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High-level expression of the Geranylgeranyl diphosphate synthase gene in the frontal gland of soldiers in Reticulitermes speratus IsopteraRhinotermitidae.

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A r t i c l e
HIGH-LEVEL EXPRESSION OF THE
Geranylgeranyl diphosphate
synthase GENE IN THE FRONTAL
GLAND OF SOLDIERS IN
Reticulitermes speratus
(ISOPTERA: RHINOTERMITIDAE)
Masaru Hojo
Faculty of Agriculture, Tamagawa University, Machida, Tokyo, Japan
Kouhei Toga, Dai Watanabe, Tomoyo Yamamoto, and
Kiyoto Maekawa
Graduate School of Science and Engineering, University of Toyama,
Toyama, Japan
Defensive strategies of termite soldiers are roughly classified as either
mechanical, using mandibles and/or the whole head, or chemical, using
frontal gland secretion. Soldiers of the genus Nasutitermes (Termitidae,
Nasutitermitinae), which is one of the most derived termite genera, use
only chemical defenses, and diterpene defensive secretions were suggested
to be synthesized through geranylgeranyl diphosphate (GGPP). On the
other hand, soldiers of the genus Reticulitermes (Rhinotermitidae,
Heterotermitinae) mainly use mechanical defenses, but also use
supplementary chemical defenses involving frontal gland secretions,
including diterpene alcohol. In this study, to confirm whether the GGPP
is used for diterpene synthesis in a representative of an earlier-branching
termite lineage, the GGPP synthase gene (RsGGPPS) was identified in
the rhinotermitid Reticulitermes speratus (Kolbe). The relative expression
level of RsGGPPS in soldiers was three-fold higher than in workers.
Furthermore, RsGGPPS gene expression was detected in epithelial class
1 gland cells around the frontal-gland reservoir. Although GGPP is used
for various essential cellular roles in animals, RsGGPPS is suggested to
Grant sponsor: Japan Society for the Promotion of Science; Grant numbers: 19770012, 20200059, 21770079.
Masaru Hojo’s present address is Tropical Biosphere Research Center, University of the Ryukyus, Okinawa
903-0213, Japan.
Correspondence to: Kiyoto Maekawa, Graduate School of Science and Engineering, University of Toyama,
Gofuku 3190, Toyama 930-8555, Japan. E-mail: kmaekawa@sci.u-toyama.ac.jp
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 77, No. 1, 17–31 (2011)
Published online in Wiley Online Library (wileyonlinelibrary.com).
& 2011 Wiley Periodicals, Inc. DOI: 10.1002/arch.20415
18
Archives of Insect Biochemistry and Physiology, May 2011
be used not only for these essential roles but also for diterpene synthesis in
order to produce defensive secretions. Chemical structures of the
diterpene identified from Reticulitermes and Nasutitermes are extremely
different from each other, and the two genera are phylogenetically distant
from each other. Thus, these two lineages may have independently
C 2011 Wiley
acquired the abilities of diterpene synthesis from GGPP. Periodicals, Inc.
Keywords: isoprenoid; terpenoid; soldier specific gene; biosynthesis;
geranylgeranyl pyrophosphate
INTRODUCTION
In the colonies of eusocial insects, there are various castes carrying out various tasks in
order to maintain the colony. Termites are a major group of eusocial insects. Termite
castes are roughly divided into reproductive castes (founding queen or king, or
supplementary reproductives) and non-reproductive castes (worker or soldier).
Within the non-reproductive castes, workers help the reproductive castes and larva,
and soldiers defend their colony (Noirot, 1989; Roisin, 2000).
Soldiers in termites are a peculiar caste among social insects in terms of their
specific morphology and production of defensive materials (Deligne et al., 1981).
Soldiers’ defensive strategies are typically classified as either mechanical, involving the
mandibles for biting or snapping and/or the whole head for blocking the enemy
invasion, or chemical, involving the frontal gland, which is an exocrine gland specially
developed in the soldier caste of termites (Deligne et al., 1981; Prestwich, 1984). The
frontal glands of soldiers develop only in derived families belonging to the
Serritermitidae, Rhinotermitidae, and Termitidae (Prestwich, 1979a; Noirot and
Darlington, 2000; Inward et al., 2007).
