conflict. Allowing offspring to wait to cannibalize eggs until all viable siblings
hatched had no effect on evolved cannibalism behaviours, because it was never optimal for
offspring to wait to cannibalize (except at low levels of relative survival benefit from
cannibalism). Finally, the results demonstrate the utility of trophic egg-laying behaviour as a
maternal mechanism to promote sibling cannibalism.
3.2 Introduction
For many egg batch-laying animals, interactions among siblings early in life have
important fitness consequences (Roitberg and Mange1 1993; Mock and Forbes 1995; Mock and
Parker 1997). Siblings can be strong competitors for limited parental and environmental
resources, yet may also make important contributions to an individual's inclusive fitness. Thus,
one should observe seemingly restrained selfishness in interactions among siblings (Mock and
Parker 1997). The outcome of sibling interactions affects parental fitness, and as such, parents
also have an evolutionary stake in the extent of sibling rivalry. Depending on the relative benefit
that an offspring receives, parental traits that facilitate or limit selfish interactions among siblings
may be favoured (O'Connor 1978). Hence, the observed degree of selfishness among siblings
should reflect a web of influence, involving traits that evolve in both parents and offspring.
Sibling cannibalism represents an extreme of sibling antagonism. Cannibalistic individuals
acquire a nutritious meal and may reduce future competition, but in doing so sacrifice a close
relative. Applying Hamilton's rule (Hamilton 1967), individuals should cannibalize siblings when
their own survival is increased by a factor at least as great as the coefficient of relatedness
between siblings (Eickwort 1973). As such, cannibalism is most likely to occur if within-clutch
relatedness is low, offspring starvation risk is high, or cannibalism provides a large survival
benefit.
Parental interests in sibling cannibalism depend critically on the relative survival benefit
that accrues to cannibal offspring. Parents should use tactics that limit or facilitate cannibalism
according to whether they gain more net offspring from the increased survival of cannibals than
they lose as victims (Chapter 2, O'Connor 1978; Crespi 1992). As an example, female Harmonia
axyridis ladybirds (Coleoptera: Coccinellidae) may use two oviposition behaviours that modulate
the opportunity for cannibalism: hatch synchrony and the production of trophic eggs (non-
developing eggs that are consumed by hatching larvae).
Current understanding of siblicide as an adaptation is based on frequency independent
inclusive fitness analysis (Chapter 2, Eickwort 1973; O'Connor 1978; Pfennig 1997), or game
theoretic models of between-sibling interactions (Dickins and Clark 1987; Godfray and Harper
1990; Forbes 1993; Godfray 1995; Rodriguez-GironCs 1996; Mock and Parker 1997; Mock and
Parker 1998) or of parent-offspring conflict over clutch size (Parker and Mock 1987). In the
sibling cannibalism scenario, however, two games are played simultaneously: between siblings
over which individuals do the cannibalizing (when it is beneficial to eat a sibling; Parker and
Mock 1987), and between parents and offspring over the occurrence of cannibalism. With the
complexity of a co-evolving, multiple-trait system, it is necessary to extend the traditional game
theory approach in order to find stable optima in the large set of possible trait combinations, i.e.,
co-evolved gene complexes. A suitable approach, which we adopt here, is the use of simulation
models with a genetic algorithm search component. A genetic algorithm (GA) is a search
algorithm that finds an optimal strategy set by allowing coded alleles to compete for space on a
population genome, over many simulated generations (Goldberg 1989). The approach
approximates a series of negotiated interactions in time (McNamara et al. 1999), as opposed to
the traditional game theory method of finding the best strategy for one player given the strategies
played by others (e.g., Parker and Mock 1987). Because GAS can simulate a game over
evolutionary time, and can search a very large strategy set quickly, they are an appropriate
technique for complex co-evolutionary games (Forrest 1993; Robertson et al. 1998; Reinhold et
al. 2002).
We ask: what is the outcome of evolutionary conflict or cooperation over sibling
cannibalism between mother and offspring? Using H. axyridis as a focal taxon, we model the co-
evolution of sibling cannibalism and two maternal behaviours: hatching synchrony (whereby
mothers may limit or encourage cannibalism among offspring) and trophic egg production (a way
for mothers to facilitate egg-eating). We begin with a frequency independent, deterministic
inclusive fitness analysis considering the conditions under which cannibalism benefits mothers
and offspring. The predicted conditions are then tested in stochastic simulations of within-clutch
cannibalistic interactions, where a GA is used to find sets of stable trait values. The simulations
also model the effects of three factors: relative versus absolute starvation risk, non-evolved egg
infertility, and time limitations on cannibalism. We demonstrate that if each trait is permitted to
evolve alone (i.e., the parent-offspring game is prevented), then the results qualitatively match the
predictions of the frequency-independent model. Adding a co-evolutionary game between
mothers and offspring, however, favours increased cannibalism and maternal response to
cannibalism. The presence of non-evolved infertile eggs also drives evolution for increased
cannibalistic tendencies among offspring. We compare the results to data available for ladybirds
and discuss the model's relevance to other taxa.
3.1 Model
Biological system
We use a model organism to focus our discussion. However, the model is generally
applicable to any taxon where parent-offspring conflict and sibling competition co-occur.
Harmonia axyridis, like many coccinellids, lays eggs in batches of 10-50. During hatching,
larvae emerge asynchronously and there is the potential for egg consumption within the clutch.
Both trophic eggs and viable siblings with delayed hatch may be consumed, with substantial
fitness benefits for the cannibal (16% to 226%, Osawa 1992; reviewed by Hodek and Honek
1996). Emerged larvae are not vulnerable to cohort cannibalism. Cannibal larvae consume a
portion of an egg or an entire egg, then become quiescent for approximately 24 hours. By that
time, other eggs have generally hatched or been eaten by other cannibals, so consumption of more
than one egg per cannibal is rare. Cannibals do not appear to discriminate between viable and
infertile eggs (Banks 1956; Pienkowski 1965; Brown 1972; Osawa 1992; Majerus 1994). In H.
axyridis, 15.2% (Kawai 1978) to 24.5% (Osawa 1992) of eggs are infertile.
Inclusive fitness argument
Eickwort (1973; see also Pfennig 1997, and Chapter 2) used Hamilton's rule to investigate
when an individual should cannibalize a sibling. To review the argument, consider a non-cannibal
with a probability of survival, p. A cannibal receives an increase in survival, p', through reduced
starvation risk, and has total survival (p + p'). By re-stating Hamilton's Rule ((Hamilton 1964))
for the spread of a selfish act ((Mock and Parker 1997)), sibling cannibalism is favoured when the
relative benefit to the cannibal exceeds the cost to the victim, weighted by relatedness - that is,
when p' > pr. This can be restated as
p'/p > r (1)
For example, a cannibal must increase survival by at least 50% to profit from consuming a
full sibling. Throughout the paper, we refer to the term (p'/p) * 100 as the relative benefit of egg-
eating (ME).
A parent favours sibling cannibalism when more net offspring are gained from the
increased survival of cannibals than are lost as victims (see O'Connor's (1978) model of brood
reduction). With pairwise sibling interactions, this occurs when
P ' > P (2)
that is, parents favour cannibalism when the survival of cannibals is at least doubled
through cannibalism (when RBE > unity).
The model demonstrates that parent and offspring interests may overlap or conflict,
depending on intra-clutch relatedness and the relative increase in survival from cannibalism.
Neither parents nor offspring favour cannibalism when RBE < r. When r < M E < 1, it is in the
interests of offspring to cannibalize siblings and the interests of parents to prevent cannibalism.
When RBE > 1, parents and offspring agree that cannibalism should proceed. The model also
shows that, for sibling cannibalism to be beneficial, a non-cannibal's probability of survival must
be low. For example, if p > 213, individuals cannot be selected to eat full siblings due to the
constraint that (p' + p) 51. Similarly, survival must be less than 50% for parents to favour sibling
cannibalism, else (p'lp) cannot exceed one (Fig. 2-1).
Genetic algorithm-based simulations: the basic model
The H. axyridis-like system contains three interdependent behaviours: the maternal
behaviours that determine hatch synchrony and the proportion of trophic eggs within egg batches,
and the offspring behaviour of eating eggs within the natal clutch. In the simulations, individuals
are represented as strings of binary information that code for the three behaviours, with 16
possible alleles for each gene. Every individual is a female that possesses all three genes, one of
which is expressed as an offspring and the other two as an adult. Details of the model and
simulations are given in the chapter appendix.
Hatching synchrony phenotypes range from complete synchrony with all offspring
hatching in one time unit, to complete asynchrony with one offspring hatching per time unit
(Table 3.1). Trophic egg alleles code for the production of zero to 15 trophic eggs. The
cannibalism delay gene codes for a lag time that offspring wait after emergence before seeking an
egg to eat within their clutch. Phenotypes for the cannibalism delay gene range from waiting one
time unit afier emergence to waiting 16 time units, by which time all viable siblings have hatched
and offspring should consume any unhatched eggs (if eggs provide a survival benefit). Because
the range of alleles allowed maximal variation in phenotypes (0 to 100% trophic egg production;
0 to maximal cannibalism events permitted through hatching synchrony; minimal cannibalism
delay to long enough that viable siblings survive), we are confident that number of alleles tested
allowed fine enough resolution of phenotypes in simulations. In the simulations, loci assort
independently.
Interactions within clutches are simulated simultaneously and independently. For each
clutch, the number of trophic eggs is determined by the mother's trophic-egg-gene allele and
trophic status is assigned randomly to offspring. In each of 16 time units, offspring are hatched
according to the maternal allele for hatch synchrony (Table 3. l), with the hatching offspring
selected randomly. Offspring wait some number of time units determined by their allele for
cannibalism delay, and then consume an egg if there is one available within their clutch. When
time ends, whether live offspring survive starvation is determined by drawing a random number
for comparison to survival parameters, which differ depending on whether the offspring has
consumed an egg.
Selection in the model. Fitness is assigned indirectly: successful alleles make more copies
of themselves and are proportionally more represented in the pool of living offspring from which
the next generation's parents are randomly drawn. We do not incorporate a clutch-level selection
component (which might arise if larger surviving clutches have higher per capita survival, as in
Breden and Wade 1989) because siblings disperse from egg batches and likely have little further
interaction after sibling cannibalism occurs in H. axyridis. There is thus no basis for group
benefits in this system, though they may be important in other systems. Mating and offspring
generation are described in Appendix I. We conducted five simulation runs for each parameter set
with different random number seeds. Convergent values in different runs allow more confidence
that the GA has found a global optimum.
The simulations are used to search for co-evolutionary outcomes under a range of
ecological conditions (i.e., starvation risk and relative benefit of egg eating), considering the
effect of biological factors (e.g., presence of non-evolved infertile eggs) and of constraints on trait
values (e.g., when hatching cannot become perfectly synchronous). We ran simulations for 1,000
generations; average gene values stabilized after approximately 100 generations. To quantify
gene evolution, we obtained the average gene value of each evolving gene in the parent
population of 400 individuals, every tenth generation for 200 generations near the end of the
simulation (750-940). The average cannibalism delay gene value corresponds directly to the
average number of time units offspring wait before eating an egg. Similarly, the average trophic
egg gene value is equal to the average number of trophic eggs produced per clutch. We analyzed
the effect of factors on gene evolution with t-tests or one- or two-way ANOVAs, as appropriate,
in full factorial designs with Tukey post-hoc tests to determine differences between groups. 95%
confidence intervals of estimates are presented as: estimate (lower bound, upper bound). The
statistics are conducted based on averages from five replicates for each parameter set - that is,
sample size was not artificially large.
Computer experiments
Experiments were conducted to test the following predictions, which are derived from the
analytical model. We refer to selection and evolution in the experiments and results to describe
processes that are analogous to organic evolution, but generated within a search algorithm.
Relative benefit of egg eating (RBE) thresholds
The RBE thresholds for mothers and offspring to favour sibling cannibalism (1 00% and
50%, respectively, when r = 0.5) should predict evolution of the three traits when each evolves
separately (Table 3.2). (Relatedness should also influence trait evolution (equation l), but was not
manipulated in our models. With single mating in randomly-drawn pairs, relatedness within
clutches is 0.5 (see Grafen 1991).) The relative benefits and absolute survival probabilities are
given in Appendix I. We examined the evolution of traits separately, when (1) cannibalism delay
was permitted to evolve, but hatching was fixed at complete asynchrony and trophic eggs were
prevented (set 1, Table 3.2); or (2) hatching synchrony (set 2, Table 3.2) or (3) trophic egg laying
(set 3, Table 3.2) could evolve, but the other maternal tactic was fixed and offspring were fixed
for extreme cannibalism.
A corollary hypothesis is that absolute survival and benefit of cannibalism should have no
impact on the evolution of sibling cannibalism traits; only relative benefit of egg eating should
matter. We tested this hypothesis by facilitating evolution of the cannibalism delay gene at 50%
relative benefits, expressed as 10: 15 or 66:99 survival probability (set 4, Table 3.2).
Game aspects
The above simulations effectively assume that offspring or mothers are constrained in their
possible evolved responses to sibling cannibalism. When all traits co-evolve, the game between
mothers and offspring or between siblings may affect trait evolution. We compared gene
evolution when there was multiple trait co-evolution (set 5, Table 3.2) to simulations where only
one trait evolved.
Non-evolved infertility
The presence of happenstance infertile eggs (hereinafter called infertile; sets 7 and 8, Table
3.2) should create increased selection for cannibalism in offspring, if RBE > 0. The predicted
maternal response is not clear: mothers may try to combat increased cannibalism among offspring
or may be selected to help offspring eat infertile eggs.
Time extension
If offspring can wait until all viable offspring hatch before attacking eggs, they may be
selected to delay cannibalism to avoid eating viable siblings. We therefore manipulated a
parameter referred to as 'time extension'. With an extension (set 5, Table 3.2), offspring could
wait until hatching was over before cannibalism. Without an extension (set 6, Table 3.2),
cannibalism was not permitted after hatching ended, so a cannibalizing offspring had to risk
consuming viable siblings. This parameter effectively permits offspring discrimination between
infertile and viable eggs; for this reason, we examined it in combination with the above parameter
of infertile eggs.
We next present and discuss results of each simulation set, before drawing more general
conclusions on parent-offspring conflict, sibling rivalry, maternal caution and the strength of
selection. To assist the reader in interpreting results, we draw attention to the existence of
multiple equivalent trait combinations (Fig. 3.1). For example, there are many combinations of
cannibalism delay and hatch synchrony behaviours that achieve the outcome of no sibling
cannibalism.
3.3 Results and discussion
Relative benefit of egg eating
For comparison with the frequency-independent predictions from the previously discussed
analytical model, we considered simulation sets where only one trait was permitted to evolve
(sets 2-3; Table 3.2). These simulations and the frequency-independent model effectively ask the
same question: given a particular offspring (or maternal) behaviour and level of benefits from egg
eating, what is the optimal behaviour for mothers (or offspring)? In the next section, we compare
these simulations to simulations where all three traits evolved to examine the effect of co-
evolution.
