JHR 45: 125-130 (2015) geFB%y, JOURNAL OF Smita
doi: 10.3897/JHR.45.4769 SHORT COMMUNICATION (ME Hymenoptera

http://jhr.pensoft.net The insertional Society of ymenoptersts. RESEARCH

Diploid males of Scaptotrigona depilis are able to join
reproductive aggregations (Apidae, Meliponini)

Ayrton Vollet-Neto', Charles Fernando dos Santos’, Leandro Rodrigues Santiago’,
Denise de Araujo Alves'*, Julia Pinheiro de Figueiredo', Marino Nanzer',
Maria Cristina Arias’, Vera Lucia Imperatriz-Fonseca'”

| Departamento de Biologia, Faculdade de Filosofia, Ciéncias e Letras de Ribeiriio Preto, Universidade de
Stéo Paulo, Ribeirio Preto, SP Brazil 2 Departamento de Biodiversidade e Ecologia, Pontificia Universidade
Catélica do Rio Grande do Sul, Porto Alegre, RS, Brazil 3 Departamento de Genética e Biologia Evolutiva,
Instituto de Biociéncias, Universidade de Sado Paulo, Sao Paulo, SP Brazil 4 Departamento de Entomologia
e Acarologia, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de Sao Paulo, Piracicaba, SP
Brazil § Instituto Tecnolégico Vale, Belém, PA, Brazil

Corresponding author: Ayrton Vollet-Neto (ayrtonneto@usp.br)

Academic editor: Jack Neff | Received 24 February 2015 | Accepted 12 April 2015 | Published 7 September 2015
http-//zoobank. org/2 BBF56 1 0-2322-4EE4-A DEF-2E3ED90AC6EB

Citation: Vollet-Neto A, dos Santos CE, Santiago LR, Alves DA, de Figueiredo JP, Nanzer M, Arias MC, Imperatriz-
Fonseca VL (2015) Diploid males of Scaptotrigona depilis are able to join reproductive aggregations (Apidae, Meliponini).
Journal of Hymenoptera Research 45: 125-130. doi: 10.3897/JHR.45.4769

Abstract

The sex determination system in the eusocial stingless bees (Apidae, Meliponini) is based on the combina-
tion of alleles at the complementary sex determination (CSD) locus. In this system, males are haploid and
females are diploid. However, diploid males can develop from fertilized eggs when they are homozygous
at single or multiple sex loci. The production of such males can negatively affect population viability, since
they are usually infertile or inviable. Moreover, when they are viable but infertile, or siring sterile triploid
offspring, this could cause another load on the population, leading the fertilized offspring of other females
to be only haploid males or triploid sterile daughters. In this context, our aim was to verify whether dip-
loid males of the stingless bee Scaptotrigona depilis do in fact join reproductive aggregations. We showed
that of 360 marked males from two different colonies, five were participating in a reproductive aggrega-
tion ca. 20 meters from their natal colonies. Using microsatellites markers, it was confirmed that three of
these five males were diploid. They were captured in the mating aggregations when they were 15 to 20
days old. Further research is necessary to determine the mating success of stingless bee diploid males under

natural conditions and to determine their impact on stingless bee population extinction risks.

Copyright Ayrton Vollet-Neto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

126 Ayrton Vollet-Neto et al. / Journal of Hymenoptera Research 45: 125-130 (2015)

Keywords
Inbreeding, diploid male load, population viability, stingless bees

Introduction

The system of sex determination in the eusocial stingless bees (Apidae, Meliponini)
has been shown to be based on the combination of alleles at the complementary sex
determination (CSD) locus (Camargo 1979), like many other Hymenoptera (Whit-
ing 1943). Under this system, individuals that are heterozygotes at the CSD locus are
females, while the hemizygotes (haploid individuals) and the homozygotes are males
(Whiting 1943; Cook and Crozier 1995).

With few exceptions (El Agoze et al. 1994; Cowan and Stahlhut 2004; Kureck et
al. 2013), diploid males are highly disadvantageous for the Hymenoptera species they
occur in, imposing fitness costs on their relatives. Most of the time they are unviable,
infertile or lead to the production of infertile triploid broods (Heimpel and de Boer
2008). They also lead to the decrease of females in the population, since the diploid
individuals were supposed to develop into females, increasing the extinction risks of
the population (Zayed and Packer 2005).

