International Journal of Molecular Sciences

Article De Novo Assembly of the Asian Psyllid (: ) Transcriptome across Developmental Stages

Chunxiao Yang 1,2,3, Da Ou 2, Wei Guo 2, Jing Lü 2, Changfei Guo 2, Baoli Qiu 2,* and Huipeng Pan 2,*

1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou 510642, China; [email protected] 2 Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, South China Agricultural University, Guangzhou 510642, China; [email protected] (D.O.); [email protected] (W.G.); [email protected] (J.L.); [email protected] (C.G.) 3 State Key Laboratory for Biology of Plant Diseases and Pests, Chinese Academy of Agricultural Sciences, Beijing 100081, China * Correspondence: [email protected] (B.Q.); [email protected] (H.P.)

 Received: 21 May 2020; Accepted: 12 July 2020; Published: 14 July 2020 

Abstract: Asian citrus psyllid Diaphorina citri Kuwayama is an important economic pest of citrus, as it transmits Candidatus Liberibacter asiaticus, the causative agent of huanglongbing. In this study, we used RNA-seq to identify novel genes and provide the first high-resolution view of the of D. citri transcriptome throughout development. The transcriptomes of D. citri during eight developmental stages, including the egg, five instars, and male and female adults were sequenced. In total, 115 million clean reads were obtained and assembled into 354,726 unigenes with an average length of 925.65 bp and an N50 length of 1733 bp. Clusters of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes analyses were conducted to functionally annotate the genes. Differential expression analysis highlighted developmental stage-specific expression patterns. Furthermore, two trehalase genes were characterized with lower expression in adults compared to that in the other stages. The RNA interference (RNAi)-mediated suppression of the two trehalase genes resulted in significantly high D. citri mortality. This study enriched the genomic information regarding D. citri. Importantly, these data represent the most comprehensive transcriptomic resource currently available for D. citri and will facilitate functional genomics studies of this notorious pest.

Keywords: Diaphorina citri; transcriptome annotation; developmental stage; differential expression; trehalase

1. Introduction The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is one of the most destructive pests of citrus, due primarily to the fact of its role as the vector of Candidatus Liberibacter asiaticus (CLas) which, worldwide, causes the highly destructive citrus disease huanglongbing (HLB) [1,2]. Huanglongbing is one of the most lethal diseases against citrus and, currently, there is no cure. Diaphorina citri was first described in Taiwan Province in 1907 [3], and the infectious nature of HLB was described in Southern China in 1956 [4]. Large populations of D. citri in HLB-endemic areas often result in high incidences and spread of HLB. Controlling D. citri to reduce its availability as a vector of CLas has been a pivotal global strategy to restrict the spread of HLB. The recent rapid spread of HLB in the Americas has motivated extensive research aimed at improving the understanding of D. citri biology, ecology, and management tactics [5].

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Several factors contribute to the status of D. citri as a serious pest species, including adaptation to a wide range of host plants [6], a short life cycle and high fecundity [7], adaptation to growth at a wide range of temperatures [8,9], high dispersal ability [5], and high capacity to evolve resistance to different types of insecticides [5,10–12]. Intense use of insecticides has led to the development of high levels of insecticide resistance in D. citri populations [12]. Therefore, the development of novel highly specific and environmentally friendly methods must be considered for controlling this pest. RNA interference (RNAi) technology is a powerful tool for functional studies that can be used to selectively suppress the expression of target genes both in vitro and in vivo [13–18]. RNA interference has been widely explored as an alternative to conventional management methods for controlling insect pests [15–18]. For example, bacterially expressed double-stranded RNA (dsRNA) from the lesswright (lwr) gene of Henosepilachna vigintioctopunctata was applied to eggplant leaves and caused 88% mortality for the 1st larvae of H. vigintioctopunctata, 66% mortality for the 3rd instar larvae, and 36% mortality to the adults after 10 d, 10 d, and 14 d of application, respectively [15]. The genome of D. citri has been sequenced in recent years (GCA_000475195.1). In addition, transcriptomes of the terminal abdomen and antennae of adult male and female D. citri (SRX1330478–SRX1330481), those of CLas-free and CLas-infected D. citri nymphs and adults (SRX525230, SRX525218, SRX525209, SRX525152), and those encompassing the three main life stages of D. citri (egg, nymph/larva, and adult) have also been sequenced [19]. Diaphorina citri undergoes gradual metamorphosis and, as noted, has three differentiated life stages. The metamorphic changes are accompanied by changes in gene expression. Molecular characterization of D. citri at its various stages of morphogenesis would likely provide important inroads into the identification of novel target sites for pest control. Furthermore, comparison of the developmental transcriptomes of D. citri from the eggs, each of the instars, and adult male and female would provide insights into the function of numerous genes and the regulation of different signaling pathways involved during the different developmental stages. Trehalose is a natural alpha-linked disaccharide formed by an α,α-1,1-glucoside bond between two α-glucose units. Trehalose can be used as an energy source by bacteria, fungi, insects, invertebrates, plants, and a large number of other organisms and plays an important role in stress tolerance [20]. Trehalose is hydrolyzed by the enzyme trehalase into glucose to rapidly provide energy when needed. Trehalase is also the first enzyme in the chitin biosynthesis pathway and significantly influences chitin metabolism by regulating the pathway. Therefore, trehalase has great potential as a target gene for controlling insects using RNAi technology [20–22]. In the current study, we attempted to obtain expression data throughout all developmental stages of D. citri by performing comprehensive RNA sequencing. In addition, two trehalase genes were characterized and their functions investigated using RNAi technology. The assembled and annotated transcriptome sequences and gene expression profiles should provide useful information for the identification of genes involved in D. citri development and will facilitate functional genomics studies on this destructive insect species.

