Fatty acid-binding protein gene is indispensable for molting process in Heortia vitessoides (Lepidoptera: Crambidae)

YE Qing-ya,LI Zhi-xing,CHEN Qing-ling,SUN Ming-xu,YIN Ming-liang,LIN Tong

College of Forestry and Landscape Architecture,South China Agricultural University,Guangzhou 510642,P.R.China

Abstract As intracellular fatty acid (FA) carriers,FA-binding proteins (FABPs) widely participate in the absorption,transport,and metabolism of FAs.It is a key protein in insect lipid metabolism and plays an important role in various physiological activities of insects.An FABP gene (HvFABP) was cloned from the transcriptional library of Heortia vitessoides Moore(Lepidoptera: Crambidae),and its expression patterns were determined using reverse transcription quantitative PCR (RTqPCR).Stage-and tissue-specific expression profiles indicated that HvFABP highly expressed from prepupal to adult stages and in larval midgut and adult wings.HvFABP expression may be induced through starvation,mRNA expression was downregulated at 24 and 48 h and upregulated at 72 h after starvation.Furthermore,20-hydroxyecdysone can induce the upregulation of its expression.RNA interference-mediated silencing of HvFABP significantly inhibited HvFABP expression,resulting in delayed development,abnormal molting or lethal phenotypes,and a significantly reduced survival rate.These results indicate that HvFABP plays a key role in the molting of H.vitessoides.

Keywords: fatty acid-binding protein,starvation,20-hydroxyecdysone,RNA interference,Heortia vitessoides Moore

1.Introduction

Fatty acid-binding proteins (FABPs) belong to an ancient genetic family,currently known as intracellular lipid-binding proteins (iLBPs),which originated nearly 930 million years ago (Schaapet al.2002).iLBPs can bind long-chain FAs,retinoids,or other hydrophobic ligands.iLBPs that bind long-chain FAs are called FABPs,which are abundant intracellular proteins with low molecular weight (Marcelinoet al.2006;Liuet al.2008).FABPs are expressed widely in vertebrates and invertebrates (Haunerland and Spener 2004).FAs play significant roles in energy metabolism and signaling.In mammals,FAs provide the primary fuel for energy production by the heart and skeletal muscles during rest as well as moderate and sustained exercise.The expression of various genes is regulated by FAs in either positive or negative way (Dupluset al.2000).FABPs are considered the important carriers of intracellular FAs (Haunerlandet al.1992).These proteins facilitate lipid transport to specific organelles in the cell for various purposes,such as the lipid droplet for storage(Furuhashi and Hotamisligil 2008).They increase the solubility of FA and actively transport FA from the plasma membrane to sites of FA oxidation (mitochondria and peroxisomes),sites of FA esterification into triacylglycerols or phospholipids,or the nucleus,possibly for regulatory functions (Zimmerman and Veerkamp 2002).The FABP family members in mammals include A-FABP (adipocyte),E-FABP (epidermal),I-FABP (intestinal),L-FABP (liver),H-FABP (heart),B-FABP (brain),Il-FABP (ileal),M-FABP(myelin),T-FABP (testis),and FABP12 (retina) (Liuet al.2008;Storch and Mcdermott 2009).The sequence similarity of amino acid varies across the FABP family members by approximately 70%,nevertheless,their three-dimensional structures are highly conserved β-barrel structure with ligand-binding cavities (Schaapet al.2002;Zimmerman and Veerkamp 2002;Marcelinoet al.2006).

