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An update of a green pesticide: Metarhizium

anisopliae

Zhe-Yu Peng, Shu-Ting Huang, Jia-Ting Chen, Ni Li, Yong Wei, Asad Nawaz &
Sheng-Qun Deng

To cite this article: Zhe-Yu Peng, Shu-Ting Huang, Jia-Ting Chen, Ni Li, Yong Wei, Asad Nawaz &
Sheng-Qun Deng (2022) An update of a green pesticide: Metarhizium anisopliae, All Life, 15:1,
1141-1159, DOI: 10.1080/26895293.2022.2147224

To link to this article: https://doi.org/10.1080/26895293.2022.2147224

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ALL LIFE
2022, VOL. 15, NO. 1, 1141–1159
https://doi.org/10.1080/26895293.2022.2147224

REVIEW

An update of a green pesticide: Metarhizium anisopliae

Zhe-Yu Penga† , Shu-Ting Huangb† , Jia-Ting Chenb , Ni Lia , Yong Weib , Asad Nawaza and Sheng-Qun Denga
a Department of Pathogen Biology, the Key Laboratory of Microbiology and Parasitology of Anhui Province, the Key Laboratory of Zoonoses of

High Institutions in Anhui, School of Basic Medical Sciences, Anhui Medical University, Hefei, People’s Republic of China; b Department of
Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University,
Guangzhou, People’s Republic of China

ABSTRACT ARTICLE HISTORY

Metarhizium anisopliae (Hypocreales: Clavicipitaceae, M. anisopliae), as an invertebrate fungal Received 17 December 2021
pathogen, has played a significant role in the control of many agricultural pests and human disease Accepted 25 September 2022
vectors. M. anisopliae is typically used as a chemical in dry or liquid formulations of large numbers KEYWORDS
of aerial conidia. The conidia can directly infect arthropod pests by penetrating their cuticular layer. Metarhizium anisopliae;
The availability of the complete genome of M. anisopliae and capable techniques for its transfor- pesticide; pathogenesis;
mation have brought several developments in its use as a pest controller. For prospects, more in- toxin; formulation; virulence
depth research to improve fermentation and formulation technologies is needed, while multistress-
tolerant and engineering entomopathogenic strains are selected to promote the widespread accep-
tance and usefulness of M. anisopliae as a cost-effective fungal biopesticide. Here, we provide an
overview of the pathogenesis and formulation strategies, as well as the host ranges and molecular
approaches of increasing virulence and efficacy. Several studies of this fungus pathogen demon-
strated that M. anisopliae can be an efficient biocontrol agent and has great potential for further
research and development.

1. Background
Agricultural pests, such as ticks, termites, grasshop-
pers, and locusts, have caused great economic losses
in many countries and regions in the world (Nour-
risson et al. 2017; Savary et al. 2019). Furthermore,
human disease vectors, such as mosquitos, are respon-
sible for about one million deaths annually (World
Health Organization 2017). To date, chemical insec-
ticides are the most frequently and widely used to
control agricultural pests and human disease vectors
(Gupta et al. 2019). However, the quantity and fre-
quency of chemical pesticide spraying have increased
annually, which has resulted in increased resistance
among pests (Damalas and Koutroubas 2018). The
large-scale use of chemical pesticides also pollutes the
environment, including soil and water, and poses great
risks to humans and animals (Deng et al. 2019a).
An eco-friendly alternative to chemical pesticides is
biopesticide, which has the characteristics of a wide
range of sources, great potential for improvement, low
drug resistance, and lower nontarget toxicity, includ-
ing lack of mammalian toxicity (Walia et al. 2017).
Entomopathogenic fungi are biological agents that do
not produce harmful effects on the environment; thus,
they could be called ‘green pesticides’ (Sandhu et al.
2017; Lovett et al. 2019a). Their high efficiency of
killing hosts and great biodiversity represent many
possibilities for biological control agents, which can
help reduce the use of chemicals and improve sustain-
able development (Damalas and Koutroubas 2018).
Metarhizium anisopliae (M. anisopliae) is a com-
mon insect pathogen, belonging to Fungi, Ascomy-
cota, Sordariomycetes, Hypocreales, Clavicipitaceae,
Metarhizium (Lovett et al. 2019a). Recently, M. aniso-
pliae has been assigned to M. anisopliae. It is widely
distributed in the soil of plant rhizosphere or arthro-
pod carcass as a saprophyte (Rehner and Kepler 2017).
M. anisopliae can infect many important crop pests
but also human disease vectors (Lovett et al. 2019a).
Moreover, this fungus is considered asexual because

CONTACT Sheng-Qun Deng dengshengqun@163.com Department of Pathogen Biology, the Key Laboratory of Microbiology and Parasitology of
Anhui Province, the Key Laboratory of Zoonoses of High Institutions in Anhui, School of Basic Medical Sciences, Anhui Medical University, Hefei, People’s Republic
of China
† Zhe-Yu Peng and Shu-Ting Huang contributed equally to this work.
This article has been corrected with minor changes. These changes do not impact the academic content of the article.
© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
1142 Z.-Y. PENG ET AL.