In termites, chemical defense is divided into three categories (Deligne et al., 1981):
(1) biting and injecting a toxic secretion into the wound; (2) daubing, using an
enlarged brush-like labrum to put a contact poison on the cuticle; and (3) ejecting an
irritant secretion against an enemy from the tip of the head. Strategy (3) is thought to
be the most advanced and is seen in soldiers of subfamily Nasutitermitinae (family
Termitidae). The chemical secretions synthesized in the frontal gland vary according to
species (Prestwich, 1979a, 1984, 1988). For example, among the soldiers using strategy
(1), the main component of the frontal gland secretion is an alkane or alkene in the
Macrotermitinae (family Termitidae) (Prestwich et al., 1977) or mucopolysaccharide in
Coptotermitinae (family Rhinotermitidae) (Moore, 1968; Blum et al., 1982). Among
soldiers using strategy (2), various ketones are included in the secretions of
Rhinotermitinae (family Rhinotermitidae) (Prestwich and Collins, 1982) or acyclic
diterpene alcohols in the secretions of Heterotermitinae (family Rhinotermitidae)
(Baker et al., 1982). Among soldiers using strategy (3), dome-shaped polycyclic
diterpenes are used in the Nasutitermitinae (family Termitidae) (Prestwich, 1979b;
Prestwich and Collins, 1981; Laurent et al., 2005). Although none of the synthetic
enzymes involving the defensive secretions in termite soldiers have been elucidated,
genes encoding geranylgeranyl diphosphates (GGPP) synthases are thought to be
related to diterpene synthesis in the Nasutitermitid species Nasutitermes takasagoensis
(Hojo et al., 2007).
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High Expression of GGPPS in Reticulitermes Soldier
19
GGPP synthase is a type of short-chain isoprenyl diphosphate synthase and is used
for GGPP synthesis, which plays an essential role in various processes such as electron
transport (e.g., ubiquinones; Kawamukai, 2002), glycosylation (e.g., dolichols; Schenk
et al., 2001), and membrane association (prenylation of proteins: e.g., geranylgeranylated proteins) (Casey, 1992; Clarke, 1992; Schafer and Rine, 1992; Omer and
Gibbs, 1994; Casey and Seabra, 1996; Zhang and Casey, 1996), as well as in secondary
metabolites such as carotenoids (Cunningham and Gantt, 1998). In many plants and
some bacteria, GGPP is the direct precursor of various diterpenes (West, 1981;
MacMillan and Beale, 1999; Croteau et al., 2000). Because most animals are not able to
synthesize cembrene-A-derived polycyclic diterpene, it is thought that GGPP synthase
genes in Nasutitermitinae have duplicated from an ancestral gene playing essential
roles as shown in ancestral termites (Hojo et al., 2007). This was accompanied by the
alteration of defensive behavior.
Because the chemical structures of acyclic diterpene alcohols used in the
Heterotermitinae and the dome-shaped polycyclic diterpene used in Nasutitermitinae
are extremely different from each other, each downstream synthetic pathway should
be different. In order to confirm whether the GGPP is also used for the diterpene
synthesis in Heterotermitinae, we performed analyses on GGPP synthase genes of
species belonging to this subfamily. In Heterotermitinae, Reticulitermes is one of the
most suitable species for these analyses, because soldiers of Reticulitermes use geranyl
linalool, a kind of acyclic diterpene alcohol, as a defensive material (Parton et al., 1981;
Baker et al., 1982; Bagnères et al., 1990; Lemaire et al., 1990; Nelson et al., 2001,
2008; Quintana et al., 2003).
In this study, we identified a GGPP synthase gene in Reticulitermes speratus. We then
analyzed the gene expression of GGPP synthase in the soldier caste, and considered the
evolution of diterpene synthesis in different species.
MATERIALS AND METHODS
Termite Samples
Colonies of R. speratus were collected from Yokohama City, Kanagawa, Japan in
February 2006 or Toyama City, Toyama, Japan in September 2008. The collected
colonies were reared in the laboratory in an air-conditioned room at 25731C until use.
