# Full Text: MutAnts

> Extracted from `2017_MutAnts.pdf`

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Leading Edge
Previews
The MutAnts Are Here
Daniel A. Friedman,1,* Deborah M. Gordon,1 and Liqun Luo1,2
1Department of Biology
2Howard Hughes Medical Institute
Stanford University, Stanford, CA 94305, USA
*Correspondence: dfri@stanford.edu
http://dx.doi.org/10.1016/j.cell.2017.07.046
The development of CRISPR/Cas9-mediated gene knockout in two ant species opens a new win-
dow into exploring how social insects use olfactory cues to organize their collective behavior.
Ant colonies have long been one of
the most accessible and fascinating ex-
amples of collective behavior in nature.
Recently, genomic, epigenomic, and tran-
scriptomic analyses have begun to unveil
the molecular underpinnings of physi-
ology and behavior in various ant spe-
cies (Yan et al., 2014; Friedman and Gor-
don, 2016). However, without the ability
to perform genetic manipulations, most
hypotheses regarding ant genetics rest
purely on correlative evidence and phylo-
genetic inference. Now, in this issue of
Cell, Trible et al. (2017) and Yan et al.
(2017) advance the field of ant genetics
into a new era by successfully performing
CRISPR/Cas9-mediated knockout of the
gene that encodes the same key olfactory
co-receptor in two different ant species.
All of the 13,000+ known ant species
are eusocial (Ward 2014): they live in col-
onies, in which females range in repro-
ductive capacity from being functionally
sterile workers to mated and reproducing
queens. Trible et al. and Yan et al. study
two
ant
species
that
diverged
over
100 million years ago, the clonal raider
ant (Ooceraea biroi) and Jerdon’s jump-
ing ant (Harpegnathos saltator). In both
studies, the authors injected freshly-laid
eggs with purified Cas9 protein and syn-
thetic guide RNAs (sgRNAs) correspond-
ing to the targeted locus, Orco. Orco is a
highly conserved and obligatory trans-
membrane co-receptor which heterodi-
merizes with a diversity of ligand-specific
olfactory receptors in the Or gene family
(Larsson et al., 2004). Among the three
families of olfactory receptors in insects,
the Or family has undergone a radical
expansion in ants (Simola et al., 2013), hy-
pothesized to reflect an increased ability
to perceive the cuticular hydrocarbons
and pheromones used in ant communica-
tion (Tsutsui 2013). However, until the ge-
netic loss-of-function studies presented
here, there was no technique that specif-
ically disrupts the function of Orco (or
any other gene) to investigate its function.
Ants use haplodiploid sex determina-
tion; haploid eggs develop as males
and diploid eggs develop as females.
In many ant species, the difference in
reproductive capacity between workers
and queens is entirely epigenetic, as
both develop from diploid eggs. This situ-
ation is analogous to the relationship be-
tween somatic and germline cells in a
multicellular organism, which differ only
in epigenomic state. The clonal raider
ant, O. biroi, is an all-female species in
which diploid females asexually produce
isogenic diploid daughters via partheno-
genesis (Figure 1A). This facilitates trans-
genics because injected G0 eggs surviv-
ing till adulthood can be screened for
successful Orco knockout (assuming the
mutant is not lethal), so mutant lines
can be quickly propagated. The obvious
downside
of
clonal
reproduction
in
O. biroi is that crosses are impossible.
In H. saltator, as in many other ant
species,
mated
diploid
females
lay
both unfertilized haploid male eggs and
fertilized diploid female eggs. However,
H. saltator workers display a form of
reproductive plasticity in which a change
in social context can induce a non-
reproductive worker to become a ‘‘game-
rgate’’—an unmated female capable of
producing sons by laying unfertilized
haploid eggs (Figure 1B). Yan et al. take
advantage of the gamergate transition
of H. saltator to generate hemizygous
mutant males, which can then be back-
crossed to receptive females, producing
heterozygous and homozygous mutant
females.
In both O. biroi and H. saltator, ho-
mozygous Orco loss-of-function individ-
uals display similar patterns of altered
behavior
and
decreased
reproductive
performance, supporting the hypothe-
sized centrality of Orco in mediating the
perception of olfactory stimuli in ants.
Trible et al. demonstrate that compared
to heterozygous Orco mutant lines of
O. biroi, homozygous Orco mutants are
less likely to be repelled by noxious odors
in a walking assay, less likely to walk in
trajectories resembling
their wild-type
nestmates
after
a
nest
disturbance,
and more likely to aimlessly wander
around the nest. Additionally, homozy-
gous mutants lay fewer eggs and have
higher mortality. Yan et al. use a more
complicated olfactory assay to find that
Orco null mutants of H. saltator have
strongly decreased sensitivity to various
pure semiochemicals delivered in puffs
of air, and also note increased levels of
wandering outside of the nest, decreased
reproductive
capacity,
and
unique
antennae-twitching behavior. These re-
sults are all consistent with the central
role of olfaction for ant behavior.
Unexpectedly, both groups also find
gross neuroanatomical defects in the
antennal lobe of Orco mutants, the initial
olfactory
processing
center
in
insect
brains. The insect antennal lobe consists
of discrete glomeruli; each glomerulus is
the target of axons of a single type of ol-
factory receptor neurons expressing a
specific olfactory receptor, an organiza-
tional principle also conserved in mam-
mals (Komiyama and Luo, 2006). The
antennal lobe of Drosophila melanogaster
Orco mutants has normal number of
glomeruli
but
exhibit
age-dependent
axon degeneration (Chiang et al., 2009).
The dramatic reduction of glomerular
Cell 170, August 10, 2017 ª 2017 Elsevier Inc.
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number in Orco mutants in both ant
species implies a novel neurodevelop-
mental role for Orco, perhaps related to
the drastic expansion of glomerular num-
ber (300–500 in ants compared to 
50 in
D. melanogaster). This is a subject worthy
of further investigation.
The work of Trible et al. and Yan et al.
together represent a watershed moment
for the budding field of eusocial insect
genetics. Ants are endemic to a wide
range of ecosystems, from the damp
leaf litter of the tropics to the desiccating
heat of the desert. Simple olfactory inter-
actions generate the collective behavior
of colonies. How has this tremendous
ecological heterogeneity been facilitated
by
neurophysiological
and
epigenetic
plasticity (Friedman and Gordon, 2016;
Gordon, 2016)? What has been the role
of genomic changes, for example the
drastic expansion of the Or gene family,
in accommodating adaptation to such
diverse environments? Exploring these
fascinating questions will elucidate the
evolution of the world’s most ecologi-
cally-dominant invertebrate societies.
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Figure 1. Reproductive diagrams for Ooceraea biroi and Harpegnathos saltator
(A) Ooceraea biroi.
(B) Harpegnathos saltator.
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Cell 170, August 10, 2017


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*Extraction method: pymupdf*