Received: 20 March 2019 | Revised: 14 August 2019 | Accepted: 2 September 2019

DOI: 10.1002/arch.21621

MICRO‐REVIEW

Genetic control of Plutella xylostella in omics era

Wei Chen1,2 | Feiying Yang1,2 | Xuejiao Xu1,2 | Uttam Kumar1,2 |

Weiyi He1,2 | Minsheng You1,2

1
State Key Laboratory for Ecological Pest
Control of Fujian and Taiwan Crops, Institute Abstract
of Applied Ecology, College of Plant Diamondback moth, Plutella xylostella (L.), is a specialist pest
Protection, Fujian Agriculture and Forestry
University, Fuzhou, China on cruciferous crops of economic importance. The large‐
2
International Joint Research Laboratory of scale use of chemical insecticides for the control of this
Ecological Pest Control, College of Plant
insect pest has caused a number of challenges to agro‐
Protection, Fujian Agriculture and Forestry
University, Fuzhou, China ecosystems. With the advent of the omics era, genetic pest
management strategies are becoming increasingly feasible
Correspondence
Weiyi He and Minsheng You, State Key and show a powerful potential for pest control. Here, we
Laboratory for Ecological Pest Control of
review strategies for using transgenic plants and sterile
Fujian and Taiwan Crops, Institute of Applied
Ecology, Fujian Agriculture and Forestry insect techniques for genetic pest management and intro-
University, 350002 Fuzhou, China.
duce the major advances in the control of P. xylostella using a
Email: wy.he@fafu.edu.cn (W. H.) and
msyou@fafu.edu.cn (M. Y.) female‐specific RIDL (release of insects carrying a dominant
lethal gene) strategy. Further, the advantages of gene drive
Funding information
National Key R&D Program of China, Grant/ developed in combination with sex determination and
Award Number: 2017YFD0200400; Natural
CRISPR/Cas9 systems are addressed, and the corresponding
Science Foundation of Fujian Province, Grant/
Award Number: 2019J01369; Innovation Fund prospects and implementation issues are discussed. It is
of FAFU, Grant/Award Numbers:
predictable that under the policy and regulation of profes-
CXZX2016128, CXZX2017321, CXZX2017471,
CXZX2018092; Special Key Project of Fujian sional committees, the genetic pest control strategy,
Province, Grant/Award Number:
especially for gene drive, will open a new avenue to
2018NZ01010013
sustainable pest management not only for P. xylostella but
also for other insect pests.

KEYWORDS
CRISPR/Cas9, diamondback moth, genetic pest management,
genome editing, omics

1 | INTRODUCTION

Diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), is a notorious pest of cruciferous crops
worldwide and has caused billions of U.S. dollar loss and cost in management per annum (Zalucki et al., 2012). The
control of P. xylostella relies heavily on the use of insecticides (Li, Feng, Liu, You, & Furlong, 2016). However,

Arch. Insect Biochem. Physiol. 2019;102:e21621. wileyonlinelibrary.com/journal/arch © 2019 Wiley Periodicals, Inc. | 1 of 6
https://doi.org/10.1002/arch.21621
2 of 6 | CHEN ET AL.

overreliance on chemicals has resulted in increased insecticide resistance, decreased natural enemies, and severe
environmental pollution (Barriuso & Koskinen, 1996; Liu, Chung, & Xiong, 2001). Integrated pest management
(IPM), including biological control, habitat management and integration of pest, and natural enemy population
dynamics, has been indicated proven to be environmentally friendly and to provide sustainable control of
P. xylostella (Shakeel et al., 2017). However, these strategies are highly dependent on the understanding of natural
ecosystems and vary with climatic and environmental conditions (Gurr et al., 2018; Sarfraz, Keddie, & Dosdall,
2005). With the advent of genomics and associated “omics” tools, genetic control of P. xylostella becomes possible.
The release of the whole‐genome sequence of P. xylostella and its derived transcriptome databases (Jouraku et al.,
2013; Tang et al., 2014; You et al., 2013) provides valuable genetic information and promotes a variety of novel
applicable techniques, such as transgenic plants, conditional lethal transgenic insects, and gene drive for the
management of P. xylostella. The main progress is reviewed as follows.

