Takahiro Shiotsuki, Invertebrate Gene Function Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan. Tel.: +81-29-838-6079; fax: +81-29-838-6028; e-mail: email@example.com
Juvenile hormone esterases (JHEs) are required for the degradation of juvenile hormones (JHs) in insects. Here, we report the cloning and analysis of the jhe gene in the red flour beetle, Tribolium castaneum, a model insect of Coleoptera. The Tcjhe gene was strongly expressed at the final instar larva, as would be expected if it functioned to decrease the JH titer at this stage. A recombinant TcJHE protein efficiently degraded JH III, suggesting that the enzyme functions in vivo as a JH-specific degradation enzyme. This is the first report describing the developmental expression profile of the jhe gene whose enzymatic activity was shown in Coleoptera, and the new data reported here will aid elucidation of the mechanism of JH titer regulation in insects.
Juvenile hormone esterase study has been focused mainly on lepidopteran species such as Heliothis virescens (Hanzlik et al., 1989), Manduca sexta (Venkatesh et al., 1990), Trichoplusia ni (Hanzlik & Hammock, 1987), Choristoneura fumiferana (Feng et al., 1999) and Bombyx mori (Shiotsuki et al., 2000; Hirai et al., 2002). This is attributable to the obvious reactivity of JH in these species. However, the gene jhehas not been extensively studied for insect orders other than Lepidoptera. Since lepidopteran JHEs are considered to be evolved independently from other insect order JHEs (Claudianos et al., 2006), cloning and functional analysis of this gene for insects besides Lepidoptera should be important for determining the evolution of JHE and JH titer regulation mechanisms.
Similarly to Lepidoptera, coleopteran insects are also considered to be appropriate for JHE study because of the clear response against JH. To date, there have been several reports of JHE in coleopteran insects such as Tribolium freemani, Tenebrio molitor and Psacothea hilaris. For T. freemani JHE, the enzymatic activity has been investigated but the gene encoding this protein has not yet been identified (Hirashima et al., 1995, 1998, 1999). However, the jhe gene was identified for the latter two species. In case of Te. molitor JHE (TmJHE), the enzyme was shown to have a strong activity for degrading JH, but the developmental expression profile of this gene has not been examined (Hinton & Hammock, 2003b). Conversely, the expression profile of P. hilaris jhe gene has been examined, but it is not known whether the enzyme has the ability to degrade JH (Munyiri & Ishikawa, 2007).
In this study, we successfully cloned a novel jhe gene from the red flour beetle, Tribolium castaneum, a species that has become increasingly important as a model coleopteran insect following the recent completion of its genome project (Tribolium Genome Sequencing Consortium, 2008). The amino acids encoded by this gene showed high similarity to TmJHE and also to P. hilaris JHE (PhJHE), suggesting that this gene functions as a JH-degrading enzyme. In accordance with this hypothesis, a recombinant T. casteneum JHE (TcJHE) retained strong activity to degrade JH in vitro. Tcjhe gene was expressed strongly at the final instar larva, as would be expected if it functioned to decrease the JH titer at this stage. This is the first report describing the developmental expression profile of the jhe gene whose enzymatic activity was revealed in Coleoptera, and our results will be of value to the elucidation of JHE-mediated regulation of JH titer in insects.
Cloning of the gene coding for Tribolium castaneum juvenile hormone esterase
To identify a candidate gene encoding a JH-specific esterase in T. castaneum, we utilized the blastp tool in the National Center for Biotechnology Information (NCBI) site by using the TmJHE amino acid sequence (Hinton & Hammock, 2003b) as a query. JHEs belong to the carboxyl/cholinesterase (CCE) family. Our search of the database revealed that a putative CCE gene (GenBank accession number is XP_969709) had remarkable identity and similarity to the TmJHE sequence, so this putative gene was named T. castaneum juvenile hormone esterase (Tcjhe).
The full-length cDNA sequence of Tcjhe was determined by rapid amplification of cDNA ends (RACE) method and used to produce the putative amino acid sequence of the encoded protein (Fig. 1). Interestingly, the gene was found to have at least five splicing variants that differed only in the 5′ untranslated region (UTR) (Tcjhe-a∼e, Fig. 2). These cDNA sequences have been submitted to the DNA Data Bank of Japan (DDBJ) nucleotide sequence database under the following accession numbers: Tcjhe-a, AB542179; Tcjhe-b, AB542180; Tcjhe-c, AB542181; Tcjhe-d, AB542182; Tcjhe-e, AB542183.
