# First report of fatty acid-derived alkaloids produced by species of the ladybird genus Scymnus (Coccinellidae: Coleoptera)

Alexandra Magro, Felipe Ramon-Portugal, David Touboul, Isabel Borges, Jean-Louis Hemptinne

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Alexandra Magro, Felipe Ramon-Portugal, David Touboul, Isabel Borges, Jean-Louis Hemptinne. First report of fatty acid-derived alkaloids produced by species of the ladybird genus Scymnus (Coccinellidae: Coleoptera). Comptes Rendus. Chimie, 2023, 26 (S2), pp.1-12. 10.5802/crchim.230. hal-04910003

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# Comptes Rendus
Chimie

Alexandra Magro, Felipe Ramon-Portugal, David Touboul, Isabel Borges and Jean-Louis Hemptinne

First report of fatty acid-derived alkaloids produced by species of the ladybird genus *Scymnus* (Coccinellidae: Coleoptera)

Published online: 14 June 2023

https://doi.org/10.5802/crchim.230

Part of Special Issue: Chemical Ecology – Chemical Mediation in the Environment

Guest editors: Anne-Geneviève Bagnères (Centre d'Ecologie Fonctionnelle et Evolutive (CEFE), Montpellier, France) and Olivier Thomas (University of Galway, Ireland)

<figure><img src="image_2.png" /></figure>
Chemical Ecology – Chemical Mediation in the Environment / Écologie chimique –
Médiation chimique dans l'environnement

First report of fatty acid-derived alkaloids produced
by species of the ladybird genus *Scymnus*
(Coccinellidae: Coleoptera)

Alexandra Magro<sup>a, *, a, b</sup>, Felipe Ramon-Portugal<sup>a, c</sup>, David Touboul<sup>d</sup>, Isabel Borges<sup>e</sup> and Jean-Louis Hemptinne<sup>a, b</sup>

<sup>a</sup> Laboratoire Evolution et Diversité biologique, UMR 5174 CNRS/UPS/IRD,
118 rt de Narbonne Bt 4R1, 31062 Toulouse cedex 9, France

<sup>b</sup> ENSFEA, University of Toulouse, 2 rt de Narbonne, Castanet-Tolosan, France

<sup>c</sup> INTHERES, Université de Toulouse, INRA, ENVT. INTHERES UMR 1436 INRA/ENVT,
23 chemin des Capelles. 31 076 Toulouse, France

<sup>d</sup> Institut de Chimie des Substances Naturelles, Université Paris-Saclay, CNRS,
UPR 2301, 91198, Gif-sur-Yvette, France

<sup>e</sup> cE3c/ABG – Centre for Ecology, Evolution and Environmental Changes/Azorean
Biodiversity Group and University of the Azores, 9501-801 Ponta Delgada, Azores,
Portugal

*E-mails*: alexandra.magro@univ-tlse3.fr (A. Magro), felipe.ramon-portugal@envt.fr
(F. Ramon-Portugal), David.TOUBOUL@cnrs.fr (D. Touboul),
isabel.mm.borges@uac.pt (I. Borges), jean-louis.hemptinne@univ-tlse3.fr
(J.-L. Hemptinne)

**Abstract.** Species in the genus *Scymnus* Kugelann are common, but there is little information available on their defence compounds. Pupae are covered with setae on tips of which there are small droplets of liquid. This study explored the patterns of the distribution of the droplets and the influence of diet on their production in five species of *Scymnus*. GC-MS analyses were used to determine the chemical identity of droplets.

All these species produced droplets, but the patterns in the distributions varied among species.
Preliminary data indicates a de novo synthesis of the droplet compounds by these ladybirds. The
results of the GC-MS analyses suggest that *Scymnus* spp. produce azamacrolides, which are fatty acid-
derived alkaloids.