As the frequency-independent analytical model predicts, the relative benefit of eating
siblings affected the evolution of sibling cannibalism and of maternal attempts to limit (via
hatching synchrony) or facilitate (via hatching asynchrony or trophic egg laying) sibling
cannibalism (Fig. 3.2, 3.3).
In the first one-trait simulation (set 2), we permitted sibling cannibalism to evolve, but
made hatching maximally asynchronous (so that offspring risked eating viable siblings) and
prevented trophic egg laying. The cannibalism delay mean phenotype decreased as RBE
increased from 0% to 75% (Fig. 3.2), when it reached a minimal value. As an example of gene
evolution over time, the cannibalism delay gene had an intermediate average value at the
beginning of simulations following random generation of individuals (Fig. 3.3), but quickly
evolved, with the direction of change dependent on the direction of selection pressure.
Cannibalism delay began to decrease before the 50% threshold predicted by the inclusive
fitness model (Fig. 3.2). In fact, the delay gene experienced its largest decline, from 11.6 to 7.2
time units, when RBE increased from 30% to 40%. There are at least two possible explanations
for the trend. First, at RBE values near 50%, fitness is similar for individuals who eat or avoid
eating siblings. The decrease in average delay at 40% RBE (Fig. 3.2) may represent weakened
selection against consumed siblings, as opposed to positive selection for increased cannibalism.
In support of this hypothesis, the LS mean delay gene value at 40% RBE was 7.2 (7.1, 7.4),
which is close to the value generated from randomly created individuals in the initial generation
(7.5). Second, the decrease in the threshold for cannibalism may be evidence of sibling rivalry.
When RBE < 50%, an individual maximizes its inclusive fitness by allowing full siblings to live.
If a second hatched sibling has an allele for cannibalism, however, the 'merciful' offspring does
not reap the kin selection gains of mercy, and does not gain the nutritional benefits from
cannibalism. It therefore loses fitness compared to a more cannibalistic sibling. In a gene pool
containing a variety of cannibalism delays, alleles will continually be paired with, and tested
against, others in the population. Sibling rivalry may drive evolution for higher cannibalistic
tendency than would otherwise be optimal.
For the second one-trait simulation, we examined evolution of the synchrony gene by
minimizing cannibalism delay (i.e., offspring readily ate eggs) and preventing trophic egg laying
(set 2, Table 3.2). Hatching became increasingly asynchronous as the RBE increased from 90% to
150% (Fig. 3.2). The first statistically detectable increase occurred as RBE increased from 90% to
1 lo%, a range encompassing the predicted 100% threshold for mothers to favour cannibalism
among offspring. The largest increases in asynchrony gene value per increment in RBE occurred
from 90% to 100% RBE (3.9 to 7.8).
Two insights can be gained from this set of simulations. First, there was evidence of
parent-offspring conflict and cooperation. At RBE values below loo%, mothers appeared to limit
sibling cannibalism with more synchronous hatching. When RBE > loo%, mothers began to
facilitate cannibalism with asynchronous hatching. Second, the strength of selection may have
affected gene evolution. Asynchrony increased slightly at 90% RBE, before the predicted 100%
threshold (Fig. 3.2), possibly because selection against asynchrony weakened near the threshold.
Similarly, in the simulation where only trophic egg laying evolved, significantly more trophic
eggs were produced at 150% RBE than 1 lo%, because the magnitude of gain from producing
more trophic eggs was greater.
We allowed the third gene, trophic egg laying, to evolve when offspring readily ate eggs
and hatching was completely synchronous, which prevented mothers from using hatching
asynchrony to facilitate cannibalism (set 3, Table 3.2). Trophic egg laying ensures that offspring
can eat eggs, and thus should not occur until the RBE equals or exceeds 100%. The mean number
of trophic eggs per clutch (equivalent to the mean gene value) increased from 0%-150%, with the
first detectable increase as RBE increased from 50-75% (Fig. 3.2). This increased trophic egg
laying when RBE < 100% was not predicted by the inclusive fitness model, and may be explained
by decreased strength of selection against trophic egg laying as RBE neared 100%. Increases in
trophic egg production became larger after RBE passed the 100% threshold: the largest increases
were from 110% to 125% (4.1 to 5.5) and 125% to 150% (5.5 to 7.0).
The relative benefit of egg eating is the increase in survival that cannibals receive
compared to non-cannibalistic individuals. We tested the effect of absolute survival by allowing
cannibalism to evolve (set 4, Table 3.2) at 50% RBE, with 10% or 66% probability of survival of
non-cannibals. There was no effect of the absolute probability of survival on the cannibalism
delay gene (F,, 8 = 0.26, P > 0.62), confirming the analytical model's prediction that the relative
increase in survival is the important parameter. This result implies that we can expect similar
sibling cannibalism behaviour in natural systems where the relative benefit that cannibalism
provides is similar. The absolute harshness of the environment does not matter.
Trait co-evolution
When all three traits were allowed to evolve simultaneously, cannibalism delays were
significantly shorter than when single traits evolved (6.2 vs. 4.0 time units, F,, 56= 97.5, P <
0.0001). The effect depended strongly on RBE (Fig. 3.5A; interaction term: F6, 56 = 10.5, P <
0.0001). There are two possible interpretations of this result. First, in the co-evolving simulations,
mothers increased hatching synchrony at RBE < 100% (Fig. 3.5B). The reduced opportunity for
cannibalism may have created selection on offspring to reduce cannibalism delay, in order to
capitalize on rare cannibalism opportunities. In effect, maternal attempts to limit cannibalism
among offspring may intensify selection for cannibalistic tendencies. Second, in the one-trait
simulations, offspring were forced to respond to the risk of eating viable siblings. In the co-
evolving simulations, mothers limited the opportunity for sibling cannibalism - effectively
weakening selection against sibling-eating individuals. The latter explanation is supported by the
results: the strongest effects of constrained maternal traits are seen at 25% RBE, a level where
both mothers and offspring disfavour cannibalism.
Game aspects significantly affected the evolution of hatching asynchrony compared to the
situation where only asynchrony evolved, with more asynchronous hatching when all traits co-
evolved (5.4 vs. 6.4, F, ,@ = 7.3, P = 0.009; mean difference 1.0 (0.3, 1.7)). The interaction
between constraints and benefits was also significant (Fig. 3.5B; F7, 64 = 7.9, P < 0.0001). The
maternal response is easily understood. When only asynchrony evolved, the gene value decreased
at 0% benefits compared to the co-evolutionary simulation, because when cannibalism delay also
evolved, delays quickly lengthened, reducing selection pressure on mothers to prevent sibling
cannibalism. On the other hand, asynchrony was significantly higher at 150% benefits when only
asynchrony evolved, compared to when all three genes evolved, because when trophic egg laying
also evolved mothers used both tactics to facilitate egg eating. When trophic egg laying was
prevented, mothers increased asynchrony to allow the same level of egg eating through increased
cannibalism of viable offspring.
More trophic eggs were produced in the one-gene simulation compared to when all three
genes evolved (3.3 vs. 2.9, F,, 72 = 37.1, P < 0.0001; mean difference 0.4 (0.3,0.5)). The
interaction between constraints and benefits was significant (Fig. 3 . X ; F8, 72 = 46.6, P < 0.0001).
Trophic egg laying increased significantly when it was the only evolving trait at 125% and 150%
RBE, for a similar reason as hatching asynchrony: when it was the only trait available for
facilitating cannibalism, levels increased.
In summary, when all three genes were permitted to evolve simultaneously, mothers used
both hatching synchrony and trophic egg laying to facilitate egg eating. When the effects of both
traits were combined and when RBE > loo%, approximately half of all eggs laid were eaten - an
optimal solution predicted by the analytical model. In this way, adding a game component to the
simulations increased cannibalistic tendencies among offspring at lower levels of RBE. At these
low levels, mothers responded to the change in offspring behaviour to limit sibling cannibalism.
However, at an RBE > loo%, when mothers attempt to facilitate cannibalism, adding a game
component did not change the result, and the optimality inclusive fitness model was a good
predictor of maternal behaviour.
Non-evolved infertility
The presence of infertile eggs affected the evolution of all three traits.
Offspring did not wait as long to eat eggs when happenstance infertile eggs were present
(3.2 vs. 4.3, Table 3.3; mean difference 1.1 time units (0.80, 1.4)). The difference depended on
the level of RBE (Fig. 3.4A; Table 3.3), such that infertile eggs increased cannibalistic tendencies
among offspring only when RBE was below the 50% threshold for cannibalizing full sibs
(significant effect at 10% and 25% levels of RBE only; Fig. 3.4A). At RBE values above the
threshold, the delay until cannibalism was minimal in the presence and absence of infertile eggs.
We can therefore infer that: (1) parent-offspring conflict over sibling cannibalism occurred,
because mothers would gain more offspring per clutch if offspring waited until viable siblings
had hatched before eating infertile eggs; (2) sibling rivalry was an important selective factor in
the evolution of cannibalism delay. Sibling rivalry is evident because, using inclusive fitness
reasoning, cannibal offspring should wait until viable siblings hatch and then consume infertile
eggs; the only reason not to wait is so that cannibals can eat infertile eggs before siblings eat the
eggs. We conclude that non-evolved infertility enhances parent-offspring conflict and increases
cannibalistic tendencies among offspring.
Hatching was significantly more synchronous in the presence of infertile eggs, as mothers
tried to limit sibling cannibalism (6.7 vs. 6.2; Table 3.3; mean difference 0.5 (0.3, 0.7)). The
response in hatch synchrony is due to a change in offspring cannibalism behaviour caused by the
presence of infertile eggs: offspring were more cannibalistic when infertile eggs were present at
levels of relative benefits where mothers did not favour sibling cannibalism, and mothers tried to
limit the cannibalism of viable offspring.
Mothers produced significantly fewer trophic eggs in the presence of infertile eggs (2.7 vs.
2.1; Table 3.3; mean difference 0.6 eggs (0.5-0.7)). The result is easily understood: with two
infertile eggs per clutch, 14 viable offspring remain. Each offspring can eat only one egg, leaving
12 offspring without an egg to eat. At most, six offspring should be trophic, lower than the
maximum of eight when all offspring are potentially viable.
Our results suggest that non-evolved infertility enhances parent-offspring conflict over
cannibalism. The increased conflict is not due to the presence of infertile eggs, per se, because
mothers should prefer that offspring eat those eggs (if there is any nutritional benefit to be
gained), rather than waste the resource. Rather, the increased conflict stems from offspring
response to infertile eggs: cannibalistic tendencies increase because there is competition among
siblings over eating the infertile eggs. Driven by sibling rivalry and the cannibal's dilemma,
offspring risk eating viable siblings, even when it is not in their inclusive fitness interests to do so
(i.e., at RBE of 10% and 25%; Fig. 3.4A) and certainly not in their mother's interests. Mothers
respond to the increased cannibalism among their offspring by using tactics that limit cannibalism
or egg eating (increased hatching synchrony; reduced trophic egg laying).
Time extension
There was generally no effect of allowing offspring to avoid eating viable siblings by
waiting until all viable eggs hatched (Table 3.3). The only exception is a significant interaction
between time extension and RBE for the trophic egg gene (Table 3.3). A time extension increased
trophic egg production at an RBE of 1 lo%, and no other benefit level (3.4 vs. 3.0, mean
difference 0.4 (0.2, 0.6); Table 3.3). A possible explanation for the result is that, for simulations
runs with a time extension, mothers happened to use the trophic egg tactic more than the hatching
asynchrony tactic. The tactics achieve the same result of allowing egg eating, so it may be a
chance result - and in fact, the P-value for the interaction was 0.04, whereas P-values for most
other significant effects were < 0.0001.
It appears that the time extension variable had no effect on the evolution of cannibalism
delay because sibling rivalry was an important selective pressure. Offspring were selected to
cannibalize siblings quickly, before other siblings ate them; the option of waiting until viable
offspring hatched before eating eggs was never optimal.
3.4 Conclusions
The frequency-independent analysis predicts RBE thresholds for optimal behaviours in
mothers and offspring. The implicit assumption is that each party responds to a fixed behaviour in
the other; the model asks: given that offspring cannibalize and benefits are x%, what should
mothers do? Results from our GA-based simulations confirm the relevance of thresholds
predicted by frequency-independent inclusive fitness theory, but also reveal further predictions
because they incorporate the game theoretic aspects of sibling cannibalism. We discuss the
evidence for these two games, the predicted optimal behaviours in parents and offspring
(especially where these differ from the analytical predictions), and compare our qualitative
predictions with natural systems.
As in the frequency-independent analytical model, the simulation results describe optimal
behaviours for mothers and offspring. Offspring should be very sensitive to the RBE in
cannibalizing siblings (Fig. 3.2); in our model, the average cannibalism delay was statistically
distinct between even small increments in RBE. If infertile eggs are present, offspring should be
more cannibalistic at RBE levels as low as 10% (Fig. 3.4A). Mothers should also respond to RE3E
in their response to offspring cannibalism. They should limit cannibalism (using a tactic such as
hatching synchrony) when RE3E < 100% (Fig. 3.2). When RBE > loo%, mothers should help
offspring to eat eggs; and in this model, hatching asynchrony and trophic egg laying were
interchangeable tactics used to accomplish this end.
Our results also suggest that mothers should err on the side of caution in promoting egg
eating. The analytical model predicts that, when RE3E > loo%, mothers benefit if half of their
offspring consume the rest. In our model, mothers promoted the cannibalism of nearly half of
their clutches, but never equal to or exceeding half (Fig. 3.2). For example, the mean number of
trophic eggs produced at an RBE of 150%, the highest tested, ranged from 6.3 to 7.3 in clutches
of 16 offspring (when hatching asynchrony could not be used to promote egg eating). When
offspring consume a maximum of one egg, the loss in fitness from overproducing trophic eggs is
more severe than from underproduction. With 150% RBE, from 30% survival of non-cannibals
and 75% survival of egg eaters, a mother producing 7 trophic eggs in a clutch of 16 gains (7 *
.75) + (2 * .3) = 5.85 surviving offspring, while a mother producing 9 trophic eggs gains (7 * .75)
= 5.25 offspring. It is best, then, to produce 7 or 8 trophic eggs, rather than 8 or 9.
When we allowed traits to co-evolve, results revealed two interacting evolutionary games
occurring over sibling cannibalism: the competition among siblings to consume eggs, and the
game between parents and offspring when there is parent-offspring conflict over cannibalism.