2. Results

2.1. Illumina Sequencing and Assembly To advance our understanding of the molecular mechanisms involved in the developmental changes that occur during D. citri life stages, complementary DNA (cDNA) libraries generated from eggs, 1st instars, 2nd instars, 3rd instars, 4th instars, 5th instars, and male and female adults were sequenced using the Illumina HiSeqTM 2000 sequencing platform. The results were deposited in the Sequence Read Archive (SRA) repository and are available using the following BioProject accession numbers: PRJNA633316, PRJNA634209, and PRJNA633417. The quality statistics of the filtered data are shown in Table S1. A total of 39,787,301 contigs were obtained with a mean length of 58.24 bp and an N50 length of 49 bp. After clustering the contigs with the nucleotide sequences available at Int. J. Mol. Sci. 2020, 21, 4974 3 of 16 Int. J. Mol. Sci. 2020, 21, 4974 3 of 16

with an average length of 925.65 bp and an N50 length of 1733 bp. The unigene length distribution the NationalInt. J. Mol. CenterSci. 2020, 21 for, 4974 Biotechnology Information (NCBI), we ultimately obtained 354,7263 unigenesof 16 indicated most of the unigenes (52.42%) were concentrated at 200–500 bp lengths (Figure 1). with an average length of 925.65 bp and an N50 length of 1733 bp. The unigene length distribution indicatedwith mostan average of the length unigenes of 925.65 (52.42%) bp and were an N50 concentrated length of 1733 at 200–500 bp. The bpunigene lengths length (Figure distribution1). indicated most of the unigenes (52.42%) were concentrated at 200–500 bp lengths (Figure 1).

Figure 1. Unigene size distribution. Unigene sizes were calculated, and their relative distribution (%) Figure 1. Unigene size distribution. Unigene sizes were calculated, and their relative distribution (%) according to size (bp) are shown. accordingFigure to 1. size Unigene (bp) aresize shown.distribution. Unigene sizes were calculated, and their relative distribution (%) according to size (bp) are shown. 2.2.2.2. Annotation Annotation of Predictedof Predicted Proteins Proteins 2.2.To Annotation annotate of the Predicted unigenes, Proteins we first searched reference sequences using BLASTX against the non- To annotate the unigenes, we first searched reference sequences using BLASTX against the redundantTo annotate (NR) NCBI the unigenes, protein database we first searched using a cut-off reference E-value sequences of 10 −using5. The BLASTX annotation against details the fornon- each non-redundant (NR) NCBI protein database using a cut-off E-value of 10 5. The annotation details unigeneredundant are provided(NR) NCBI in protein Table databaseS2, with usingthe matching a cut-off E-valueprotein oflisted 10−5. wasThe annotationthe− top hit details for each for unigene each for each unigene are provided in Table S2, with the matching protein listed was the top hit for each (Tableunigene S2). are A totalprovided of 120,902 in Table of S2,all withthe annotated the matching unigenes protein (34.08%) listed was provided the top hita BLAST for each result. unigene Gene unigenenumber(Table (Table S2).distribution A S2). total A of among total 120,902 of the of 120,902 allbest the match ofannotated all top the 10unigenes annotated species (34.08%) is unigenes shown provided in (34.08%)Figure a BLAST 2. providedA total result. of Gene a101,114 BLAST result.unigenesnumber Gene numberweredistribution annotated distribution among to thethe amongbest 10 matchtop-hit the besttop specie 10 match speciess of topinsects is 10shown species of inwhich Figure is shown 86,146 2. A in totalannotated Figure of 101,1142. Agenes total of 101,114(approximatelyunigenes unigenes were 85.20%) annotated were annotatedwere to matchedthe 10 to top-hit theto D. 10 citrispecie top-hit, thes of speciesnumber insects ofone of insects whichtop-hit of 86,146sp whichecies. annotated The 86,146 other annotatedgenes top-hit genesspecies(approximately (approximately were Zootermopsis 85.20%) 85.20%) were nevadensis were matched matched, toCimex D. citri to lectulariusD., the citri number, the, Halyomorpha number one top-hit one halyssp top-hitecies., Acyrthosiphon The species. other top-hit The pisum other , top-hitDiuraphisspecies species werenoxia were , ZootermopsisTriboliumZootermopsis castaneum nevadensis nevadensis, Pediculus, Cimex, Cimexlectulariushumanus lectularius ,corporis Halyomorpha, ,CandidatusHalyomorpha halys , ProfftellaAcyrthosiphon halys ,armatura,Acyrthosiphon pisum and, pisumNeodiprionDiuraphis, Diuraphis leconteinoxia noxia, Tribolium (Figure, Tribolium 2).castaneum castaneum, Pediculus, Pediculus humanus humanus corporis corporis, Candidatus, Candidatus ProfftellaPro armatura,fftella armatura, and and NeodiprionNeodiprion lecontei (Figure(Figure 2).2).

80000 80000

60000 60000

4000040000 Number of genes of Number Number of genes of Number 2000020000

00 i s s s a s a i itri nsis riu s ly s um xi um ori s tur nte i a citr de si la iu ha ly pis um no xia ane um rp ri a uraco te rina c vaden ctular ha ha on pis his no ast ne co rpoarm at le con horin neva lectu orp ha ph n ap is c sta us colla armion le iapho sis ne ex le omorp osi ho iur aphium ca an us fte la ipr on Diap opsis Cimex aly m rth sip D iur ol um hum anrof el od pri D ermop im H lyo cy tho D rib oli us ums P offtNe di otrm C Ha Acyr T rib ul s h tu Pr eo Zote A T dic ulu ida tus N Zo Pe ic and da Ped C ndi Ca

Figure 2. Top 10 species distribution of the BLASTX top hits results. Species distribution of the Figure 2. Top 10 species distribution of the BLASTX top hits results. Species distribution of the unigeneFigure BLASTX 2. Top 10 top species hits results distribution against of the the National BLASTX Center top hits for results. Biotechnology Species Informationdistribution of (NCBI) the unigene BLASTX top hits results against the National Center5 for Biotechnology Information (NCBI) non-redundantunigene BLASTX protein top database hits results using against a cuto theff E-valueNational of Center 10− andfor Biotechnol the proportionsogy Information of each species (NCBI) are

shown. Each bar represents a different species. Int. J. Mol. Sci. 2020, 21, 4974 4 of 16

−5 Int. J. Mol.non-redundant Sci. 2020, 21, protein 4974 database using a cutoff E-value of 10 and the proportions of each species are4 of 16 shown. Each bar represents a different species.

