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Cold tolerance strategies of the fall armyworm, Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae)

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The fall armyworm (FAW), Spodoptera frugiperda, is native to the tropical and subtropical areas of the American continent and is one of the world's most destructive insect pests and invaded Africa and spread to most of Asia in two years. Glycerol is generally used as a cryoprotectant for overwintering insects in cold areas. In many studies, the increase in glycerol as a main rapid cold hardening (RCH) factor and enhancing the supercooling point was revealed at low temperatures. There are two genes, including glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK), that were identified as being associated with the glycerol synthesis pathway. In this study, one GPDH and two GK sequences (GK1 and GK2) were extracted from FAW transcriptome analysis. RNA interference (RNAi) specific to GPDH or GK1 and GK2 exhibited a significant down-regulation at the mRNA level as well as a reduction in survival rate when the RNAi-treated of FAW larvae post a RCH treatment. Following a cold period, an increase in glycerol accumulation was detected utilizing high-pressure liquid chromatography and colorimetric analysis of glycerol quantity in RCH treated hemolymph of FAW larvae. This research suggests that GPDH and GK isozymes are linked to the production of a high quantity of glycerol as an RCH factor, and glycerol as main cryoprotectant plays an important role in survival throughout the cold period in this quarantine pest studied.
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Cold tolerance strategies of the fall
armyworm, Spodoptera frugiperda
(Smith) (Lepidoptera: Noctuidae)
Mohammad Vatanparast & Youngjin Park*
The fall armyworm (FAW), Spodoptera frugiperda, is native to the tropical and subtropical areas of
the American continent and is one of the world’s most destructive insect pests and invaded Africa and
spread to most of Asia in two years. Glycerol is generally used as a cryoprotectant for overwintering
insects in cold areas. In many studies, the increase in glycerol as a main rapid cold hardening (RCH)
factor and enhancing the supercooling point was revealed at low temperatures. There are two
genes, including glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK), that were
identied as being associated with the glycerol synthesis pathway. In this study, one GPDH and two
GK sequences (GK1 and GK2) were extracted from FAW transcriptome analysis. RNA interference
(RNAi) specic to GPDH or GK1 and GK2 exhibited a signicant down-regulation at the mRNA level
as well as a reduction in survival rate when the RNAi-treated of FAW larvae post a RCH treatment.
Following a cold period, an increase in glycerol accumulation was detected utilizing high-pressure
liquid chromatography and colorimetric analysis of glycerol quantity in RCH treated hemolymph of
FAW larvae. This research suggests that GPDH and GK isozymes are linked to the production of a high
quantity of glycerol as an RCH factor, and glycerol as main cryoprotectant plays an important role in
survival throughout the cold period in this quarantine pest studied.
e fall armyworm (FAW), Spodoptera frugiperda (Smith), is native to the American continent’s tropical and
subtropical regions1. It is a polyphagous insect, and due to its wide host range, it is one of the most dangerous
pests aecting tropical annual crops2,3. ey are usually composed of two genetically distinct strains, such as
rice (R-strain) and corn (C-strain)46. FAW was reported in a number of Southeast Asian countries in 2018 and
2019, including India, ailand, Myanmar, China, Japan, the Philippines, Indonesia, and most recently, Aus-
tralia. e rst invaded populations of FAW in South Korea were genetically conrmed using a mitochondrial
cytochrome oxidase subunit I (COI) gene in 20197. e presence of ideal climatic conditions for FAW in many
parts of Africa and Asia, as well as an abundance of suitable host plants, indicates that the pest can produce many
generations in a single season, and that the pest is likely to become endemic8. e chance of FAW spreading
would be greatly increased by its long-distance migration. e rst conrmation of the invasion of FAW in Yun-
nan Province (western area) of China was documented on January 11, 2019. FAW had spread to most provinces
in southern China by May 20199. In reality, due to low temperatures, FAW can only successfully breed in the
summer and cannot survive the winter in most areas of mainland China, Japan, and the Korean, so these areas
will need to be reinvaded on an annual basis10,11. e East Asian migration area includes the Japanese Islands,
Korea, and eastern China. e geographical place, ecological climate, and climatic conditions of these areas are
all intertwined. Many seasonal pests, such as rice plant hoppers (Nilaparvata lugens (Stål), Sogatella furcifera
(Horváth), and Laodelphax striatellus (Fallén)) and the oriental armyworm, Mythimna separate (Walker), can y
from China to Japan and the Korea1214. Now that FAW has made its way into Southeast Asia and southern China
and southern Korea, the pest has a better possibility of invading Japan and Korea9. FAW has posed a signicant
threat to local corn and other crop production, as well as food security. Modeling the insect’s rate of expansion
and future potential migratory range using a trajectory analytical method and meteorological data during ve
years (2014–2018) revealed a very high probability that FAW will annually invade Korea, potentially causing a
substantial decrease in agricultural productivity.
Since FAW do not diapause, they migrate to areas with better environmental conditions4. Even though Sparks
estimated the minimum temperature for survival to be 10°C15, it was discovered in 1979 that temperatures below
13°C at FAW overwintering sites do not enable larvae and pupae to survive1. Aer exposing all stages of FAW
to low temperatures for three hours, it was discovered that the egg was the most resistant, with a 30% survival
Plant Quarantine Technology Center, Animal and Plant Quarantine Agency, Gimcheon 39660, Republic of Korea.
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rate at –10°C16. Temperature is a vital abiotic variable that inuences organisms’ geographic distribution and
seasonal activity patterns15 and it has a big impact on pest biology, and abundance17. With behavioral avoidance,
migration, diapause or in an extremely altered physiological state, insects escape extreme temperatures18. Since
insect development takes place within a certain temperature, a change in temperature can aect the development
rate, lifespan and ultimately survival of the insects19. Because of their poikilothermic nature, low temperatures
act as a physical barrier preventing insects from expanding their habitats20. e insect’s survival capability is
characterized as cold hardiness aer exposure to low temperature levels. is procedure leads to the develop-
ment of particular compounds known as cryoprotectants, which are polyols and sugars21. Insects withstand
cold temperatures by holding their body uids liquid below their normal melting point (freeze-intolerant) or by
avoiding ice formation in their tissues (freeze-tolerance)2224. e primary strategy of freeze-intolerant insects
is to avoid exposure to lethal temperatures. In contrast, freeze-tolerance insects are able to overcome freezing
by employing a variety of mechanisms, such as reducing ice formation in cells or delaying ice formation21,25.
Another strategy, termed ‘supercooling’ is focused on the ability of insects to be cooled until spontaneous ice
nucleation happens within their body uids. e supercooling point (SCP) is the temperature at which body
water spontaneously freezes24,26. While body uid cools below its freezing point during the supercooling state,
no crystallization occurs. However, in many situations, death is likely to happen at temperatures far above the
SCP27,28. Insects may quickly change their response to low temperatures, either by preventing chilling injury or
by modifying their behavior, a process known as rapid cold hardening (RCH) that it is associated with chemical
changes in hemolymph composition to increase polyols29. Exposure to 5°C for 6h in Spodoptera exigua (Hüb-
ner) caused a major RCH in all developmental stages, from egg to adult, which was accompanied by a strong
increase in glycerol titers in hemolymph30. RCH for 2h at 5°C of the newly-emerged adult of ve coleopteran
grain-related species substantially increased the survival at dierent temperatures below zero as compared to
the non-acclimated period31.
