1. Introduction
Chitin is found in fungi, nematodes and arthropods and plays key roles in maintaining morphology and protecting organisms against external attacks [
1,
2]. In insects, chitin is a vital component of the cuticles of the epidermis and trachea, as well as the peritrophic matrix (PM) in the midgut lumen [
3,
4]. During the molting process, some chitin in the old cuticle and PM are degraded and replaced by the newly synthesized chitin [
5]. Chitin synthesis is catalyzed by chitin synthases (
CHSs), which are cataloged into two classes:
CHS1 and
CHS2. In contrast, chitin degradation is hydrolyzed by the chitinase, which catalyzes the random hydrolysis of
N-acetyl-β-
d-glucosamine β-1,4-glycosidic linkages in chitin and chitodextrins. Insect chitinases belong to the family 18 glycosyl hydrolases, based on the conservation of amino acid sequences and many conserved motifs [
6].
Insect chitinases are crucial enzymes responsible for the degradation of the chitin in the cuticle and presumably in the PM during molting [
7,
8]. To date, it is known that there are rather large and diverse groups of chitinase genes identified in several insect species. For instance, the model genetic organism,
Drosophila melanogaster, has 22 chitinase and chitinase-like genes.
Anopheles gambiae has 17 genes encoding chitinase, and
Tribolium castaneum, a member of the order of Coleoptera, has 16 chitinase family genes [
9]. Based on phylogenetic analysis of the catalytic domains, insect chitinase and chitinase-like proteins are classified into eight groups [
10]. Multiple chitinase family genes may have various functions during insect growth and development. Based on the technology of RNA interference (RNAi) to ascertain the function of different chitinases during the development of
T. castaneum [
7] and
Spodoptera exigua [
11], it is suggested that a part of the chitinases are essential for cuticle turnover, regulating abdominal contraction and wing expansion. In addition, chitinases may be involved in other physiological processes, such as immune defense [
12] and disease control [
13].
Chitinase activity in insects is at least in part under hormonal regulation, its activity being regulated by hormones, such as juvenile hormone (JH) and 20-hydroxyecdysone (20E) [
14,
15]. The influence of the hormones on the expression of chitinase genes has been evaluated in some lepidopteran insects [
16,
17]. 20E has been shown to stimulate the expression of these chitinase genes, but some of them could be suppressed by the simultaneous application of JH [
18].
During insect larval development, a restricted supply of nutrients is critical for metamorphosis. In lepidopteran insects, starvation induces supernumerary molts associated with a high level of hemolymph JH titers [
19]. However, in
Psacothea hilaris, starvation of the fourth instar larvae can lead to precocious metamorphosis exceeding a threshold weight with formatting of unusually small, but morphological normal adults [
20]. A previous study also found that the
OnCht expression level can be decreased by feeding in
Ostrinia nubilalis, suggesting that this chitinase gene may play important roles in the program of molting [
21]. Therefore, it is urgent to clarify the relation between chitinase expression and starvation during the insect molting process.
The polyphagous tephritid fruit fly,
Bactrocera dorsalis, is an insect pest in the tropical and subtropical areas, damaging more than 250 plant species, including numerous fruits and vegetables [
22,
23]. At present, it is one of the most destructive pests, mainly because of its extreme ability to evolve resistance to many classes of insecticides [
24,
25]. There is an urgent need to develop new pest control strategies by targeting vital genes. In recent years, it has been documented that chitinases are essential for insect growth and development, and since chitin is not present in vertebrates, it has been considered as a potential target for insect pest management.
In this study, the cloning of the cDNA and genomic DNA sequences of a chitinase gene from B. dorsalis (BdCht2), as well as the expression patterns in different developmental stages and different tissues were described. The changes in the BdCht2 expression patterns after the treatment of 20E and starvation were also investigated. This study may provide some insights for further investigation about the chitin-degrading mechanism in the oriental fruit fly and other insects.