A totaltotal ofof 120,902 120,902 unigenes unigenes were were annotated annotated based ba onsed four on major four databases,major databases, the NCBI the Nr NCBI database, Nr SwissProtdatabase, database,SwissProt Clustersdatabase, of OrthologousClusters of GroupsOrthologous (COG) Groups database, (COG) and Kyotodatabase, Encyclopedia and Kyoto of GenesEncyclopedia and Genomes of Genes (KEGG) and Genomes database (KEGG) (Table1 databa) (Figurese 3(Table). Among 1) (Figure them, 3). 119,683 Among unigenes them, 119,683 were matchedunigenes inwere the Nrmatched database in includingthe Nr database 47,191 unigenes including that 47,191 matched unigenes only to that this matched database. only In addition, to this 64,503database. unigenes In addition, had significant 64,503 unigenes matches inhad the signi SwissProtficant matches database in of whichthe SwissProt 116 unigenes database only of matched which this116 unigenes database. only Moreover, matched 63,429 this unigenesdatabase. had Moreover, specific matches63,429 unigenes in the COG had database specific matches and 10,121 in unigenesthe COG haddatabase specific and matches 10,121 unigenes in the KEGG had database,specific matche with 507s in and the 485KEGG unigenes database, being with unique 507 and to each 485 unigenes database, respectivelybeing unique (Figure to each3). database, respectively (Figure 3).

Table 1.1. Statistics of annotation results.

DatabaseDatabase Nr Nr SwissProt SwissProt COG COG KEGG KEGG Total Total UnigenesUnigenes 119,683 119,683 64,503 64,503 63,429 63,429 10,121 10,121 120,902 120,902

Figure 3. Comparison of unigene annotation in the NationalNational CenterCenter forfor BiotechnologyBiotechnology InformationInformation (NCBI) non-redundantnon-redundant (Nr), (Nr), SwissProt, SwissProt, Clusters Clusters of Orthologous of Ortholog Groupsous (COG), Groups and (COG), Kyoto Encyclopedia and Kyoto ofEncyclopedia Genes and Genomeof Genes (KEGG)and Genome databases. (KEGG) databases. 2.3. Functional Annotation Results 2.3. Functional Annotation Results The functional classification of D. citri unigenes was predicted by performing Gene Ontology (GO), The functional classification of D. citri unigenes was predicted by performing Gene Ontology COG, and KEGG analyses. A total of 33,800 unigenes were assigned to three main categories of GO (GO), COG, and KEGG analyses. A total of 33,800 unigenes were assigned to three main categories classification, molecular function (12,876 unigenes), cellular component (8548 unigenes), and biological of GO classification, molecular function (12,876 unigenes), cellular component (8548 unigenes), and process (12,376 unigenes). The terms “binding” and “catalytic activity”, “cell part” and “organelle”, biological process (12,376 unigenes). The terms “binding” and “catalytic activity”, “cell part” and and “metabolic process” and “cellular process” were the top two dominant GO terms for each of the “organelle”, and “metabolic process” and “cellular process” were the top two dominant GO terms three main categories (Figure4). In total, 58 sub-categories were divided from the primary categories, for each of the three main categories (Figure 4). In total, 58 sub-categories were divided from the 23 categories for “biological process,” 18 categories for “cellular component”, and 17 categories for primary categories, 23 categories for “biological process,” 18 categories for “cellular component”, and “molecular function.” 17 categories for “molecular function.”

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Figure 4. Gene Ontology (GO) functional categories of the unigenes annotated in the current study. FigureFigure 4. GeneGene Ontology Ontology (GO) (GO) functional functional categories categories of the of unigenes the unigenes annotated annotated in the in current the current study. Unigenes were annotated to three main categories: molecular function, cellular component, and study.Unigenes Unigenes were annotated were annotated to three to threemain maincategori categories:es: molecular molecular function, function, cellular cellular component, component, and biological process. andbiological biological process. process. ForFor COGCOG functional functional classification, classification, a total a total of 71,145 of 71,145 unigenes unigenes were annotated were annotated to 25 COG to categories 25 COG For COG functional classification, a total of 71,145 unigenes were annotated to 25 COG (Figurecategories5). Among (Figure them, 5). Among the largest them, group the largest was the gr “generaloup was function the “general prediction” function (9856 prediction” genes, 13.85%), (9856 categories (Figure 5). Among them, the largest group was the “general function prediction” (9856 followedgenes, 13.85%), by the large followed groups by (i.e., the >large2000 groups genes), “signal(i.e., >2000 transduction genes), “signal mechanisms” transduction (9776 genes, mechanisms” 13.74%), genes, 13.85%), followed by the large groups (i.e., >2000 genes), “signal transduction mechanisms” “transcription”(9776 genes, 13.74%), (6216 genes, “transcription” 8.74%), “posttranslational (6216 genes, 8.74%), modification, “posttranslational protein turnover, modification, chaperones” protein (9776 genes, 13.74%), “transcription” (6216 genes, 8.74%), “posttranslational modification, protein (5522turnover, genes, chaperones” 7.76%), and (5522 “lipid genes, transport 7.76%), and and metabolism” “lipid transport (3470 and genes, metabolism” 4.88%). (3470 genes, 4.88%). turnover, chaperones” (5522 genes, 7.76%), and “lipid transport and metabolism” (3470 genes, 4.88%).

