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\DOI{10.5802/crchim.230}
\datereceived{2023-02-16}
\daterevised{2023-04-06}
\dateaccepted{2023-04-11}
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\dateposted{2023-06-14}
\begin{document}

\begin{noXML}
%
%\TopicEF{Chemical Ecology -- Chemical Mediation in the
%Environment}{\'Ecologie chimique -- M\'ediation chimique dans
%l'environnement}

\title{First report of fatty acid-derived alkaloids produced by species
of the ladybird genus \protect\textit{Scymnus} (Coccinellidae:
Coleoptera)}

\author{\firstname{Alexandra} \lastname{Magro}\CDRorcid{0000-0002-7043-0845}\IsCorresp}
\address{Laboratoire Evolution et Diversit\'{e} biologique, UMR 5174
CNRS/UPS/IRD, 118~rt~de~Narbonne Bt 4R1, 31062 Toulouse cedex 9,
France}
\address{ENSFEA, University of Toulouse, 2 rt de Narbonne,
Castanet-Tolosan, France}
\email[A. Magro]{alexandra.magro@univ-tlse3.fr}

\author{\firstname{Felipe} \lastname{Ramon-Portugal}\CDRorcid{0000-0002-9519-9707}}
\addressSameAs{1}{Laboratoire Evolution et Diversit\'{e} biologique,
UMR 5174 CNRS/UPS/IRD, 118 rt de Narbonne Bt 4R1, 31062 Toulouse cedex
9, France}
\address{INTHERES, Universit\'{e} de Toulouse, INRA, ENVT. INTHERES UMR
1436 INRA/ENVT, 23 chemin des Capelles. 31 076 Toulouse, France}
\email[F. Ramon-Portugal]{felipe.ramon-portugal@envt.fr}

\author{\firstname{David} \lastname{Touboul}\CDRorcid{0000-0003-2751-774X}}
\address{Institut de Chimie des Substances Naturelles, Universit\'{e}
Paris-Saclay, CNRS, UPR~2301, 91198, Gif-sur-Yvette, France}
\email[D. Touboul]{David.TOUBOUL@cnrs.fr}

\author{\firstname{Isabel} \lastname{Borges}\CDRorcid{0000-0003-1807-0659}}
\address{cE3c/ABG -- Centre for Ecology, Evolution and Environmental
Changes/Azorean Biodiversity Group and University of the Azores,
9501-801 Ponta Delgada, Azores, Portugal}
\email[I. Borges]{isabel.mm.borges@uac.pt}

\author{\firstname{Jean-Louis} \lastname{Hemptinne}\CDRorcid{0000-0003-3875-2497}}
\addressSameAs{1}{Laboratoire Evolution et Diversit\'{e} biologique,
UMR 5174 CNRS/UPS/IRD, 118 rt de Narbonne Bt 4R1, 31062 Toulouse cedex
9, France}
\addressSameAs{2}{ENSFEA, University of Toulouse, 2 rt de Narbonne,
Castanet-Tolosan, France}
\email[J.-L. Hemptinne]{jean-louis.hemptinne@univ-tlse3.fr}

\shortrunauthors

\keywords{\kwd{Azamacrolides}\kwd{Defensive
compounds}\kwd{Pupae}\kwd{Insects}\kwd{Diet}\kwd{GC-MS}}

\begin{abstract} 
Species in the genus \textit{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 \textit{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
\textit{de novo} synthesis of the droplet compounds by these ladybirds.
The results of the GC-MS analyses suggest that \textit{Scymnus} spp.
produce azamacrolides, which are fatty acid-derived alkaloids. 
\end{abstract} 

\maketitle
\vfill\pagebreak

\twocolumngrid

\end{noXML}

\section{Introduction}

{\advance\baselineskip-.17pt
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~\cite{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~\cite{2}. Paradoxically, although there
has been a longstanding research interest in this group (e.g. review
by~\cite{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~\cite{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.~\cite{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~\ref{tabI}). 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~\cite{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~\cite{3}.}

\begin{table*} %tab1
\caption{\label{tabI}Defence strategies of ladybird pupae}
\tabcolsep10pt
\begin{tabular}{lcc}
\thead
Strategy &amp; Taxa &amp; Reference\\
\endthead
\morerows{2}{Gin traps} &amp; Coccinellini &amp; \cite{8}\\
&amp; Coccidulini and Chilocorini &amp; \cite{3}\\
&amp; \textit{Rhyzobius chrysomeloides} (Herbst) &amp; ($^*$)\vspace*{6pt}\\