According to a recent study on the cold hardiness of invasive FAW species in China, pupae and older larvae
have a much higher survival rate than eggs and younger larvae, and FAW can live in some southern areas of
China’s subtropical zone during the winter based on the SCPs of developmental stages at low temperatures and
China’s climatic regionalization. Supercooling capacity of S. exigua32 and its RCH33,34 allows it to live at low
temperatures in temperate areas during the winter. Based on high pressure liquid chromatography (HPLC)
analysis of glycerol titers in response to pre-exposure to a low temperature, it was demonstrated that glycerol is
a key cryoprotectant in RCH in S. exigua30.
Because of FAW’s dispersal ability and high spreading eciency, as well as its large reproductive capacity and
wide host plant range, the pest is likely to become one of the most important migratory insect pests in South
Korea that already categorized as a quarantine pest. FAW may increase cryoprotectant contents in hemolymph,
such as glycerol, and may be able to endure cold seasons, but its high spreading eciency and dispersal ability
may also assist it in migrating from harsh to moderate environments. In this study we hypothesized that FAW use
RCH and glycerol as an associated factor to survive to low temperatures. To investigate the function of glycerol,
we used RNA interference (RNAi) to knock down genes involved in glycerol biosynthesis and then examined
the intensity of RCH and glycerol accumulation.
Glycerol content in plasma in response to low temperatures and exposure time. When h
instar larvae of FAW were incubated at low temperatures (5 and 10°C) compared to higher temperatures, their
glycerol content increased more than threefold (15 and 20°C) (Fig.1A). e exposure period was also linked to
an increase in the amount of glycerol in plasma, with the most glycerol found aer 24h of incubation (Fig.1B).
e high glycerol level indicated that it is a major component of plasma aer cold stress.
Molecular architecture of glycerol biosynthesis genes. To explain the increase in glycerol content,
we attempted to identify the enzymes involved in glycerol biosynthesis (Fig.1C). Based on a previous study30 we
chose dihydroxyacetone-3-phosphate (DHAP) as a precursor of glycerol biosynthesis from glycolysis intermedi-
ates. e catalytic activity of GPDH and GK converts DHAP to glycerol (Fig.1D). As a result, the GPDH and
GK genes were predicted to be involved in the synthesis of glycerol, which is an important cryoprotectant in
insects when temperatures are extremely low. e transcriptome of FAW (NCBI accession number: GSE175545)
were used to determine full open reading frames (ORFs) of GPDH (Sf-GPDH) and GK (Sf-GK1 and Sf-GK2) of
FAW. Sf-GPDH, Sf-GK1 and Sf-GK2 ORFs encode for 353, 343, and 332 amino acid residues, respectively. Sf-
GPDH protein contains a bi-domain protein structure, as illustrated in Fig.2A that it encoded NAD+-dependent
GPDHs with an N-terminal NAD+-binding domain and a C-terminal NAD+-dependent GPDH domain. Both
identied FAW glycerol kinases shared N-terminal (FGGY-N) and C- terminal (FGGY-C) domains, which are
colored blue and red, respectively, as shown in Fig.2B, conrming that the targeted proteins are members of
the FGGY carbohydrate kinase family. Based on comparisons with other well-known insect proteins, the three-
dimensional structures of Sf-GPDH, Sf-GK1, and Sf-GK2 proteins were predicted using the homology modeling
method (Fig.3). ese ndings indicated that the sequences of Sf-GPDH and two Sf-GKs closely matched the
homologous templates on the server, indicating that these protein models were reliable. e GPDH domain
structure of Sf-NAD+-binding revealed two key components: a spatially symmetric β-sheet core and multiple
helices (α1–α17) wrapping on both sides of the β-sheet core. e bioinformatics analysis indicated that four
functional amino acids including Arg99, Glu100, Phe155, and Asn266 in Sf-GK1 and Arg92, Glu93, Phe148, and
Asp258 in Sf-GK2 which are as glycerol binding residues (Fig.3C, E). ree-dimensional analysis indicates 66%
homology of Sf-GPDH with Tribolium castaneum (Herbert) GPDH under 73% coverage. When the Sf-GK1 and
Sf-GK2 were compared by Spodoptera litura (Fabricus) glycerol kinase, the homology was 45 and 48% under 54
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Incubation temperature (
Free Glycerol (nmol/ml) of L5 haemolymph
5 10 15 25
Incubation time (h) at 5
0 1.5 3 6 12 24
Free Glycerol (nmol/mL) of L5 haemolymph
Figure1. Measurement of free glycerol in plasma of L5 of Spodoptera frugiperda (A) at dierent temperature when the
larvae incubated for 24h. (B) Eect of exposure time on glycerol content of plasma when the larvae were incubated at 10°C.
Each treatment was replicated three times with 10 larvae per replication. Dierent letters indicate signicant dierences
among means at (Type I error = 0.05, LSD test). (C) Chromatograms of hemolymph extracted from larvae exposed to 5°C
for 24h. (D) A putative glycerol production pathway. Glycerol is formed by catabolizing glucose to dihydroxyacetone-3-
phosphate (DHAP), which is then reduced to glycerol-3-phosphate (G3P). TRE, PGM, PGI, and GPP represent for trehalase,
phosphoglucomutase, phosphoglucoisomerase, and glycerol3phosphate phosphatase, respectively.
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and 59% coverage (Fig.3D, F). In these two glycerol kinases, several amino acids were conserved including ATP-
binding motif and FGGY signature motives (Figs.3, 4). A phylogenetic analysis indicated that the Sf-GPDH and
Sf-GK1 were clustered with lepidopteran insects quite distinct from other insect orders. However, interestingly
Sf-GK2 was clustered with Homopteran insect (Fig.5).
Expression prole of glycerol biosynthesis genes and inducible expression in response to
low temperature in FAW. ree glycerol biosynthesis genes were expressed in FAW (Fig.6). ey were
expressed from egg to adult in whole stages of development (Fig.6A, C, E). In larval stage, they were expressed
in dierent tissues such as hemocytes, fat body, midgut, and epidermis (Fig.6B, D, F). However, their expression
levels were varied among treatments during the developmental stages. All the three genes showed high expres-
sion level at adult stages of female insects. e highest expression level of all three genes was detected at midgut
tissue. e expression levels of all three glycerol biosynthesis genes were inducible in response to low tempera-
ture (5°C), and they showed a positive correlation with increasing incubation time. (Fig.6G).
Glycerol content is reduced by RNAi targeting glycerol biosynthesis genes following
RCH. RNAi was done on each glycerol biosynthesis gene (Sf-GPDH, Sf-GK1, and Sf-GK2) by injecting gene
specic double-stranded RNAs (dsRNAs) into L5 larvae (Fig.7). All three genes showed signicant decreases
(P < 0.05) with incubation time when one µg of dsRNA for each gene was injected into each larva. In all three
genes, the strongest RNAi eect was observed at 48h post injection, with a 40–80 percent drop in mRNA
expression levels (Fig.7A).