COG Function Classification COG Function Classification 12000 A: RNA processing and modification 12000 A:B: RNAChromatin processing structure and and modification dynamics B:C: ChromatinEnergy production structure and and conversion dynamics C:D: CellEnergy cycle production control, cell and division, conversion chromosome partitioning 10000 D:E: AminoCell cycle acid control, transport cell and division, metabolism chromosome partitioning 10000 E:F: NucleotideAmino acid transport transport and and metabolism metabolism F:G: NucleotideCarbohydrate transport transport and and metabolism metabolism G:H: CoenzymeCarbohydrate transport transport and and metabolism metabolism 8000 H:I: Lipid Coenzyme transport transport and metabolism and metabolism 8000 I:J: LipidTranslation, transport ribosomal and metabolism structure and biogenesis J:K: Translation,Transcription ribosomal structure and biogenesis K:L: Replication,Transcription recombination and repair 6000 L:M: Replication,Cell wall/membrane/envelope recombination and biogenesis repair 6000 M:N: Cell motilitywall/membrane/envelope biogenesis N:O: CellPosttranslational motility modification, protein turnover, chaperones O:P: InorganicPosttranslational ion transport modification, and metabolism protein turnover, chaperones

Number of Genes of Number 4000 P:Q:Secondary Inorganic ion metabolites transport andbiosynthesis, metabolism transport and catabolism R: General function prediction only

Number of Genes of Number 4000 Q:Secondary metabolites biosynthesis, transport and catabolism R:S: FunctionGeneral functionunknown prediction only S:T: SignalFunction transduction unknown mechanisms 2000 T:U: SignalIntracellular transduction trafficking, mechanisms secretion, and vesicular transport 2000 U:V: DefenseIntracellular mechanisms trafficking, secretion, and vesicular transport V:W: Defense Extracellular mechanisms structures W:X: Nuclear Extracellular structure structures 0 X:Y: NuclearCytoskeleton structure 0 ABCDEFGH I JKLMNOPQRSTUVWYZ Y: Cytoskeleton ABCDEFGH I JKLMNOPQRSTUVWYZ Function Class Function Class FigureFigure 5.5. ClustersClusters ofof Orthologous Orthologous Groups Groups (COG) (COG) functional functional categories categories of theof the unigenes unigenes annotated annotated in the in Figure 5. Clusters of Orthologous Groups (COG) functional categories of the unigenes annotated in currentthe current study. study. The namesThe names of each of classeach definitionclass definition (A to Y)(A areto Y) provided are provided on the on right the side right of theside figure. of the the current study. The names of each class definition (A to Y) are provided on the right side of the figure. figure.

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Diaphorina citri unigene sequences that mapped to reference canonical pathways in the KEGG databaseDiaphorina were citrialsounigene analyzed. sequences We specifically that mapped assigned to reference 31,909 canonicalunigene sequences pathways into the44 KEGGKEGG databasepathways. were The also top analyzed. three pathways We specifically were assignedsignal transduction, 31,909 unigene endocrine sequences system, to 44 KEGG and translation pathways. The(Figure top three6). pathways were signal transduction, endocrine system, and translation (Figure6).

FigureFigure 6.6. KyotoKyoto EncyclopediaEncyclopedia ofof GenesGenes andand GenomeGenome (KEGG)(KEGG) functionalfunctional categoriescategories ofof thethe unigenesunigenes annotatedannotated inin thethe currentcurrent study.study.Unigenes Unigeneswere wereannotated annotatedand andassigned assignedto to sixsix mainmain categories,categories, cellularcellular processes,processes, environmentalenvironmental informationinformation processing,processing, geneticgenetic informationinformation processing,processing, humanhuman diseases,diseases, metabolism,metabolism, andand organismalorganismal systems.systems. 2.4. Differentially Expressed Genes (DEGs) 2.4. Differentially Expressed Genes (DEGs) To identify DEGs among the different developmental stages, the number of clean tags for each To identify DEGs among the different developmental stages, the number of clean tags for each gene was calculated. Gene expression variation was analyzed between different stage combinations gene was calculated. Gene expression variation was analyzed between different stage combinations (Figure7). The top 20 upregulated and downregulated DEGs expressed genes at each stage are shown (Figure 7). The top 20 upregulated and downregulated DEGs expressed genes at each stage are shown in Table S3. in Table S3.

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A UP E DOWN Egg-VS-Male

Egg-VS-Female 4th instar-VS-Male

Egg-VS-5th instar

Egg-VS-4th instar 4th instar-VS-Female

Egg-VS-3rd instar

Egg-VS-2nd instar 4th instar-VS-5th instar

Egg-VS-1st instar

0 5000 10000 15000 20000 25000 0 2000 4000 6000 8000 10000 12000 14000

B F 1st instar-VS-Male

1st instar-VS-Female 5th instar-VS-Male

1st instar-VS-5th instar

1st instar-VS-4th instar

5th instar-VS-Female 1st instar-VS-3rd instar

1st instar-VS-2nd instar

0 2000 4000 6000 8000 10000 12000 14000 16000 18000 0 2000 4000 6000 8000 10000 12000 14000

C G

2nd instar-VS-Male

2nd instar-VS-Female

2nd instar-VS-5th instar Female-VS-Male

2nd instar-VS-4th instar

2nd instar-VS-3rd instar

0 2000 4000 6000 8000 10000 12000 14000 0 1000 2000 3000 4000 5000

D

3rd instar-VS-Male

3rd instar-VS-Female

3rd instar-VS-5th instar

3rd instar-VS-4th instar

0 2000 4000 6000 8000 10000 12000 14000 Figure 7. Comparison of differentially expressed gene (DEG) numbers in each. Upregulated (UP) and Figuredownregulated 7. Comparison (DOWN) of differentially unigenes were expressed quantified. gene The (DEG) results numbers of 28 comparisons in each. Upregulated are shown. (UP) (A )and the downregulatedcomparison of DEGs (DOWN) between unigenes egg and were other quantifi stages;ed. (B The) the results comparison of 28 comparisons of DEGs between are shown. 1st instar and other stages; (C) the comparison of DEGs between 2nd instar and other stages; (D) the comparison of 2.5. DevelopmentalDEGs between Gene 3rd instar Expression and other of Trehalase stages; (E 1) theand comparison Trehalase 2 of DEGs between 4th instar and other Resultsstages; (Ffrom) the the comparison gene expression of DEGs between analysis 5th show instared and that other trehalase stages; 1 ( Gwas) the differently comparison expressed of DEGs between female and male. across different development stages (F7,16 = 62.363, p < 0.0001). Specifically, the trehalase 1 gene exhibited the highest expression level during the egg stage, while the expression level was lowest in the male and female adults. In addition, expression of trehalase 1 gene was lower in the 5th instar compared to that in the other four instar stages. There was no significant difference in trehalase 1 gene expression among the other development stages (Figure 8A). Similar gene expression profiles were found for the trehalase 2 gene across the different developmental stages (Figure 8B).