Several results suggest that sibling rivalry over sibling cannibalism is a selective pressure
influencing the co-evolved game outcome. For example, offspring began eating siblings before an
inclusive fitness analysis predicted they should. Furthermore, when infertile eggs were present,
cannibalism delays decreased (Fig. 3.4A) as offspring risked consuming viable eggs - even when
it was possible to wait until viable siblings hatched. An inclusive fitness analysis would predict
that offspring should, optimally, wait until viable siblings hatched before consuming eggs, but
sibling rivalry caused offspring to eat eggs quickly before other cannibals did so.
The existence of parent-offspring conflict over sibling cannibalism was evident from
several results. When 50% < RBE < loo%, where parent-offspring conflict is expected, offspring
tried to eat viable siblings to their mothers' detriment, and mothers tried to prevent cannibalism
by making hatching synchronous (Fig. 3.2).
The co-evolved outcome, in terms of number of viable offspring cannibalized per clutch,
depended on two things: who had power to control sibling cannibalism, and the strength of
selection on both parties.
Simulation sets 1 and 2, where only cannibalism delay or hatching synchrony could evolve,
are akin to natural systems where offspring or parents are constrained in trait evolution. For
example, in predatory insects, offspring may experience strong selection for early predatory
ability; selection may not be able to separate the behaviours of attacking prey or siblings soon
after emergence, if the behaviours are genetically correlated. Hatching synchrony can also be
constrained: mothers may be unable to make hatching perfectly synchronous, simply because egg
formation and oviposition are not perfectly precise processes. The presence of constraints gives
control of sibling cannibalism to the unconstrained party. Thus, in natural systems, the
observation of sibling cannibalism does not imply that parents permit it (in an evolutionary
sense); they may simply be unable to prevent it.
The importance of the strength of selection was clearly seen in the GA model. In the
analytical analysis, strength of selection is not considered: individuals are predicted to select the
optimal tactic based on a simply inequality, even if it is only slightly better than the next-best
option. In our results, evolved outcomes depended on the magnitude of selective advantage. For
example, mothers produced significantly more trophic eggs at 125% and 150% than at 110%
RBE (Fig. 3.2) - even though 1 10% is above the cannibalism facilitation threshold for mothers.
In a second example, when cannibalism and hatching synchrony co-evolved, more viable
cannibalized offspring occurred as offspring became more cannibalistic (RBE 50-go%), even
though this worked against maternal interests and mothers could have prevented cannibalism
through hatch synchrony. We suggest that mothers did not do so because, at levels of RBE
approaching loo%, there was little fitness difference for mothers between facilitating or limiting
cannibalism; but for offspring there was a large fitness gain to be had from eating a sibling. It is
not clear what these results imply for the evolution of sibling cannibalism in natural systems,
where natural selection in the long term may allow slightly optimal strategies to dominate (see
Roitberg 1990; Roitberg and Mange1 1997). On the other hand, Roitberg and Mangel (1997)
suggest that when fitness functions are fairly flat, variation in traits should persist. Thus,
persistence may depend on the exact shape of the fitness function and strength of selection. We
may predict that variation is more likely to persist in this case, where the fitness function appears
flat, than where fitness functions appear steep based on the results we obtained.
A final point concerns the effect of relatedness in the model. The coefficient of relatedness
expresses the ratio of the difference between the frequency of a gene of concern in the recipient
of an action and population gene frequency, compared to the difference between the gene
frequency of the actor and population gene frequency (Grafen 1991). This ratio is always 0.5
between full siblings. However, the magnitude of the difference between both actors and
recipients and the population gene frequency will decrease as genetic variation for the gene
decreases. For a fixed gene, relatedness must be zero or undefined. Thus, as one allele comes to
dominate the locus, efforts by bearers of the allele to help siblings have less effect in increasing
the allele in the population - the benefit to the allele that is the basis of kin selection. In our
model, this might mean weaker selection to avoid eating siblings - the action that promotes the
spread of copies of long cannibalism delay alleles in the population - towards the end of
simulations as gene values converged on one or a few alleles. The effect would only be relevant
in simulations where offspring should be selected to avoid eating siblings (RBE < 50%), because
the importance to alleles of promoting copies of themselves in others will be greatest in these
cases.
Trophic egg laying
One goal of this study was to investigate the evolution of trophic egg laying as a tactic for
facilitating egg eating. That is, when cannibalism is in mothers' interests (when RBE > loo%),
mothers do best by ensuring that egg eating occurs; for example, by neglecting to fertilize an egg.
This may benefit mothers even if trophic eggs cost the same to produce as viable eggs, as
assumed in our model. It may seem counter-intuitive that a mother's best option may be to stop
an egg's development altogether: why would she not, instead, increase hatching asynchrony so
that an egg may continue development if it happens to escape cannibalism? In our simulations
where both hatching asynchrony and trophic egg laying evolved, mothers used both tactics to
provide eggs to offspring - it was not better to increase hatching asynchrony rather than
producing trophic eggs. We suggest that mothers should produce trophic eggs in order to
guarantee that egg eating occurs; if hatching asynchrony alone is used, there is some chance that
delayed-hatch eggs will hatch before cannibalism occurs.
Our results also suggest that, even if animals produce non-evolved infertile eggs, they may
be selected to produce additional trophic eggs when Rl3E is high. If infertile eggs are initially
produced by an uncontrollable process, however, it may be difficult for mothers to evolve
production of additional trophic eggs (Crespi 1992). Whether the occurrence of infertile eggs
inhibits the evolution of trophic eggs across systems is a question for empirical investigation.
The feasibility of an adaptation whereby mothers feed some eggs to others has been treated
skeptically by some authors (e.g., Dixon 2000). However, our model shows that use of trophic
eggs as a provisioning tactic is reasonable. In Chapter 4 , l suggest that the ladybird Harmonia
axyridis produces trophic eggs to provision offspring, and demonstrate that mothers adjust trophic
egg production to predicted starvation risk for offspring. In this species, trophic eggs appear
identical to viable eggs to the eye, and their weight is not different from viable eggs (unpublished
data). Previous work shows that the Rl3E in H. axyridis can be very high (reviewed in
'Methods'). Thus, evidence is accumulating that some portion of the apparently infertile eggs
produced by this animal has evolved to provision offspring. A diverse assortment of other
animals produce infertile eggs that are eaten by other offspring (reviewed in Chapter 2). Whether
they are truly adaptively produced by mothers is an untested question in most systems.
Future directions
It would be of interest to test several additional parameters predicted to affect co-
evolutionary outcomes regarding sibling cannibalism. First, the frequency-independent inclusive
fitness model presented in this paper analyzes a painvise interaction between cannibal and victim
siblings. Thus, a simulation where clutch size = 2 should exactly match the frequency-
independent predictions. However, most animals that exhibit sibling cannibalism lay eggs in
larger clutches, and as our results suggest, this may influence the evolution of cannibalistic
tendencies through sibling rivalry. Second, the frequency-independent model predicts two
variables that drive evolution of sibling cannibalism: RBE and within-clutch relatedness.
Polygamous mating systems would create lower relatedness, expected to enhance selection for
sibling cannibalism, while assortative mating (by any phenotypic trait) would increase relatedness
and reduce selection for sibling cannibalism. Finally, in natural systems offspring may be selected
to discriminate between viable and infertile eggs. It is, however, hard to predict which they
should prefer eating. Perhaps offspring should avoid eating viable siblings with delayed hatch in
favour of eating infertile eggs - or perhaps offspring should consume viable siblings first to
prevent their hatch! Our GA approach allows testing of a variety of ecological parameters
predicted to be relevant to the evolution of sibling cannibalism.
3.5 Appendix
Details of genetic algorithm and simulations
Model organisms
In the model, haploid individuals are represented as 12-byte chromosomes. We use a gray
coding translation of genotype to phenotype that allows the GA to find adjacent solutions more
easily (Forrest 1993). Each chromosome has three four-byte genes (hatching asynchrony,
proportion of trophic eggs per clutch, delay until cannibalism) with 16 possible alleles.
Simulations
In the initialization of simulations, a population of 400 chromosomes is created with
randomly drawn byte sequences for the trophic egg and cannibalism delay genes. For the hatch
synchrony gene, allele values from one to nine are randomly drawn because values of nine and
above create the same number of opportunities for cannibalism (Table 3.1).
The GA introduces new alleles and new allele combinations from which to search for
optimal strategy sets through mutation- and crossover-like processes during reproduction. In our
model, 200 pairs of individuals are randomly selected to mate and generate 16 offspring with a
1% chance of mutation at each byte locus. There is a 10% chance of crossover for each pair of
offspring created. When crossover occurs, genotypes of mother and father are spliced at a
randomly drawn location to create offspring. When there is no crossover, half of the offspring are
clones of the mother and half of the father. Individuals do not have a sex and can be drawn as a
mother or father in the next generation.
Each individual has three states: alive or dead, the time period of its hatch, and the number
of eggs it has consumed (0 or 1). Individuals may be killed in several ways during intra-clutch
interactions: by being randomly selected as an infertile or trophic egg; by being cannibalized by
an earlier hatching sibling; or by starvation, with risk weighted by 'fed' state.
Relative benefit of egg-eating
The RBE levels tested in simulations (shown here as probability of survival of a non-
canniba1:cannibal) were 0% (75:75), 10% (70:77), 25% (60:75), 30% (70:91), 40% (70:98), 50%
(60:90), 75% (40:70), 90% (50:95), 100% (49:98), 125% (40:90), and 150% (30:75). Not all
simulations tested all values.
3.6 Literature cited
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Breden, F., and M. J. Wade. 1989. Selection within and between kin groups of the imported willow leaf beetle. American Naturalist 134:35-50.
Brown, H. D. 1972. The behaviour of newly hatched coccinellid larvae (Coleoptera: Coccinellidae). Journal of the Entomological Society of South Africa 35:149-157.
Crespi, B. J. 1992. Cannibalism and trophic eggs in subsocial and eusocial insects. Pages 176-2 13 in M. A. Elgar, and B. J. Crespi, eds. Cannibalism: ecology and evolution among diverse taxa. Oxford University Press, Oxford.
Dickins, D. W., and R. A. Clark. 1987. Games theory and siblicide in the kittiwake gull, Rissa tridactyla. Journal of Theoretical Biology 125:301-305.
Dixon, A. F. G. 2000. Insect predator-prey dynamics. Cambridge University Press, London.
Eickwort, K. R. 1973. Cannibalism and kin selection in Labidomera clivicollis (Coleoptera: Chrysomelidae). American Naturalist 107:452-453.
Forbes, L. S. 1993. Avian brood reduction and parent-offspring "conflict". American Naturalist 142:82-117.
Forrest, S. 1993. Genetic algorithms: principles of natural selection applied to computation. Science 261:872-878.
Godfray, H. C. J. 1995. Evolutionary theory of parent-offspring conflict. Nature 376: 133-138.
Godfray, H. C. J., and A. B. Harper. 1990. The evolution of brood reduction by siblicide in birds. Journal of Theoretical Biology 145: 163-1 75.
Goldberg, D. E. 1989, Genetic algorithms in search, optimization, and machine learning. Addison-Wesley, Reading, Massachusetts.
Grafen, A. 1991. Modelling in behavioural ecology. Pages 5-3 1 in J. R. Krebs, and N. B. Davies, eds. Behavioural Ecology, 3rd ed. Blackwell Science, Oxford.
Hamilton, W. D. 1964. The genetical evolution of social behaviour. I, 11. Journal of Theoretical Biology 7: 1-52.
-. 1967. Extraordinary sex ratios. Science 156:477-488.
Hodek, I., and A. Honek. 1996. Ecology of Coccinellidae. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Kawai, A. 1978. Sibling cannibalism in the first instar larvae of Harmonia axyridis Pallas (Coleoptera: Coccinellidae). Kontyu 46: 14-1 9.
Majerus, M. E. N. 1994. Ladybirds. HarperCollins, London.
McNamara, J. M., C. E. Gasson, and A. I. Houston. 1999. Incorporating rules for responding into evolutionary games. Nature 401 :368-371.
Mock, D. W., and L. S. Forbes. 1995. The evolution of parental optimism. Trends in Ecology and Evolution 10: 130-134.
Mock, D. W., and G. A. Parker. 1997. The evolution of sibling rivalry. Oxford University Press, Oxford.
-. 1998. Siblicide, family conflict and the evolutionary limits of selfishness. Animal Behaviour 56:l-10.
O'Connor, R. J. 1978. Brood reduction in birds: selection for fratricide, infanticide and suicide. Animal Behaviour 26:79-96.
Osawa, N. 1992. Sibling cannibalism in the ladybird beetle Harmonia axyridis: fitness consequences for mother and offspring. Researches in Population Ecology 34:45-55.
Parker, G. A., and D. W. Mock. 1987. Parent-offspring conflict over clutch size. Evolutionary Ecology 1:161-174.
Pfennig, D. W. 1997. Kinship and cannibalism. Bioscience 47:667-775.
Pienkowski, R. L. 1965. The incidence and effect of egg cannibalism in first instar Coleomegilla maculata lengi (Coleoptera: Coccinellidae). Annals of the Entomological Society of America 58:150-153.
Reinhold, K., J. Kurtz, and L. Engqvist. 2002. Cryptic male choice: sperm allocation strategies when female quality varies. Journal of Evolutionary Biology 15:201-209.
Robertson, I. C., W. G. Robertson, and B. D. Roitberg. 1998. A model of mutual tolerance and the origin of communal associations between unrelated females. Journal of Insect Behaviour 11 :265-286.
Rodriguez-GironCs, M. A. 1996. Siblicide: the evolutionary blackmail. American Naturalist 148:lOl-122.
Roitberg, B.D.. 1990. Optimistic and pessimistic fruit flies: evaluating fitness consequences of estimation errors. Behaviour 1 14:65-82.
Roitberg, B., and M. Mangel. 1993. Parent offspring conflict in butterflies and wasps. American Naturalist 142:443-456.
Figure 3-1 The average number of offspring cannibalized per clutch with various combinations of cannibalism delay and hatching synchrony values. Cannibalism delay alleles correspond to the number of time units offspring wait before eating an egg. Phenotypes for hatching synchrony alleles are shown in Table 3.1. The shaded area indicates combinations of traits that yield no sibling cannibalism. The number of cannibalized offspring increases diagonally to a maximum of eight (in a clutch of 16), as indicated by the arrow.
Offspring cannibalism delay allele
Figure 3-2 The evolution of cannibalism delay, hatching asynchrony and trophic egg laying, when other genes were prevented from evolving (Table 3.2). Least square means are from a one- way ANOVA with the factor 'relative benefit of egg eating', which was a significant factor for all three genes; P < 0.0001 in each test. Means with different letters are statistically different. The bars represent standard error. The y-axis is reduced for the trophic egg laying gene because average allele value never exceeded 8.
f \ b c Cannibalism delay
P\d , e
\ k-.. ---. f
z.,, g g i? g 5--- x-+- ---*
Relative benefit of egg eating
Figure 3-3 Evolution of the cannibalism delay gene over 1000 generations, for different levels of relative benefit of egg-eating, when the evolution of other traits was prevented (Table 3.2). Results from one representative simulation are shown for each level of relative benefit. Mean gene value was taken for the parental population every tenth generation. With the random generation of individuals in generation zero, initial average gene values were approximately 7.5.