\morerows{3}{\parbox[t]{5.5cm}{Pupa enclosed in larva exuvium but visible
through dorso-longitudinal slit}} &amp; Chilocorini &amp; \cite{8}\\
&amp; \textit{Rodolia} spp, \textit{Anovia circumclusa} (Gorham) &amp; \cite{9}\\
&amp; \textit{Hyperaspis maindroni} Sicard &amp; \cite{10}\\
&amp; \textit{Thalassa saginata} Mulsant &amp; \cite{11}\vspace*{6pt}\\

\morerows{2}{\parbox[t]{5cm}{Pupa enclosed in waxy \cite{12} larva
exuvium}} &amp; \textit{Azya} spp &amp; \cite{13}\\
&amp; \textit{Hyperaspis silvestrii} Weise &amp; \cite{14}\\
&amp; \textit{Cryptolaemus} spp, \textit{Nephus} spp &amp; ($^*$)

\botline
\end{tabular}
\tabnote{($^*$)~Authors pers. obs.\vspace*{-1.2pc}}
\end{table*}

Furthermore, several species of the tribe Epilachnini~\cite{15}, as
well as \textit{Delphastus catalinea} (Horn) of the tribe
Serangiini~\cite{16}, \textit{Platynaspis luteorubra} Goeze of the
tribe Platynaspidini~\cite{17}, and \textit{Nephaspis oculatus}
(Blatchley), \textit{Scymnus sinuanodulus} Yu and Yao \cite{18,19},
\textit{S.~ningshanensis} Yu and Yao~\cite{20} and
\textit{S.~posticalis} Sicard~\cite{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~\cite{16,21,22}. For
species of Epilachnini, the droplets are reported to contain molecules
of the azamacrolides family~\cite{7} as well as tocopheryl
acetates~\cite{23,24}. The droplets produced by \textit{D.~catalinea}
contain polyketides~\cite{16}. For the remaining species, the nature of
the chemicals in the droplets is \mbox{unknown}.

The present study investigated the chemical defence of the pupae of
five species of \textit{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 \textit{Scymnus nubilus} Mulsant is examined.

\section{Materials and methods} \label{sec2}
\subsection{Rearing methods and comparison of the\newline pupae of
different species of \textit{Scymnus}} \label{sec2.1}

Adults of \textit{S.~interruptus} (Goeze), \textit{S.~rubromaculatus}
(Goeze), \textit{S.~apetzi} Mulsant, and \textit{S.~subvillosus}
(Goeze) were collected near Toulouse (France), and \textit{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 $22
\pm 1$~{\textdegree}C. Ladybird adults were kept in 2-L plastic boxes
with a piece of corrugated filter paper and were fed three times a
week, \textit{ad libitum}, with either \textit{Acyrthosiphon pisum}
(Harris) or \textit{Rhopalosiphum padi}~L, common prey of
\textit{Scymnus} spp. (e.g.~\cite{25}). Two stems of broad bean,
\textit{Vicia faba}~L. or \textit{Triticum turgidum}~L. were added to
the boxes to improve the survival of \textit{A.~pisum} or
\textit{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 \textit{Scymnus}~\cite{12}
and some of which can be present on the pupae.

\subsection{Collection of droplets from pupae of\newline
\textit{Scymnus nubilus}} \label{sec2.2}

The mean diameter of the droplets on the tips of setae on the pupae of
\textit{S.~nubilus} is about $50~\upmu$m and they were collected using
Solid Phase Micro-extraction (SPME) fibers (50/$30~\upmu$m
DVB/CAR/{\ubreak}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~{\textdegree}C, to remove contaminants
that might result in a high background reading in\vadjust{\pagebreak}
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. \vspace*{-.6pc}

\subsection{GC-MS analysis of droplets collected from pupae of
\textit{Scymnus nubilus}} \label{sec2.3}