Simulation models have shown that extinction risks increase when diploid males
are viable and can mate (Zayed and Packer 2005). This is expected because females
mortality would increase over two generations, first in the diploid offspring that was
supposed to be female but ends as an infertile diploid male, and secondly because the
females that mate with diploid male would produce triploid daughters or only haploid
males (Harpur et al. 2013). Indeed it has been shown that in several hymenopteran
species the diploid males are able to mate (reviewed in Heimpel and de Boer 2008).

The extinction risks are even more extreme for stingless bees, since their nests are
sparse and, together with habitat fragmentation, there are few effective population sizes
(Packer and Owen 2001; Zayed 2009). It is well known that the queens mate only once
in this group (Peters et al. 1999), causing half of their diploid brood to develop into dip-
loid males when there is a matched mating (queen mating with a male with the same sex
allele), greatly reducing their fitness (Cook and Crozier 1995) and increasing popula-
tion extinction risks when there is low population size or low sex alleles variability (Alves
et al. 2011). Therefore, to assess the effects of the diploid males on stingless bee con-
servation, it is important to know the viability of diploid males and their participation
on the reproductive events. Despite some previous observations of aggressive behaviour
of workers towards the diploid males in Melipona interrupta, suggesting that although
the diploid males are viable they would be killed before leaving the nest (Francini et al.
2012), diploid males of Tetragonisca angustula were found in a reproductive aggregation
close to the entrance of a queenless colony (Santos et al. 2013).

Here we investigate the fate of diploid males of Scaptotrigona depilis and aimed to
answer the question: do diploid males leave their colonies to join mating aggregations?

Diploid males of Scaptotrigona depilis are able to join reproductive aggregations... 127

Table |. Age at collection and genotypes of diploid males of Scaptotrigona depilis in a reproductive aggregation.

Colony Males’ genotypes at locus
Age (d:
and male | “8° #79) [qq T3 TA T8__| _Sxant 06 | Sxant 18

l-a 18 140/142 125/129 132/140 147/153 VSNL 172/192
1-b 15 140/142 1257129 132/138 153/153 177/181 L072
2-c 19 138/142 1 2SL129 132/140 147/153 177/179 172/174

In order to answer this question, two free-foraging colonies kept in wooden boxes in
the Biology Department at Sao Paulo University (Ribeirao Preto, Brazil) were utilised.
These colonies were chosen because their queens were producing diploid males, as pre-
viously confirmed through genotyping ten male pupae from each colony following the
methods described below. We collected brood combs containing mature pupae and
let the bees emerge in an incubator. Thirty newly emerged males of each colony were
marked with nontoxic paint (Revell GmbH & Co. KG, Germany) on their thorax
for six days, using a different colour for each day (total 360 marked males) and all the
males were reintroduced into their natal nest on the day of marking. During the time
of the study, the mating aggregations were observed twice a day, and any marked males
present were collected. They were preserved in absolute ethanol for genetic analysis.
The DNA was extracted using the Chelex method and males were genotyped at six
microsatellite /oci, T1, T3, T4 and T8 (Paxton et al. 1999), and Sxant06 and Sxant18
(Duarte et al. 2011). Microsatellite amplification and visualization were done as de-
scribed previously by Francisco et al. (2011). Males were categorized as diploid if they
were heterozygous at one or more loci (Alves et al. 2011).

Five marked males were found at two mating aggregations which were approxi-
mately 20 meters from their natal colonies. Three of them were diploid, since almost
all loci showed two different alleles (Table 1), whereas the other two were likely hap-
loid. The age of diploid males within the aggregation ranged from 15 to 20 days old
(Table 1), and the haploid were from 16 and 20 days old.