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2.5. Developmental Gene Expression of Trehalase 1 and Trehalase 2 Results from the gene expression analysis showed that trehalase 1 was differently expressed across different development stages (F7,16 = 62.363, p < 0.0001). Specifically, the trehalase 1 gene exhibited the highest expression level during the egg stage, while the expression level was lowest in the male and female adults. In addition, expression of trehalase 1 gene was lower in the 5th instar compared to that in the other four instar stages. There was no significant difference in trehalase 1 gene expression amongInt. J. Mol. the Sci. other 2020, development21, 4974 stages (Figure8A). Similar gene expression profiles were found for8 theof 16 trehalase 2 gene across the different developmental stages (Figure8B).

1.2 A a 1.0 b b b 0.8 b c 0.6

0.4

0.2 Relative trehalase1 expression d d

0.0

1.2 B a 1.0 b b 0.8 b bc c 0.6

0.4

0.2 Relative trehalase2 expression trehalase2 Relative d d 0.0 gg tar tar tar tar tar ale ale E ns ns ns ns ns m M t i d i d i h i h i Fe 1s 2n 3r 4t 5t

FigureFigure 8.8. Expression patterns of trehalase 1 ((AA)) andand trehalasetrehalase 22 ((BB)) inin D.D. citricitri acrossacross didifferentfferent developmentaldevelopmental stages.stages. The values shown areare meansmeans ++ SE.SE. DiDifferentfferent lettersletters indicateindicate significantsignificant didifferencesfferences in in gene gene expression expression across across di differentfferent developmental developmental stages stages (p (p< <0.05). 0.05). 2.6. Impact of the silencing of trehalase 1 and trehalase 2 on Gene Expression 2.6. Impact of the silencing of trehalase 1 and trehalase 2 on Gene Expression After two days of treatment, gene expression of trehalase 1 in instars treated with trehalase1-specific After two days of treatment, gene expression of trehalase 1 in instars treated with trehalase1- dsRNA (dstrehalase1) was significantly suppressed (F2,6 = 77.681, p < 0.0001). The expression of specific dsRNA (dstrehalase1) was significantly suppressed (F2,6 = 77.681, p < 0.0001). The expression trehalase 1 was reduced 2.495-fold compared to that of the ddH2O-treated control (Figure9A). Similarly, of trehalase 1 was reduced 2.495-fold compared to that of the ddH2O-treated control (Figure 9A). gene expression of trehalase 2 in instars treated with trehalase2-specific dsRNA (dstrehalase2) was also Similarly, gene expression of trehalase 2 in instars treated with trehalase2-specific dsRNA significantly suppressed (F2,6 = 24.181, p = 0.001). The expression of trehalase 2 was reduced 2.232 fold (dstrehalase2) was also significantly suppressed (F2,6 = 24.181, p = 0.001). The expression of trehalase compared to that of the ddH2O-treated control (Figure9B). 2 was reduced 2.232 fold compared to that of the ddH2O-treated control (Figure 9B).

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1.4 A 1.2 a a 1.0

0.8

0.6 b 0.4

0.2 Relative trehalase1 expression trehalase1 Relative

0.0 H2O dsGFP dstrehalase1

1.2 B a a 1.0

0.8

0.6 b 0.4

0.2 Relative trehalase2 expression trehalase2 Relative

0.0 H2O dsGFP dstrehalase2

Figure 9. Relative gene expression in D. citri 2nd instars after treatment with specific double-stranded FigureRNA 9. (dsRNA). Relative Trehalasegene expression 1 (A) and in trehalase D. citri 2 2nd (B). instars The 2nd after instars treatment were treated with with specific trehalase1-specific double-stranded RNAdsRNA (dsRNA). (dstrhalase1), Trehalase trehalase2-specific 1 (A) and trehalase dsRNA (dstrhalase2),2 (B). The 2nd control instars green were fluorescent treated protein-specific with trehalase1-

specificdsRNA dsRNA (dsGFP), (dstrhalase1), or water (H2 O)trehalase2-specific and the gene expression dsRNA levels (dstrhalas were measured.e2), control Values green shown fluorescent are protein-specificmeans + SE. DidsRNAfferent letters(dsGFP), indicate or water significant (H2O) di andfferences the gene among expression treatments levels (p < 0.05, were Tukey’s measured. Valuesmultiple shown comparisons are means test). + SE. Different letters indicate significant differences among treatments (p < 2.7.0.05, Toxicity Tukey’s of inmultiple Vitro Synthesized comparisons Dstrehalase1 test). and Dstrehalase2

2.7. ToxicityDiaphorina of in Vitro citri Synthesimortalityzed in Dstrehalase1 the dstrehalase1- and Dstrehalase2 and dstrehalase2-treated groups was observed starting on the first day of dsRNA bioassay (Figure 10). Specifically, knockdown of trehalase 1 andDiaphorina trehalase citri 2 using mortality their respective in the dstrehalase1- specific dsRNAs and each dstrehalase2-treated resulted in significant groups mortality was ofobservedD. startingcitri compared on the first to thatday inof the dsRNA dsGFP-treated bioassay and (Fig Hure2O-treated 10). Specifically, control groups knockdown on day 1 (Fof3,8 trehalase= 28.152, 1 and trehalasep < 0.0001 2 using), day their 2 (F3,8 respective= 217.502, pspecific< 0.0001 dsRNAs), day 3 (F each3,8 = 15.672,resultedp = in0.001), significant and day mortality 4 (F3,8 = 7.958, of D. citri comparedp = 0.009 to) post-treatmentthat in the dsGFP-treated (Figure 10). There and was H2 noO-treated significant control difference groups in D. on citri daymortality 1 (F3,8 between= 28.152, p < the dstrehalase1-treated and dstrehalase2-treated groups at any time point. 0.0001), day 2 (F3,8 = 217.502, p < 0.0001), day 3 (F3,8 = 15.672, p = 0.001), and day 4 (F3,8 = 7.958, p = 0.009) post-treatment (Figure 10). There was no significant difference in D. citri mortality between the dstrehalase1-treated and dstrehalase2-treated groups at any time point.