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 ${\times}$ 0.25~mm, $0.25~\upmu$m film
thickness, 5\% diphenyl and 95\% dimethylpolysiloxane) and a splitless
injector (280~{\textdegree}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{\break}
composition that is used to improve the annotation of the
chromatographic peaks. In both GC-MS, ionization was done by electron
ionization (70~eV, source temperature 200~{\textdegree}C). The mass
spectra were scanned from \textit{m/z}~20 to \textit{m/z}~700. Helium
was the carrier gas ($1.2~\text{mL}{\cdot}\text{min}^{-1}$). The
initial oven temperature was set at 50~{\textdegree}C for 1~min. Then,
the temperature was increased by 20~{\textdegree}C${\cdot}$min$^{-1}$ to
140~{\textdegree}C, then by 3~{\textdegree}C${\cdot}$min$^{-1}$ to
300~{\textdegree}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.

\looseness=-1
The purity of the chromatographic peaks was checked with the help of
the Mass Spectral Deconvolution and Identification System program
(AMDIS 2.70). For each of the fractions the most likely elemental
composition was calculated~\cite{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~\cite{27} and by comparing the
peaks to an external standard consisting of a solution of $n$-alkanes
($n$-C$_{7}$H$_{16}$--$n$-C$_{40}$H$_{82}$) of known concentrations.
The degree of unsaturation was used to determine how many rings and/or
double bonds are present in a compound~\cite{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~\cite{29}, and
(iii)~the ACD/MS Fragmenter software (2016, 1.1, Advanced Chemical
Development, Toronto, ON, Canada).\vspace*{-.6pc}

\section{Results} \label{sec3}
\subsection{Observations on the pupae of five species of
\textit{Scymnus}} \label{sec3.1}\vspace*{-.15pc}

Observations of the droplets (Figure~\ref{fig1}) produced by the pupae
of 5 species of \textit{Scymnus} whose larvae were reared on two
different diets are presented in \mbox{Table}~\ref{tabII}. These
results indicate that all of these species of \textit{Scymnus} can
produce droplets, but their distribution on pupae varies among species
with three main patterns: (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 covered 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 individuals of \textit{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.

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}Photograph on the left, dorsal view of a pupa of
\textit{Symnus nubilus}, and on the right, a high magnification
photograph of glandular setae on the dorsal integument of a pupa.}
\end{figure*}

\begin{table*} %tab2
\caption{\label{tabII}Distribution of droplets and presence of wax on
pupae of five species of \textit{Scymnus} when fed on two different
aphid diets (\textit{Acyrthosiphon pisum} or \textit{Rhopalosiphum
padi})\vspace*{-2pt}}
\tabcolsep7pt
\begin{tabular}{cccl}
\thead
\xmorerows{1}{Species} &amp; \multicolumn{2}{c}{Diet} &amp; 
\xmorerows{1}{Distribution of droplets and presence of wax}\\\cline{2-3} 
&amp; \textit{A.~pisum} &amp; \textit{R.~padi} &amp; \\
\endthead
\textit{S.~nubilus} &amp; 20/20 &amp; -- &amp; Large droplets, all over the body\\

\textit{S.~apetzi} &amp; 20/20 &amp; 20/20 &amp; Large droplets, all over the body\\

\textit{S.~interruptus} &amp; 3/22 &amp; 0/10 &amp; 
\parbox[t]{20.5pc}{\raggedright Droplets, when present, small
and on the pronotum\Lbreak Wax from the last larval exuviae partially
covering the body}\vspace*{4pt}\\

\textit{S.~rubromaculatus} &amp; $^*$ &amp; 14/32 &amp; 
\parbox[t]{20.5pc}{\raggedright Droplets, when present,
small and on the pronotum\Lbreak Wax from the last larval exuviae
partially covering the body}\vspace*{4pt}\\

\textit{S.~subvillosus} &amp; 2/2 &amp; 7/7 &amp; Small droplets present on the
pronotum
\botline
\end{tabular}
\tabnote{Number of individuals with droplets divided by the total
number of individuals examined. $^*$~Non-accepted prey; --~rearing not
done.\vspace*{-.55pc}}
\end{table*}

\subsection{Chemical analyses of the droplets produced by pupae of
\textit{Scymnus nubilus}} \label{sec3.2}

