royalsocietypublishing.org/journal/rspb
Research
Cite this article:
Brückner A, Kaltenpoth M,
Heethoff M. 2020 De novo biosynthesis of
simple aromatic compounds by an arthropod
(
Archegozetes longisetosus
).
Proc. R. Soc. B
287
: 20201429.
http://dx.doi.org/10.1098/rspb.2020.1429
Received: 16 June 2020
Accepted: 11 August 2020
Subject Category:
Evolution
Subject Areas:
biochemistry, evolution, molecular biology
Keywords:
biosynthetic pathways, Benzenoids,
chemical ecology, Oribatid mites,
Chemical defence
Authors for correspondence:
Adrian Brückner
e-mail: bruckner@caltech.edu
Michael Heethoff
e-mail: heethoff@bio.tu-darmstadt.de
Electronic supplementary material is available
online at https://doi.org/10.6084/m9.figshare.
c.5095275.
De novo biosynthesis of simple aromatic
compounds by an arthropod
(
Archegozetes longisetosus
)
Adrian Brückner
1,2
, Martin Kaltenpoth
3,4
and Michael Heethoff
1
1
Technische Universität Darmstadt, Ecological Networks, Schnittspahnstraße 3, 64287 Darmstadt, Germany
2
California Institute of Technology, Division of Biology and Biological Engineering, 1200 East California
Boulevard, Pasadena, CA 91125, USA
3
Evolutionary Ecology, Institute of Organismic and Molecular Evolution, Johannes Gutenberg University,
Johann-Joachim-Becher-Weg 13, 55128 Mainz, Germany
4
Department of Insect Symbiosis, Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8,
07745 Jena, Germany
AB, 0000-0002-9184-8562; MK, 0000-0001-9450-0345; MH, 0000-0003-3453-4871
The ability to synthesize simple aromatic compounds is well known from
bacteria, fungi and plants, which all share an exclusive biosynthetic
route
—
the shikimic acid pathway. Some of these organisms further evolved
the polyketide pathway to form core benzenoids via a head-to-tail conden-
sation of polyketide precursors. Arthropods supposedly lack the ability to
synthesize aromatics and instead rely on aromatic amino acids acquired
from food, or from symbiotic microorganisms. The few studies purportedly
showing de novo biosynthesis via the polyketide synthase (PKS) pathway
failed to exclude endosymbiotic bacteria, so their results are inconclusive.
We investigated the biosynthesis of aromatic compounds in defence
secretions of the oribatid mite
Archegozetes longisetosus
. Exposing the mites
to a diet containing high concentrations of antibiotics removed potential
microbial partners but did not affect the production of defensive benzenoids.
To gain insights into benzenoid biosynthesis, we fed mites with stable-
isotope labelled precursors and monitored incorporation with mass
spectrometry. Glucose, malonic acid and acetate, but not phenylalanine,
were incorporated into the benzenoids, further evidencing autogenous
biosynthesis. Whole-transcriptome sequencing with hidden Markov model
profile search of protein domain families and subsequent phylogenetic
analysis revealed a putative PKS domain similar to an actinobacterial PKS,
possibly indicating a horizontal gene transfer.
1. Introduction
Simple aromatic compounds (i.e. chemicals containing a benzene ring) are
important products in chemical science and industry, but also in nature [1,2].
Overall, about 550 different simple and many more complex aromatics have
been described from bacteria, fungi, plants and animals [3,4]. The unique elec-
tronic structure of the benzene ring
—
a delocalized
π
-electron system with a
six-ringed carbon skeleton
—
provides aromatics with key structural motifs
that shape interactions on both molecular and organismal levels [5,6]. In the pri-
mary metabolism of arthropods, aromatic amino acids are important building
blocks of proteins, but also are used in cuticle formation, sclerotization and
melanization [7,8]. Among secondary metabolites, arthropods use simple benze-
noids in pheromonal communication and as defensive compounds, while the
more complex polyketide aromatics also function as potent antibiotics [3,9,10].
Bacteria, fungi and plants evolved two biosynthetic routes to form benzene
rings: the shikimic acid pathway [1,10] and the polyketide pathway [3]. In the
latter benzene rings form via a head-to-tail condensation of poly-
β
-carbonyl
© 2020 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution
License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original
author and source are credited.
intermediates followed by an intramolecular condensation
forming the aromatic system [11,12]. Sponges, sea urchins
and some vertebrates also can synthesize simple aromatics
via the polyketide pathway [3,13,14]. Among arthropods, aro-
matics are important for chemical interactions in insects,
arachnids and myriapods [9], yet it is still unclear whether
these benzenoids are acquired from diet or from endosymbiotic
bacteria, or are synthesized de novo by the animals [3,10,15].