Int. J. Mol. Sci. 2020, 21, 4974 10 of 16

Int. J. Mol. Sci. 2020, 21, 4974 10 of 16

100 * * * 80 *

60

40 Survival rate (%)

H2O 20 dsGFP dstrehalase1 dstrehalase2

0 01234 Assay time (day)

FigureFigure 10. 10.E ffEffectect of of trehalase1-specific trehalase1-specific double-stranded double-stranded RNA RNA (dstrehalase1) (dstrehalase1) and and trehalase2-specific trehalase2-specific double-strandeddouble-stranded RNA RNA (dstrehalase2) (dstrehalase2) treatment treatment on D. on citri D. 2ndcitri instars2nd instars mortality. mortality.D. citri D.2nd citri instars 2nd instars were treatedwere treated with dstrehalase1, with dstrehalase1, dstrehalase2, dstrehalase2, green fluorescent green fluorescent protein-specific protein-specific dsRNA dsRNA (dsGFP), (dsGFP), or water or (H O) and monitored for mortality. Values shown in the figure are means SE. * Indicates statistically water2 (H2O) and monitored for mortality. Values shown in the figure± are means ± SE. * Indicates significantstatistically di ffsignificanterences (p differences< 0.05; ANOVA, (p < 0.05; Tukey’s ANOVA, multiple Tukey’s comparisons multipletest). comparisons test). 3. Discussion 3. Discussion In the current study, we compared the transcriptomes of eight developmental stages of D. citri. In the current study, we compared the transcriptomes of eight developmental stages of D. citri. A better understanding of the molecular mechanisms regulating the life cycles and developmental A better understanding of the molecular mechanisms regulating the life cycles and developmental stages of this important pest may aid in its control by facilitating the development of more sustainable stages of this important pest may aid in its control by facilitating the development of more sustainable and environmentally friendly interventions. Our results significantly advance the molecular resources and environmentally friendly interventions. Our results significantly advance the molecular available for the study of this and other insect pests and provide a framework to understand resources available for the study of this and other insect pests and provide a framework to changes in gene expression during insect development. We assembled transcriptomes from all eight understand changes in gene expression during insect development. We assembled transcriptomes developmental stages and identified a total of 354,726 unigenes with an average length of 925.65 bp. from all eight developmental stages and identified a total of 354,726 unigenes with an average length A total of 120,902 unigenes were annotated using four major databases. of 925.65 bp. A total of 120,902 unigenes were annotated using four major databases. Interestingly, transcriptome sequence analysis revealed that Z. nevadensis, which belong to the Interestingly, transcriptome sequence analysis revealed that Z. nevadensis, which belong to the order Blattaria, shared the highest similarity with D. citri based on the BLAST annotation. This was order Blattaria, shared the highest similarity with D. citri based on the BLAST annotation. This was in contrast to H. halys, A. pisum, and D. noxia, which like D. citri belong to the order Hemiptera but in contrast to H. halys, A. pisum, and D. noxia, which like D. citri belong to the order Hemiptera but showed lower best-match percentages. These unexpected results were not likely due to the availability showed lower best-match percentages. These unexpected results were not likely due to the of more sequence resources for Z. nevadensis in the NCBI protein database compared to those for availability of more sequence resources for Z. nevadensis in the NCBI protein database compared to the other organisms, as the number of protein sequences for A. pisum (53,285) was higher than that those for the other organisms, as the number of protein sequences for A. pisum (53,285) was higher for Z. nevadensis (49,273). It appears that D. citri may have a closer relationship to Z. nevadensis than that for Z. nevadensis (49,273). It appears that D. citri may have a closer relationship to Z. than to the other Hemipteran insects. The precise relationship among these insect species needs nevadensis than to the other Hemipteran insects. The precise relationship among these insect species further investigation. needs further investigation. One of the top 10 species regarding distribution of the best match by BLASTX against the Nr One of the top 10 species regarding distribution of the best match by BLASTX against the Nr database was Ca. Profftella armature. D. citri possesses a specialized organ called a bacteriome, database was Ca. Profftella armature. D. citri possesses a specialized organ called a bacteriome, which which may harbor the vertically transmitted intracellular mutualists Ca. Carsonella ruddii and Ca. may harbor the vertically transmitted intracellular mutualists Ca. Carsonella ruddii and Ca. Profftella Profftella armature. Carsonella is a typical nutritional symbiont while Profftella is an unprecedented armature. Carsonella is a typical nutritional symbiont while Profftella is an unprecedented type of type of toxin-producing defensive symbiont, unusually sharing organelle-like features with nutritional toxin-producing defensive symbiont, unusually sharing organelle-like features with nutritional symbionts [23,24]. Furthermore, many strains of D. citri are infected with Wolbachia, which manipulates symbionts [23,24]. Furthermore, many strains of D. citri are infected with Wolbachia, which the reproductive and developmental processes in various hosts [25]. In our study, Ca. manipulates the reproductive and developmental processes in various arthropod hosts [25]. In our Carsonella ruddii, Ca. Profftella armature, and Wolbachia were each found in the transcriptome, with