Figure~\ref{fig2} 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.

\begin{figure*}
\includegraphics{fig02}
\vspace*{-.2pc}
\caption{\label{fig2}A high resolution GC-MS chromatographic profile of
the droplets collected from glandular setae on the pupa of
\textit{S.~nubilus}.\vspace*{-.5pc}}
\end{figure*}

Analysis of the mass spectra of the two major peaks (Figure~\ref{fig3})
indicates a similar fragmentation pattern with ion signals at
\textit{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.
Nonetheless, the peak with a retention time of 24.99~min exhibits a
molecular ion at \textit{m/z}~478.37, while for the peak with a
retention time of 28.33~min a molecular ion at \textit{m/z}~506.40 is
detected. Although the two mass spectra show a difference in the
intensity of the ion at \textit{m/z}~268, this can be explained by the
difference in the size of the carbon chain~\cite{22} and the two peaks
can therefore be considered as belonging to the same chemical family. 

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Mass spectra of the two major chromatographic
peaks of the secretion produced by glandular setae on the pupae of
\textit{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.}
\end{figure*}

Table~\ref{tabIII} shows the \textit{m/z} values and chemical
formula/possible structures of each ion detected by high-resolution
GC-MS. According to the accurate 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
C$_{28}$H$_{50}$N$_{2}$O$_{4}$ and C$_{30}$H$_{54}$N$_{2}$O$_{4}$,
respectively. The two chemical formulas have five degrees of
unsaturation, which~may indicate the presence of double bonds and/or
the presence of a ring. The difference between the two molecules can be
attributed to a C$_{2}$H$_{4}$ increment typical of an increment in a
fatty acid chain.

\begin{table*} %tab3
\caption{\label{tabIII}$m/z$ 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 \textit{S.~nubilus} (cf.
Figure~\ref{fig2})}
\tabcolsep8.5pt
\begin{tabular}{cccc}
\thead
\multicolumn{2}{c}{\textit{m/z} value in positive mode} &amp; 
\xmorerows{1}{Elementary composition} &amp; \xmorerows{1}{Structure}\\\cline{1-2}
\parbox[t]{7pc}{\centering Peak RT $=$ 24.99~min\Lbreak KI $=$ 3644} &amp; 
\parbox[t]{7pc}{\centering Peak RT $=$ 28.83 min\Lbreak KI $=$ 3831}&amp;&amp;
\vspace*{2pt}\\
\endthead
30.0115 &amp;&amp; CH$_{2}$O\raisebox{1.8pc}{} &amp; \inlinefig{fx01}\vspace*{.8pc}\\
30.0344 &amp; 30.0350 &amp; CH$_{4}$N &amp; \inlinefig{fx02}\vspace*{.8pc}\\
44.0493 &amp; 44.0493 &amp; C$_{2}$H$_{6}$N &amp; \inlinefig{fx03}\vspace*{.8pc}\\
\raisebox{15pt}{55.0176} &amp; \raisebox{15pt}{55.0176} &amp; \raisebox{15pt}{C$_{3}$H$_{3}$O} &amp; \inlinefig{fx04}\vspace*{.8pc}\\

\raisebox{15pt}{55.0540} &amp; \raisebox{15pt}{55.0540} &amp; \raisebox{15pt}{C$_{4}$H$_{7}$} &amp; \inlinefig{fx05}\vspace*{.8pc}\\

\raisebox{15pt}{69.0997} &amp; \raisebox{15pt}{69.0997} &amp; \raisebox{15pt}{C$_{5}$H$_{9}$} &amp; \inlinefig{fx06}\vspace*{.8pc}\\

84.0816 &amp; 84.0813 &amp; C$_{5}$H$_{10}$N &amp; \inlinefig{fx07}\vspace*{.8pc}\\