RNAi downregulation of glycerol biosynthesis gene expression signicantly suppressed glycerol amount
(P < 0.05) in plasma at 48h post-dsRNA injection aer RCH treatment (Fig.7B). e larvae treated with dsRNA
for three genes had a basal amount of glycerol (29–35mmol/mL), but control larvae (injected with dsRNA to
enhanced green uorescent protein (EGFP) gene) had approximately 73mmol/mL glycerol (Fig.7C). Aer
RCH treatment and RNAi, the cryoprotectant(s) was monitored in hemolymph of h instar larvae using HPLC
(Figs.1C, 7B). Glycerol content signicantly increased from 17.1 to 44.0mM (Table1) when the larvae were
incubated at 5°C. RNAi treatment larvae also showed a reduction in glycerol level when compare with control
treatment (EGFP). Injection of dsGK2 resulted in a signicant reduction in glycerol levels of more than seven
times (6.08mM) (Table1, Fig.7B).
RNAi of glycerol biosynthesis genes increases the mortality of treated larvae of FAW. Larvae
at 48h post-dsRNA injection did increase their mortality aer RCH treatment (Fig.7C, D). ere was no sig-
nicant dierence in mortality between RCH and control (no RCH) treatment aer RNAi of either Sf-GPDH or
Figure2. Protein domain analysis of glycerol biosynthesis genes of Spodoptera frugiperda. (A) Prediction of
signature motifs of Sf-GPDH. (B) Prediction of signature motifs of Sf-GK1 and Sf-GK2. e functional domains
were predicted using the NCBI conserved domain database and the EMBL-EBI HMMER database.
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Sf-GK2, However the mortality decreased signicantly when the larvae injected with dsRNA specic to Sf-GK1
aer RCH treatment than to control (Fig.7C).
Following RCH treatment, the SCP increased. e eect of RCH on SCP was evaluated in all devel-
opmental stages including both sexes in pupal and adult stages (Table1). Egg, rst instar and pupal stages exhib-
ited SCP at lowest temperature than other developmental stages. e data showed that supercooling capacity
was unaected by RCH treatment in egg, rst and second instar, male pupae, and female adult whereas SCP
temperature in the others was signicantly reduced (Table2). From these data, we found that RCH treatment
is oen accompanied by elevated SCPs. To investigate the involvement of glycerol biosynthesis genes in SCP,
RNAi-treated larvae (L3 to L6) were incubated at RCH conditions and their SCP was assessed (Table3). e
SCPs of larvae injected with dsRNA specic to glycerol biosynthesis genes, specically dsGPDH and dsGK2,
were signicantly lower than those of dsEGFP-injected larvae, suggesting that glycerol biosynthesis genes elevate
Figure3. ree-dimensional analysis of glycerol biosynthesis genes of Spodoptera frugiperda. (A, C, and E)
e functional domains of Sf-GPDH, Sf-GK1, and Sf-GK2 were demonstrated, respectively. (B, D, and F) the
Sf-GPDH, Sf-GK1, and Sf-GK2 proteins respectively, were compared with same protein from another well-
known insect, including Tribolium castaneum and Spodoptera litura. Blue and pink region in (A) indicate
beta sheet and alpha helices, respectively. In (C and E), the glycerol binding residues were indicated with blue
atoms as well as yellow part that showing ATP-binding domains. N and C are an abbreviation for N-terminus
and C-terminus of amino acid sequences. ese models were made using SWISS-model web database. ree
dimensional constructs were made using Chimera, version 1.13.1.
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Figure4. Alignment of amino acid sequences of glycerol biosynthesis genes including (A) glycerol-3-
phosphate dehydrogenase (GPDH) and (B) two glycerol kinases (GK1 and GK2) of Spodoptera frugiperda with
other well-known insect. Sequence alignment used Clustal W program of MegAlign (DNASTAR, Version 7.0).
e abbreviation explanation and GenBank accession numbers of sPLA2 amino acids are listed in TableS2.
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Figure5. Phylogenetic analysis of glycerol biosynthesis genes including (A) glycerol-3-phosphate
dehydrogenase (GPDH) and (B) two glycerol kinases (GK1 and GK2) of Spodoptera frugiperda with other insect
species from dierent orders. e analysis was performed using MEGA6.06. Each node contains bootstrap
value aer 1000 replications to support branching and clustering. Accession numbers and the abbreviation
explanation are shown in TableS2.
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(B) (A)
Time (h) at 5°C
Relative mRNA expression level
(C) (D)
Figure6. Expression analysis of glycerol biosynthesis genes from Spodoptera frugiperda. RT-PCR analysis of Sf-GPDH, Sf-GK1, and
Sf-GK2 in dierent developmental stages (A, C, and E, respectively) and tissues (B, D, and F, respectively). e gels in (A, C, and
E) were cropped from various gels and were cleared with vertical white space. e full-length gels are included in Supplementary
Information (Fig.S2). EF1 was used to validate cDNA integrity. Dierent developmental stages included Egg (‘Eg’), larval instars
(ʻL1–L6ʼ), pre-pupa (‘PP’), pupa of female (ʻPFʼ), pupa of male (‘PM’), adult of female (ʻAFʼ) and adult of male (‘AM’). Dierent
tissues included hemocyte, fat body, midgut, and epidermis. (G) RT-PCR analysis of Sf-GPDH, Sf-GK1 and Sf-GK2 expression at low
temperature (5°C) at dierent exposure time (h). Agarose gel (1%) was used for electrophoresis. Each treatment was replicated three
times. Dierent letters above standard deviation bars indicate signicant dierence among means at Type I error = 0.05 (LSD test).
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Figure7. e eect of RNA interference (RNAi) specic to genes linked with glycerol biosynthesis, GPDH and GK of
Spodoptera frugiperda. All dsRNAs specic to target glycerol biosynthesis genes were constructed at ~ 300–400bp and injected
to each L5 larva at 3μg. Control RNAi (‘dsEGFP’) was injected with dsRNA specic to EGFP gene. (A) Quantitative real-time
PCR to monitor changes in mRNA levels of Sf-GPDH, Sf-GK1, and Sf-GK2 aer RNAi. EF1 was used to validate cDNA
integrity. (B) Chromatogram of HPLC that shows the eect of RNAi specic to glycerol biosynthesis genes, (48h post dsRNAs
injection) and RCH treatment (5°C for 6h), on glycerol content in hemolymph of h instar larvae. (C) Suppression of cold
tolerance aer RNAi treatment of either GPDH or GK1 or GK2. e glycerol content in hemolymph of h instar larvae was
measured aer 48h of dsRNAs injection. (D, E) Aer RNAi injection (48h post injection) and RCH treatment (5°C for 6h),
the larvae were incubated at 10°C for 1h and the mortality was recorded. Each treatment was replicated three times with 10
individuals per replication. Asterisks indicate signicant dierence between RCH and no RCH treatments (Type I error = 0.05,
LSD test). n.s. means no signicant dierence.
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Relative glycerol content in hemolymph of L5
dsS f-GK 1
dsSf-G K2
dsEGFP dsGPDH dsGK1dsGK2
Relative mortalityof treated L5 with dsRNAs (%)
n.s. n.s.
Figure7. (continued)
Table 1. Change polyol content in Spodoptera frugiperda h instar hemolymph in response to exposure
to 5°C. Each treatment was replicated three times with 10 individuals per replication. Dierent superscript
letters indicate signicant dierence between means for each polyol (Type I error = 0.05, LSD test). *Dierent
superscript letters indicate signicant dierence between means for each polyol (Type I error = 0.05, LSD test).