no teleomorph has been observed, and conidia are its 3. Pathogenesis of M. anisopliae
form of infection (Lovett et al. 2019a). The infection
M. anisopliae infects susceptible hosts by penetrating
process of M. anisopliae includes adhesion, spore ger-
the cuticle directly (Freimoser et al. 2005). For ease
mination, appressorium formation, host penetration,
of description, the infection process can be divided
in vivo development, and host death (Gao et al. 2011).
into: (1) conidia adherence to the host cuticle; (2) coni-
However, compared to chemical pesticides, M. aniso-
dia germination and development; (3) appressorium
pliae has a slower killing speed, which takes approx-
formation; (4) cuticle penetration; (5) colonization of
imately five days under ideal conditions (Fang et al.
hemolymph; (6) extrusion and sporulation (Figure 1)
2007). Furthermore, pest control efficiency is easily
(Gao et al. 2011). Metarhizium spp. has species-specific
affected by environmental conditions, such as humid-
genes in its infection pattern, such as the M. anisopliae
ity, temperature, and ultraviolet radiation (Gašić and
adhesin-like protein 1 (Mad1) and high osmolarity
Tanović 2013). With a better understanding of the
glycerol (Hog) pathway Hog1 kinase genes during ger-
processes and molecular mechanisms of host infec-
mination (San and Mun 2017). The major obstacle to
tion, the virulence of M. anisopliae can be enhanced
insect infection is the complexity of the host’s cuticle,
by genetic engineering or the development of a suit-
which consists mainly of proteins and chitin (Charnley
able formulation, which could greatly improve the
2003). The secretion of protease is considered to be an
application and efficacy of M. anisopliae (Lovett et al.
important pathogenic factor for fungi attached to the
2019b). This review mainly introduces the host infec-
cuticle layer (Butt et al. 2016).
tion pathogenesis of M. anisopliae and the application
and development of M. anisopliae in different regions
of the world. 3.1. Conidia adhesion
In the first stage of infection, conidia deposit on the
2. Historical perspective host cuticle and then adhere (San and Mun 2017;
Schrank and Vainstein 2010). There are hydropho-
The Metarhizium genus was initially composed of four
bic proteins in the outer layer of conidia, which pro-
varieties, including M. anisopliae, M. guizhouense, M.
mote the adhesion of the hydrophobic epidermis and
pingshaense, and M. taii (Bischoff et al. 2009). With the
conidia (Ment et al. 2010a; Greenfield et al. 2014).
improvement of DNA sequencing technology, more
The cell walls of conidia of M. anisopliae also con-
than 30 species of the genus Metarhizium have been
tain exogenous lectins, which combine with epider-
isolated and identified (Fernández-Bravo et al. 2021).
mal glycoprotein of the host to form a specific bond
In Russia, Elie Metchnikoff studied an insect disease
and play a role in identifying the host (Samson et al.
of wheat cockchafers, and he called the disease green
1988). Adhesion refers to hydrophobic interactions
muscardine and determined that the infecting agent
between lipid layers covering the cutin of arthropods
was M. anisopliae (Vega et al. 2009). Subsequent stud-
and spore surface proteins (such as hydrophobic pro-
ies showed that the cereal cockchafer and sugar beet
teins). Lipids in the cuticular layer are the first barrier
weevil could be killed via direct infection of M. aniso-
of the arthropod against pathogenic microorganisms
pliae conidia (Zhang et al. 2018). Since then, hundreds
and strengthen the importance of lipid enzymes in the
of fungi have been identified and developed as biocon-
early stages of infection (Schrank and Vainstein 2010).
trol agents. M. anisopliae has been extensively stud-
Moreover, lipid degradation is related to the prepene-
ied because of its environmental friendliness, safety
tration growth of M. anisopliae on the host epidermis
for humans and mammals, narrow host range, and
(Schrank and Vainstein 2010). The degradation of the
ease of mass production (Zhang et al. 2018). It has
host lipid layer is of great significance for the identifica-
been reported that M. anisopliae can infect a variety
tion of susceptible hosts and the production of the first
of arachnid and insect hosts, including agricultural
nutrient molecules that support the germination of
pests and human disease vectors (Gopal et al. 2006).
conidia (Pedrini et al. 2007). Furthermore, lipase can
As we learn more about the pathogens, their hosts
be identified on the surface of conidia (Schrank and
and their environment, microbial control has also
Vainstein 2010). The conidia of less-virulent strains
evolved.