1.1 | Transgenic plants

Transgenic plants overexpressing genes encoding protease inhibitors (Qiu et al., 2013) or protein toxins from
Bacillus thuringiensis (Wang, Zhang, Wang, Liu, & Liu, 2014) and nuclear‐ or chloroplast‐derived double‐stranded
RNA (dsRNA) targeting developmentally key genes (Mao et al., 2007; Zhang et al., 2015) exert negative effects on
the biological fitness of target pests. A transgenic broccoli cultivar expressing a cry1Aa gene of B. thuringiensis
caused 100% mortality of P. xylostella after a three‐day treatment, which indicated a great potential of practical
utility in pest management (Kumar et al., 2018). Similarly, the expression of long dsRNAs in plastids of potato plants
against the β‐actin gene of Leptinotarsa decemlineata resulted in the repelling of herbivores and mortality of the
larvae (Zhang et al., 2015). However, the increase in insect resistance to B. thuringiensis toxins and the low stability
and efficiency of dsRNAs restrict the large‐scale use of transgenic plants for P. xylostella control.

1.2 | Sterile insect technique (SIT)

The SIT involves mass rearing, sterilization, and release of males that compete to mate with wild females, and,
consequently, fail to produce progeny. This approach was the first genetic pest management strategy for insect pest
control and has been highly successfully applied for the control of the New World screwworm, Cochliomyia hominivorax, a
major pest of livestock (L. Alphey, 2016; Concha et al., 2016). For the lepidopteran insects, it was found that high doses of
60
Co‐γ radiation treatments altered the testis development of P. xylostella (X. Li et al., 2019). No successful application in
the field was reported in P. xylostella because of the lack of a quick and reliable way to separate sexes before release and
insensitivity to radiation, making it often costly, labor‐consuming, and inefficient (Furlong, Wright, & Dosdall, 2013).

1.3 | Release of insects carrying dominant lethal genes

A management strategy of conditional lethal transgenes, named RIDL (release of insects carrying a dominant lethal gene),
also requires mass‐rearing and releasing processes and renders progeny nonviable in the field. This has been carried out
for many insects (Black, Alphey, & James, 2011; Thomas, Donnelly, Wood, & Alphey, 2000), especially invasive pests
(Harvey‐Samuel, Ant, & Alphey, 2017). The first step is to establish a line integrated with an expression cassette containing
a lethal gene (tTAV, etc.) under the control of the “Tet‐off” system (Martins et al., 2012). In this system, insects can only
survive on the diet containing antidote (tetracycline or suitable analogues), which is able to suppress the expression of the
lethal gene, whereas they die in the wild where no antidote was provided. To improve the efficiency of RIDL in insect
pests, such as pink bollworm, Pectinophora gossypiella, gender‐specific splicing variants of the sex determination gene
doublesex (dsx) were used to drive the expression of the lethal gene in females, ultimately achieving male‐only releasing
called female‐specific RIDL strategy (fsRIDL; Jin et al., 2013). To date, the self‐limitation strategy of fsRIDL has
been successfully applied in netted caged P. xylostella, leading to rapid population decline and ultimate elimination
CHEN ET AL. | 3 of 6

(Harvey‐Samuel, Ant, Gong, Morrison, & Alphey, 2014). By the introduction fsRIDL adults of P. xylostella at a glasshouse,
it was found that the OX4319 strain of P. xylostella became dominant after competition with the natural population
(Harvey‐Samuel et al., 2015). Moreover, the small‐scale field experiment evaluating the performance of the OX4319 strain
is ongoing (http://shelton.entomology.cornell.edu/2017/07/13/2015‐diamondback‐moth‐field‐cage‐trials/).