RNA Extraction, cDNA Synthesis, and Reverse Transcription Polymerase Chain Reaction
(RT-PCR)
For the determination of the partial sequence of GGPP synthase genes from R. speratus,
total RNA was extracted from the head of soldiers using Isogen (Nippon Gene, Tokyo,
Japan). First-strand cDNAs were synthesized using SuperScript III transcriptase
(Invitrogen, Carlsbad, CA) with oligo(dT) primer. For RT-PCR, we designed a forward
primer (50 -GGA AGA ACA TGT GAA AGG AAA CGG-30 : Fig. 1, narrow downward
arrow) and a reverse primer (50 -CTC TGA ATC AGC TGC TTG TAT TCC-30 : Fig. 1,
narrow forward arrow) based on one of the GGPP synthase gene sequences of
N. takasagoensis (NtGGPPS1: GenBank accession no. AB266077). PCR was performed
using TaKaRa EX Taq (Takara Bio Inc., Otsu, Shiga, Japan) under the following
conditions: 941C for 5 min; 941C for 30 s, 501C for 1 min, and 721C for 1 min for 35
cycles; and then 721C for 7 min.
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Archives of Insect Biochemistry and Physiology, May 2011
Figure 1. Full-length cDNA sequence and the deduced amino acid sequence of the Reticulitermes speratus
geranylgeranyl diphosphate synthase (RsGGPPS) protein (2,036 bp, ORF encoding 323 amino acid
residues). The deduced amino acid sequence is shown under the nucleotide sequence. The termination
codon is indicated by an asterisk. The possible polyadenylation signal (AATAAA) is double underlined. The
region determined by RT-PCR is shaded, and narrow arrows indicate the regions of the primers. The dotted
arrow indicates the region of the primer for 30 -RACE. The double-lined arrow indicates the region of the
primer for 50 -RACE. The region used for RT-qPCR is underlined, and the regions of the primers are
indicated by broad arrows. The region of the probe for in situ hybridization is boxed.
Subcloning and Sequencing
The PCR product was purified using a Wizard SV Gel and PCR Clean-Up System
(Promega, Madison, WI), ligated into a pGEM-T Easy Vector (Promega) using a
DynaExpress DNA Ligation Kit ver. 2 (BioDynamics Laboratory Inc., Tokyo, Japan),
and was transfected into Escherichia coli JM109 competent cells (Takara Bio Inc.). The
inserted DNA fragment was amplified by PCR using primers SP6 and T7, and was
purified using a ExoSAP-IT For PCR Product Clean-Up kit (USB Corporation,
Cleveland, OH). The purified product was used as a template for sequencing. The
sequencing reactions were performed using the dideoxy-nucleotide cycle sequencing
procedure with a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems,
Foster, CA). Data collection was performed using an automatic DNA sequencer
(Applied Biosystems, ABI PRISM 3100 Genetic Analyzer).
Rapid Amplification of cDNA Ends (RACE)
For the determination of the full-length sequence of the GGPP synthase gene of
R. speratus, first-strand cDNAs for 30 -RACE and 50 -RACE were each synthesized from
Archives of Insect Biochemistry and Physiology
High Expression of GGPPS in Reticulitermes Soldier
21
total RNA described above by using SMART RACE cDNA Amplification Kit (BD
Biosciences Clontech, Palo Alto, CA). RACE-PCR was performed with Advantage 2
Polymerase Mix (BD Biosciences Clontech) using the adaptor primer in the kit and a
specific primer designed based on the determined partial sequences of the R. speratus
GGPP synthase gene. The cycle parameters of the PCR were in accordance with the
manufacturer’s instructions (BD Biosciences Clontech). The amplified cDNA
fragments were purified using the Wizard SV Gel and PCR Clean-Up System
(Promega), ligated into a TOPO TA-cloning vector (Invitrogen), and used to transform
E. coli One Shot Mach1-T1 competent cells (Invitrogen) using TOPO Cloning of Taq
polymerase-amplified PCR products into an entry vector for the Gateway System
(Invitrogen). The inserted DNA was amplified by PCR using primers M13 forward and
T7 and was purified using ExoSAP-IT For PCR Product Clean-Up (USB Corporation).