3. Discussion
Chitinase is considered as a potential target for insect control, due to its importance in chitin degradation during insect molting [
2]. A variety of chitinase and chitinase-like proteins have been reported in different insect species, with emphasis on protein activities and specific functions of individual chitinases and their response to inhibitors [
10]. To date, the cDNAs encoding chitinase and chitinase-like proteins have been identified in at least 15 insect species, including: five dipterans,
Glossina morsitans morsitans [
12],
Aedes aegypti,
A. gambiae,
D. melanogaster [
26],
Chironomus tentans [
27]; eight lepidopterans,
Spodoptera frugiperda [
8],
S. exigua [
11],
Manduca sexta [
17],
O. nubilalis [
21],
Spodoptera litura [
28],
Choristoneura fumiferana [
29],
Helicoverpa armigera [
30],
Tenebrio molitor [
31]; and two coleopterans,
T. castaneum [
8] and
Apriona germari [
32]. Analyses of these cloned genes will provide us with an opportunity to study the biological functions in different insect species. In this study, we identified and characterized
BdCht2 from the oriental fruit fly,
B. dorsalis. The
BdCht2 cDNA has a molecular structure consisting of a single peptide, a single catalytic domain and no CBD, which is similar to the domain architecture of Group IV chitinases [
10]. The presence of a conserved motif in the catalytic domain that has the consensus, FDGDLDWE motif, suggested that it belonged to the family of 18 glycosyl hydrolases. Based on the typical structure and phylogenetic analyses,
BdCht2 belong to Group VII chitinases.
Chitinases have different expression patterns at different developmental stages depending on specific and distinct functions. In
B. dorsalis, a higher expression level of
BdCht2 was detected during the larval-pupal and pupal-adult transitions, which suggested that
BdCht2 may be involved in the degradation of chitin for insect molting. Interestingly, the chitin synthase 1 of
B. dorsalis was also mainly expressed during this molting phase, suggesting that chitin degradation occurs almost at the same time as chitin biosynthesis [
33]. Previous studies in
Tribolium castaneum showed that
TcCht2 was expressed at developmental stages from larval to pupal, but not in embryonic stages or adults [
7]. In
A. gambiae,
AgCht2 was expressed at almost all developmental stages, including eggs, four different larval instars, pupae and adults and mainly expressed in pupae and third-instar larvae [
34]. Developmental expression of different chitinase genes exhibited substantial differences in expression patterns of individual groups of chitinase proteins. In
S. exigua, for example, two chitinase genes were found to be increased substantially before each molting and in the prepupae period, as well as in the eclosion stage [
11]. Therefore, further investigations are needed for the elucidation of the roles of other chitinases in
B. dorsalis.
The
BdCht2 transcript was abundant in the integument of third-instar larvae and lower levels in the midgut, fat body, Malpighian tubules and trachea. It was found that
AgCht2 was expressed in the foregut [
34], and
TcCht2 was expressed in the carcass [
10]. Taken together, these data demonstrated that
Cht2 in different species may have different functions. There are several reports showing that the integument is the primary site of chitinase expression, including
T. castaneum [
8],
M. sexta [
17],
S. litura [
28] and
C. fumiferana [
29]. These integument-specific chitinases may be involved in cuticle chitin degradation and, subsequently, affect the insect development. Moreover, several chitinases were found to be mainly expressed in the gut of many insect species and presumed to responsible for regulating the chitin content of the PM [
7,
21,
31]. Besides, in
G. morsitans, a fat body-specific chitinase gene was identified, and it may play a role in immune defense against chitin-containing pathogens [
12].
It was reported that gut-specific chitinase expression could be regulated by starvation in many insects. Specifically, in blood-feeding insects, such as
A. gambiae [
35] and
Lutzomyia longipalpis [
36], the expression of chitinase genes was upregulated substantially in response to feeding. In
O. nubilalis, the transcript level of
OnCht increased significantly through feeding and was downregulated through starvation [
21]. These results suggested that chitinases were involved in changing the gut chitin contents during feeding. However, there has been no report on integument-specific chitinase in response to feeding to date. As starvation induces precocious metamorphosis in the larvae, we propose that the expression of
BdCht2 is precisely coordinated to control the degradation of chitin during the molting process. In the present study, the expression of
BdCht2 was increased by the starvation treatment, but decreased again by the re-feeding treatment, suggesting that
BdCht2 may play roles in the molting process.