The first insect FABP was discovered in the flight muscles of the adult desert locustSchistocerca gregaria(Haunerland and Chisholm 1990).Two abundant FABPs(MFB1 and MFB2) were isolated from the midgut of larvalManduca sexta(Smithet al.1992).The expression of SlFABP1 protein in the midgut tissue ofSpodoptera liturais upregulated during feeding and inhibited during starvation,and SlFABP1 is indispensable to feed (Huanget al.2012).FABPs affect insect physiological metabolism by regulating intracellular FAs.FABPs found in the flight muscles of locusts are strictly adult-specific proteins that provide sustained energy for long-distance flight activities through participating in the uptake and transport of lipids(Haunerlandet al.1992;Zhang and Haunerland 1998;Rajapakseet al.2019).InDrosophila melanogaster,FABP is involved in the regulation of sleep and longterm memory consolidation (Gerstneret al.2011a,b).FABP affects lipid accumulation,which is an important part of the diapause preparatory program and is crucial for insect diapause (Sim and Denlinger 2009;Tanet al.2017).FABPs produced byAphidius erviteratocytes play complementary roles in the nutritional exploitation of the host (Falabellaet al.2005;Cacciaet al.2012).FABP may also be involved in the caste division ofApis mellifera(Evans and Wheeler 1999).Thus,the function of FABP is diverse and may vary across insects.

Heortia vitessoidesMoore (Lepidoptera: Crambidae)is an oligophagous pest found in tropical and subtropical areas.It causes serious damage toAquilaria sinensiswhich is a precious tree species,and leads to severe economic loss (Qiaoet al.2012;Wanget al.2019).Although FABP has been systematically studied in mammals,studies in insects are limited;to the best of our knowledge,no relevant study has exploredH.vitessoidesFABP (HvFABP).This study investigated the expression pattern ofHvFABPand explored its transcription pattern under starvation and 20-hydroxyecdysone (20E)treatment.Also,in this study,we silenced this gene with RNA interference (RNAi) to determine the important role of FABP in insect metamorphosis and provide theoretical evidence for the application of FABP-based insecticides.

2.Materials and methods

2.1.Experimental insects and sample treatments

Heortia vitessoideswas reared in a laboratory at 26°C,with a relative humidity of 70-75% and a photoperiod of 14 h light and 10 h dark.The larvae were fed with freshA.sinensisleaves,and mature larvae were transferred to the soil with a relative humidity of 12% to pupate until they emerged into adults.Whole bodies of the first to fifth instar larvae (L1-L5),prepupae (PP),pupae (P),and adults (A) were collected to detect stage-specific expression profiles ofHvFABP.Fifth instar larval and adult tissues were dissected and collected to detect tissue-specific expression profiles ofHvFABP.In the starvation experiment,1-day-old fifth instar larvae (L5D1)were randomly selected as the experimental group.They were starved for 72 h and sampled every 24 h (three replicates).All samples were stored at -80°C.

2.2.Preparation and injection of 20E

We purchased 20E from Shanghai Yuanye Biotechnology Co.,Ltd.(Shanghai,China).This was diluted to 10 mg mL-1of dimethyl sulfoxide and 1 μg μL-1of 1× phosphatebuffered saline (PBS).The injection volume was 1 μL,and the injection site was the area between the membrane of the seventh and eighth segments of fifth instar larvae.Each group comprised 15 20E-injected larvae,and each experiment included three biological and three technical replicates.PBS (1×) of the same volume was used as the control treatment.Insects were collected 72 h after injection and stored at -80°C.

2.3.Sequence characterization and phylogenetic tree analysis

A full-length FABP cDNA was obtained from theH.vitessoidestranscriptome (SRX3035102) (Chenget al.2017) band identified through BLAST search in the GenBank database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).cDNA sequences of FABP open reading frames (ORFs)were acquired using ORF finders (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).Corresponding gene-specific primer pairs were designed to amplify the FABP ORF for sequence verification (Table 1).Corresponding pairs ofHvFABP-specific primers were designed using Primer Premier 5.0 (Premier Biosoft International,Palo Alto,CA,USA) to verify the sequences.PCR amplification conditions were as follows: 95°C for 5 min and 34 cycles of 95°C for 30 s,58°C for 30 s,and 72°C for 2 min,andthen 72°C for 10 min.The PCR product was gel-purified and ligated into a pClone007 simple vector (Tsingke Bio.,Guangzhou,China),transformed intoEscherichia coliDH5α-competent cells (TaKaRa Bio.,Otsu,Japan),and sequenced (Tsingke Bio).The physicochemical properties of FABP amino acid sequences were predicted using the ExPASy server (http://web.expasy.org/compute_pi/).MAFFT version 7 (https://mafft.cbrc.jp/alignment/server/)was used to align multiple sequences.Phylogenetic tree analysis was performed using MEGA 7.0viathe neighborjoining method and a bootstrap value of 1 000 times.

