Beetle Biology and Tinbergen’s Four Questions and Ecology

11/11/22, 2:04 AM
HW 11: Burying Beetle Biology
HW 11: Burying Beetle Biology
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Introduction
This is a group assignment. It asks you to explore important
aspects of the scientific method (formulating a question,
hypothesis, and conclusion) using material from Chapter 51 in
the textbook on evolution of behavior as a case study. You may
find it useful to reference section 51.1.
Please read and view the material presented below, then
complete the questions on this worksheet
(https://canvas.pitt.edu/courses/158231/files/10966051?wrap=1)
(https://canvas.pitt.edu/courses/158231/files/10966051/download?
download_frd=1) and submit it on Gradescope. You will have to
indicate where your responses to each question are when you
submit. Be sure to include the names of participating group
members on the sheet and add them to your submission on
Gradescope.
In this assignment, you will explore the biology of American burying beetles (Nicrophorus
americanus). They are a critically endangered insect that used to range across the entire eastern
US but are now confined to a few isolated populations (Figure 1).
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HW 11: Burying Beetle Biology
Figure 1. Historical (light gray) and current (dark gray) range
of the American burying beetle. From Lomolino et al. 1995.
First, for some visual background, watch this brief video on how
burying beetles got their common name.
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HW 11: Burying Beetle Biology
Burying Beetles | National Geographic
Next, read about this recent study on the burying beetle’s
microbiome (https://www.inverse.com/article/49899-buryingbeetles-microbial-flora) and the role it plays in their reproduction.
Finally, read the following about some additional research on
burying beetle reproduction.
Burying beetles are called nature’s undertakers because they bury the remains of small dead
animals. These animal bodies serve as food for the beetles’ young. A mating pair of beetles
typically meets over a fresh animal carcass, copulates, and buries the carcass. The female
lays fertilized eggs in the soil above the carcass. Some 50–60 hours later, the eggs hatch and
both parents spend the next 3 days eating from the carcass and regurgitating the partially
digested material to feed their young. After 3 days, the larvae feed themselves.
Researchers used these beetles to study trade-offs in time and energy investments between
brood care and production of new broods. Both activities are demanding, so how do the
beetles control their apportionment of energy? The researchers studied this system in 3
ways:
A. They provided female beetles with mating partners and carcasses to stimulate egg
laying. There were three treatment groups: (1) no larvae group: just as the eggs
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HW 11: Burying Beetle Biology
hatched, the new hatchlings were removed and the parents were presented with a new
carcass; (2) new larvae group: just after their eggs hatched, the parents were left with
10 larvae and given a new carcass; and (3) old larvae group: 4 days after their eggs
hatched and they had cared for their hatchlings, the parents were presented with a new
carcass. The researchers measured the number of females in each group that
produced eggs. The results are shown in Figure A.
B. They measured juvenile hormone (JH) levels present at various times in the breeding
cycles of females with and without larvae. Juvenile hormone is known to suppress the
fertility of the females. The results are shown in Figure B.
C. They measured the effects of a possible pheromone, methyl geranate, on the
beetles’ sexual behavior. Methyl geranate is a volatile compound that shares the same
biosynthetic pathway with JH. The researchers recorded whether or not males
copulated with females and the amount of methyl geranate emitted by those females.
Figure C summarizes these results. The data points represent meetings resulting in
copulation or not.
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HW 11: Burying Beetle Biology
Original Paper: Engel, K. C. et al. 2016. A hormone-related female anti-aphrodisiac signals
temporary infertility and causes sexual abstinence to synchronize parental care. Nature
Communications 7: 1–10.
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ARTICLE
Received 31 Jul 2015 | Accepted 12 Feb 2016 | Published 22 Mar 2016
DOI: 10.1038/ncomms11035
OPEN
A hormone-related female anti-aphrodisiac signals
temporary infertility and causes sexual abstinence
to synchronize parental care
Katharina C. Engel1, Johannes Sto¨kl2, Rebecca Schweizer1, Heiko Vogel3, Manfred Ayasse1, Joachim Ruther2 &
Sandra Steiger1
The high energetic demand of parental care requires parents to direct their resources towards
the support of existing offspring rather than investing into the production of additional young.