The purified product was used as a template for the sequencing reaction. The
sequencing reactions and data collection were performed with the same methods as
described above.
Homology Search and Phylogenetic Analysis
To find homologous proteins, homology searches were performed with the amino acid
sequences predicted from the nucleotide sequences using the Basic Local Alignment
Search Tool (BLAST) database on the National Center for Biotechnology Information
(NCBI) server (http://www.ncbi.nlm.nih.gov/BLAST/). Then, the deduced amino acid
sequences were aligned with similar proteins using the Clustal W ver. 1.83 package
(Thompson et al., 1994) and shaded using Boxshade version 3.21 on the Swiss
EMBnet server (http://www.ch.embnet.org/software/BOX_form.html), and the alignments were corrected manually. Estimations of tree topology were obtained using
maximum parsimony (MP) criteria and by the neighbor-joining (NJ) method using the
program PAUP4.0b10 (Swofford, 2000). MP trees were estimated heuristically in
PAUP using default options with 100 random addition replicates. Fifty percent
majority-rule bootstrap trees were also obtained using PAUP (1000 replicates, 10
random addition replicates per bootstrap replicate). All characters were weighed
equally, and gaps were treated as a new state. For NJ, mean character differences
implemented in PAUP were used for genetic distances, and gaps were treated as
missing. Bootstrap confidence intervals on each branching pattern were calculated
from 1,000 replicates of resampling.
Reverse Transcription Quantitative Real-Time PCR (RT-qPCR)
For RT-qPCR, total RNA from the head of 10 soldiers or 10 workers was isolated using
Isogen (Nippon Gene). Each biological sample was replicated three times, using three
different colonies. The quality and quantity of extracted RNA were determined by
spectroscopic measurements at 230, 260, and 280 nm using a NanoVue spectrophotometer (GE Healthcare Bio-Sciences, Tokyo, Japan). For single-strand cDNA
synthesis, DNase-treated RNA (1 mg per sample) was transcribed using High Capacity
cDNA Reverse Transcription Kit as indicated by the manufacturer (Applied
Biosystems). Real-time PCR (200 nM of each primer) was performed using SYBR
Green I reagent and a MiniOpticon Real-Time System (Bio-Rad Laboratories,
Hercules, CA). For determining endogenous control of constitutive expression, the
suitability of three reference genes, i.e. EF1-alfa (Accession No. AB602838), NADH-dh
(No. AB602837), and b-actin (No. AB520714; Maekawa et al., 2010) of R. speratus, were
Archives of Insect Biochemistry and Physiology
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Archives of Insect Biochemistry and Physiology, May 2011
evaluated with the appropriate softwares, i.e. GeNorm (Vandesompele et al., 2002) and
NormFinder (Andersen et al., 2004). Primers for the target and reference genes were
designed using Primer Express software (Applied Biosystems); for EF1-alfa: forward,
50 -GGT GAT GCG GCT ATT GTT AAC C-30 , reverse, 50 -GTG GTG GGA ATT CTG
AGA AAG ATT-30 ; for NADH-dh: forward, 50 -GCT GGG GGG GTT ATT CAT TCC AT-30 ,
reverse, 50 -GGC ATA CCA CAA AGG GCA AAA-30 ; for b-actin: forward, 50 -AGC GGG
AAA TCG TGC GTG AC-30 , reverse, 50 -CAA TGG TGA TGA CCT GGC CAT-30 ; for
GGPP synthase (RsGGPPS; see results): forward, 50 -CTC TTC TCT CCG TAG AGG
T-30 , reverse, 50 -GCC AGT ATA TTT CCA TCC CA-30 . The thermal cycling program
consisted of 3 min at 951C followed by 39 cycles of 20 s at 951C, 20 s at 601C, and 30 s at
721C. The production of single products was confirmed by dissociation curve analysis
conducted using the MiniOpticon system. These analyses were performed with
reference to the MIQE guidelines (Minimum Information for Publication of
Quantitative Real-time PCR Experiments) (Bustin et al., 2009). Statistical analysis
was performed using Student’s t-test using statistical software Mac Statistical Analysis
ver. 1.5 (Esumi, Tokyo, Japan).
