# Recent Advances in the Understanding of Molecular Mechanisms of Resistance in Noctuid Pests

Gaelle Le Goff, Ralf Nauen

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insects

Recent Advances in
the Understanding
of Molecular
Mechanisms of
Resistance in
Noctuid Pests

Edited by

Gaelle Le Goff and Ralf Nauen

Printed Edition of the Special Issue Published in *Insects*
Recent Advances in the Understanding
of Molecular Mechanisms of
Resistance in Noctuid Pests
<figure><img src="image_4.png" /></figure>
Recent Advances in the Understanding
of Molecular Mechanisms of
Resistance in Noctuid Pests

Editors

Gaelle Le Goff
Ralf Nauen

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

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Editors

<table><tr><td>Gaelle Le Goff</td><td>Ralf Nauen</td></tr><tr><td>INRAE, CNRS, Institut Sophia Agrobiotech</td><td>Bayer AG, Crop Science Division Molecular Entomology & Toxicology</td></tr><tr><td>Université Côte d'Azur</td><td>Toxicology</td></tr><tr><td>Sophia Antipolis</td><td>Monheim</td></tr><tr><td>France</td><td>Germany</td></tr></table>

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This is a reprint of articles from the Special Issue published online in the open access journal *Insects* (ISSN 2075-4450) (available at: www.mdpi.com/journal/insects/special_issues/molecular_resistance_Noctuid).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

Last Name, A.A.; Last Name, B.B.; Last Name, C.C. Article Title. *Journal Name Year, Volume Number, Page Range*.

ISBN 978-3-0365-1758-2 (Hbk)

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© 2021 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

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# Contents

About the Editors vii

Gaëlle Le Goff and Ralf Nauen

Recent Advances in the Understanding of Molecular Mechanisms of Resistance in Noctuid Pests
Reprinted from: *Insects* **2021**, *12*, 674, doi:10.3390/insects12080674 1

David G. Heckel

The Essential and Enigmatic Role of ABC Transporters in Bt Resistance of Noctuids and Other Insect Pests of Agriculture
Reprinted from: *Insects* **2021**, *12*, 389, doi:10.3390/insects12050389 3

Debora Boaventura, Macarena Martin, Alberto Pozzebon, David Mota-Sanchez and Ralf Nauen

Monitoring of Target-Site Mutations Conferring Insecticide Resistance in *Spodoptera frugiperda*
Reprinted from: *Insects* **2020**, *11*, 545, doi:10.3390/insects11080545 19

Torranis Ruttanaphan, Georges de Sousa, Anchulee Pengsook, Wanchai Pluempanupat, Hannah-Isadora Huditz, Vasakorn Bullangpoti and Gaëlle Le Goff

A Novel Insecticidal Molecule Extracted from *Alpinia galanga* with Potential to Control the Pest Insect *Spodoptera frugiperda*
Reprinted from: *Insects* **2020**, *11*, 686, doi:10.3390/insects11100686 35

Hwa Yeun Nam, Min Kwon, Hyun Ju Kim and Juil Kim

Development of a Species Diagnostic Molecular Tool for an Invasive Pest, *Mythimna loreyi*, Using LAMP
Reprinted from: *Insects* **2020**, *11*, 817, doi:10.3390/insects11110817 51

Khouloud KLAI, Benoît CHÉNAIS, Marwa ZIDI, Salma DJEBBI, Aurore CARUSO, Françoise DENIS, Johann CONFAIS, Myriam BADAWI, Nathalie CASSE and Maha MEZGHANI KHEMAKHEM

Screening of *Helicoverpa armigera* Mobilome Revealed Transposable Element Insertions in Insecticide Resistance Genes
Reprinted from: *Insects* **2020**, *11*, 879, doi:10.3390/insects11120879 63

Andre Gergs and Christian U. Baden

A Dynamic Energy Budget Approach for the Prediction of Development Times and Variability in *Spodoptera frugiperda* Rearing
Reprinted from: *Insects* **2021**, *12*, 300, doi:10.3390/insects12040300 75

Sudeeptha Yainna, Nicolas Nègre, Pierre J. Silvie, Thierry Brévault, Wee Tek Tay, Karl Gordon, Emmanuelle dAlençon, Thomas Walsh and Kiwoong Nam

Geographic Monitoring of Insecticide Resistance Mutations in Native and Invasive Populations of the Fall Armyworm
Reprinted from: *Insects* **2021**, *12*, 468, doi:10.3390/insects12050468 85

Macarena Martin, Debora Boaventura and Ralf Nauen

Evaluation of Reference Genes and Expression Level of Genes Potentially Involved in the Mode of Action of Cry1Ac and Cry1F in a Susceptible Reference Strain of *Chrysodeixis includens*
Reprinted from: *Insects* **2021**, *12*, 598, doi:10.3390/insects12070598 97
# About the Editors

## Gaelle Le Goff

Gaëlle Le Goff is a senior scientist at the Institut Sophia Agrobiotech, French National Institute for Agriculture, Food and Environment (INRAE). She has a PhD in Molecular Biology and Pharmacology from the University of Nice Sophia Antipolis, France. She did a post-doc at the University of Bath in UK with a European Marie Curie grant and was then recruited at INRA, where she still carries out her research in functional genomics in insects. Her current projects focus on i) insect adaptation to their chemical environment (adaptation to plant secondary metabolites/insecticide resistance), ii) regulation of detoxification genes expression, and iii) annotation of insect genomes (detoxification genes).

## Ralf Nauen

Ralf Nauen is an insect toxicologist working on functional (toxico)genomics, molecular entomology, fundamental and applied aspects of insecticide/acaricide mode of action, selectivity and detoxification, as well as biochemical and molecular mechanisms of insecticide resistance and its management. He received his PhD from the University of Portsmouth (UK) and is a Bayer Distinguished Science Fellow. In 2013 he was awarded to become Fellow of the Entomological Society of America and in 2014 he received the prestigious American Chemical Society International Award for Research in Agrochemicals, in recognition of his outstanding and influential research into insecticide and acaricide modes of action and resistance. He authored more than 230 scientific papers/book chapters with more than 22,000 citations. He received the Highly Cited Researcher award by the Web of Science 2018-2020. He is appointed Visiting Professor by the Chinese Academy of Agricultural Sciences (Beijing, China) and lecturer at the University of Bonn (Germany).
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Editorial

# Recent Advances in the Understanding of Molecular Mechanisms of Resistance in Noctuid Pests

Gaëlle Le Goff <fnref n="1" />,* and Ralf Nauen <fnref n="2" />,*

Noctuid moths are among the most devastating crop pests on the planet. Many of them are polyphagous pests capable of feeding on numerous host plants. Their larval stages cause significant damage to plants of agronomic importance, including corn, cotton, soybean, and rice, which result in severe yield losses if not kept under economic damage thresholds. Control of these pest insects is mainly based on the use of chemical insecticides and/or the cultivation of transgenic plants expressing *Bacillus thuringiensis* (Bt) pore-forming insecticidal proteins (Cry toxins). However, the efficacy of these control measures is often jeopardized by the development of resistance due to frequent applications of insecticides and long-term exposure to a relatively small set of Cry toxins expressed in transgenic crops. Understanding the molecular mechanisms of resistance that these pest insects have developed is essential for the implementation of sustainable control methods and resistance management strategies. This special issue is dedicated to this topic and includes 10 papers presenting either original results or reviews.

Several species of the noctuid moth family are invasive species that have recently spread to various parts of the world. The most problematic is probably *Spodoptera frugiperda* (Smith, 1797), which originated from the American continent and recently invaded the Western hemisphere. It was first detected in Africa in 2016 and later in Asia (2018) and Australia (2020). It is approaching Europe and was detected in the Canary Islands (Spain) in July 2020. The Food and Agriculture Organization (FAO) has this year proclaimed it as "one of the most destructive pests jeopardizing food security across vast regions of the globe". The importance of this species, also known as the fall armyworm (FAW), is reflected in the Special Issue, with half of the publications dedicated to it. Some of the papers focus on the diagnosis to differentiate the two sympatric host plant species of FAW, maize and rice [1], or on the most suitable rearing conditions for this non-model species [2], while others tackle the development of molecular tools for the detection of mutations in insecticide targets conferring resistance [3]. Other studies have monitored the presence of known mutations in the targets of major insecticide classes by comparing native and invasive populations [1,4]. Marked differences exist between FAW populations, which can provide guidance for appropriate resistance management decisions. Indeed, these studies show the presence of mutations in acetylcholinesterase, the target of carbamates and organophosphates, in both native and invasive populations, whereas only native populations show a deletion in ABCC2, the target of some Bt proteins [1,4]. In this case, diamide insecticides could be useful in the fight against FAW since none of the known mutations in the target of diamides, the ryanodine receptor, have been detected. However, it should not be forgotten that resistance can also be acquired through increased capacity to metabolize insecticides and involves detoxification enzymes, such as cytochromes P450 monooxygenases, carboxylesterases, glutathione S-transferases, or transporters like ATP binding cassette (ABC) transporters. Some of the roles of these detoxification genes are highlighted in a review on species in the genus *Spodoptera* [5]. Nevertheless, the fight against insect pests must also include research and discovery of new molecules with

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check for
updates

**Citation:** Le Goff, G.; Nauen, R.  
Recent Advances in the  
Understanding of Molecular  
Mechanisms of Resistance in Noctuid  
Pests. *Insects* **2021**, *12*, 674. https://  
doi.org/10.3390/insects12080674

Received: 16 July 2021

Accepted: 24 July 2021

Published: 27 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_9.png" /></figure>

**Copyright:** © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

<footnote n="1">¹ Université Côte d'Azur, INRAE, CNRS, ISA, F-06903 Sophia Antipolis, France</footnote>

<footnote n="2">² Bayer AG, Crop Science Division, R&D Pest Control, Alfred Nobel Str. 50, 40789 Monheim, Germany</footnote>

<footnote>* Correspondence: gaëlle.le-goff@inrae.fr (G.L.G.); ralf.nauen@bayer.com (R.N.); Tel.: +33-4-9238-6578 (G.L.G.); +49-21-7338-4441 (R.N.)</footnote>
different modes of action to counteract resistance mechanisms. Such research should also
take into account a societal demand for the development of more environmentally friendly
molecules [6].

The other noctuid moth species covered in this Special Issue are also important pests.
Klai et al. proposed an analysis of the presence of transposable elements in the genome
of *Helicoverpa armigera* and focused on their presence in detoxification genes as they may
cause changes in the expression level and confer resistance [7]. Other authors have sought
to develop molecular tools either to differentiate between two closely related species, such
as *Mythimna loreyi* and *Mythimna separata*, with the LAMP (loop-mediated isothermal
amplification) assay [8], or by designing primers to monitor the expression of Bt toxin
target genes and reference genes in soybean looper, *Chrysodeixis includens* [9].

ABC transporters are important Bt protein receptors and there is an increasing body of
evidence that mutations in ABC transporters (e.g., ABCC2) confer high levels of resistance
to Cry toxins as reviewed in [10]. In his review, Heckel [10] highlights a number of research
questions that remain open, such as how potential Cry toxin receptor proteins and Cry
toxins interact to form pores. However, our knowledge and understanding regarding the
function and regulation of genes conferring resistance in pests is constantly increasing and
particularly facilitated by the exploitation of new technologies, such as genome editing by
CRISPR/Cas9.

Finally, we would like to thank all authors for their commitment to contribute their
articles and the many reviewers for their valuable comments and suggestions helping all
of us to shape this interesting Special Issue, which hopefully attracts a broad readership
of *Insects*.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

References

1. Boaventura, D.; Martin, M.; Pozzebon, A.; Mota-Sanchez, D.; Nauen, R. Monitoring of Target-Site Mutations Conferring Insecticide Resistance in *Spodoptera frugiperda*. *Insects* **2020**, *11*, 545. [CrossRef] [PubMed]

2. Gergs, A.; Baden, C. A Dynamic Energy Budget Approach for the Prediction of Development Times and Variability in *Spodoptera frugiperda* Rearing. *Insects* **2021**, *12*, 300. [CrossRef] [PubMed]

3. Schlum, K.; Lamour, K.; Tandy, P.; Emrich, S.; de Bortoli, C.; Rao, T.; Dillon, D.V.; Linares-Ramirez, A.; Jurat-Fuentes, J. Genetic Screening to Identify Candidate Resistance Alleles to Cry1F Corn in Fall Armyworm Using Targeted Sequencing. *Insects* **2021**, *12*, 618. [CrossRef]

4. Yainna, S.; Nègre, N.; Silvie, P.; Brévault, T.; Tay, W.; Gordon, K.; Dalençon, E.; Walsh, T.; Nam, K. Geographic Monitoring of Insecticide Resistance Mutations in Native and Invasive Populations of the Fall Armyworm. *Insects* **2021**, *12*, 468. [CrossRef] [PubMed]

5. Hilliou, F.; Chertemps, T.; Maïbèche, M.; Le Goff, G. Resistance in the Genus *Spodoptera*: Key Insect Detoxification Genes. *Insects* **2021**, *12*, 544. [CrossRef] [PubMed]

6. Ruttanaphan, T.; De Sousa, G.; Pengsook, A.; Pluempanupat, W.; Huditz, H.-I.; Bullangpoti, V.; Le Goff, G. A Novel Insecticidal Molecule Extracted from *Alpinia galanga* with Potential to Control the Pest Insect *Spodoptera frugiperda*. *Insects* **2020**, *11*, 686. [CrossRef] [PubMed]

7. Klai, K.; Chénais, B.; Zidi, M.; Djebbi, S.; Caruso, A.; Denis, F.; Confais, J.; Badawi, M.; Casse, N.; Khemakhem, M.M. Screening of *Helicoverpa armigera* Mobilome Revealed Transposable Element Insertions in Insecticide Resistance Genes. *Insects* **2020**, *11*, 879. [CrossRef] [PubMed]

8. Nam, H.Y.; Kwon, M.; Kim, H.J.; Kim, J. Development of a Species Diagnostic Molecular Tool for an Invasive Pest, *Mythimna loreyi*, Using LAMP. *Insects* **2020**, *11*, 817. [CrossRef] [PubMed]

9. Martin, M.; Boaventura, D.; Nauen, R. Evaluation of Reference Genes and Expression Level of Genes Potentially Involved in the Mode of Action of Cry1Ac and Cry1F in a Susceptible Reference Strain of *Chrysodeixis includens*. *Insects* **2021**, *12*, 598. [CrossRef] [PubMed]

10. Heckel, D. The Essential and Enigmatic Role of ABC Transporters in Bt Resistance of Noctuids and Other Insect Pests of Agriculture. *Insects* **2021**, *12*, 389. [CrossRef] [PubMed]
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Review

# The Essential and Enigmatic Role of ABC Transporters in Bt Resistance of Noctuids and Other Insect Pests of Agriculture

David G. Heckel

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Max Planck Institute for Chemical Ecology, Hans-Knoell-Str. 8, D-07745 Jena, Germany; heckel@ice.mpg.de;
Tel.: +49-3641-57-1599

**Simple Summary:** The insect family, Noctuidae, contains some of the most damaging pests of agriculture, including bollworms, budworms, and armyworms. Transgenic cotton and maize expressing Cry-type insecticidal proteins from *Bacillus thuringiensis* (Bt) are protected from such pests and greatly reduce the need for chemical insecticides. However, evolution of Bt resistance in the insects threatens the sustainability of this environmentally beneficial pest control strategy. Understanding the interaction between Bt toxins and their targets in the insect midgut is necessary to evaluate the risk of resistance evolution. ABC transporters, which in eukaryotes typically expel small molecules from cells, have recently been proposed as a target for the pore-forming Cry toxins. Here we review the literature surrounding this hypothesis in noctuids and other insects. Appreciation of the critical role of ABC transporters will be useful in discovering counterstrategies to resistance, which is already evolving in some field populations of noctuids and other insects.