**Keywords.** Azamacrolides, Defensive compounds, Pupae, Insects, Diet, GC-MS.

*Published online: 14 June 2023*

<footnote>* Corresponding author.</footnote>
# 1. Introduction

The egg and pupa are critical life stages in holometabolous insects because they are immobile and therefore relatively vulnerable to attack by natural enemies. Eggs generally contain antioxidants or defence molecules and are laid where they are less likely to be found and eaten by predators. However, parental care of hatchings is rare, with a few exceptions (e.g. some Hymenoptera and the burying beetles Silphidae), and care after nutritional independence is not reported other than for vertebrates. Nevertheless, pupae are not defenceless. In Coleoptera, the most diversified group of insects, pupae have evolved several physical, behavioural and/or chemical strategies that enable them to avoid being attacked [1].

The Coccinellidae are a large family in the order Coleoptera with a worldwide distribution, comprising two sub-families, 38 tribes and about 6000 described species [2]. Paradoxically, although there has been a longstanding research interest in this group (e.g. review by [3,4]), there is no systematic study of the ecology and defence strategies of ladybird pupae. There is only one comparative analysis of the morphology of pupae [5], which includes 32 genera and 57 species of North American ladybirds. However, this seminal work does not refer to any structural or other mechanism of defence. Apart from that study, the information on pupae is scattered, although it could be valuable for phylogenetic studies, similar to those based on larval characters (e.g. [6]). Not surprisingly, the absence of information leads to conflicting views on pupal defences.

The available information on the subject refers to several ladybird pupal enemy avoidance strategies involving physical and behavioural mechanisms (Table 1). However, the Coccinellidae are mainly known for their chemical defences. A wide range of molecules have been identified, among which there are several alkaloids (reviewed in [7]), including those derived from fatty-acids. Many pupae, like the other ladybird stages, are chemically protected by an aposematic system associating alkaloids in the haemolymph with bright and contrasting colours and/or strong odours. In addition, all developmental stages reflex bleed when attacked, instantaneously expelling droplets through the femorotibial articulations or dorsal glands that contain high concentrations of toxic or repulsive alkaloids [3].

Furthermore, several species of the tribe Epilachnini [15], as well as *Delphastus catalinea* (Horn) of the tribe Serangiini [16], *Platynaspis luteorubra* Goeze of the tribe Platynaspidini [17], and *Nephaspis oculatus* (Blatchley), *Scymnus sinuanodulus* Yu and Yao [18,19], *S. ningshanensis* Yu and Yao [20] and *S. posticalis* Sicard [21] belonging to the tribe Scymnini, have a more specific chemical defence. In these species, the larval exuviae folds back and total or partially uncovers the pupa. The pupal cuticle that is visible bears setae on the tips of which droplets can be seen. Behavioural experiments indicate that these droplets are extremely efficient in deterring attacks by ants [16,21,22]. For species of Epilachnini, the droplets are reported to contain molecules of the azamacrolides family [7] as well as tocopheryl acetates [23,24]. The droplets produced by *D. catalinea* contain polyketides [16]. For the remaining species, the nature of the chemicals in the droplets is unknown.

The present study investigated the chemical defence of the pupae of five species of *Scymnus*. One of the objectives was to determine whether pupae of all the five species produce droplets. Another was to determine if droplet production by the insect pupae is influenced by larval diet or is of autogenous origin. Finally, based on the annotation of GC-MC analyses, the chemical composition of the droplets produced by *Scymnus nubilus* Mulsant is examined.

# 2. Materials and methods

## 2.1. Rearing methods and comparison of the pupae of different species of *Scymnus*

Adults of *S. interruptus* (Goeze), *S. rubromaculatus* (Goeze), *S. apetzi* Mulsant, and *S. subvillosus* (Goeze) were collected near Toulouse (France), and *S. nubilus* in the Azores (Portugal). These species were chosen because of their availability.