Figure 1. Overview of the basic infection cycle depicted by M. anisopliae in invertebrates. The infection process can be divided into: (1)
conidia adherence to the host cuticle; (2) conidia germination and development; (3) appressorium formation; (4) cuticle penetration; (5)
colonization of hemolymph; (6) extrusion and sporulation.
have poor adhesion to the host epidermis (Beys-
Da-Silva et al. 2013). Adhesin protein Mad1 is of
great significance in the attachment of swelling coni-
dia to the host surface (Wang and St Leger 2007b).
It was speculated that Mad1 could replace degraded
hydrophobins upon changes in the conidia cell wall
(Wang and St Leger 2007b).
3.2. Conidia germination and development
When conidia adhere to the host cuticle, the germi-
nation of conidia is initiated, and different lengths of
germ tubes are produced (Ment et al. 2010a). Tre-
halase can be observed in the early stage of germi-
nation and it utilizes trehalose, which is common in
host hemolymph. The activity of trehalase may pro-
vide glucose for energy production (San and Mun
2017). Spores swell and produce germ tubes that dif-
ferentiate into appressorium after germination (Lovett
and St Leger 2015). The growth of the germ tube is
strongly oriented to the epidermis, and the germ tube
adheres to the upper epidermis surface through mucus
secreted by the hypha apex. Then, the germ tube grad-
ually elongates to produce infection pegs (Hui et al.
2004). Amino acids, proteins, sugars, lipids, and other
general nutrients can germinate the conidia of M.
anisopliae. Two adhesin-encoding genes, Mad1 and
Mad2, are respectively related to the pathogenesis of
insects or the colonization of plant roots (Butt et al.
2013). Mad1 protein is expressed in the early stage of
insect infection, while Mad2 protein is expressed only
ALL LIFE 1143
after fungal hyphae emerge and conidiate on the insect
cadaver. Mad1 protein is also in charge of cell division
and proper cytoskeleton organization, and the deletion
of the Mad1 gene causes a delay in the germination of
conidia (Barelli et al. 2011).
3.3. Appressorium formation
The germ tube differentiates into the appresso-
rium before penetrating the epidermis (Senthil-
Nathan 2015). Appressorium contains a large number
of mitochondria, Golgi bodies, endoplasmic reticu-
lum, and ribosomes. Through exuberant attachment
metabolism, M. anisopliae can concentrate mechanical
and dissolve energy into a small part of the host epi-
dermis, thus promoting fungal invasion. The appres-
sorium is covered with a thick mucilage layer, and the
morphology of the appressorium varies greatly, rang-
ing from elliptic to irregular, presenting a mulberry
shape due to mucous accumulation (Hui et al. 2004).
The number of appressoria and its structure are related
to the thallus itself, host, and nutritional conditions.
It has been demonstrated that the ornithine decar-
boxylase (ODC1) gene and Metarhizium perilipin-like
protein (MPL1) gene are responsible for the formation
of appressoria. The ODC1 gene is upregulated during
the germination of conidia and the differentiation of
the germ tube to form the appressorium (Wang and
St. Leger 2007c; Pulido et al. 2011). The MPL1 gene
regulates lipid homeostasis and appressorium differen-
tiation (San and Mun 2017). MPL1 is mainly expressed
1144 Z.-Y. PENG ET AL.
when the fungus is engaged in lipid accumulation
(Wang and St Leger 2007b). Fang et al. proved that
specific inhibitors of protein kinase A could delay the
expression of cuticle degrading enzymes and the for-
mation of appressorium (Fang et al. 2009). In addition,
it is reported that protein kinase A is involved in ergos-
terol biosynthesis and maintains turgor pressure in the
appressorium. This expansion pressure produces the
pressure required to penetrate the host cuticle (San and
Mun 2017).
3.4. Cuticle penetration
In the process of penetration, physical pressure and
cuticle degradation are two key factors. Besides, the
efficiency of physical pressure mainly depends on
appressorium formation, and lipid droplet formation
provides the major physical pressure. Protein, chitin,
and wax are important compositions in insect cuticles.
Protease plays an important role in the degradation
of the host epidermis, and its activity is the main fac-
tor determining the infection ability of M. anisopliae
(Fang et al. 2009). M. anisopliae produces subtilisin-
like enzyme (PR1), trypsin-like enzyme (PR2), acid
protease (PR3), chymotrypsin, and carboxypeptidase
with different catalytic properties, which digest the
arthropods’ protein-rich plasma membrane (Moraes
et al. 2003). The types and amount of protein pro-
duced by M. anisopliae are unique to each host, which
can explain its ability to infect many different hosts
(San and Mun 2017). Trypsins are only produced
in specific hosts, such as cockroaches and beetles
(Santi et al. 2010a). Chitinase activity is regulated by
chitin degradation products, that is, induction inhi-
bition mechanism. Pretreatment of insect epidermis
with PR1, which is related to the pathogenesis of M.
anisopliae, can enhance the subsequent chitinase activ-
ity (Lin et al. 2011). Different chitinase subtypes are
produced in different hosts (Senthil-Nathan 2015).
P450 monoxygenase is a key factor for cuticle pen-
etration. The importance of monoterminal oxidation
to M. anisopliae is due to the alkyl branch at the β
position causing steric inhibition of terminal oxidiz-
ing enzymes. The single cytochrome P450 monoxy-
genase family 52 (MrCYP52) is required by M. aniso-
pliae for rapid hydrolysis of alkanes and that this
provides nutrition for germination and formation of
infection structures (St. Leger et al. 1986). In addi-
tion, besides destroying host antifungal proteins that
block pathogen invasion, release amino acids, and gen-
erate amines to regulate pH, epidermal degradation
enzymes can also use host proteins as nutrients (Hui
et al. 2004). The optimal pH values of PR1 and PR2
are approximately 8, which are generated only under
alkaline conditions (Stleger et al. 1987). Therefore, the
alkalinity of the infected epidermis is a physiological
signal of toxicity factors (Hui et al. 2004). In the case
of nutritional-deficiency, PR1 penetrates the cuticle
by hydrolyzing the cuticular proteins (Carneiro et al.
2015). Furthermore, the production of PR1 was inhib-
ited by exogenous carbon and nitrogen sources (Santi
et al. 2010a). PR3 is an acidic protease with low iso-
electric point. At present, its composition and action
mechanism are still unclear. Chitinase is also the main
determinant of pathogenicity (Hui et al. 2004). Car-
bon competition between pathogens and hosts may
determine the rate and extent of infection (Seyoum
et al. 2002). Lipase exists on the surface of conidia,
which can release free fatty acids by enhancing the
hydrophobic interaction between conidia and host,
and enhance the adhesion between conidia and host
through its lipolytic activity, so as to promote the
adhesion between conidia and host (San and Mun
2017). Protease, lipase, and chitinase degrade the cuti-
cle together, enabling effective penetration and uti-
lization of nutrients in the hemocoel of the host for
successful infection (Santi et al. 2010b). St Leger et al.
concluded that the epidermal degradation enzymes of
M. anisopliae are only synthesized at a pH that can
play an effective role, regardless of the presence of
epidermal substances in the culture medium (Leger
et al. 1998). In addition, the pH of the surrounding
area changes during M. anisopliae infection, which
enables the production of extracellular enzymes and
their activities. Environmental pH also regulates the
expression of toxic genes (Hui et al. 2004).
3.5. Colonization of hemolymph
Once the fungus breaks through the cuticular layer
and the lower epidermis, it starts to invade and pro-
liferate in the hemolymph, which may be restricted
to the hemolymph until the death of the host (Sey-
oum et al. 2002). In vivo and in the culture of infected
insects, M. anisopliae produces a family of cyclic pep-
tide toxins called destruxins (DTXs) (Hsiao and Ko
2001). DTXs of M. anisopliae are the first cyclic peptide
insect-borne fungal toxins to be systematically studied.