These findings suggest that the presence of stingless bee diploid males in mating
ageregations may be more common than previously suspected. Our data are in ac-
cordance with those obtained by Santos et al. (2013), who also found diploid males
in a mating aggregation of 7: angustula. Although the workers of stingless bees are
supposed to recognize and kill diploid males inside the nests (Francini et al. 2012),
it is clear that some of these escape worker policing, depart from their colonies, and
become a member of a reproductive aggregation (Santos et al. 2013; this study). It is
important to note that removing the brood combs and allowing the pupae to emerge
out of the colony context might have enhanced their survival. However, no aggressive
behaviour towards males in colonies producing diploid males during their emergence
was observed (this study). Camargo (1982) verified that the average lifespan of diploid
males of M. quadrifasciata is only three days, while haploid males live 15 days on aver-
age, both in artificial conditions. The average lifespans of haploid and diploid males of

128 Ayrton Vollet-Neto et al. / Journal of Hymenoptera Research 45: 125-130 (2015)

S. depilis were not compared, but if there are any physiological constraints that limit
the diploid males’ life span, at least some of them must live long enough to be able to
join mating aggregations.

Some new information about the mating strategies of S. depilis males was discov-
ered. Despite the fact that stingless bee males disperse away from their natal nests to
avoid inbreeding (Paxton 2000; Cameron et al. 2004; Kraus et al. 2008), some of
them (haploid or diploid) join aggregations very close to their original colonies. It
is important to note that this is a managed population kept in a stingless bee apiary
maintaining up to 100 nests in a small area. Further research on this topic is needed to
draw stronger conclusions.

These results add more precision as to the age of sexual maturity age of male sting-
less bees, since previous studies considered only the age that males of leave the nest (an
average of 18.6 days old in M. favosa; van Veen et al. 1997). It is showed here that both
haploid and diploid males join mating aggregations from 15 to 20 days old. It was not
possible to ascertain that this was their first experience in a mating aggregation since
they could have already visited one previously. However, considering that Pech-May
et al. (2012) found that M. beecheii males are sexually mature at ca. 14 days old, it is
possible that males of S. depilis also mature later in life, and would join reproductive
ageregations only when mature.

Despite the few diploid males found in mating aggregation compared to the
marking effort and presence of approximately one thousand individuals therein,
there is a great chance that a much higher fraction of diploid males produced by
S. depilis leave the nest to search for an aggregation site, since there is good evidence
that males disperse from their natal nests (Cameron et al. 2004; Kraus et al. 2008).
This could also explain why only ca. 1.4% of the marked males were recaptured.
Probably, the majority of males had dispersed to distant aggregations that we did not
sample. However, some did not disperse and thus increased the risks of inbreeding.
It is important to note that our manipulation may have allowed the males to live
normally by minimizing worker policing. Additionally, diploid male production in
social insects under monandry is predicted to be very high, since half of the diploid
brood are diploid males when a matched mating is performed. As this species pro-
duces approximately 200 or more new brood cells per day (this study), even if just
a small fraction of the diploid males succeeded in leaving the nest, the total amount
could be high.

All these factors together (high rate of production of diploid males, survivorship,
and visit to mating sites) strongly suggest that diploid males could be dangerous to
stingless bee populations from the females’ point of view (Harpur et al. 2013). Further
research is needed to verify the potential mating success of S. depilis diploid males,
since we had no success when testing this under controlled conditions, following the
protocol established by Engels and Engels (1984). If it is established that diploid males
of S. depilis can successfully mate, this would suggest that this species is under a higher
risk of extinction due to the decreased fitness of queens that mate with them (Zayed
2009; Harpur et al. 2013).

Diploid males of Scaptotrigona depilis are able to join reproductive aggregations... 129)

Acknowledgements

We thank FAPESP (Fundagao de Amparo a Pesquisa do Estado de Sao Paulo,
2012/11144-0 to AVN; 2010/19717-4 to DAA; 2012/16716-2 to JPF), CNPq (Con-
selho Nacional de Desenvolvimento Cientifico e Tecnolégico, 470372/2013-3 to
DAA; 500458/2013-8 to CFS), PNPD/CAPES (Programa Nacional de Pdés-Dout-
orado da Coordenacao de Aperfeigoamento de Pessoal de Nivel Superior, to DAA)
and BioComp - NAP-USP (Research Center on Biodiversity and Computing of the
Universidade de Sao Paulo, to JPE MN and DAA) for financial support.

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