Int. J. Mol. Sci. 2020, 21, 4974 11 of 16

Profftella having the most abundant transcripts (Table S2). The function of these symbionts in D. citri should be investigated in future studies. Compared to that of the previous study in which the D. citri transcriptomes of eggs, nymphs, and adults were sequenced with no biological replicate [19], our transcriptome and gene expression profiling data greatly enrich the current D. citri transcriptome database and will benefit research with respect to the identification of novel genes, chemical targets, developmental mechanisms, and sex difference of D. citri. During the developmental stages, most DEGs were Upregulated and downregulated in adults compared to that in eggs. In contrast, the expression of few genes was changed among different larvae stages. Each stage library contained large numbers of genes that showed specific expression and were likely involved in developmental differentiation. This is consistent with other insects that also show their larvae stage development to be tightly regulated by a few key genes, and this regulation is important for developmental differentiation [26,27]. The success of RNAi-based strategies in future pest management will rely on the identification of essential target genes that confer lethal phenotypic effects upon knockdown [28]. Generally, functional genes involved in insect development, metamorphosis, or key metabolic processes may be suitable RNAi targets as part of control strategies of insects [15–18,29–33]. Previous studies have shown that trehalase plays pivotal roles in various physiological processes, including energy metabolism [34], chitin synthesis during molting [35], and diapause [36]. Recently, trehalases from several insects have been characterized and their biological roles investigated, including those from Nilaparvata lugens [21], Tribolium castaneum [22], and Harmonia axyridis [36]. Suppressing the expression of trehalase can lead to the developmental inhibition and death of the insects [21,22]. In the current study, treating D. citri nymphs with a solution containing dsRNAs specific for trehalase 1 and trehalase 2 genes resulted in a robust RNAi response. When dsRNA is administered by soaking the insect with the solution, the dsRNA can be taken up through spiracles or by cuticle permeation [37]. This is then followed by the spread of the dsRNA/siRNA molecules throughout the body of the insect. The individual insect cells can then take up the dsRNA/siRNA from their nearby environment, thereby inducing the RNAi activity, which is process referred to as environmental RNAi silencing [28]. Ultimately, we found that silencing trehalases induced high mortality in D. citri, which lays the foundation for developing RNAi-based biopesticides aimed at controlling D. citri. In general, we developed a comprehensive sequence resource of D. citri consisting of a desirable quality. It was built based on preparing and analyzing eight different developmental transcriptomes of D. citri eggs, larvae, and adults. Future analyses of genes related to insect development or death will be useful for pest control. Furthermore, this study enriches the genomic platform of D. citri which should facilitate our understanding of the metamorphosis and development of this important pest insect.

4. Materials and Methods

4.1. Insect Rearing and Sample Preparation Diaphorina citri adults were collected from Murraya exotica (L.) and reared on host M. exotica in an incubator at 25 0.5 C, a 16 h light/8 h dark photoperiod and 50% relative humidity. ± ◦ Eight developmental stages of D. citri were sampled, including eggs, 1st instars, 2nd instars, 3rd instars, 4th instars, 5th instars, and male and female adults. Each developmental sample was collected during the first two days of their respective stages. First to fifth instars were discriminated by appearance and body size. All the samples were placed in TRIzol reagent (Invitrogen, Carlsbad, CA, USA), rapidly frozen in liquid nitrogen, and then transferred to 80 C. All D. citri developmental samples were − ◦ represented by three biological replicates that were independently processed. Int. J. Mol. Sci. 2020, 21, 4974 12 of 16

4.2. mRNA Sequencing by ILLUMINA HiSeq Total RNA was extracted from the D. citri samples using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. After quantification of the RNA using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and 1% agarose gel electrophoresis, the extracted RNA with RNA integrity number (RIN) values >8 was used for generation of cDNA libraries. Next generation sequencing libraries were constructed using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® according to the manufacturer’s protocol (New England Biolabs, Ipswich, MA, USA). Libraries with different indices were then multiplexed and loaded onto an Illumina HiSeqTM 2000 instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was carried out using a 2 150 bp paired-end (PE) platform (GENEWIZ, Suzhou, China). × 4.3. Functional Annotation Analysis of Sequencing Data After sequencing, all sequence data were cleaned using Trimmomatic with default parameters [38]. Two approaches were then used for data analysis. For the first approach, expression profiles of each gene at different developmental stages were determined. During this step, both assembly and annotations of D. citri were downloaded from NCBI and used as references. Briefly, the clean data were mapped to the D. citri genome using Hisat2 [39]. Gene values, represented as transcripts per kilobase million (TPM), were then calculated using StringTie [40]. Differentially expressed genes between every two samples were also determined using DESeq with a cutoff for fold change set at > 2 and false discovery rate (FDR) < 0.05 [41]. For the second approach, a transcript dataset of D. citri was constructed. Briefly, all the clean reads were assembled using Trinity [42] and the transcripts generated were clustered by CD-Hit software [43]. Ultimately, transcript datasets of D. citri were constructed using the above approaches. A database consisting of NR, SwissProt, COG, GO, and KEGG gene sequences and related parameters were analyzed using the appropriate software (Table S4).

4.4. Temporal Gene Expression Analysis of Trehalase RNAs from different developmental stages used for Illumina HiSeq sequencing were used for gene expression analysis. First-strand cDNA was prepared using 1 µg of total RNA and a PrimeScript RT Kit with gDNA Eraser (Perfect Real Time) from TaKaRa (Dalian, China) following the manufacturer’s instructions. The synthesized first-strand cDNA was diluted 10 fold and was immediately used or stored at 20 C until used. − ◦ Real-time reverse transcription polymerase chain reaction (RT-qPCR) was conducted according to the manufacturer’s recommendations using SYBR® Premix Ex Taq™ (Tli RNaseH Plus) purchased from TaKaRa. PCR amplification in 15 µL reactions were performed using the cycling program and PCR system described in our previous study [44]. Briefly, PCR reaction mixtures contained 5.25 µL ddH O, 7.5 µL 2 SYBR Green MasterMix (Bio-Rad Laboratories, Hercules, CA, USA), 4 µM each 2 × specific primer, and 1.0 µL first-strand cDNA template. The RT-qPCR program included an initial denaturation at 95 ◦C for 3 min followed by 40 cycles of denaturation at 95 ◦C for 10 s, annealing for 30 s at 55 ◦C, and extension for 30 s at 72 ◦C. The gene-specific primers used for the RT-qPCR are ∆∆Ct listed in Table2. Relative quantification was calculated using the 2 − method [45] and data were normalized to expression of reference genes GAPDH and EF1α [46]. Three technical replicates were used for each sample.

Table 2. Assembly statistics for Diaphorina citri transcriptomes across developmental stages.