**Abstract:** In the last ten years, ABC transporters have emerged as unexpected yet significant contributors to pest resistance to insecticidal pore-forming proteins from *Bacillus thuringiensis* (Bt). Evidence includes the presence of mutations in resistant insects, heterologous expression to probe interactions with the three-domain Cry toxins, and CRISPR/Cas9 knockouts. Yet the mechanisms by which ABC transporters facilitate pore formation remain obscure. The three major classes of Cry toxins used in agriculture have been found to target the three major classes of ABC transporters, which requires a mechanistic explanation. Many other families of bacterial pore-forming toxins exhibit conformational changes in their mode of action, which are not yet described for the Cry toxins. Three-dimensional structures of the relevant ABC transporters, the multimeric pore in the membrane, and other proteins that assist in the process are required to test the hypothesis that the ATP-switch mechanism provides a motive force that drives Cry toxins into the membrane. Knowledge of the mechanism of pore insertion will be required to combat the resistance that is now evolving in field populations of insects, including noctuids.

**Keywords:** *Bacillus thuringiensis*; Cry protein; ATP-Binding Cassette; ABC Transporter; ATP switch model; pore-forming toxin; resistance; genetics; Noctuidae; *Helicoverpa*; *Spodoptera*; *Heliothis*; *Chloridea*; *Trichoplusia*

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check for
updates

**Citation:** Heckel, D.G. The Essential and Enigmatic Role of ABC Transporters in Bt Resistance of Noctuids and Other Insect Pests of Agriculture. *Insects* **2021**, *12*, 389.
https://doi.org/10.3390/insects12050389

Academic Editors: Gaelle Le Goff
and Ralf Nauen

Received: 5 April 2021
Accepted: 26 April 2021
Published: 28 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_13.png" /></figure>

**Copyright:** © 2021 by the author.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

## 1. Introduction

ABC proteins are a huge and ancient superfamily of proteins that are defined by the presence of a domain called the *ATP-binding cassette* [1]. Binding to ATP causes this domain to change its shape, and this conformational change has been harnessed for many purposes. The ABCE1 protein is required for termination of protein translation, where its lever-like action separates the large and small ribosomal subunits [2]. The Rad50 protein is invoked when chromosomes are damaged, by clamping onto DNA like tweezers to bring the strands together for repair [3]. The ABC transporters possess membrane-spanning helices that change orientation to squeeze small molecules across membranes when the ABC domain binds to ATP [4]. The most recently discovered property of ABC proteins
is their role in the mode of action of pore-forming three-domain Cry toxins from *Bacillus thuringiensis* (Bt), the subject of this review.

The Cry toxins are important in agriculture because cotton, maize, and other crops have been transformed with genes from Bt to enable the plants to manufacture enough toxins to kill certain pest caterpillars that feed on them. See [5] for a comprehensive list of Bt toxins. When ingested along with plant tissue, the toxins form pores in the larval midgut epithelium, lysing the cells and eventually killing the insect. In 2019, 108.7 million hectares of transgenic Bt crops were grown worldwide [6], greatly reducing the use of chemical insecticides. The sustainability of this strategy is threatened by the evolution of resistance, which is already appearing in field populations of some pest insects (see [7] for a useful overview). Studies of Bt-resistant insects provided the first evidence for ABC transporters in the Cry toxin mode of action.

Cry1-type toxins are particularly potent against polyphagous noctuid pests, primarily the heliothines and the armyworms. The cotton bollworm *Helicoverpa armigera* (Hübner) was formerly distributed in Asia, Africa, and Australia, but has recently invaded South America. Its resistance to chemical insecticides and its ability to hybridize with the native corn earworm *Helicoverpa zea* (Boddie) has promoted its spread. *H. zea* populations in North America are evolving resistance to Cry1Ab-expressing maize. *Chloridea* (formerly *Heliothis* *virescens* (Fabricius), the tobacco budworm [8], is a pest of cotton and other crops that has been well-controlled by Bt-cotton up to now. We will use the genus name *Heliothis* in this review for continuity with the literature cited. The fall armyworm *Spodoptera frugiperda* (J. E. Smith) has recently invaded Africa, India, Asia, and Australia from its home in the New World to become a worldwide pest of maize. Its congeners, the beet armyworm *Spodoptera exigua* (Hübner) and cotton leafworm *Spodoptera litura* (Fabricius), are also polyphagous pests. The cabbage looper *Trichoplusia ni* (Hübner) has evolved resistance to Dipel in greenhouses. Cry1 toxins are also effective against non-noctuid Lepidoptera, such as the diamondback moth *Plutella xylostella* (L), a worldwide pest of crucifer crops and the first insect to evolve Bt resistance in open field populations, and the domesticated silkworm *Bombyx mori* (L), a model insect for which powerful genetic tools have been developed. Cry1 resistance has appeared in some populations of the European corn borer *Ostrinia nubilalis* (Hübner) and the Asian corn borer *Ostrinia furnacalis* (Guenée). Cry2 toxins are active against many of the same Lepidoptera. The pink bollworm *Pectinophora gossypiella* has evolved resistance to Cry2-expressing Bt-transgenic cotton. The Cry3 toxins are potent against Coleoptera, such as *Diabrotica virgifera* (LeConte), the corn rootworm. ABC transporters of these non-noctuids will also be discussed as they illustrate the common features of the mode of action of the three-domain Cry toxins. Cell lines from noctuids have also been important in Bt research, including SI-HP from *S. litura*, Sf9 from *S. frugiperda*, Hi5 from *T. ni*, and QB-Ha-E5 from *H. armigera*.

Unfortunately, there is no standard nomenclature yet for insect ABC transporters, which is becoming a problem for comparative studies as more and more are implicated as Bt targets. Gene and protein names provided by automatic annotation at NCBI do not uniquely identify them or reveal their relationship to the ABC transporters described here. The most reliable point of reference is the genome of *B. mori*, the first to be sequenced among Lepidoptera. Figure 1 shows the genomic context of the *B. mori* orthologs of the proteins discussed in this review. Figure 2 shows their predicted membrane topology.
<figure><img src="image_14.png" /><figcaption>**Figure 1.** Genomic location of *Bombyx mori* (A–C) transporters orthologous to those interacting with three-domain Cry proteins described in the text. Coordinates are based on the chromosome view on NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 2 April 2021) and are the same as shown in the new version of Kaikobase [9]. (D) Names for ABCC6 and ABCC7 are defined here for the first time, to avoid confusion with the previously named ABCC1.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Figure 2.** Phobius [10,11] predictions of membrane topology in the lipid bilayer of four ABC transporters from *Bombyx mori* orthologous to those interacting with three-domain Cry proteins described in the text (not to scale). The external lumenal surface is on top. Numbers of residues in each predicted external loop are shown. Transmembrane domains: TM0, TM1, and TM2. Nucleotide-binding domains: NBD1, NBD2. The N-terminus and C-terminus of each polypeptide is indicated.</figcaption></figure>

**2. Initial Discoveries**

ABC transporters were first discovered as targets of Bt toxins by positional cloning
in strains of resistant insects. Despite decades of research on Bt resistance, there was
no previous biochemical or physiological evidence that ABC transporters could be in-
volved. The discovery of their role was a surprise, and only the independent efforts by
three groups that converged on the same protein has convinced the research community
that understanding their role in resistance is important, as described in several recent
reviews [5,12–23]. Solving this problem holds the key to developing strategies to combat
the increasing problem of Bt resistance.
A mutation in the ABCC2 protein was identified in a Cry1Ac-resistant strain of *H. virescens*, by positional cloning using markers from the early versions of the *B. mori* genome sequence, well before a genome sequence for *H. virescens* was available [24]. Evidence that this mutation was important for resistance came from mapping, binding studies, and an allele frequency change correlated with the increase of resistance over time [24]. Like most ABC mutations subsequently found in other species, and like the cadherin mutation previously found in *H. virescens* [25] it introduced a frameshift and prevented expression of the full-length protein in the membrane. This contrasts with many cases of chemical insecticide resistance, where deletion of the target would be lethal.

Even stronger evidence came from analysis of a more subtle mutation in the ABCC2 protein in *B. mori* [26]. Positional cloning using the genome sequence converged on ABCC2, but no incapacitating mutation could be found. Instead, the protein in the Cry1Ab-resistant strain had several amino acid substitutions and an insertion of a tyrosine in an extracellular loop. Germline transformation was used to prove that one copy of the susceptible allele inserted in the resistant genetic background could confer Cry1Ab-susceptibility on the resistant strain [26], and subsequent experiments [27] proved that the inserted tyrosine was necessary and sufficient for resistance.

A similar approach using bulked segregant analysis based on cDNA markers identified ABCC2 and its neighboring gene ABCC3 as contributing to Cry1Ac and Cry1Ca resistance in *S. exigua* [28]. In contrast to the previous mutations, ABCC2 carried a lesion in an intracellular nucleotide-binding domain (NBD). Suppression of ABCC2 and ABCC3 by RNA inhibition (RNAi) increased the tolerance of susceptible larvae to Cry1Ac and Cry1Ca. This independent and unbiased positional cloning approach extended the phenomenon to a third genus, and to a third type of mutation in ABCC2, proving that ABC transporters could no longer be ignored in the mode of action of Cry toxins.

### 3. Search for ABC Mutants in Resistant Strains from Field and Laboratory

These early findings motivated the search for the involvement of ABC transporters in other Bt-resistant strains of noctuids and other Lepidoptera. Comparative linkage mapping with markers, previously shown to be linked to ABCC2 in *H. virescens*, was used to identify a mutation that eliminated the last transmembrane domain of ABCC2 in Cry1Ac-resistant *P. xylostella* from Hawaii [29]. The same study localized Cry1Ac resistance in *T. ni* from British Columbia to a region containing ABCC2, although a specific mutation was not identified [29]; a different resistance mechanism, altered aminopeptidase expression, was also identified in the same strain of *T. ni* [30]. Mis-spliced transcripts of ABCC2 generating a truncated protein were found in a Cry1Ac-resistant strain of *H. armigera* from China [31]. Another comparative linkage mapping approach identified a genomic region containing ABCC2 in a laboratory-selected Cry1F-resistant strain of *O. nubilalis* from collections in the Corn Belt of the USA [32], although involvement of ABCC2 has not yet been confirmed in field-evolved Cry1F resistance in *O. nubilalis* from Nova Scotia [33]. In Puerto Rico, rapid appearance of Cry1F resistance in *S. frugiperda* stimulated withdrawal of the transgenic maize variety from the market, and was found to be associated with mutations in ABCC2 [34]. Additional mutations in *S. frugiperda* ABCC2 were associated with Cry1Fa and Cry1A.105 resistance in Puerto Rico [35] and Brazil [36]. Screening for some of these was included in surveys using DNA diagnostics for resistance to chemical insecticides as well as Bt [37,38].

The only member of the ABCB family to be investigated in Lepidoptera as a target of Cry toxins initially came to attention because it was down-regulated in a Cry1Ac-resistant strain of *P. xylostella*. *PxABCB1* expression was found to be lower in other resistant strains, was further reduced by additional Cry1Ac selection, and was suppressed by RNAi in susceptible strains, which increased their tolerance to Cry1Ac [39].
4. Functional Studies

Heterologous expression of ABC transporters in insect cell lines has been extensively used to probe their function. The crucial role of the tyrosine insertion in loop 2 of *B. mori* ABCC2 in conferring Cry1Ab resistance was convincingly shown by expression in Sf9 cells [27]. The same study demonstrated the synergy of the cadherin BtR175 and ABCC2 for the first time; co-expression of both made the cells much more susceptible to Cry1Ab than either one alone. These results were recapitulated using proteins from *H. virescens* in Sf9 cells [40], with the added information that synergy was observed only when both proteins were expressed in the same cell; i.e., not from a mixture of cells expressing one or the other, as might be expected from the sequential binding hypothesis [41]. The mechanism of synergy was further probed by comparing the ability of the cadherin from *H. armigera* or *S. litura* to synergize Cry1Ac action on *H. armigera* ABCC2 expressed in Hi5 cells [42]. Although the *S. litura* cadherin was an ineffective synergist, when cadherin repeat 11 from *H. armigera* was swapped in, synergistic activity increased. The authors hypothesized that specific binding sites on the cadherin localized the toxin to a good position for interaction with ABCC2 in a species-specific manner [42]. A similar species-specific synergism with ABCC2 from *S. exigua* and the cadherin from *S. exigua* (but not *H. armigera*) was observed with the Cry1C toxin in Sf9 cells [43]. Domain-swapping between Cry1C and Cry1Ac was used to infer that domains II and III of Cry1Ac have different binding sites on ABCC2 of *S. exigua* [44]. ABCC2 from *S. frugiperda* expressed in Hi5 cells conferred sensitivity to Cry1Ab and Cry1Fa, while the cadherin did not, but synergism was not investigated in this study [45]. ABCC3 from *S. frugiperda* was also found to confer sensitivity to Cry1Ab and Cry1Fa under similar conditions [46].

A wide-ranging study explored the specificity of the toxin-target interaction by expressing ABC transporters from Lepidoptera, Coleoptera, Diptera, and humans in Sf9 cells and testing them with lepidopteran- or coleopteran-active toxins [47]. ABCC2 or ABCC3 from *B. mori* conferred sensitivity to Cry1Aa, but not Cry1Ca or Cry1Da. The latter two must have different, unknown targets because they are active on caterpillars of some lepidopteran species. Human and dipteran ABC transporters tested did not respond to lepidopteran- or coleopteran-active toxins.

*D. melanogaster* is not normally susceptible to Cry1Ac, but when ABCC2 was expressed in the midgut of transgenic larvae, Cry1Ac in the artificial diet killed them [48]. Moreover, the genome of *D. melanogaster* lacks the ortholog of the 12-cadherin domain protein found in all Lepidoptera, so the killing mechanism did not rely on the same type of synergism from the cadherin. However, synergism could be observed when the transgenic larvae were fed peptide fragments from lepidopteran cadherins along with Cry1Ac [48].

The most sensitive measurements of the interaction between Cry toxins and their receptors have been made using heterologous expression in *Xenopus* oocytes [49]. Messenger RNA experimentally injected into these huge cells is translated and the proteins (e.g., ABCC2 or cadherin) are incorporated into the egg membrane. This technique is often used to investigate the properties of ion channels using the voltage-clamp technique. The current through the channel is measured as a function of the experimentally-fixed voltage gradient across the membrane and the resulting graph characterizes the electrophysiological properties of the channel. In this case, the channel is the Cry toxin pore inserted into the membrane, which allows inward cation flux. The dynamics of current flow depend on the details of pore insertion and structure. Using this sensitive technique, it was shown that expression of the cadherin alone produced almost no current, expression of ABCC2 allowed abundant current, and expression of both produced even more current—the most convincing demonstration of synergism to date.

The mechanism of synergism is still obscure, but a number of hypotheses can be envisaged, which are not mutually exclusive (Figure 3). These can be classified into trans-acting mechanisms where synergism can occur when the cadherin and ABC transporter may be separated from each other, and cis-acting mechanisms where synergism requires a close physical interaction. According to the sequential binding hypothesis [41], toxin
monomers bind to the cadherin and are further processed by cleavage of the N-terminal α1-helix, whereupon they form oligomeric pre-pores in solution (Figure 3A). However, toxin monomer binding to the cadherin is not an absolute requirement for toxicity; cadherin knockouts can still be killed by higher toxin amounts [24,50–52] and Cry1Ac is still lethal to *T. ni* despite not being able to bind to the *T. ni* cadherin [53]. Synergism is due to presence of the cadherin, which speeds up a process that happens at a slower rate in its absence. Here the pre-pore structure can diffuse away from the cadherin to interact with a remote ABC transporter, so this mechanism is classified as *trans*-acting. If, however the cadherin traps the toxin and brings it to the ABC transporter, this would be a *cis*-acting mechanism (Figure 3B). This could be synergistic if there are many more cadherin molecules in the membrane than ABCC2 molecules. Another previously suggested *cis*-acting mechanism [40] is shown in Figure 3C, where the cadherin pulls the pre-pore away from the ABC transporter, enabling it to insert into the membrane and freeing up the ABC transporter for the next pre-pore.