Many chitinase genes in various insects are at least, in part, under hormone regulation, their expressions being regulated by hormones, such as 20E and the JH analog (JHA) [
15,
16,
29]. Notably, for minimum endogenous interference, the ideal stage in which the endogenous hormone titers are low should be selected to investigate the precise effect of the treatment of hormone. 20E titers have been shown to be correlated with the abundance of transcripts for the chitinase gene in
T. molitor during metamorphosis. Application of 20E in pupae alone or in combination with JHA resulted in an induction of transcripts for the
TmChit5 gene [
16]. Here, we also found that the
BdCht2 mRNA level could be induced by 20E 8 h after treatment. However, in
B. mori, upregulation of chitinase expression by 20E can be interfered with simultaneously by JHA treatment [
15]. No effect of the treatment of JH alone on chitinase expression was found in
M. sexta [
17]. Besides, the 20E agonist, tebufenozide (RH5992), induced expression of
Cfchitinase in the integument during the early stages of the sixth instar and caused larvae to undergo an incomplete molting into an extra larval stage [
29]. In insects, the expression of more than one chitinase gene was regulated by 20E, and these effects were indirect and might be due to some transcription factors [
37].
RNAi has been successfully used to study the gene function of chitinases in diverse groups of insects. In this regard, reports on RNAi-mediated knockdown of various chitinase genes resulting in lethal phenotype are encouraging in coleopteran [
7]. Moreover, knockdown of two chitinase genes (
SeChi and
SeChi-h) in
S. exigua resulted in different degrees of survival at the pupation and eclosion stages [
11]. Although we performed RNAi for
BdCht2 in
B. dorsalis by injection of dsRNA into the third-instar larvae, no phenotypic abnormalities were observed, due to a limited RNAi response in the larvae (data not shown), consistent with observations in
A. gambiae [
34]. Similarly, the knockdown efficiency of dsRNA for
TcCht2 was low; there was also no observable adverse effect in the treated larvae [
7]. However, whether and how chitinase 2 contributes to insect development is inconclusive and requires further investigation.
4. Experimental Section
4.1. Test Insect
The stock colony of the oriental fruit fly,
B. dorsalis, was reared in the laboratory at 27 ± 1 °C and 70% ± 5% relative humidity on a 14 h Light:10 h Dark photoperiod using an artificial diet, as described previously [
38]. Insects at different developmental stages were collected, immediately frozen in liquid nitrogen and stored at −80 °C.
4.2. Cloning of the Full-Length BdCht2 cDNA
Total RNA was isolated from the third-instar larvae using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. An additional DNase digestion was performed using DNase (TaKaRa, Dalian, China). First-strand cDNA was synthesized by PrimeScript™ RT enzyme (TaKaRa, Dalian, China) using random hexamer primers and oligo dT as reverse transcript primers and was used as a template for PCR.
Based on the high-throughput transcriptome sequencing of
B. dorsalis [
39], we identified a chitinase homologous sequence. This sequence was 1310 bp in length, but lacked a coding sequence at the 3′ end. To obtain the full-length cDNA sequence of
BdCht2, 3′-RACE (rapid amplification of cDNA ends) first strand cDNA were synthesized and a PCR system was constructed according to the instructions of the SMARTer
TM RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA). Specific primers (
Table 1) were used for 3′-RACE, which were synthesized based on the known fragments. The 3′-RACE used 2 μL of 3′-ready-cDNA with Universal Primer Mix (UPM, Clontech, Palo Alto, CA, USA) and
BdCht2-3GSP1 primer. Then, the nested PCR was carried out with Nested Universal Primer (NUP, Clontech, Palo Alto, CA, USA) and
BdCht2-3GSP2 primer. The PCR conditions were as follows: 3 min at 95 °C, followed by 34 cycles of 30 s at 95 °C, 30 s at 60 °C and 90 s at 72 °C, then 10 min at 72 °C. After that, to verify the full-length of cDNA,
BdCht2-F and
BdCht2-R (
Table 1) were designed to amplify the open reading frame (ORF) of
BdCht2. The PCR products were separated by 1.5% agarose gel electrophoresis and stained with GoodView
™ (SBS Genetech, Beijing, China).