Table 1 Primers used for RT-qPCR and synthesis of dsFABP and dsGFP1)

2.4.RNA extraction and cDNA synthesis

The E.Z.N.A.Total RNA Kit II (Omega Biotec,Norcross,GA,USA) was used to extract total RNA according to the manufacturer’s instructions.The quality of RNA samples was evaluated through agarose gel electrophoresis.Total RNA (2 μg) was used to synthesize first-strand cDNA using the PrimeScript?RT Reagent Kit with gDNA Eraser(TaKaRa BioInc.,Japan) and stored at -20°C.

2.5.Reverse transcription quantitative PCR

Total RNA was extracted from all stored insect samples,and first-strand cDNA was synthesized using the PrimeScript?RT Reagent Kit with gDNA Eraser.This cDNA was then used as a template for reverse transcription quantitative PCR (RT-qPCR).The primers used are shown in Table 1.qPCR was performed using a LightCycler?Real-time PCR System (Roche Diagnostics,Indianapolis,IN) and TB Green? Premix ExTaqTM(TaKaRa Bio).β-actinwas used as an internal reference gene (Chenget al.2018),and the negative control received sterile ultrapure water.cDNA samples were derived from three biological replicates and three technical replicates using the 2-ΔΔCtmethod on the basis of the Ct value obtained at the end of the reaction (Livak and Schmittgen 2001).

2.6.Preparation of double-stranded RNA and injection

All reagents used for double-stranded RNA (dsRNA)synthesis in RNAi experiments were derived from the T7 RiboMAXTMExpress RNAi System (Promega,Madison,WI,USA).Primers with T7 polymerase promoter sequences were designed for conventional PCR to obtain DNA templates.HvFABPand green fluorescent protein were subsequently used in a transcription reaction with T7 RNA polymerase to generate dsFABP(293 bp) and dsGFP(400 bp) fragments.dsGFPwas used as a negative control for the nonspecific effects of dsRNA.The DNA template was removed,followed by dsRNA annealing and single-stranded RNA digestion using nuclease.dsRNA was purified according to the manufacturer’s purification protocol (Promega).After purification,dsRNA was dissolved in nuclease-free water.In addition,NanoDrop 2 000 Spectrophotometer(Thermo Fisher Scientific,Waltham,MA,USA) was used for quantification of dsRNA,which was then confirmed through 1.5% agarose gel electrophoresis to ensure purity and integrity.All samples were stored at -80°C.

The solution containing the dsRNA ofHvFABP(dsHvFABP) was diluted to 4 μg μL-1,and 1 μL dsHvFABPwas injected into abdominal cavity at the lateral internode between the seventh and eighth segments of each L5D1 larva with a FemtoJet(Eppendorf,Hamburg,Germany).The control group received dsGFPat the same concentration and volume.At least 40 larvae were included in each group.Survival rates and phenotypic changes were recorded.The transcription levels ofHvFABPat 12,24,36,48,and 72 h were detected using RT-qPCR.Three biological repeats per treatment were used to evaluate the efficiency of RNAi treatment.

2.7.Statistical analysis

Excel (Microsoft) was used for preliminary statistical analysis.Experimental data were presented as the mean±standard error (SE) of three independent replicates.SPSS version 18.0 (IBM,Armonk,NY,USA)was used for statistical analysis.Significance analysis of the data between two samples was performed usingt-test,and the data among multiple samples were analyzed using one-way analysis of variance (ANOVA) and Tukey’s test.P＜0.05 were considered statistically significant.