<figure><img src="image_16.png" /><figcaption>**Figure 3.** Hypothetical models of the mechanism of synergy between the 12-cadherin domain protein and an ABC transporter. (A) *Trans*-acting synergy, due to acceleration of oligomer formation following monomer binding to the cadherin. (B) *Cis*-acting synergy, due to the cadherin trapping the pre-pore and moving it to the ABC transporter. (C) *Cis*-acting synergy, due to the cadherin pulling the pre-pore away from the ABC transporter, freeing it to interact with another toxin pre-pore. Dotted lines represent movement of the cadherin within the membrane.</figcaption></figure>
**5. Extracellular Loops**

Most of the ABC protein is out of reach of Bt toxins approaching the cell, since the
nucleotide binding domains are entirely cytoplasmic, and most of the transmembrane
domains are buried within the lipid bilayer (Figure 2). Extracellular loops connecting
adjacent transmembrane helices are very short in ABCC proteins, but larger in ABCB
and especially ABCA proteins. Detailed analyses of the interaction between extracellular
loops of *B. mori* ABC transporters and various domains of Cry1A toxins have been carried
out by the group of Ryoichi Sato in Tokyo. Following the discovery that insertion of a
single tyrosine in Loop 2 of ABCC2 conferred resistance to Cry1Ab in larvae, swapping
other amino acids for the inserted tyrosine blocked pore formation in cells expressing the
transporter, while amino acid substitutions at other positions in the non-inserted loop
did not [54]. Thus, the size of the loop, rather than its amino acid composition, was the
more important determinant of sensitivity. Domain-swapping within the toxin implicated
Domain II as most important in this interaction. Subsequent mutagenesis of Domain II
of Cry1Aa revealed a region that bound both to ABCC2 and another important receptor,
the cadherin BtR175 [55]. While ABCC3 had much lower binding affinities to Cry1Aa and
Cry1Ab than ABCC2, binding was increased in constructs containing partial loops from
ABCC2 [56]. Another group pointed out an amino acid difference within Loop 1 of ABCC2
of *S. frugiperda* and *S. litura* that correlated with binding affinity to Cry1Ac, and they also
replicated the binding difference by creating two versions of the *H. armigera* ABCC2, one
with each amino acid [57].

**6. Regulatory Changes**

Regulatory changes involving ABC transporters were also found to confer resistance. In a Cry1Ac-resistant strain of *P. xylostella*, resistance mapped to the vicinity of ABCC2 but no disruptive mutations in the gene could be found [58]. Instead, the expression level of ABCC2 and ABCC3 was found to be controlled by the mitogen-activated protein kinase (MAPK) signaling pathway, with the MAP4K4 gene located close by on the same genomic scaffold, accounting for the mapping results. Constitutive expression of MAP4K4 in the resistant strain suppressed ABCC2, ABCC3, and alkaline phosphatase, another Cry1Ac-binding protein. RNA interference (RNAi) suppression of MAP4K4 transiently restored susceptibility by upregulating ABCC2 and ABCC3. Thus, resistance in this case was due to higher expression of a negative regulator of ABCC2 transcription.

The Forkhead Box Protein A (FOXA) transcription factor was found to stimulate
transcription of ABCC2 and ABCC3 in *H. armigera*, as predicted from FOXA binding sites
in the promoters [59]. RNAi silencing of FOXA downregulated ABCC2 and ABCC3 and
increased the tolerance of susceptible larvae to Cry1Ac. Parallel results were obtained by
expression in SI-HP cells in the same study. A different study screened several members
of the GATA transcription factor family from *H. armigera* and found that GATAe caused
Sf9, QB-Ha-E5, and Hi5 cell lines to increase their expression of ABCC2, conferring greater
Cry1Ac sensitivity [60]. If either mechanism were to be found in resistant strains from
the field, resistance would be due to lower expression of a positive regulator of ABCC2
transcription.

Comparison of the ABCC2 coding sequence across many species of Lepidoptera identified a conserved region targeted by the microRNA miR-998-3p [61]. MicroRNAs bind to messenger RNAs in a sequence-specific fashion and target them for destruction or inhibit translation. Injection of an agomir (a chemically modified RNA that mimics the effect of the miRNA) into susceptible larvae of *P. xylostella*, *S. exigua*, or *H. armigera* increased their tolerance of Cry1Ac and decreased the abundance of ABCC2 mRNA. Injection of an antagonir (a single-stranded molecule designed to block the effect of the miRNA) into larvae of Cry1Ac-resistant *P. xylostella* reduced their tolerance of Cry1Ac and increased their ABCC2 mRNA levels.
7. Cell Lines

The influential colloid-osmotic lysis theory to explain how Cry toxins kill cells was
developed using different cell lines that naturally differed in their susceptibilities to two
different toxins [62]. It would be interesting to determine which ABC transporters are
naturally expressed by those cells. Sl-HP cells from *S. litura* are susceptible to activated
Cry1Ac even though *S. litura* larvae are not. ABCC3 was found to be expressed in this cell
line, and RNA inhibition of ABCC3 decreased Sl-HP sensitivity to Cry1Ac [63]. In another
study, comparison of Cry1Ac sensitivities of cell lines from different tissues produced the
order midgut > fat body > ovary as expected, but unexpectedly fat body-derived cells were
most susceptible to Cry2Ab toxin [64]. Surveys of heterologous expression of candidate
targets in cell lines [65] should also take their native expression patterns into account.

8. Cry2A Toxins

An extensive sampling effort in Australia employing the F2 screen [66] yielded strains of *H. armigera* and *H. punctigera* with high levels of resistance to the Cry2Ab toxin. Linkage mapping with these strains revealed several different mutations in the ABCA2 gene that prevented expression of the full-length protein [67]. Unlike the ABCC proteins, ABCA2 has two very large ectodomains (Figure 2), and because the mutants are extremely resistant to Cry2Ab, it was speculated that the single ABCA2 protein functions similarly to the sequential binding model for the cadherin and ABCC2 [67]. Linkage mapping in a strain of *T. ni* that was selected with Dipel in British Columbia greenhouses [68] eventually resulted in the identification of a transposable element in ABCA2 conferring Cry2Ab resistance [69]. Mis-splicing mutants in ABCA2 were associated with Cry2Ab resistance in *P. gossypiella* in a laboratory-selected strain from Arizona and field populations from India [70]. Additional crosses confirmed these mutations but suggested that an additional, uncharacterized mechanism was also involved in Cry2Ab resistance in this species [71].

A different member of the ABCC family from *H. armigera* (GenBank Accession No. KY796050) was also found to bind Cry2Ab, identified by the authors as “HaABCC1” [72], although it is not the ortholog of the ABCC1 (BGIBMGA007737+38) on *B. mori* Chromosome 15 next to ABCC2 and ABCC3 described previously [24,26,28]. The ortholog of “HaABCC1” in *B. mori* is part of a small cluster on Chromosome 12 (Figure 1D) of ABCC proteins with 5 additional N-terminal transmembrane domains as well as two very large ectodomains, unlike the Chromosome 15 ABCC1-ABCC2-ABCC3 cluster in *B. mori* and other Lepidoptera (Figure 2). Although the authors speculated on the role of ABCC proteins in cross-resistance between Cry1Ac and Cry2Ab, no binding or toxicity studies were performed with Cry1Ac, and the strain of *H. armigera* used was susceptible to both Cry1Ac and Cry2Ab [72]. The use of a name already assigned to another gene has unfortunately created the false impression that the results are relevant to cross-resistance. Here we designate this gene as ABCC6 (Figures 1D and 2).

9. CRISPR/Cas9 Knockouts

CRISPR/Cas9 knockouts provide a very useful tool to investigate gene function in
non-model organisms. The first use of the technique to knock out ABC transporters
in Lepidoptera targeted the half-transporter genes *white*, *scarlet*, and *ok* in *Helicoverpa armigera* [73]. These are homologs of the well-known pigment transporters *white*, *scarlet*,
and *brown* in *Drosophila melanogaster*, and as expected, the knockouts affected adult eye
color and larval skin pigmentation. Homozygotes for the *white* knockout, however, were
embryonic lethal in *H. armigera* and in the milkweed bug [74], which was unexpected
because these are viable in *Drosophila*, *Aedes*, and *Tribolium*. Lethality has also complicated
the interpretation for some knockouts of Cry toxin targets.

Knockouts of ABCA2 in a susceptible strain of *H. armigera* conferred >100-fold resistance to Cry2Aa and Cry2Ab, and eliminated Cry2Ab binding to BBMV, but did not affect resistance or binding to Cry1Ac [75]. After mapping Cry2Ab resistance in *T. ni* to ABCA2, where a transib mobile element was found to disrupt the gene, either ABCA1 or ABCA2
were knocked out in a susceptible strain and only ABCA2 was found to affect Cry2Ab
tolerance [69]. Knockouts of the ABCA2 gene in *B. mori* (using the TALEN technique)
conferred Cry2A resistance on larvae, and heterologous expression of ABCA2 in HEK239
cells confirmed the absence of cross-resistance to Cry1A, Cry1Ca, Cry1Da, Cry1Fa, and
Cry9Aa toxins [76].

In *P. xylostella*, a knockout of ABCC2 conferred 724-fold resistance to Cry1Ac and a knockout of ABCC3 conferred 413-fold resistance to the same toxin. Each knockout greatly reduced BBMV binding to Cry1Ac, but the double knockout was not made in this study [77]. Somewhat different results were obtained in another study of the same species [78], in which single knockouts were weakly resistant (~4-fold) and only the double knockout was >8000-fold resistant to Cry1Ac. So far there is no explanation for the differing results in *P. xylostella*. A study in *H. armigera*, however, produced results closer to the second study, in that single knockouts of ABCC2 and ABCC3 were weakly resistant to Cry1Ac, while the double knockout was >15,000-fold resistant [79].

Knocking out ABCC2 in *O. furnacalis* conferred >300-fold resistance to Cry1Fa but less
than 10-fold resistance to Cry1Ab or Cry1Ac and no resistance to Cry1Aa [80]. Knocking
out ABCC2 in *S. frugiperda* increased tolerance to either Cry1Fa or Cry1Ab >120-fold,
while knocking out ABCC3 increased tolerance by a lesser amount (>16-fold); in this
study the double knockout was reported to be lethal [46]. In *S. exigua*, an ambitious study
created single knockouts of ABCC1, ABCC2, or ABCC3, as well as the cadherin and an
aminopeptidase, and examined susceptibility to Cry1Ac, Cry1Fa, and Cry1Ca. Among the
15 pairwise comparisons, ABCC2 had a strong effect and the cadherin a weak effect on
Cry1Ac or Cry1Fa tolerance, and ABCC2 also had a weak effect on Cry1Ca tolerance [52].

CRISPR/Cas9 knockout experiments are useful in confirming the role of a given ABC transporter in susceptibility to a given toxin, and when more than one knockout and more than one toxin are compared, in assessing their relative importance. The possibility of non-target effects needs more investigation, in order to reconcile studies where double knockouts are lethal with those where they confer even more resistance, since these studies make diametrically opposed recommendations for resistance management. Targeting the nucleotide binding domains would increase the probability of nontarget effects, since these are more highly conserved across ABC family members. In addition, no knock-ins have been reported yet for ABC transporters, as in the case of another Bt resistance gene, tetraspanin [81].

10. Negative Cross-Resistance with Chemical Insecticides

The intriguing possibility that mutations in ABC transporters could interfere with
the insect's ability to use them to rid itself of other toxins has motivated many recent
studies. With the increasing use of Bt sprays and transgenic plants in the 1990s, the issue of
cross-resistance between Bt and chemical insecticides had achieved some attention, but Bt
resistance mechanisms had not been characterized at the molecular level. More recently, in
2016 when several chemical insecticides were screened against an ABCC2-mutant strain
of *H. armigera*, abamectin and spineotram were more toxic compared with their activity
against a Cry1Ac-susceptible strain [82]. Measurements of higher abamectin concentrations
in mutant larvae and transfected cells were consistent with the bioassay results. RNAi
silencing of ABCC2 decreased susceptibility to Cry1Ac and increased susceptibility to
abamectin [82]. However, the selective differential exerted by abamectin on the Cry1Ac-
resistant versus Cry1Ac-susceptible strains was small, and not all subsequent studies have
confirmed the effect.

Single and double knockouts of ABCC2 and ABCC3 in *H. armigera* produced in a
different study were not more susceptible to abamectin or spinetoram [79]. The knockout
of ABCC2 in *O. furnacalis* was not more susceptible to abamectin or chlorantraniliprole [80].
On the other hand, single knockouts of ABCC2 or ABCC3 in *S. frugiperda* were more
susceptible to abamectin and spinosad (while the double knockout was reported to be
lethal and could not be compared) [46]. Another study on *S. frugiperda* found that a
Cry1F-resistant strain isolated from the field with a frameshift mutation in ABCC2 had lower sensitivity to bifenthrin and higher sensitivity to spinetoram; yet when ABCC2 was knocked out in a different strain, Cry1F resistance increased 25-fold but sensitivity to chlorantraniliprole, bifenthrin, spinetoram, and acephate was unchanged [83]. Knocking out the P-glycoprotein ABCB1 in *S. exigua* increased, rather than decreased, susceptibility to abamectin and emamectin benzoate [84]; whether this protein is orthologous to the coleopteran ABCB1 (see below) has not been determined. Contradictory results were obtained in a study of ABCC2 in *P. xylostella* [85]; HEK-293 cells stably transformed with ABCC2 accumulated less avermectin, but down-regulating ABCC2 in vivo with RNAi had no effect on avermectin or chlorfenapyr tolerance.

Although results are suggestive in some studies, the changes in tolerance to the conventional insecticides examined are small and would not be useful in a resistance-breaking approach against ABCC mutations, as mortality of Bt-resistant insects would not be much greater than their Bt-susceptible counterparts. Only a few insecticides have been examined so far, and some with a greater effect are likely to be found eventually in a wider screen.

## 11. ABCB (P-glycoprotein) and Cry3 Toxins in Coleoptera

A different family of ABC transporters, the P-glycoproteins (ABCB), is involved in toxicity of the Cry3 toxins in Coleoptera. Linkage mapping in a strain of the poplar leaf beetle *Chrysomela tremula* (Fabricius) resistant to transgenic Cry3Aa-expressing poplar identified a frameshift in the ABCB1 protein, and heterologous expression of ABCB1 in Sf9 cells conferred susceptibility to Cry3Aa in vitro [86]. In the western corn rootworm *D. virgifera* virgifera, heterologous expression of the orthologous ABCB1 protein also conferred Cry3A sensitivity on Sf9 and HEK-293 cells in vitro, and an mCry3A-resistant strain was found to have deletions in the ABCB1 gene [87].

Whether the beetle ABCB1 genes are orthologous to P*ABCB1* from *P. xylostella* mentioned in Section 3 above [39] is not known; Cry3 toxins were not experimentally tested in that study. The authors pointed out structural similarities among Cry1 and Cry3 toxins, and searched for but could not find P*ABCB1* in the fragmented *Plutella* genome sequence. We have found that the *Bombyx* ortholog maps to Chromosome 15, about 1 MB away from the ABCC cluster (Figure 1B). More studies are required to confirm the involvement of the P-glycoproteins in Cry1A toxin interactions in Lepidoptera, and to establish the generality of the P-glycoproteins as targets of the beetle-active Cry3 toxins. Cross-resistance studies suggest the existence of additional, different targets in beetles [88].

## 12. Other ABC Transporters in Lepidoptera

Suppression of the *white* gene in *P. xylostella* by RNAi reduced Cry1Ac susceptibility but was not lethal [89]; as pointed out earlier, CRISPR/Cas9 knockouts of *white* were lethal in *H. armigera* [73]. Suppression of ABCH1 in *P. xylostella* caused larval mortality but did not affect Cry1Ac resistance [90]. A gene in *O. furnacalis* identified as ABCG was down-regulated in Cry1Ab- and Cry1Ac-resistant strains [91]; it is evidently not orthologous to either of the two genes in *P. xylostella*. No mutations in half-transporter ABCG or ABCH family genes have yet been identified in Bt-resistant Lepidoptera.