Stock cultures of all five species were maintained in the laboratory for at least 2 generations (around 23 days from egg to adult stage) before the study was initiated. Rearing conditions were LD 16:8 and 22 ± 1 °C. Ladybird adults were kept in 2-L plastic boxes with a piece of corrugated filter paper and were fed three times a week, *ad libitum*, with either *Acyrthosiphon pisum* (Harris) or *Rhopalosiphum padi* L, common prey of *Scymnus* spp. (e.g. [25]).
<table><caption>**Table 1.** Defence strategies of ladybird pupae</caption><thead><tr><th>Strategy</th><th>Taxa</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="3">Gin traps</td><td>Coccinellini</td><td>[8]</td></tr><tr><td>Coccidulini and Chilocorini</td><td>[3]</td></tr><tr><td><i>Rhyzobius chrysomeloides</i> (Herbst)</td><td>(*)</td></tr><tr><td rowspan="4">Pupa enclosed in larva exuvium but visible through dorso-longitudinal slit</td><td>Chilocorini</td><td>[8]</td></tr><tr><td><i>Rodolia</i> spp, <i>Anovia circumclusa</i> (Gorham)</td><td>[9]</td></tr><tr><td><i>Hyperaspis maindroni</i> Sicard</td><td>[10]</td></tr><tr><td><i>Thalassa saginata</i> Mulsant</td><td>[11]</td></tr><tr><td rowspan="3">Pupa enclosed in waxy [12] larva exuvium</td><td><i>Azya</i> spp</td><td>[13]</td></tr><tr><td><i>Hyperaspis silvestrii</i> Weise</td><td>[14]</td></tr><tr><td><i>Cryptolaemus</i> spp, <i>Nephus</i> spp</td><td>(*)</td></tr></tbody></table>

Two stems of broad bean, *Vicia faba* L. or *Triticum turgidum* L. were added to the boxes to improve the survival of *A. pisum* or *R. padi*, respectively. Adult ladybirds were transferred to new boxes every 2 weeks and the eggs and larvae that remained in the old boxes were reared in similar conditions to the adults (see above). For each species, all the fourth instar larvae available in the stock cultures were collected and isolated in 3 cm Petri dishes, kept at the same temperature and photoperiod as the stock cultures and fed three times a week an excess of aphids according to the diet treatment. On reaching the pupal stage, they were observed daily to check for the presence of droplets. In addition, pupae were checked for the presence of wax that typically covers the larvae of *Scymnus* [12] and some of which can be present on the pupae.

## 2.2. Collection of droplets from pupae of *Scymnus nubilus*

The mean diameter of the droplets on the tips of setae on the pupae of *S. nubilus* is about 50 µm and they were collected using Solid Phase Micro-extraction (SPME) fibers (50/30 µm DVB/CAR/ PDMS, Stableflex 24Ga, Supelco, Bellefonte, PA, USA). This enabled us to exclusively analyse droplets without collecting other cuticular compounds.

The fibers were conditioned before use by insertion in the GC-MS injector port for 1 h at 270 °C, to remove contaminants that might result in a high background reading in the chromatogram. They were

then gently rubbed over the tip of the setae to collect the droplets. The droplets of 45 pupae were collected. For pupae with a dense covering of setae, it is difficult to determine exactly how many droplets were collected per individual; each fiber was loaded with the droplets from 15 pupae and therefore 3 fibers (samples) were used for analyses. Simultaneously, a blank test to check for possible laboratory environment contaminations was performed by leaving another fiber close to the stereomicroscope. Each fiber was then injected directly in a GC-MS.