Generation (1 0s)
Figure 3-4 Effect of infertility on the evolution of three traits. For each figure, least squares mean gene values are given for different levels of relative benefit of egg eating, given by the different lines, in the absence and presence of infertile eggs. (a) Cannibalism delay. The mean difference in gene value in the presence and absence of infertile eggs was 6.7 (4.8,8.5) at 10% relative benefit and 3.8 (1.9, 5.6) at 25% relative benefit. The difference was not significant for any other level of benefits. For clarity, levels above 75% are not shown. (b) Hatching synchrony. The presencetabsence of infertile eggs affected the mean difference in gene values at 10% benefits (2.0 (0.9, 3.2)). The difference was not significant at any other level of relative benefit. For clarity, not all of the tested levels are shown. (c) Trophic egg-laying. Infertile eggs significantly decreased trophic egg production at the following levels of relative benefit, with the mean difference in gene value given: 0.9 (0.5, 1.3) at 75%; 0.8 (0.4, 1.2) at 90%; 1.1 (0.7, 1.5) at 100%; 0.8 (0.4, 1.2) at 1 10%; 0.9 (0.5, 1.3) at 125%; 0.9 (0.5, 1.3) at 150%. The y-axis range is reduced to show differences between the curves. For clarity, not all levels of relative benefit are shown.
1
Infertile eggs absent Infertile eggs present
A - -
Infertile eggs absent Infertile eggs present
0 ' I
Infertile eggs absent Infertile eggs present
Figure 3-5 Trait evolution when other traits are constrained. Levels of relative benefit of egg eating are given by the different lines. (a) The evolution of cannibalism delay in offspring when maternal traits affecting cannibalism are free to evolve or constrained (set 1, Table 3.2). Least squares mean gene values are obtained from an ANOVA with the factors relative benefit, presence of constraints, and the interaction term. Constraining traits lead to delayed cannibalism; see discussion in text. Significant increases in delay times occurred at relative benefit levels of 25% (mean difference 6.6 time units (4.5, 8.7)) and 50% (2.2 time units (0.1,4.3)). The differences were not significant at other levels of relative benefit. (b) The effect of constraints on cannibalism delay and trophic egg laying on the evolution of hatching synchrony (set 2, Table 3.2). The difference in gene value when other traits were constrained, or free to evolve, was significant at 0% relative benefit (mean difference 5.2 (1.6, 8.8)) and 150% relative benefit (mean difference 3.8 (0.2, 7.4)). (c) The effect of constrained traits (set 3, Table 3.2) on the evolution of trophic egg laying. The mean difference between the trophic egg gene when traits were constrained, or free to evolve, was 0.7 (0.1, 1.4) at 75% relative benefit; 1.6 (0.9,2.2) at 125%; and 3.2 (2.6, 3.9) at 150%. Note that the y-axis scale for this trait differs from that in graphs of the other genes.
I
Maternal traits not constrained Maternal traits constrained
All traits permitted Trophic egg laying
to evolve and cannibalism delay constrained
All traits free Synchrony and to evolve cannibalism delay
constrained
Table 3-1 The range in hatching synchrony phenotypes, in terms of number of offspring hatching in each time period for each allele, that were tested in simulations. Mothers with each non-binary allele value produce clutches with the temporal hatching pattern indicated in the table.
condition that offspring eat, at most, one egg. With a clutch size of 16, a maximum of eight cannibalism events can occur. Alleles closer to 16 allow maximal cannibalism at a wider range of offspring cannibalism delay phenotypes.
Table 3-2 Conditions for each set of simulations of the evolution of sibling cannibalism and maternal traits that influence cannibalism: the range of relative benefits that cannibal offspring receive; whether infertile eggs are present or absent; whether cannibalism is time-limited; and the presence of constraints, if any, on the evolution of any of the tested traits
Constrained evolution of.. . Simulation Relative Non- Can Cannibalism Hatching Trophic set benefit of evolved offspring delay asynchrony egg laying
egg eating infertile wait for eggs viable present? eggs to
hatch?
2 0-1 50 3 0-1 50 4 50%, with
10% or 66% survival when no eggs were eaten
5 0-1 50 6 0-150 7 0-150
No Yes Asynchronous Prevented
No Yes Minimal Prevented No Yes Minimal Synchronous No Yes Asynchronous Prevented
No Yes No No Yes Yes
8 0-150 Yes No Note: Each set consists of five replicated simulations. The values of relative benefit of egg eating tested in the model fall within the range given; see Appendix I. There were two non-evolved infertile eggs per clutch, when they were present. When traits were constrained, the fixed phenotype is listed; blank cells indicate that the trait was permitted to evolve.
Table 3-3 The effect of the relative benefit of egg eating, infertile eggs, and a time extension (whereby offspring could wait for viable siblings to hatch before eating eggs) on the evolution of the three genes
Source Relative benefit of egg eating Time extension Relative benefit * Time extension Infertile eggs Relative benefit * Infertile eggs Time extension * Infertile eggs
Cannibalism delay
F ratio P 225.0 <0.0001
Hatching synchrony
F ratio P 71.0 <0.0001
Note: Average gene values for the parental population were taken from gener:
Trophic egg laying
F ratio P 293.8 <0.0001
ions 750-940, every tenth generation, then averaged across the five replicated simulations for each set. Whole model result: for cannibalism delay, F30, 169 = 75.0, P < 0.0001; for asynchrony, F30, 169 = 24.0, P < 0.0001; for trophic eggs, F30, 169 = 102.2, P < 0.0001. The three-way interaction term was not significant for any gene and was dropped from the models.
Chapter 4 Ladybird Mothers Mitigate Offspring Starvation Risk
by Laying Trophic Eggs
4.1 Abstract
A large proportion of ladybird eggs are apparently infertile - they are identical in
appearance to viable eggs, but do not develop an embryo and are consumed by larvae hatching
within the same egg batch. The predicted benefits of egg consumption for larvae are empirically
well-supported. An important question, however, remains: are these infertile eggs a maternal
strategy to feed offspring (i.e., trophic eggs) or did egg cannibalism evolve secondarily to exploit
the infertile eggs already present for non-adaptive reasons (e.g., gamete incompatibility or sperm
limitation)? We investigated the former explanation in laboratory experiments with multi-spotted
Asian ladybirds (Harmonia axyridis). Female H. axyridis were assigned to low and high resource
environments for brief intervals; we predicted that tactics to facilitate egg cannibalism, such as
infertile egg production and hatching asynchrony, would be adopted in low food environments
where starvation risk for offspring is greater. We conducted two experiments in this manner that
provided ladybirds with information about resource levels in different ways, through prey feeding
or scent. We also observed female oviposition patterns and tested for infertile egg distributions
that departed from random. Females produced 56% more infertile eggs in the low compared to
the high food environment; however, hatching synchrony did not change. We consider a potential
confound between information and nutrition state unlikely in this experiment because, first,
ladybirds are well able to tolerate low food for 24 hours, the duration of trials; second, females
were in similar physiological condition when each trial began. Results suggest that female
ladybirds use information from the amount of prey they encounter, but not from other cues about
prey levels, to manipulate the proportion of trophic eggs produced, but not hatch synchrony, in a
manner consistent with the adaptive hypothesis.
4.2 Introduction
Parents in many species face the problem of high starvation risk for offspring. Furthermore,
some parents (e.g., most insects) do not interact with their offspring at all after oviposition; hence
mothers in this situation are limited to starvation-reduction behaviours that occur at the egg
production or deposition stage. In the extreme case, a mother's best option might be to provide
food for offspring in the form of eggs themselves (Alexander 1974; Kawai 1978; Polis 1981;
1984; Crespi 1992; Mock and Forbes 1995; Mock and Parker 1997; Pfennig 1997). Mothers
should sacrifice some offspring to others when they gain more offspring from the increased
survival of the consumers than they lose as the consumed (Chapter 3, Crespi 1992) - thus, when
starvation risk is high and eating a sibling provides a large benefit.
One way for mothers to ensure that offspring have a sibling to eat is to produce 'trophic'
offspring that serve as a meal (Chapter 2, Alexander 1974; Crespi 1992; Mock and Forbes 1995).
This hypothesis provides a foundation for the study of trophic eggs - ovariole-produced
structures, formed to feed offspring, that do not otherwise develop into viable offspring (Crespi
1992) - which are noted across diverse taxa (reviewed in Chapter 2), but rarely treated in an
adaptive context (but see Crespi 1992). The hypothesis and definition distinguish trophic eggs
from infertile eggs that did not evolve for offspring provisioning, though they may be consumed
by offspring. In studying trophic eggs, it is therefore necessary to ask the functional question: is
there an adaptive maternal strategy to feed offspring? The alternative hypothesis is that offspring
cannibalize infertile eggs produced through some constraint (e.g., sperm limitation); hence, the
approach of demonstrating that cannibal offspring benefit from consuming eggs does not
distinguish between the two explanations. In some taxa, the adaptive nature of trophic eggs seems
clear; for example, when they have a unique morphology, clearly different from viable eggs (e.g.,
West and Alexander 1963; Henry 1972), or when parents actively feed them to offspring (e.g.,
MacKaye 1986; Nakahira 1994; Crump 1996; Gobin et al. 1998; Kim and Roland 2000; Heying
2001; Matsuko 2003). In other taxa, trophic eggs are simply unfertilized eggs consumed by
offspring in the same egg batch (e.g., Peck and Whitcomb 1970; Valerio 1974; Crespi 1992;
Dickinson 1992), as in ladybird beetles (Banks 1956; Pienkowski 1965; Brown 1972; Kawai
1978; Osawa 1989; 1992; Stevens 1992; Majerus 1994). When this is the case, the adaptive
nature of trophic eggs must be empirically investigated (see Chapter 2).
A second way for mothers to sacrifice some offspring to others is to manipulate hatching
synchrony (O'Connor 1978; Polis 198 1; Dickins and Clark 1987; Magrath 1990; Forbes et al.
2002). If cannibalism is directed towards siblings still in the defenceless egg stage, as in many
insects (e.g., Crespi 1992; Branquart et al. 1997; Via 1999; Sigsgaard et al. 2002), then mothers
can decrease hatching synchrony in low-food environments to allow early-hatching offspring to
cannibalize siblings. Lacewing mothers appear to manipulate hatching synchrony in this way
(Frechette and Coderre 2000).
One way to determine whether mothers adaptively use these two provisioning strategies -
trophic eggs and hatch synchrony adjustment - is to show that they are adjusted to starvation risk,
which determines the benefit of sibling cannibalism from a mother's perspective (Chapter 3,
Frechette and Coderre 2000). The ladybird beetle Harmonia axyridis is a good candidate for
examining flexible use of the behaviours for several reasons. First, females produce many
apparently infertile eggs (15.2%, Kawai 1978; 24.5%, Osawa 1992) that are consumed by larvae
hatching in the same egg batch. Second, first instar larvae face a high starvation risk due to
oviposition sites that are some distance from aphid prey (Majerus 1994) and their poor predatory
abilities (Hodek and Honek 1996). Furthermore, egg cannibalism dramatically reduces the risk of
starvation in H. axyridis (up to 226%, Osawa 19%; see also Kawai 1978) and other ladybirds
(reviewed in Hodek and Honek 1996). Finally, female ladybirds frequently disperse to new sites
between bouts of egg laying (Evans 2003) and are thus likely to deposit offspring at sites that
vary in resource availability. Therefore, female ladybirds should be selected to use plastic
strategies to mitigate offspring starvation risk.
Two possibilities, then, are (1) that the infertile eggs of H. axyridis are trophic offspring
adaptively designed to be eaten (Polis 198 1, 1984; Mock 1994; Pfennig 1997; Osawa 2003) or (2)
that sibling cannibalism behaviour takes advantage of an available food source. We also looked
for evidence that females use hatching synchrony to alter the opportunity for sibling cannibalism
among offspring (Godfray 1987; Godfray and Harper 1990). Assuming that these are plastic
behaviours, we hypothesized that female ladybirds would lay clutches with fewer trophic eggs
and more synchronous hatching when food resources for offspring were high, and more trophic
eggs and less synchronous hatching in low food environments. We conducted two experiments to
test the hypotheses that differed in how ladybirds acquired information about food levels, through
direct contact with prey or indirect cues. Female ladybirds used in experiments were in similar
physiological condition, minimizing a potential confound between information about resource
levels and physiological state. Finally, if the infertile eggs are adaptively produced then
deposition of such eggs should optimize their contribution to sibling survival. Thus, we examined
the spatial distribution and order of oviposition of infertile eggs, suggesting that a uniform
distribution indicates adaptive production (e.g., if it maximizes the chance that cannibals attack an
infertile egg first rather than a viable offspring). Results suggest that females use trophic eggs, but
not hatching synchrony, as an offspring provisioning strategy. However, the distribution of
trophic eggs within clutches and oviposition sequences was not different from random.
4.3 Methods I: Food level experiments
Study system and experimental animals
Harmonia axyridis and other ladybirds produce three types of eggs: apparently infertile
eggs, viable eggs and 'inviable' eggs where a larva develops but does not emerge from the egg
capsule (Ng 1986). After a period of quiescence (mean: 141 min; 95% CI: 126-155; N = 76;
Appendix I. 1) following hatching, larvae consume any unhatched eggs, without discriminating
between viable and infertile or inviable eggs (Banks 1956; Pienkowski 1965; Brown 1972;
Osawa 1992; Majerus 1994). We do not consider inviable eggs to be potentially trophic eggs
because some embryo development occurs. Generally, other unhatched eggs are hatched or
consumed by the time a cannibal is ready to eat a second egg. Larvae remain at the egg batch
about 24 hours, then disperse in search of aphid prey (Osawa 1989), the food source for juveniles
and adults.
Adult H. axyridis were obtained from Applied Bionomics (Victoria, British Columbia,
Canada) and pupae were collected from the Simon Fraser University campus (Burnaby, British
Columbia, Canada). Females used in experiments were maintained individually in petri dishes
and supplied daily with an excess of pea aphids (Acyrthosiphon pisum reared on broad bean,
Vicia faba) and a water source. They were mated to haphazardly chosen males from laboratory
colonies every ten days.
Food level experiments
To observe behavioural plasticity in trophic egg and hatching synchrony behaviours, we
needed to provide ladybirds with information about local food availability. Ladybirds might
assess resource levels by (1) using the internal cue of their own food intake (because adults and
offspring consume the same prey), andlor (2) using an external cue from other senses, e.g. the
scent of aphids (Stubbs 1980; Evans and Dixon 1986) or their honeydew excretions (Carter and
Dixon 1984; Evans and Dixon 1986). We conducted two experiments that provided ladybirds
with information about food resources through internal and external cues, in a paired design such
that females experienced both low and high food treatments in randomized order.