However, how such a resource ?ow is channelled appropriately is poorly understood. In this
study, we provide the ?rst comprehensive analysis of the physiological mechanisms
coordinating parental and mating effort in an insect exhibiting biparental care. We show a
hormone-mediated infertility in female burying beetles during the time the current offspring is
needy and report that this temporary infertility is communicated via a pheromone to the male
partner, where it inhibits copulation. A shared pathway of hormone and pheromone system
ensures the reliability of the anti-aphrodisiac. Female infertility and male sexual abstinence
provide for the concerted investment of parental resources into the existing developing
young. Our study thus contributes to our deeper understanding of the mechanisms
underlying adaptive parental decisions.
1 Institute of Evolutionary Ecology and Conservation Genomics, University of Ulm, 89081 Ulm, Germany. 2 Institute of Zoology, University of Regensburg,
93053 Regensburg, Germany. 3 Department of Entomology, Max Planck Institute for Chemical Ecology, 07745 Jena, Germany. Correspondence and requests
for materials should be addressed to S.S. (email: sandra.steiger@uni-ulm.de).
NATURE COMMUNICATIONS | 7:11035 | DOI: 10.1038/ncomms11035 | www.nature.com/naturecommunications
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11035
amily life creates a venue for cooperation and con?ict1,2.
Parents of many species cooperate to rear offspring, but
nevertheless there can be intense con?icts between males
and females over mating rate or how much each sex should invest
in raising the young. Moreover, due to the high energetic and
temporal costs of parental care, parents are ultimately faced with
the reproductive dilemma of whether to continue to invest into
the current offspring or to produce additional ones3. In general,
parental care, such as offspring guarding or provisioning
increases offspring lifetime reproductive success and thus can
enhance parental ?tness4. Consequently, as long as the bene?ts to
the parents outweigh the costs of parental care, the decision
should be made in favour of the current brood. However,
the proximate mechanisms regulating resource ?ow into the
appropriate direction are poorly understood. Especially, in
mothers that provision their offspring with food, mechanisms
should exist that direct nutrients and energy either into feeding
the offspring or the development of new eggs/foetuses. Moreover,
the entire family might bene?t from parents synchronizing their
sexual activity and only investing in costly matings when new or
additional offspring are to be produced but not during the period
of intensive parental care. However, coordination of parental and
mating effort requires effective communication systems as the
female needs to inform her male partner about whether she
intends to reproduce or care. Although cues and signals of
female receptivity or fertility are known to exist in some animal
species5, our current knowledge of whether and how sexual
activity and parental care is synchronized between partners is
very limited. Mammals are one animal taxon where at least
some information about the regulation of the resource trade-off
within females as well as coordination of sexual activity between
individuals is available. In many mammalian species, including
humans, mothers are characterized by a postnatal infertility
during the time of lactation6. The hormone prolactin secreted
during lactation appears to be one factor suppressing ovulation7,8.
Consequently, nutritional resources are allocated towards the
existing young as long as they are needed, instead of developing
new foetuses. In addition, many female mammals produce visual
(for example, changes in colouration and/or swelling of the skin
of the external genitalia) or chemical signals indicating the time of
fertility9,10. These signals might help to synchronize mating
activities, but also other functions have been discussed, especially
as these signals often do not reveal the exact time of ovulation and
do not necessarily coincide with the fertile phase11.
In insects, parental care including offspring provisioning has
evolved many times12, but it is unknown how the con?icting
energetic and nutritional demand of a current brood and sexual
activity, including the development of new eggs, is balanced. To
shed light on the physiological basis of parental investment
strategies as well as on how sexual activity and parental care is
coordinated between breeding partners, we studied burying
beetles. These beetles are a prime example of insects
performing elaborate biparental care13–16 and are known for
their complex recognition and communication systems17.
Burying beetles reproduce on small dead vertebrates and feed
their developing young with pre-digested carrion food. The larvae
of the burying beetle Nicrophorus vespilloides beg for food until
about 3 days after hatching18. Afterwards they continue to feed
independently on the carrion. The bene?ts of parental care in N.
vespilloides are substantial. Not only larval survival rate but also
larval mass at dispersal are signi?cantly increased in broods
receiving parental care compared with broods without parental
attendance19. Particularly larval mass at dispersal is a measure of
offspring quality, because it is highly correlated with adult body
size which in turn enhances the effectiveness of securing a carcass
needed for reproduction20,21. Hence, mothers are predicted to
F
2
allocate their energy and nutritional resources into their existing
larvae as long as they are needy instead of investing into the
production of new eggs. Also time- and energy-consuming sexual
activity distracting both parents from caring for their offspring
should be avoided during this crucial time period of offspring
feeding. Reliably communicating the reproductive status of the
female to the male partner would therefore be advantageous for
both parents and offspring.