3.Results

3.1.Sequence analysis of HvFABP and phylogenetic analysis

A putativeFABPgene with a complete ORF was identified from the transcriptome library ofH.vitessoidesand namedHvFABP(GenBank accession number OK267275).The cDNA sequence was 798 bp in length.The ORF ofHvFABPwas 399 bp length and encoded a 132-amino acid protein with a theoretical molecular mass of 14.82 kDa and a predicted isoelectric point of 5.62 (Fig.1).The deduced amino acid sequence ofHvFABPwas such sequences of other insects such asBombyx mori(FABP1/FABP2,90.91%/35.07%,identity),Papilio xuthus(84.85% identity),Eumeta japonica(69.70% identity),Helicoverpa zea(33.33%identity),S.gregaria(45.93% identity),Harpegnathos saltator(27.94% identity),andDrosophila yakuba(52.27% identity) (Fig.2).The phylogenetic tree showed a clear separation of the insect FABP family from the vertebrate FABP family,which included FABPs from human (Homo sapiens) and mouse(Rattus norvegicus).In the insect part of the tree,the relationships among Diptera,Lepidoptera,Orthoptera,Hymenoptera,and Coleoptera were shown.HvFABPwas most closely related toBmFABP1,and the bootstrap value was 88 (Fig.3).

Fig.1 Nucleotide and deduced amino acid sequences of HvFABP.The start codon ATG and stop codon TAA are marked with the dotted line.

Fig.2 Sequence alignment of HvFABP with insect homologs.The amino acid residues that are identical in all sequences are shown in dark-gray shadow,whereas light-gray shadow indicates at least 75% identical amino acids in all sequences.The aligned sequences are the predicted amino acid sequences of FABPs from Heortia vitessoides (HvFABP GenBank accession no.OK267275),Bombyx mori (BmFABP1/2,BAE96009.1/BAE96010.1),Papilio xuthus (PmFABP,NP_001298777.1),Eumeta japonica (EjFABP,GBP00683.1),Helicoverpa zea(HzFABP,AAC25674.1),Schistocerca gregaria (SgFABP,AAK20174.1),Harpegnathos saltator (HsFABP,EFN85977),and Drosophila yakuba (DyFABP,XP002097988).

Fig.3 The phylogenetic tree of HvFABP was constructed using the neighbor-joining method;the GenBank accession no.of protein sequences used is shown in the corresponding brackets.The exogroups are from the mammals Homo sapiens and Rattus norvegicus.The two Fatty acid-binding proteins (FABPs) of Bombyx mori are BmFABP1 (BAE96009.1) and BmFABP2(BAE96010.1).Bootstrap analyses of 1 000 replications are shown;and node support values of ＜70% are not shown.

3.2.Spatial and temporal expression of HvFABP

Larval tissue-specific expression analyses revealed thatHvFABPtranscripts were widely produced in all detected tissue,and the expression levels varied significantly across larval tissues.HvFABPwas expressed at the highest level in the midgut (Fig.4-A).Adult tissuespecific expression analyses revealed thatHvFABPtranscripts were widely expressed in all detected tissues,with the highest expression level noted in wings (Fig.4-B).Stage-specific expression analyses revealed thatHvFABPtranscript expression levels were low during the entire larval stage (L1-L5) but increased before entering the pupal stage.HvFABPexhibited relatively high mRNA expression in the prepupal-to-adult molting stage (Fig.5).

Fig.4 Relative expression levels of HvFABP in larval and adult tissues.A,relative expression levels of HvFABP in larval tissues obtained from head (HE),epidermis (EP),fat body (FB),foregut (FG),midgut (MG),and hindgut (HG).B,relative expression levels of HvFABP in adult tissue from HE,thorax (TH),abdomen (AB),wing (WI),and foot (FO).Data are the mean±SE of three biological repeats.Different letters indicate significant differences at P＜0.05 (one-way ANOVA followed by Tukey’s test).