## 2.3. GC-MS analysis of droplets collected from pupae of *Scymnus nubilus*

Analyses were done on a two-step basis. Firstly, two fibers were injected into a low-resolution mass spectrometer quadrupole detector (ISQ QD) coupled to a Trace 1300 gas chromatography (Thermo Fisher Scientific Inc., Illkirch, France) fitted with an apolar capillary column (Restek RTX-5MS 30 m × 0.25 mm, 0.25 µm film thickness, 5% diphenyl and 95% dimethylpolysiloxane) and a splitless injector (280 °C). Then, a third fiber was injected into a high-resolution GCT Premier TOF mass spectrometer (Waters, Manchester, UK) coupled to a gas chromatography 6890 series (Agilent Technologies, Palo Alto, CA, USA) fitted with a similar capillary column to the one described above and a splitless injector. This high-resolution MS enables the determination of the exact mass of the molecular ion and fragments, giving additional information on the elemental

<footnote>(*) Authors pers. obs.</footnote>
composition that is used to improve the annota-
tion of the chromatographic peaks. In both GC-MS,
ionization was done by electron ionization (70 eV,
source temperature 200 °C). The mass spectra were
scanned from *m/z* 20 to *m/z* 700. Helium was the
carrier gas (1.2 mL·min⁻¹). The initial oven temper-
ature was set at 50 °C for 1 min. Then, the tempera-
ture was increased by 20 °C·min⁻¹ to 140 °C, then by
3 °C·min⁻¹ to 300 °C. This final temperature was held
for 3 min. Prior to each injection of a fiber loaded
with droplets, a control empty fiber, i.e. exposed to
the same environmental conditions, was injected.

The purity of the chromatographic peaks was
checked with the help of the Mass Spectral Decon-
volution and Identification System program (AMDIS
2.70). For each of the fractions the most likely ele-
mental composition was calculated [26]. The limits
of each fragment were fixed as follows: C—from 1 to
50 atoms; H—from 0 to 100 atoms; O—from 0 to 7
atoms; N—from 0 to 3 atoms. All data was processed
using MassLynx software (Dat version 4.0, Waters
SAS, Guyancourt, France). Accurate masses were
calculated and used for the determination of the
elementary composition of analytes. Moreover, the
Kovat's retention indices were calculated using [27]
and by comparing the peaks to an external stan-
dard consisting of a solution of *n*-alkanes (n-C<sub>7</sub>H<sub>16</sub>-
n-C<sub>40</sub>H<sub>82</sub>) of known concentrations. The degree
of unsaturation was used to determine how many
rings and/or double bonds are present in a com-
pound [28]. The structure of each fragment ion was
identified using (i) the exact mass of the fragment in
high resolution (ii) the most common fragment ion
tables [29], and (iii) the ACD/MS Fragmenter soft-
ware (2016, 1.1, Advanced Chemical Development,
Toronto, ON, Canada).

**3. Results**

3.1. *Observations on the pupae of five species of*
*Scymnus*

Observations of the droplets (Figure 1) produced by
the pupae of 5 species of *Scymnus* whose larvae were
reared on two different diets are presented in Table 2.
These results indicate that all of these species of
*Scymnus* can produce droplets, but their distribution
on pupae varies among species with three main pat-
terns: (a) all pupae present droplets and there are
droplets all over them, (b) most pupae don't produce

droplets and, when they do, these are present on the
pronotum and the rest of the body is partially cov-
ered by the exuvium of the last larval instar, and (c) all
pupae produce droplets, which are present on the
pronotum and the rest of the body is not covered by
the exuvium of the last larval instar. The 40 individ-
uals of *S. apetzi* reared on the different diets did not
differ in droplet production, but there are insufficient
results for the remaining species to make it a general
assertion.

3.2. *Chemical analyses of the droplets produced by pupae of Scymnus nubilus*

Figure 2 shows the chromatographic profile of a direct injection of the pupal secretions from a SPME fiber, obtained using the high-resolution GC-MS. This analysis reveals two main peaks at retention times of 24.99 and 28.83 min, which make up 87% of the total area of all the peaks. Analysis of these two major peaks reveals the absence of co-elution, indicating that they are each for a single compound. The small peaks from 0 to 24 min are mainly hydrocarbons and siloxanes eluted from the chromatographic column during the temperature gradient.