The same procedure was employed in preparing females for all experiments. Before trials,
females were kept separately in petri dishes and fed a set mass of pea aphids daily for five days
(0.03 10 g * 10% if alone; 0.0445 g * 10% if kept with a male for mating), to ensure a similar
physiological condition. This amount is approximately as much as an adult female can consume
in 24 hours (Soares et al. 2001).
Experimental trials began on the sixth day after controlled feeding. Each female was placed
in an empty petri dish (90 x 23 mm) for one hour to emphasize the transition from maintenance to
experimental conditions. Females were then transferred to a new petri dish that contained the
randomly-assigned treatment (low or high aphid masses), a piece of cotton wick moistened with
distilled water, and filter paper as an oviposition surface. During the 24-hour trials, egg batches
were removed from petri dishes every three hours and aphid levels were replenished every six
hours (in the internal cue experiment, by adding three second or third instar aphids to the low
food treatment and a small mass of aphids to the high food treatment; in the external cue
experiment, by adding three second or third instar aphids to both treatments). After first trials,
females were fed the same aphid mass daily for five days before the second trial with the reverse
treatment.
During hatching (c. 80 hours after each trial), we monitored egg batches every ten minutes,
recording temperature and removing emerged larvae to prevent egg cannibalism. Larvae were
considered emerged when all six legs were free of the egg capsule.
The experiments are summarized in Table 4.1.
Internal cue experiment
In the first of two replications of the experiment, all ladybirds came from the supply
company; in the second, 47 were purchased and 23 were collected. In an initial analysis, origin of
female did not significantly affect the results and it was dropped from the final models.
We did not use a 'no aphid' treatment to signal a low food environment because
preliminary trials indicated that females would not oviposit in the absence of prey. Instead, we
used a 'low food' treatment of the smallest mass of aphids necessary to stimulate oviposition
(0.0040-0.0070 g in preliminary trials). The 'high food' treatment was over nine times that
amount, 0.05 16 g + lo%, 1.7 times the amount of pea aphids a female consumes daily (Table 4.1,
Soares et al. 2001).
External cue experiment
Of the 116 females used, 89 were purchased and 27 collected. The experimental chamber
was designed to allow ladybirds to smell the aphids of the high food treatment but to prevent
contact with aphids. It consisted of two petri dish bottoms (90 x 23 mm) separated by two pieces
of nylon mesh secured to the bottom dish with a rubber band (Fig. 4.1; Table 4.1). To minimize
absorbed odors, the mesh had been washed with soap and water, rinsed with 70% ethanol, and
rinsed with distilled water. The upper dish was secured to the lower with cellulose transparent
tape. The lower compartment contained a 1 dram vial of distilled water with a bean leaf pair
inserted through Parafilm03 (Pechiney Plastic Packaging, Wisconsin, USA) into the vial. The leaf
pairs were cut with a razor and inserted through the Parafilm@ under distilled water within two
hours prior to the experiment, to minimize distress odors released by the plant (Petrescu et al.
2001). For the high food treatment, aphids (0.0806 g h 10%) were placed in the lower
compartment, whereas the low food treatment was no aphids (Table 4.1). Ladybirds were placed
in the upper compartment with a small mass of aphids (0.0040-0.0070 g) to stimulate oviposition.
Statistical analyses
We excluded egg batches from analysis when: (1) they were laid in the first six hours of a
trial, because ladybirds may have been responding to the food levels of maintenance conditions
rather than to the new experimental conditions; (2) they had more than 40% non-developing eggs,
because H. axyridis females can carry a male-killing bacteria (Majerus et al. 1998) and killed
male embryos appear as infertile eggs (taking 40% as a conservative level for exclusion); (3) they
had fewer than 5 eggs, because small egg batches are oviposited in an atypical way - scattered
rather than clumped, with eggs on their sides rather than upright; (4) females produced more than
one egg batch during a trial; then we chose one randomly to avoid pseudoreplication.
We used nested analysis of variance (ANOVA) tests to analyze the effect of food level on
the number of eggs per batch, the proportion of trophic eggs weighted by clutch size, and
hatching synchrony, with the factors order of treatment, treatment, the order by treatment
interaction, and the random factor of female nested in order. Exceptions to this form are noted.
The models were built using the 'Fit Model' platform and the restricted maximum likelihood
function in the program JMP 5.0 (SAS 2002). Some females produced egg batches under one
treatment and not the other; these singleton data were included in analysis.
We used three indices of hatching synchrony (Frechette and Coderre 2000): (1) Total hatch
time, standardized to mean batch size by the equation: Standardized hatch time = Mean batch size
(25.9) * (Total hatch time)/(Number of eggs). (2) The average interval between two sequential
hatching larvae. (3) The proportion of eggs per batch, weighted by batch size, that were
vulnerable to sibling cannibalism (i.e., because they emerged later than 141 min after the first
larva in their egg batch, and thus could have been attacked by that larva; see Study system and
experimental animals and Appendix I. 1). We refer to this variable as the 'proportion of delayed
hatch eggs'.
We report least squares means * standard error. As an indication of the magnitude of
difference between treatments, we report the estimated difference and the 95% confidence
interval of the difference (based on the Tukey honestly significant difference function) in the
format: mean difference (confidence interval). For non-significant results, the confidence interval
indicates our ability to detect a real difference between groups (Hoenig and Heisey 2001;
Colegrave and Ruxton 2003).
4.4 Results I: Food level experiments
Internal cue experiment
In the first replication of the experiment, 155 egg batches were produced (77 low food, 78
high food) and 100 were excluded from analysis - 44 were laid in the first six hours, 59 had more
than 40% trophic eggs, 13 had fewer than five eggs, and two were from the same female in the
same treatment - leaving 55 batches (27 low food, 28 high food). In the second replication, 50
egg batches were laid (17 low food, 33 high food) and 34 were excluded: 15 were laid in the first
six hours, 27 had more than 40% trophic eggs, three had fewer than five eggs, and seven were
from the same female in the same treatment, leaving six low food and 10 high food batches.
Some batches had a combination of excluded features. Results did not change when egg batches
produced in the first six hours were included.
Females produced similar sized batches in both replications (first replication: 26.5 eggs * 1.6; second replication: 30.1 eggs * 2.9; mean difference: 3.6 (-3.1 - 10.3); F = 1 . 2 ; ~ = 0.28), so
the factor 'Replication' was removed from the model. Egg batches were also of similar size under
both treatments (low food: 25.7 * 2.3; high food: 26.4 * 2.1; mean difference: 0.7 eggs (-6.0 -
7.4); F = 0.05; p = 0.82).
Trophic egg-laying.
The proportion of trophic eggs was not affected by the factor 'Replication' (first run:
18.4% * 1.4%; second run: 23.1 * 2.4%; mean difference = 4.8% (-0.9% - 10.4%; F = 3.0; p =
0. lo), so it was dropped from the final model.
Females produced 56% more trophic eggs in a low compared to a high food environment
(Fig. 4.2a; Table 4.2). There was little effect of order (low food first, 18.1% * 1.8%; high food,
20.0% * 2.3%; F = 0.38; p = 0.54) and the interaction term was not significant ( F = 0.95; p =
0.35).
Hatch synchrony
We analyzed hatch synchrony data for the first replication only for two reasons. First, order
significantly affected synchrony in most models - that is, there was a day effect of duration of
hatching. We interpret this to mean that temperature affected hatch synchrony, because
temperature was slightly warmer during hatching in the second trial of the first replication (first
trial: 24.4 "C * 0.6, N=116; second trial: 25.8 "C * 0.6, N=116). Second, we lacked hatch
synchrony data for seven batches from the second replication, so excluding the nine remaining
batches did not sacrifice much information. In some batches, a few larvae hatched much later
than the rest; we excluded their hatch times from analysis.
Hatching was not statistically different under low and high food treatments by all three
measures of synchrony (Table 4.2), although hatching was less synchronous in the low food
treatment for all measures, as predicted. The wide range of values in the 95% confidence intervals
indicates a low ability to detect differences (Table 4.2). There was an order effect, such that
females that experienced 'low food' first produced egg batches that took significantly longer to
hatch, with a trend toward longer intervals between hatching eggs (Low vs. high food: Total
hatch time: 227 min * 22 vs. 143 min * 30, F = 5 . 1 0 , ~ = 0.03; Hatching interval: 16.4 min * 2.1
vs. 9.4min*2.8,F=3.87,p=0.06).
There was thus no effect of food availability on any measure of hatching synchrony,
though the trend was in the predicted direction; however, more trophic eggs were produced under
the low food treatment.
External cue experiment
In this experiment, females produced a similar number of egg batches under both
treatments (74 low food, 70 high food). Ninety-six were laid in the first six hours, 15 had more
than 40% trophic eggs, one had fewer than five eggs, and 1 1 were produced by the same females
in the same treatment. Some egg batches fell into more than one excluded category, leaving 38
for analysis (1 7 low food, 21 high food). Because we were unable to monitor eggs for the entire
hatching period, some hatching time data were lost, leaving 17 batches for synchrony analysis (1 1
low food, 6 high food).
Egg batches were similar in size for both low and high food treatments (low food: 24.2 * 3.3; high food: 3 1.8 St 3.3; mean difference: 7.5 (-5.1 - 20.1); F = 3.624; p = 0.15).
Trophic egg-laying
When information regarding resource levels was provided through an external cue, trophic
egg production was not different between treatments (Fig. 4.2b; Table 4.2). There was no effect
of order (F = 0.25; p = 0.62).
Hatch synchrony
There were too few hatching synchrony data to include all factors in the nested models of
hatching synchrony in this experiment, so Order and Treatment were used.
Food level did not affect total hatch time or average hatching interval (Table 4.2). There
were, however, significantly more larvae that were vulnerable to cannibalism from delayed
hatching (Table 4.2). Order did not significantly affect total hatch time (58 min * 44 vs. 113 min
22; F = 1.28; p = 0.28), the interval between hatching eggs (8.7 min St 2.6 vs. 6.4 min St 1.4; F =
0.64; p = 0.44), or the proportion of vulnerable eggs (-2.4% * 3.1 % vs. 3.6% rt 1.4%; F = 3.35; p
= 0.09), but there was limited ability to detect differences between the groups (mean differences:
total hatching time, 54.6 (-49 - 158); interval between hatching eggs, 2.3 (-8.4 - 3.8); proportion
of vulnerable eggs, 0.60 (-0.01 - 13)).
Thus, there was no effect of food availability on trophic egg production in the external cue
experiment. And, while there were more larvae with delayed hatch in the low food treatment, as
predicted, the statistical model did not include the interaction or nested terms, limiting the
confidence we can place in the result.
4.5 Methods 11: Non-random patterns of infertile egg production
Experimental animals were obtained and maintained as described under 'Methods 1'.
Spatial patterns
To examine the spatial distribution of infertile eggs, we photographed egg batches from 20
females with a digital microscope (Scalar USB Microscope M2, 50X magnification). Larvae were
removed from the egg batch after all viable eggs hatched and egg cannibalism occurred. It is easy
to distinguish trophic eggs from embryonated eggs because trophic eggs contain visible yolk, or
traces of yolk if they have been eaten. It is unlikely that consumed infertile eggs would have
developed an embryo because viable and inviable eggs show visible embryo development by the
time emerged larvae are ready to eat an egg.
Following the approach of Aviles et al. (1999), we obtained the coordinates of the centre of
each egg (estimated visually) from the program NIH Image 1.62 (available on the Internet at
http://rsb.info.nih.gov/nih-image/), then calculated the distance in pixels between each pair of
eggs in the batch. After hatching occurred, we determined the average distance between each pair
of the N infertile eggs. Using a computer-coded bootstrap procedure, we sampled randomly with
replacement from all pairwise distances in the batch to generate average distances between N
randomly drawn eggs. We repeated the sampling 10,000 times to generate a null distribution and
calculated the proportion of randomly generated values that were greater or less than observed
average distance between infertile eggs, following usual randomization test procedures
(Edgington 1995). Using a = 0.05, proportions less than 0.025 indicate a clumped distribution and
values greater than 0.975 indicate a uniform distribution.
Oviposition patterns
To examine the dispersion of infertile eggs within the oviposition sequence, we videotaped
ovipositing females kept individually in petri dishes. When the eggs hatched, we associated each
type of egg with its position in the oviposition sequence. We tested for non-random pattern with a
runs test, which uses the null hypothesis of randomness in the order of occurrence of members of
two groups (Zar 1999).
4.6 Results 11: Non random patterns of infertile egg production
Spatial patterns
Of the twenty egg batches collected from different females, six had at least two infertile
eggs, a condition necessary to examine their distribution. The spatial distribution of infertile eggs
was not different from random in any batch (Table 4.3).
Oviposition patterns
Oviposition sequences from four females were recorded. The distribution of infertile eggs
was not different from random in any sequence (Table 4.4).
4.7 Discussion
This study is the first to test the hypothesis that the infertile eggs of ladybirds are an
evolved maternal strategy (Polis 198 1 ; 1984; Mock 1994; Pfennig 1997; Osawa 2003). We found
evidence that trophic egg production is a plastic maternal behaviour, used adaptively depending
on resource availability: in low food conditions, mothers produced 56% more trophic eggs than in
high food conditions (Fig. 4.2; Table 4.2). Our results are consistent with models of siblicide
(Chapter 3, O'Connor 1978; Mock and Parker 1997), which predict that when offspring starvation
risk is great enough, and the relative benefit of eating a sibling exceeds loo%, mothers should
facilitate cannibalism among their offspring. One way to ensure cannibalism is to lay a trophic
egg (e.g., to neglect to fertilize an offspring). Our results are also consistent with the empirical
work of Osawa (1992), which used survival data from a field experiment with H. axyridis to show
that sibling cannibalism often supported the inclusive fitness interests of mothers. Our
contribution is showing maternal control of a behaviour that appeared adaptive; taken together,
these studies suggests some amount of infertility is an adaptation in this species - a maternally-
controlled sacrifice of some offspring to improve the survival of others. This suggestion does not
claim that all ladybird infertility is adaptive for mothers, for some level of infertility may be
unavoidable.
The potential for confound between a female's nutritional and information states merits
consideration. A female might lay more infertile (trophic) eggs in a low food environment
because she is in poor condition, not due to an offspring provisioning strategy. This 'condition
hypothesis' suggests that the short period in the low food treatment in this study made females so
physiologically weak that fertility plummeted. We consider this explanation unlikely for
ladybirds, which feed on an ephemeral prey and should be well adapted to surviving a day with
less food (and can survive much longer under laboratory conditions). Furthermore, to ensure
similar condition, we fed all females an equal mass of aphids for five days prior to each trial.