1.4 | CRISPR/Cas9‐based gene drive

On the basis of the CRISPR/Cas9 system, the self‐sustaining method (or gene drive strategy) shows great potential
for pest management. This method requires much less initial release of gene‐modified insects than the fsRIDL
method (L. Alphey, 2014). In the scenario of the original global gene drive system, the expression cassette
containing a single‐guide RNA (sgRNA) and Cas9 gene is first inserted into one allele of a target gene. Then, this
cassette drives itself to cut and insert into the opposing locus, converting heterozygotes to homozygotes via a
homology‐directed DNA repair pathway (or homing; Gantz et al., 2015). Theoretically, the release of transgenic
insect lines carrying a gene drive system can result in a sharp decline in wild populations.
To improve the control efficiency of a global gene drive by releasing the male individuals, the sex‐determination
system can be applied. If the target gene is essential for female viability, the disruption of gene function caused by
insertion will lead to the male‐only generation and a further population crash. In a study of Anopheles gambiae,
based on the CRISPR/Cas9 gene drive system, three genes that confer a recessive female‐sterility phenotype were
utilized, and stable populations with transmission rates to progeny ranging from 91.4% to 99.6% were successfully
established (Hammond et al., 2016). Similarly, a caged A. gambiae population was suppressed by targeting the
doublesex gene (Kyrou et al., 2018). In addition, the technique of homing endonuclease genes (such as Y‐linked
X‐shredder), which can cut a specific nucleotide sequence on the X chromosome and result in the male‐biased
progeny by the elimination of X‐bearing sperm, has been successfully applied in A. gambiae (Galizi et al., 2014).
However, the sex determination is still a “black box” in lepidopteran insects, except that the sex determination gene
Masculinizer in Bombyx mori was recently reported (Kiuchi et al., 2014). Therefore, the available omics data of
P. xylostella of sex determination are the fundamental supports for gene drive (Figure 1).
Our team has successfully established a CRISPR/Cas9‐based genome‐editing system and identified RNA polymerase
type III (Pol‐III) promoters for expressing sgRNA in P. xylostella (Huang et al., 2016, 2017), though no case of gene drive
strategy has been reported in P. xylostella. Nevertheless, the potential extinction of species (impacts on food webs or
increased abundance of secondary pests in crops) and related ecologic risks (spread of gene drive systems into related
taxa via hybridization) of global gene drive have raised widespread attention due to the concern of “virus‐like” spread of
expression cassettes without limitation in wild populations (N. Alphey & Bonsall, 2018; Harvey‐Samuel et al., 2017). In
addition, the resistance to sgRNA caused by mutation at the target site is also worthy of concern. To solve this problem,
improved strategies such as daisy drive, daisy field drive or daisy quorum drive combined with an underdominance
effect have also been proposed and applied in other pests (Deredec, Burt, & Godfray, 2008; Reeves, Bryk, Altrock,
Denton, & Reed, 2014). This system involves the separate integration of expression cassettes into different
chromosomes of the host genome, resulting in the limited spread of expression cassettes in the wild population.

2 | CONC LU DIN G REMARKS

Omics‐based data have played an important role in promoting the development of a variety of novel applicable
techniques for the control of the destructive pest P. xylostella. The latest CRISPR/Cas9‐based gene drive strategy
has shown potential for pest control with high efficiency and low requirement of the initial release population.
Although some potential risks caused by the release of transgenic pests in the sociocultural, politicoeconomic, and
ecological dimensions are emerging, the in‐depth digging of omics‐based data and the establishment of professional
committees from scientists, the public and the government would help solve these issues to some extent.
4 of 6 | CHEN ET AL.