Analysis of the mass spectra of the two major
peaks (Figure 3) indicates a similar fragmentation
pattern with ion signals at *m/z* 55, 69, 81, 83, 95, 102,
123, 210, 240 and 268. In addition, the mass spectra
of the two major peaks show a loss of 29. Nonethe-
less, the peak with a retention time of 24.99 min ex-
hibits a molecular ion at *m/z* 478.37, while for the
peak with a retention time of 28.33 min a molecular
ion at *m/z* 506.40 is detected. Although the two mass
spectra show a difference in the intensity of the ion
at *m/z* 268, this can be explained by the difference in
the size of the carbon chain [22] and the two peaks
can therefore be considered as belonging to the same
chemical family.

Table 3 shows the *m/z* values and chemical for-
mula/possible structures of each ion detected by
high-resolution GC-MS. According to the accu-
rate mass measurement, all ions are composed
only of carbon, hydrogen, oxygen and nitrogen.
The proposed chemical formulas for the ion signal
at 24.99 min and 28.83 min are C₂₈H₅₀N₂O₄ and
C₃₀H₅₄N₂O₄, respectively. The two chemical formu-
las have five degrees of unsaturation, which may
indicate the presence of double bonds and/or the
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Photograph on the left, dorsal view of a pupa of *Symnus nubilus*, and on the right, a high magnification photograph of glandular setae on the dorsal integument of a pupa.</figcaption></figure>

<table><caption>**Table 2.** Distribution of droplets and presence of wax on pupae of five species of *Scymnus* when fed on two different aphid diets (*Acyrthosiphon pisum* or *Rhopalosiphum padi*)</caption><thead><tr><th rowspan="2">Species</th><th colspan="2">Diet</th><th rowspan="2">Distribution of droplets and presence of wax</th></tr><tr><th><i>A. pisum</i></th><th><i>R. padi</i></th></tr></thead><tbody><tr><td><i>S. nubilus</i></td><td>20/20</td><td>–</td><td>Large droplets, all over the body</td></tr><tr><td><i>S. apetzi</i></td><td>20/20</td><td>20/20</td><td>Large droplets, all over the body</td></tr><tr><td><i>S. interruptus</i></td><td>3/22</td><td>0/10</td><td>Droplets, when present, small and on the pronotum<br>Wax from the last larval exuviae partially covering the body</td></tr><tr><td><i>S. rubromaculatus</i></td><td>*</td><td>14/32</td><td>Droplets, when present, small and on the pronotum<br>Wax from the last larval exuviae partially covering the body</td></tr><tr><td><i>S. subvillosous</i></td><td>2/2</td><td>7/7</td><td>Small droplets present on the pronotum</td></tr></tbody></table>

Number of individuals with droplets divided by the total number of individuals examined. * Non-accepted prey; – rearing not done.

presence of a ring. The difference between the two molecules can be attributed to a C₂H₄ increment typical of an increment in a fatty acid chain.

Based on the elementary composition and with the help of the ACD Fragmenter software and the most common fragment ion table, it is reasonable to propose the structure of fragment ions below *m/z* 110 (Table 3). The following identification of the unsaturation sites is proposed: (i) Fragment at *m/z* 69.0997, present in both compounds (C₂₈H₅₀N₂O₄—24.99 min; C₃₀H₅₄N₂O₄—28.83), can be attributed to C₅H₉⁺ indicating the presence of C=C double bond; (ii) The two compounds present

a fragment ion at *m/z* 30.01 related to a carbonyl function C=O; (iii) a neutral loss of 29 amu from both molecular ions is detected. This can be attributed to an ethyl group —CH₂—CH₃ leading to the fragment ion at *m/z* 449.33 for the peak at 24.99 min, and at *m/z* 477.36 for the peak at 28.83 min.

