Received: 31 May 2022 Revised: 29 August 2022 Accepted: 19 October 2022

DOI: 10.1111/phen.12398

ORIGINAL ARTICLE

Between-partner concordance of vertically transmitted gut

microbiota diminishes reproductive output in the dung beetle
Onthophagus taurus

Anna L. M. Macagno | Armin P. Moczek

Department of Biology, Indiana University, Abstract

Bloomington, Indiana, USA
Correspondence
Anna L. M. Macagno, Department of Biology,
Indiana University, 915 East Third Street,
102 Myers Hall, Bloomington, IN 47405-7107,
USA.
Email: amacagno@indiana.edu
Funding information
John Templeton Foundation, Grant/Award
Number: 61369; National Science Foundation,
Grant/Award Number: 1901680
In this study, we explored the potential contribution of the gut microbiome to reproduc-
tive isolation in tunnelling dung beetles, using Onthophagus taurus (Schreber, 1759) and
its sister species O. illyricus (Scopoli, 1763) as a model system (Coleoptera: Scarabaeidae:
Scarabaeinae: Onthophagini). Gut microbiota play critical roles in normative develop-
ment of these beetles, and are vertically inherited via a maternally derived faecal pellet
called the pedestal. We first compared the developmental outcomes of individuals reared
with pedestals derived from either the same or the sister species (Self and Cross inocula-
tion treatments, respectively). We then crossed the resulting adult O. taurus in three
combinations (Self female X Self male; Self female X Cross male; Cross female X Self male).
We predicted that if the vertically transmitted gut microbiome plays a role in reproduc-
tive isolation by facilitating species recognition, the Self X Self line would have improved
reproductive outcomes compared to the lines in which partners had mismatched gut
microbiomes. Instead, we found that between-partner concordance of maternally trans-
mitted gut microbiota resulted in fewer offspring, and that this reduction was due to par-
tial pre-copulatory isolation as evidenced by reduced sperm transfer in the Self X Self
line. This pattern is consistent either with microbiome-mediated familiarity/kin recogni-
tion, or with absence of mate choice in crosses with mismatched microbiomes. We dis-
cuss our results in the light of recent research on the influence of extracellular microbial
symbionts over insects’ mating preferences.

KEYWORDS
kin recognition, mate choice, microbial symbionts, Onthophagus illyricus, prezygotic isolation,
reproductive isolation

I N T R O D U CT I O N ranging from nutrient metabolism, immunological and physiological

Microbial symbionts and their hosts are increasingly recognized to
interact in ways able to shape each other’s evolutionary history (Gilbert
et al., 2012; Lim & Bordenstein, 2020; McFall-Ngai et al., 2013). Among
insects, the gut microbiome in particular contributes to host fitness by
playing diverse roles in several fundamental physiological functions,
homeostasis, detoxification, and cuticle synthesis (Engl & Kaltenpoth,
2018; Jang & Kikuchi, 2020; Lee et al., 2017; Ma et al., 2021; Pernice
et al., 2014). Interestingly, insect gut-inhabiting microbes often exhibit
phylosymbiosis, i.e. “patterns of microbiome composition that can be
predicted by host phylogeny” (Lim & Bordenstein, 2020; Mallott &
Amato, 2021). Several mechanisms have been proposed as drivers of

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provided the original work is properly cited.
© 2022 The Authors. Physiological Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.

14 wileyonlinelibrary.com/journal/phen Physiological Entomology. 2023;48:14–23.