It appears that especially the complex, large ringed polyketide
aromatics are mostly produced by bacteria [16
–
20], while
simple aromatics may originate from a potential arthropod
polyketide pathway [21
–
24]. Pankewitz & Hilker [15] reviewed
available studies on polyketides and found no unequivocal
evidence for de novo biosynthesis of aromatics in arthropods,
since potential endosymbiont contributions were not excluded.
A decade later, this still holds true [10,25,26]. Only the studies
by Bestmann
et al
. [21] on aromatic trail pheromones of
formicine ants and Pankewitz
et al
. [27] on anthraquinones iso-
lated from leaf beetle eggs indicated that bacteria may not be
involved in the biosynthesis of these chemicals, yet in both
studies the success of antibiotic treatment was not confirmed.
Also, leaf beetle anthraquinones are formed via a eukaryo-
tic polyketide folding mode, rendering a direct bacterial
involvement unlikely [28].
The oribatid mite
Archegozetes longisetosus
Aoki (figure 1
i
)
produces defensive secretions containing two simple aro-
matics, 2-hydroxy-6-methyl-benzaldehyde (2,6-HMBD) and
3-hydroxybenzene-1,2-dicarbaldehyde (
γ
-acaridial), in a pair
of opisthonotal exocrine glands [26]. We reared animals in a
controlled, sterile environment to investigate their ability to
synthesize these benzene-ringed compounds de novo. We
performed stable isotope labelling experiments with different
precursors under intensive antibiotic treatment to maintain
symbiont-free animals and to uncover the biosynthetic path-
way of these aromatics. We demonstrate that 2,6-HMBD and
γ
-acaridial are both synthesized de novo, probably from poly-
β
-carbonyl (e.g. acetyl-CoA and malonyl-CoA). Furthermore,
whole-transcriptome sequencing (RNAseq) uncovered puta-
tive PKS domains similar to those of bacteria, indicating a
potential horizontal gene transfer.
2. Results and discussion
The defensive chemicals of the oribatid mite
A. longisetosus
consist of a blend of ten compounds (figure 1
a
,
i
) including
two terpenes (approx. 45%), six hydrocarbons (approx. 15%)
and two aromatic compounds (approx. 40%) [29,30]. While
the hydrocarbons probably serve as solvents, the terpenes
and aromatics are bioactive compounds used as alarm
pheromones and predator repellents [31].
In a first feeding experiment, we tested if elimination of
potentially symbiotic gut bacteria influences the biosynthesis
of 2,6-HMBD and
γ
-acaridial by feeding the mites with food
containing 10% antibiotics (a mixture of amoxicillin, strepto-
mycin and tetracycline; see methods below). We found that
production of 2,6-HMBD and
γ
-acaridial was unaffected by
treatments with individual antibiotics or with all three in con-
cert (figure 1
b
; U-test:
z
=
−
1.3,
p
= 0.19;
n
= 32; electronic
supplementary material, figure S1). The combined treatment,
which was used in further incorporation experiments with
labelled precursors, effectively eliminated bacteria in the
mites (figure 1
c
;
U
-test:
z
=
−
2.1,
p
= 0.027;
n
= 13). These
results also were supported by fluorescence
in situ
hybridiz-
ation (FISH), revealing that all detectable bacteria found on
the food and in the alimentary tract (figure 1
d
) were elimi-
nated in the antibiotic-treated mites (figure 1
e
). We detected
no bacteria in the glands (figure 1
f
), and no or only single
bacteria in the digestive caeca (figure 1
g
), irrespective of anti-
biotic treatment, further supporting the general absence of
endosymbiotic bacteria. By contrast, bacteria on the outer
cuticle of the mites remained mostly unaffected by antibiotic
treatment and served as an internal control for FISH (elec-
tronic supplementary material, figure S2). Hence, the gland
does not house bacteria involved in the production of defen-
sive compounds or precursors, as described for defensive
symbioses in other arthropods [16,18,32]. The paired diges-
tive caeca, which are directly connected with the glandular
tissue via a plasma mass [33], do not appear to be brood
chambers for bacteria or yeasts (electronic supplementary
material, figure S2 andS3). Also, in none of the numerous
histological and ultrastructural studies on the ovarial ultra-
structure, egg development, vitellogenesis and cleavage was
there any indication of vertically transmitted prokaryotic or
eukaryotic symbionts [34
–
38].