We tested another hypothesis about the level of control of trophic egg production,
predicting that trophic eggs would be uniformly distributed to maximize the probability that a
cannibal attacks a trophic rather than a viable egg. The lack of evidence for any non-random
pattern may indicate that, while females manipulate the proportion of trophic eggs in an egg
batch, they do not control fertility on an egg-by-egg basis. It is possible that the distribution of
trophic eggs is not important for provisioning offspring or preventing the cannibalism of viable
eggs. However, the number of egg batches examined was small, so further work on this question
is warranted.
A second way mothers may facilitate egg consumption is to decrease hatch synchrony so
that emerged offspring can consume siblings with delayed hatching. We did not find a statistical
difference in hatching synchrony under low and high food conditions. However, for most
measures of hatching synchrony in the two experiments, the difference between low and high
food groups was in the predicted direction. More work is needed to make a conclusion about the
plastic use of this tactic in ladybirds.
Mothers who lay eggs and leave their offspring are limited in avenues for parental care.
Still, in addition to facilitating sibling cannibalism, mothers might reduce offspring starvation risk
by adjusting egg size to put more yolk resources into eggs in high starvation risk environments.
However, there is no evidence of egg size plasticity in the ladybird Coccinella septempuctata
(Dixon and Guo 1993), and other studies in ladybirds suggest that egg size an inflexible species
trait (Stewart et al. 1991a; 1991b; Soares et al. 2001), perhaps inflexibly tied to ovariole size. If
this is correct, then egg size should be linked to starvation risk across species or populations; and,
in fact, a recent study of two goby fish populations with different offspring starvation risk found
that mothers laid larger eggs in the high risk population (Maruyama et al. 2003). Furthermore, if
egg size is highly constrained, it leaves mothers with little ability for adaptive adjustments
between environments, whereas manipulating the opportunity for offspring to consume each other
offers the advantage of plasticity between environments of differing starvation risk.
We therefore expect the use of the trophic egg tactic in taxa where use of other potential
tactics is constrained. For example, ladybird mothers may have limited control over hatching
synchrony, e.g. if it is temperature dependent, as it appeared to be in our study. With imperfect
control, hatching may be too synchronous, preventing cannibalism when it works for a mother's
interests. Then the solution is to ensure a meal for offspring by laying trophic eggs. As a second
example of a constrained behaviour, a possible tactic for reducing offspring starvation would be
to refrain from oviposition in low food environments. However, ladybirds appear to be unable to
delay oviposition in poor environments: they mature eggs continually and do not resorb eggs
(Evans 2003), and in our experiments, they did not reduce clutch size in low food treatments (but
see Dixon and Guo 1993).
Our study should be compared with the work of Frechette and Coderre (2000), who found
that female lacewings increased the hatching synchrony of egg batches in the presence of aphid
prey, but did not change the proportion of infertile eggs. Infertile egg production was low in both
the presence (3%) and absence (4%) of aphid prey; thus one possibility is that these animals
produced too few infertile eggs to allow detection of a difference. Lacewings and ladybirds have
similar life histories - both are batch-laying aphid predators showing sibling cannibalism
behaviour - and both seem to have evolved maternal strategies to promote sibling cannibalism
among offspring. Taken together, their study and ours suggest that manipulating hatching
synchrony and producing trophic eggs are alternate solutions to the problem of reducing offspring
starvation risk when there is no or little parental care.
We have focused on a scenario of parent-offspring agreement over sibling cannibalism,
proposing that mothers help cannibal offspring by neglecting to fertilize some eggs (Mock and
Forbes's (1995) trophic facilitation hypothesis; Crespi 1992). An alternative explanation for
trophic egg evolution is the conflict reduction hypothesis (Crespi 1992), which proposes that
trophic eggs may have evolved to reduce parent-offspring conflict over cannibalism of viable
eggs, in systems where trophic eggs are less costly to produce than viable offspring. However,
parent-offspring agreement is a more likely explanation for the trophic eggs of ladybirds for two
reasons. First, the survival benefit to cannibals of consuming eggs are substantial (reviewed in
Hodek and Honek 1 996), and in most studies exceed loo%, the threshold for mothers to benefit
from sibling cannibalism (Chapter 3, O'Connor 1978). Second, there is no evidence that ladybird
trophic eggs are energetically cheaper to produce than viable eggs. Ladybird trophic eggs and
viable eggs appear identical to the eye (until the last few hours before hatching when the
developing embryo is visible in viable eggs) and there is no difference in their mass (Appendix
1.2). It thus seems reasonable to assume that they require similar energy input from mothers,
though this assumption requires biochemical analysis to be tested. If trophic and viable eggs truly
have equal cost, then mothers should produce trophic eggs only if sibling cannibalism is also in
their interests, because producing a trophic egg has the same effect as a mother killing a
potentially viable offspring. Thus, there should be no conflict over the cannibalism of viable
offspring - mothers and cannibals agree that it should proceed.
Our study is the first to test the assumption that trophic eggs are an adaptive maternal
strategy in a nonsocial insect. Another approach to demonstrating the adaptive nature of trophic
eggs is a cross-taxa comparison of infertile egg production in species with relevant
characteristics, such as those that do or do not lay eggs in batches and where larvae do or do not
cannibalize siblings.
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Stevens, L. 1992. Cannibalism in beetles. Pages 156-175 in M. A. Elgar, and B. J. Crespi, eds. Cannibalism: ecology and evolution among diverse taxa. Oxford University Press, Oxford.
Stewart, L. A., A. F. G. Dixon, Z. Ruzicka, and G. Iperti. 1991a. Clutch and egg size in ladybird beetles. Entomophaga 36:329-333.
Stewart, L. A., J.-L. Hemptinne, and A. F. G. Dixon. 1991b. Reproductive tactics of ladybird beetles: relationships between egg size, ovariole number and developmental time. Functional Ecology 5:380-385.
Stubbs, M. 1980. Another look at prey detection by coccinellids. Ecological Entomology 5: 179- 182.
Valerio, C. E. 1974. Feeding on eggs by spiderlings of Achaeranea tepidariorum (Araneae, Theridiidae), and the significance of the quiescent instar in spiders. Journal of Arachnology 2:57-63.
Via, S. 1999. Cannibalism facilitates the use of a novel environment in the flour beetle, Tribolium castaneum. Heredity 82:267-275.
West, M. J., and R. D. Alexander. 1963. Sub-social behaviour in a burrowing cricket Anurogryllus muticus (De Geer). Ohio Journal of Science 63: 19-24.
Zar, J. H. 1999. Biostatistical analysis. Prentice Hall, New Jersey.
Figure 4-1 External cue experimental chamber. Ladybirds were placed in the upper compartment with filter paper as an oviposition surface, a water source, and a small mass of pea aphids. The bottom compartment contained a bean leaf pair, inserted through Parafilm0 into a vial of water, and the treatment: either no or many pea aphids.
Figure 4-2 Proportion of trophic eggs produced when females were given information that there was low or high food availability through (a) the internal cue of food consumption or (b) the external cue of prey scent; see text for details. The mean proportion was smaller under the low food treatment in the internal cue but not the external cue experiment (Table 4.2). Horizontal lines represent means. Whiskers indicate the range of observations.
0.00 ' I I -- I I
Low food High food
Treatment
No food signal High food signal
Treatment
Table 4-1 Comparison of protocols for the internal and external cue experiments
Experiment Number of Low food High food Conditions subjects treatment treatment
Internal 106 in first 0.0040-0.0070 0.05 16 g * - Females could contact cue replicate, 70 in g pea aphids 10% pea treatment aphids
second replicate aphids External 116 No aphids 0.0806 g * - Females were separated cue 10% pea from treatment aphids by
aphids mesh - Females could contact a small amount of pea aphids (0.0040-0.0070 g)
Table 4-2 Effect of food level on proportion of trophic eggs and three measures of hatching synchrony (see text) when females were given information about food conditions through feeding (internal cue) or other senses (external cue). Sample sizes were: in the internal cue experiment, 7 1 for the proportion of trophic eggs and 55 for hatching synchrony; for the external cue experiment, 38 for the proportion of trophic eggs and 17 for hatching synchrony.
Least squares mean (SE) Reproductive tactic Low food High food Mean difference F P
(95% CI) Internal cue experiment
Proportion oftrophic eggs
Hatching synchrony Total hatch time (min) Interval between hatching eggs (min) Proportion of delayed hatch eggs
External cue experiment Proportion of trophic eggs
Hatching synchrony Total hatch time (min) Interval between hatching eggs (min) Proportion of delayed hatch eggs (0.016) (0.027)
Proportions are weighted by number of eggs per batch. Total hatch time is standardized to mean number of eggs per batch.
Table 4-3 Randomization tests for the spatial distribution of trophic eggs within egg batches produced by different females. P values were generated from simulations of possible distributions, given the spatial arrangement of each batch; values less than 0.025 indicate a clumped distribution while values greater than 0.975 indicate a uniform distribution.
Egg batch Number of eggs P 1 18 0.03
Table 4-4 Runs tests (Zar 1999) of the temporal distribution of infertile eggs within oviposition sequences of different females
Egg batch Number of eggs Test statistic P 7 5 8 -1.29 0.20 8 2 1 1.13 0.26 9 9 -1.63 0.10 10 3 2 -0.29 0.77
Chapter 5 Summary of the Thesis
and Suggestions for Future Work
5.1 Introduction
Trophic egg laying - the production of infertile eggs that are consumed by offspring -
occurs in diverse taxa (see Chapter 2; Crespi 1992). The goals of this thesis were to outline a
behavioural ecological framework for the study of trophic eggs; to investigate the co-evolution of
maternal and offspring traits related to trophic eggs; and to assess the adaptive function of trophic
eggs in the ladybird Harmonia axyridis. The present chapter summarizes the thesis and suggests
approaches for the further study of trophic eggs.
5.2 Thesis summary
This thesis comprised a literature review and synthesis, a model of trophic egg laying and
related behaviours, and empirical study of trophic eggs in H. axyridis. Chapter 1 reviews the
approach taken in each chapter.
In Chapter 2, I collected hypotheses for the adaptive function of trophic eggs and reviewed
the evidence necessary to distinguish among hypotheses. One set of hypotheses suggests that
trophic eggs function to reduce parent-offspring conflict over sibling cannibalism (Crespi 1992):
mothers might produce trophic eggs if offspring will consume them instead of viable siblings. A
second set of hypotheses posits that trophic eggs are an offspring-provisioning tactic. An
alternative hypothesis is that trophic eggs are not a maternal adaptation, but simply infertile eggs
that mothers cannot avoid producing. If trophic eggs are to be studied in an evolutionary
ecological context, the hypotheses of adaptation versus constraint, and the adaptive functions of
parent-offspring conflict reduction or cooperation, must be distinguished. I reviewed the value of
evidence from natural history observations, behavioural experiments, and comparative analyses.
Chapter 3 presented the results of a simulation-based model of the co-evolution of trophic
egg laying, hatching synchrony, and sibling cannibalism. I compared the results to predictions of
a frequency-independent inclusive fitness analysis of sibling cannibalism that did not incorporate
game aspects. The model predicts that competition among siblings over the opportunity to
cannibalize increases cannibalistic tendencies. I also analyzed the effect of three biological
factors. First, as predicted by the frequency-independent model, the relative increase in survival
that cannibals receive determined the optimal behaviour for offspring and mothers. Second, when
infertile eggs were present and there was even a small benefit to eating eggs, offspring risked
eating viable siblings in order to consume infertile eggs. Third, allowing offspring to wait until
viable siblings hatched before eating eggs had no effect on optimal behaviours. The model
suggests that using trophic eggs to provision offspring is a feasible maternal tactic.
In Chapter 4, I presented the results of laboratory experiments on trophic eggs and hatching
synchrony in H. axyridis. I hypothesized that the infertile eggs of H. axyridis are a maternal
adaptation for provisioning offspring (thus, that they are trophic eggs), and that mothers would
produce more trophic eggs in low food environments where starvation risk for offspring was
greater. I also predicted that mothers would create asynchronously hatching clutches in low food
environments so that first-hatching offspring could consume siblings with delayed hatch. I
conducted two experiments to test the hypotheses that differed in how females were given
information about local food conditions. The results showed that females used information from
their own consumption of aphids to adjust trophic egg production as predicted. There was no
statistically significant decrease in hatching synchrony in the low food treatment; however,
because the trend was in the predicted direction, more work is needed in this area. I controlled
for a potential confound between the nutrition and information states of females by ensuring that
females were in similar condition before the experiments and exposing them to low or high food
for a short period. Finally, it might benefit mothers to over-disperse trophic eggs within clutches,
if offspring are then more likely to eat a trophic egg rather than a viable sibling. However, there
was no evidence for a non-random distribution of trophic eggs within clutches or oviposition
sequences.
Appendix I, following this chapter, contains additional data collected during this thesis
research. The data demonstrate that first instar H. axyridis larvae will cannibalize sibling eggs
when given the opportunity; that trophic and viable eggs do not differ in mass; and that field-
collected egg batches have a lower proportion of trophic or infertile eggs than laboratory egg
batches. I also review the levels of infertility observed in other studies of ladybirds infected with
male-killing bacteria and suggest that female ladybirds may adjust trophic egg production
depending on infection status.
5.3 Suggestions for future work
Before commenting on the study of trophic eggs in general, I note further work needed to
broaden the understanding of trophic eggs in H. axyridis. Chapter 4 showed that female H.
axyridis adjust the proportion of trophic eggs to local food abundance in the laboratory. This
result must be repeated in the field to confirm that it occurs under natural conditions. Such a field
study will be a difficult undertaking. Investigators would need to find egg batches deposited near
small and large aphid colonies within the same local environment - but if large aphid colonies are
available, ladybirds will not oviposit near small colonies (pers. obs.). Alternatively, investigators
might compare the proportion of trophic eggs in egg batches from low- and high-food sites.
However, this approach cannot eliminate the possibility that females in low-food environments
are in poor condition and produce eggs with low hatchability for that reason alone (the
information-nutrition confound; see Chapter 3). Another approach would be to place ladybirds on
plants with small or large aphid colonies and to prevent ladybirds from departing by clipping their
wings or placing nets over plants. This experimental design would also be subject to the
information-nutrition confound, unless experimental trials were short to prevent female condition
from deteriorating in the low-food treatment.
A second prediction for the H. axyridis system relates to the senescence pattern of aphid
colonies. Colonies of large mature aphids are on the wane and may disappear before foraging
larvae mature to adulthood (Majerus 1994). Ladybirds prefer to oviposit near young aphid
colonies (Evans 2003). Thus, the age of aphid colonies is an index of offspring starvation risk,
and ladybirds should increase the proportion of trophic eggs when laying near old, compared to
young, aphid colonies.