Comparison of the amino acid sequence of TcJHE with those of TmJHE, Drosophila melanogaster JHE (DmJHE) and B. mori JHE (BmJHE) revealed the conservation of important motifs for JHE activity, in particular, the RF, DQ, GQSAG, E and GxxHxxD/E motifs (Fig. 1). TcJHE was also predicted to retain putative signal peptides at the N-terminus (Fig. 1). The deduced protein without putative signal peptide has a theoretical molecular weight of 62.9 kDa and a theoretical pI of 6.29. TcJHE showed 67 % sequence identity to TmJHE and 49 % to PhJHE, while the identity between TcJHE and BmJHE or TcJHE and DmJHE is 30.6 % or 36.2 %, respectively.
Intron-exon structure of Tcjhe
The cDNA sequence of Tcjhe was applied to the T. castaneum genome sequence and the intron-exon structures were determined (Fig. 2). Tcjhe-a–e all have eight exons, and they utilize a different first exon that corresponds to a part of 5′UTR as described above (Fig. 2). The intron positions and splicing site phases with other insect JHEs in the alignment were also compared (Fig. 3). The phase 0 intron at position 95 and the phase 2 intron at position 571 are conserved in all the JHEs analysed (Fig. 3). The phase 0 introns of Tcjhe at position 148 and 467 are shared with JHE of Aedes aegypti and D. melanogaster, suggesting the close relationship between coleopteran and dipteran JHE (Fig. 3). However, coleopteran JHE might also be close to hymenopteran JHE since the phase 1 intron at position 400 and 524 are common between Tcjhe and Apis mellifera jhe (Fig. 3).
Phylogenetic analysis of jhe
To investigate the evolutionary relationship of TcJHE to JHEs of other insect species, a phylogenetic analysis was carried out using the amino acid sequence of each gene (Fig. 3). TcJHE clustered with other coleopteran JHEs such as TmJHE or PhJHE (Fig. 3). This coleopteran cluster was close to those of dipteran and orthopteran JHEs but, as has been reported previously (Claudianos et al., 2006), was independent of that of lepidopteran JHEs (Fig. 3).
Expression of Tcjhe during development
Tcjhe mRNA levels were measured at various developmental stages by quantitative reverse transcription-PCR (qRT-PCR). Tcjhe levels were low in first to third larval instars (Fig. 4A), but substantially higher levels were detected at the beginning of the sixth (penultimate) and seventh (final) instars (Fig. 4B). For pupal and adult stages the expression was examined individually for males and females (Fig. 4C). High levels were detectedon the final day of pupal development in both sexes and, interestingly, the levels in females were greater than those in males by a factor of more than three on the seventh day of adult life (Fig. 4C).
Expression of recombinant proteins of TcJHE
To confirm the enzymatic activity of Tcjhe gene product, a recombinant protein was expressed in Sf9 cells using an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) baculovirus expression system. Since TcJHE was predicted to have signal peptide at the N-terminus (Fig. 1) and to be a secreted type of protein, the culture medium was collected and the expression of the recombinant protein was examined with Western blotting using an antibody against the V5-tag fused to the C-terminus (Fig. 5). The expression of specific protein approximated the predicted size (Fig. 5), suggesting that TcJHE is expressed properly and secreted to the extracellular environment.
Enzymatic activity of TcJHE
The recombinant protein showed a strong ability to degrade JH III. Km and kcat values of TcJHE for JH III of 657 ± 89 nM and 0.164 ± 0.017 s−1, respectively, were obtained (Table 1). We also found that the recombinant protein had a strong ability to degrade methyl hepthylthioacetothioate (HEPTAT), a synthetic substrate of JHE (Table 1).