In a second experiment, the diet of
A. longisetosus
was
supplemented with food containing 25% of a stable isotope-
labelled precursor
—
[
13
C
6
,d
7
] D-glucose, [
13
C
3
] malonic acid,
sodium [
13
C
1
] acetate or [
13
C
6
] phenylalanine
—
and 10%
antibiotics. To examine the incorporation and possible biosyn-
thesis of 2,6-HMBD (figure 2
a
) and
γ
-acaridial (figure 2
b
), we
compared selected fragment ions using mass spectrometry
and calculated an enrichment factor based on relative intensi-
ties. Both aromatics showed consistently enriched [M+1]
+
to
[M+8]
+
-ion series for [
13
C
6
,d
7
] D-glucose, [
13
C
3
] malonic acid
and sodium [
13
C
1
] acetate. Enrichment was strongest for the
most general source, the heavily labelled D-glucose, while
the more specific
β
-carbonyl precursors showed less enrich-
ment (electronic supplementary material, table S1) for both
compounds (see figure 2
a
for 2,6-HMBD and figure 2
b
for
γ
-acaridial). This stepwise enrichment pattern indicates that
short
β
-carbonyls joined to form complex molecules via
multiple head-to-tail condensation reactions, which is indica-
tive of a PKS-like mechanism [22
–
24]. When feeding [
13
C
6
]
phenylalanine, we found no enrichment of the [M+6]
+
-ion
(electronic supplementary material, table S1; figure 2), which
would be expected for the incorporation of the completely
13
C-labelled benzene ring [39]. Instead, we again found
a stepwise enrichment in the [M+1]
+
to [M+8]
+
-ion series
(electronic supplementary material, table S1; figure 2), indicat-
ing that only fragments of the benzene ring of [
13
C
6
]
phenylalanine had been incorporated into 2,6-HMBD
and
γ
-acaridial via a PKS-like reaction after the mites had
catabolized the amino acids to poly-
β
-carbonyl units [40,41].
As we can exclude a direct influence of bacteria (antibiotics
treatment, qPCR, microscopy; figure 1; electronic supple-
mentary material figure S2), fungi (microscopy; electronic
supplementary material figure S3) and protists (microscopy)
[34
–
38], this strongly supports a de novo biosynthesis of both
aromatic compounds.
Given this mass spectrometric evidence, the mite must
ultimately possess the necessary enzymes to produce
2,6-HMBD and
γ
-acaridial via a PKS-like pathway. To explore
whether
A. longisetosus
expresses the respective genes encoding
for such an enzyme and to trace down its potential evolutionary
origin, we performed whole-body RNAseq of adult mites,
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assembled the transcriptome and predicted the longest open
reading frame (ORFs) of the transcripts. Subsequent searching
of the predicted protein sequences for ketoacyl-synthase (KS)
domains
—
one module of both fatty acid (FAS) and polyketide
synthases (PKSs) commonly used for phylogenetic analyses of
these enzyme families [13,14]
—
revealed 10 candidate KS-
domains in
A. longisetosus
(see Material and methods; electronic
supplementary material, table S2). A phylogenetic analysis of
the mite
’
s KS-domains using a maximum-likelihood (ML)
approach
—
including a pre-existing dataset of 139 KS-domains
[13,14] of bacterial, fungal and animal PKS as well as animal
FAS
—
revealed that eight of the mite
’
s KS-domains were well
nested within the animal FAS-clade (see electronic supplemen-
tary material, figure S4 for full ML tree), while two KS-domains
were nested with a clade of bacterial PKS, specifically PKS from
the actinobacterial genus
Streptomyces
(figure1
h
). As expected, a
SPARCLE (=Subfamily Protein Architecture Labelling Engine)
[42] search identified both protein sequences as beta-ketoacyl-
synthases known from both FAS and PKS, yet based on their
phylogenetic placement with bacterial KS-domains (figure 1
h
),
0
12.5
13.5
neral
HO
HO
O
O
O
n.s.
C13
neryl
formate
14.5
(%) aromatic compounds
bacteria/host copy ratio (
n
× 10
3
)
15.5 (min)
control
control
antibiotics
antibiotics
5
10
15
20
25
30
*
0
7.5
15
22.5
30
0.5
Streptomyces cinnamonensis
(bacterial polyketide synthase II)
Streptomyces coelicolor
(actinorhodin polyke tide synthase II)
Streptomyces coelicolor
(actinorhodin polyke tide synthase I)
Streptomyces violaceoruber
(granaticin polyketide synthase II)
Streptomyces cinnamonensis
(bacterial polyketide synthase I)
Streptomyces rimosus
(oxytetracycline polyketid esynthase I)
mycocerosic acid synthase (
Mycobacterium bovis
)
Botrytis cinerea
(fungal polyketidesynthaseI)
Streptomyces violaceoruber
(granaticin polyketide synthase I)
37
100
99
100
100
100
100
100
98
77
99
91
36
100
100
100
99
95
100
100
mixed PKS
fungal PKS
animal FAS
bacterial PKS
Archegozetes longisetosus
bacterialV PKS
with nested mite PKS
(
a
)
(
h
)
(
i
)
(
d
)
(
e
)
(
f
)
(
g
)
(
b
)(
c
)
Figure 1.