RNAi treatment groups Glycerol
(mM)/hemolymph of h instar Trehalose
(mM)/hemolymph of h instar
dsEGFP-noRCH 17.10 ± 0.98d*4.42 ± 0.10d
dsEGFP-RCH 44.07 ± 0.95a5.99 ± 0.13c
dsGPDH-RCH 22.58 ± 0.82c6.89 ± 0.08b
dsGK1-RCH 40.36 ± 0.30b5.70 ± 0.17c
dsGK2-RCH 6.08 ± 0.03e21.53 ± 0.23a
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their SCP by accumulating extracellular cryoprotectant including glycerol in their bodies. In compared to the
dsEGFP treatment group, the SCPs of larvae injected with dsGK1 were signicantly lower than to dsGPDH and
dsGK2 injected larvae (Table3).
Table 2. Changes in supercooling points of Spodoptera frugiperda aer rapid cold hardening treatment
(5°C for 6h). Each treatment was replicated three times with dierent individuals per replication. Dierent
superscript letters indicate signicant dierence between means for each SCP (Type I error = 0.05, LSD test).
*Dierent superscript letters indicate signicant dierence between means for each SCP (Type I error = 0.05,
LSD test).
Stage RCH treatment N SCP (°C)
Egg No 90 − 17.50 ± 0.56a*
Yes 90 − 17.80 ± 0.35a
L1 No 90 − 17.20 ± 1.40a
Yes 90 − 17.50 ± 0.64a
L2 No 60 − 14.60 ± 1.10a
Yes 60 − 15.80 ± 0.85a
L3 No 10 − 10.62 ± 0.59b
Yes 10 − 14.12 ± 0.73a
L4 No 5 − 12.15 ± 0.62b
Yes 5 − 14.15 ± 0.45a
L5 No 5 − 8.58 ± 0.93b
Yes 5 − 13.88 ± 0.48a
L6 No 5 − 8.35 ± 0.49b
Yes 5 − 13.39 ± 0.45a
Pre-pupae No 5 − 14.62 ± 0.67b
Yes 5 − 16.19 ± 0.57a
Pupae (male) No 5 − 18.47 ± 1.19a
Yes 5 − 18.87 ± 0.53a
Pupae (female) No 5 − 16.90 ± 0.35b
Yes 5 − 18.20 ± 1.12a
Adult (male) No 5 − 12.33 ± 1.20b
Yes 5 − 14.15 ± 0.58a
Adult (female) No 5 − 13.23 ± 1.52a
Yes 5 − 14.53 ± 0.82a
Table 3. Changes in supercooling points of Spodoptera frugiperda (third to sixth instar) aer RNAi and
rapid cold hardening treatment. All dsRNAs specic to target glycerol biosynthesis genes were constructed
at ~ 300–400bp and injected to each L5 larva at 3μg. Control RNAi (‘dsEGFP’) was injected with dsRNA
specic to EGFP gene. e supercooling point was measured aer RNAi injection (48h post injection) and
RCH treatment (5°C for 6h). Each treatment was replicated three times with 18 individuals per replication.
Dierent superscript letters indicate signicant dierence between means for each SCP (Type I error = 0.05,
LSD test). *Dierent superscript letters indicate signicant dierence between means for each SCP (Type I
error = 0.05, LSD test).
Stage RCH treatment N
SCP (°C)
dsEGFP dsGPDH dsGK1 dsGK2
L3 No 18 − 17.20 ± 0.53a* − 12.30 ± 0.87c − 15.76 ± 0.45b − 8.65 ± 0.44d
Yes 18 − 17.30 ± 0.51a − 13.70 ± 0.73b − 16.95 ± 0.34a − 9.75 ± 0.61c
L4 No 18 − 16.80 ± 2.40a − 10.24 ± 0.70b − 16.21 ± 0.42a − 7.88 ± 0.68c
Yes 18 − 17.70 ± 0.62a − 10.32 ± 1.12b − 16.80 ± 1.10a − 8.54 ± 0.48c
L5 No 18 − 14.10 ± 0.80a − 8.76 ± 0.92c − 12.76 ± 0.62b − 7.36 ± 0.74c
Yes 18 − 15.70 ± 1.85a − 10.23 ± 0.65b − 13.48 ± 0.74a − 9.87 ± 0.56c
L6 No 18 − 11.82 ± 0.32a − 7.94 ± 0.62c − 9.54 ± 0.76b − 7.24 ± 0.23c
Yes 18 − 13.02 ± 0.52a − 9.39 ± 0.45b − 10.88 ± 0.33b − 7.56 ± 0.36c
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Many insect species can develop cold-hardiness well below freezing temperatures, and various features of insect
cold-hardiness have been studied23,35. e most signicant part of acclimatization for cold resistance is low
temperature exposure22,36. Low-weight molecular molecules, oen known as cryoprotectant, such as polyols
and sugars, are produced during this procedure21. e most prevalent cryoprotectants include polyols (glycerol,
sorbitol, and manitol), sugars (glucose, trehalose, and fructose), and amino acids3740. High polyol concentrations
not only lower the temperature at which an insect’s body uids crystallize but also stabilize the state of proteins,
even when collected in relatively low concentrations41. Polyols regulate the amount of water accessible for freez-
ing, which reduces the amount of cell dehydration caused by extracellular freezing. ey protect biological
membranes and proteins from freezing-induced dehydration by preserving their structures41,42. In the present
work, the tolerance of FAW was analyzed by rapid cold hardening (RCH). In insects without diapause, RCH
is especially important for overcoming lethal cold shock by rapidly increasing cold tolerance20. RCH has been
induced in a variety of insects at temperatures ranging from 0 to 5°C30,4347. Glycerol production is divided into
two distinct pathways, depending on the insect. In Epiblema scudderiana (Clemens), a moth belongs to Tortrici-
dae family, polyol dehydrogenase catalyzes the reaction of glyceraldehyde with NADPH + H+ in one route48. e
other pathway converts dihydroxyacetone-3-phosphate to glycerol via GPDH/GK (S. exigua)30. Identication of
key genes associated with overwintering in Anoplophora glabripennis (Motschulsky) larva, a coleopteran species,
using gene co-expression network analysis, was demonstrated that, fatty acid desaturase, glycerol phosphate
dehydrogenase, glycerol kinase, and trehalose phosphate synthase were among the 15 genes implicated in the
control of antifreeze protectants49. We studied on GPDH and GK genes expression to investigate the glycerol
production pathway. In the FAW transcriptome, we discovered two GK isoforms and one GPDH isoform. It
was discovered that both genes expressed and associated with glycerol biosynthesis pathway. e whole Plutella
xylostella (Linnaeus) genome was used to predict four GKs and one GPDH50. e genome of FAW contains only
one type of GPDH, indicating that it is a unigene with a conserved biological function in metabolism. Because
we obtained these sequences from transcriptome data and there are likely no other endogenous genes of GPDH
and GK, we believe our expression and functional analysis are associated with these isozymes. GPDH and both
GK isoforms were discovered to be widely expressed in dierent studied tissues. As we know at low temperatures,
most gene expression decreases51. However, in 5°C, real-time PCR of cold-exposed larvae revealed that GPDH,
GK1, and GK2 were expressed at relatively high levels (Fig.6G). is suggests that these proteins are important
for cold tolerance to the low temperature by RCH. As found in other insects52,53, cold tolerance rose as acclima-
tization time increased, which could be in line with our ndings, that mRNA expression levels of analyzed genes
increased as incubation time increased (Fig.6G).