## 13. Hypotheses on the Mechanism of Pore Insertion

The lack of three-dimensional structures of the ABCC2 or ABCA2 proteins, the Cry toxin pore embedded in the membrane, and the toxin-binding region of the cadherin has inhibited the development of detailed hypotheses on the manner by which ABC transporters facilitate pore insertion. ABC transporters could simply be another binding site on the membrane surface, increasing the local toxin concentration and increasing the rate of pore insertion, due to a concentration effect. It has been hypothesized that ABCC2 facilitates the formation of the pre-pore oligomer, in a manner similar to the cadherin [92]. It was hypothesized that active opening and closing of the ABC transporter channel would
be required to pull the pre-formed pore into the membrane [13]. This hypothesis would seem to be refuted by results with a mutant ABCC2 from *S. exigua* lacking the second nucleotide-binding domain [93], as well as engineered mutants of ABCC2 from *B. mori* lacking nucleotide-binding domains [94].

As previously pointed out [15], it is difficult to explain how evolution in *Bacillus thuringiensis* has resulted in Cry2A-type toxins that target ABCA proteins, Cry1A-type toxins that target ABCC proteins, and Cry3B-type toxins that target ABCB proteins, without invoking some fundamental property that unites these very different ABC transporters. If the shared ATP-switch mechanism powering substrate transport [4] is not such a property, then we are left without a mechanistic explanation of the pore-insertion process for the three-domain Cry proteins [13]. Many other bacterial pore-forming toxins enter the membrane with dynamic conformational changes, for example Vip3A [95] or the Membrane Attack Complex [96], or the Tc toxin [97]. Whether such a dynamic process is required for Cry toxin pore formation deserves more investigation. A reasonable hypothesis at this point is that the dynamism comes from the toxin-target interaction, not just the toxin.

**14. Future Perspectives**

Since the first description in 2010, mutations in ABC transporters have emerged as the most important type of mutation causing resistance to the three-domain Cry toxins of *Bacillus thuringiensis*. Yet mechanistic studies have lagged behind those on other pore-forming toxins with much more complicated structures. Why? Not enough effort has been expended on determining the three-dimensional structures that will be required for a full understanding of how the toxin interacts with membrane proteins to form a membrane pore. Since the first two structures of trypsin-activated monomers of the three-domain Cry toxins revealed their structural similarity [98,99], many more have appeared confirming that this similarity is fundamental. Recently a structure of the entire protoxin was determined [100], revealing additional domains that might potentiate pore formation in some way. The low-hanging fruit has been harvested. Lacking is a structure of any ABC transporter known to interact with a Cry toxin. Lacking is a structure of any cadherin known to interact with a Cry toxin. Lacking is a structure of the Cry toxin pore in the membrane. Without these structures, theorizing about how Bt toxins work is fantasy. Recent advances in electron cryo-microscopy (cryo-EM) have made these structures attainable. It is now time to attain them.

**Funding:** Preparation of this review was funded by the Max-Planck-Gesellschaft. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Data Availability Statement:** NCBI GenBank: www.ncbi.nlm.nih.gov, accessed on 2 April 2021. Kaikobase: kaikobase.dna.affrc.go.jp, accessed on 2 April 2021.

**Acknowledgments:** I thank Linda J. Gahan for years of fruitful collaboration at Clemson University, and the U. S. National Science Foundation for finally funding our grant proposal for positional cloning in *Heliothis virescens* on the seventh submission.

**Conflicts of Interest:** The author declares no conflict of interest.

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Article

# Monitoring of Target-Site Mutations Conferring Insecticide Resistance in *Spodoptera frugiperda*

Debora Boaventura<fnref n="1" />,<fnref n="2" />, Macarena Martin<fnref n="3" />, Alberto Pozzebon<fnref n="3" /> ID, David Mota-Sanchez<fnref n="4" /> and Ralf Nauen<fnref n="2" />,* ID

Received: 22 July 2020; Accepted: 13 August 2020; Published: 18 August 2020

<figure><img src="image_18.png" /></figure>

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**Simple Summary:** Fall armyworm, *Spodoptera frugiperda*, is an invasive moth species and one of the most destructive pests of maize. It is native to the Americas but recently invaded (sub)tropical regions in Africa, Asia and Oceania. Fall armyworm larvae feeding on maize plants cause substantial economic damage and are usually controlled by the application of insecticides and genetically modified (GM) maize expressing *Bacillus thuringiensis* (Bt) proteins, selectively targeting fall armyworm. It has developed resistance to many different classes of insecticides and Bt proteins as well; therefore, it is important to check field populations for the presence of mutations in target proteins conferring resistance. Here, we developed molecular diagnostic tools allowing us to test the frequency of resistance alleles in field-collected populations, either alive or preserved in alcohol. We tested 34 different populations collected on four different continents for the presence of mutations conferring resistance to common classes of insecticides and Bt proteins. We detected resistance mutations which are quite widespread, whereas others are restricted to certain geographies or even completely absent. The established molecular methods show robust results in samples collected across a broad geographical range and can be used to support decisions for sustainable fall armyworm control and applied resistance management.

**Abstract:** Fall armyworm (FAW), *Spodoptera frugiperda*, a major pest of corn and native to the Americas, recently invaded (sub)tropical regions worldwide. The intensive use of insecticides and the high adoption of crops expressing *Bacillus thuringiensis* (Bt) proteins has led to many cases of resistance. Target-site mutations are among the main mechanisms of resistance and monitoring their frequency is of great value for insecticide resistance management. Pyrosequencing and PCR-based allelic discrimination assays were developed and used to genotype target-site resistance alleles in 34 FAW populations from different continents. The diagnostic methods revealed a high frequency of mutations in acetylcholinesterase, conferring resistance to organophosphates and carbamates. In voltage-gated sodium channels targeted by pyrethroids, only one population from Indonesia showed a mutation. No mutations were detected in the ryanodine receptor, suggesting susceptibility to diamides. Indels in the ATP-binding cassette transporter C2 associated with Bt-resistance were observed in samples collected in Puerto Rico and Brazil. Additionally, we analyzed all samples for the presence of markers associated with two sympatric FAW host plant strains. The molecular methods established show robust results in FAW samples collected across a broad geographical range and can be used to support decisions for sustainable FAW control and applied resistance management.

**Keywords:** fall armyworm; insecticide resistance; target-site mutations; Bt resistance; corn strain; rice strain; resistance management; Indonesia; Kenya

<footnote n="1">¹ Institute of Crop Science and Resource Conservation, University of Bonn, 53115 Bonn, Germany; de.boaventura@hotmail.com</footnote>

<footnote n="2">² Bayer AG, Crop Science Division, R&D Pest Control, 40789 Monheim, Germany</footnote>

<footnote n="3">³ Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, 35020 Padova, Italy; macamar_20@hotmail.com (M.M.); alberto.pozzebon@unipd.it (A.P.)</footnote>

<footnote n="4">⁴ Department of Entomology, Michigan State University, East Lansing, MI 48824, USA; motasanc@msu.edu</footnote>

<footnote>* Correspondence: ralf.nauen@bayer.com; Tel.: +49-(0)2173-38-4441</footnote>
# 1. Introduction

The fall armyworm (FAW), *Spodoptera frugiperda* (J.E. Smith) (Lepidoptera: Noctuidae), is an important agricultural pest of several crops in the western hemisphere [1,2]. Since 2016, FAW distribution expanded globally by invading different continents, first reported in Africa and later reaching Southeast Asia and, more recently, Australia, totalizing its presence in 107 countries worldwide [3–6]. The success of FAW spread is due to many factors, such as the high reproductive capacity, long-distance migration and high polyphagia [7,8].

Two sympatric host plant strains of *S. frugiperda* have been previously described: the corn strain, which feeds on large grasses such as corn and sorghum, and the rice strain, preferring small grasses such as rice [9,10]. The two strains differ not only in their host preferences but also regarding their physiology [11], insecticide susceptibility [12] and composition of genes involved in chemoreception, detoxification and digestion [13].

In South America, both strains have been already identified in field populations using molecular markers, and most populations were structured in agreement with their host preferences [14,15]. Although initial studies on the genetic structure of FAW populations from the newly invaded countries suggest a common source of origin, probably from Florida or the Caribbean [16,17], there are differences in the strain haplotypes and disagreements regarding the molecular marker and host plant that may imply inter-population movement of FAW populations from African and Asian countries [18].

An understanding of the genetic background of FAW is essential for resistance management strategies in different regions. Besides the intrinsic variation in insecticide susceptibility associated with FAW strains [11,12,19,20], the impact of migration on insecticide resistance will depend on the pre-existence of resistance alleles in the starting population and selection pressure on the newly invaded areas and spread [14,17].

At present, the Arthropod Pesticide Resistance Database (APRD) reports 144 cases of insecticide resistance in FAW globally. Among the 41 different active substances affected, 45% of the cases belong to proteins produced by *Bacillus thuringiensis* (Bt), 26% and 19% to insecticides targeting the voltage-gated sodium channel (VGSC), and acetylcholinesterase (AChE), respectively [21]. The high number of cases reported for Bt proteins, particularly those expressed in transgenic corn, reflects the intensive adoption of transgenic crops, which corresponded to 191.7 million ha worldwide in 2018 [22]. The adoption of transgenic crops expressing insect-resistant traits to control lepidopteran pests is most advanced in the United States and Brazil. Nevertheless, in Asia, the adoption of Bt-corn is high, particularly in China and India, while it is rather limited to just a few countries in Africa [22].

Many resistance cases are reported for pyrethroid insecticides targeting the VGSC and inhibitors of AChE (i.e., carbamates and organophosphates). This is due to low application costs, a high number of compounds registered for decades and frequent applications [23]. Nevertheless, together, they still account for around 30% of the global insecticide market share [23]. The most modern chemical class used to control lepidopteran pests are the diamide insecticides, acting on the ryanodine receptor (RyR) and used in different agronomic settings [23].

It is unclear whether FAW populations present in Africa were already resistant to old chemical compounds [24]. However, farmers have complained about the efficacy of pyrethroids and organophosphate insecticides under field conditions [25]. Hence, this has led to misuse by increasing rates, application frequency or even the use of unregistered compounds [25,26]. On the other hand, if no control measures against FAW are adopted, the yield losses for corn could reach up to 20.6 m tons per annum for only 12 corn-producing countries in Africa [24].

Insecticide resistance is usually conferred by the insensitivity of the target receptor and/or pharmacokinetic processes modifying the rate or the properties of the insecticides delivered to the target site [27]. Amino acid substitutions/indels at the VGSC (T929I, L932F, and L1014F), AChE (A201S,
G227A, and F290V), RyR (I4790M and G4946E) and ATP-binding cassette subfamily C2 transporter (ABCC2) (GC insertion and GY deletion) have been linked to resistance in *S. frugiperda* to pyrethroids, carbamates and organophosphates, diamides and Bt proteins (e.g., Cry1F), respectively [28–30].

In the present study, we monitored the frequency of the above-mentioned target-site mutations in the VGSC, AChE, RyR and ABCC2 in 34 populations of *S. frugiperda* collected in Brazil, Puerto Rico, Kenya and Indonesia by PCR-based allelic discrimination assays as well as pyrosequencing diagnostics. We validated and established robust diagnostic tools based on genomic DNA, which can be implemented to support decisions for appropriate resistance management strategies.

**2. Materials and Methods**

2.1. *Insect Collection*

Larvae of FAW were collected from different sites in Brazil, Puerto Rico, Kenya and Indonesia (Figure 1 and Table S1) and kept in 70% ethanol or RNAlater® (Life Technology, Carlsbad, CA, USA) until DNA extraction and genotyping.

<figure><img src="image_19.png" /><figcaption>**Figure 1.** Map showing the origin of 34 fall armyworm populations collected in Brazil, Puerto Rico, Kenya and Indonesia (more details about collection sites in Table S1). All samples were used for the genotyping of target-site mutations. The schematic map was created using EasyMap software (Lutum + Tappert DVBeratung GmbH, Bonn, Germany).</figcaption></figure>

2.2. *DNA Extraction*

Genomic DNA was extracted from individual larvae (whole body for second/third instar and abdominal fragments of fifth instar larvae). At least five individuals per FAW population (Table S1) were used for gDNA extraction and the total sample number used for the genotyping analysis is shown in Table 1. Agencourt DNAdvance™ (Beckmann Coulter, Beverly, CA, USA) and DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) were used to extract gDNA for the pyrosequencing and fluorescent probe assay, respectively. Both kits were used according to the suppliers' recommended protocols.
<table><caption>**Table 1.** Genotyping by pyrosequencing for different target-site mutations in major insecticide targets. In total, larvae of 34 populations from Brazil, Puerto Rico, Kenya and Indonesia were analyzed. Homozygous susceptible (SS), heterozygotes (RS) and homozygous resistant (RR).</caption><thead><tr><th>Target</th><th>Country</th><th>Mutation</th><th>N</th><th>SS (%)</th><th>RS (%)</th><th>RR (%)</th></tr></thead><tbody><tr><td rowspan="10">Voltage-gated sodium channel (VGSC)</td><td>Brazil</td><td rowspan="4">L1014F</td><td>140</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Puerto Rico</td><td>70</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Kenya</td><td>76</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Indonesia</td><td>110</td><td>98.2</td><td>1.8</td><td>0.0</td></tr><tr><td>Brazil</td><td rowspan="4">L932F</td><td>143</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Puerto Rico</td><td>64</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Kenya</td><td>75</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Indonesia</td><td>88</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Brazil</td><td rowspan="4">T929I</td><td>143</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Puerto Rico</td><td>64</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Kenya</td><td>75</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Indonesia</td><td>88</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td rowspan="10">Acetylcholinesterase (AChE)</td><td>Brazil</td><td rowspan="4">A201S</td><td>147</td><td>92.5</td><td>4.1</td><td>3.4</td></tr><tr><td>Puerto Rico</td><td>29</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Kenya</td><td>76</td><td>89.5</td><td>10.5</td><td>0.0</td></tr><tr><td>Indonesia</td><td>85</td><td>77.6</td><td>22.4</td><td>0.0</td></tr><tr><td>Brazil</td><td rowspan="4">F290V</td><td>127</td><td>55.1</td><td>44.9</td><td>0.0</td></tr><tr><td>Puerto Rico</td><td>70</td><td>4.3</td><td>10.0</td><td>85.7</td></tr><tr><td>Kenya</td><td>76</td><td>26.3</td><td>47.4</td><td>26.3</td></tr><tr><td>Indonesia</td><td>86</td><td>19.8</td><td>55.8</td><td>24.4</td></tr><tr><td>Brazil</td><td rowspan="4">G227A</td><td>161</td><td>55.3</td><td>32.3</td><td>12.4</td></tr><tr><td>Puerto Rico</td><td>29</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Kenya</td><td>76</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Indonesia</td><td>86</td><td>83.7</td><td>16.3</td><td>0.0</td></tr><tr><td rowspan="10">Ryanodine receptor (RyR)</td><td>Brazil</td><td rowspan="4">G4946E</td><td>140</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Puerto Rico</td><td>70</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Kenya</td><td>76</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Indonesia</td><td>90</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Brazil<sup>a</sup></td><td rowspan="4">I4790M</td><td>140</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Puerto Rico</td><td>70</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Kenya</td><td>76</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Indonesia</td><td>90</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td rowspan="4">ATP-binding cassette transporter subfamily C (ABCC2)</td><td>Brazil<sup>b</sup></td><td rowspan="4">GY del</td><td>211</td><td>39.83</td><td>14.30</td><td>45.87</td></tr><tr><td>Puerto Rico</td><td>19</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Kenya</td><td>70</td><td>100.0</td><td>0.0</td><td>0.0</td></tr><tr><td>Indonesia</td><td>79</td><td>100.0</td><td>0.0</td><td>0.0</td></tr></tbody></table>

## 2.3. PCR and qPCR Conditions

PCR for pyrosequencing, PCR-RFLP and PCR for sequencing were performed in 30 µL reaction mixture containing 15 µL JumpStart™ Taq ReadyMix™ (Sigma-Aldrich, St. Louis, MO, USA), 500 nM of forward and reverse primers (Table S2), around 20 to 50 ng gDNA and nuclease-free water. The cycling conditions comprised 95 °C for 3 min, followed by 40 cycles of 95 °C for 30 s, the respective annealing temperature according to Table S2 for 30 s and 72 °C for 45 s, and a final elongation step at 72 °C for 5 min.