Trehalases are produced to convert trehalose from host
hemolymph into glucose for energy production, which
may represent an adaptation to the host environment
(Santi et al. 2010b). In addition, to protect conidia
from ultraviolet radiation and reactive oxygen species
formed at high temperatures, catalase and peroxidases
are found on the surface of conidia (San and Mun
2017). Fungi can use sugar as nutrition and consume
trehalose, thus reducing the utilization rate of sugar as
a host nutrient (Santi et al. 2010b). Moreover, Mad1
protein initiates gene expression involved in the cell
cycle, enabling hyphae to rapidly proliferate and dif-
ferentiate in the hemolymph of the host (Wang and
St. Leger 2007c). These proteins target the cytoskele-
ton and regulate cell division during the cell cycle (San
and Mun 2017).
3.6. Extrusion and sporulation
With the development of host colonization, the nutri-
ents are exhausted and the fungus produces hyphae,
which will appear and produce conidia on the surface
of the dead host (Gabarty et al. 2014). M. anisopliae
then forms a denser network of green spores on the
cadavers of infected hosts, ending the infection (San
and Mun 2017).
4. Toxins
M. anisopliae produces a large number of fungal sec-
ondary metabolites, including DTXs, which induce
membrane depolarization by opening Ca2+ channels,
resulting in paralysis and death of the host insects
(Hsiao and Ko 2001). DTXs are a class of insecticidal,
phytotoxic, and anti-viral cyclic peptide insect-borne
fungal toxins and are the most deeply studied toxins
of the entomopathogenic fungi (Schrank and Vain-
stein 2010). These compounds are composed of an
α-hydroxy acid and five amino acids to form a cyclic
hexadepsipeptide (Gabarty et al. 2014). To date, 38
DTXs have been reported and the majority are pro-
duced by M. anisopliae (Schrank and Vainstein 2010).
DTXs can be divided chemically into five basic groups
labeled A to E (Pedras et al. 2002). Destruxins A, E,
and B show significant insecticidal activity (Sree et al.
2008). Both destruxins B (DB) and desmethyl-DB have
phytotoxicity to the plants of Brassica (Hu et al. 2006).
Moreover, it has been reported that DB can suppress
the expression of the hepatitis B viral surface antigen
ALL LIFE 1145
in human hepatoma Hep3B cells, while the chromo-
somes of Hep3B cells carry integrated viral genes,
which suggests that DB may have a promising future as
a specific anti-HBV drug (Chen et al. 1997). Further-
more, these toxins assume a significant role in weaken-
ing host immune defense by damaging the Malpighian
tubules and the muscular system, leading to feeding
and affecting excretion as well as mobility difficulties
(Schrank and Vainstein 2010). Infected insects often
seek out warmer places to raise their body temper-
ature, thereby inhibiting the growth of the infected
microbes (Elliot and Thomas 2002). DTXs can reduce
host mobility to undermine behavior defense mecha-
nisms (Schrank and Vainstein 2010).
Swainsonine, a sugar analogue from M. anisopliae
isolate F-3622, was first reported by Hino et al. (1985;
Batta 2003; Roberts and Leger 2004). Swainsonine has
great potential as a chemotherapeutic drug because
its glycosylation changes are associated with the pro-
cesses of some diseases (Sim and Perry 1997). In addi-
tion to its direct effects on oligosaccharide processing
in cancer cells, it also has immunostimulating activ-
ity (Roberts and Leger 2004). Effects of swainsonine,
such as enhancing lymphocyte proliferation and aug-
menting cytotoxicities of lymphokine-activated killer
and natural killer cells, have been demonstrated (Sim
and Perry 1997). In addition to its therapeutic poten-
tial, swainsonine is also widely used as a biochemical
tool in studies of the nature and biological roles of N-
linked oligosaccharides and alpha mannosidases (Sim
and Perry 1997).
5. M. anisopliae as a Biological Control Agent
Mosquitoes transmit various pathogens, such as Plas-
modium and dengue virus, causing illness and death in
humans (Alkhaibari et al. 2016); World Health Organi-
zation 2020). Longevity, fecundity, and blood-feeding
are the most important factors affecting the possibil-
ity of mosquitoes transmitting diseases (Scholte et al.
2006). It has been reported that M. anisopliae infection
can reduce the survival time of mosquitoes, includ-
ing Anopheles, Aedes, and Culex (Daoust and Roberts
1982; Riba et al. 1986a; Riba et al. 1986b; Scholte
et al. 2003; Scholte et al. 2007; Lwetoijera et al. 2010;
Paula et al. 2019; Choi et al. 2020; Shoukat et al.
2020; Vivekanandhan et al. 2020) (Figure 2). Scholte
et al. contaminated Anopheles gambiae (Diptera: Culi-
cidae, An. gambiae) with the oil-formulated conidia
1146 Z.-Y. PENG ET AL.
Figure 2. Mycotoxic effects of M. anisopliae. (A) M. anisopliae cultured on Potato Dextrose Agar Medium. At the initial stage, the colonies
were white and hairy. In the sporulation stage, there were clumps of green conidia in the middle of the colony. (B) The cadavers of Aedes
aegypti infected by M. anisopliae or not. (C) The cadavers of Anopheles stephensi infected by M. anisopliae or not.
of M. anisopliae, and they found that M. anisopliae
infection reduced fecundity and blood-feeding of the
adult female malaria vector An. gambiae (Scholte et al.
2006). Similarly, Scholte et al. and Shoukat et al.
reported that the fecundity of the female dengue
virus vector Aedes aegypti (Diptera: Culicidae, Ae.
aegypti) and Aedes albopictus (Diptera: Culicidae, Ae.
albopictus) was reduced by M. anisopliae infection
(Scholte et al. 2006; Shoukat et al. 2020). They also
found that when Ae. aegypti females were coinfected
with M. anisopliae and dengue virus type 2 (DENV-
2), the infection rates of DENV-2 in the heads and
midguts were significantly reduced (Scholte et al. 2007;
Garza-Hernández et al. 2013). Moreover, a transgenic
approach can be used to improve the virulence of M.
anisopliae for mosquitoes or interfere with the devel-
opment of pathogens in the mosquito (Wassermann
et al. 2016). In addition, ticks are considered to be
the main vectors of animal diseases, second only to
mosquitoes as human disease vectors. These arthro-
pods are distributed all over the world, and diseases
spread by human ticks range from bacteria to proto-
zoa, viruses, and even fungi (Parola and Raoult 2001).
It has been demonstrated that M. anisopliae can kill
the larvae, nymphs, and adults of many genera and
species of ticks (Table 1) (Mwangi et al. 1995; Zhioua
et al. 1997; Kirkland et al. 2004; Ment et al. 2010b; Ren
et al. 2014; Beys-da-Silva et al. 2020; Molaei et al. 2020;
Abuowarda et al. 2020). Therefore, M. anisopliae may
have potential as a biocontrol agent for vector-borne
disease control.
Termites are important pests that cause severe dam-
age to crops, living trees and wood-based materials
(Rath 2000). It has been reported that M. anisopliae
can be used to control many termites (Table 1) (Hänel
1982; Zober 1995; Moino et al. 2002; Shimizu and
Yamaji 2003; Yanagawa and Shimizu 2007; Dong et al.
2009; Pikkheng et al. 2009; Kin et al. 2017; Mitaka
et al. 2017; Keppanan et al. 2018; Fu et al. 2020).
The first report of termite fungal biological control
agent is a single isolate C4-B, which is identified as
M. anisopliae taxonomically (Wright et al. 2005). In
addition, locusts represent probably the most striking
of all insect pests and are abundant insects of deserts
and dry grasslands (Scanlan et al. 2001). Tradition-
ally, chemical pesticides have been used to control
swarms and bands, but this is under strict scrutiny. M.
anisopliae has been tested successfully against Schis-
tocerca gregaria (Orthoptera: Acrididae), Chortoicetes
terminifera (Orthoptera: Acrididae), Locusta migra-
toria (Orthoptera: Oedipodidae) and Oxya chinensis
(Orthoptera: Acrididae) (Table 1) (Zober 1995; Xia
et al. 2000; Hunter et al. 2001; Peng et al. 2008; Abro
et al. 2019; Liu et al. 2019; Abdellaoui et al. 2020). In
Australia, field trials have shown that efficacy depends
on the application rate and vegetation cover, while
the rate of development of both Metarhizium and the
locust is affected by temperature (Hunter et al. 2001).
 ALL LIFE 1147

Table 1. Agricultural pests and human disease vectors sensitive to M. anisopliae.