Fig.5 Relative expression levels of HvFABP during different developmental stages.The stages collected were first to fifth instar larvae (L1-L5),prepupae (PP),pupae (P),and adults (A).Data are the mean±SE of three biological repeats.Different letters indicate significant differences at P＜0.05 (one-way ANOVA followed by Tukey’s test).

3.3.Expression of HvFABP after starvation treatment

The relative expression ofHvFABPin fifth-instar larvae was detected after 24,48,and 72 h of starvation.The relative expression ofHvFABPwas inhibited after 24 and 48 h of starvation.However,the relative expression ofHvFABPincreased significantly after 72 h of starvation(Fig.6).

Fig.6 Expression profiles of HvFABP after 72 h of starvation.Expression levels at 24,48,and 72 h after starvation were normalized compared with those at 24,48,and 72 h after feeding (control).Data are the mean±SE of three biological repeats.＊＊,P＜0.01 (t-test).

3.4.Expression of HvFABP after 20E injection

Fifth instar larvae received 20E injection or the same volume of 1× PBS,and the expression levels ofHvFABPwere detected using RT-qPCR.At 48 and 72 h after the injection of 20E,the expression levels were significantly higher than those noted in the concurrent control,and comparisons within the experimental group also showed an upward trend (Fig.7).

Fig.7 Expression profiles of HvFABP after 20-hydroxyecdysone(20E) treatment.Expression levels after 20E injection were normalized compared with those after 1× PBS injection (control).Data are the mean±SE of three biological repeats.＊,P＜0.05;＊＊,P＜0.01 (t-test).

3.5.Efficiency and phenotypic analysis after RNAi

Each fifth instar larvae was injected with 1 μL dsHvFABP(4 μg μL-1).Total RNA was extracted from dsRNAinjected larvae,andHvFABPexpression after injection was detected using RT-qPCR.The expression level ofHvFABPwas relatively reduced,and its highest expression levels were lower than the corresponding dsGFPexpression levels.TheHvFABPexpression level was the lowest at 36 h after injection (Fig.8-A).After dsHvFABPinjection,the survival rate of the larva to the pupal stage was 65.7%,and that of the pupal to the adult stage was 31.3%,which was significantly lower than the rates of the control group (Fig.8-B).Compared with the controls injected with dsGFP,individuals injected with dsHvFABPshowed significant developmental abnormalities or lethal phenotypes.Delayed larval development was observed by 1 or 2 days,and the larvae were thinner than those in the control group (Fig.8-C).Nearly 50% of the pupae were abnormal or dead.The old cuticle had trouble shedding,particularly that on the abdomen,and was even trapped within exuviae until death.Notably,after dsHvFABPinjection,adult individuals emerged with deformed wings,as manifested through wrinkles on the wing membrane.Lethal phenotype occurs in one-third of the emerging adults.

Fig.8 Effects of HvFABP RNAi on larval to pupal and pupal to adult molting.A,dsRNAi efficiency decreases HvFABP expression levels at 12,24,48,36,and 72 h.＊,P＜0.05;＊＊,P＜0.01 (t-test).B,survival,from fifth-instar larval stage to the adult stage,rates of insects after dsHvFABP injection(＊,P＜0.05,Kaplan-Meier survival analysis with log-rank test).C,developmental abnormalities or lethal phenotypes due to the RNAi treatment of HvFABP.Data are the mean±SE of three biological repeats.