The fluorescent probe assays for detection of mutations F290V, I4790M and a GC insertion in the ABCC2 consisted of reactions set up at a final volume of 10 µL, with 5 µL SsoAdvanced™ Universal Probes Supermix (Bio-Rad, Hercules, CA, USA), 700 nM of forward and reverse primers (Table S2), 200 nM of probes, 20–50 ng of gDNA and nuclease-free water, and the reactions were run in duplicate. The conditions of PCR amplification were 95 °C for 5 min and 40 cycles at 95 °C for 15 s and 60 °C

<footnote><sup>a</sup> Data published by Boaventura et al. (2020a) [30]; <sup>b</sup> Data published by Boaventura et al. (2020b) [28].</footnote>
for 30 s. The real-time PCR was conducted in a CFX-384 real-time thermocycler (Bio-Rad, Hercules, CA, USA) and the end-point fluorescence values, taking cycle 35 as a threshold, were plotted in a scatter-plot using Bio-Rad qPCR analysis software CFX Maestro 1.0 (Bio-Rad, Hercules, CA, USA).

## 2.4. Characterization of *S. frugiperda* Strains

### 2.4.1. Characterization of COI Haplotypes Using PCR-RFLP

Corn and rice strain genotyping were performed using the molecular markers based on mitochondrial cytochrome oxidase subunit I (COI) with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), according to Nagoshi et al. (2007, 2012) [31,32]. Three to five individuals from different populations of *S. frugiperda* collected in Kenya (EP-K, KV-K, NJ-K, MJ-K and MD-K), Indonesia (WS-I, DS-I, S-I, WC-I and BC-I), Brazil (Sf_Bra, Sf_Cor, MT-PL1-2, BA-SD and PR-PG) and Puerto Rico (PR60, PR61, PR62, PR63 and PR64) (Table S1) were characterized. PCR reactions were carried out according to Section 2.3, using primer JM76 and JM77 (Table S2). After amplification, 1.0 µL of FastDigest MspI (Thermo Scientific, Vilnius, Lithuania) was added to 10 µL of each PCR reaction and incubated at 37 °C for 10 min. The PCR products were verified by an automated gel electrophoresis system, according to the AL320 method (QIAxcel DNA Screening Kit v2.0, QIAGEN, Hilden, Germany). In order to validate the results, a second PCR spanning another restriction site was performed using designed forward (891F_COI) and reverse (c1303R_COI) primers (Table S2). After the amplification, the digestion step was performed by adding EcoRV (New England Biolabs, Frankfurt, Germany), according to the manufacturer's instructions.

### 2.4.2. Characterization of Tpi Haplotypes Using DNA Sequencing

Plant host strain identification was additionally performed using the triosephosphate isomerase (*Tpi*) gene as a genetic marker, according to Nagoshi et al. (2019) [18]. The PCR amplification was performed according to Section 2.3, using the forward (TpiE4) and reverse (850R) primers described in Table S2. The PCR products were verified by an automated gel electrophoresis system, according to the OM500 method (QIAxcel DNA Screening Kit v2.0, QIAGEN), purified using PCR Clean-up Gel Extraction kit (Macherey-Nagel, Düren, Germany) and Sanger-sequenced by Eurofins Genomics (Cologne, Germany). The obtained *S. frugiperda* *Tpi* nucleotide sequences were aligned with the *Tpi* sequences for corn and rice variants according to the reference genome [13] (https://bipaa.genouest.org/data/public/sfrudb/), using Geneious software v. 10.2.3 (Biomatters Ltd., Auckland, New Zealand).

## 2.5. Target-Site Resistance Diagnostics by Pyrosequencing

Amino acid substitutions in the VGSC (T929I, L932F and L1014F), AChE (A201S, G227A and F290V), RyR (I4790M and G4946E) and ABCC2 (GC insertion and GY deletion) result in resistance to pyrethroid, carbamate/organophosphate, diamide and Cry1F Bt protein, respectively. Mutation sites in the VGSC, AChE and RyR are numbered according to *Musca domestica* (GenBank X96668), *Torpedo californica* (PDB ID: 1EA5) and *Plutella xylostella* (GenBank AET09964), respectively.

A pyrosequencing based genotyping assay was designed for targeting each mutation separately and performed across 34 FAW populations (see Table S1 for details about FAW populations).

Primer pairs were designed with Assay Design Software (QIAGEN, Hilden, Germany), according to sequences deposited at the National Center for Biotechnology Information (NCBI) for FAW para-type VGSC (GenBank KC435025) and *ace-1* (GenBank KC435023). Primers targeting FAW *RyR* (GenBank MK226188) and ABCC2 (GenBank KY489760) were described elsewhere [28,30], as indicated in Table S2.

The PCR conditions for pyrosequencing were performed as described in Section 2.3, using primers given in Table S2. The pyrosequencing reaction was carried out as described elsewhere [33], using a sequencing primer specific for every target-site mutation analyzed, according to Table S2.
## 2.6. Fluorescence Based Allelic Discrimination Assays

### 2.6.1. F290V Mutation in AChE

Primers were designed using the OligoArchitect™ Assay Design (Sigma-Aldrich, St. Louis, MO, USA) for the detection of the F290V mutation in ace1. Allele-specific probes were labeled with FAM (Sf_F290_FAM) or HEX (Sf_F290_mut_HEX) at the 5' end for the detection of the wildtype and mutant allele, respectively (Table S2). Five individuals from populations from Brazil (Sf_Bra, MT-PL1 and PR-PG), Puerto Rico (PR60, PR61, PR62 and PR63), Kenya (EP-K, KV-K, MJ-K, KF-K and NW-K) and Indonesia (WS-I, DS-I, S-I, WC-I and JL-I) were tested (Table S1). PCR reactions and allele discrimination analysis were performed as described in Section 2.3.

### 2.6.2. GC Insertion in ABCC2

The GC insertion in ABCC2 was detected according to Banerjee et al. (2017) [34], with slight modifications. Briefly, reactions were composed of a HEX-labeled probe (SfABCC2mut allele) that is SfABCC2 mutant allele-specific and a FAM-labeled probe (SfABCC2), specific to the SfABCC2 wildtype allele, gDNA (around 50 ng), the forward (Sf_ABCC2_F) and the reverse (Sf_ABCC2_R) primers (Table S2). The populations tested were the same as described in Section 2.6.1 and PCR reactions were prepared as mentioned in Section 2.3.

### 2.6.3. I4790M Mutation in the RyR

The detection of the RyR I4790M mutation was performed as described by Boaventura et al. (2020) [30] using forward (Sf_taq_I4790_F) and reverse (Sf_taq_I4790_R) primers, the mutant allele-specific FAM-labeled probe (Sf_I4790_mut_FAM) and a HEX-labeled probe (Sf_I4790_HEX) that is wildtype allele-specific (Table S2). Individuals with known genotype from strain Chlorant-R (homozygote for M4790) as well as artificial heterozygotes (a mixture of gDNA from Chlorant-R and Sf_Bra individuals) were used as internal controls. The assay was validated with populations collected in Brazil, Puerto Rico, Kenya and Indonesia, as described above (Section 2.6.1).

# 3. Results

## 3.1. Characterization of S. frugiperda Strains

The mitochondrial COI and nuclear Tpi molecular markers were employed for the identification of sympatric FAW rice and corn strain according to Nagoshi et al. [17,31,32]. The amplification of the respective COI fragment resulted in a PCR product of around 569 bp for both strains, but the fragment amplified from corn strain contained a MspI restriction site; therefore, after digestion, the PCR product was cut into two fragments (approximately 487 and 72 bp) (Figure 2A and Figure S1A). According to this method, all samples tested from Brazil and Puerto Rico were characterized as corn strain, whereas in Kenya and Indonesia, most of the individuals were characterized as rice strain, i.e., 70% and 91.7%, respectively (Figure 2A).

On the other hand, when using the EcoRV restriction site, the rice strain fragment was cut into two bands of around 350 and 150 bp and the corn strain fragment of 500 bp remained uncut (Figure 2B and Figure S1B). Again, all samples tested from Brazil and Puerto Rico were corn strain and most of the samples from Kenya and Indonesia were rice strain, i.e., 61 and 92%, respectively.

Host plant strain characterization using the *Tpi* gene was performed according to Nagoshi et al. (2019) [17]. The genetic markers used in this method are all single nucleotide substitutions present in the *TpiE4* exon. When using the primers 412F and 850R (Table S2), most of the *TpiE4* exon is amplified, producing a fragment of about 199 bp. Trimmed sequences were deposited in NCBI (GenBank MT706015-MT706018). The strain is defined by the gTpi183Y site, where the corn strain has a cytosine and the rice strain a thymine. All the samples tested from the four countries were corn strain, having a cytosine at the position gTpi183Y. It is worth mentioning that, at the position gTpi192Y,
an adenine or thymine was observed in some samples from Brazil, Puerto Rico and Kenya (1, 1 and 2, respectively), while Nagoshi et al. (2019) [17] reported only a cytosine or a thymine at position Tpi192Y.

<figure><img src="image_20.png" /><figcaption>**Figure 2.** Schematic representation of amplified COI and TpiE4 fragments used for Spodoptera frugiperda host plant strain identification in field samples collected in Brazil, Puerto Rico, Kenya and Indonesia. COI polymorphism in S. frugiperda was determined by RFLP-PCR. (A) PCR product containing a MspI restriction site in the corn strain and PCR fragments obtained after digestion with FastDigest MspI. (B) PCR product that contains an EcoRV strain-specific site. After digestion with EcoRV, the corn strain remains uncut, whereas the rice strain is cut. (C) TpiE4 fragment with different polymorphic sites was Sanger-sequenced. Position marked H defines whether it is a rice strain (thymine; TpiR) or a corn strain (cytosine; TpiC).</figcaption></figure>

## 3.2. Detection of Target-Site Mutations by Pyrosequencing

The pyrosequencing assay used to genotype the mutations in the VGSC revealed that almost all analyzed larvae (n = 396) were wildtype, with no mutations at those sites analyzed. Only strain NB-KA from Indonesia included a few individuals heterozygous for the L1014F mutation, corresponding to 1.8% of all samples analyzed from Indonesia (Table 1). On the other hand, the mutations T929I and L932F were not detected at all in any population tested (Table 1), suggesting the lack of target-site resistance to pyrethroids in almost all samples analyzed. Resistant AChE alleles were found at much higher frequencies across countries in many populations analyzed. The mutation F290V was detected at the highest frequency (Table 1). In Brazil, 45% of the samples genotyped (57 out of 127 larvae) were heterozygote, whereas most samples from Puerto Rico (except strain PR65) were homozygote for V290, representing 85.7% of all samples tested (60 out of 70 larvae). Populations collected in Kenya and Indonesia also carried the F290V mutation in AChE and, on average, 47% and 56% of the samples were heterozygotes, respectively. The other AChE mutation sites analyzed, A201S and G227A, were not detected in Puerto Rico, while G227A was absent also in Kenya. RyR mutations G4946E and
I4790M, conferring resistance to diamide insecticides, were not detected in any of the populations tested (Table 1); all individuals tested were homozygous wildtype at both positions. We also tested 379 individuals for the presence of a GY deletion in ABCC2, known to confer resistance to Cry1F in FAW [28]. This functionally validated target-site mutation was absent in samples collected in Puerto Rico, Kenya and Indonesia but detected in many of the tested Brazilian larvae, as recently described [28] (Table 1).

### 3.3. *Detection of Target-Site Mutations by Fluorescence Based Allelic Discrimination Assays*

As the target-site, mutation F290V in AChE was the most frequent mutation found in all populations tested. We decided to develop a PCR-based allelic discrimination assay using fluorescent probes, which could be performed at larger-scale worldwide using a qPCR machine, because pyrosequencing-based diagnosis is more expensive and less common. All larvae analyzed from Puerto Rico were homozygote for the V290 resistance allele (Figure 3A). In Kenya and Indonesia, five populations were tested and, on average, 40% and 56% of the larvae were heterozygotes, respectively (Figure 3C,D). Individuals from the two field populations from Brazil (MT-PL1 and PR-PG) were homozygous for the V290 resistance allele (Figure 3B). The population Sf_Bra was kept for 15 years under laboratory conditions without insecticide exposure and all larvae were homozygotes for the susceptible wildtype allele F290 (Figure 3B).

<figure><img src="image_21.png" /><figcaption>**Figure 3.** Bivariate plot showing the discrimination of different acetylcholinesterase alleles in *Spodoptera frugiperda* samples by an allele-specific real-time PCR fluorescent probe assay. Each dot represents a single larva. Blue squares represent mutant RR homozygotes (V290; allele 1), orange circles susceptible SS homozygotes (F290; allele 2) and green triangles SR heterozygotes (F290/V290). Analysis of fall armyworm field samples collected in (A) Puerto Rico, (B) Brazil, (C) Kenya and (D) Indonesia.</figcaption></figure>

The I4790M mutation in the RyR was assessed using Chlorant-R resistant FAW larvae as a positive control for M4790. The resistant allele was not present in any other sample analyzed (Figure 4). For the detection of GC insertion at the ABCC2 causing resistance to Cry1F protein in *S. frugiperda* in Puerto Rico, the assay described by Banerjee et al. (2017) [34] was used, however substituting the VIC fluorescent probe with FAM, as described in Section 2.6.2. The GC insertion was only observed in Puerto Rican samples (Figure S2).
<figure><img src="image_22.png" /><figcaption>**Figure 4.** Detection of the RyR I4790M mutation using an allele-specific real-time PCR fluorescent probe assay, as recently described by Boaventura et al. (2020). (A) Genotyping of *Spodoptera frugiperda* collected in Puerto Rico represented by blue squares (wildtype SS homozygotes, I4790 allele), orange circles represent strain Chlorant-R mutant RR homozygotes from Brazil (M4790; allele 1) and green triangles artificial SR heterozygotes (I4790/M4790). All individuals tested from (B) Kenya and (C) Indonesia were susceptible homozygotes for I4790 (blue squares).</figcaption></figure>

**4. Discussion**

The highly invasive nature and the potential economic impact of FAW have raised a lot of concerns across continents. Changes in agricultural practices and biological control are among a diverse range of measures implemented in recently invaded African countries and India by smallholder farmers and at rather low FAW infestation levels [25,26,35–39]. In countries with significant agricultural input subsidy programs, synthetic insecticides have been used to control FAW outbreaks [25,26,38]. However, in some countries, farmers claimed rather low efficacy of some of the insecticide classes used, such as organophosphates and pyrethroids [25,35]. It remains unclear whether the low field efficacy of insecticides against FAW in Africa is due to resistance or poor application technology affecting plant coverage.

Our genotyping study was conducted to shed some light on the presence of target-site insecticide resistance mechanisms in 34 populations collected in Kenya, Indonesia, Puerto Rico and Brazil. Our results from FAW populations collected in Kenya showed a relatively high frequency of the F290V mutation in AChE, the target of organophosphate and carbamate insecticides. The chance that alleles conferring resistance to these rather old chemical classes were already present at high frequency in the invasive population is quite high. Their frequency was likely augmented by further selection, using applications of cheap products based on organophosphate and carbamate chemistries. Similar findings have been reported for the tomato leafminer (*Tuta absoluta*) in Iran, where this pest has been recently introduced. Resistance to pyrethroids and organophosphates was expected in the invading populations and this expectation was supported by the identification of target-site mutations in VGSC and ace1, respectively [40].

As a result of frequent insecticide applications, multiple resistance cases have been described for field populations in regions where FAW is native [41,42]. In Brazil and Puerto Rico, for instance, resistance has been reported to pyrethroids, organophosphates, carbamates, spinosyns, benzoylureas and, most recently, diamides [29,42–47]. The genetic inheritance of insecticide resistance in FAW has been investigated, and cases of FAW resistance were described as polygenic and metabolic [46–48].