RNAi is a non-invasive way of delivering dsRNA into insects to knockdown specic gene expression5457. We
have shown that injecting RNAi is feasible and can suppress the transcription level of target genes in FAW larvae.
Our system conrmed the eective knockdown of three genes at the mRNA expression level.
eir expression was knocked-down by specic dsRNAs associated with glycerol biosynthesis genes. In
response to pre-exposure to a low temperature, this RNAi treatment reduced RCH and prevented glycerol
accumulation. According to the RNAi experiments, sorbitol dehydrogenase, trehalose-6-phosphate synthase,
and glycerol kinase are all involved in the overwintering stage of Chinese white pine larvae (Dendroctonus
armandi (Tsai and Li))58. Glycerol phosphorylation, which is essential for glycerol consumption, is catalyzed by
GK59,60. GK has a function in overwintering termination in Hyalophora cecropia (Linnaeus) eggs that accumulate
glycerol by converting glycerol to glycerol-3-phosphate for other intermediary metabolism61. In A. glabripennis
larvae, the gene expression level of glycerol kinase increased sharply at the midpoint of the overwintering stage,
and then declined at the latter, which corresponded to the change in glycerol content. e ndings suggest that
glycerol kinase is involved in the synthesis of glycerol, which could help this insect adapt to low temperatures49.
Because RNAi targeting GK1 and GK2 signicantly reduced glycerol accumulation in a 5°C pretreatment,
it was shown that both GKs catalyze the dephosphorylation of glycerol-3-phosphate to generate glycerol, as
reported earlier in S. exigua, a near Noctuidae species to FAW30,62. However, it was discovered that GK2 has a
greater eect on glycerol production based on RNAi data for mortality, glycerol accumulation, and HPLC results.
e signicant increase in Sf-GK2 expression vs Sf-GK1 shows that it has physiological signicance in RCH, as
evidenced by the RNAi functional study. P. xylostella GK1 showed a signicant increase in expression in response
to 5°C exposure vs other three isozyme of GKs50. In Bombyx mori (Linnaeus), at least three GK isozymes have
been discovered, but only one, GK3, appears to be connected to glycerol utilization63. e knockdown of the
target genes Sf-GPDH, Sf-GK1, and Sf-GK2 not only reduces their transcription levels but also aects larval
cold-tolerance capacity, leading to an increase in low-temperature mortality. e most obvious explanation for
these ndings is that these genes are necessary for overwintering larvae’s cold tolerance. As there is no existing
evidence of systemic spread in Lepidoptera64, we were unable to totally silence these three genes, however, the
partial knockdown had a clear eect on low-temperature mortality.
e hemolymph polyol analysis revealed that trehalose was the primary blood sugar, with a concentration of
4.42 mmol−1 in hemolymph and a slight increase with low temperature exposure (5.99 mmol−1 aer 6h at 4°C).
Trehalose titers in insect hemolymph are relatively high in general, but very considerably between insects (rang-
ing from 0.1 to 133 mmol−1)30. We detected 5.7mM of trehalose following dsGK1-RCH treatment, whereas the
titer increased considerably to 21.53mM following dsGK2-RCH treatment. is may be a compensating eect
of the glycerol depletion. However, we believe that in order to obtain more precise results, we need incorporate
trehalose(s) (which catalyzes the conversion of trehalose to two glucose monomers) into our future studies.
In conclusion, due to a lack of a diapause mechanism, FAW cannot overwinter in area with a cold winter,
despite the fact that they can disperse thousands of kilometers north during the growth season65,66. However,
in this study, RNAi investigation of two types of important genes linked to glycerol production and their eects
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Scientic Reports | (2022) 12:4129 |
on glycerol accumulation and insect mortality in response to low temperature pre-exposure, revealed that glyc-
erol is a substantial cryoprotectant in RCH in FAW. Increased glycerol concentrations may contribute to whole
animal freeze tolerance by enhancing cell survival by freeze-tolerant. However, each cryoprotectant may have
a distinct non-overlapping function and contribute to freeze tolerance through memchanisms distinct from
those of others with dierent potency. In addition, the permeability of dierent tissues to cryoprotectants can
be vary, aecting their ability to protect cells and this constituents. Supercooling data clearly demonstrated that
FAW can endure very low temperatures, and as a key agricultural pest, it may be able to become one of most
important migratory insect pests in Korea. To limit the impact of this pest, it is critical to create pest manage-
ment strategies and detecting systems. In addition, more research on migration behavior is needed to predict
source areas and migration times.
Materials and methods
Insect rearing, exposure temperatures and sample preparation. e larvae of FAW were obtained
from Frontier Agriculture Sciences (Newark, DE, USA) and used F4 and F5 generations, which were main-
tained in laboratory. ey were raised until pupation under laboratory-controlled conditions (26°C, 70% RH,
and a photoperiod of 14h:10h [L:D]). e larvae were fed an articial diet (Newark, DE, USA) during their
development67 and larval instar (L1-L5) were determined by head capsule sizes and molting times. e diet
was changed every day for larvae. ese larvae were grown in plastic containers with aerated lids measuring
40 × 20 × 15cm. From the third instar onwards, larvae were reared separately to prevent cannibalism. is was
carried out in Petri dishes (8.5cm diameter).
Bioinformatics to predict Glycerol synthase genes. e mRNA sequences for all genes were obtained
from a previous study of transcriptome analysis of whole body of FAW using TruSeq RNA Sample Prep Kit v2
(Macrogen, Seoul, South Korea). e predicted amino acid sequences were aligned using Clustal W program of
MegAlign (DNASTAR Version 7.0). e phylogenetic trees were constructed with Neighbor-joining method and
Poisson correction model (1,000 bootstrap repetitions to support branching clusters) using MEGA 7.0 soware
(www. megas owa re. net). Conserved domains of glycerol synthase genes were predicted using NCBI Conserved
Domain Database (www. ncbi. nlm. nih. gov/ cdd). UCSF Chimera (https:// www. cgl. ucsf. edu/ chime ra/) was used
for protein motif analysis and making 3D structure.
RNA extraction and RT-qPCR. RNAs samples were extracted from FAW larvae using Trizol reagent (Inv-
itrogen, Carlsbad, CA, USA)68. Aer RNA extraction, it was resuspended in nuclease-free water and quantied
using a spectrophotometer (NanoDrop, ermo Scientic, Wilmington, DE, USA). cDNA was then synthesized
from RNA (1μg) using RT PreMix (Intron Biotechnology, Seoul, Korea) containing oligo dT primer according
to the manufacturer’s instruction. All quantitative PCRs (qPCRs) in this study were determined using a Real
time PCR machine (CFX Connect Real-Time PCR Detection System, Bio-Rad, Hercules, CA, USA) and iQ
SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) according to the guideline of manufacture. e reaction
mixture (20 μL) contain 10 μL of iQ SYBR Green Supermix, 1 μL of cDNA template (100ng), and 1 μL each of
forward and reverse primers (TableS1) and 7 μL nuclease free water. RT-qPCR cycling began with 95°C heat
treatment for 10min followed by 40 cycles of denaturation at 94°C for 30s, annealing at 55°C temperature for
30s, and extension at 72°C for 20s. Expression level of EF1 as reference gene was used to normalize target gene
expression levels69 under dierent treatments. PCR products were assessed by melting curve analysis. Quantita-
tive analysis was performed using comparative CT (2−ΔΔCT) method70.