In Situ Hybridization
The heads of soldiers (n 5 12) were dissected from the bodies and fixed with 4%
paraformaldehyde in phosphate-buffered saline. Then, the heads were embedded in
TissueTek O.C.T. Compound (Sakura Finetek USA Inc., Torrance, CA) and 12-mm
cryosections were prepared and collected on Mas-coated glass slides (Matsunami Glass
Ind., Osaka, Japan) using a CM1510S cryostat (Leica, Tokyo, Japan). The sections
were hybridized with DIG-11-UTP-labeled single-strand sense (n 5 6) or antisense
(n 5 6) RNA probes using the ISHR Starting Kit (Nippon Gene) in accordance with the
instructions provided by the manufacturer. DIG-labeled RNA probes were prepared
using a DIG RNA Labeling Kit (Roche, Grenzacherstrasse, Basel, Switzerland). After
hybridization, the slides were washed using a ISHR Starting Kit in accordance with the
manufacturer’s instructions. Immunocytochemical detection of DIG-labeled RNA was
carried out with an alkaline phosphatase-conjugated anti-DIG antibody and NBT/
BCIP as a substrate using a DIG Nucleic Acid Detection Kit (Roche) in accordance with
the manufacturer’s instructions.
RESULTS
Determination of the Partial GGPP synthase Gene Sequence of R. speratus
As a result of RT-PCR, a single, approximately 500 bp band was detected. The PCR
product was purified, and the purified product was subcloned and sequenced.
Consequently, we obtained one identical 435 bp sequence from nine clones (Fig. 1,
shaded). The amino acid sequence was translated from the obtained nucleotide
sequence and subjected into BLAST P search. Three GGPP synthases of N. takasagoensis
were retrieved with the highest score. We concluded that this sequence is a GGPP
synthase gene of R. speratus and named it RsGGPPS.
Full-Length Sequence of RsGGPPS
In order to determine the full-length sequence of RsGGPPS, 30 -RACE was performed
with primer (50 -CCT CAC AGG GCT GAA GAA GGC GCA GAG-30 : Fig. 1, dotted
Archives of Insect Biochemistry and Physiology
High Expression of GGPPS in Reticulitermes Soldier
23
arrow) designed from the partial sequence obtained by RT-PCR. This revealed five
clones with identical sequences of approximately 1.6 kbp with part of the sequence
identical to that of RsGGPPS already determined. Next, we performed 50 -RACE with
another primer (50 -CAC CTG ATG GTC ATC TGG-30 : Fig. 1, double-lined arrow)
designed from the sequence obtained by 30 -RACE. We obtained four identical clones of
approximately 0.9 kbp with identical sequence of RsGGPPS. Finally, we obtained one
cDNA fragment of 2,036 bp with an ORF encoding 323 amino acid residues. The
polyadenylation signals (AATAAA) were found 848-bp downstream of the predicted
stop codon (Fig. 1). The determined nucleotide and putative amino acid sequence are
available at DDBJ/EMBL/GenBank databases (GenBank accession no. AB548355).