The above results confirm that the two major ion signals on the chromatogram belong to the same chemical family. As it was not possible to exclude the presence of small quantities of other molecules of the same chemical family in the sample, a more thorough analysis of the chromatogram was carried out focused on the three fragment ions at *m/z* 102,
<table><caption>**Table 3.** *m/z* values and some structures of major fragments of the mass spectra of the two major compounds in the secretion of the glandular setae on pupae of *S. nubilus* (cf. Figure 2)</caption><thead><tr><th colspan="2">m/z value in positive mode</th><th rowspan="2">Elementary composition</th><th rowspan="2">Structure</th></tr><tr><th>Peak RT = 24.99 min<br>KI = 3644</th><th>Peak RT = 28.83 min<br>KI = 3831</th></tr></thead><tbody><tr><td>30.0115</td><td></td><td>CH<sub>2</sub>O</td><td>O•CH<sub>2</sub><sup>+</sup></td></tr><tr><td>30.0344</td><td>30.0350</td><td>CH<sub>4</sub>N</td><td>H<sub>2</sub>C<sup>+</sup>NH<sub>2</sub></td></tr><tr><td>44.0493</td><td>44.0493</td><td>C<sub>2</sub>H<sub>6</sub>N</td><td>H<sub>2</sub>NCH<sub>2</sub><sup>+</sup></td></tr><tr><td>55.0176</td><td>55.0176</td><td>C<sub>3</sub>H<sub>3</sub>O</td><td>OCH<sub>2</sub>CH<sub>2</sub><sup>+</sup></td></tr><tr><td>55.0540</td><td>55.0540</td><td>C<sub>4</sub>H<sub>7</sub></td><td>CH<sub>3</sub><sup>+</sup></td></tr><tr><td>69.0997</td><td>69.0997</td><td>C<sub>5</sub>H<sub>9</sub></td><td>CH<sub>3</sub>CH<sub>2</sub><sup>+</sup></td></tr><tr><td>84.0816</td><td>84.0813</td><td>C<sub>5</sub>H<sub>10</sub>N</td><td>H<sub>2</sub>C<sup>+</sup>NCH<sub>2</sub>CH<sub>3</sub></td></tr><tr><td>95.0858</td><td>95.0855</td><td>C<sub>7</sub>H<sub>11</sub></td><td>CH<sub>3</sub>CH<sub>2</sub>CH<sup>+</sup></td></tr><tr><td>102.0910</td><td>102.0910</td><td>C<sub>5</sub>H<sub>12</sub>NO</td><td>H<sub>3</sub>C<sup>+</sup>NH<sub>2</sub>OH</td></tr><tr><td>210.1481</td><td>210.1513</td><td>C<sub>12</sub>H<sub>20</sub>NO<sub>2</sub></td><td></td></tr><tr><td>240.1947</td><td>240.1964</td><td>C<sub>14</sub>H<sub>26</sub>NO<sub>2</sub></td><td></td></tr><tr><td>268.2283</td><td>268.2283</td><td>C<sub>16</sub>H<sub>30</sub>NO<sub>2</sub></td><td></td></tr><tr><td>449.3379</td><td></td><td>C<sub>26</sub>H<sub>45</sub>NO<sub>2</sub></td><td></td></tr><tr><td></td><td>477.3689</td><td>C<sub>28</sub>H<sub>49</sub>N<sub>2</sub>O<sub>4</sub></td><td></td></tr><tr><td>478.3771 (M+)</td><td></td><td>C<sub>28</sub>H<sub>50</sub>N<sub>2</sub>O<sub>4</sub></td><td></td></tr><tr><td></td><td>506.4084 (M+)</td><td>C<sub>30</sub>H<sub>54</sub>N<sub>2</sub>O<sub>4</sub></td><td></td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** A high resolution GC-MS chromatographic profile of the droplets collected from glandular setae on the pupa of *S. nubilus*.</figcaption></figure>

