Iridoid Sex Pheromone Biosynthesis in Aphids Mimics Iridoid-Producing Plants

Article abstract of DOI:10.1002/chem.202001356

Biosynthesis of (1R,4aS,7S,7aR)-nepetalactol (1) and (4aS,7S,7aR)-nepetalactone (2) in plants involves iridoid synthase (ISY), an atypical reductive cyclase that catalyses the reduction of 8-oxogeranial into the reactive enol of (S)-8-oxocitronellal, and cyclization of this enol intermediate, either non-enzymatically or by a nepetalactol-related short chain dehydrogenase enzyme (NEPS) that yields the nepetalactols. In this study, we investigated the biosynthesis in vivo of 1 and 2 in the pea aphid, Acyrthosiphon pisum, using a library of isotopically-labelled monoterpenoids as molecular probes. Topical application of deuterium-labelled probes synthesized from geraniol and nerol resulted in production of ²H₄?lactol 1 and ²H₄?lactone 2. However, deuterium incorporation was not evident using labelled probes synthesized from (S)-citronellol. These results suggest that iridoid biosynthesis in animals, specifically aphids, may follow a broadly similar route to that characterised for plants.

Full text of DOI:10.1002/chem.202001356

Communication
doi.org/10.1002/chem.202001356
Chemistry—A European Journal
Iridoid Sex Pheromone Biosynthesis in Aphids Mimics
Iridoid-Producing Plants
Suzanne J. Partridge,[a, b] David M. Withall,[a] John C. Caulfield,[a] John A. Pickett,[c]
Robert A. Stockman,[b] Neil J. Oldham,[b] and Michael A. Birkett*[a]
Abstract: Biosynthesis of (1R,4aS,7S,7aR)-nepetalactol (1)
and (4aS,7S,7aR)-nepetalactone (2) in plants involves iridoid
synthase (ISY), an atypical reductive cyclase that catalyses
the reduction of 8-oxogeranial into the reactive enol of (S)-
8-oxocitronellal, and cyclization of this enol intermediate,
either non-enzymatically or by a nepetalactol-related short
chain dehydrogenase enzyme (NEPS) that yields the
nepetalactols. In this study, we investigated the biosyn-
thesis in vivo of 1 and 2 in the pea aphid, Acyrthosiphon
pisum, using a library of isotopically-labelled monoterpe-
noids as molecular probes. Topical application of deute-
rium-labelled probes synthesized from geraniol and nerol
2
2
resulted in production of H4À lactol 1 and H4À lactone 2.
However, deuterium incorporation was not evident using
labelled probes synthesized from (S)-citronellol. These
results suggest that iridoid biosynthesis in animals, specifi-
cally aphids, may follow a broadly similar route to that
characterised for plants.
Figure 1. Upper: Aphid sex pheromone components (1R,4aS,7S,7aR)-nepeta-
lactol (1), (4aS,7S,7aR)-nepetalactone (2), (1S,4aR,7S,7aS)-nepetalactol (3) and
(1R,4aR,7S,7aS)-nepetalactol (4). Lower: Iridoid biosynthetic pathway in
Nepeta spp plants (modified from Lichman et al., 2019).
Aphids (Homoptera: Aphididae) are major pests of arable and
horticultural crops throughout the world, causing damage both
directly and indirectly through their feeding behaviour and
plant virus transmission.[1] Pheromones and other semiochem-
icals (naturally-occurring behaviour- or development-modifying
chemical signals) play a critical role in the life-cycle of aphids.[2]
The female-produced sex pheromone for many pest aphid
species mainly comprises the iridoid bicyclic monoterpenoids
(1R,4aS,7S,7aR)-nepetalactol (1) and (4aS,7S,7aR)-nepetalactone
(2), but other diastereoisomers of the nepetalactol 1, i.e.
(1S,4aR,7S,7aS)-nepetalactol (3) and (1R,4aR,7S,7aS)-nepetalactol
(4) are also utilized as sex pheromone components by the
damson-hop aphid, Phorodon humuli (Figure 1, upper panel).[3]
Practical development of the aphid sex pheromone for pest
management has met with success in the trapping of male
aphids during the mating season but has mainly focused on the
recruitment of beneficial natural enemy wasps which utilize 2
as a cue for the location of aphid hosts.[4–6] Biosynthesis of 1
and 2 in plants has received much attention,[7–14] with elegant
mechanistic studies showing that iridoid synthase (ISY), an
atypical reductive cyclase identified from Nepeta spp, Cathar-
anthus roseus, Olea europaea and Antirrhinum majus, catalyses
the reduction of 8-oxogeranial into the reactive enol of (S)-8-
oxocitronellal.[14] Cyclization of this enol intermediate, either
non-enzymatically or by a nepetalactol-related short chain
dehydrogenase enzyme (NEPS) yields the nepetalactols, which
can undergo a final oxidation to nepetalactones (Figure 1
lower).[14] Concurrent to these studies, we have investigated the
biosynthesis in vivo of 1 and 2 in aphids, using the pea aphid,
Acyrthosiphon pisum, as the model species, and a library of
isotopically-labelled monoterpenoids as molecular probes, to
test the hypothesis that biosynthesis of 1 and 2 in animals,
specifically aphids, follows a similar route to that determined in
plants.