SYMBIONTS AFFECT DUNG BEETLE MATING RATE
such patterns, including microbial effects on host viability and
selection—instances where microbial symbionts affect host physiology in
ways that alter host responses to selective pressures, thereby shaping
host evolution (Mallott & Amato, 2021). In principle, these associations
should be particularly evolutionarily consequential in the context of
reproduction, potentially leading to host diversification and ultimately
speciation. For example, Morimoto et al. (2017) found both direct and
transgenerational effects on mating and reproduction following gut
microbiota manipulations in Drosophila melanogaster. Strikingly, the gut
microbiome was also found to be responsible for hybrid breakdown in
Nasonia parasitoid wasps (Brucker & Bordenstein, 2013). However, no
clear evidence of a direct, post-zygotic effect of extracellular gut
microbes on hybrid viability has been found in other insects so far (Miller
et al., 2021). At the same time, a growing research body has focused on
microbe-induced traits that associate with changes in the hosts’ behav-
iour, with the potential to create premating or post-mating-prezygotic
barriers to interbreeding (reviewed in Shropshire & Bordenstein, 2016;
Engl & Kaltenpoth, 2018). In this context, the gut microbiome has been
found to affect mating preference in a few select host systems. For
example, Lizé et al. (2014) provided evidence for a role of gut bacteria in
kin recognition in Drosophila. Here, the microbiome appears to affect
copulation preference as a function of familiarity (D. bifasciata) and diet
(D. melanogaster), potentially via changes in the composition of the hosts’
cuticular hydrocarbon compounds (García-Roa et al., 2022; Lewis et al.,
2014). Intriguingly, Sharon et al. (2010, 2011) also showed that some
components of the gut microbiome of two D. melanogaster lines reared
on different food sources were responsible for assortative mating—an
observation that hints at a potential role for microbial symbionts in
reproductive isolation and ecological speciation. However, the signifi-
cance of these findings for host divergence and speciation is debated,
because associations between host and gut microbes in Drosophila are
rather labile and mainly driven by diet, which varies across an individual’s
lifetime. An evolutionary host/gut microbiota association that has the
potential to initiate reproductive isolation, therefore, appears more likely
in hosts where there is either stable vertical transmission of gut-
associated microbial symbionts, or recurrent exposure to parental gut
microbes in each subsequent generation (Leftwich et al., 2017, 2018), as
has recently been documented for several dung beetle species.
Tunnelling dung beetles (Scarabaeidae: Scarabaeinae) mainly uti-
lize mammalian dung for feeding and breeding, relying on their gut
microbiome to digest their recalcitrant diet (Ebert et al., 2021; Estes
et al., 2013; Frank et al., 2017; Schwab et al., 2016; Schwab et al.,
2017; Shukla et al., 2016). In the Onthophagini tribe, which encom-
passes high species diversity including many closely-related species
complexes (e.g. Breeschoten et al., 2016; Génier & Moretto, 2017;
Joaqui et al., 2019; Macagno et al., 2011; Moctezuma & Halffter,
2020; Pizzo et al., 2006; Roy et al., 2016), dams provision dung for
developing offspring in the form of buried brood balls. Within each
brood ball, mothers position a single egg on top of a pedestal—a
maternal faecal secretion which is consumed by newly hatched larvae
(Estes et al., 2013; Halffter & Edmonds, 1982). Onthophagini dung
beetles thus provide a promising model system to investigate the con-
servation and diversification in host–microbiome interactions at the
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15
early stages of macroevolutionary divergence (Parker et al., 2021), and
the effects of the vertically transmitted gut microbiome on host repro-
duction. Recent work has shown that (i) the microbiota contained in
the pedestal are crucial for the offspring’s normative development and
fitness, especially under stressful conditions (Schwab et al., 2016); that
(ii) the benefits conferred to the offspring are at least partially species-
specific, as inoculation with a different species’ pedestal fails to fully
rescue the fitness of developing hosts (Parker et al., 2019, 2021); and
that (iii) adult gut microbiota composition is shaped by both the local
environment as well as the maintenance of a putative core microbiome,
providing partial evidence of phylosymbiosis (Ebert et al., 2021;
Mallott & Amato, 2021; Parker et al., 2020). Lastly, well-documented
occurrences of kleptoparasitism in this tribe (e.g. Cambefort & Hanski,
1991; Crowe et al., 2013; Davis & Huijbregts, 2000; Emlen et al., 2005;
Martín Piera & Lobo, 1993) implicate that larval development in the
presence of an alien gut microbiome—and its potential consequences
on reproduction—are likely to occur in the wild.
In this study, we leveraged a well-characterized Onthophagus sis-
ter species pair—O. taurus and O. illyricus—to start exploring the
potential contribution of the vertically transmitted gut microbiome to
reproductive isolation in tunnelling dung beetles. Although ecologi-
cally and phenotypically similar, and frequently co-occurring within
the same dung pads in areas of sympatry (Macagno et al., 2016), these
sister species last shared a common ancestor 3–4 Mya, and do not
appear to hybridize in the wild (Pizzo et al., 2006). To explore whether
the gut microbiota may be contributing to host reproductive isolation
in Onthophagus, we performed reciprocal gut microbiome transplants
between O. taurus and O. illyricus and subsequent controlled crosses.
First, we compared the developmental outcomes of beetles reared
with pedestals derived from either the same (self-inoculated, Self ) or
the sister species (cross-inoculated, Cross). We then crossed self- and
cross-inoculated O. taurus in three combinations (Self female X Self
male; Self female X Cross male; Cross female X Self male). We pre-
dicted that if the vertically transmitted gut microbiome plays a role in
reproductive isolation by facilitating species recognition (e.g. Fischer
et al., 2017), the line in which both partners were provided an
O. taurus pedestal would have improved reproductive outcomes com-
pared to those in which partners had mismatched gut microbiomes.
Alternatively, reproductive isolation via familiarity/kin recognition
(Lizé et al., 2014) may manifest in improved reproductive outcomes of
the lines pairing self-inoculated with cross-inoculated partners.
M A T E R I A L S A N D M ET H O D S
Beetle collection and common garden rearing
Adult Onthophagus illyricus and O. taurus F0 were field-collected in
June 2021 on cattle pastures in central Italy (Pian di Rolla, Scheggia e
Pascelupo, Province of Perugia) and North Carolina, USA (Maple View
Farm, Hillsborough), respectively. Beetles were shipped to Blooming-
ton, IN, and kept at 24 C and 16:8-h light–dark cycle in monospecific
laboratory colonies provided with standardized soil (one part sifted