210, and 240, respectively. The low-resolution GC-MS chromatograms were used for this analysis because the instrument has a higher sensitivity in terms of signal-to-noise ratio (S/N) compared to the high-resolution GC-MS. The limit of detection was set at a S/N of 10:1. This enabled the detection of four smaller chromatographic peaks (Figure 4), whose mass spectra are presented in Figure 5. The spectra are characteristic of ions from the same molecular family according to the intense common fragment ion at *m/z* 240 but are differenced by the number of insaturations (difference of 2 mass units). The signal-to-noise ratio for these peaks was higher than 25 indicating they cannot be interpreted as noise. They all present similarities in their fragmentation profiles indicating that all 6 peaks belong to the same chemical family. The relative amounts of these peaks (for a total area of 100% under those peaks) were 74.71, 23.7, 0.61, 0.59, 0.25, and 0.14%, respectively, for peaks 1 to 6.

**4. Discussion**

Species of *Scymnus* are distributed worldwide and are very common in guilds of aphid and coccid predators (e.g. [30]). However, they are small and dull

coloured, which probably explains why there is so little information available on their chemical ecology and life history in general, compared to ladybirds in some other tribes [25].

Pupae of the *Scymnus* are covered in setae with droplets of defensive compounds at their tips. In this paper, the chemical defences of the pupae of five species of *Scymnus* were studied. The production and distribution of droplets on pupae was described and the chemical composition of droplets produced by pupae of *S. nubilus* was determined.

Droplets were produced by the pupae of all the species studied and in addition another 3 *Scymnus* species are reported producing droplets in the literature [18–21]. Therefore, it is a widespread phenomenon in this genus. It is also noteworthy that the distribution of droplets on pupae of the 5 species varied, with three different patterns corresponding to the three clades: *S. apetzi/S. nubilus*, *S. interruptus/S. rubromaculatus*, and *S. subvillosus*. These clades correspond to the species phylogenetic relationships highlighted by Magro *et al.* [31] and presented in Figure 6. These patterns do not seem to be related to the distribution of setae, but might be related to their morphology, which was not studied.
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Mass spectra of the two major chromatographic peaks of the secretion produced by glandular setae on the pupae of *S. nubilus*. 1 = mass spectra of the peak with retention time 24.99 min; 2 = mass spectra of the peak with retention time 28.83 min.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 4.** Low resolution GC-MS chromatographic profile (scan filter *m/z* 240, 210 and 102) showing the six compounds in the droplets produced by the glandular setae on the pupa of the ladybird *S. nubilus*.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Mass spectra of the four minor chromatographic peaks of the secretion produced by the glandular setae on the pupae of *S. nubilus*. 3, 4, 5 and 6 are the mass spectra of the chromatographic peaks in Figure 4.</figcaption></figure>

Diet does not seem to influence droplet production, suggesting that *Scymnus* make a *de novo* synthesis of the compounds in the droplets, which is generally the case for the defensive compounds in the Coccinellidae [32], but this needs to be verified.

Furthermore, the chemical composition of droplets produced by pupae given different diets was not analysed, which could be interesting as there is a putative case of a ladybird sequestering defensive chemicals, and an additive effect of autogenous
<figure><img src="image_8.png" /><figcaption>**Figure 6.** Phylogenetic relationships of the 5 species of *Scymnus* studied, based on Magro *et al.* [31].</figcaption></figure>

defences and chemicals sequestered via certain
foods cannot be excluded [33].