[a] Dr. S. J. Partridge, Dr. D. M. Withall, Dr. J. C. Caulfield, Dr. M. A. Birkett
Biointeractions and Crop Protection Department
Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ (UK)
E-mail: mike.birkett@rothamsted.ac.uk
[b] Dr. S. J. Partridge, Prof. R. A. Stockman, Prof. N. J. Oldham
School of Chemistry, University of Nottingham
University Park, Nottingham, NG7 2RD (UK)
[c] Prof. J. A. Pickett
School of Chemistry, Cardiff University, Cardiff CF10 3AT (UK)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/chem.202001356
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Scheme 1. Production of deuterium-labelled putative sex pheromone precursors and intermediates. Boxed compounds indicate deployment in aphid sex
pheromone collections from female pea aphids, Acyrthosiphon pisum.
Synthesis of isotopically-labelled putative precursors was
undertaken via multi-step synthesis starting from unlabelled
geraniol, nerol and (S)-citronellol (Scheme 1) with the aim of
incorporating labelling at the C3 and C8 positions of the
nepetalactone skeleton.[15,16,17] Elaboration of [2H6]-labelled ace-
tates furnished [2H6]-alcohols, [2H6]-aldehydes, [2H5]-8-hydroxyal-
cohols, [2H5]-8-hydroxyaldehydes, [2H4]-8-oxoalcohols and [2H4]-
oxoaldehydes. Employing standard topical application method-
ology of the labelled probes onto the upper (dorsal) surface of
female pheromone-producing A. pisum at two doses (0.1 mg
and 1 mg, Supporting Information) was followed by pheromone
collection from aphids using dynamic headspace collection.[18]
Coupled gas chromatography – mass spectrometry (GC-MS,
70 eV EI+) analysis of pheromone collections was undertaken
to provide evidence of deuterium incorporation at the C3 and
C8 positions in the final nepetalactone products (molecular ion
m/z 172 for [2H4]-nepetalactol 1, m/z 170 for [2H4]-nepetalactone
2) (Figure 2).
Coupled GC-MS analysis of pheromone collections following
application of [2H6]-alcohols (14) and [2H6]-aldehydes (15)
showed no evidence of deuterium incorporation (Table 1, see
Supporting Information). Application of [2H5]-8-hydroxygeraniol
and [2H5]-8-hydroxynerol led to the production of [2H4]-1 and
[2H4]-2. The presence of [2H4]-1 and [2H4]-2 was observed again
when [2H5]-8-hydroxygeranial, [2H5]-8-hydroxyneral, [2H4]-8 oxo-
geraniol, [2H4]-8 oxonerol, [2H4]-8 oxogeranial and [2H4]-8-
oxoneral were applied. By contrast, no deuterium labelling was
Figure 2. Typical coupled gas chromatography from feeding [2H5]-8-hydrox-
ygeraniol 20a – mass spectrometry (GC-MS, 70 eV EI+) analysis of
pheromone collections from female pea aphids, Acyrthosiphon pisum,
following application of synthesized deuterium-labelled compounds listed in
Table 1. Insert: mass spectra of unlabelled and deuterium-labelled pher-
omone components, correlating to unsuccessful and successful deuterium
incorporation respectively i) unlabelled nepetalactol 1 ii) unlabelled
nepetalactone 2 iii) labelled nepetalactol [3,8,8,8-2H4]-1 and iv) labelled
nepetalactone [3,8,8,8-2H4]-2. [3,8,8,8-2H4]-1 shows a molecular ion m/z 172
and a fragment ion m/z 154 following loss of H2O whilst [3,8,8,8-2H4]-2 shows
a molecular ion m/z 170 and a fragment ion m/z 142 following loss of CO.
observed for the pheromone components when any of the
labelled forms of (S)-citronellol were applied. In the cases where
deuterium incorporation was observed,
a high level of
deuterium incorporation was observed (>87%), determined by
comparison of the isotopomer peak area.
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Table 1. Incorporation of deuterium labelling in sex pheromone collected from female pea aphids, Acyrthosiphon pisum.