The objectives of the current study were to investigate (1) how
the resource trade-off between caring for current offspring and
producing additional ones is regulated in females, (2) whether
females signal their reproductive state to facilitate the
coordination of sexual activity and parental care and (3) how
the signal is reliably linked to fertility. Our ?rst experiments
addressed the question whether the fertility of female burying
beetles is—similar to mammals—depressed during the time of
offspring feeding and if this transient infertility is mediated by a
hormone. Therefore, we determined egg production, hormone
pro?les and gene expression pattern of offspring provisioning and
non-feeding females and manipulated the females’ hormone titres
experimentally. We demonstrate that females’ fertility is indeed
temporarily depressed when caring for nutritionally dependent
larvae and this infertility is directly or indirectly linked to juvenile
hormone (JH). In a previous study Engel et al.22 have shown that
during the time of intensive offspring tending males desist from
mating with their female partner. This observation and the
?nding that breeding females release a volatile chemical23 led us
to the hypothesis that females produce a chemical signal linked to
hormone production to coordinate sexual activity during periods
of parental care. By conducting quantitative chemical analyses, we
show that the emission of the volatile methyl geranate coincides
with the presence of nutritionally dependent larvae and high JH
levels. We further reveal that methyl geranate and JH production
are linked via a shared pathway implying that females advertise
their hormonal and therefore reproductive state. Finally,
by means of electrophysiology and behavioural assays we
demonstrate that methyl geranate serves as an antiaphrodisiac
inhibiting male mating attempts. Overall, our results uncover
mechanisms underlying parental care decisions, and illustrate
how a physiological interplay between hormone and pheromone
systems guarantees that both parents draw their attention towards
the existing young as long as they are needy.
Results
Egg production and JH III during parental care. To investigate
whether oviposition is suppressed during provisioning of young,
we provided females under three different parenting conditions
with a new carcass to determine whether egg laying can be
triggered. Females of the ?rst treatment group (without larvae)
had been allowed to lay eggs, but then were denied access to their
larvae before they were provided with a new carcass (see
Methods). Consequently, these females represented mothers that
did not engage in offspring provisioning. Females of the second
treatment group (old larvae) were caring for old, and already
nutritionally independent larvae (4 days after hatching), when
given a new carcass. Although these females had larvae, they also
represented non-feeding mothers. Females of the third treatment
group (young larvae) were caring for nutritionally dependent
larvae (1 day after hatching; Fig. 1a) when a new carcass was
provided. Hence, those females represented offspring provisioning mothers. Consistent with our hypothesis, mothers who were
denied access to their larvae as well as mothers caring for older
and already nutritionally independent larvae responded to the
new carcass with egg laying (Fig. 1b). In contrast, in the majority
of mothers with access to their newly hatched and therefore
NATURE COMMUNICATIONS | 7:11035 | DOI: 10.1038/ncomms11035 | www.nature.com/naturecommunications
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11035
a
b
A
% of females which laid eggs
100
A
60
40
20
0
B
Without larvae
Old larvae
Young larvae
d
250
JH acid methyltransferase**
***
200
Without larvae
JH esterase NS
With larvae
150
Larvae hatch
JH epoxide hydrolase 1 NS
*
Protein takeout-like **
100
Takeout jhbp like protein ***
*
50
Protein takeout **
0
0
1
2
3
4
5
6
7
Day in breeding cycle
8
Protein chiffon ***
9
Female sterile m3 ***
40
Chorion protein a *
RPKM value
Number of larvae
Female sterile nasrat ***
30
10
0
10
Vitellogenin **
5
Vitellogenin **
0
Vitellogenin **
–5
Acetone
Pyriproxyfen
Egg- & chorionrelated
R-T dna helicase recq4 ***
e
20
JH synthesis & regulation
Amount of JH III (ng)
c
80
With Without
larvae larvae
Figure 1 | Offspring production and JH III. (a) Female N. vespilloides feeding a larva (reproduced with permission from the author, Heiko Bellmann).