Pyrethroid insecticides are supposed to bind in the domain IIS4-S5 linker and domain IIIS6 of *para*-type sodium channels [49] and the common L1014F mutation has been reported to confer pyrethroid resistance ratios of 10-20-fold [29,50–52]. More than 30 unique resistance-associated mutations including L1014F or combinations thereof have been described in VGSC in many other different species [53]. Three mutations (T929I, L932F and L1014F) at the VGSC have been recently described in pyrethroid-resistant *S. frugiperda* from Brazil [29] and one of the mutations, L932F, was detected in FAW populations from China [54]. Our genotyping results revealed the absence of the L1014F mutation in almost all analyzed samples, except for one population from Indonesia (K-I, Table S1), where only two heterozygotes out of 30 individuals were detected. No other mutation conferring pyrethroid resistance and described for *S. frugiperda* was detected in the populations tested.
However, other mechanisms such as enhanced metabolism by elevated levels of cytochrome P450 monooxygenases are known to confer pyrethroid resistance in FAW [29] but were not tested in our study as we used gDNA of alcohol preserved FAW samples as we did not have access to living insects.

Organophosphates and carbamates target AChE and resistance is often associated with mutations in the *ace-1* gene, leading to amino acid substitutions at the enzyme's active site [55]. Our genotyping results confirmed the presence of the following amino acid substitutions A201S, G227A and F290V in populations collected in Brazil, as described by Carvalho et al. (2013) [29]. The detected point mutations co-exist, at least in heterozygous individuals, in populations BA-SD, PR-PG and MT-PL1-2. Moreover, we also detected the F290V mutation in samples from Puerto Rico, Indonesia and Kenya. Point mutations linked to organophosphate resistance have been described for *Cydia pomonella* (F399V), *Chilo suppressalis* (A314S) and *P. xylostella* (D131G, A201S, G227A and A441G) [56–60]. Moreover, heterologous expression of AChE mutants (A303S, G329A and L554S) from the silkworm (*Bombyx mori*) have supported the reduction in AChE sensitivity towards carbamate and organophosphate insecticides [61].

Diamide insecticides comprised two chemotypes, the phthalic (flubendiamide) and anthranilic acid diamides (e.g., chlorantraniliprole), which were shown to be affected differently by the presence of point mutations leading to amino acid substitutions, particularly G4946E and I4790M in the lepidopteran RyR (numbering according to the *P. xylostella* RyR)—recently reviewed by Richardson et al. [62]. Phthalic diamides are less potent against pests carrying a methionine at position 4790 [63]. In Puerto Rico, resistance ratios of 160 to 500-fold have been reported to chlorantraniliprole and flubendiamide, respectively, but the mechanisms of resistance were not studied in detail [42]. However, in our genotyping assays, G4946E and I4790M were not detected in samples from Puerto Rico or any other country. So far, the I4790M mutation in *S. frugiperda* has been detected only in one FAW strain (Chlorant-R) from Brazil, selected with chlorantraniliprole under laboratory conditions and showing resistance ratios of >230 and >42,000-fold against chlorantraniliprole and flubendiamide, respectively [30,43]. Our genotyping data suggest that the frequency of those resistance alleles (G4946E and I4790M) is low under field conditions in those locations here investigated.

Mutations in the ABCC2 transporter have been associated with Cry1F and Cry1A.105 resistance in FAW: a GC insertion causing a premature stop codon has been found in Cry1F-resistant FAW strains from Puerto Rico [34,64], whereas a functionally validated GY deletion was very recently described in Cry1F-resistant populations from Brazil [28]. While we identified the described GC insertion and GY deletion in many samples from Puerto Rico and Brazil, respectively, none of the above-mentioned mutations were found in populations from Kenya or Indonesia, supporting the absence/very low frequency of these mutations in the field and a lack of selection pressure by transgenic corn expressing Cry1F in those countries. Recent whole-genome sequencing of FAW samples collected in China, Malawi, Uganda and Brazil revealed a novel ABCC2 resistance allele in FAW collected in Brazil, leading to a truncated and likely non-functional protein [65].

Two sympatric host plant strains of *S. frugiperda* have been previously described: the corn strain and the rice strain, which prefers forage grasses and rice [9–11]. Recent studies have reported that *S. frugiperda* populations present in Asia and Africa are an inter-strain hybrid, with the genetic background mostly from the corn strain [54,66]. Therefore, we were interested in the host-plant strain composition of our samples and analyzed individual larvae of different populations using recently described markers by RFLP and PCR. Our results using COI and Tpi genetic markers confirmed that the corn strain is the most abundant in Brazil and Puerto Rico, as shown in previous studies [14,15,67,68]. The COI genetic marker used in this study revealed dominance of the rice strain in those populations that we collected in Kenya (70%, though collected from corn plants) and Indonesia (91.7%). However, to avoid any identification bias, we used a second marker, *Tpi*, and the obtained data revealed that all samples, including those from Kenya and Indonesia, resemble corn strain and none rice strain. This discrepancy between COI and *Tpi* markers has already been noticed by other authors, especially with samples from Africa and Asia, where strain characterization is dependent on the molecular marker
used [16,18,65]. However, the exclusive identification of the corn strain in our samples by using the *Tpi*
marker is in accordance with the preferred host, suggesting that it is a more accurate strain marker
than COI. In terms of insecticide susceptibility, there is not much difference between host-plant strains,
at least when considering the efficacy of recommended label rates of many insecticides. A slightly
higher level of cytochrome P450 activity in corn-adopted FAW [9] may render the corn strain slightly
more tolerant, but this is unlikely to result in reduced efficacy of insecticides at their recommended label
rates in the absence of resistance. The rice strain has been reported to be more susceptible to diazinon,
carbaryl and Bt toxins, whereas corn strain larvae were shown to be more susceptible to the carbamate
carbofuran [12,19,20]. Recently, Arias et al. (2019) [14] have tested the possible influence caused by the
migration of individuals from hot spots—characterized by higher LC₅₀ values against flubendiamide
and lufenuron. The authors concluded that migration did not play the key role but, rather, the pest
management measures adopted and cropping strategies in the respective region. Therefore, we want
to reinforce that, although high frequencies of alleles conferring resistance to organophosphate and
carbamates were detected, the choice of the appropriate management strategy to be adopted based on
regionally registered insecticides and alternative measures is likely to be the key factor for sustainable
FAW control. The practical relevance of the presence of alleles conferring resistance is determined
by the selection pressure adopted in the field and whether the mutations present carry any fitness
cost. The resistance alleles might decrease in frequency in the absence of selection pressure or increase
when the application of specific insecticides increases [14]. Therefore, strategies to slow down the
development of insecticide resistance should be driven by the application of insecticides with different
modes of action [14,69]. Compounds such as diamides, emamectin benzoate and spinosyns [25,70–72]
have mostly shown good control of several lepidopteran pests and would be valuable tools in FAW
resistance management strategies in the newly invaded countries.

**5. Conclusions**

Based on our genotyping results described in this study, the field efficacy of organophosphate and
carbamate insecticides is likely to be compromised by the presence of the AChE V290 allele in hetero-
and homozygous form in Brazil, Kenya, Indonesia and Puerto Rico. To achieve successful integrated
pest management of FAW and reduce the risk of economic losses, resistance management strategies
will need to be implemented at regional levels in the newly invaded countries and can be supported by
using the presented diagnostic tools to detect and monitor the early presence of resistance alleles in
the field.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2075-4450/11/8/545/s1,
Table S1: Populations of *Spodoptera frugiperda* collected in different countries and years used for genotyping of
target-site mutations, Table S2: List of primers for pyrosequencing and dual fluorescence probe assay used for the
identification of different target-site mutations and Spodoptera frugiperda strain identification by RFLP-PCR and
Sanger sequencing, Figure S1: Automated analysis of DNA fragments showing COI polymorphism in Spodoptera
frugiperda. (A) PCR product containing a strain specific MspI site that was amplified using the JM76 and JM77
primers (Table S2) followed by products obtained after the digestion with FastDigest MspI. Corn-strain is cut and
rice-strain remains uncut as it does not have the MspI site. (B) PCR product amplified with the primers 891F_COI
and c1303R_COI (Table S2) that contains a EcoRV strain specific site. After digestion with EcoRV the corn-strain
amplicon remains uncut whereas it is cut in the rice-strain. Details about samples, see Table S1, Figure S2:
Detection of GC insertion allele at the ATP-binding cassette subfamily C2 (ABCC2) conferring resistance to
Bacillus thuringiensis Cry1F toxin using PCR fluorescent probe assay described by Banerjee et al [34]; Blue squares
represent mutant ABCC2 homozygotes for the GC insertion, orange circles ABCC2 wildtype SS homozygotes,
and green triangles SR representing heterozygotes. Analysis of fall armyworm field samples collected in (A)
Brazil, (B) Puerto Rico, (C) Kenya, and (D) Indonesia.

**Author Contributions:** R.N. and D.B. conceived the study. R.N. supervised the project. D.B. and M.M. performed the research and analyzed data. D.M.-S. supported the study with biological material. A.P. supervised M.M. and acquired funding. D.B. and R.N. wrote the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** M.M. thanks the Erasmus+—KA1 Erasmus Mundus Joint Master’s Degree Programme of the European Commission as this paper has been developed as a result of a mobility stay funded by the PLANT HEALTH Project.
**Acknowledgments:** The authors thank numerous field researchers from Bayer for collecting FAW samples in Brazil, Kenya and Indonesia.

**Conflicts of Interest:** R.N. is employed by Bayer AG, a manufacturer of insecticides and transgenic maize. This work was partially financed by Bayer AG. The authors declare no additional conflicts of interest.

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Article

# A Novel Insecticidal Molecule Extracted from *Alpinia galanga* with Potential to Control the Pest Insect *Spodoptera frugiperda*

Torranis Ruttanaphan <fnref n="1" />,<fnref n="2" />, Georges de Sousa <fnref n="2" />, Anchulee Pengsook <fnref n="3" />, Wanchai Pluempanupat <fnref n="3" />, Hannah-Isadora Huditz <fnref n="2" />, Vasakorn Bullangpoti <fnref n="1" /> and Gaëlle Le Goff <fnref n="2" />,*

Received: 9 September 2020; Accepted: 8 October 2020; Published: 11 October 2020

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**Simple Summary:** The fall armyworm is an insect pest that feeds on many plants, including plants of agronomic importance, such as corn and rice. In addition, it has developed resistance to the main families of synthetic insecticides. There is, therefore, a need to find new, more environmentally friendly molecules to control this pest. We have extracted a molecule from greater galangal and tested its activity as an insecticide on the fall armyworm. This natural molecule causes larval growth inhibition and larval developmental abnormalities. To understand its action, a cell model with Sf9 cells was used. The molecule is much more toxic to insect cells than to human cells. It affects cell proliferation and induces cell death. This study demonstrates that a molecule extracted from an edible plant may have potential in the future development of botanical insecticides for the control of insect pests.

**Abstract:** *Spodoptera frugiperda*, a highly polyphagous insect pest from America, has recently invaded and widely spread throughout Africa and Asia. Effective and environmentally safe tools are needed for successful pest management of this invasive species. Natural molecules extracted from plants offer this possibility. Our study aimed to determine the insecticidal efficacy of a new molecule extracted from *Alpinia galanga* rhizome, the 1'S-1'-acetoxychavicol acetate (ACA). The toxicity of ACA was assessed by topical application on early third-instar larvae of *S. frugiperda*. Results showed that ACA caused significant larval growth inhibition and larval developmental abnormalities. In order to further explore the effects of this molecule, experiments have been performed at the cellular level using Sf9 model cells. ACA exhibited higher toxicity on Sf9 cells as compared to azadirachtin and was 38-fold less toxic on HepG2 cells. Inhibition of cell proliferation was observed at sublethal concentrations of ACA and was associated with cellular morphological changes and nuclear condensation. In addition, ACA induced caspase-3 activity. RT-qPCR experiments reveal that ACA induces the expression of several caspase genes. This first study on the effects of ACA on *S. frugiperda* larvae and cells provides evidence that ACA may have potential as a botanical insecticide for the control of *S. frugiperda*.

**Keywords:** 1'S-1'-Acetoxychavicol acetate; *Alpinia galanga*; *Spodoptera frugiperda*; Sf9 cells; botanical pesticide

<footnote n="1">¹ Animal Toxicology and Physiology Specialty Research Unit, Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; torranis40@gmail.com (T.R.); fscivkb@ku.ac.th (V.B.)</footnote>

<footnote n="2">² Université Côte d'Azur, INRAE, CNRS, ISA, F-06903 Sophia Antipolis, France; georges.de-sousa@inrae.fr (G.d.S.); hannah.huditz@hotmail.de (H.-I.H.)</footnote>

<footnote n="3">³ Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, and Special Research Unit for Advanced Magnetic Resonance, Kasetsart University, Bangkok 10900, Thailand; a_ampere@hotmail.com (A.P.); fsciwcp@ku.ac.th (W.P.)</footnote>

<footnote>* Correspondence: gaelle.le-goff@inrae.fr; Tel.: +33-492-386578</footnote>
# 1. Introduction

*Spodoptera frugiperda* (Smith) (Lepidoptera: Noctuidae), a major agricultural insect pest native to the American continent, has recently invaded Africa and Asia [1,2]. *S. frugiperda* causes severe yield losses in various species of economically important crops, such as maize, rice, cotton and peanut [3]. Synthetic insecticides are largely used for its management; however, their use has negative impacts on the environment and human health and led to the development of resistance. *S. frugiperda* has developed resistance against 41 different molecules (https://www.pesticideresistance.org/display.php? page=species&arId=200), resulting in pest control failures [4]. Such problems necessitate finding safer and innovative alternatives to the existing synthetic insecticides. Plants can be sources of organic molecules with insecticidal potential and advantages such as low toxicity to humans and environmental safety [5].

Plant secondary metabolites are organic compounds produced by plants and are known to play a role in adaptation to their environment [6]. They are usually subdivided according to their biosynthetic pathways in three main groups, including alkaloids, phenolics and terpenoids [6]. Plant secondary metabolites can act as insecticidal, antifeedant, repellency, oviposition deterrence and growth regulation agents against insects [5]. They have been intensively studied for the past 20 years to develop alternatives to synthetic insecticides due to their low ecological side effects [5,7].

*Alpinia galanga* (L.) Willd is a plant belonging to the Zingiberaceae family of Zingiberales order. Its rhizome has several active compounds, such as 1'S-1'-acetoxychavicol acetate (ACA), p-hydroxycinnamaldehyde, 1'-acetoxychavicol acetate, β-pinene, β-bisabolene and 1,8-cineole [8]. It has been used for various purposes, including antibacterial, antifungal, antiulcer, antitumor, antiallergic and antioxidant purposes. For example, it has been proposed as a new therapeutic agent in certain human cancers, such as myeloid leukemia [9], where it acts by inducing apoptosis, a natural occurring cell death process. ACA also has insecticidal activity against insect pests, such as *Bactrocera dorsalis* (Hendel), *Coptotermes gestroi* (Wasmann), *Coptotermes curvignathus* (Holmgren) and *Spodoptera litura* (Fabricius) [8,10–12]. Moreover, *A. galanga* oil could enhance the toxicity of a synthetic pyrethroid against *S. litura* [13]. In addition, *A. galanga* is a plant widespread in South East Asian countries and can be found throughout the year [14].

The aim of this study was to determine whether a compound extracted from *A. galanga*, 1'S-1'-acetoxychavicol acetate (ACA), could be used as a botanical insecticide against a major agricultural pest, *S. frugiperda*. It was a good candidate as it is a major constituent of *A. galanga* and its stereochemistry exhibited insecticidal activity [15]. The effects of this molecule and its possible mode of action were then investigated in the cell model of Sf9 cells by biochemical and molecular approaches.

# 2. Materials and Methods

## 2.1. Chemicals

Azadirachtin (95% purity), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), RNase A, propidium iodide (PI), penicillin-streptomycin, trypan blue solution (0.4%) and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich, Steinheim, Germany. Alanyl-glutamine, trypsin/EDTA and fetal bovine serum were purchased from Sigma-Aldrich, St. Louis, MO, USA.