Deuterium-labelled compound applied to female A. pisum
Evidence of deuterium incorporation
in nepetalactol 1
Evidence of deuterium incorporation
in nepetalactone 2
[8,8,8,10,10,10-2H6]-labelled compounds
2H6À Geraniol (14a)
×
×
×
×
×
×
×
×
×
×
×
×
*
2H6À Nerol (14b)
*
2H6-(S)-Citronellol (14c)
*
2H6À Geranial (15a)
*
2H6À Neral (15b)
*
2H6-(S)-Citronellal (15c)
*
[8,8,10,10,10-2H5]-labelled compounds
p
p
p
p
2H5-8-Hydroxygeraniol (20a)
*
2H5-8-Hydroxynerol (20b)
*
2H5-(S)-8-Hydroxycitronellol (20c)
×
×
*
p
p
2H5-8-Hydroxygeranial (24a)
*
p
p
2H5-8-Hydroxyneral (24b)
*
2H5-(S)-8-Hydroxycitronellal (24c)
×
×
*
[8,10,10,10-2H4]-labelled compounds
p
p
p
p
2H4-8-Oxogeraniol (17a)
*
2H4-8-Oxonerol (17b)
*
2H4-8-(S)-Oxocitronellol (17c)
×
×
*
p
p
2H4-8-Oxogeranial (18a)
*
p
p
2H4-8-Oxoneral (18b)
*
2H4-(S)-8-Oxocitronellal (18c)
×
×
*
Biosynthesis of nepetalactones from 8-oxogeranial in plants
is thought to be a multi-step process involving reduction and
cyclization to generate an activated non-isolable enol inter-
mediate, followed by oxidation to a corresponding lactone.
Recently, Lichman et al suggested that these steps are
uncoupled and catalysed by different enzymes (ISY, NEPS) with
the enol intermediate diffusing between enzyme active sites.[14]
Our data suggest that female A. pisum aphids are not only able
to incorporate 8-oxogeranial as a substrate for nepetalactone
production, similar to that observed for plants, but are also able
to utilize closely related compounds which only differ in their
oxidative state. Incorporation of neryl-derived substrates in
nepetalactone biosynthesis as described in our work has not
been reported in vivo elsewhere, even in plants although in vitro
activity has been observed,[8] suggesting that further work on
enzyme acceptability of substrates in this biosynthetic pathway
needs to be undertaken, as well as confirmation of the
stereochemistry of the resulting nepetalactol/nepetalactone.
The unacceptability of [2H6]-alcohols and [2H6]-aldehydes sug-
gests that A. pisum is unable to use non-ω-oxygenated
monoterpenoid precursors for sex pheromone biosynthesis.
Given the high degree of deuterium incorporation observed for
probes that are accepted, unacceptability could be due to
either a lack of oxygenase in A. pisum limiting hydroxylation of
the [2H6]-alcohols and [2H6]-aldehydes, inability of compounds
to pass through the haemolymph, or, less likely, aphid depend-
ence on the host plants for availability of the intermediates.
Lichman et al.[14] reported that (S)-8-oxocitronellal is not
incorporated as a substrate for NEPS enzymes, and that high
concentrations of buffer are required for the formation of the
enol intermediate which either undergoes cyclization sponta-
neously or acts as a substrate for NEPS3, a multi-functional
cyclase-dehydrogenase. In our experiments, none of the
citronellyl-based labelled compounds (including the 8-oxo
variants) resulted in production of labelled nepetalactones,
despite the fact that they were performed in vivo, with potential
for the presence of all biosynthetic enzymes utilized by the
insect. This implies that either (i) unlike for plants, nepetalac-
tone biosynthesis in A. pisum occurs via tandem reduction/
cyclization of 8-oxogeranial, as postulated in Figure S2 or (ii) if
nepetalactone biosynthesis occurs via a non-concerted process,
as for plants, then exogenously added 8-oxocitronellal cannot
insert into the pathway, possibly due to the absence of
spontaneous enol formation. Interestingly, Lichman et al.
showed that cyclization of (S)-8-oxocitronellal i.e. via the enol in
high buffer conditions leads to formation of nepetalactols with
relative stereochemistry similar to 3 and 4, which comprises the
sex pheromone for P. humuli. It could be that P. humuli
possesses the enzymology for incorporating citronellyl-derived
compounds in pheromone biosynthesis whereas A. pisum does
not.
Assuming that scenario (ii) above is correct, as A. pisum
does not produce nepetalactol 3, one could reasonably expect
that citronellyl derivatives will not be accepted. Our early work
on sex pheromone identification in aphids detected the
presence of (S)-citronellol in pheromone collections, with the
implication being that it was involved in the biosynthesis of 1
and 2.[16] However, its presence in collections alongside 1 and 2
could also be explained by a lack of enzyme acceptability/
suitable enzyme to catalyse cyclization. Further work is required
to confirm if P. humuli, which utilises 3 and 4 as a sex
pheromone, can incorporate any of the citronellyl derivatives
used in this study in sex pheromone production, and to confirm
the enzymology involved in aphid sex pheromone biosynthesis.
In summary, we provide evidence for the biosynthesis of
(1R,4aS,7S,7aR)-nepetalactol 1 and (4aS,7S,7aR)-nepetalactone 2
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Acknowledgements
This work was supported by a BBSRC Rothamsted-University of
Nottingham Doctoral Training Programme (DTP) studentship,
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Conflict of Interest
The authors declare no conflict of interest.
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Keywords: biosynthesis
· mass spectrometry · molecular
probe · pea aphid · sex pheromone
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Manuscript received: March 18, 2020
Version of record online: April 13, 2021
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