RCH bioassay. RCH was measured according to a previous method30. e each developmental stage from
eggs to adults was exposed to 5°C for 6h prior to − 10°C for 1h. For each treatment group, test individuals were
placed in a Petri dish (10 × 15mm). Aer 2h of recovery at 25°C aer cold treatment, the survival rates of all
developmental stages were determined. Aer gentle probing on the abdomen with a stick, autonomous move-
ment of individuals was the criterion for being categorized as alive. Hatching in the 25°C recovery state was used
to assess egg survival. Adult emergence in the 25°C recovery state was used to assess pupal survival.
SCP measurement. SCPs were measured using a thermocouple (BTM-4208SD, LT Lutron, Taipei, Taiwan)
to detect the release of the latent heat of fusion as body water froze, as described previously32,71. In SCP meas-
urement, all developmental stages of FAW were examined aer RCH treatment (exposed to 5°C for 6h prior
to 10°C for 1h). e thermocouples were kept in contact with the cuticle by putting the insect in a 1.5mL
tube and lling it with cotton wool to keep the insect and thermocouple together (FigureS1). ey were then
put in a styrofoam box (30 × 30 × 20cm), and the box was placed into a freezer at − 80°C. e cooling rate was
measured as 1°C min−1.
Glycerol analysis in FAW hemolymph. e glycerol content of the samples was determined using the
Glycerol Assay Kit (BioVision, Milpitas, CA, USA). We followed the manufacturer’s instruction for uorometric
measurements. In summary, a hemolymph from 10 h instar larvae (Day 1) was collected by cutting prologs
of the treated larvae and mixed with a 100 µL volume of anticoagulant buer (ACB). ACB was prepared with
186mM NaCl, 17mM Na2EDTA, and 41mM citric acid72. e ACB was adjusted to pH 8.0 by the addition of
NaOH. e resultant hemolymph was centrifuged at 13,500rpm for 10min at 4°C. e supernatant (100 µL)
were mixed with 100 µL of glycerol assay buer (GAB) (provided by the kit) for 10min on ice. e amount of 10
µL resulted supernatant (12,000rpm, 5min) was mixed with 86 µL GAB followed by 2 µL Probe (provided by
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Scientic Reports | (2022) 12:4129 |
the kit) and 2 µL glycerol enzyme mix (GEM) (provided by the kit) in a 96 well plate. e background control
mixture was prepared as described above without GEM. In this assay, glycerol in the presence of glycerol enzyme
mix is converted to an intermediate aer incubation at 37°C for 60min, which reduces a colorless probe to a
colored product with strong absorbance at 450nm.
Down-regulation of associated glycerol biosynthesis genes by RNA interference (RNAi). RNAi
was performed using dsRNA prepared with Megascript RNAi Kit (Ambion, Austin, TX, USA) according to the
manufacturer’s instruction and a previous method73. Partial segments were amplied with gene-specic primers
containing T7 promoter sequence at 5 end (TableS1). dsRNAs (dsGPDH, dsGK1 and dsGK2) were synthesized
at 37°C for 4h and then le at 70°C for 5min to inactivate T7 RNA polymerase. As control dsRNA (‘dsCON’),
300bp fragment of enhanced green uorescent protein (EGFP) was synthesized74. ree µg of dsRNA (1µg/
µL) was injected into each h instar larva with a Hamilton micro syringe. RNAi eciency was determined by
qPCR described above at 24, 48, 72, and 96h post injection. For each treatment, at least 10 larvae were used.
Each treatment was replicated three times.
Sample preparation and HPLC condition. Hemolymph of 10 h instar (Day 1) was collected by cut-
ting prologs of the treated larvae and mixed with a 100 µL volume of ACB. e resultant hemolymph was centri-
fuged at 13,500rpm for 10min at 4°C. e supernatant (500 µL) was transferred to a new 1.5mL tube, and then
the same volume of acetonitrile (ACN) was added into the tubes and were shaken for 15s. e tubes were incu-
bated at room temperature for 10min and then centrifuged as described above. e upper phase was collected
in a new 1.5mL tube. e previous step was repeated with the addition of 250 µL ACN to increase the purica-
tion. e nal supernatant was ltered out by 0.22µM syringe lters. e puried samples were directly used
for HPLC in Metabolomics Research Center for Functional Materials, Kyungsung Univeristy (Busan, Korea).
A reversed-phase HPLC connected to an evaporative light scattering detector (ELSD) (ELSD-LT II, Shimadzu,
Japan) was optimized for simultaneous determination of cryoprotectant. HPLC separation was achieved using a
Unison UK-Amino column (250 × 4.6mm). Water and acetonitrile were used as the mobile phase. e ideal ow
rate of 0.7mL/min and a proportion of acetonitrile of 90% over 30min were used to optimize the separation of
cryoprotectant using isocratic elution conditions. e temperature of the ELSD detector was set at 30°C with a
temperature of column oven at 64°C. Calibration curves were generated in GraphPad Prism by plotting the area
against cryoprotectant concentration. Averages of the areas for each standard were calculated and plotted against
the known concentrations.
Data analysis. All studies were performed in three independent biological replicates. Results were plotted
using Sigma plot 10.0. Means were compared by least squared dierence (LSD) test of one-way analysis of vari-
ance (ANOVA) using PROC GLM of SAS program75,76 and discriminated at Type I error = 0.05.
Received: 14 November 2021; Accepted: 3 March 2022
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is research was supported by funds (PQ20180A006 and B-1543086-2021-23) by Research of Animal and Plant
Quarantine Agency, Korea.
Author contributions
Y.P. and M.V. conceived the idea, designed the experiments; M.V. and Y.P. performed the experiments; M.V.
and Y.P. analyzed the data; M.V. and Y.P. co-wrote the manuscript; M.V. and Y.P. discussed the results and com-
mented on the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 022- 08174-4.
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... In other words, field trials are confirming the power of spray-induced gene silencing (SIGS) based bioinsecticides and subsequently promoting their transition to the market, although further improvement is needed to boost the field efficacy of SIGS and sustain RNAi technology (R odriguez et al., 2021;DeSchutter et al., 2021). Currently, promising results in gene lockdown or silencing with a recognized side effect in normal biological activities are being reported in S. frugiperda (Rodríguez-Cabrera et al., 2010;Vatanparast and Park, 2022), signifying its potential as a component of pest management. However, the sustainability of these technologies is threatened by increased field resistance in S. frugiperda (Tabashnik, and Carriere, 2017;Wang et al., 2017). ...