(b) Percentage of mothers laying eggs. Mothers were either denied access to their larvae after hatching (without larvae) or were caring for young needy
larvae (young larvae) or older nutritionally independent larvae (old larvae). Different letters represent signi?cant differences between groups at Po0.05
(w2-test following Benjamini–Hochberg; N ¼ 18-20). (c) JH III pro?le in ng per individual (mean±s.e.) over an entire breeding cycle. Females were either
allowed to care for their larvae (with larvae; N ¼ 191) or withheld from their hatched larvae (without larvae; N ¼ 186). There was an interaction effect of
treatment group and day (Gaussian GLM: F9,357 ¼ 9.91, Po0.0001). Note: on day 3 larvae hatched, but had not yet arrived on the carcass. (*Po0.05,
**Po0.01, ***Po0.001). (d) Expression patterns of genes (heatmap based on geometric means) related to JH III biosynthesis/regulation and ovarian
activity of females caring for larvae (day 4 in the breeding cycle) and females who were withheld from their larvae on hatching (day 4 in the breeding
cycle; for each group N ¼ 3). Transcript abundances are expressed as log2 transformed normalized values (RPKM). (Student’s t-test following
Benjamini–Hochberg; *Po0.05, **Po0.01, ***Po0.001). (e) Number of larvae produced by females treated with pyriproxyfen or acetone (control).
Boxplots show median, interquartile range, minimum/maximum range; N ¼ 19, Poisson GLM: Wald-w21;27 ¼ 18.34, Po0.0001.
needy larvae oviposition was signi?cantly suppressed (w2-test,
N ¼ 58, w22;56 ¼ 29.1, Po0.0001, Fig. 1b).
Juvenile hormone III (JH III), a multifunctional hormone of
insects, is well known to regulate female fertility by controlling
the biosynthesis of vitellogenin (Vg) and its uptake by the
growing oocyte24. JH III has been found to be elevated during
parental care in the nearctic burying beetle N. orbicollis15,25, but
its function remained unclear. In ants26,27, wasps28 and bees29
very high titres of JH or its analogues repress ovarian activity. We
thus hypothesized that high JH III levels are associated with
offspring feeding as well as suppression of ovarian activity and
offspring production in N. vespilloides females. We indeed found
that the JH III titres of mothers increased to very high levels
directly after the hatching of their larvae (Fig. 1c; Supplementary
Fig. 1). Corresponding to previous ?ndings in N. orbicollis30,
JH III peaked when offspring begging and provisioning is known
to be most intense (24 h after hatching, day 4 in the breeding
cycle)18,31, while it declined to pre-hatching levels when larvae
can feed independently from the carrion resource (72 h after
hatching, day 6 in the breeding cycle)18. In contrast, mothers
whose larvae hatched but were experimentally prevented from
accessing their offspring had signi?cantly lower JH III titres and
never reached the high levels of provisioning mothers (Fig. 1c;
Supplementary Fig. 1). Sixty-six per cent of these females
resumed egg laying, in contrast to zero per cent of offspring
provisioning mothers. To verify the link between JH III and
ovarian activity, we analysed the expression pattern of genes
associated with JH biosynthesis/regulation and ovarian activity.
Consistent with our prediction, offspring-provisioning mothers
showed signi?cantly higher expression of genes associated with
NATURE COMMUNICATIONS | 7:11035 | DOI: 10.1038/ncomms11035 | www.nature.com/naturecommunications
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JH III synthesis but lower expression of genes involved in JH
degradation and egg production than non-provisioning mothers,
whose larvae were withheld (Fig. 1d). Moreover, when females
were treated with pyriproxyfen (PPN), a potent JH analogue29,
reproduction was signi?cantly reduced, and females treated with
PPN produced fewer offspring than control females (Fig. 1e).
Consequently, our results strongly suggest that needy larvae
stimulate high JH levels in brood-caring mothers, which directly
or indirectly lead to oviposition suppression.