Mites (
Archegozetes longisetosus
) contain two simple aromatic compounds in their defensive secretions, and reduction of bacteria did not affect their
production. (
a
) Representative GC trace of mite gland extracts; in order of retention time: 2-hydroxy-6-methyl-benzaldehyde (2,6-HMBD), neral, neryl formate,
tridecane, 3-hydroxybenzene-1,2-dicarbaldehyde (
γ
-acaridial). Pentadec-7-ene, pentadecane, heptadeca-6,9-diene, heptadec-8-ene, heptadecane are not shown.
(
b
) Supplementing a wheat grass diet (control) with a mixture of antibiotics (10% w/w; combined amoxicillin, streptomycin and tetracycline;
‘
antibiotics
’
) did
not affect the relative amount of aromatic compounds but resulted in significantly lower bacterial load in the mite (
c
). Fluorescence
in situ
hybridization
(FISH) revealed a high bacterial prevalence in the food bolus of control group mites (
d
), while no detectable bacteria (
e
) were found in similar bolus in anti-
biotic-treated mites. Even in the control group, no bacteria were detected in the gland (
f
); the white arrowhead marks the centre of the gland) or in the
caeca (i.e. pairwise sac-like organ that are located close to the glandular tissue; (
g
). Maximum-likelihood tree (
h
) based on an alignment of the KS-domains
of fatty-acid and PKSs from animals (including those of the mite
—
depicted in dark red), fungi and bacteria. Bootstrap values (based on 1000 replicates) are
indicated along branches and the scale bar below the tree denotes substitutions per site. The tree was rooted by the outgroup mycocerosic acid synthas
e
from
Mycobacterium bovis
. Frontal view (
i
) of a μCT-reconstruction of
A. longisetosus
. In the FISH images, bacteria are stained in green with the general bacterial
probe EUB338-Cy5; the green arrow heads mark bacterial signals detected in the food bolus (
d
) or on the cuticle (
f
). The scale bar in FISH images is 20 μm.
U
-test:
n.s. = not significant,
p
> 0.05; *= significant,
p
< 0.05. (Online version in colour.)
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80
90
100
110
120
122
93
80
79
77
82
93
99
136
140
136
136
137
139
143
144
127
121
99
96
121
136
143
93
93
82
82
77
77
144
122
127
labelled glucose +
antibiotics + wheat grass
labelled sodium acetate +
antibiotics + wheat grass
labelled phenylalanine +
antibiotics + wheat grass
labelled malonic acid +
antibiotics + wheat grass
77
0
100
130
140
150
160
137
136
HO
2-hyroxy-6-methyl-benzaldehyde
3-hyroxybenzene-1,2-dicarbaldehyde
O
HO
O
O
136
control (wheat grass)
control (wheat grass)
relative abundance (%)
0
100
relative abundance (%)
0
100
relative abundance (%)
0
100
relative abundance (%)
0
100
relative abundance (%)
0
100
relative abundance (%)
0
100
relative abundance (%)
0
100
relative abundance (%)
0
100
relative abundance (%)
0
100
136
77
82
93
99
122
143
relative abundance (%)
m/z
80
90
100
110
120
130
140
150
160
160
m/z
80
90
100
110
120
130
140
150
160
m/z
80
90
100
110
120
130
140
150
m/z
80
90
100
110
120
130
140
150
m/z
160
80
90
77
78
77
80
81
77
93
121
labelled sodium acetate +
antibiotics + wheat grass
labelled phenylalanine +
antibiotics + wheat grass
labelled malonic acid +
antibiotics + wheat grass
labelled glucose +
antibiotics + wheat grass
150
150
150
153
156
136
99
93
82
77
121
151
153
155
157
80
93
93
95
95
121
121
150
150
126
126
136
155
141
158
93
95
121
150
151
153
150
136
100
110
120
130
140
150
m/z
160
80
90
100
110
120
130
140
150
m/z
160
80
90
100
110
120
130
140
150
m/z
80
90
100
110
120
130
140
150
m/z
160
80
90
100
110
120
130
140
150
m/z
(
a
)
(
b
)
Figure 2.
Representative mass spectra of 2-hydroxy-6-methyl-benzaldehyde (
a
) and 3-hydroxybenzene-1,2-dicarbaldehyde (
b
) extracted from defensive glands of
mites fed with unlabelled wheatgrass (control), or wheatgrass infused with
13
C/d-labelled glucose,
13
C-malonic acid,
13
C-acetate and
13
C-phenylalanine recorded in
single-ion mode. Inserts show the [M + 1]
+
-ion series in the control and
13
C-acetate. Mites fed with wheatgrass infused with a mixture of labelled precursors and
antibiotics show enriched ions.