Total GC Max Min Average Total Assembled Type N50 Sequences Percentage Length Length Length Bases Contig 39,787,301 42.54% 49 19,067 25 58.24 2,317,181,757 Unigene 354,726 39.43% 1733 19,592 201 925.65 328,351,381 Int. J. Mol. Sci. 2020, 21, 4974 13 of 16

4.5. dsRNA Synthesis

Specific dsRNA primers containing a T7 promoter sequence at the 50end targeting trehalase 1 and trehalase 2 were designed using E-RNAi. The sequences of the primers are listed in Table3. The PCR template and cycling program used were described in our previous study [44]. PCR products were purified using a Universal DNA Purification Kit (TIANGEN, Beijing, China) and used as template with a T7 MEGAscript Kit (Thermo Fisher Scientific) following the manufacturer’s protocol to generate the dsRNA. The primers for dsGFP synthesis were used according to our recent study [16]. Synthesized dsRNAs were purified and suspended in ddH2O and their size and integrity confirmed by 1.5% agarose/TAE gel electrophoresis stained with GoldView I. Concentrations of the purified dsRNAs were determined using a NanoDrop One spectrophotometer (Thermo Fisher Scientific), and the purified dsRNAs were stored at 20 C until use. − ◦ Table 3. Primers used in this study.

Name of Primers Primer Sequences (50-30) dstrehalase1-F TAATACGACTCACTATAGGGGGGCGTGATCGAGAACATAA dstrehalase1-R TAATACGACTCACTATAGGGGATGAACCACCGACTGGAAA dstrehalase2-F TAATACGACTCACTATAGGGCTTTGAAGCAGGCAATGAGC dstrehalase2 -R TAATACGACTCACTATAGGGGCACAATCTGTTGCCGATTT RT-qPCR-trehalase1-F TTTCCAGTCGGTGGTTCATC RT-qPCR-trehalase1-R CCACCATTCGCTCAGATAGTT RT-qPCR-trehalase2-F CGAGCCTTGCTCACCAATAA RT-qPCR-trehalase2-R CTCGGGTTGGAGGTGAAATC

4.6. RNA Interference Assays on D. citri In our preliminary investigation, the effect of dsRNA on D. citri 2nd instars mortality was determined using dstrehalase concentrations of 10, 25, 50, 75, and 100 ng/µL. We found that D. citri 2nd instars had the highest mortality rates when exposed to 75 ng/µL dstrehalase and the mortality did not increase when the dstrehalase concentration was increased to 100 ng/µL. Therefore, the concentration of dsRNA used in this study was 75 ng/µL. RNA interference assays were conducted according to a recent study [14] with modification. Specifically, D. citri 2nd instars were starved for two hours and then completely soaked with a 4 µL droplet containing 75 ng/µL dsRNA. The dsRNA droplet was removed using a pipette after soaking for approximately 2 min. As controls, additional D. citri 2nd instars were treated with ddH2O or dsGFP. After drying on filter paper, 20 newly emerged D. citri 2nd instars were transferred onto 5 to 7 M. paniculata seedlings of a true leaf stage (tender leaf). The petiole was then fixed in agar at the bottom of a glass cylindrical container (4 cm diameter and 20 cm high), the upper opening of the cylinder was covered with a mesh screen for ventilation, and the agar was covered with a piece of filter paper and cotton to prevent water loss. The feeding chambers were placed incubated at 25 0.5 C with a 16 h light/8 h dark photoperiod and 50% relative humidity. ± ◦ Each treatment was performed using three biological replicates. Mortality was monitored and recorded for four days. For analysis of gene suppression caused by the cognate mRNAs, D. citri 2nd instars were treated as noted above. A total of 100 instars were used for each treatment. After two days, 50 living nymphs were collected, flash-frozen in liquid nitrogen, and stored at –80 ◦C in 1.5 mL centrifuge tubes until processed for total RNA extraction. Each treatment was independently repeated three times. For RT-qPCR analysis, total RNA was extracted, cDNA synthesized, and RT-qPCR amplification performed as described above. Three technical replicates were used for each sample.

4.7. Data Analysis One-way analysis of variance (ANOVA) was used to detect significant differences in trehalase 1 and trehalase 2 expression levels among different treatments, followed by a Tukey’s multiple range test Int. J. Mol. Sci. 2020, 21, 4974 14 of 16 with p < 0.05 being considered statistically significant. One-way ANOVA was also used to compare mortality rates each day of D. citri treated with H2O, dsGFP, dstrehalase1, and dstrehalase2. Mortality means were compared using Tukey’s tests and again p < 0.05 was considered statistically significant. Proportional data were arcsine square root transformed before analyses. All statistical analyses were performed using SPSS version 21.0 software (SPSS Inc., Chicago, IL, USA).

5. Conclusions Our data greatly improved our genetic understanding of D. citri and provides a large number of gene sequences for future studies. Our findings also provide comprehensive insight into gene expression profiles across different developmental stages of D. citri. Dietary RNAi toxicity assays demonstrated that trehalase 1 and trehalase 2 may be effective molecular targets for controlling D. citri. However, prior to the practical application of RNAi to control D. citri, further studies are necessary, such as determining an effective delivery system of dsRNA to citrus trees.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/14/4974/s1. Table S1. The quality statistics of the filtered sequencing data. Table S2. The Nr annotation information for each unigene. Table S3. The top 20 upregulated and downregulated differential expression genes expressed genes at each stage. The stage on the right for each DEG comparison served as the control. Table S4. Gene function analysis software and parameters. Author Contributions: Conceptualization, H.P. and C.Y.; methodology, H.P.; validation, D.O., W.G., J.L. and C.G.; formal analysis, H.P.; investigation, C.Y., D.O., W.G., J.L. and C.G.; resources, B.Q.; data curation, D.O., W.G., J.L. and C.G.; writing—original draft preparation, H.P., C.Y.; writing—review and editing, H.P. and C.Y.; supervision, H.P.; funding acquisition, H.P. All authors have read and agreed to the published version of the manuscript Funding: This research was funded by the State Key Laboratory for Biology of Plant Diseases and Insect Pests (SKLOF201808), the Science and Technology Program of Guangzhou (201804020070), a project supported by GDUPS (2017), and a start-up fund from the South China Agricultural University. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations

Nr Non-redundant COG Clusters of Orthologous Groups of proteins GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes RT-qPCR reverse transcriptase-quantitative polymerase chain reaction

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