Linking hormone and pheromone production. During the time
when the offspring are still needy, not only egg production but
also costly male mating attempts distracting both parents from
caring for offspring should be avoided. Copulations, for example,
might divert the attention from protecting the offspring
against enemies and restrain from offspring feeding. Therefore,
reliably signalling their hormonal state to the male should be
advantageous for females. Fathers in turn should respond to the
signal by ceasing sexual activities, as they do not gain any bene?ts
from copulating during the female’s period of infertility. An
excellent candidate for such a reliable signal is methyl geranate,
a volatile that has been shown to be emitted by breeding females
but has been studied hitherto only in the context of partner
recognition23. Methyl geranate is structurally related to JH III
(Fig. 2a) and has been hypothesized to share the same
biosynthetic pathway as the hormone, although experimental
evidence is missing23. To test for a functional linkage of the
hormone system and methyl geranate biosynthesis, we injected
deuterium-labelled geranyl pyrophosphate32, a known precursor
of JH III in insects33 and putative key component in the
b
OH
Amount of methyl geranate (ng)
Mevalonate pathway
O
c
OPP
Geranyl diphosphate
OPP
Farnesyl diphosphate
OH
Farnesol
Geraniol
O
Farnesal
OH
Farnesoic acid
O
Methyl farnesoate
O
O
O
O
Juvenile hormone lll
Haemolymph
Corpora allata
O
Geranial
O
Geranic acid
OH
O
O
Methyl geranate
Amount of methyl geranate (ng)
a
postulated shared pathway (Fig. 2a), into offspring-provisioning
females. Subsequent headspace analyses revealed that
injected females emitted deuterium-labelled methyl geranate
(Supplementary Fig. 2). Consequently, the volatile substance is
indeed a suitable candidate for reliably re?ecting the female’s
hormone level and therefore her reproductive state, as it shares
the same metabolic pathway with the hormone. In fact, when
measuring JH III levels and released methyl geranate quantities of
females during an entire breeding cycle, we found a strong
correlation between the hormone and the volatile substance
(Pearson correlation, N ¼ 332, r ¼ 0.5, Po0.0001). In females
caring for larvae, methyl geranate emission tightly followed JH
levels and peaked along with the hormone titre at the time when
offspring begging and feeding is known to be most intense
(Fig. 2b). In contrast, mothers who were denied access to their
larvae emitted little or no methyl geranate (Fig. 2b). Furthermore,
methyl geranate emission closely matched the observed mating
pattern during a breeding cycle22. Males discontinue mating with
mothers during the time of offspring provisioning but do not stop
mating with females whose larvae had been withheld and
therefore resume egg laying22. To further con?rm that methyl
geranate reliably re?ects the female’s reproductive state, we
exploited the fact that females are less likely to resume egg laying
the more offspring they have to care for. An earlier study found
that one larva prevented about 20% of the mothers from
producing a new clutch, and more larvae prevented a
signi?cantly higher percentage34. Consequently, we manipulated
the amount of larvae a female had to care for and quanti?ed
methyl geranate emission. Indeed, methyl geranate quantity
reliably re?ected oviposition probability: the more larvae the
mother had to care for, the higher was the amount of methyl
50
***
40
Without larvae
With larvae
30
20
**
Larvae hatch
10
0
0
1
2
3
4
5
6
7
Day in breeding cycle
60
8
9
B
B
40
A
20
A
0
1
5
10
Number of larvae
20
Figure 2 | Methyl geranate biosynthesis and emission. (a) Putative biosynthesis of methyl geranate and its link to JH III. (b) Methyl geranate emission in
ng per individual per 20 min (mean±s.e.) over an entire breeding cycle. Females were either allowed to care for their larvae (with larvae; N ¼ 170) or
withheld from their hatched larvae (without larvae; N ¼ 169). There was an interaction effect of treatment group and day (Gaussian GLM: F9,319 ¼ 11.35,
Po0.0001). Note: on day 3 larvae hatched, but had not yet arrived on the carcass. (*Po0.05, **Po0.01, ***Po0.001). (c) Amount of methyl
geranate (mean±s.e.) emitted over a period of 20 min by mothers caring for 1, 5, 10 or 20 larvae. Different letters represent signi?cant differences
(Gaussian GLM following Benjamini–Hochberg; N ¼ 20–30).
4
NATURE COMMUNICATIONS | 7:11035 | DOI: 10.1038/ncomms11035 | www.nature.com/naturecommunications
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geranate emitted (Gaussian GLM, N ¼ 105, F3,101 ¼ 7.50,
Po0.001; Fig. 2c). Furthermore, those mothers that resumed
egg laying were characterized by a lower JH III titre than mothers
that did not engage in oviposition (N ¼ 103, mean±s.e.,
egg-laying mothers: 84.52±8.91 ng, non-egg-laying mothers:
122.17±8.63 ng; Gaussian GLM, F1,101 ¼ 4.29, P ¼ 0.04).
Function of methyl geranate as anti-aphrodisiac. Having
established that methyl geranate reliably re?ects hormone
level and consequently the reproductive state of females, we
further evaluated its putative function as an anti-aphrodisiac,
a chemical substance that inhibits copulation35,36. Using gas
chromatography coupled with electroantennographic detection
(GC-EAD), we found that male antennae respond to synthetic
methyl geranate (Fig. 3a). Furthermore, we observed the
copulation behaviour of pairs of breeding N.

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