The following section reviews some interesting questions for systems where the adaptive
function of trophic eggs is known. Some points have been covered in previous chapters; for
synthesis, they are brought together here.
Future work - both theoretical and empirical - should address the following questions.
(1) What ecological circumstances and life history characteristics drive selection for
trophic egg laying, rather than other functionally equivalent tactics?
(2) What does the evolution of trophic eggs imply for selection on related traits?
(3) What is the evolutionary origin of trophic egg laying?
Approaches to answering question (1)
From a behavioural ecological perspective, an important research objective is to understand
the occurrence of trophic eggs across taxa. What ecological factors or species characteristics
should be correlated with the use of trophic eggs, rather than other tactics? Alternative tactics
mothers might use to facilitate or limit cannibalism among offspring include the manipulation of
hatching synchrony (Chapter 3), clutch size, and egg size .(Crespi 1992).
Consider the situation in which mothers are selected to reduce starvation risk for offspring.
I suggest that the tactic that evolves in any particular system will depend on environmental
variability and predictability. In an environment of constant offspring starvation risk across
habitats and seasons, there is no need for flexibility in maternal behaviour. Egg size, for example,
appears to be a fixed species characteristic with little phenotypic plasticity (Chapters 2,4). Across
species, then, I predict that egg size will be inversely related to the mean starvation risk that
offspring experience. When environments are heterogeneous and unpredictable, parents may
manipulate clutch size to create insurance offspring. In poor circumstances, senior offspring can
kill (and/or cannibalize) junior offspring, while if good food conditions occur, parents can
successfully rear junior offspring. Examples of the use of this tactic include facultatively
siblicidal birds, such as great egrets (Mock and Parker 1997) and blue-footed boobies
(Drummond and Garcia Chavelas 1989). In environments that are heterogeneous for starvation
risk, but predictable at the time of egg deposition, parents can use plastic tactics to adjust the
opportunity for sibling cannibalism among offspring, such as hatch synchrony and trophic eggs.
In order to use trophic egg tactics - either to reduce conflict over sibling cannibalism or to
provision offspring - species must already exhibit certain characteristics. Offspring must occur in
a physically confined and inescapable space for some period of their lives (Mock and Parker
1997). Offspring must also have the ability to digest eggs or siblings. We might, then, expect
trophic eggs to occur more frequently in carnivorous species.
The above comments are related to understanding the use of trophic egg laying rather than
alternative tactics. We can also gain understanding of the selective pressures that drive trophic
egg laying by comparing species that lay trophic eggs to related species that do not. If trophic
eggs function to provision offspring (see Chapter 2), several predictions can be made. First,
species in which offspring face high starvation risk - where the chance of surviving starvation is
150% (Chapter 2) - should exhibit trophic egg laying more often than species with lower
starvation risk. Suitable indices of starvation risk include average food resource abundance
experienced by foraging offspring or food acquiring ability (e.g., success as predators) in young
offspring. Spatial and temporal variability in resource abundance may make the first index
difficult to quantify.
A second prediction is that species that lay eggs singly should experience lower rates of
infertility than batch-layers. Solitary offspring have no opportunity to consume a sibling or
trophic egg and therefore mothers do not derive benefit from the increased survival of egg-eating
offspring.
A good group for testing these predictions is the coccinellid beetles. Starvation risk ranges
from 16% to 226% (Osawa 1992) in laboratory studies. Many species, but not all species, lay
trophic eggs. Some species lay eggs singly while others are batch-layers. A phylogeny for the
group exists (Mawdsley 2001) and they are easily reared.
A suggested approach for question (2)
In Chapter 3, I claim that trophic egg laying is necessarily related to other maternal and
offspring behaviours that affect sibling cannibalism. The suite of traits that co-evolve with trophic
egg laying should be tested in a selection experiment. As an example, female H. axyridis that
produce very low or high proportions of trophic egg production could be used to create sib-mated
lines. Each generation would be artificially selected for further divergence in trophic egg
proportion. I predict that hatch synchrony and cannibalistic tendencies in offspring will co-evolve
with trophic egg laying, though the expected direction of change is not clear (see discussion in
Chapter 2).
A suggested approach for question (3)
The only considerations of the evolutionary origins of trophic eggs occur in Crespi (1992),
Weygoldt (1980) in reference to the Dendrobatidae, and Chapter 2 of this thesis. As discussed in
Chapter 2, trophic eggs may have arisen via increased production of already-present infertile eggs
or from a novel process to stop egg development. A study of the genetic and biochemical
differences between trophic eggs, other infertile eggs, and viable eggs may suggest the
evolutionary precursors from which they arose, yet no biochemical or genetic work has been done
on trophic eggs outside of eusocial insects. For example, chromosomal analysis could determine
whether trophic eggs are not fertilized, or fertilized but halted at an early stage of development
(as in the trophic eggs of fire ants, Voss et al. 1987). Biochemical differences between trophic
and viable eggs could be examined by conducting a percentage fatkarbohydratelprotein analysis;
the result might show similarities between trophic eggs and infertile or viable eggs.
5.4 Conclusions
In Chapter 2, I emphasized the need for progress in theory of the ecology and evolution of
trophic egg laying, and the need for empirical studies to demonstrate that purported trophic eggs
are not simply infertile eggs produced by constraint. I attempted to pursue these goals in my own
study of trophic eggs in Chapters 3 and 4.
An important point, emphasized in chapter 1, is that there are likely varied adaptive
functions and evolutionary origins of trophic eggs across systems. No single answer exists to any
of the questions of interest I have suggested here. Thus, research programs should concentrate on
answering questions for particular trophic egg-laying groups, such as the dendrobatid frogs or the
coccinellid beetles.
Further work in the ecology and evolution of trophic eggs is warranted because the topic is
of general interest to ecological theory. Answers to questions about the evolution and adaptive
function of trophic eggs may shed light on other areas in behavioural and evolutionary ecology
research, such as parent-offspring conflict, forms of parental care, maternal effects, sibling
rivalry, and brood reduction theory. In general terms, the study of trophic eggs shows that an
unusual parenting strategy, killing off some offspring to benefit others, can be adaptive.
5.5 Literature cited
Crespi, B. J. 1992. Cannibalism and trophic eggs in subsocial and eusocial insects. Pages 176-213 in M. A. Elgar, and B. J. Crespi, eds. Cannibalism: ecology and evolution among diverse taxa. Oxford University Press, Oxford.
Drummond, H., and C. Garcia Chavelas. 1989. Food shortage influences sibling aggression in the blue-footed booby. Animal Behaviour 37:806-8 19.
Evans, E. W. 2003. Searching and oviposition behaviour of female aphidophagous ladybirds (Coleoptera: Coccinellidae): a review. European Journal of Entomology 100: 1-10.
Kawai, A. 1978. Sibling cannibalism in the first instar larvae of Harmonia axyridis Pallas (Coleoptera: Coccinellidae). Kontyu 46: 14-1 9.
Majerus, M. E. N. 1994. Ladybirds. HarperCollins, London.
Mawdsley, J. R. 2001. Mitochondria1 cytochrome oxidase I DNA sequences and the phylogeny of Coccinellidae (Insecta: Coleoptera: Cucujoidea). Journal of the New York Entomological Society 109:304-308.
Mock, D. W., and G. A. Parker. 1997. The evolution of sibling rivalry. Oxford University Press, Oxford.
Osawa, N. 1992. Sibling cannibalism in the ladybird beetle Harmonia axyridis: fitness consequences for mother and offspring. Researches in Population Ecology 34:45-55.
Voss, S. H., J. F. McDonald, J. H. D. Bryan, and C. H. Keith. 1987. Abnormal mitotic spindles: developmental block in fire ant trophic eggs. European Journal of Cell Biology 45:9-15.
Weygoldt, P. 1980. Complex brood care and reproductive behaviour in captive poison-arrow frogs, Dendrobatespumilio 0. Schmidt. Behavioural Ecology and Sociobiology 7:329- 332.
APPENDIX I
This Appendix summarizes additional data collected during and relevant to this thesis
research. Each section below describes the procedure followed for an experiment or observation
set, presents the results, and briefly comments on the relevance to the rest of the thesis (see
Chapter 1).
1.1 Frequency of sibling cannibalism in Harmonia q r i d i s
Cannibalism is a ubiquitous behaviour in coccinellid beetles (e.g., Majerus 1994).
However, the frequency of sibling cannibalism within species has seldom been studied. Because
trophic egg-laying is related to sibling cannibalism, the question arose: how often mothers faced
with cannibalism among offspring? I assayed the tendency of newly emerged larvae to consume a
sibling egg.
Methods
I created pairs of sibling eggs, consisting of taking an egg from each of two batches laid by
the same female. Females were maintained under laboratory conditions as described in Chapter 4.
Eggs were collected at least one day after oviposition, when they can be handled without damage.
I glued eggs upright (as they occur normally) in the center of the bottom of 5 cm petri dishes,
using a dilute mixture of UHS glue and water. Pairs of eggs were in contact with each other, as
they would occur in a normal egg batch.
Developing eggs turn grey several hours before hatching. When the first-laid egg of each pair
turned grey, I monitored the pair every 15 minutes and noted the hatch time. Hatched larvae could
consume the egg next to them or disperse away, i.e., move at least 3 cm from the egg pair. I
monitored eggs until cannibalism or dispersal occurred. The assays were conducted at 24 C * 1.
I monitored 76 egg pairs from eleven paired egg batches from eight females, with a mean
of 10 egg pairs from each female (range 1, 3 1). Four paired egg batches were tested from one
female. To obtain the mean latency time until cannibalism, I averaged the latency time of each
female and calculated the weighted mean of this average.
Results and summary
All 76 larvae consumed an adjacent egg upon hatching. The result suggests that cannibalism
is frequent in first instar H. axyridis. Thus, we may expect mothers to evolve tactics to manage
cannibalism among offspring.
The weighted mean latency to cannibalism was 14 1 minutes h 12.9 (S.E.). In Chapter 4 , I
compared the mean latency time to the mean total hatch time for egg batches to check whether
larvae delay cannibalism until after viable eggs have hatched.
1.2 Does egg viability or hatching time depend on egg mass?
Trophic eggs may be produced to reduce parent-offspring conflict over sibling cannibalism or
to provision offspring. As argued in Chapter 2, the former explanation is possible only if trophic
eggs are less costly to produce than viable eggs - for example, if they contain less yolk than
viable eggs. To test whether egg mass was related to egg type (i.e., viable, inviable, or trophic), I
weighed eggs and followed their development.
The experiments discussed in Chapter 4 tested whether female ladybirds manipulated
hatching synchrony within batches of offspring. Females might control synchrony by
differentially provisioning eggs, given that egg mass affects development time across coccinellid
species (Stewart et al. 1991). Here, I tested whether egg mass affected development time within
H. axyridis.
Methods
(a) I collected egg batches from H. axyridis females maintained under laboratory conditions
as described in Chapter 4.1 weighed individual eggs to the nearest 0.001 pg using a cahnTM
microbalance, taking care to avoid damaging eggs. Eggs were weighed approximately 12 hours
after oviposition ('initial mass') and again in the 24 hours preceding hatch ('final mass'), which
occurs approximately 78 hours after oviposition. 1 weighed 288 eggs from 11 egg batches laid by
eight females (mean batch size * SE, 26 * 3.4; mean eggs per female, 36 * 8.7): a single batch
from five females and two batches from three females.
In preliminary analysis, weight did not differ greatly among batches within females, so the
factor 'egg batch' was dropped from the final models. I tested whether egg mass (independent
variable) affected embryo development and the likelihood of egg hatching with logistic
regression, in which 'initial mass' and 'female' were main factors. Reported statistics for these
tests are in the form (Wald Chi Square, df, P). I report means * S.E.
Ladybirds produce viable eggs, infertile trophic eggs where no embryo develops, and inviable
eggs where an embryo develops but does not emerge from the egg capsule. I tested whether egg
type affected proportionate mass lost between initial and final weighings, using a two-way
ANOVA that also included the factor 'female'.
(b) To examine the relationship between egg mass and development time, I haphazardly
selected four weighed egg batches in which to observe hatching. Batches are referred to in the
form 'female-batch number'. When eggs were close to hatching (evident from the colour change
several hours before hatch), I monitored them every 10 minutes and noted the time that larvae
emerged (i.e., when all six appendages were free of the egg capsule). I weighed larvae two hours
after emergence, when they are sclerotized enough to handle without damage. I tested whether
egg mass affected development time by the mean square successive difference test (Zar 1999),
which tests the null hypothesis of randomness in the order of continuous data collected serially.
The test generates a C statistic; the null hypothesis is rejected if C equals or exceeds the
appropriate critical C value obtained from Table B.30 in Zar (1 999, p. App 180).
Results and summary
Across females, the mean initial and final masses of ladybird eggs were 0.222 pg * 0.001 and
0.203 p + 0.001, respectively.
The initial mass of eggs did not affect embryo development (0.13, 1, P < 0.72) or hatching
likelihood (0.90, 1, P < 0.34). The eight females tested produced eggs that did not differ
significantly in likelihood of embryo development (12.6, 7, P = 0.080; minimum and maximum
proportion of non-developing eggs per female: 0.07,0.67), but eggs from different females
differed in likelihood of hatching (17.7, 7, P = 0.013; minimum and maximum proportions of
non-hatching eggs per female: 0.10, 0.78).
Egg type affected the proportion of mass lost (Figure I. 1; F Ratio 6.4, df 2, P = 0.002).
Females differed in the proportion of weight loss that their eggs experienced (F Ratio 4.2, df 7, P
= 0.0002; minimum and maximum LS mean, 0.06,O. 10).
Thus, the trophic eggs of ladybirds do not differ in initial mass from viable or inviable
eggs. However, viable eggs lose more mass during development than do trophic eggs, probably
due to yolk metabolism in developing embryos. Some females were more likely than others to
produce inviable eggs; the reason is not known.
The initial mass of eggs was not related to hatching order in three of four tested batches
(27-4: C, 0.04; C,",, 0.45; 32-33: C, -0.23; C,,, 0.49; 60-16: C, -0.25; C,",, 0.41). In batch 60-15,
heavier eggs hatched significantly earlier than lighter eggs (C, 0.49; C,",, 0.41; Fig. 1.2). The
amount of weight loss that eggs experienced was not related to hatching order (27-4: C, 0.1 1;
C,~,, 0.45; 32-33: C, 0.41; C,~,, 0.49; 60-15: C, -0.36; Cca, 0.41; 60-16: C, -0.12; Cc,it, 0.41).
There is some indication from these data that mothers can control hatch synchrony via the
proximate mechanism of within-clutch variation in egg mass. Mothers might increase the
opportunity for sibling cannibalism among offspring by differentially provisioning eggs within
batches; then heavier, earlier hatching offspring can cannibalize lighter, later hatching siblings.