F I G U R E 1 Genetic control of Plutella xylostella in omics era. (a) The integration of omics‐based data enables a
quick identification for candidate genes involved in the molecular mechanism of sex determination and provides
ideal targets, like maleness or doublesex (dsx) genes, for CRISPR/Cas9‐based gene drive. (b) Schematic diagrams of
gene drive strategy integrating with maleness genes. The expression cassette consisting of single‐guide RNA
(sgRNA), Cas9, and maleness gene is first inserted into one allele of a target site. The complex of sgRNA and Cas9
protein will cleavage the opposing locus. The homing process occurs through homology‐directed DNA repair
pathway. The ectopic expression of maleness gene results in male‐only offspring. (c) Schematic diagrams of gene
drive strategy based on sex determination gene doublesex (dsx). The expression cassette consisting of sgRNA and
Cas9 is inserted into the intron–exon boundary of dsx, which causes the disruption of a female‐specific isoform of
dsx whereas a functional male‐specific isoform due to a different splicing way. In this system, theoretically, the
population can only produce male offspring as well

Compared with the successful application of releasing the transgenic line of fsRIDL in P. xylostella to the wild
population, CRISPR/Cas9‐based gene drive will be a candidate of the next‐generation strategy that is effective and
promising not only in the management of P. xylostella but also in the management of other insect pests.
Further interests would be focused as follows:

1. The omics‐based understanding of the molecular mechanism of sex determination will provide ideal targets for
CRISPR/Cas9‐based gene drive strategies.
2. A high‐quality genome map is fundamental to improving the precision of the CRISPR/Cas9 system by avoiding
the potential off‐target effect in gene drive systems.
3. For the application of genetic pest control technology, a combination of multidisciplinary or interdisciplinary
approaches, especially those involving the concept of integrated pest management, is necessary for making a
release decision.
4. The positive cooperation and establishment of professional committees among local or worldwide scientists, the
public and the government will enhance the safety and reliability of genetic pest control technology.

A C K N O W L E D GM E N T S

The work was supported by National Key R&D Program of China (2017YFD0200400), Special Key Project of Fujian
Province (2018NZ01010013), Natural Science Foundation of Fujian Province (2019J01369) in China, and
Innovation Fund of FAFU (CXZX2016128, CXZX2017321, CXZX2017471 and CXZX2018092).
CHEN ET AL. | 5 of 6