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The fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) is among the invasive insect pests that damages maize and sorghum, the high-priority crops in newly colonized agro-ecologies, including African contexts. Owing to the increasing infestation of the pest and the limitations of current conventional methods for its management, there is a call for discovering advanced pest management approaches. RNA interference (RNAi) is an emerging molecular tool showing flexible potential for the management of S. frugiperda. We conducted a search of the recent application of RNAi literature using Google Scholar and Mendeley to find advanced papers on S. frugiperda management using RNAi molecular tools that led to growth inhibition, developmental aberrations, reduced fecundity, and mortality, mainly by disruption of normal biological processes of the pest. Although efforts have been made to accelerate the utility of RNAi, many factors limit the efficiency of RNAi to achieve successful control over S. frugiperda. Owing to RNAi’s potential bioactivity and economic and ecological acceptability, continued research efforts should focus on improving its broad applicability, including field conditions. Screening and identification of key target genes should be a priority task to achieve effective and sustainable management of this insect via RNAi. In addition, a clear understanding of the present status of RNAi utilization in S. frugiperda management is of paramount importance to improve its efficiency. Therefore, in this review, we highlight the biology of S. frugiperda and the RNAi mechanism as a foundation for the molecular management of the pest. Then, we discuss the current knowledge of the RNAi approach in S. frugiperda management and the factors affecting the efficiency of RNAi application. Finally, the prospects for RNAi-based insect pest management are highlighted for future research to achieve effective management of S. frugiperda.
The fall armyworm (FAW), Spodoptera frugiperda (Smith), is known as a destructive moth pest of agricultural crops such as corn, rice, and sorghum. Recently, its damage threatens crops in Asian temperate regions due to its high migration ability. Two genetic types of FAW, a corn strain mostly found on corn and a rice strain associated with rice, have been reported. The objective of this study was to evaluate whether migrated corn-collected population of FAW could maintain its population on various seasonal plants including non-preferred Poaceae plants, with special emphasis on Jeju, Korea. Development-related parameters were compared after switching host plants including corn (C), Sudan grass (S), proso millet (M), and rye (R) when rearing FAW. Most developmental parameters showed dynamic changes according to sequential alternation of host plants with the progress of generations. When all R0 (net reproductive rate) values were scaled by the R0 in C to C alteration, the scaled values of other alterations were less than “1.0”, indicating less fitness than C to C alteration (R0 value: 0.57 for C to S, 0.49 for C to M in F3 generation; 0.82 for S to S, 0.65 for S to M, and 0.58 for S to C in F4 generation, 0.50 for M to M, 0.59 for M to R, and 0.83 for M to C in F5 generation). Although our results did not show that S. frugiperda adapted perfectively on new host plants, this pest was able to successfully reproduce up to five generations with non-preferred host plants. Results indicate that winter crops such as rye might be hosts for migrated corn-preferred populations of S. frugiperda during winter after breeding three to four cycles on other host plants before winter if weather condition is allowed for its survival.
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Repat (=response to pathogen) is proposed for an immune-associated gene family from Spodoptera exigua, a lepidopteran insect. In this gene family, 46 members (Repat1–Repat46) have been identified. They show marked variations in their inducible expression patterns in response to infections by different microbial pathogens. However, their physiological functions in specific immune responses and their interactions with other immune signaling pathways remain unclear. Repat33 is a gene highly inducible by bacterial infections. The objective of this study was to analyze the physiological functions of Repat33 in mediating cellular and humoral immune responses. Results showed that Repat33 was expressed in all developmental stages and induced in immune-associated tissues such as hemocytes and the fat body. RNA interference (RNAi) of Repat33 expression inhibited the hemocyte-spreading behavior which impaired nodule formation of hemocytes against bacterial infections. Such RNAi treatment also down-regulated expression levels of some antimicrobial genes. Interestingly, Repat33 expression was controlled by eicosanoids. Inhibition of eicosanoid biosynthesis by RNAi against a phospholipase A2 (PLA2) gene suppressed Repat33 expression while an addition of arachidonic acid (a catalytic product of PLA2) to RNAi treatment recovered such suppression of Repat33 expression. These results suggest that Repat33 is a downstream component of eicosanoids in mediating immune responses of S. exigua.
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Epoxyoctadecamonoenoic acids (EpOMEs) are epoxide derivatives of linoleic acid (9,12-octadecadienoic acid) and include 9,10-EpOME and 12,13-EpOME. They are synthesized by cytochrome P450 monooxygenases (CYPs) and degraded by soluble epoxide hydrolase (sEH). Although EpOMEs are well known to play crucial roles in mediating various physiological processes in mammals, their role is not well understood in insects. This study chemically identified their presence in insect tissues: 941.8 pg/g of 9,10-EpOME and 2,198.3 pg/g of 12,13-EpOME in fat body of a lepidopteran insect, Spodoptera exigua. Injection of 9,10-EpOME or 12,13-EpOME into larvae suppressed the cellular immune responses induced by bacterial challenge. EpOME treatment also suppressed the expression of antimicrobial peptide (AMP) genes. Among 139 S. exigua CYPs, an ortholog (SE51385) to human EpOME synthase was predicted and its expression was highly inducible upon bacterial challenge. RNA interference (RNAi) of SE51385 prevented down-regulation of immune responses at a late stage (> 24 h) following bacterial challenge. A soluble epoxide hydrolase (Se-sEH) of S. exigua was predicted and showed specific expression in all development stages and in different larval tissues. Furthermore, its expression levels were highly enhanced by bacterial challenge in different tissues. RNAi reduction of Se-sEH interfered with hemocyte-spreading behavior, nodule formation, and AMP expression. To support the immune association of EpOMEs, urea-based sEH inhibitors were screened to assess their inhibitory activities against cellular and humoral immune responses of S. exigua. 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) was highly potent in suppressing the immune responses. The addition of AUDA to a pathogenic bacterium significantly increased bacterial pathogenicity by suppressing host immune defense. In sum, this study demonstrated that EpOMEs play a crucial role in facilitating anti-inflammatory responses in S. exigua.
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Background: Anoplophora glabripennis (Coleoptera: Cerambycidae) is a major quarantine pest in forestry industry. It is widely distributed throughout many regions such as Asia, Europe, and North America, and has enormous destructive potential to forests. The larvae of A. glabripennis overwinter in a dormant state with strong cold tolerance, and whether the larvae survive the winter determines the population density in the following year. However, the molecular mechanisms of this process are not clear. Results: RNA sequencing (RNA-Seq) analysis of A. glabripennis larvae at five overwintering stages identified 6876 differentially expressed genes (DEGs). Among which, 46 functional genes that might respond to low temperature across the overwintering process were identified. Weighted gene co-expression network analysis revealed that the MEturquoise module was correlated with the overwintering process. The STPK, PP2A, DGAT, and HSF genes were identified as hub genes using the visualization of gene network. Besides, four genes related to sugar transport, gluconeogenesis and glycosylation were screened, which may involved in the metabolic regulation of overwintering larvae. Protein-protein interaction network indicated that ribosomal protein and ATP synthase may play an important role in connecting with other proteins. Fifteen hub genes were further validated by quantitative RT-PCR, and the results were consistent with RNA-Seq. Conclusion: This study demonstrates key genes that may reveal the molecular mechanism of overwintering in A. glabripennis larvae. The genes may be the potential targets to prevent larvae from surviving the cold winter by developing new biological agents using genetic engineering. This article is protected by copyright. All rights reserved.