16
topsoil, two parts sand) and antibiotic-free cow dung ad lib., as
described in Macagno et al. (2015). All soil utilized in rearing was pre-
viously sterilized in autoclave at 121 C for 60 minutes. Dung from
grass-fed cattle was collected in the spring and summer 2021 from
pastures within Marble Hill Farm (Monroe Co, IN) and preserved at
20 C for at least four weeks. Prior to the experiment, dung was
thawed and thoroughly homogenized using a hand-held cement mixer
to avoid differences in offspring size and fitness that might be due to
uncontrolled differences in food quality (Hunt & Simmons, 2002).
First generation: Developmental outcomes of self- and
cross-inoculated Onthophagus taurus and O. illyricus
After a two-month acclimation period within laboratory colonies, F0
females of both species were transferred to individual 1.4 L polypro-
pylene containers (Stor-All solutions, 27 cm  8 cm  8 cm) filled
with soil to a height of 21 cm and provided with 200 g dung, and
allowed to produce brood balls for five days. Each female was marked
individually as in Macagno et al. (2021) and used in 1–4 weekly exper-
imental rounds, until egg depletion, over the span of seven rounds in
total. Twenty-one (out of 46) O. illyricus and 23 (out of 39) O. taurus
females produced brood balls containing an egg. Following previously
established protocols (Parker et al., 2019, 2021; Schwab et al., 2016),
we sifted brood balls out of the soil, opened them, and extracted F1
eggs and pedestals with flame-sterilized tweezers and scalpels,
respectively. We treated eggs with 150 μl of a sterilizing solution of
1% bleach (final concentration 0.06% sodium hypochlorite) and 0.1%
Triton-X 100 in deionized water, and rinsed them twice with 1 ml of
deionized water. We then placed surface-sterilized eggs individually
at the centre of standardized, artificially constructed brood balls
within 12-well plates (Shafiei et al., 2001), providing them with either
a pedestal from a different, conspecific mother (self-inoculation, Self )
or that of a mother of the sister species (cross-inoculation, Cross).
Eggs, pedestal donors, and individual wells within plates were

assigned at random. Plates were stored at 24 C throughout beetle
development and shuffled at least twice a week within the incubator
to also randomize any potential microclimatic variation. Individuals
were checked daily until egg hatching or failure, and then weekly on
days 1, 3, and 5, starting seven days before adult emergence was
expected. We measured body size of all newly emerged F1 adults as
the width of the prothorax to the nearest 0.01 cm, using a digital calli-
per. We then marked O. taurus by treatment, by clipping the inner dis-
tal corner of either the left (Self ) or right elytron (Cross). We pooled
marked adults in 8.5 L single-sex maturation containers with moist soil
and unlimited access to dung for a minimum of 10 days.
Second generation: Reproductive outcomes of crosses
among self- and cross-inoculated Onthophagus taurus
To explore whether between-partner concordance of vertically trans-
mitted microbiome may influence reproductive outcomes, we crossed
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MACAGNO AND MOCZEK
F I G U R E 1 Experimental design. Onthophagus taurus F1
developed in the presence of either the maternally transmitted
microbiome of an O. taurus mother different than their own (self-
inoculated, self ), or of that of an O. illyricus mother (cross-inoculated,
Cross). (a) Three or four mature, virgin F1 females and males were
housed in three replicate mating containers per line (SelfXSelf,
SelfXCross, CrossXSelf ) for at least one week. (b) Over the course of
two consecutive ovipositing rounds, one or two dams from each
replicate were transferred into individual 1.4 L containers filled with
moist soil, provided with unlimited access to dung and allowed to
construct brood balls for either four (round 1) or seven days (round 2).
(c) All brood balls (BBs) produced were harvested and incubated in
individual 30 ml containers filled with moist soil. Upon adult
emergence, brood balls were removed and the F2 offspring left in
moist soil without access to food. Number of brood balls generated
and F2 offspring are reported for each line
O. taurus that were either self-inoculated with an O. taurus pedestal
(Self ) or cross-inoculated with an O. illyricus pedestal (Cross) following
the design outlined in Figure 1. We had originally planned to also exe-
cute the reciprocal approach in O. illyricus after a period of artificially-
induced diapause (obligate in this species but not in North Carolinian
O. taurus; pers. obs.; Parker et al., 2021). However, higher-than-
anticipated mortality reduced individual availability to a degree that
prevented us from carrying out this portion of the original study
design. Instead, three or four mature, virgin F1 female and male
O. taurus were housed in three replicate 1 L mating containers per line
(SelfXSelf: 12 females in total; SelfXCross: 10 females; CrossXSelf:
12 females) for at least one week. Mating containers were one-third
filled with moist soil and provided with 200 g dung that was replaced
once a week. Over the course of two consecutive ovipositing rounds,
one or two dams from each replicate were transferred into individual
containers set up as described above, and allowed to construct brood
balls for either four (round 1) or seven days (round 2). We measured
dam’s body size to the nearest 0.01 cm with a digital calliper, as the
width of the prothorax. Each dam was used either in the first or in the
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SYMBIONTS AFFECT DUNG BEETLE MATING RATE 17