4.3. Cloning of the Genomic Sequences and 5′-Flanking Region of BdCht2
Genomic DNA was extracted from adults using an EasyPure Genomic DNA Extraction Kit (TransGen, Beijing, China) according to the manufacturer’s instructions. One pair of gene-specific primers (BdCht2-F/BdCht2-R) was used to produce the genomic DNA sequence of BdCht2 with B. dorsalis genomic DNA as a template. Exons and introns were identified by comparing and analyzing the cDNA and genomic DNA sequences. The 5′-flanking region of BdCht2 was obtained by a Genome Walking Kit (TaKaRa, Dalian, China), and PCR reactions were carried out as per the manufacturer’s instruction.
4.4. Cloning and Sequencing
A band of the expected size was excised, and the fragment was purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and, then, cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA). Ligation reactions were used for transformation of DH5α competent cells (Transgen, Beijing, China). Several recombinant clones were identified by PCR with the primers used before and further sequenced in both directions with an ABI (Applied Biosystems, Foster City, CA, USA) Model 3100 automated sequencer (Life Technologies, Shanghai, China).
4.6. Developmental and Tissue-Specific Expression of BdCht2
To investigate the expression of
BdCht2 in different developmental stages and tissues, the insects from newly-molted third-instar larvae to adults were collected, and five selected tissues, including the integument, trachea, midgut, Malpighian tubules and fat body, were dissected from the third-instar larvae. Total RNA extraction, DNase treatment and cDNA synthesis were performed as described in Section 4.2. Gene-specific qPCR primers were shown in
Table 1. The qPCR was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the following thermo-cycler conditions: 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. Finally, a melting curve analysis from 60 °C to 95 °C was applied to all reactions to ensure the consistency and specificity of the amplified product. The PCR amplifications were performed in 20-μL reaction systems containing 1 μL of template cDNA, 10 μL iQ
™ SYBR
® Green Supermix (Bio-Rad, Hercules, CA, USA) and 0.2 mM each of the primers. The relative expression analysis for qPCR was performed by using α-Tubulin gene (GU269902) as an internal reference based on our previous evaluations [
41]. The ΔΔ
Ct method was used to analyze the relative differences in the expression levels [
42]. Three biological replicates, each with two technical replications were used for qPCR analysis. Data were statistically analyzed using one-way analysis of variance (ANOVA), and the means were separated by Duncan’s new multiple range test (DMRT) for significance (
p < 0.05) by using SPSS 16.0 for windows (SPSS Inc., Chicago, IL, USA, 2008).
4.7. 20E Treatment
The 20-hydroxyecdysone (20E; Sigma, St. Louis, MO, USA) was dissolved in 95% ethanol to make a storage concentration of 10 μg/μL. The storage solutions were then diluted to 1 μg/μL with distilled water. The day 2 third-instar larvae were used for 20E treatment. The treatment method following the micro-topical application technique was performed as previously reported [
33]. For the 20E treatment group, the insects were injected with three different doses of 20E (100, 500 and 1000 ng/larva). For the control group, the larvae were only injected with an equivalent volume of 0.1% ethanol. All the treated larvae were reared as described above. In 1, 4, 8 and 12 h after the topical application of 20E, the insects were collected and the
BdCht2 mRNA level was determined as described above.
4.8. Gene Expression Patterns in Feeding and Starvation Treatment
In the starvation experiments, the day 2 third-instar larvae were used and divided into three groups each with 30 larvae. The first group was used as the control and maintained on the artificial diet until sample collection. The second group was starved for 24 h or 48 h before sample collection. The third group was starved for 24 h and then re-fed for the next 24 h. All of the treated larvae were collected to determine the BdCht2 mRNA level as described above. Three biological replications, each with two technical replications, were used in this analysis.