Family Species Selected references
Culicidae Anopheles gambiae adults and larvae Scholte et al. (2006); Opisa et al. (2019)
Anopheles stephensi larvae Riba et al. (1986b); Vivekanandhan et al. (2020)
Anopheles arabiensis adults and larvae Choi et al. (2020)
Aedes aegypti adults and larvae Riba et al. (1986b); Scholte et al. (2007); Vivekanandhan et al. (2020)
Aedes albopictus adults and larvae Scholte et al. (2007); Shoukat et al. (2020)
Culex pipiens adults and larvae Daoust and Roberts (1982); Choi et al. (2020); Bilal et al. (2012)
Culex quinquefasciatus adults and larvae Lwetoijera et al. (2010); Wassermann et al. (2016); Mohanty et al. (2008)
Ixodidae Dermacentor variabilis adults Ren et al. (2014)
Rhipicephalus microplus larvae and adults Ojeda-Chi et al. (2010); Beys-da-Silva et al. (2020)
Rhipicephalus sanguineus nymphs and adults Kirkland et al. (2004); Abuowarda et al. (2020)
Rhipicephalus appendiculatus larvae, nymphs and adults Mwangi et al. (1995)
Ixodes scapularis larvae, nymphs and adults Zhioua et al. (1997); Inyang et al. (2000)
Haemaphysalis qinghaiensis adults, nymphs and adults Ren et al. (2014); Albernaz et al. (2009)
Hyalomma excavatum adults Ment et al. (2010b)
Rhinotermitidae Coptotermes formosanus Yanagawa and Shimizu (2007); Keppanan et al. (2018)
Coptotermes curvignathus Pikkheng et al. (2009); Kin et al. (2017)
Reticulitermes flavipes Zober (1995)
Reticulitermes speratus Shimizu and Yamaji (2003); Mitaka et al. (2017)
Heterotermes tenuis adults Moino et al. (2002)
Termitidae Odontotermes formosanus Dong et al. (2009); Fu et al. (2020)
Nasutitermes exitiosus Hänel (1982)
Acrididae Schistocerca gregaria adults, nymphs, eggs Xia et al. (2000); Abdellaoui et al. (2020)
Chortoicetes terminifera adults Hunter et al. (2001)
Locusta migratoria nymphs Peng et al. (2008); Liu et al. (2019); Bilal et al. (2012)
Oxya chinensis nymphs Zober (1995); Abro et al. (2019)
Pyralidae Ostrinia furnacalis larvae Chiuo and Hou (1993)
Ostrinia nubilalis larvae Riba et al. (1986a)
Diatraea saccharalis larvae De Oliveira et al. (2018); Destéfano et al. (2004)
Cnaphalocrocis medinalis larvae Hong et al. (2017); Destéfano et al. (2004)
Chilo partellus larvae Tefera and Pringle (2003)
Galleria mellonella larvae Keppanan et al. (2017); Sbaraini et al. (2021)
Noctuidae Spodoptera litura larvae Sree et al. (2008)
Spodoptera littoralis larvae El Husseini (2019)
Helicoverpa armigera larvae Lawo et al. (2008); Santi et al. (2011)
Hyblaea puera larvae Remadevi et al. (2010)
Carposinidae Carposina niponensis larvae Yaginuma (1990)
Lymantriidae Pantana phyllostachysae adults Zhang et al. (2002)
Blattellidae Blattella germanica adults Lopes and Alves (2011); Chao et al. (2020)
Lasiocampidae Dendrolimus punctatus larvae Zhang et al. (2002)
Reduviidae Triatoma infestans nymphs and adults Luz et al. (1998)
Triatoma sordida nymphs and adults Rangel et al. (2020)
Panstrongylus megistus nymphs and adults Rangel et al. (2020)
Delphacidae Nilaparvata lugens adults Tang et al. (2019)
Sogatella furcifera adults Tang et al. (2019)
Tephritidae Anastrepha ludens adults Toledo-Hernández et al. (2018)
Zeugodacus cucurbitae adults Onsongo et al. (2019)
Bruchidae Callosobruchus maculatus adults Cherry et al. (2005); Ozdemir et al. (2020)
Caryedon serratus adults Ekesi et al. (2001)
House flies Musca domestica larvae and adults Oreste et al. (2016); Baker et al. (2018)
Tenebrionidae Alphitobius diaperinus larvae and adults Ment et al. (2010a); Cassiano et al. (2008); Inyang et al. (2000)
Pyrrhocoridae Dysdercus peruvianus nymphs and adults Santi et al. (2011)
Plutellidae Plutella xylostella larvae Zafar et al. (2020)
Aleyrodidae Trialeurodes vaporariorum adults Oreste et al. (2016)
Aphelinidae Encarsia formosa larvae and adults Oreste et al. (2016)
Thripidae Frankliniella occidentalis adults Li et al. (2021)
Curculionidae Curculio nucum larvae Sharififard et al. (2011)
Cerambycidae Monochamus alternatus larvae and adults Li et al. (2021)
Delphacidae Peregrinus maidis adults Toledo et al. (2010)
Bostrychidae Rhyzopertha dominica adults Ashraf et al. (2017); Saeed et al. (2020)
Tenebrionidae Tribolium castaneum adults Ashraf et al. (2017); Saeed et al. (2020)
Laemophloeidae Cryptolestes ferrugineus adults Ashraf et al. (2017); Saeed et al. (2020)
Liposcelididae Liposcelis paeta adults Ashraf et al. (2017); Saeed et al. (2020)
Phycitidae Ephestia kuehniella larvae Athanassiou et al. (2017)
Tortricidae Thaumatotibia leucotreta adults Mkiga et al. (2020); Acheampong et al. (2020)
Crambidae Spoladea recurvalis adults Opisa et al. (2019)
Gelechiidae Tuta absoluta pupae Contreras et al. (2014)
Tettigoniidae Anabrus simplex nymphs Rangel et al. (2021)
1148 Z.-Y. PENG ET AL.
However, one challenge of using M. anisopliae is to
overcome the behavioral fever of locusts, which can