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we refer to them as putative PKS domains. Both putative PKS
domains had a GC content of approximately 37%, which is
slightly higher than the usual GC content of known oribatid
mite genomes (approx. 30%) [43], but much lower than in
Strep-
tomyces
genomes (approx. 70%) [44]. Furthermore, the putative
PKS domains have an amino acid composition more similar to
those of other mitesthan to those of
Streptomyces
(electronic sup-
plementary material, figure S5). Thus, the putative PKS domain
genes are most likely integrated into the mite
’
s genome and not
due to
Streptomyces
contamination. The nested position within a
Streptomyces
clade (figure 1
h
) suggests an ancient horizontal
gene transfer (HGT) of PKS-encoding genes from bacteria
to
A. longisetosus
, as previously discussed for arthropods by
Pankewitz & Hilker [15].
We were not able to isolate the entire biosynthetic PKS gene
cluster for
A. longisetosus
, if such clusters indeed exist in
animals. This might be due to the many short transcripts in
the transcriptome and a lack of confirmed, contiguous gene
models, but also to the diffuse genomic organization of
pathway loci within the genomes of animals. Unlike in
prokaryotes, where components of a biosynthetic cluster are
conveniently organized as operons [45], identifying all com-
ponents of biosynthetic pathways is much more challenging
in animals [46]. Enzymes (or domains) are seldom clustered
as tandems, but instead are scattered across the genome, and
regulatory elements controlling expression are cryptic and
sometimes even very distant from the open reading frame
[47]. Furthermore, since there is no functional arthropod
PKS-cluster for reference, it remains unknown whether PKS
gene clusters of arthropods
—
in case they exist
—
are similar to
those of bacteria, fungi or other animals. Our study provides
evidence that HGT
—
a common mechanisms of acquiring
new genes in mites [48] as well as other arthropods [49,50]
—
could explain the presence of putative PKS-domains and the
mode of benzenoid synthesis via head-to-tail condensation in
A. longisetosus
(figure 3).
Since all experimental data (mass spectrometry of stable
isotopes and RNAseq) point to a polyketide-like mechanism,
we propose the following as the most likely biochemical
pathway (figure 3): the mites harness (poly)-
β
-carbonyls
like malonyl-CoA and/or acetyl-CoA produced via glycoly-
sis and gluconeogenesis from sugars and amino acids,
respectively, to form a C8-polyketid-like intermediate via a
head-to-tail condensation. Subsequently, a ring closing
cyclization via an aldol-reaction yields a second intermediate,
which eventually undergoes several
–
H
2
O enolization and
–
H
2
O reduction reactions [22], inducing aromatization of the
ring and the formation of the final product 2,6-HMBD. The
second aromatic compound (3-hydroxybenzene-1,2-dicarbal-
dehyde) may be biosynthesized from 2,6-HMBD by an
enzymatic oxidation of the methyl group to the corresponding
aldehyde or via a different head-to-tail condensation.
This biosynthetic scenario accords well with studies on
benzenoid ant pheromones [21,23,24] and benzoquinoid
defensive chemicals of harvestmen [22] that indicated a poly-
ketide origin of these compounds, but did not exclude or
specifically test for an involvement of symbiotic microorgan-
isms [51
–
53]. A more closely related arachnid, the storage
mite
Chortoglyphus arcuatus
Troupeau, produces an aliphatic
polyketide-derived aggregation pheromone
—
(4
R
,6
R
,8
R
)-
4,6,8-trimethyldecan-2-one
—
synthesized from one acetate
and four propionates [54], further supporting the ability of
mites to produce polyketides de novo, yet potential bacterial
participation was not excluded, as well.
Overall, the mass spectrometric analysis of labelled
precursors under controlled conditions, as well as the mol-
ecular evolutionary assessment, indicates that this oribatid
mite produces small aromatic compounds using a horizon-
tally acquired putative PKS. With more examples, we may
find that ancient horizontal gene transfer had a more general
role in the evolution of aromatic compound synthesis across
different arthropod groups.
3. Material and methods
(a) Mites
The lineage
‘
ran
’
[55] of the pantropical, parthenogenetic oribatid
mite
Archegozetes longisetosus
was used in this study. Experimen-
tal cultures were established from an already existing line fed on
wheat grass (
Triticum
sp.) powder from Naturya (Bath, UK) as
follows: first, we collected eggs and surface-washed them with
sodium hypochlorite solution (3% w/v), ethanol (70% v/v) and
sterilized water for 5 s, 15 s and 30 s, respectively. Eggs were
then transferred to sterile Petri dishes (diameter 45 mm) lined
with 1 cm sterilized plaster of Paris. Sterile cultures were main-
tained in a laminar-flow closet at 28°C and 90% relative
humidity. Sterilized water and 3
–
5 mg wheat grass or different
food mixtures (see below) were provided three times each week.