## 2.2. Extraction and Isolation of 1'S-1'-Acetoxychavicol Acetate

Sun-dried *A. galanga* (1.6 kg) rhizomes were powdered and sequentially extracted with hexane and ethyl acetate by soaking at room temperature for seven days. Ethyl acetate crude extract was filtered using a Buchner funnel and evaporated by a rotary evaporator. Ethyl acetate crude was fractionated by column chromatography (Kiesel gel 60 G cat No. 7731, Merck, Molsheim, France) with a gradient elution system of hexane-ethyl acetate (7:1). Eleven fractions (Fr. I–XI) were obtained. Fr. VI
(250 mg) was purified using thin layer chromatography (Silica gel 60 PF254, Merck) with hexane and ethyl acetate. 1'S-1'-acetoxychavicol acetate (Figure 1) was obtained and verified by spectral analysis. The analytical data of 1'S-1'-acetoxychavicol acetate are described as below. High resolution mass spectra were recorded using a Maxis Bruker spectrometer (Karlsruhe, Germany). ¹H and ¹³C NMR spectra were recorded using a Bruker 400 MHz Advance III HD spectrometer (Karlsruhe, Germany).

<figure><img src="image_27.png" /><figcaption>**Figure 1.** Chemical structure of 1'S-1'-acetoxychavicol acetate.</figcaption></figure>

$[\alpha]_D^{20} - 50$ ($c$ = 0.5, dichloromethane). ¹H NMR (CDCl₃, 400 MHz), $\delta$: 7.37 (2H, d, $J$ = 8.7 Hz), 7.07 (2H, d, $J$ = 8.7 Hz), 6.26 (1H, d, $J$ = 5.7 Hz), 5.98 (1H, ddd, $J$ = 17.1, 10.5, 5.8 Hz), 5.30 (1H, dt, $J$ = 17.2, 1.3 Hz), 5.25 (1H, dt, $J$ = 10.5, 1.2 Hz), 2.30 (3H, s), 2.11 (3H, s). ¹³C NMR (CDCl₃, 100 MHz), $\delta$: 170.0, 169.4, 150.7, 136.4, 136.3, 128.6, 121.7, 117.3, 75.8, 21.1, 21.0. HRMS (ESI) Calcd. for C₁₃H₁₄NaO₄ 257.0790 ([M + Na]⁺), Found 257.0825.

## 2.3. Insect Rearing and Insecticide Treatments

*Spodoptera frugiperda* populations were provided by Dr. Emmanuelle d'Alençon (DGIMI, Université de Montpellier, INRAE, Montpellier, France). The larvae were fed ad libitum on an artificial diet [16] and maintained under controlled conditions at 24 °C and 65% relative humidity with a 16 h light: 8 h dark photoperiod as described earlier [17].

The effects of ACA on early third-instar larvae of *S. frugiperda* were determined by topical application. Serial dilutions (0.005, 0.01, 0.02 µg/µL) of ACA were prepared in acetone. Doses were applied to the dorsal thoracic region of each larva using a micropipette. Each larva received 1 µL of solution per treatment, with acetone alone as the negative control. In each experiment, 10 larvae/treatment in three replicates were used ($n$ = 30 per treatment). Treated larvae were placed in a sealed plastic container with an artificial diet under controlled conditions as described above. Larval weight, abnormal development and mortality were recorded after 24, 48 and 72 h. Larvae that did not move after being prodded were considered dead. Abnormal larvae were observed as described by [18]. Relative growth rate was calculated based on the following formula described by [19] as:

$$ \text{Relative growth rate (\%)} = (\text{Mean larval weight of treated group}/\text{Mean larval weight of control group}) \times 100\% $$

## 2.4. Cell Culture

Sf9 cells (from Invitrogen), derived from the ovarian tissue of *S. frugiperda* pupa, were cultured in 75-cm² cell culture flasks (TPP, Trasadigen, Switzerland) with the Insect-XPRESS™ protein-free medium (Lonza, Verviers, Belgium) and routinely maintained at 26 °C in a cell culture incubator (Memmert GmbH & Co., Schwabach, Germany). Cells were subcultured after achieving 90% confluence, and cell media were changed every 4 days.

HepG2 cells, the human hepatocellular carcinoma cell line, were provided by Dr. Georges de Sousa (INRAE, Sophia Antipolis, France). Cells were routinely maintained in William's E Medium (Sigma-Aldrich, Steinheim, Germany) supplemented with 1% alanyl-glutamine, 1% penicillin-streptomycin and 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO₂. After achieving 80-90% confluence, the cells were washed using PBS (Sigma-Aldrich,
Steinheim, Germany) and trypsinized with 0.25% Trypsin/EDTA. Cells were plated in 75-cm² cell culture flasks (TPP, Trasadigen, Switzerland), and cell media were changed every 3 days.

## 2.5. Cell Viability Assay

For cell viability assays, Sf9 cells ($5 \times 10^5$ cells/mL) and HepG2 cells ($2 \times 10^5$ cells/mL) were seeded onto 96-well plates (100 µL/well). Sf9 cells were treated with 0, 0.25, 0.875, 1.125, 1.75, 2.625, 3.25, 4, 5, 7.5, 10, 12.5, 15, and 17.5 µM of ACA in DMSO while HepG2 cells were treated with 5, 10, 20, 40, 80, 160, and 320 µM of ACA. After 24 h of incubation, each well received 100 µL of solution mixed with medium per treatment, with 0.25% DMSO as the negative control and azadirachtin (0–100 µM), the pure molecule, as the positive control. After 24 h of treatment, 100 µL MTT dissolved in Insect-XPRESS™ medium (0.5 mg/mL) was added to each well. After an additional 2 h of incubation, the medium was discarded and DMSO was added to dissolve the formazan crystal, and the absorbance was measured at 570 and 690 nm using a microplate reader (SpectraMax, Molecular Devices). (Wokingham, UK). The relative percentage of cell viability was calculated as:

$$ \text{Cell viability rate (\%)} = \left[ \frac{(A - 0.02)}{(B - 0.02)} \right] \times 100\% $$

where A and B are the absorbances of the ACA treated cells and DMSO treated cells, respectively.

## 2.6. Cell Proliferation Assay

Sf9 cells ($3.5 \times 10^5$ cells/mL) were seeded onto 6-well plates (2 mL/well). Serial dilutions of 0.38, 0.57, 0.88 and 1.17 µM (Inhibitory concentration (IC) IC$_5$, IC$_{10}$, IC$_{20}$ and IC$_{30}$, respectively) ACA were prepared using DMSO. After 24 h of incubation, each well received 2 mL of solution mixed with medium per treatment, with 0.25% DMSO as the negative control. After 24, 48 and 72 h of treatment, cell density was evaluated using a Malassez hemocytometer (Marienfeld, Harsewinkel, Germany) counted with 0.4% Trypan blue solution staining. The morphologic characteristics of Sf9 cells were observed with an inverted phase contrast microscope (Nikon Eclipse TE2000-U, Tokyo, Japan) at 72 h after 1.17-µM ACA treatment. To examine the reversible arrest of cellular proliferation, the medium containing ACA was removed after 72 h of treatment. The cells were washed using PBS and fresh medium without ACA was added. After an additional 72 h of incubation, the cells were counted.

## 2.7. Caspase-3 Activity Assay

Caspase-3 activity was measured using a fluorometric activity assay kit (Molecular probe, Eugene, OR, USA) following the manufacturer's protocol. The substrate, rhodamine 110 bis-(*N*-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide) (Z-DEVD-Rho 110), was cleaved proteolytically by caspase-3. After 24 h treatment with ACA (0.88, 1.17, and 1.75 µM corresponding to IC$_{20}$, IC$_{30}$ and IC$_{40}$, respectively), the cells were harvested and lysed in 1 mL lysis buffer. The sample was incubated with the substrate Z-DEVD-Rho 110. Fluorescence intensity was determined using a LightCycler 480® (Roche Diagnostic, Inc., Mannheim, Germany) at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. Values were expressed as arbitrary units of Rhodamine-110 (Rho 110) fluorescence.

## 2.8. Hoechst 33342 Staining

Sf9 cells ($1 \times 10^5$ cells/mL) were seeded in 96-well plates, incubated for 24 h and then treated with ACA (0.88, 1.17, and 1.75 µM corresponding to IC$_{20}$, IC$_{30}$ and IC$_{40}$, respectively) and incubated for 24 h. The cells were stained with Hoechst 33342 (Thermo Fisher Scientific Inc. Waltham, MA, USA) and incubated for 1 h at 26 °C in a cell culture incubator. The stained cells were observed using an ArrayScan™ XTI High Content Analysis (Thermo Fisher Scientific Inc. Waltham, MA, USA).
## 2.9. Cell Cycle Analysis

The Sf9 cell cycle analysis was carried out using fluorescence-activated cell sorting (FACS) assay (FACSCalibur, BectonDickinson, Eysins, Switzerland). The Sf9 cells were incubated for 24, 48 and 72 h with 1.17 µM of ACA (IC₃₀). Cells were resuspended and fixed with cold 80% ethanol/PBS (10 mM Na₂HPO₄, 138 mM NaCl, 2.7 mM KCl, pH 7.4). Fixed Sf9 cells were incubated in a PBS solution containing 50 µg/mL propidium iodide and 50 µg/mL RNAse A at 37 °C for 20 min. At least 10,000 cells were counted in each assay. The percentage of cells in each cell cycle phase were measured and classified in G0/G1, S and G2/M phases, depending on the intensity of the fluorescence peaks. Cells incubated with 0.25% DMSO at the same time were used as a control.

## 2.10. RNA Extraction, cDNA Synthesis and Real-Time Quantitative PCR (RT-qPCR)

To study gene expression of apoptosis-related genes induced by ACA, Sf9 cells were harvested after 24 h exposure to 1.17 µM ACA corresponding to IC₃₀. Total RNA was extracted using the TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. RNA extractions were performed in six-well plates with three biological replicates.

An amount of 1 µg total RNA was reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad). RT-qPCR reactions were performed using an Opticon monitor 2 (Bio-Rad) with the qPCR MasterMix for SYBR® Green I No ROX (Eurogentec, Seraing, Belgium). The PCR conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. Each reaction was performed in two technical replicates and the mean of the three independent biological replicates was calculated. All results were normalized to the glucose 6-phosphate dehydrogenase (G6PDH), ribosomal protein L4 (RpL4) and ribosomal protein L18 (L18) mRNA expression levels, identified, in one of our previous studies, as the most stably expressed reference genes under different conditions [20]. Relative expression values were calculated in R using the SATQPCR (http://satqpcr.sophia.inra.fr) described in [21]. Primer sequences and efficiencies are listed in Table 1.

<table><caption>**Table 1.** Primers used in qRT-PCR.</caption><thead><tr><th>Name</th><th>Primer Sequence</th><th>Fragment Length (bp)</th><th>PCR Efficiencies (%)</th></tr></thead><tbody><tr><td>Sf-Caspase-1-F</td><td>5'-AATGCTGGACGGAAAACAAG-3'</td><td>139</td><td>99</td></tr><tr><td>Sf-Caspase-1-R</td><td>5'-AACTGGCATCCTAGCGACAC-3'</td><td></td><td></td></tr><tr><td>Sf-Caspase-2-F</td><td>5'-TCCAGTCCACCCTGATTTTC-3'</td><td>83</td><td>103</td></tr><tr><td>Sf-Caspase-2-R</td><td>5'-ACCAAGATCCACGTTTACGG-3'</td><td></td><td></td></tr><tr><td>Sf-Caspase-5-F</td><td>5'-GGCCTCTACGAGTGATGGAC-3'</td><td>89</td><td>99</td></tr><tr><td>Sf-Caspase-5-R</td><td>5'-CGGAAGACACGTCAGTCAAA-3'</td><td></td><td></td></tr><tr><td>Sf-Caspase-6-F</td><td>5'-ACCACAAGGAATGGAAGTGG-3'</td><td>149</td><td>101</td></tr><tr><td>Sf-Caspase-6-R</td><td>5'-GTGCTGTGTCCGGTACTTCA-3'</td><td></td><td></td></tr><tr><td>Sf-Cathepsin B-F</td><td>5'-AACGGTGACTCCAAAACACC-3'</td><td>98</td><td>109</td></tr><tr><td>Sf-Cathepsin B-R</td><td>5'-GAGTACACGTGCTTGCCGTA-3'</td><td></td><td></td></tr><tr><td>Sf-Cathepsin L-F</td><td>5'-AGTGCAGGTACAACCCCAAG-3'</td><td>146</td><td>100</td></tr><tr><td>Sf-Cathepsin L-R</td><td>5'-CTGGAAGGTCTCCTGTGAGG-3'</td><td></td><td></td></tr><tr><td>Sf-DnaJ1-F</td><td>5'-TGAGAGAGGGAGGAGTTGGA-3'</td><td>93</td><td>95</td></tr><tr><td>Sf-DnaJ1-R</td><td>5'-GTCTACGACCACCGCTGAAT-3'</td><td></td><td></td></tr><tr><td>Sf-p53-F</td><td>5'-GCACTTGATATCGGTGGAGAA-3'</td><td>150</td><td>96</td></tr><tr><td>Sf-p53-R</td><td>5'-GATCCTACAGTCACCCAGCA-3'</td><td></td><td></td></tr><tr><td>Sf-TCTP-F</td><td>5'-GGACATCCTTGGCAGGTTTA-3'</td><td>147</td><td>105</td></tr><tr><td>Sf-TCTP-R</td><td>5'-TCCTCCTCAAGACCATGCTT-3'</td><td></td><td></td></tr><tr><td>G6PD-F</td><td>5'-GGCCCTGTGGCTAACAGAAT-3'</td><td>142</td><td>98</td></tr><tr><td>G6PD-R</td><td>5'-CATCGTCTCTACCAAAAGGCTTC-3'</td><td></td><td></td></tr><tr><td>L18-F</td><td>5'-CGTATCAACCGACCTCCACT-3'</td><td>126</td><td>108</td></tr><tr><td>L18-R</td><td>5'-AGGCACCTTGTAGAGCCTCA-3'</td><td></td><td></td></tr><tr><td>RpL4-F</td><td>5'-CAACAAGAGGGGTTCACGAT-3'</td><td>149</td><td>98</td></tr><tr><td>RpL4-R</td><td>5'-GCACGATCAGTTCGGGTATC-3'</td><td></td><td></td></tr></tbody></table>

G6PD, glucose 6-phosphate dehydrogenase; L18, ribosomal protein; RpL4, ribosomal protein.
## 2.11. Statistical Analysis

All experiments were conducted in three biological replications and the values are described as mean ± SE (standard error). Probit analysis was used to estimate contact toxicity as the median lethal dose (LD₅₀) values. Statistical comparisons between various groups were analyzed using Tukey's multiple range tests (*p* < 0.05 being considered significant) by the program Stat Plus v.Pro 6.5.0.0 (AnalystSoft Inc, Walnut, CA, USA). The half maximal inhibitory concentration (IC₅₀) values were calculated by log-probit analysis using the GraphPad Prism 5 program. For RT-qPCR analysis, statistical comparisons between DMSO and ACA groups were analyzed using *t*-test by the SATQPCR (http://satqpcr.sophia.inra.fr/cgi/home.cgi), described in Rancurel et al. [21].

# 3. Results

## 3.1. Extract Yield

The percentage yield of *A. galanga* for each crude extract derived from 1.6 kg of dry material was measured, and we found that ethyl acetate crude extract resulted in the highest yield percentage (1.19% yield), followed by hexane crude extract (0.43% yield). ACA (156 mg) was derived from 1 g of ethyl acetate crude extract. Ethyl acetate crude extract was brown gum, while ACA was pale yellow solid.