second ovipositing round—never in both—and frozen at 20 C there- First generation

after. All brood balls produced were harvested, cleaned of soil, and
weighed to the closest 0.001 g using a Mettler Toledo (AL 54) scale.

We incubated each brood ball at 24 C, in a 30 ml container (SOLO
cup) filled with moist soil, and checked them weekly on days 1, 3, and
5 for emergence. Upon adult emergence, F2 offspring were weighed
to the closest 0.0001 g and measured with a calliper as above. Brood
balls were removed and adult beetles left without access to food in
moist soil, to prevent dehydration. We checked adults weekly on days
1, 3, and 5 until death. Dams that did not produce offspring were sub-
sequently thawed and dissected in phosphate buffered saline (PBS) to
check for the presence of sperm in their reproductive tract. To do so,
their spermatheca and bursa copulatrix were ruptured in 10 μl PBS on
a microscope slide, covered with a cover slip, and observed under a
Nikon Labophot-2 microscope at x200 (Appendix S1).
Data analysis
We used the lme4 package (Bates et al., 2015) in R 3.5.2 (R Core
Team, 2018) to fit general/generalized linear mixed models to our
data, specifying the appropriate link function (identity for continuous
variables, that is, size, mass, time; binomial for binary variables, that is,
survival, sperm transfer, sex ratio; Poisson for count variables;
Crawley, 2007). We obtained type II ANOVA tables with the Anova()
function in the car package (Fox & Weisberg, 2019), and summary
tables for model fits with the lmerTest package (Kuznetsova
et al., 2017).
Survival to adult eclosion, development time (time elapsed between
egg transfer and adult eclosion), body size (measured as prothoracic
width), and sex ratio of the F1 were modelled as a function of species,
inoculation treatment (Self vs Cross), and their interaction. We entered
maternal identity and twelve-well plate nested within experimental
round as random factors.
Second generation
We modelled sperm transfer (as evidenced by presence of spermato-
zoa in the female F1 reproductive tract, or successful reproduction) as
a function of line (SelfXSelf; SelfXCross; CrossXSelf ). Brood ball
(BB) mass, BB count, and F2 count were modelled as a function of line
and maternal body size. Mass at emergence, development time (time
between F1 setup in ovipositing container and adult F2 emergence),
time survived as adult, and body size of the F2 were modelled as a
function of line, F2 sex, and their interaction, with BB mass as a covar-
iate. Ovipositing round was included as a random factor in all models.
For traits with only one measure per dam (BB and F2 count, sperm
transfer), we entered mating container replicate as a random factor. In
the analysis of all traits including multiple measures from each dam,
we grouped observations by dam nested within mating container
replicate.
To avoid overfitting, random factors explaining 0 variance were
removed starting from the higher order (Barr et al., 2013; Pasch et al.,

T A B L E 1 Coefficients of statistical models testing for the significance of species (O. taurus, O. illyricus) and inoculation treatment (inoculated
with a pedestal from a mother of the same species—Self—or from a mother of the sister species—Cross) on developmental outcomes of F1 reared
on standardized food quality/quantity. Rows show chi-square (χ 2) test statistics, resulting test probabilities (P), and estimated effect sizes (β) plus
or minus standard error (SE) for each response variable