\raisebox{16pt}{95.0858} &amp; \raisebox{16pt}{95.0855} &amp; \raisebox{16pt}{C$_{7}$H$_{11}$} &amp; \inlinefig{fx08}\vspace*{.8pc}\\
102.0910 &amp; 102.0910 &amp; C$_{5}$H$_{12}$NO &amp; \inlinefig{fx09}\vspace*{.6pc}\\
210.1481 &amp; 210.1513 &amp; C$_{12}$H$_{20}$NO$_{2}$ &amp;\vspace*{.2pc}\\
240.1947 &amp; 240.1964 &amp; C$_{14}$H$_{26}$NO$_{2}$ &amp;\vspace*{.2pc}\\
268.2283 &amp; 268.2283 &amp; C$_{16}$H$_{30}$NO$_{2}$ &amp;\vspace*{.2pc}\\
449.3379 &amp;&amp; C$_{26}$H$_{45}$NO$_{2}$ &amp;\\
&amp; 477.3689 &amp; C$_{28}$H$_{49}$N$_{2}$O$_{4}$ &amp;\vspace*{.2pc}\\
478.3771 (M+) &amp;&amp; C$_{28}$H$_{50}$N$_{2}$O$_{4}$ &amp;\\
&amp; 506.4084 (M+) &amp; C$_{30}$H$_{54}$N$_{2}$O$_{4}$ &amp;
\botline
\end{tabular}
\end{table*}

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
\textit{m/z}~110 (Table~\ref{tabIII}). The following identification of
the unsaturation sites is proposed: (i)~Fragment at
\textit{m/z}~69.0997, present in both compounds
(C$_{28}$H$_{50}$N$_{2}$O$_{4}$---24.99~min;
C$_{30}$H$_{54}$N$_{2}$O$_{4}$---28.83), can be attributed to
C$_{5}$H$_{9}^{+}$ indicating the presence of C${=}$C double bond; (ii)~The
two compounds present a fragment ion at \textit{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
$-$CH$_{2}$--CH$_{3}$ leading to the fragment ion at
\textit{m/z}~449.33 for the peak at 24.99~min, and at
\textit{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 \mbox{focused} on the three fragment ions
at \textit{m/z}~102, 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~\ref{fig4}), whose mass spectra are
presented in Figure~\ref{fig5}. The spectra are characteristic of ions
from the same molecular family according to the intense common fragment
ion at \textit{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.\looseness=-1 \vspace*{-.4pc}

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Low resolution GC-MS chromatographic profile (scan
filter \textit{m/z}~240, 210 and 102) showing the six compounds in the
droplets produced by the glandular setae on the pupa of the ladybird
\textit{S.~nubilus}.}
\end{figure*}

\begin{figure*}
\includegraphics{fig05}
\vspace*{-.3pc}
\caption{\label{fig5}Mass spectra of the four minor chromatographic
peaks of the secretion produced by the glandular setae on the pupae of
\textit{S.~nubilus}. 3, 4, 5 and 6 are the mass spectra of the
chromatographic peaks in Figure~\ref{fig4}.}
\vspace*{-.5pc}
\end{figure*}

\section{Discussion} \label{sec4}
\vspace*{-.1pc}

Species of \textit{Scymnus} are distributed worldwide and are very
common in guilds of aphid and coccid predators (e.g.~\cite{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~\cite{25}. 

Pupae of the \textit{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 \textit{Scymnus} were studied. The
production and distribution of droplets on pupae was described and the
chemical composition of droplets produced by pupae of
\textit{S.~nubilus} was determined. 

Droplets were produced by the pupae of all the species studied and in
addition another 3 \textit{Scymnus} species are reported producing
droplets in the literature~\cite{18,19,20,21}. 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:
\textit{S.~apetzi}/\textit{S~nubilus},
\textit{S.~interruptus}/\textit{S.~rubromaculatus}, and
\textit{S.~subvillosus}. These clades correspond to the species
phylogenetic relationships highlighted by Magro {\etal}~\cite{31} and
presented in Figure~\ref{fig6}. These patterns do not seem to be
related to the distribution of setae, but might be related to their
morphology, which was not studied. 

\begin{figure*}
{\vspace*{-2pt}}
\includegraphics{fig06}
{\vspace*{-4pt}}
\caption{\label{fig6}Phylogenetic relationships of the 5 species of
\textit{Scymnus} studied, based on Magro {\etal}~\cite{31}.}
{\vspace*{-3pt}}
\end{figure*}

Diet does not seem to influence droplet production, suggesting that
\textit{Scymnus} make a \textit{de novo} synthesis of the compounds in
the droplets, which is generally the case for the defensive compounds
in the Coccinellidae~\cite{32}, but this needs to be verified.
\mbox{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{\break}
defences and chemicals sequestered via certain foods cannot be
excluded~\cite{33}.