(b) Antibiotics feeding experiment
In the first experiment, we fed four different mixtures of antibiotic-
laden wheat grass (10% antibiotics, w/w) and pure wheat grass as
a control to different groups of 150 mites for one generation
head-to-tail condensation
(PKS-like)
phenylalanine
D-glucose
no direct incorporation of Phe into mite aromatics
pyruvate
gluconeogenesis
glycolysis
pyruvate
decarboxylation
acetyl-CoA
aldol reaction
–H
2
O enolization
/
–H
2
O reduction
2,6-HMBD
malonyl-CoA
OH
OH
OH
H
H
H
H
H
HO
HO
O
O
OH
NH
2
O
OO
O
O
OH
O
O
–2 H
2
O
O
O
O
HO
S-Enz
S-Enz
O
SCoA
SCoA
–
O
O
O
–
Figure 3.
Proposed biosynthetic scenario leading to 2-hydroxy-6-methyl-benzaldehyde (2,6-HMBD) in the oribatid mite
Archegozetes longisetosus
starting with
D-glucose and phenylalanine as primary substrates. Common metabolic pathways are labelled in green, pathway-specific reactions are depicted in pu
rple. Multiple
arrows indicate multiple reaction steps. (Online version in colour.)
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(approx. 50 days). We prepared 10% (w/w) mixtures of sterilized
wheat grass powder with amoxicillin, streptomycin and tetra-
cycline as well as a mixture of all three antibiotics (3.3% w/w for
each). One week after the adult eclosion, the defensive glands
were extracted in hexane and chemically analysed (see below).
(c) Feeding experiments with labelled precursors
For the second experiment, we used only the 10% (w/w) mixture
which contained all three antibiotics. Additionally, we added 25%
(w/w) stable isotope-labelled precursors. We prepared four differ-
ent mixtures with [
13
C
6
,d
7
] D-glucose, [
13
C
3
] malonic acid, sodium
[
13
C
1
] acetate and [
13
C
6
] phenylalanine (all greater than 99%
enrichment, Sigma-Aldrich, St Louis, USA) as well as a control
with untreated wheat grass. Again, cultures were maintained for
one generation and glands of adult specimens were extracted
one week after eclosion using hexane (see below). Furthermore, a
subset of these mites was used for FISH and qPCR experiments
(see below).
(d) Fluorescence
in situ
hybridization
For the control as well as the [
13
C
6
,d
7
] D-glucose and [
13
C
6
] phenyl-
alanine treatments (both with 10% antibiotics, see above) three
entire specimens of
A. longisetosus
were fixated in 4% paraformalde-
hyde in PBS, and FISH was performed on semi-thin sections as
described previously [56,57]. The fixated samples were dehydrated
in a graded ethanol series and then embedded in cold-polymeriz-
ing resin (Technovit 8100; Heraeus Kulzer, Hanau, Germany)
according to manufacturer
’
s instructions. Sections of 7 μm thick-
ness were obtained with a steel knife on a HM355S microtome
(Leica, Germany) and mounted on microscope slides coated with
poly-
L
-lysine (Kindler, Freiburg, Germany). FISH was done with
the general eubacterial probes EUB388-Cy5 (5
0
-GCTGCCTCC
CGTAGGAGT-3
0
) [58] and EUB784-Cy3 (5
0
-TGGACTACCAGGG
TATCTAATCC-3
0
) [59] (two individuals each), or a combination
of EUB338-Cy3 and the general yeast probe PF2-Cy5 (5
0
-CTCT
GGCTTCACCCTATTC-3
0
) [60] (one individual each). Samples
were incubated for 90 min at 60°C in 100 μl hybridization buffer
(0.9 M NaCl, 0.02 M Tris/HCl pH 8.0, 0.01% SDS) containing 5 μl
of each probe (500 nM) as well as DAPI (4
0
,6-diamidino-2-phenylin-
dole) for counterstaining of host cell nuclei. Two wash steps with
pre-warmed washing buffer (0.1M NaCl, 0.02M Tris/HCl pH8.0,
0.01% SDS, 5 mM EDTA), the second for 20 min at 60°C, as well
as rinsing with dH
2
O, served to remove residual probes. After
drying at room temperature, slides were covered with VectaShield
and inspected on an AxioImager.Z1 fluorescence microscope
(Zeiss, Jena, Germany).