Identification of Homologous Proteins
The putative amino acid sequence of the RsGGPPS protein was subjected to an NCBI
database search (performed in February 2010). This search identified the following
matches in decreasing order of sequence similarity: GGPP synthase-1-B-isoform of
N. takasagoensis (GenBank accession no. BAF42686, Identities 66%, Score (bits) 441,
e-value 5e-122); GGPP synthase-3-B-isoform of N. takasagoensis (BAF42694, 65%, 431,
5e-119), GGPP synthase-2-B-isoform of N. takasagoensis (BAF42692, 64%, 426, 1e-117),
predicted GGPP synthase homolog of Nasonia vitripennis (XP_001605679, 64%, 423,
1e-116), putative GGPP synthase of Pediculus humanus corporis (XP_002429245, 60%,
403, 1e-110), putative trans-isoprenyl diphosphate synthase (trans-IPPS) of Drosophila
mojavensis (XP_002007505, 60%, 399, 3e-109), putative trans-IPPS of Drosophila
erecta (XP_001971464, 61%, 396, 1e-108), putative trans-IPPS of Drosophila virilis
(XP_002046879, 60%, 396, 2e-108), putative trans-IPPS of Anopheles gambiae
(XP_308860, 62%, 396, 2e-108), GGPP synthase of Drosophila melanogaster (quemao)
(NP_523958, 60%, 395, 4e-108), putative trans-IPPS of Apis mellifera (XP_001122899,
61%, 392, 3e-107), GGPP synthase of Aedes aegypti (XP_001650014, 61%, 387, 8e-106),
putative trans-IPPS of Tribolium castaneum (XP_971444, 60%, 380, 1e-103), GGPP
synthase of Ixodes scapularis (XP_002415093, 57%, 350, 8e-95), GGPP synthase of Homo
sapiens (AAH67768, 55%, 335, 5e-90), and GGPP synthase of Danio rerio (NP_956329,
55%, 333, 1e-89) (Fig. 2). The putative amino acid sequence of the RsGGPPS protein
has two characteristic aspartate-rich domains (Fig. 2A, underlined); the first aspartaterich motif is thought to be involved in the product chain-length determination, and
second aspartate-rich motif is thought to be critical for the enzyme’s catalytic efficiency
for short-chain isoprenyl diphosphate synthases (Wang and Ohnuma, 2000; Liang
et al., 2002).
Inferred Phylogenetic Tree
There were two ambiguous alignment regions in the amino acid sequences of the
GGPP synthases from animals (21 amino acids in the N-terminal region, and 44 amino
acids in C-terminal region; Fig. 2A), thus the data were analyzed without these regions.
Based on the remaining 272 characters, five MP trees were found; the strict consensus
of these trees is shown in Figure 2B. The GGPP synthases of termites, including
R. speratus, formed a monophyletic group with strong support in the bootstrap analysis
(100% in MP and NJ). The GGPP synthases of insects were all grouped in a
monophyletic group (over 95% bootstrap support).
Archives of Insect Biochemistry and Physiology
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Archives of Insect Biochemistry and Physiology, May 2011
Figure 2. (A) Comparison of the deduced amino acid sequence of RsGGPPS with those of three retrieved
termite GGPP synthase proteins of Nasutitermes takasagoensis (NtGGPPS1B, GenBank accession no.
BAF42686; NtGGPPS2B, BAF42692; NtGGPPS3B, BAF42694) and GGPP synthase-like proteins from
P. humanus corporis (Pediculus, XP_002429245), T. castaneum (Tribolium, XP_971444), A. mellifera (Apis,
XP_001122899), and D. melanogaster (DmGGPPS, NP_523958). The two characteristic, highly conserved
aspartate-rich domains for short-chain isoprenyl diphosphate synthase are underlined. Filled boxes indicate
the presence of conserved residues in all sequences and shaded boxes indicate the same characteristic
residues in all sequences. (B) Phylogenetic relationship of RsGGPPS proteins; three termite GGPP synthase
proteins of N. takasagoensis (NtGGPPS1B, GenBank accession no. BAF42686; NtGGPPS2B, BAF42692;
NtGGPPS3B, BAF42694), and GGPP synthase-like protein from insects: P. humanus corporis (XP_002429245),
T. castaneum (XP_971444), N. vitripennis (XP_001605679), A. mellifera (XP_001122899), D. melanogaster
(NP_523958), Drosophila erecta (XP_001971464), D. virilis (XP_002046879), Drosophila mojavensis
(XP_002007505), A. gambiae (XP_308860), and A. aegypti (XP_001650014); other arthropod: I. scapularis
(XP_002415093); and vertebrates: Homo sapiens (AAH67768), and D. rerio (NP_956329). The tree shown is a
strict consensus of the five most parsimonious trees of RsGGPPS and other homologous proteins. Bootstrap
values (%) from MP and NJ analyses are shown above and below branches, respectively. Asterisks indicate
nodes that were not supported in greater than 50% of MP and NJ bootstrap replicates.