Table 1. Km and kcat values of the TcJHE protein for JH III, methyl hepthylthioacetothioate (HEPTAT), 1-NA and p-NPA
To investigate whether TcJHE displays general esterase activity with low substrate selectivity, native-polyacrylamide gel electrophoresis (PAGE) and activity staining with 1-naphthyl acetate (1-NA) or 2-NA, the general substrates of esterases, was conducted (Fig. 6A). The migration patterns of both substrates were similar, although more intense staining was present for 2-NA (Fig. 6A). The TcJHE showed weak activity for 1-NA and also for p-nitrophenylacetate (p-NPA), another substrate for detecting a general esterase (Table 1). TcJHE activity was not suppressed by the addition of diisopropyl fluorophosphate (DFP), a serine enzyme inhibitor that does not inhibit lepidopteran JHEs (Shiotsuki et al., 2000); however, it was inhibited by 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which is known to be a specific inhibitor of JHE (Fig. 6B). The IC50 value of OTFP for TcJHE was calculated as 2.0 ± 0.58 nM.
In this study, a novel jhe gene in T. castaneum was successfully identified and cloned. Enzymatic analysis of a recombinant protein from this gene showed that it was able to degrade JH (Table 1). In comparison with other insect JHEs, TcJHE had a kcat value for JH III approximately six to 10 times less than those of TmJHE and lepidopteran JHEs (Ward et al., 1992; Hinton & Hammock, 2003a,b; Tsubota et al., 2010b). Nevertheless, we strongly believe that TcJHE functions as a JH-specific esterase in vivo as it has high affinity for JH III. The Km value of TcJHE for JH III was 657 ± 89 nM (Table 1); this value is lower than the recently described titer of JH III in the haemolymph of adult T. castaneum (Parthasarathy et al., 2009). A biological role for TcJHE as a JH-specific degradation enzyme is also supported by the presence of enzymatic properties that are common in JHEs of other insects, such as a strong ability to degrade HEPTAT, weak activity as a general CCE, and strong inhibition by OTFP (Table 1, Shiotsuki et al., 2000; Hirai et al., 2002; Hinton & Hammock, 2003a; Tsubota et al., 2010b).
This is the first report to describe the developmental expression profile of jhe gene whose enzymatic activity was revealed in the Coleoptera. Tcjhe was strongly expressed at the final larval instar stage (Fig. 4B), as expected of an esterase that functions to reduce the JH titer at this stage. However, comparatively strong expression of the gene was also observed at the penultimate instar stage (Fig. 4B). Expression of jhe at this stage has also been observed in the fat body of the silkworm larva, but in this case JHE protein is supposed not to be translated or secreted into the haemolymph (Hirai et al., 2002). Whether this is also the true for T. castaneum remains to be determined.
Previous analysis examined the developmental JHE activity at the penultimate larval stage, last larval stage and pupal stage in T. molitor (Connat, 1983). According to this study, the strongest JHE activity was detected at the beginning of the pupal stage; rather, the JHE activity at the final larval instar was not so strong (Connat, 1983). A peak of Tcjhe expression at the beginning of pupal stage was also detected in our analysis, but the expression level at this stage was weaker than that in several other stages, including the beginning of sixth larval instar, the beginning of seventh larval instar and the end of pupal stage (Fig. 3). This discrepancy might be due to the difference of the species, but further analysis is required to demonstrate this possibility.
Another interesting character of Tcjhe expression profile is that there was a considerable difference of the expression level between females and males on the seventh day of adulthood: the level in females was greater than that in males by a factor of more than three (Fig. 3C). Since it is known that jhe expression could be induced by the application of JH in several insect species (Wroblewski et al., 1990; Venkataraman et al., 1994; Feng et al., 1999; Vermunt et al., 1999; Kethidi et al., 2005; Kamimura et al., 2007), the higher Tcjhe expression level in adult females might be attributable to the higher JH titer. In T. castaneum whether this is the case is not known, but in B. mori, it is known that JHAMT, a rate-limiting enzyme in JH biosynthesis, is expressed more strongly in females than in males at the adult stage, suggesting that JH titer in females is higher than that in males at the adult stage in this species (Kinjoh et al., 2007). Comparison of JH titer between females and males in T. castaneum might provide the explanation for the distinct expression level of Tcjhe.