4.Discussion

The proposed functions of FABPs include the long-chain FA uptake and transport in cells,interactions with other transporters,gene transcription regulation,and cellular protection (Liuet al.2008).FABPs indirectly participate in the regulation of FA-mediated gene expression (Yuet al.2013).Although FABPs have been systematically studied in mammals,such studies are scarce in insects.In this study,an FABP gene (HvFABP) was identified from the transcriptome of adultH.vitessoides.The deduced amino acid sequence ofHvFABPwas highly homologous withBmFABP1which plays an important role in lipid accumulation (Akiduki and Imanishi 2007),but less homologous withBmFABP2from the same species.Phylogenetic tree analysis revealed the relationship among the FABPs of different orders of insects,and FABPs of Lepidoptera were clearly divided into two clusters.HvFABPhad a high degree of homology with FABPs from the same cluster of Lepidoptera,Diptera,and Orthoptera,but low homology with FABPs from another cluster of Lepidoptera,Coleoptera,and Hymenoptera.This is consistent with the results of FABP grouping in phylogenetic tree analysis performed by Huanget al.(2012),which shows possible differences in the gene function between the two clusters of Lepidoptera FABPs.

Expression patterns vary across developmental stages and insect species (McPhee and Baehrecke 2009).Expression profiles analyses revealed that the expression ofHvFABPwas higher in the larval midgut and adult wings than in other tissues,implying that this protein functions in the midgut and wings.High expression in the midgut was also observed in the larval tissue ofS.litura(Huanget al.2012;Wenet al.2020) andM.sexta(Smithet al.1992)and showed significant midgut-specific expression.Midgut tissue is the main digestive organ of insects,and its functions include secreting digestive enzymes,absorbing nutrients,and processing lipids (Smithet al.1992).HighHvFABPexpression in the midgut implies thatHvFABPmay be involved in lipid uptake and transport from food.InS.gregaria,SgFABPis highly expressed in the flight muscles of adults,which provides sustained energy for flight activities and is regulated by hormones (Haunerlandet al.1992).HighHvFABPexpression in wings implies that it may have a similar effect.The expression levels ofHvFABPwere higher during the prepupal-adult stage and lower during the larval stage.InApis cerana cerana,AccFABPshowed relatively high expression levels during prepupal-adult (20-day-old) stages (Yuet al.2013),similar to the expression profiles ofHvFABP.InS.litura,the peak expression of SlFABP2 protein in the midgut occurs during the prepupal stage,during which the larvae stop feeding and use the substances and energy inside the body to reconstruct pupal and adult structures (Wenet al.2020).InH.vitessoides,HvFABPexpression significantly increased during the prepupal stage,and mature larvae stopped feeding during the prepupal stage.Lipid metabolism can provide essential energy for insect growth and development in the nonfeeding state.This expression profile noted in the present study might have been because of the need for more energy for development during the prepupal-to-adult stage,and FABPs are the primary substrates for energy production.As an important lipid carrier,highHvFABPexpression may contribute to the transport and utilization of FAs in the prepupal and pupal stages.

In mammals andCaenorhabditis elegans,starvation mediates the induction of genes involved in β-oxidation and FABP production;these genes produce proteins that convert stored fat into energy (Van Gilstet al.2005).During the normal life cycle,larval feeding is interrupted by molting or sometimes by the lack of food and then mobilizes the reserves to provide energy for molting or finding a new plant (Ziegler 1991).Previous studies have shown that starvation leads to extra larval stages or earlier pupation (Fischer and Fiedler 2001;Munyiriet al.2003;Munyiri and Ishikawa 2005).Starvation affects FABP expression inS.litura(Huanget al.2012;Wenet al.2020).The expression of SlFABP1 protein was downregulated by starvation (Huanget al.2012),whereas that of SlFABP2 protein was induced by starvation (Wenet al.2020).FABP may assist in the absorption and transport of FAs in the midgut,which is the main organ for digesting food in insects.In this study,HvFABPexpression was inhibited at 24 and 48 h after starvation,which indicates thatHvFABPcould be involved in the absorption and transport of lipid from food.After 72 h,the mature larvae entered the prepupal stage and stopped feeding.