The GC-MS analyses of the chemical composi-
tion of *S. nubilus* droplets indicated the presence
of 6 compounds, the two major ones corresponding
to C28H50N2O4 and C30H54N2O4 elemental compo-
sitions. Four characteristics of these molecules are
important: (i) presence of oxygen, (ii) for every two
molecules of nitrogen there are 4 molecules of oxy-
gen, (iii) there are five degrees of unsaturation related
to C=C and C=O double bonds and (iv) the com-
pounds are volatile (low polarity) in the GC-MS with-
out the need of derivatization, indicating that the ni-
trogen atom probably lies within a ring. As the vol-
ume of droplet liquid available is very small it is not
possible at this stage to carry out further analyses,
such as purification and complete structural identi-
fication by NMR and circular dichroism. Searches in
the Pherobase database [34], however, indicate the
azamacrolides family, i.e. fatty acid-derived alkaloids
(Figure 7). Moreover, the chemical analyses of this
study may have missed some compounds because
GC-MS cannot detect chemicals with high boiling

points and there are some reports of such high-
boiling chemicals in the droplets produced by pupae
of ladybirds (e.g. [35]). Thus, an analysis of droplets
using HPLC-MS is needed, which will require much
larger volumes of droplet liquid than available in the
present study.

It is noteworthy that azamacrolides are present
in the liquid on the tips of setae on pupae of the
ladybird tribe Epilachnini [15,22]. Comparison of the
mass spectra of the liquid collected from *S. nu-
bilus* with that of the mono-azamacrolide epi-
lachnene reported by Attygalle *et al.* [22] for *Epi-
lachna varivensis* Mulsant (Figure 7A, B) revealed
they both have six fragment ions at *m/z* 44.093,
55.0176, 55.0540, 69.0997, 84.0816 and 95.0858. The
mono-azamacrolides, however, only have three de-
grees of unsaturation whereas in the case of *S. nu-
bilus* it is five degrees of unsaturation. Moreover,
the ions at *m/z* 268 and 240 are complementary
and the sum of the two fragments leads to the
molecular ions at *m/z* 478 (= 240 + 240 - 2) and
*m/z* 506 (= 268 + 240 - 2) indicating the presence
of a macrocycle resulting from the condensation
<figure><img src="image_9.png" /><figcaption>**Figure 7.** (A, B) Mono-Azamacrolides produced by *Epilachna varivensis* [22]; (C) di-Azamacrolide produced by *Epilachna borealis* and *Subcoccinella vigintiquatuorpunctata* [35]; (D) structure suggested in this study for those produced by pupae of *Scymnus nubilus*, where R₁ and R₂ are two CH₃-CH₂-ethyl chains, R₃ and R₄ are two mono-unsatured chains, and R₅ and R₆ are methyl or ethyl chain.</figcaption></figure>

of two subsequent molecules. Thus, it is likely that *S. nubilus* produces di-azamacrolides as it is the case for the Epilachnini *Subcoccinella vigintiquatuorpunctata* (L) (Figure 7C, D). A full comparison is not possible as there are no GC-MS mass spectra of di-azamacrolides. The lack of available reference compounds remains a problem for studies on natural product for which the size of the sample available is very small.

The study by Seago *et al.* [6] based on the simultaneous analysis of molecular data and morphological characters of both ladybird adults and larvae shows that an integrative approach can be important to resolve evolutionary relationships within the Coccinellidae. Nevertheless, the evolutionary history of the Coccinellidae is blurred by rapid radiation and interfering the major relationships of the ladybird tribes remains a challenge [2,36]. As specialized metabolites have been used in studies on many taxa as complementary tools to traditional morphological and gene-based phylogenetic exploration [37], we recommend additional studies on the defence traits of ladybirds

to help elucidate the internal phylogenetic relationships within the Coccinellidae.

**Conflicts of interest**

Authors have no conflict of interest to declare.

**Acknowledgements**

We thank G. Espinasse and J.-F. Garrigues for technical support in collecting droplets and rearing the insects. We are also grateful to J. Bouajila for helpful comments on an early version of the manuscript. Finally, we would like to thank T. Dixon who assisted with manuscript editing. The laboratory "Evolution et Diversité Biologique" is part of the "Laboratoire d'Excellence" LABEX TULIP (ANR-10-LABX-41) and LABEX CEBA (ANR-10-LABX-25-01).

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