OR CID

Minsheng You http://orcid.org/0000-0001-9042-6432

REFERENC ES

Alphey, L. (2014). Genetic control of mosquitoes. Annual Review of Entomology, 59, 205–224.
Alphey, L. (2016). SIT 2.0: 21st‐century genetic technology for the screwworm sterile‐insect program. BMC Biology, 14(1),
80.
Alphey, N., & Bonsall, M. B. (2018). Genetics‐based methods for agricultural insect pest management. Agricultural and Forest
Entomology, 20(2), 131–140.
Barriuso, E., & Koskinen, W. C. (1996). Incorporating nonextractable atrazine residues into soil size fractions as a function
of time. Soil Science Society of America Journal, 60(1), 150–157.
Black, W. C., Alphey, L., & James, A. A. (2011). Why RIDL is not SIT. Trends in Parasitology, 27(8), 362–370.
Concha, C., Palavesam, A., Guerrero, F. D., Sagel, A., Li, F., Osborne, J. A., … Scott, M. J. (2016). A transgenic male‐only
strain of the New World screwworm for an improved control program using the sterile insect technique. BMC Biology,
14(1), 72.
Deredec, A., Burt, A., & Godfray, H. C. J. (2008). The population genetics of using homing endonuclease genes in vector and
pest management. Genetics, 179(4), 2013–2026.
Furlong, M. J., Wright, D. J., & Dosdall, L. M. (2013). Diamondback moth ecology and management: Problems, progress, and
prospects. Annual Review of Entomology, 58, 517–541.
Galizi, R., Doyle, L. A., Menichelli, M., Bernardini, F., Deredec, A., Burt, A., … Crisanti, A. (2014). A synthetic sex ratio
distortion system for the control of the human malaria mosquito. Nature Communications, 5, 3977.
Gantz, V. M., Jasinskiene, N., Tatarenkova, O., Fazekas, A., Macias, V. M., Bier, E., & James, A. A. (2015). Highly efficient
Cas9‐mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proceedings
of the National Academy of Sciences of the United States of America, 112(49), E6736–E6743.
Gurr, G. M., Reynolds, O. L., Johnson, A. C., Desneux, N., Zalucki, M. P., Furlong, M. J., … You, M. (2018). Landscape ecology
and expanding range of biocontrol agent taxa enhance prospects for diamondback moth management. A review.
Agronomy for Sustainable Development, 38(3), 23.
Hammond, A., Galizi, R., Kyrou, K., Simoni, A., Siniscalchi, C., Katsanos, D., … Nolan, T. (2016). A CRISPR‐Cas9 gene drive
system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nature Biotechnology, 34(1),
78–83.
Harvey‐Samuel, T., Ant, T., & Alphey, L. (2017). Towards the genetic control of invasive species. Biological Invasions, 19(6),
1683–1703.
Harvey‐Samuel, T., Ant, T., Gong, H., Morrison, N. I., & Alphey, L. (2014). Population‐level effects of fitness costs associated
with repressible female‐lethal transgene insertions in two pest insects. Evolutionary Applications, 7(5), 597–606.
Harvey‐Samuel, T., Morrison, N. I., Walker, A. S., Marubbi, T., Yao, J., Collins, H. L., … Alphey, L. (2015). Pest control and
resistance management through release of insects carrying a male‐selecting transgene. BMC Biology, 13(1), 49.
Huang, Y., Chen, Y., Zeng, B., Wang, Y., James, A. A., Gurr, G. M., … You, M. (2016). CRISPR/Cas9 mediated knockout of the
abdominal‐A homeotic gene in the global pest, diamondback moth (Plutella xylostella). Insect Biochemistry and Molecular
Biology, 75, 98–106.
Huang, Y., Wang, Y., Zeng, B., Liu, Z., Xu, X., Meng, Q., … You, M. (2017). Functional characterization of Pol III U6 promoters
for gene knockdown and knockout in Plutella xylostella. Insect Biochemistry and Molecular Biology, 89, 71–78.
Jin, L., Walker, A. S., Fu, G., Harvey‐Samuel, T., Dafa’alla, T., Miles, A., … Alphey, L. (2013). Engineered female‐specific
lethality for control of pest Lepidoptera. ACS Synthetic Biology, 2(3), 160–166.
Jouraku, A., Yamamoto, K., Kuwazaki, S., Urio, M., Suetsugu, Y., Narukawa, J., … Noda, H. (2013). KONAGAbase: A genomic
and transcriptomic database for the diamondback moth, Plutella xylostella. BMC Genomics, 14(1), 464.
Kiuchi, T., Koga, H., Kawamoto, M., Shoji, K., Sakai, H., Arai, Y., … Katsuma, S. (2014). A single female‐specific piRNA is the
primary determiner of sex in the silkworm. Nature, 509(7502), 633–636.
Kumar, P., Gambhir, G., Gaur, A., Sharma, K. C., Thakur, A. K., & Srivastava, D. K (2018). Development of transgenic broccoli
with cryIAa gene for resistance against diamondback moth (Plutella xylostella). 3 Biotech, 8(7), 299.
Kyrou, K., Hammond, A. M., Galizi, R., Kranjc, N., Burt, A., Beaghton, A. K., … Crisanti, A. (2018). A CRISPR–Cas9 gene drive
targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Nature
Biotechnology, 36(11), 1062–1066.
Li, X., Zhang, K., Deng, Y., He, R., Zhang, X., Zhong, G., … Weng, Q. (2019). Effects of 60Co‐γ radiation on testis physiological
aspects of Plutella xylostella (Linnaeus). Ecotoxicology and Environmental Safety, 169, 937–943.
6 of 6 | CHEN ET AL.