inactivate the conidia (Hunter et al. 2001). It has been
reported that heat-tolerant genes can be transferred
into M. anisopliae by genetic engineering to overcome
this challenge (San and Mun 2017; Keppanan et al.
2017). Thus, M. anisopliae has desirable attributes for
the development of a mycoinsecticide against pests.
In addition, M. anisopliae has been applied to
control Spodoptera litura (Lepidoptera: Noctuidae),
Carposina niponensis (Lepidoptera: Carposinadae),
Galleria mellonella (Lepidoptera: Pyralidae), Prode-
nia litura (Lepidoptera: Noctuidae), Pantana phyl-
lostachysae (Lepidoptera: Lymantriidae), Blattella ger-
manica (Blattodea: Ectobiidae), Dendrolimus puncta-
tus (Lepidoptera: Lasiocampidae), Triatoma infestans
(Hemiptera: Reduviidae), Helicoverpa armigera (Lep-
idoptera: Noctuidae), Hyblaea puera (Lepidoptera:
Hyblaeidae), Callosobruchus maculatus (Coleoptera:
Chrysomelidae), Musca domestica (Diptera: Mus-
cidae), Dysdercus peruvianus (Hemiptera: Pyrrho-
coridae), Anastrepha luden (Diptera: Tephritidae),
Chilo partellus (Lepidoptera: Crambidae), Panstrongy-
lus megistus (Hemiptera: Reduvidae), Monochamus
alternatus (Coleoptera: Cerambycidae), Curculio
nucum (Coleoptera: Curculionidae), Frankliniella occi-
dentalis (Thysanoptera: Thripidae), Encarsia Formosa
(Hymenoptera: Aphelinidae), Trialeurodes vaporar-
iorum (Hemiptera: Aleyrodidae), Plutella xylostella
(Lepidoptera: Plutellidae), Zeugodacus cucurbitae
(Diptera: Tephritidae), Sogatella furcifera (Hemiptera:
Delphacidae), Nilaparvata lugens (Hemiptera: Del-
phacidae), Ostrinia furnacalis (Lepidoptera: Cram-
bidae), Ostrinia nubilalis (Lepidoptera: Crambidae),
Diatraea saccharalis (Lepidoptera: Crambidae), Tri-
atoma sordida (Hemiptera: Triatominae), Cnaphalo-
crocis medinalis (Lepidoptera: Pyralidae), Spodoptera
littoralis (Lepidoptera: Noctuidae), Alphitobius diaper-
inus (Coleoptera: Tenebrionidae), Pieris rapae (Lep-
idoptera: Paleidae), Plutella xylostella (Lepidoptera:
Plutellidae), Dorysthenes granulosus (Coleoptera: Cer-
ambycidae), Caryedon serratus (Coleoptera: Bruchi-
dae), Peregrinus maidis (Hemiptera: Delphacidae),
Rhyzopertha dominica (Coleoptera Bostrichidae), Tri-
bolium castaneum (Coleoptera: Tenebrionidae), Cryp-
tolestes ferrugineus (Coleoptera: Laemophloeidae),
Liposcelis paeta (Psocoptera: Liposcelididae), Eph-
estia kuehniella (Lepidoptera: Pyralidae), Thauma-
totibia leucotreta (Lepidoptera: Tortricidae), Spoladea
recurvalis (Lepidoptera: Crambidae), Tuta absoluta
(Lepidoptera: Gelechiidae), Anabrus simplex
(Orthoptera: Tettigoniidae) (Table 1) (Sree et al. 2008;
Ment et al. 2010b; Keppanan et al. 2017; Yaginuma
1990; Rustiguel et al. 2018; De Oliveira et al. 2018; Luz
et al. 1998; Hong et al. 2017; Kirubakaran et al. 2014;
Chiuo and Hou 1993; Destéfano et al. 2004; Tefera and
Pringle 2003; Zhang et al. 2002; Guogui 2003; Cherry
et al. 2005; Cassiano et al. 2008; Lawo et al. 2008;
Remadevi et al. 2010; Lopes and Alves 2011; Santi et al.
2011; Sharififard et al. 2011; Cheng et al. 2016; Oreste
et al. 2016; Baker et al. 2018; Toledo-Hernández et al.
2018; El Husseini 2019; Onsongo et al. 2019; Tang et al.
2019; Chao et al. 2020; Fite et al. 2020; Kim et al. 2020;
Li et al. 2021; Ozdemir et al. 2020; Rangel et al. 2020;
Rice et al. 2020; Zafar et al. 2020; Bilal et al. 2012;
Mohanty et al. 2008; Ojeda-Chi et al. 2010; Sbaraini
et al. 2021; Ekesi et al. 2001; Toledo et al. 2010; Ashraf
et al. 2017; Saeed et al. 2020; Athanassiou et al. 2017;
Mkiga et al. 2020; Acheampong et al. 2020; Opisa et al.
2019; Contreras et al. 2014; Rangel et al. 2021). In sum-
mary, due to its safety and lack of arthropod resistance,
M. anisopliae has good potential in the control of agri-
cultural pests and vectors of human diseases (Wang
and St Leger 2007a).
6. Molecular approaches to improve the
virulence and efficacy of M. anisopliae
Poor efficacy has hindered the application of Metarhiz-
ium; hence, we urgently need to find methods to
maximize environmental persistence and virulence
of Metarhizium (Wang and St Leger 2007a). One
approach to improve virulence is genetic manipula-
tion. M. anisopliae provides a large list of genes that
can be combined in a single pathogen vector because
they use different proteins to adhere to the host, pene-
trate host cuticles, and overcome host defenses (Lovett
and St Leger 2018). Many natural and synthetic genes
have been inserted into the genome of M. anisopliae
to enhance its virulence and efficacy against pests
(Table 2).
The scorpion excitatory sodium channel blocker,
Androctonus australis Hector insect toxin (AaIT), is
an insect-specific neurotoxin (Lovett and St Leger
2018). The recombinant expression of AaIT in fungi
can increase their virulence against insect pests and
disease vectors (Deng et al. 2019b). M Pava-Ripoll
et al. demonstrated that recombinant M. anisopliae
 ALL LIFE 1149