Archives of Insect Biochemistry and Physiology
High Expression of GGPPS in Reticulitermes Soldier
25
Figure 2. Continued
Figure 3. Relative expression level of RsGGPPS gene measured by reverse transcription quantitative realtime polymerase chain reaction. The quantity of transcripts from the soldiers was normalized to that of
workers as 1.0. Data are expressed as means7SD. The biological replicate numbers are shown in
parentheses (worker: n 5 3; soldier: n 5 3). The P-values were determined using Student’s t-test.
Relative Expression of RsGGPPS Gene
Both GeNorm (Vandesompele et al., 2002) and NormFinder (Andersen et al., 2004)
analyses showed that the expression levels of EF1-alfa were the most stable. Therefore,
EF1-alfa was used as the reference gene for quantification in this study. Results from
RT-qPCR experiments with the RsGGPPS gene showed that the expression level in
soldiers was about three-fold higher than that in workers (Fig. 3). A single peak on the
melting curve was observed, showing that a specific PCR product was amplified and
that primer dimers were not formed. Furthermore, the sequence analysis showed that
the amplified band was RsGGPPS only (data not shown).
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Localization of the RsGGPPS Gene
To examine the expression site of the RsGGPPS gene, in situ hybridization analysis was
performed using DIG-labeled RNA probes (Fig. 1, boxed) on cryosections of soldier
heads. RsGGPPS gene expression was detected in the thick epithelial cell layer around
the frontal-gland reservoir (Fig. 4, arrows), and the signal was uniformly present
throughout the glandular cells. This glandular epithelium of the frontal gland reservoir
in Reticulitermes is only composed of class 1 gland cells. On the other hand, there are class
3 gland cells around the frontal pore discharging the frontal gland secretion (Quennedey,
1984). These gland cell types were classified as secretory epithelium cell classes by Noirot
and Quennedey (1974). Class 1 gland cells are a common epidermal cell simply covered
by the cuticle; the secretion is released through the cuticle. Class 3 gland cells have a
cuticular ductule composed of ductule cells, and the secretion is released through the
ductule. No signal was detected in vacuolar class 3 gland cells (Fig. 4, arrowheads).
DISCUSSION
The frontal gland in soldiers is only observed in the relatively derived termite families
(Serritermitidae, Rhinotermitidae, and Termitidae). Therefore, chemical defenses using
frontal gland secretions are thought to be derived (Prestwich, 1983). The most derived
Nasutitermes soldiers use polycyclic diterpenes as defensive secretions (Prestwich, 1979b;
Prestwich and Collins, 1981; Laurent et al., 2005). Although animals cannot usually
synthesize compounds such a cembrene-A derived polycyclic diterpene, it is known that
the soldiers of Nasutitermes can synthesize this complicated polycyclic diterpene
(Prestwich et al., 1981, 1987). On the contrary, the defensive secretion in Reticulitermes,
whose soldiers use mechanical and chemical defense, is diterpene alcohol with a simple
acyclic structure (Parton et al., 1981; Baker et al., 1982; Bagnères et al., 1990; Lemaire
et al., 1990; Nelson et al., 2001, 2008; Quintana et al., 2003).
All kinds of diterpenes are synthesized through the acyclic 20 carbon direct
precursor, GGPP (West, 1981; MacMillan and Beale, 1999; Croteau et al., 2000), and
GGPP is synthesized from isopentenyl diphosphate (IPP) with dimethylallyl diphosphate, an interconvertible isomer of IPP (Chen et al., 1994; Ogura and Koyama, 1998;
Wang and Ohnuma, 1999). The sequential condensation of IPP is carried out by shortchain isoprenyl diphosphate synthases, such as geranyl diphosphate (GPP) synthase,
farnesyl diphosphate synthase, and GGPP synthase, and two characteristic aspartate-rich
sequences of these enzymes (Fig. 2, underlined) serve not only as substrate binding sites,
but also as essential sites for the catalytic reactions (Song and Poulter, 1994; Tarshis et al.,
1996; Koyama, 1999). In animals, GGPP is mainly used for various essential roles in cells
(Clarke, 1992; Casey, 1992; Schafer and Rine, 1992; Omer and Gibbs, 1994; Casey and
Seabra, 1996; Zhang and Casey, 1996; Cane, 1999). However, in derived Nasutitermes, it
was suggested that GGPP synthases were also used in connection with the synthesis of
diterpene-containing defensive secretions (Hojo et al., 2007).