F1 developmental outcome Species Inoculation

Survival to adulthood all individuals (Y/N) χ2 10.849 0.392

P <0.001 0.531
β  SE 0.929  0.285 (taurus) 0.190  0.305 (Self )
Survival to adulthood excluding unhatched eggs (Y/N) χ2 0.61164 0.19625
P 0.4342 0.6578
β  SE 0.313  0.398 (taurus) 0.188  0.428 (Self )
Development time (days) χ2 21.383 17.078
P <0.0001 <0.0001
β  SE 2.205  0.477 (taurus) 1.639  0.3965 (Self )
Body size (mm) χ2 109.339 0.001
P <0.0001 0.972
β  SE 0.333  0.032 (taurus) 0.001  0.032 (Self )
Sex ratio (F/M) χ 2
1.091 0.006
P 0.296 0.939
β  SE 0.307  0.294 (taurus) 0.023  0.300 (Self )

Note: Notations in parentheses following β  SE estimates reflect the change in value from one category to another (e.g. –2.205  0.477 (taurus) means
that “development time” decreases by 2.205  0.477 days in O. taurus, when compared to O. illyricus). Significant results are reported in bold type.
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18 MACAGNO AND MOCZEK

2013). We simplified the resulting models by removing nonsignificant
interactions as determined by χ 2 tests (Bolker et al., 2009). Final
models are included in Appendix S1.
RESULTS
First generation: Developmental outcomes of self- and
cross-inoculated Onthophagus taurus and O. illyricus
When reared in artificial brood balls made of dung obtained from
exclusively grass-fed cattle, Onthophagus taurus survived overall at a
higher rate, attained larger adult body size, and had shorter develop-
ment time than its sister species O. illyricus, regardless of inoculation
treatment (Table 1). The lower survival of O. illyricus was mainly due
to lower egg hatching rate, because we detected no between-species
difference in survival once unhatched eggs were removed from the
analysis (Table 1). Final sex ratio was approximately 50–50 in both
species (Table 1). In keeping with previous results (Parker et al., 2019,
2021), cross-inoculation significantly lengthened development in both
species (Table 1).
Second generation: Reproductive outcomes of crosses
among self- and cross-inoculated Onthophagus taurus
In line with earlier studies (Hunt & Simmons, 2002), maternal body
size positively influenced brood ball mass (Table 2). Higher brood ball

T A B L E 2 Coefficients of statistical models testing for the significance of line (SelfXSelf; SelfXCross; CrossXSelf) and other predictors (dam’s
body size, F2 sex, brood ball mass) on reproductive outcomes. Rows show chi-square (χ 2) test statistics, resulting test probabilities (P), and
estimated effect sizes (β) plus or minus standard error (SE) for each response variable

Reproductive Maternal
outcome Line body size F2 sex BB mass

Sperm transfer, χ2 16.886 NA NA NA

F1 (Y/N) P <0.001 NA NA NA
β  SE 39.08  12.34 (SelfXSelf ), 13.94  14.49 (SelfXCross) NA NA NA
BB mass (g) χ2 0.241 4.387 NA NA
P 0.887 0.036 NA NA
β  SE 0.138  0.389 (SelfXSelf ), 0.052  0.362 (SelfXCross) 1.329  0.635 NA NA
BB count χ2 8.331 3.531 NA NA
P 0.015 0.06 NA NA
β  SE 0.704  0.346 (SelfXSelf ), 0.308  0.320 (SelfXCross) 1.178  0.627 NA NA
F2 mass at χ2 4.9106 NA 0.197 15.062
emergence (g) P 0.086 NA 0.657 <0.001
β  SE 0.002  0.003 (SelfXSelf ), 0.003  0.002 (SelfXCross) NA 0.001  0.001 0.004  0.001
(male)
F2 count χ2 10.493 3.261 NA NA
P 0.005 0.071 NA NA
β  SE 0.859  0.329 (SelfXSelf ), 0.207  0.294 (SelfXCross) 1.138  0.630 NA NA
F2 development χ2 0.746 NA 11.633 3.826
time (days) P 0.689 NA <0.001 0.05
β  SE 0.234  0.497 (SelfXSelf ), 0.200  0.414 (SelfXCross) NA 1.031  0.302 0.419  0.214
(male)
F2 body size (mm) χ2 4.394 NA 0.007 16.723
P 0.111 NA 0.934 <0.001
β  SE 0.078  0.077 (SelfXSelf ), 0.084  0.064 (SelfXCross) NA 0.002  0.032 0.101  0.025
(male)
F2 adult survival to χ2 3.178 NA 2.49 1.383
starvation (days) P 0.204 NA 0.115 0.24
β  SE 0.756  0.662 (SelfXSelf ), 0.954  0.558 (SelfXCross) NA 0.575  0.365 0.313  0.266
(male)

Note: Notations in parentheses following β  SE estimates reflect the change in value from one category to another (e.g. when compared to the CrossXSelf
line, F2 count of the SelfXSelf line decreases by 0.859  0.329 individuals, whereas the SelfXCross line does not differ significantly). Significant results are
in bold type.
Abbreviation: NA, not available (predictor not included in model).