(e) Quantitative PCR
To assess the effect of antibiotic treatment on absolute numbers of
bacteria associated with the mites, bacterial 16S rRNA copy num-
bers were determined by quantitative PCR (qPCR). Since FISH
experiments had revealed the presence of bacteria on the surface,
the mites were surface washed in 5% (v/v) sodium dodecyl sulfate
solution before bacterial quantification. For the control and the
[
13
C
6
,d
7
] D-glucose + 10% antibiotics treatment, DNA was
extracted from eight replicates of 15
–
25 mites each, using the
MasterPure DNA purification kit (Epicentre Technologies) accord-
ing to manufacturer
’
s instructions. As a quality control of the DNA
extracts and for later standardization of bacterial titres, the DNA
extracts were subjected to a qPCR with primers targeting the
host 28S rRNA gene (D3A_F: 5
’
-GACCCGTCTTGAAA
CACGGA-3
0
; and D3B_R: 5
’
-TCGGAAGGAACCAGCTACTA-3
0
)
[61]. Subsequently, samples that showed amplification for the
host 28S (five of the control replicates and all eight of the antibiotic
treatment) were subjected to a qPCR with general eubacterial 16S
rRNA gene primers (Univ16SRT-F: 5
’
-ACTCCTACGGGAGG
CAGCAGT-3
0
; Univ16SRT-R: 5
’
-TATTACCGCGGCTGCTGGC
-3
0
) [62]. qPCRs were done on a RotorGene-Q cycler (Qiagen,
Hilden, Germany) in final reaction volumes of 25 μl, including
the following components: 1 μl of DNA template, 2.5 μl of each
primer (10 μM), 6.5 μl of autoclaved distilled H
2
O, and 12.5 μl of
SYBR Green Mix (Qiagen, Hilden, Germany). PCR conditions
included 95°C for 5 min, followed by 40 cycles of 95°C for 10 s,
70°C for 15 s, and 72°C for 10 s. A melting curve analysis was per-
formed by increasing the temperature from 60°C to 95°C within
20 min. Standard curves were established for the host 28S and
bacterial 16S assays by using 10
3
–
10
10
copies of PCR product as
templates. A Qubit fluorometer (Thermo Fisher Scientific) was
used to measure DNA concentrations for the templates of the
standard curve. The ratio between absolute copy numbers of bac-
terial 16S and host 28S (= bacterial/host copy ratio) was used as a
standardized measure of bacterial abundance per mite sample.
(f) Chemical analysis
Gland exudates, containing the two studied aromatics, were
extracted from living mites by submersing individuals (antibiotics
feeding experiment) or groups of 15 (labelling experiment) in 50 μl
hexane for 3 min, which is a well-established method to obtain
oil gland compounds from mites [29,30]. Crude hexane extracts
(2
–
5 μl) were analysed with a GCMS-QP2010 Ultra gas chromato-
graphy
—
mass spectrometry (GCMS) system from Shimadzu
(Kyo
̄
to, Japan) equipped with a ZB-5MS capillary column
(0.25 mm × 30 m, 0.25 μm film thickness) from Phenomenex (Tor-
rance, USA). Hydrogen was used a carrier gas with a flow rate of
3.00 ml min
−
1
, with splitless injection and a temperature ramp was
set to increase from 50°C (5 min) to 210°C at a rate of 6°C min
−
1
,
followed by 35°C min
−
1
up to 320°C (for 5 min). Electron ioniz-
ation mass spectra were recorded at 70 eV and characteristic
fragment ions were monitored in single ion mode.
(g) Data analysis
For the antibiotic feeding experiment, we quantified the ion abun-
dance and calculated the relative composition of aromatics
(2,6-HMBD and
γ
-acaridial combined) compared to ion abundance
of the other compounds on an individual base. Then we compared
the (%) aromatic compounds among groups or treatments with a
Kruskal
–
Wallis test or Mann
–
Whitney
U
-tests, respectively.
The bacterial/host copy ratio was analysed with a Mann
–
Whitney
U
-test as well. Statistics were performed in PAST 3.17 [63]. For
stable isotope enrichment, we compared the four treatment
groups with the control and calculated the enrichment factors
(EF) as EF = (
r
treatment
−
r
control
)/
r
control
, where
r
treatment
is the rela-
tive abundance (% relative to M
+
) of a respective ion in the
treatment GCMS analyses and
r
control
is the relative intensity (%)
of the same ion in the control group.
(h) DNA/RNA extraction, genome/RNA sequencing
For both extractions, about 200 adult mites were taken from the
non-treated stock culture, starved for 24 h to avoid possible con-
tamination from food in the gut, subsequently washed with 1%
SDS for 10 s. Finally, DNA or RNA was extracted from living
specimens using the
‘
Quick-DNA Miniprep Plus Kit
’
or the
‘
Quick-RNA MiniPrep Kit
’
from Zymo Research (Irvine, CA,
USA) according to the manufacturer
’
s protocols, respectively.