However, with the small sample size and inconsistent relationship between egg mass and
hatching order, more work is needed on this topic.
1.3 Trophic egg production in the laboratory and field
Some portion of the infertile eggs of H. axyridis observed in this thesis may be an artefact
of laboratory conditions. For example, most laboratory settings involve periodic feeding, rather
than the continuous food supply available in nature; this may disrupt the addition of yolk to eggs
(Dixon 2000). To test whether infertile egg production was higher under laboratory conditions, I
compared the proportion of trophic eggs in field- and laboratory-collected egg batches.
Methods
I collected 24 H. axyridis egg batches from aphid-infested trees on the Simon Fraser
University campus in Burnaby, BC, in July 2003. Egg batches were maintained in laboratory
conditions (described in Chapter 4) until hatch, which occurred within three days. The proportion
of trophic eggs was noted for each batch. Hatched larvae were reared to the second instar, when
they can be identified as H. axyridis. Cannibalism likely occurred within most batches. I recorded
cannibalized eggs that showed no sign of development as trophic eggs, because viable eggs begin
to turn grey by the time hatched larvae are ready to eat an egg.
For comparison, I randomly selected 24 egg batches from H. axyridis females collected from
the field as adults and maintained under laboratory conditions. Egg batches had to contain at least
five eggs to be selected, because batches containing fewer eggs are oviposited in an atypical
manner. Field-collected batches all contained at least five eggs.
Female H. axyridis can be infected with a male-killing bacteria that prevents the development
of male eggs (Majerus et al. 1998). Killed eggs would appear as trophic or infertile eggs. To
eliminate the possibility that infection status influenced the result, I conducted a second analysis
excluding egg batches with more than 40% non-developing eggs. Twenty-three field-collected
batches had at least 60% of embryos develop. For comparison, I randomly selected 23 egg
batches with at least 60% embryo development from laboratory-maintained females.
I compared the mean proportion of trophic eggs among field- and laboratory-collected
batches ('egg origin') with one-way ANOVA in JMP 5.0. In separate analyses, I considered the
response variables as the weighted proportion of trophic eggs per egg batch or the number of eggs
per batch. Least square means are reported * SE. Estimated differences are reported with the 95%
confidence interval of the difference.
Results and summary
When egg batches with more than 40% trophic eggs were included, field-collected egg
batches were composed of 14.9% * 3.4% trophic eggs, while laboratory-collected batches had
21.3% * 3.9% trophic eggs. The difference was not statistically significant (F Ratio 1.5, df 1, P =
0.23). The confidence interval (-4.1%, 16.8%) around the estimated difference of 6.3% is wide,
indicating that the variation in the data is too large for the sample size of this study to allow
detection of a difference, if one existed.
When egg batches containing more than 40% trophic eggs were excluded, the difference in
proportion of trophic eggs between laboratory and field collected batches decreased (laboratory:
13.3% * 2.5%; field: 12.5% * 2.3%; F Ratio 0.1, df 1, P = 0.81). The relatively smaller
confidence interval around the estimated difference (3.4% (-7.6%, 6.0%)) indicates a better
ability to detect a difference that actually existed.
One of 24 field-collected batches and six of 24 randomly selected laboratory batches
contained more than 40% trophic eggs. The higher incidence of egg batches with over 40%
trophic eggs in laboratory batches is statistically significant (h2 = 14.7, df 1, P < 0.001).
The data therefore suggest that laboratory conditions do not generally cause higher rates of
infertile egg production, but do cause some females to produce a high proportion of trophic eggs
(> 40%). The effect may be related to the greater diet diversity in the field than in the laboratory
or to the restricted number of mates. Considering these results, some portion of the infertile eggs
produced in the experiments described in Chapter 4 were likely caused by laboratory conditions.
This factor is not likely to bias the observed increase in trophic egg proportion in low food
conditions.
Egg batches were significantly smaller in the laboratory than in the field when egg batches
with more than 40% trophic eggs were included (laboratory: 20.2 eggs * 2.2; field: 27.0 eggs %
2.2; F Ratio 5.0, df 1, P = 0.03). When these egg batches were excluded, the difference was not
statistically significant (laboratory: 23.1 eggs * 2.0; field: 27.0 * 2.0; F Ratio 1.8, df I, P = 0.18).
The estimated difference was 3.8 eggs (-1.9, 9.5). It seems that, for some females, laboratory
conditions induce smaller batch sizes and higher rates of infertility.
1.4 Male-killing bacteria and the proportion of trophic eggs laid
Female ladybirds are frequently infected with cytoplasmic male-killing (MK) agents that
kill male embryos at an early stage of development (e.g., Majerus et al. 1998; Hurst et al. 1999).
Non-developed male eggs are eaten by their hatching sisters. An additional fraction of ladybird
eggs normally fails to develop and may be a maternal adaptation to feed offspring (trophic eggs;
see Chapter 4). The null expectation for the percent hatch in egg batches from infected females is
therefore half of the normal hatch rate (because, with the 5050 sex ratio observed in ladybirds
(Majerus 1994), half of normally viable eggs would not develop). If female ladybirds could detect
their infection status, and if they control trophic egg production, then infected females might
benefit from reducing trophic egg production because half of their egg batches will be effectively
'trophic'. I review studies of MK bacteria in coccinellids that report hatching success in infected
and uninfected females to test whether infected females reduce trophic egg production. The null
hypothesis is that the proportion of hatching eggs from infected females will not differ from half
of the hatching rate in eggs of uninfected females.
Methods
Six studies report hatchability in MK-infected and uninfected ladybirds (Tables I. 1 and
1.2). Some data from Hurst et al. (1999) could not be used because only one uninfected female
line was followed, so a confidence interval could not be calculated. A hrther three studies report
hatch rates of MK-infected lines, but do not report rates for non-infected lines for comparison
(Hurst et al. 1993; Majerus et al. 2000; Schulenburg et al. 2001).
Two studies report hatch rates and number of eggs for field-collected, laboratory-reared
lines of infected and uninfected females (Table 1.1). For these studies, I calculated a weighted
mean proportion hatch for uninfected lines and divided it in half to obtain the expected proportion
hatch for infected lines. I then tested whether the 95% CI of the weighted mean proportion hatch
in uninfected females encompassed the predicted value. Hurst et al. (1999) report hatch rates for
field-collected ladybirds maintained in the laboratory and for subsequent crosses within infected
and uninfected lines. I analyzed these datasets separately. Two additional studies report hatch
rates, but not number of eggs. For these studies, I followed an equivalent procedure without
weighting the mean proportion hatch.
Another two studies report hatch rates for individual females before and after an antibiotic
treatment that cures MK infection. I used a paired t-test to compare pre-antibiotic hatch rate with
half of the post-antibiotic hatch rate. It is possible that the antibiotic treatment influenced hatch
rate independently of the cure of MK infection. I compared pre- and post-antibiotic hatch rates for
uninfected females (which were reported in both studies) using a paired t-test. The rates should
not differ if antibiotics do not affect females other than curing MK infection.
Results and summary
In four of the seven tested datasets, females appeared to reduce trophic egg production
when infected with MK agents (Tables I. 1,1.2). When cured of MK infection by antibiotic
treatment, females increase trophic egg production (Table 1.2) as predicted if females control
trophic egg production and are sensitive to their infection status. However, in one of the studies
the antibiotic appeared to affect hatching rate independent of the cure of MK infection.
The results do not allow the conclusion that females can detect MK infection status or that
females control trophic egg production. However, that the trend appears in four of seven datasets
is suggestive. If the hypothesis of reduction in trophic egg production with MK infection is
correct, then this is another line of evidence demonstrating that trophic eggs are a maternal
adaptation in ladybirds (see Chapter 2). An alternative hypothesis is that infection confers some
fertility benefit such that non-adaptively infertile eggs are eliminated in infected females. This
might occur because the vertically transmitted MK agents have an interest in promoting host
fertility. More work is required in this respect.
1.5 Literature cited
Dixon, A. F. G. 2000. Insect predator-prey dynamics. Cambridge University Press, London.
Hurst, G. D. D., C. Bandi, L. Sacchi, A. G. Cochrane, D. Bertrand, I. Karaca, and M. E. N. Majerus. 1999. Adonia variegata (Coleoptera: Coccinellidae) bears maternally inherited Flavobacteria that kill males only. Parasitology 1 18: 125-1 34.
Hurst, G. D. D., T. C. Hammarton, J. J. Obrycki, T. M. 0. Majerus, L. E. Walker, D. Bertrand, and M. E. N. Majerus. 1996. Male-killing bacterium in a fifth ladybird beetle, Coleomegilla maculata (Coleoptera: Coccinellidae). Heredity 77: 177-185.
Hurst, G. D. D., M. E. N. Majerus, and L. E. Walker. 1992. Cytoplasmic male killing elements in Adalia bipunctata (Linnaeus) (Coleoptera: Coccinellidae). Heredity 69:84-91.
-. 1993. The importance of cytoplasmic male killing elements in natural populations of the two spot ladybird, Adalia bipunctata (Linnaeus) (Coleoptera: Coccinellidae). Biological Journal of the Linnean Society 49: 195-202.
Hurst, G. D. D., E. L. Puwis, J. J. Sloggett, and M. E. N. Majerus. 1994. The effect of infection with male-killing Rickettsia on the demography of female Adalia bipunctata L. (two spot ladybird). Heredity 73:309-3 16.
Majerus, M. E. N. 1994, Ladybirds. HarperCollins, London.
Majerus, M. E. N., J. H. G. V. D. Schulenburg, and I. A. Zakharov. 2000. Multiple causes of male-killing in a single sample of the two-spot ladybird, Adalia bipunctata (Coleoptera: Coccinellidae) from Moscow. Heredity 84:605-609.
Majerus, T. M. O., M. E. N. Majerus, B. Knowles, J. Wheeler, D. Bertrand, V. N. Kuznetzov, H. Ueno et al. 1998. Extreme variation in the prevalence of inherited male-killing microorganisms between three populations of Harmonia axyridis (Coleoptera: Coccinellidae). Heredity 8 1 :683-691.
Matsuka, M., H. Hashi, and I. Okada. 1975. Abnormal sex-ratio found in the lady beetle, Harmonia axyridis Pallas (Coleoptera: Coccinellidae). Applied Entomology and Zoology 10:84-89.
Schulenburg, J. H. G. V. D., M. Habig, J. J. Sloggett, K. M. Webberley, D. Bertrand, G. D. D. Hurst, and M. E. N. Majerus. 2001. Incidence of male-killing Rickettsia spp. (a- Proteobacteria) in the ten-spot ladybird beetle Adalia decempunctata L. (Coleoptera: Coccinellidae). Applied and Environmental Microbiology 67:270-277.
Stewart, L. A., J.-L. Hemptinne, and A. F. G. Dixon. 1991. Reproductive tactics of ladybird beetles: relationships between egg size, ovariole number and developmental time. Functional Ecology 5:380-385.
Zar, J. H. 1999, Biostatistical analysis. Prentice Hall, New Jersey.
Figure 1-1 Proportion of egg mass lost during the development of H. axyridis egg types. Trophic eggs lose significantly less mass than viable eggs (estimated difference and 95% CI: 0.025 (0.009,0.042)). Inviable eggs, where an embryo develops but does not emerge, lost an intermediate amount of mass. The initial mass of eggs was taken 12 hours after oviposition; final mass was taken in the 24 hours before hatching.
I I
Viable Inviable Trophic
Figure 1-2 Hatching time in relation to egg mass within egg batch 60-15. Hatching time is expressed relative to the first hatching egg in the batch, which has a hatch time of zero. The initial mass was taken within 12 hours following oviposition. Heavier eggs tended to hatch sooner than lighter eggs, though clearly the trend has exceptions.
Relative hatch time (minutes)
Table 1-1 The evidence, across studies, that female ladybirds adjust trophic egg production depending on male-killing infection status. The expected proportion hatch is a weighted mean proportion of hatching eggs from uninfected lines, based on data reported in each study. The observed proportion is also a weighted mean. The null hypothesis is that the observed hatching proportion does not differ from half of the hatching proportion in uninfected lines. If females can control the production of trophic eggs, infected females may benefit from reducing trophic egg production because hatching larvae will have dead male eggs to eat. When the lower limit of the 95% CI of the observed proportion exceeds the expected proportion, the evidence is taken to support the alternate hypothesis.
Species N eggs from N eggs Expected Observed Evidence for Source uninfected from proportion proportion hypothesis of lines (N MK hatch hatch in female lines) lines infected lines control of
(N (95% CI) trophic eggs lines)
Harmonia 3598 (33) 4762 0.376 0.413 (0.383, Supports Majerus axyridis (30) 0.442) et al.
(1 998) Harmonia 3968 (12) 3598 0.341a 0.360 (0.261, Does not Matsuka axyridis (12) 0.458) support et al.
(1 975) Coleomegilla 1206 (20) 1056 0.432 0.533 (0.444, Supports Hurst et maculata (6) 0.590) al.
(1 996) Coleomegilla 1440 (12) 1590 0.340 0.477 (0.42 1, Supports Hurst et maculata (1 1) 0.533) al.
(1 996) Adalia - (31) - (31) O.43ga 0.423a Does not Hurst et bipunctata (0.40 1, support al.
0.445) (1 994) a Proportions not weighted because sample sizes not given. Field-collected lines. Laboratory crosses of field-collected lines.
Table 1-2 Evidence that females control trophic egg production from studies in which male- killing (MK) infected ladybirds were cured with antibiotic. The expected proportions for MK- infected lines before antibiotic treatment are calculated as half of the proportion of hatching eggs after antibiotic treatment. If the observed proportion is greater than the expected proportion, females appear to reduce trophic egg production when infected. For lines that were uninfected before treatment, the expected hatching proportion is the proportion observed following antibiotic treatment. If the observed proportion is greater than the expected, the antibiotic causes some reduction in trophic egg production regardless of MK infection status.
Study N eggs N eggs Mean Expected P-value Evidence for from lines from lines proportion proportion (Test hypothesis of before after hatched (SE) statistic) female antibiotic antibiotic before control of treatment treatment treatment trophic eggs - -
(N lines) (N lines) (SE) ( a ) Adonia variegata (Hurst et al. 1999) MK- 1084(6) 1014(6) 0.401 0.349 0.014 Supports infected (0.032) (0.044) (t5=3.3) lines Uninfected 591 (5) 728 (5) 0.901 0.854 0.04 1 Does not lines (0.01 6) (0.023) (t4=2.3) support (b) Adalia bipunctata (Hurst et al. 1992) MK- - (9) - (9) 0.464 0.341 0.004 Supports infected (0.038) (0.020) (te=3 .2) lines Uninfected - (5) - (5) 0.826 0.850 0.287 Supports lines (0.036) (0.032) (t4=0. 7)