## 3.2. Effects of ACA on S. frugiperda Larvae

The effects of ACA on the growth inhibition and abnormality phenotype of third instar larvae of *S. frugiperda* were determined. Larval weights and phenotypic changes were examined after treatment with ACA and acetone (negative control). After 72 h, the larvae treated with ACA exhibited growth inhibition and abnormal phenotypes, such as a shrunk body, wrinkled body, deformed body and parts of the old cuticle remaining in the ACA-treated region (Figure 2A). The size of ACA-treated larvae was obviously smaller as compared to the larvae of the negative control. Larvae treated with 0.02 µg/µL ACA showed a significant reduction in relative growth rate of 34% (*p* < 0.05, Figure 2B) and a significant increase in abnormal phenotypes of around 27% (*p* < 0.05, Figure 2C). Additionally, the larvae treated with 0.02 µg/µL ACA weighed significantly less, with a 31.79% decrease (13.55 mg ± 1.15 vs. 19.87 mg ± 1.19, *p* < 0.05, *n* = 30).
<figure><img src="image_28.png" /><figcaption>**Figure 2.** Effects of 1'S-1'-acetoxychavicol acetate (ACA) on *Spodoptera frugiperda* larvae; (A) Phenotypic changes of *S. frugiperda* larvae. (A1) Larval body size of *S. frugiperda* at 72 h after ACA treatment compared with acetone-treated larvae (negative control). (A2) Abnormalities of *S. frugiperda*. (B) Relative growth rate (%). (C) Larval abnormality rate (%). Arrow indicates larval abnormality region. Different lowercase letters indicate significantly different groups using Tukey's honestly significant difference (HSD) test (p < 0.05). In each experiment, 10 larvae/treatment in three replicates were used (n = 30 per treatment).</figcaption></figure>

### 3.3. Cytotoxicity of ACA on Sf9 and HepG2 Cells

In order to further investigate the effect of ACA at the cellular level, we used a cellular model, Sf9, an *S. frugiperda* cell line. Cell viability was measured by MTT assay after 24 h of compound exposure. ACA exhibited a high toxicity on Sf9 cells in a dose-dependent manner (IC₅₀ = 1.84 µM, Figure 3A), while azadirachtin (a commercial botanical pesticide used to control lepidopteran pests in particular), a positive control, was less toxic than ACA (percent inhibition of 100 µM azadirachtin = 11%). In order to explore the selective toxicity of ACA, experiments were performed on the non-target cellular model HepG2 cells. ACA was toxic on HepG2 cells in a dose-dependent manner (IC₅₀ = 70.40 µM, Figure 3B), however this toxicity was 38-fold lower than the one observed on Sf9 cells.
<figure><img src="image_29.png" /><figcaption>**Figure 3.** Effects of 1′-S-1′-acetoxychavicol acetate on (A) Sf9 and (B) HepG2 cell viabilities at 24 h after treatment. IC₅₀ represents the half maximal inhibitory concentration.</figcaption></figure>

3.4. *Sf9 Cell Proliferation Is Inhibited by ACA*

The effect of ACA on cell proliferation was examined by staining to assess the cell membrane integrity after treatment with sublethal concentrations (IC<sub>5</sub>, IC<sub>10</sub>, IC<sub>20</sub> and IC<sub>30</sub>) of ACA, at various time intervals from 24 h to 72 h. The cell density in the DMSO control corresponds to the 100% proliferation rate set for each time. The proliferation rate of ACA-treated cells was calculated relative to this control. The cell density increased in the control as follows: 4.58 × 10<sup>5</sup> cells/mL, 9.75 × 10<sup>5</sup> cells/mL and 23.33 × 10<sup>5</sup> cells/mL at 24, 48, and 72 h, respectively. All treatments showed a dose-dependent decrease in the rate of cell proliferation (Figure 4A). A significant inhibition of cell proliferation was observed in the concentration range between 0.88 and 1.17 µM, with no significant difference between proliferation rate at 24 h or 72 h. Cell densities were 1.62 × 10<sup>5</sup> cells/mL, 4.83 × 10<sup>5</sup> cells/mL and 7.10 × 10<sup>5</sup> cells/mL at the concentrations of 1.17 µM after 24, 48 and 72 h of treatment, respectively. Inverted phase contrast microscopy observation revealed that Sf9 cells were irregular in shape, became larger in size and lost adhesion after 72 h treatment with 1.17 µM of ACA. In order to further confirm that ACA inhibited cell proliferation, the reversible arrest of cell proliferation was examined. The proliferation rate of the cells at 72 h after removal of ACA (1.17 µM) was significantly higher than the cells with the medium containing ACA, suggesting that the inhibitory effect of ACA on Sf9 cell proliferation is reversible (Figure 4B).

In order to evaluate the possible mechanisms involved the inhibition of cell proliferation, cell cycle
monitoring and measurements of apoptosis-associated caspase activities were determined. The cell cycle
was examined after 24, 48, and 72 h of 1.17 µM ACA treatments using FACS analysis. The distribution
of cells in the different phases of the cell cycle was the same between control and ACA-treated cells (no
significant difference) and no cell cycle arrest was observed, indicating that ACA did not affect the
cell cycle (data not shown). Caspase-3 activity was evaluated in Sf9 cells after 24 h of IC20, IC30 and
IC40 of ACA treatments (0.88, 1.17 and 1.75 µM, respectively) using Z-DEVD-Rho 110 (Figure 4C).
The results demonstrated an increase in Rho 110 fluorescence following ACA treatments, indicating
that ACA induced caspase-3 activity in Sf9 cells in a dose-dependent manner. Furthermore, Hoechst 33342 staining revealed nuclear morphologic changes (Figure 4D). Nuclei of ACA-treated cells showed deeper white staining with nuclear condensation after 24 h treated with 0.88, 1.17 and 1.75 µM of ACA. The number of nuclear condensations increased in a dose-dependent manner. Altogether, these results suggest that the inhibition of cell proliferation by ACA was not due to cell cycle arrest but appeared to involve apoptosis, as evidenced by caspase activity and nuclear condensation.

<figure><img src="image_30.png" /><figcaption>**Figure 4.** Effects of 1′-S-1′-acetoxychavicol acetate on Sf9 cell proliferation, caspase-3 activity and nuclear condensation; (A) anti-proliferative effect of ACA at various concentrations and time points. (B) Sf9 cell proliferation was observed at 72 and 144 h after treatment by 1.17 µM of ACA. Reversible arrest of cell proliferation was observed in IC₃₀R where the molecule was removed after 72 h. R stands for removal. All data were expressed as mean ± SE of three independent experiments. A t-test was performed to determine significance of the results as compared to the control group (0.25% dimethyl sulfoxide DMSO). \* denotes significant difference at *p* < 0.05. Tukey's honestly significant difference (HSD) test was used to compare the groups of each time intervals. Different lowercase (for 72 h) or uppercase (for 144 h) letters indicate significantly different groups (*p* < 0.01). (C) Caspase-3 activity of Sf9 cells after 24 h treatment with ACA. Values were expressed as arbitrary units of Rhodamine-110 (Rho 110) fluorescence. (D) Nuclear morphologic changes of Sf9 cells was detected by Hoechst 33342 staining.</figcaption></figure>

### 3.5. Gene Expression Profile of Apoptosis-Related Genes after ACA Treatment of Sf9 Cells

To investigate the effect of ACA at the transcriptional level, mRNA expression levels of nine genes that play important roles in apoptosis were measured by RT-qPCR. Of these nine genes, six showed a significant differential expression after ACA treatment (Figure 5). The expression of *caspases 1, 2, 6, cathepsin B* and *p53* was significantly up-regulated compared to the DMSO-treated group following exposure to 1.17 µM ACA for 24 h (Figure 5A,B,D,E,H), while *DnaJ1* was significantly downregulated (Figure 5G). These results indicate that ACA was involved in apoptosis of Sf9 cells at the transcriptional level.
<figure><img src="image_31.png" /><figcaption>**Figure 5.** Effects of 1'S-1'-acetoxychavicol acetate on gene expression of Sf9 cells at 24 h after treatment; (A) Caspase-1 gene; (B) caspase-2; (C) caspase-5; (D) caspase-6; (E) cathepsin B; (F) cathepsin L; (G) DnaJ1 gene; (H) p53; (I) TCTP gene. Gene expression levels were normalized to the three reference genes; glucose 6-phosphate dehydrogenase (G6PD), ribosomal protein (L18) and ribosomal protein (RpL4). All data were expressed as mean ± SE of three independent experiments. A t-test was performed to determine significance of the results compared with the control group (DMSO-treated cells).</figcaption></figure>

**4. Discussion**

Plant secondary metabolites with insecticidal properties have long been of interest as alternatives to synthetic insecticides for insect pest management [7]. Many plant secondary metabolites appear highly effective with multiple mechanisms of action while having low toxicity to non-target organisms [22,23]. So far, *A. galanga*, a plant belonging to the Zingiberaceae family, has been known for its wide range of medicinal properties [8] but little is known regarding its potential use as source of insecticide molecules. In our study, a major constituent, the 1'S-1'-acetoxychavicol acetate (ACA), was extracted from *A. galanga* and its insecticidal potency was evaluated on an insect pest, *S. frugiperda*.

The sustainable development of agriculture requires insecticides that are effective in insect pest management and less toxic to non-target organisms and humans [24]. ACA toxicity was evaluated in parallel on insect cells (Sf9) and mammalian cells (HepG2). HepG2 are duman hepatocellular carcinoma cells that have retained many activities found in normal hepatocytes [25,26]. Hepatocytes are increasingly being used as a cellular model for assessing the toxic potential of substances, as the liver is the principal organ associated with metabolic processes and xenobiotic toxicity [27]. Many insecticides have been tested on HepG2 cells to assess their effects on non-target organisms [28,29]. In our study, ACA was significantly less toxic to HepG2 cells than to Sf9 cells, consistent with what is expected in terms of selectivity for an insecticide molecule.

The ACA effects on the third-instar larvae of *S. frugiperda* showed an inhibition of growth and phenotypic abnormalities, such as a shrunk body, wrinkled body, deformed body and the remaining of parts of the old cuticle in the ACA-treated region. These effects are similar to those observed with insect growth disruptors, such as juvenile hormone analogues, inhibitors of chitin synthesis and ecdysone agonists. These chemicals interfere with the molecular, biochemical and physiological processes
associated with insect growth and development [30–32]. However, similar mechanisms of inhibition of insect growth have also been observed in insects exposed to plant secondary metabolites. Indeed, azadirachtin is a tetranortriterpenoid limonoid isolated from *A. indica* and it could regulate growth and cocooning in *Bombyx mori* L. by inducing apoptosis in the prothoracic gland [33]. Furthermore, Shu et al. [34] reported that azadirachtin inhibited the larval growth of *S. litura* by inducing apoptosis in the midgut cells (including intestinal wall cracking, abnormal cell structure and cell death). In order to investigate the mechanisms involved in this growth inhibition, ACA effects have been evaluated on the cellular model of *S. frugiperda*, the Sf9 cells. Our results indicated that ACA had a higher toxicity than the widely used botanical insecticide azadirachtin. At sublethal concentrations, ACA caused inhibition of cell proliferation. The observed effects, including changes in cell morphology, nuclear condensation and induction of caspase-3 activity, suggested that the inhibition of cell proliferation is due to apoptosis. Similar effects of another plant compound, harmine, isolated from *Peganum harmala* were observed on Sf9 cells [35]. The apoptosis in this case had been highlighted by DNA fragmentation, cellular morphological changes, nuclear condensation, and increased caspase-3 activity [35]. Apoptosis is a naturally occurring cell death process responsible for the elimination of damaged or unwanted cells to maintain homeostasis and normal development in multicellular organisms in response to stimuli such as heat, hormones, hypoxia, radiation, virus and chemical agents [36]. Apoptosis is regulated by multiple proteins, such as caspases, inhibitors of apoptosis proteins (IAP) (including IAP, IAP-2, Survivin and Survivin-1), IAP antagonist proteins (including IBM1 and Grim-19), anti-apoptotic proteins (including DnaJ1 and TCTP), pro-apoptotic proteins (including Cathepsin B and Cathepsin L), p53 protein, etc. [37]. Among these proteins, caspases (Cysteinyl aspartate specific proteinase) form a family of cysteine proteases playing important roles in programmed cell death and can be classified, according to their biological functions, into two groups—initiator and effector caspases [38]. Once activated, initiator caspases initiate the apoptosis processes and effector caspases cleave various cellular substrates, ultimately leading to apoptosis [39]. Six caspases of lepidopteran insects have been classified based on phylogenetic analyses (caspase-1 to -6) [40]. Among them, caspase-3 is known as an effector caspase in apoptosis because of its pivotal roles, such as catalyzing the cleavage of various cellular proteins during apoptosis, coordinating the dismantling of cellular structures, chromatin condensation, DNA fragmentation, cellular morphological changes and the formation of apoptotic bodies [36]. The differential expression of caspases after exposure to chemicals or pathogen infection illustrated their various responses to external stimuli [41]. Moreover, the apoptosis, process also occurs via caspase-independent apoptotic pathways. For example, azadirachtin can induce mitochondria-mediated apoptosis by causing the opening of mitochondrial permeability transition pores and loss of mitochondrial membrane potential, ultimately leading to the release of cytochrome-c in the cytoplasm and activating caspases in the Sf9 cells [42]. The lysosomal signaling pathway also plays a role in the process of apoptosis. It was shown that azadirachtin induced lysosomal membrane permeabilization, cathepsin L releasing to cytosol and activation of caspase-3 in Sf9 cells [43]. The induced apoptosis in insect cells will help to elucidate the inhibition mechanism of cellular proliferation that could also inhibit the growth of insect pests by preventing metamorphosis [44]. Our results were in favor of caspase-dependent apoptosis with, in particular, the increased caspase-3 activity and the over-expression of p53 and several caspases at the transcriptomic level. The regulation of caspase expression has been shown to be different depending on the chemical compounds that induce apoptosis [41]. In addition, the overexpression of cathepsin B also suggested that lysosomal apoptotic pathways are activated. One gene, DnaJ1, was down-regulated after ACA exposure. DnaJ homolog subfamily A member1 (DnaJ1) belongs to the heat shock 40 kDa protein family, known to play an important role in neuroprotection and hematopoiesis of *Drosophila melanogaster* Meigen [45,46]. Recently, Shu and colleagues [47] discovered a new role for DnaJ1 as an anti-apoptotic protein. In their study, the level of DnaJ1 expression was down-regulated in Sf9 cells undergoing apoptosis following treatment with azadirachtin. When DnaJ1 is knocked out, induced apoptosis is increased. A similar
decrease in the mRNA expression of DnaJ1 was observed in our study following exposure to ACA,
confirming that ACA induces apoptosis in Sf9 cells.

The present study revealed that ACA from *A. galanga* exhibited an insecticidal activity against *S. frugiperda*. However, to achieve a more efficient integrated pest management, further studies are required to determine the efficacy and stability of ACA in field trails and its effects on natural enemies.

**5. Conclusions**

Fall armywormhas developed resistance to many insecticide molecules. Its invasiveness makes it a major pest worldwide. The invasive populations already present resistance [48], so there is a real need to find new molecules, and ACA could potentially be one of them. It responds to the renewed interest in natural molecules that are not harmful to humans and the environment. This first study on the insecticidal activity of ACA under laboratory conditions is a necessary step for a future optimization and before authorization of its usage in agriculture.

**Author Contributions:** Conceptualization, G.L.G., T.R., and G.d.S.; methodology, G.L.G., G.d.S., A.P., W.P., T.R., and H.-I.H.; validation, G.L.G., T.R., G.d.S., W.P., and V.B.; formal analysis, T.R.; investigation, T.R. and A.P.; resources, G.L.G., G.d.S., and A.P.; writing—original draft preparation, T.R.; writing—review and editing, G.L.G.; supervision, G.L.G.; funding acquisition, G.L.G. and V.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Thailand Research Fund (Grant No. PHD/0043/2558), the French Government's Contribution from the Embassy of France and PHC Siam Mobility Grant (Grant No. 42852PL) and by the French Ministries of Europe and Foreign Affairs (MEAE) and of Higher Education, Research and Innovation (MESRI), as well as from the Office of the Commission for Higher Education of the Thai Ministry of Education.

**Acknowledgments:** The authors thank Julie Cazareth from Institut de Pharmacologie Moleculaire et Cellulaire, Sophia Antipolis, France, for technical assistance on flow cytometry and Dries Amezian for his assistance on molecular biology technique. We would like to thank Emmanuelle d'Alençon and Magali Eychenne from DGIMI, Université de Montpellier, INRAE, Montpellier, France for supplying *S. frugiperda* populations.

**Conflicts of Interest:** The authors declare no conflict of interest.

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