The expression profiles of several genes related in JH biosynthesis or JH signalling in T. castaneum, such as TcJHAMT, Tcbroad, TcMet and TcKr-h1, were determined previously (Minakuchi et al., 2008, 2009; Suzuki et al., 2008; Parthasarathy et al., 2008a,b). Among them, TcKr-h1 is considered to be an early JH-response gene that mediates JH action, and its expression was shown to correlate well with that of TcJHAMT (Minakuchi et al., 2009). The expression of TcKr-h1 declined by day1 of the seventh larval instar (Minakuchi et al., 2009), and our analysis revealed that Tcjhe was expressed strongly at this stage (Fig. 4B), suggesting that the degradation of JH by TcJHE might be significant for the decrease of TcKr-h1 expression at the beginning of the final larval instar. TcKr-h1 was expressed strongly in the early larval stages (Minakuchi et al., 2009), and this is also in accordance with the low expression level of Tcjhe in those stages (Fig. 4A).
A phylogenetic analysis indicated that TcJHE clustered with other coleopteran JHEs such as TmJHE and PhJHE (Fig. 3). Among them, the enzymatic activity of PhJHE has not been investigated (Munyiri & Ishikawa, 2007), but the result that PhJHE is placed in the same phylogenetic cluster with other coleopteran JHEs suggest that this enzyme also functions as JH-degrading enzyme in vivo. Recently the phylogenetic analysis of insect CCEs was carried out (Oakeshott et al., 2010) and only TcJHE (corresponding to Tc13193 in (Oakeshott et al., 2010)) was shown to be located in this coleopteran JHE cluster among 49 putative T. castaneum CCEs found in its genome. From this result, it is considered that Tcjhe might be the only gene that functions as JH-specific esterase in T. castaneum.
A phylogenetic and intron-exon structure analysis also showed that the coleopteran JHE was close to those of dipteran, orthopteran and hymenopteran JHEs, but was distant from that of lepidopteran JHEs (Fig. 3). Such a phylogenetic relationship is inconsistent with those normally seen in insects. Thus, for example, the JHE of Orthoptera, hemimetabolous insects, was located with those of holometabolous insect JHEs (Fig. 3). This unique relationship might be correlated with the particular type of JH used by each insect group: JH III is used predominantly in most insects, but in the Lepidoptera, JH I and JH II are apparently the major JHs (Goodman & Granger, 2005). The phylogenetically distant relationship of lepidopteran JHEs to those of other insects might reflect the preferential use of different JH types in these groups. Interestingly, hemipteran insects, whose JHE is located at the most distant position of the phylogenetic tree (Fig. 3), use a novel type of JH (JHSB3) that has a second epoxide next to the carboxylic ester, a different position from Dipteran JHB3 (Kotaki et al., 2009).
In this study, we succeeded in identifying a novel jhe gene that was enzymatically active in T. castaneum, and examined its developmental expression profile. Our recent analysis also identified novel genes encoding JH epoxide hydrolase, another JH-degradation enzyme in this species (Tsubota et al., 2010a), and both results provide significant novel information that will aid elucidation of the mechanisms of developmental regulation by JH titer change in insects.
RNA isolation, molecular cloning, phylogenetic analysis and quantitative reverse transcription-PCR
The wild-type strain of T. castaneum was reared as described previously (Minakuchi et al., 2008). The RNA isolation, molecular cloning, phylogenetic analysis and qRT-PCR were performed as described previously (Minakuchi et al., 2008; Tsubota et al., 2010b). For RACE of Tcjhe, 43 clones were sequenced for 5′RACE while 12 clones for 3′RACE. The primers used for cloning and qRT-PCR are listed in Table 2.
Table 2. Primers used for cloning and quantitative reverse-transcription-PCR (qRT-PCR) of Tcjhe
(A) primers for cloning
(B) primers for qRT-PCR
Expression and enzymatic assays of recombinant TcJHE
A recombinant TcJHE was expressed as a C-terminal V5-His tagged protein using the baculovirus system in Sf9 cells as described previously (Tsubota et al., 2010b). The culture medium was collected and expression of the recombinant protein was confirmed and quantified by Western blotting as described previously (Tsubota et al., 2010b). Enzymatic analyses were performed using [10-3H(N)]- JH III, HEPTAT, 1-NA, 2-NA and p-NPA as substrates, with a slightly modified version of the method (800 nM JH III and 0.1–10 nM OTFP) described previously (Shiotsuki et al., 2000; Tsubota et al., 2010b).
We thank Dr K. Furuta (Shimane University) for kindly providing HEPTAT. This work was supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).