Li, Z., Feng, X., Liu, S. S., You, M., & Furlong, M. J. (2016). Biology, ecology, and management of the diamondback moth in
China. Annual Review of Entomology, 61, 277–296.
Liu, Y. H., Chung, Y. C., & Xiong, Y. (2001). Purification and characterization of a dimethoate‐degrading enzyme
of Aspergillus niger ZHY256, isolated from sewage. Applied and Environmental Microbiology, 67(8), 3746–3749.
Mao, Y. B., Cai, W. J., Wang, J. W., Hong, G. J., Tao, X. Y., Wang, L. J., … Chen, X. Y. (2007). Silencing a cotton bollworm P450
monooxygenase gene by plant‐mediated RNAi impairs larval tolerance of gossypol. Nature Biotechnology, 25(11),
1307–1313.
Martins, S., Naish, N., Walker, A. S., Morrison, N. I., Scaife, S., Fu, G., … Alphey, L. (2012). Germline transformation of the
diamondback moth, Plutella xylostella L., using the piggyBac transposable element. Insect Molecular Biology, 21(4),
414–421.
Qiu, L., Wu, T., Dong, H., Wu, L., Cao, J., & Huang, L. (2013). High‐level expression of sporamin in transgenic Chinese
cabbage enhances resistance against diamondback moth. Plant Molecular Biology Reporter, 31(3), 657–664.
Reeves, R. G., Bryk, J., Altrock, P. M., Denton, J. A., & Reed, F. A. (2014). First steps towards underdominant genetic
transformation of insect populations. PLoS One, 9(5), e97557.
Sarfraz, M., Keddie, A. B., & Dosdall, L. M. (2005). Biological control of the diamondback moth, Plutella xylostella: A review.
Biocontrol Science and Technology, 15(8), 763–789.
Shakeel, M., Farooq, M., Nasim, W., Akram, W., Khan, F. Z. A., Jaleel, W., … Jin, F. (2017). Environment polluting
conventional chemical control compared to an environmentally friendly IPM approach for control of diamondback
moth, Plutella xylostella (L.), in China: A review. Environmental Science and Pollution Research, 24(17), 14537–14550.
Tang, W., Yu, L., He, W., Yang, G., Ke, F., Baxter, S. W., … You, M. (2014). DBM‐DB: The diamondback moth genome
database. Database, 2014, bat087.
Thomas, D. D., Donnelly, C. A., Wood, R. J., & Alphey, L. S. (2000). Insect population control using a dominant, repressible,
lethal genetic system. Science, 287(5462), 2474–2476.
Wang, Y., Zhang, Y., Wang, F., Liu, C., & Liu, K. D. (2014). Development of transgenic Brassica napus with an optimized
cry1C* gene for resistance to diamondback moth (Plutella xylostella). Canadian Journal of Plant Science, 94(8),
1501–1506.
You, M., Yue, Z., He, W., Yang, X., Yang, G., Xie, M., … Wang, J. (2013). A heterozygous moth genome provides insights into
herbivory and detoxification. Nature Genetics, 45(2), 220–225.
Zalucki, M. P., Shabbir, A., Silva, R., Adamson, D., Shu‐Sheng, L., & Furlong, M. J. (2012). Estimating the economic cost of one
of the world's major insect pests, Plutella xylostella (Lepidoptera: Plutellidae): Just how long is a piece of string? Journal
of Economic Entomology, 105(4), 1115–1129.
Zhang, J., Khan, S. A., Hasse, C., Ruf, S., Heckel, D. G., & Bock, R. (2015). Full crop protection from an insect pest by
expression of long double‐stranded RNAs in plastids. Science, 347(6225), 991–994.

SU P P ORT IN G INF O RM A TIO N

Additional supporting information may be found online in the Supporting Information section.

How to cite this article: Chen W, Yang F, Xu X, Kumar U, He W, You M. Genetic control of Plutella xylostella
in omics era. Arch. Insect Biochem. Physiol. 2019;102:e21621. https://doi.org/10.1002/arch.21621