SYMBIONTS AFFECT DUNG BEETLE MATING RATE
F I G U R E 2 Presence (dark grey, Y) or absence (light grey, N) of
sperm transfer in females of each line, expressed as percentage.
Successful sperm transfer was detected either by the presence of
sperm in the F1 dam’s genital tract, or by successful reproduction.
Sperm transfer occurred less often in the SelfXSelf line (*p < 0.001,
Table 2). Twelve females were included in both the SelfXSelf and the
CrossXSelf lines, whereas the SelfXCross line included 10 females
F I G U R E 3 Number of brood balls (top panel) and offspring
(bottom panel) produced by each line. Dams in the SelfXSelf line
constructed fewer brood balls (*p < 0.05, Table 2) and produced
fewer offspring (*p < 0.01, Table 2)
mass resulted in higher mass at emergence, larger body size, and lon-
ger development time in the offspring—with males taking longer than
females to develop to adulthood (Table 2). Intriguingly, and contrary
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19
to our initial prediction, sperm transfer occurred less often in self-
inoculated females paired with self-inoculated males (SelfXSelf line)
than in the two lines in which partners had a mismatched microbiome
(SelfXCross and CrossXSelf ) (Figure 2, Table 2). This resulted in fewer
brood balls and fewer offspring produced by the SelfXSelf line
(Figure 3, Table 2). By contrast, adult survival to starvation did not
appear to be influenced by any of the predictors included in the model
(Table 2).
DI SCU SSION
In this study, we aimed to investigate whether the vertically transmit-
ted gut microbiome may be involved in reproductive isolation in
Onthophagus beetles (Scarabaeinae). To do so, we reared O. taurus in
the presence of either the pedestal-derived, maternally transmitted
gut microbiota of the same species (self-inoculated), or that of its sis-
ter species, O. illyricus (cross-inoculated). We then crossed these indi-
viduals in three combinations (self-inoculated female X self-inoculated
male; self-inoculated female X cross-inoculated male; cross-inoculated
female X self-inoculated male). We predicted that if the microbiome
plays a role in reproductive isolation via species recognition, the line
in which both partners were provided with an O. taurus microbiome
would have improved reproductive outcomes compared to those in
which partners had mismatched microbiomes. Instead, we found the
opposite outcome. Between-partner concordance of the maternally
transmitted gut microbiome resulted in fewer offspring due to partial
pre-copulatory isolation. We discuss our results, the limitations of our
study, and the future directions they indicate in the light of the grow-
ing body of work on the influence of extracellular microbes on mating
preferences in insects.
Inoculation with a species-specific, maternally
transmitted gut microbiome promotes normative
development
Previous studies showed that (i) Onthophagini reared in the presence
of a vertically transmitted gut microbiota of a different host species
had worse developmental and fitness outcomes than those reared
with their own, that (ii) these detrimental effects appeared of greater
magnitude when the microbiome was exchanged between distantly
related species (Parker et al., 2019, 2021), and that (iii) the contribu-
tion of the gut microbiome to the maintenance of normative develop-
ment appears especially important under stressful rearing conditions
(Schwab et al., 2016). Specifically, Parker et al. (2021) showed that
inoculation with the gut microbiome of a different host species slows
growth in the third larval instar—a period critical for mass gain
(Moczek & Nijhout, 2002)—leading to subsequent developmental
delays as cross-inoculated larvae likely spend more time feeding to
become competent to initiate pupation (Shafiei et al., 2001). Despite
the relatively benign rearing conditions used in our study (constant
optimal temperature, individuals left undisturbed during development,