Reducing the abundance of FABP transcripts during the preparation for diapause substantially decreases lipid accumulation,and FABP plays a crucial role in accumulating nutrients required for diapause (Tanet al.2017).In locust flight muscles,when metamorphosis is blocked through treatment with ecdysone antagonists,the insects remain in their final nymphal stage for several weeks.FABP is not expressed in these overage nymphs,suggesting that FABP expression requires final molting(Haunerland 1994).InB.mori,BmFABP1induces lipid accumulation,and ecdysone or its analogs are more likely to activate theBmFABP1promoter indirectlyviatranscription factors (Akiduki and Imanishi 2007).In the present study,fifth-instar larvae were treated with 20E,and the expression level ofHvFABPincreased significantly after 48 and 72 h of treatment,indicating thatHvFABPis regulated by 20E.

RNAi has become a widely used reverse genetic tool to knock out and analyze the function of genes and may contribute to new strategies for the selective control of agricultural pests (Whyardet al.2009;Wynantet al.2012).The degree of sensitivity to RNAi varies strongly across insect species (Tereniuset al.2011).RNAi shows a high silencing effect inH.vitessoides(Lyuet al.2019).In this study,the expression level ofHvFABPwas minimized after 36 h of RNAi,suggesting that RNAi can effectively and significantly reduceHvFABPexpression.Adverse effects onH.vitessoidesindividuals were observed after dsHvFABPinjection.These adverse effects included delayed development,ecdysis disorder,and wrinkled wing formation.Furthermore,prepupae with knocked-outHvFABPwere thinner than the control group,which might have resulted from the limited of lipid accumulation.Silencing FABP with RNAi can significantly reduce lipid accumulation in diapause-destined individuals and inhibit hypertrophy of the fat body (Tanet al.2017).BmFABP1which had the highest homology withHvFABPwas confirmed to be a positive relationship between the expression level ofBmFABP1and the quantity of lipid(Akiduki and Imanishi 2007).After RNAi treatment,the rate of survival during the larval-to-pupal and pupal-toadult molting stages decreased significantly,indicating thatHvFABPsilencing considerably reduced the number of successful pupation and eclosion ofH.vitessoides.In the nonfeeding stage,the main energy substrate is the lipid stored in the body,which is mainly used to reconstruct the structure of pupal and adult structures.We hypothesized thatHvFABPsilencing affects the construction of new epidermis,resulting in a significant reduction in the survival rate during the larval-to-pupal and pupal-to-adult molting stages and the failure to shed the old cuticle -the insects strapped within their exuviae until they die.Thus,HvFABPis indispensable for the larval-topupal and pupal-to-adult molting stages ofH.vitessoides.Interestingly,the lethal phenotype ofH.vitessoidesinjected with dsHvFABPwas similar to that of the insects injected with dsRNA with chitin deacetylases (Wanget al.2019).Chitin deacetylases are involved in epidermal chitin degradation and remodeling (Xiet al.2014).HvFABPmay be directly or indirectly involved in insect molting.Taken together,all evidence support the view thatHvFABPplays a key role in molting and is regulated by ecdysone.

5.Conclusion

This study identified and characterized FABP fromH.vitessoides.HvFABPis present and expressed inH.vitessoidesat different developmental stages and in different larval and adult tissues.HvFABPexpression is high in the prepupal-to-adult molting stage in the midgut tissue of larvae and wings of adults.After 72 h of starvation,HvFABPexpression was upregulated.HvFABPexpression was induced by 20E injection.RNAimediatedHvFABPsilencing significantly suppressedHvFABPexpression,resulting in developmental abnormalities or lethal phenotypes and a sharp decline in survival during the larval-to-pupal and pupal-to-adult molting stages.These results suggest thatHvFABPis an indispensable gene in the metamorphosis ofH.vitessoidesand participates in the molting process.This study expands the existing knowledge on FABP in insects and also provides a reference for RNAi targets for insect control.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (32070012).

Declaration of competing interest

The authors declare that they have no conflict of interest.