Table 2. The major approaches for modifying entomopathogens In addition, wild M. anisopliae isolates showed no
to enhance the virulence of M. anisopliae. pathogenicity to Spodoptera litura larvae (Lee et al.
Modified strain Principal targeted species Selected references 2006). Zhang et al. genetically modified M. anisopliae
Ma-AaIT Hypothenemus hampei Pava-Ripoll et al. (2008) by inserting the insect midgut-specific toxin Vip3Aal
larvae
Manduca sexta caterpillars Wang and St Leger (2007a) gene of Bacillus thuringiensis. The expression of this
larvae toxin allows M. anisopliae to infect S. litura larvae
Aedes aegypti adults Wang and St Leger (2007a)
Ma-[SM1]8 Plasmodium falciparum Fang et al. (2011) through the ingestion of conidia rather than through
and Anopheles gambiae the cuticle penetration (Zhang et al. 2014).
adults
Ma-scorpine Plasmodium falciparum Fang et al. (2011)
and Anopheles gambiae
adults 7. M. anisopliae formulations
Ma-PfNPNA-1 Plasmodium falciparum Fang et al. (2011)
and Anopheles gambiae
adults In addition to strain selection and genetic modifica-
Ma-[SM1]8 :scorpine Plasmodium falciparum Fang et al. (2011) tion, formulations can also have a significant impact
and Anopheles gambiae
adults on improving the efficacy of biological pesticides. An
Ma-Cat1 Plutella xylostella larvae Morales Hernandez et al. ideal formulation is helpful to the handling and appli-
(2010)
Ma-Vip3Aa1 Spodoptera litura larvae Zhang et al. (2014) cation of biopesticides and reduces the chances of
nontarget organisms coming into contact with fungal
spores.
AaIT-Ma549 was dramatically more virulent than Bukhari et al. devised formulations of M. aniso-
wild-type against Hypothenemus hampei larvae (Pava- pliae spores in aqueous, dry, synthetic oil (ShellSol
Ripoll et al. 2008). Wang et al. also showed that the T and Ondina oil 917) for the control of Anopheles
high-level expression of AaIT toxin in M. anisopliae larvae (Table 3) (Bukhari et al. 2011). Among these for-
increased its fungal toxicity to larval Manduca sexta mulations, synthetic oil is easily miscible and suitable
caterpillars by 22-fold and Ae. aegypti adults by 9-fold for water surfaces and improves persistence (Abdul-
(Table 2) (Wang and St Leger 2007a). Ghani et al. 2012). Their results have shown that the
Fang et al. engineered M. anisopliae to express the delivery system of fungal conidia suspended in Shell-
eight repeats of salivary gland and midgut peptide 1 Sol T solvent was significantly more virulent to An.
([SM1]8 ); a single-chain human antibody that aggluti- gambiae and Anopheles stephensi (Diptera, Culicidae)
nates sporozoites; an antimicrobial toxin scorpine; or larvae than that of unformulated conidia (Bukhari
a fusion protein of [SM1]8 and scorpine. These recom- et al. 2011). However, dry and aqueous formulated M.
binant fungi not only killed An. gambiae, but also anisopliae spores lost their pathogenicity quickly after
reduced sporozoite counts of An. gambiae by 70% to being applied to the water surface (Bukhari et al. 2011).
90%. M. anisopliaeexpressing scorpion and an [SM1]8 : Inyang et al. took the third instar larvae of Phae-
scorpion fusion protein reduced the number of sporo- don cochleariae as the target host to study the effect of
zoites by 98%, indicating that M. anisopliae-mediated simulated rain on the persistence of oil formulations
inhibition of Plasmodium development may be a pow- of M. anisopliae conidia when applied to oilseed rape
erful weapon for malaria control (Fang et al. 2011). foliage (Inyang et al. 2000). Their results suggested that
M. anisopliae exhibits an increase in catalase- sunflower oil formulations enhanced the infectivity of
peroxidase activity during its germination and growth M. anisopliae for P. cochleariae in both laboratory and
(Kawasaki and Aguirre 2001). Morales Hernandez field experiments (Inyang et al. 2000). This viscous oil
et al. found that catalase overexpression can reduce the carrier could improve the adhesion of M. anisopliae
germination time and increase the pathogenicity of M. conidia to plant surfaces and reduce displacement by
anisopliae (Morales Hernandez et al. 2010). Thus, they rain (Inyang et al. 2000).
isolated the genomic copy of M. anisopliae catalase-1 Oil carriers, such as sunflower, peanut, Ondina, soy-
(cat1) gene and overexpressed it in the fungus. They bean, canola and mineral oil, as well as cornstarch oil
found that increased cat1 expression was accompanied can transport the inoculum to parts of the insect’s
by increased H2 O2 tolerance and germination rate, body, which is more conducive to germination than
all of which enhanced the virulence against Plutella the application of conidia in Tween (Table 3) (Batta
xylostella larvae (Morales Hernandez et al. 2010). 2003; Paula et al. 2019; Sharififard et al. 2011; Pereira
1150 Z.-Y. PENG ET AL.