20
high quality nutrition: Rohner & Moczek, 2021), the phylogenetic
closeness between hosts, and the randomization of pedestal donors
resulting in all individuals developing with a gut microbiota other than
their own mother’s, both Onthophagus taurus and O. illyricus showed
significantly slowed development following cross-inoculation. Our
results, therefore, further support the role of vertically transmitted
dung beetle microbiota as host species-specific nutritional symbionts
(Estes et al., 2013; Parker et al., 2021; Schwab et al., 2016; Shukla
et al., 2016).
Between-partner microbiome concordance causes
partial pre-copulatory isolation in Onthophagus taurus
The insect gut microbiome has been shown to facilitate the mainte-
nance of sister species within the same ecological niche in phytopha-
gous beetles (Zhang et al., 2020). Here, we explored the possibility
that the vertically transmitted gut microbiome may also be involved in
host mating preference, predicting elevated mating success in
instances in which hosts possessed matching microbiota. Contrary to
our predictions, we found that both Onthophagus taurus lines in which
dams and sires had developed in the presence of mismatched
pedestal-derived gut microbiomes (Self X Cross, Cross X Self ) produced
more offspring than the line in which both partners had developed in
the presence of O. taurus gut microbiota (Self X Self ), and that this
effect was due to reduced mating frequency in the Self X Self cross.
Specifically, between-partner microbial concordance resulted in partial
pre-copulatory isolation, as evidenced by lack of sperm transfer in
approximatively 30% of Self females paired with Self males. These
observations thus reject the hypothesis that the gut microbiome
transmitted via the pedestal may facilitate mating preference via spe-
cies recognition in this system. Because O. taurus is known to faculta-
tively parasitize the brood balls of other dung beetle species in the
wild (e.g. Martín Piera & Lobo, 1993), an evolved role for pedestal-
transmitted microbes in species recognition may indeed be unlikely in
this species.
Instead, our results are consistent with the possibility that the gut
microbiome may mediate familiarity/kin recognition (Lizé et al., 2013,
2014), to the extent that mating avoidance between individuals with
similar microbial profiles may promote outcrossing. Interestingly, in
Drosophila, the gut microbiota appears to influence kin recognition via
modifications of cuticular hydrocarbons, thereby shaping the scent
profiles used in mate choice (García-Roa et al., 2022; Lewis et al.,
2014). Bacterial symbionts support cuticle synthesis and have the
potential to interfere with the host’s cuticular hydrocarbon profile in
several insects, including beetles (Engl & Kaltenpoth, 2018). Further
studies are needed to confirm this possibility in dung beetles
specifically.
Importantly, if the gut microbiome is found to influence assorta-
tive mating via modifications of the host’s cuticular hydrocarbon pro-
file as shown in Drosophila (Sharon et al., 2010, 2011), a second
mechanism may also contribute to explain the patterns found in our
study. In O. taurus, sexual selection is characterized not only by
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MACAGNO AND MOCZEK
male–male competition (Moczek & Emlen, 2000), but also by female
choice based on both a male’s courtship behaviour (Kotiaho et al.,
2001) and cuticular hydrocarbon profile (Berson et al., 2019;
Berson & Simmons, 2018). If females are able to choose whether or
not to mate only in cases where there is concordance of vertically
transmitted microbiota between partners, female choice may manifest
as the observed decreased mating rate in the Self X Self line, where
both partners developed in the presence of the gut microbiota derived
from O. taurus. The absence of female choice would instead translate
into higher mating frequency in the mismatched (Self X Cross and
Cross X Self ) lines, even though these matings may yield poorer repro-
ductive outcomes. Intriguingly, even though the Self X Self line did
produce fewer offspring, those offspring possessed higher mass at
emergence—a trait linked to higher fitness (Macagno et al., 2015). This
trend was however only close to significance (Table 2) and should,
therefore, be confirmed in future studies.
Limitations of the study and future directions
The scope of our study was limited by low fecundity of field-collected
beetles and high mortality throughout development and sexual matu-
ration of experimental animals, especially in Onthophagus illyricus. Our
egg sterilization treatment followed by re-inoculation with a different
dam’s pedestal may not have replenished symbiont microbial presence
to the extent observed in natural vertical transmission, with detrimen-
tal effects throughout the experimental individuals’ life cycle. Because
of these constraints, we were unable to execute a reciprocal design in
O. illyricus, and had to forgo establishing a Cross X Cross line, which
would have allowed us to corroborate our results on the reproductive
outcomes of microbiome-matched vs mismatched partners. Specifi-
cally, observing lower fecundity in a Cross X Cross line would have fur-
ther supported a contribution of the microbiome to familiarity/kin
recognition. By contrast, observing elevated fecundity in a Cross X
Cross line would have provided additional support for the hypothesis
of microbiome-mediated mate choice ability that becomes disrupted
when either one or both partners receive a mismatched microbiota.
We were also prevented from exploring the possibility that the verti-
cally transmitted microbiome may affect hybridization between these
Onthophagus sister species (Brucker & Bordenstein, 2013). These
directions thus remain to be probed in future studies, including addi-
tional species and using larger sample sizes. Further, although we did
not find evidence for a contribution of pedestal-derived microbes in
host species recognition in the present study, this result was obtained
by exchanging pedestals between O. taurus and O. illyricus collected in
disparate geographical regions (North Carolina, USA and Italy, respec-
tively). Because these populations never come into contact in nature,
the implications for reproductive isolation in sympatry clearly warrant
further investigation. Finally, reciprocal microbial transplants would
only allow to experimentally validate narrow-sense symbiont-induced
reproductive isolation, that is, host-microbe or microbe-microbe asso-
ciations resulting in immediate reproductive barriers that can be
ameliorated by eliminating or replacing symbionts. Host genetic