The GC-MS analyses of the chemical composition of \textit{S.~nubilus}
droplets indicated the presence of 6 compounds, the two major ones
corresponding to C$_{28}$H$_{50}$N$_{2}$O$_{4}$ and
C$_{30}$H$_{54}$N$_{2}$O$_{4}$ elemental compositions. Four
characteristics of these molecules are important: (i)~presence of
oxygen, (ii)~for every two molecules of nitrogen there are 4 molecules
of oxygen, (iii)~there are five degrees of unsaturation related to C${=}$C
and C${=}$O double bonds and (iv)~the compounds are volatile (low polarity)
in the GC-MS without the need of derivatization, indicating that the
nitrogen atom probably lies within a ring. As the volume 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 identification by NMR and circular dichroism. Searches in
the Pherobase database~\cite{34}, however, indicate the azamacrolides
family, i.e. fatty acid-derived alkaloids (Figure~\ref{fig7}).
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.~\cite{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.

\begin{figure*}
\includegraphics{fig07}
\caption{\label{fig7}(A, B)~Mono-Azamacrolides produced by
\textit{Epilachna varivensis}~\cite{22}; (C)~di-Azamacrolide produced
by \textit{Epilachna borealis} and \textit{Subcoccinella
vigintiquatuorpunctata}~\cite{35}; (D)~structure suggested in this
study for those produced by pupae of \textit{Scymnus nubilus}, where
R$_{1}$ and R$_{2}$ are two CH$_{3}$--CH$_{2}$-ethyl chains, R$_{3}$
and R$_{4}$ are two mono-unsatured chains, and R$_{5}$ and R$_{6}$ a
methyl or ethyl chain.}
\vspace*{-.15pc}
\end{figure*}

It is noteworthy that azamacrolides are present in the liquid on the
tips of setae on pupae of the ladybird tribe Epilachnini~\cite{15,22}.
Comparison of the mass spectra of the liquid collected from
\textit{S.~nubilus} with that of the mono-azamacrolide epilachnene
reported by Attygalle {\etal}~\cite{22} for \textit{Epilachna
varivensis} Mulsant (Figure~\ref{fig7}A,~B) revealed they both have
six fragment ions at \textit{m/z}~44.093, 55.0176, 55.0540, 69.0997,
84.0816 and 95.0858. The mono-azamacrolides, however, only have three
degrees of unsaturation whereas in the case of \textit{S.~nubilus} it
is five degrees of unsaturation. Moreover, the ions at \textit{m/z}~268
and 240 are complementary and the sum of the two fragments leads to the
molecular ions at \textit{m/z}~478 ($=240+240-2$) and
\textit{m/z}~506 ($=268+240-2$) \mbox{indicating} the \mbox{presence} of a
macrocycle resulting from the condensation of two \mbox{subsequent}
molecules. Thus, it is likely that \textit{S.~nubilus} produces
di-azamacrolides as it is the case for the Epilachnini
\textit{Subcoccinella vigintiquatuorpunctata} (L) (Figure~\ref{fig7}C,~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 \mbox{small.}\looseness=1

The study by Seago {\etal}~\cite{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 inferring the major relationships of the
ladybird tribes remains a challenge~\cite{2,36}. As specialized
metabolites have been used in studies on many taxa as complementary
tools to traditional morphological and gene-based phylogenetic
exploration~\cite{37}, we recommend additional studies on the defence
traits of ladybirds to help elucidate the internal phylogenetic
relationships within the Coccinellidae.

\section*{Conflicts of interest}

Authors have no conflict of interest to declare.

\section*{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.
\mbox{Finally,} we would like to thank T.~Dixon who assisted with manuscript
editing. The laboratory ``Evolution et Diversit\'{e} Biologique'' is
part of the ``Laboratoire d'Excellence'' LABEX TULIP (ANR-10-LABX-41)
and LABEX CEBA (ANR-10-LABX- 25-01).

\CDRGrant[ANR]{ANR-10-LABX-41}
\CDRGrant[ANR]{ANR-10-LABX-25-01}

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