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The fall armyworm, Spodoptera frugiperda (Smith, 1797), originated from tropical and subtropical America is one of sporadic agricultural pests in the world. Since the moth has high migration capacity, it rapidly expanded the world distribution such as Africa in 2016, India in 2018, and East-Asian countries in 2019. In Korea, this species was firstly found at maize fields of Jeju Island, in early June 2019, and subsequently detected at many counties of Jeolla-do and Gyeongsang-do in June and July 2019. The first invaded populations of S. frugiperda in Korea were genetically confirmed as one species, S. frugiperda by using a mitochondrial cytochrome oxidase subunit I (COI) gene, and analyzed to be comprised of two haplotypes (hap-1 and hap-2) each belonging to different clades. Among 31 COI sequences, the hap-1 sequence was predominant, accounting for 93.5%.
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Exploiting the RNA interference (RNAi) gene mechanism to silence essential genes in pest insects, leading to toxic effects, has surfaced as a promising new control strategy in the past decade. While the first commercial RNAi-based products are currently coming to market, the application against a wide range of insect species is still hindered by a number of challenges. In this review, we discuss the current status of these RNAi-based products and the different delivery strategies by which insects can be targeted by the RNAi-triggering double-stranded RNA (dsRNA) molecules. Furthermore, this review also addresses a number of physiological and cellular barriers, which can lead to decreased RNAi efficacy in insects. Finally, novel non-transgenic delivery technologies, such as polymer or liposomic nanoparticles, peptide-based delivery vehicles and viral-like particles, are also discussed, as these could overcome these barriers and lead to effective RNAi-based pest control.
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Sterile insect release technique (SIT) is effective for eradicating quarantine insects including various tephritid fruit flies. When SIT is used for fruit flies, it is challenging to remove females from sterile males due to oviposition-associated piercing damage. This study developed a sex transition technique by feeding double-stranded RNA (dsRNA) specific to a sex-determining gene, Transformer-2 (Zs-Tra2) of the striped fruit fly, Zeugodacus scutellata. Zs-Tra2 is homologous to other fruit fly orthologs. It is highly expressed in female adults. RNA interference (RNAi) of Zs-Tra2 by injecting or feeding its specific dsRNA to larvae significantly increased male ratio. Recombinant Escherichia coli cells expressing dsRNA specific to Zs-Tra2 were prepared and used to feed larvae to suppress Zs-Tra2 gene expression levels. When these recombinant bacteria were fed to larvae during the entire feeding stage, the test population was significantly male-biased. Some females treated with such recombinant E. coli exhibited mosaic morphological characters such as the presence of male-specific abdominal setae in females. This study proposes a novel technique by feeding dsRNA specific to Transformer-2 to reduce female production during mass-rearing of tephritid males for SIT
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The fall armyworm (Spodoptera frugiperda) is a pest of tropical origin which recently invaded Africa, the Far East and Australia. Temperature, therefore, plays an important role in its invasion biology, since this pest does not go into diapause. The aim of this study was to determine the development rate of S. frugiperda at different temperatures and to calculate the number of degree-days (• D) required for each stage to complete its development. This study was conducted at five different temperatures-18, 22, 26, 30 and 32 ± 1 • C. Larvae were reared individually in Petri dishes with sweetcorn kernels provided as food. The development rate of S. frugiperda increased linearly with increasing temperatures between 18 and 30 • C and larval survival was the highest between 26 and 30 • C. The optimal range for egg, larval and egg-to-adult development was between 26 and 30 • C. The optimum temperature with the fastest larval development rate and lowest mortality was at 30 • C. The pupal development period ranged between 7.82 and 30.68 days (32-18 • C). The minimum temperature threshold for egg and larva development was 13.01 and 12.12 • C, respectively, 13.06 • C for pupae and 12.57 • C for egg-to-adult development. Degree-day requirements for the development of the respective life cycle stages of S. frugiperda were 35.68 ± 0.22 for eggs, 204.60 ± 1.23 • D for larvae, 150.54 ± 0.93 • D for pupae and 391.61 ± 1.42 • D for egg-to-adult development.
Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), the cotton bollworm, is a destructive pest which is famous for its resistance to a variety of insecticides. RNA interference is a posttranscriptional gene silencing mechanism that has become a popular tool to control insect pests, triggered by double-stranded RNAs (dsRNAs). The effect of ingestion and injection delivery methods of dsRNA related to some protease genes including Trypsin (Ha-TRY39 and Ha-TRY96), Chymotrypsin (Ha-CHY), and Cathepsin L (Ha-CAT) on growth and development of H. armigera was investigated in this study. All protease genes encoded full ORFs and were expressed in all H. armigera larvae stages and tissues. In both injection and feeding bioassays, Ha-RNAi CHY's performance outperformed that of other protease genes. CHY enzyme activity in the midgut of larvae was significantly reduced after treatment with ds-HaCHY. Oral administration of ds-CHY also resulted in significant mortality of H. armigera larvae. However, because of the high RNase activity in the midgut lumen of lepidoptera, a large amount of dsRNA was needed to effectively kill instars of H. armigera. To reduce dsRNA degradation, bacterial expression and dsRNA formulation were used. After oral administration, it was toxic to H. armigera larvae. Before oral administration, bacterial cells were sonicated to increase dsRNA release. The RNA interference efficiency of sonicated bacteria was significantly increased, resulting in higher larval mortality when administered orally. All of these findings point to Ha-CHY as a new candidate for developing an effective dsRNA-based pesticide for H. armigera control.
Protection from parasitism by a virus Parasitoid wasps have developed myriad systems to overcome the defense mechanisms of their hosts as they lay their eggs in the bodies and eggs of targeted species. Gasmi et al . report how the host can fight back when infected by a virus that expresses a protein conferring resistance to the parasitoid. When members of the butterfly and moth family are targeted by wasps, a protein family has evolved that is horizontally carried by viruses—and sometimes is incorporated into the host genome—and impairs the ability of parasitoid offspring to fully develop and emerge. Characterizing the ability of this protein to protect hosts against specific parasites, the authors document an ongoing host-parasite evolutionary arms race. —LMZ
The fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) is a polyphagous pest with 353 plant species as its hosts, including maize, sorghum, cotton, and rice. Azadirachtin is one of the most effective botanical insecticides. The effect of azadirachtin against S. frugiperda remains to be determined. Here we report strong growth inhibition of azadirachtin on S. frugiperda larvae under either 1.0 or 5.0 μg/g azadirachtin. To explore the relevant mechanisms, the larvae fed with normal artificial diet and with 1.0 μg/g azadirachtin exposure for 3 days were collected as samples for RNA-Seq. RNA-Seq on S. frugiperda larvae under different treatments identified a total of 24,153 unigenes, including 3494 novel genes, were identified. Among them, 1282 genes were affected by 1.0 μg/g azadirachtin exposure, with 672 up-regulated and 610 down-regulated. The impacted genes include 61 coding for detoxification enzymes (31 P450s, 7 GSTs, 11 CarEs, 7 UGTs and 5 ABC transporters), 31 for cuticle proteins, and several proteins involved in insect chitin and hormone biosynthesis. Our results indicated that azadirachtin could regulate the growth of S. frugiperda by affecting insect chitin and hormone biosynthesis pathway. The enhanced expression of detoxification enzymes might be related to detoxifying azadirachtin. These findings provided a foundation for further delineating the molecular mechanism of growth regulation induced by azadirachtin in S. frugiperda larvae.