SYMBIONTS AFFECT DUNG BEETLE MATING RATE
divergence and reproductive isolation evolved instead in response to
selection to accommodate microbial symbionts (broad-sense symbiont-
induced R.I.) could still be present and go undetected with such
transplant experiments, because in this case, microbial alterations are
not expected to affect mating preference (Shropshire & Bordenstein,
2016). Despite its limitations, however, our study provides the first
evidence for an effect of microbial concordance between partners on
reproductive success in the Onthophagini. Molecular-based investiga-
tions into the composition and vertical transmission of pedestal-
inoculated microbiota are currently underway and are poised to shed
more light on which microbial symbionts may be causally involved in
such patterns. Further studies are also clearly needed to elucidate any
potential relationship between gut microbiome, cuticular hydrocarbon
profile, and mate choice in these beetles.
C O N CL U S I O N S
Progressing towards a holistic understanding of how organisms and
associated microbes are able to influence each other’s evolutionary
history is at the forefront of integrative biology (Miller et al., 2021).
However, experimental microbiome manipulations to assess the con-
tribution of symbionts on hosts’ reproductive isolation and ultimately
speciation have been limited to select few study systems so far
(reviewed in Shropshire & Bordenstein, 2016; Miller et al., 2021).
Additionally, studies performed in highly artificial germ-free conditions
(e.g. Brucker & Bordenstein, 2013) may provide only limited insight
into how microbial symbionts come to influence reproductive isola-
tion in the wild.
Prior studies have shown that Onthophagini dung beetle species
rely on host species-specific maternally-transmitted microbiomes to
support their development and enhance their survival (Parker et al.,
2019). Such disparate, non-equivalent host–microbiota associations
appear to be maintained even in closely-related host species, with
longstanding coexistence in the same geographical areas and overlap-
ping autecology (Parker et al., 2021). Here, we have shown for the
first time that such pedestal-derived microbiota can also affect the
hosts’ pre-copulatory isolation and reproductive output. With its
extraordinary species diversity, including many closely-related line-
ages that could presumably still hybridize (e.g. Breeschoten et al.,
2016; Génier & Moretto, 2017; Joaqui et al., 2019; Moctezuma &
Halffter, 2020; Pizzo et al., 2006; Pizzo et al., 2013; Roy et al., 2016),
the Onthophagini tribe thus emerges as an ideal microcosm to investi-
gate the role of microbial symbionts in mate attraction, mate choice,
aggregation, and kin recognition (Engl & Kaltenpoth, 2018). Further
research in this system, therefore, promises to offer valuable insights
into the potential of microbial symbionts to contribute to reproductive
isolation and evolution of their hosts.
ACKNOWLEDGMENTS
We would like to extend our gratitude to Maura Bocci and Tracy
 n-Rettig for collecting the beetles used in this study; to Levi Bur-
Ledo
dine, Mikayla Higgins, and Jackson Norman for beetle husbandry; to
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21
Whitney and Kip Schlegel, for granting us access to their farm and cat-
tle dung therein; to Erik Parker (Indiana University Biostatistics Con-
sulting Center) for helpful discussions over the experimental design
and data analysis; and to two reviewers and the associate editor for
constructive feedback on a previous draft. This work was funded in
part by the National Science Foundation (grant 1901680) and the
John Templeton Foundation (grant 61369). The opinions, interpreta-
tions, conclusions, and recommendations are the authors’ own and
are not necessarily endorsed by the National Science Foundation nor
the John Templeton Foundation.
ET HIC STATEMENT
Onthophagus beetles were obtained for research use in accordance
with USDA-APHIS guidelines.
CONFLIC T OF INT ER E ST
The authors declare no conflict of interest.
DATA AVAILABILITY STAT EMEN T
The data that support the findings of this study are available from the
corresponding author upon request.
ORCID
Anna L. M. Macagno https://orcid.org/0000-0003-4133-2098
Armin P. Moczek https://orcid.org/0000-0002-3478-9949
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SUPPORTING INF ORMATION
Additional supporting information can be found online in the Support-
ing Information section at the end of this article.
Appendix S1: Supporting Information
How to cite this article: Macagno, A.L.M. & Moczek, A.P.
(2023) Between-partner concordance of vertically transmitted
gut microbiota diminishes reproductive output in the dung
beetle Onthophagus taurus. Physiological Entomology, 48(1),
14–23. Available from: https://doi.org/10.1111/phen.12398