Amounts and quality of DNA/RNA were accessed using a
Qubit fluorometer and NanoDrop One (Thermo Fisher Scientific),
respectively. Extracted DNA/ RNAwas shipped to Omega Bioser-
vices (Norcross, GA, USA) on dry ice for library preparation and
sequencing. DNA library preparation followed the KAPA Hyper-
Prep Kit protocol, RNA was used for poly-A selection, cDNA
synthesis and library preparation followed the Illumina TruSeq
royalsocietypublishing.org/journal/rspb
Proc. R. Soc. B
287
: 20201429
6
mRNA Stranded Kit protocol. Both libraries were 150 bp paired-
end sequenced on a HighSeq4000 platform.
(i) Genome and transcriptome assembly
Quality of reads was assessed using FastQC v. 0.11.8, Illumina
adapters were trimmed with Cutadapt v. 1.18 [64]. Illumina short
reads for the genome were assembled using the Platanus
Genome Assembler pipeline v. 1.2.4 [65], yielding an assembly
with a total length of 172.0 Mbp, an N50 = 25 196 bp and a
BUSCO score [66] of C:94.4%[S:92.6%, D:1.8%], F:1.8%, M:3.8%.
For the genome-guided assembly of the transcriptome, a bam-file
was created from the genome fasta-file using STAR [67], while
RNAseq reads were
in silico
normalized and subsequently used
together with the bam-file to assemble the transcripts using Trinity
v. 2.8.4 [68], yielding an assembly with a total length of 162.8 Mbp,
an N50 = 2994 bp and a BUSCO score of C:96.3%[S:36.5%,
D:59.8%], F:1.3%, M:2.4%. TransDecoder (v. 5.5.0) [69] was used
to identify putative candidate coding regions in the transcriptome
data and translate them into protein sequences.
( j) HMMR search, alignment and phylogenetic analysis
One way of identifying putative PKS domains is a phylogenetic
analysis of the KS-domain. Therefore, we isolated potential
KS-domain sequences of fatty acid and PKSs, by searching the
transdecoded protein sequences with profile hidden Markov
models as implemented in HMMER v3 [70] using the pfam
domains
‘
PF16197
’
,
‘
PF00109
’
and
‘
PF02801
’
as queries. Sub-
sequently, protein sequences of fatty acid and PKS s previously
used for phylogenetic analyses [13,14], as well as sequences from
the acariform mite species
Tetranychus urticae
[48] were down-
loaded and the same HMMER searches were performed to
extract the KS-domain sequences from the respective fasta-files.
All putative KS-domain protein sequences were concatenated
and sequences shorter than 300 amino acids were removed. All
remaining sequences were aligned using MUSCLE [71]; ends
were manually inspected and trimmed. The resulting final protein
sequence alignment (electronic supplementary material, table S2;
GenBank MT849380) was used to construct a maximum-likelihood
(ML) phylogenetic tree for the KS domains using the iqtree pipe-
line [72], including automated model selection. The ML tree was
constructed under the LG + R7 model using 1000 bootstrap runs
and rooted by the outgroup sequence for mycocerosic acid
synthase from
Mycobacterium bovis
. The similarity of amino acid
frequency of the mite and
Streptomyces
KS-domains was assessed
using kmer counting and principal component analysis in R [73].
Ethics.
There are no legal restrictions on working with mites.
Data accessibility.
The datasets supporting this article have been
uploaded as part of the electronic supplementary material.
Authors
’
contributions.
A.B. designed research, performed chemical
analysis and transcriptomic work, analysed the data and took lead
in drafting the manuscript; M.K. performed molecular and micro-
scopic research, analysed the data and helped drafting the
manuscript; M.H. designed research, performed chemical analysis,
analysed the data and drafted the manuscript. All authors gave
final approval for publication and agree to be held accountable for
the work performed therein.
Competing interests.
The authors declare no conflict of interest.
Funding.
A.B. is a Simons Fellow of the Life Sciences Research Foun-
dation and was previously supported by a PhD scholarship from
the German National Academic Foundation. This study was sup-
ported by the German Research Foundation (DFG; HE 4593/5-1) to
MH, a pilot grant of Caltech
’
s Center for Environmental Microbial
Interactions (CEMI; CEMI-19-028) to A.B. and a Consolidator Grant
of the European Research Council (ERC CoG 819585
‘
SYMBeetle
’
)
to M.K.
Acknowledgements.
We thank Roy A. Norton for improving the manu-
script and Benjamin Weiss, Dagmar Klebsch and Maximilian
Maschler for their technical assistance. We are further grateful to
Julian Wagner who helped with bioinformatics.
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