Table 3. Selected examples of formulations using M. anisopliae in various habitats.

Formulation Habitat Principal targeted species Selected references
Oil ShellSol T plastic containers Anopheles gambiae larvae Bukhari et al. (2011)
plastic containers Anopheles stephensi larvae Bukhari et al. (2011)
experimental plots Phaedon cochleariae larvae Inyang et al. (2000)
Ondina oil 917 plastic containers Anopheles gambiae larvae Bukhari et al. (2011)
plastic containers Anopheles stephensi larvae Bukhari et al. (2011)
Mineral oil culture medium Rhipicephalus microplus Camargo et al. (2012)
larvae, nymphs and
adults
Mineral oil and plastic cups Aedes aegypti adults Rodrigues et al. (2019)
diatomaceous earth
Cornstarch-oil petri dishes Diabrotica undec- Pereira and Roberts (1991)
impunctata
adults
Sunflower oil and petri dishes Conotrachelus psidii adults Brito et al. (2008)
Imidacloprid (IMI)
Sunflower oil-in-water paper cups Aedes aegypti eggs Albernaz et al. (2009)
Oil-in-water petri plates RhipicephalusMicroplus Muniz et al. (2020)
females
Soybean oil agar plates Dysdercus peruvianus Rangel et al. (2020)
Coconut/soybean oil capped graduated tubes Tetranychus cinnabarinus Batta (2003)
adults
capped graduated tubes Bemisia tabaci nymphs Batta (2003)
Peanut oil potted grass Rhipicephalus appendicula- Kaaya and Hedimbi (2012)
tus larvae, nymphs and
adults
potted grass Amblyomma variegatum Kaaya and Hedimbi (2012)
larvae, nymphs, and
adults
Canola oil field paddock RhipicephalusMicroplus Leemon et al. (2008)
Neem oil natural field Aedes aegypti larvae Paula et al. (2019)
natural field Culex quinquefasciatus Seye et al. (2012)
larvae and adults
experimental cage Anopheles stephensi female Dhar et al. (1996)
experimental cage Anopheles culicifacies Dhar et al. (1996)
female
greenhouse conditions Anopheles gambiae larvae Okumu et al. (2007)
Dust Diatomaceous earth glass-capped dishes Tribolium confusum larvae Michalaki et al. (2006)
and adults
small cylindrical glass vial Rhyzopertha dominica Rice et al. (2019)
adults
small cylindrical glass vial Sitophilus oryzae adults Rice et al. (2019)
Other Alginate tightly sealed plastic tubes Diabrotica undec- Pereira and Roberts (1991)
impunctata
adults
Aqueous potted grass Rhipicephalus appen- Kaaya and Hedimbi (2012)
diculatus adults and
eggs
recipientes plásticos Alphitobius diaperinus Rohde et al. (2006)
larvae and adults
Granular formulations petri dishes Lesser mealworm larvae Rice et al. (2019)
consisting of ground
chicken feed pellets
with conidial
powder
Polymerized cellulose Polypropylene bags Rhipicephalus microplus Rice et al. (2019)
gel combined with
surfactants

and Roberts 1991; Dhar et al. 1996; Okumu et al.
2007; Brito et al. 2008; Leemon et al. 2008; Albernaz
et al. 2009; Kaaya and Hedimbi 2012; Seye et al. 2012;
Camargo et al. 2012; Rodrigues et al. 2019; Muniz
et al. 2020). Oil carriers also seem to improve spore
germination at low relative humidities and prolong
spore viability, even at high temperatures (Inyang et al.
2000).
Michalaki et al. utilized diatomaceous earth (DE)
in combination with an M. anisopliae formulation to
treat wheat/flour. Their research is the first work on the
combination of M. anisopliae and DE (Michalaki et al.
2006). Kavallieratos et al. demonstrated that the com-
bined use of DE and M. anisopliae could be of practi-
cal significance to pest control, and this combination
could be used against adults of three stored-grain

beetle species (Table 3). DEs can remove certain epi-
dermis lipids from insects, allowing germinated M.
anisopliae conidia to more easily infiltrate insect blood
(Michalaki et al. 2006).
Alginates have been used to encapsulate fungal
structures for the biocontrol of entomopathogens and
weeds Pereira and Roberts (1991), (Walker and Con-
nick 1983) found that the alginate formulations are
very effective in preventing fungi from inactivation
due to high temperatures and artificial solar radia-
tion. Thus, they used an Alginates formula to control
adult Diabrotica undecimpunctata (Table 3) (Pereira
and Roberts 1991).
In addition to the above formulations, there are also
other formulations that can improve the efficacy of
M. anisopliae, such as aqueous, granular formulations
consisting of ground chicken feed pellets with coni-
dial powder and polymerized cellulose gel combined
with surfactants (Kaaya and Hedimbi 2012; Walker
and Connick 1983; Rohde et al. 2006; Reis et al. 2008;
Rice et al. 2019).
8. Conclusions and future perspectives
M. anisopliae has been considered an important ento-
mopathogenic fungus in the biocontrol of many pests
and human disease vectors throughout the world since
it was isolated and identified nearly 150 years ago. In
this review, we mainly summarized the historical per-
spective, pathogenesis, toxins, M. anisopliae as a bio-
logical control, and molecular approaches to increase
its virulence as well as the formulations of M. aniso-
pliae. Generally, conidia, also known as asexual spores,
are the main form of M. anisopliae that infects sus-
ceptible hosts. Once conidia touch the cuticular layer
of a sensitive host, the infection begins with adhe-
sion and germ tube formation. Then the expression of
genes related to fungal infection structures, hydrolytic
enzymes, and host defense mechanisms is triggered
by both the insect pathogen and host. Normally, a
sign of a successful fungal infection is the cleavage
of the hosts’ epidermis. When the fungus arrives in
the hemolymph through the growing hyphal, the host
body will become colonized. M. anisopliae also pro-
duces numerous toxins, including DTXs and swainso-
nine. DTXs play an important role in weakening host
immune defense, damaging the muscle system and
Malpighian tubule, affecting host excretion, and result-
ing in feeding and mobility difficulties. The discoveries
ALL LIFE 1151
about the ecological roles displayed by M. anisopliae
with multiple effects will help its eventual commercial-
ization.
Although great progress has been made in the con-
trol of diseases and pests by using biopesticides, the
development of fungal biological control agents still
faces some problems. First, most biopesticide formu-
lations, including spores, filaments, and other living
preparations, cannot be easily combined with anti-
corrosive ingredients, which affect the use and preser-
vation of fungal pesticides. Second, the use of biopes-
ticides is greatly affected by environmental conditions,
thus leading to unstable efficacy. Third, compared
with that of chemical pesticides, the price of bio-
logical pesticides is relatively high. Thus, researchers
should make full use of the rich microbial resources
in the world to screen out and select efficient strains
and make continuous breakthroughs in technology to
cultivate biocontrol strains with high virulence and
productivity.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This research was supported by National Natural Science Foun-
dation of China (82102432), Anhui Provincial Natural Science
Foundation (2108085QH347), Research Fund Project of Anhui
Medical University (2020xkj005) to DSQ.
Declaration of interest statement
No potential conflict of interest was reported by the author(s).
Author contributions statement
Conception and design: SQD; analysis and interpretation of
the data: ZYP, STH, JTC, NL, YW and AN; writing – original
draft preparation: STH and SQD; writing – review and editing:
ZYP, AN and SQD. All authors have agreed to be accountable
for all aspects of the work and the published version of the
manuscript.
Data availability statement
Data sharing is not applicable to this article as no new data were
created or analysed in this study.
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