In this study, we showed that the RsGGPPS gene in soldiers was expressed at higher
levels than in workers of R. speratus (Fig. 3), and the expression was shown in class 1
gland cells around the frontal gland reservoir (Fig. 4). Although the defensive secretions
of R. speratus soldiers have not been investigated, these results suggest that RsGGPPS is
concerned with the synthesis of diterpene alcohol contained in the defensive secretion
synthesized in frontal gland. Consequently, GGPP synthase is suggested to be involved in
Archives of Insect Biochemistry and Physiology
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Figure 4. In situ hybridization of RsGGPPS mRNA in the soldier head of Reticulitermes speratus. (A) and (B)
are hematoxylin-eosin stained paraffin-embedded vertical sections (6-mm thick) from a soldier. The head is
on the left side in (A). The frontal gland reservoir (R) is located in the middle of thorax. (B) is a magnified
photograph of (A) around the frontal pore (P) discharging the frontal gland secretion. The class 1 gland cell
layer surrounding the frontal gland reservoir is indicated by an arrow. The vacuolar class 3 gland cell unit
around the frontal pore (P) is indicated by arrowhead. (C) and (D) are cryosections (12-mm thick) of a soldier
head subjected to in situ hybridization with an antisense DIG-labeled RNA probe. Only the upper side of the
soldier head is shown in (C). The front of the head is on the left side. (D) is a magnified photograph of (C)
around the frontal pore (P). The class 1 gland cell layer is stained dark (arrow) in contrast to the vacuolar
class 3 gland cell unit (arrowhead). The inset of (D) indicates epithelial cells hybridized with a sense probe
(negative control). Bars 5 0.5 mm (A), 0.05 mm (B and D), and 0.25 mm (C).
Archives of Insect Biochemistry and Physiology
28
Archives of Insect Biochemistry and Physiology, May 2011
diterpene synthesis as well as for cellular essential roles even in more ancestral species.
To know whether RsGGPPS identified in this study have these dual roles, further
analyses on the paralogous genes and their function are needed.
RsGGPPS expression was not detected in vacuolar class 3 gland cells around the
frontal pore (Fig. 4). In Nasutitermitinae, monoterpenes are thought to be synthesized
as a solvent for diterpene in the class 3 gland cells around the frontal pore, because
diterpenes are viscous and monoterpenes prevent plugging of the pore (Deligne et al.,
1981). Some monoterpenes are also included in the frontal gland secretions in
Reticulitermes (Parton et al., 1981; Zalkow et al., 1981; Bagnères et al., 1990; Nelson
et al., 2001, 2008; Quintana et al., 2003). These monoterpenes are thought to be
concerned with the volatility of the secretions (Reinhard et al., 2003). Because the
precursors of all monoterpenes are GPP, further analyses of the expression of GPP
synthase genes are needed to determine whether monoterpene secretions are also
synthesized only in class 3 gland cells in Reticulitermes.
It is thought that GGPP synthase is associated with the synthesis of diterpene, and that
this gene evolved in parallel with the acquisition of chemical defense mechanisms found
in the most derived termites (Hojo et al., 2007). The chemical structures of acyclic
diterpene alcohol synthesized by Reticulitermes and of dome-shaped polycyclic diterpene
synthesized by Nasutitermes are substantially different. Reticulitermes (Heterotermitinae,
Rhinotermitidae) and Nasutitermes (Nasutitermitinae, Termitidae) are phylogenetically
distantly related to each other (Inward et al., 2007), and the soldiers of other taxa, which
are more closely related to each subfamily [for example, Macrotermitinae (Termitidae),
Coptotermitinae and Rhinotermitinae (Rhinotermitidae)], do not use diterpenes as a
defensive secretion (Prestwich, 1984). Thus, diterpene synthesis abilities have probably
been acquired independently at least in Heterotermitinae and Nasutitermitinae. GGPP
synthase-mediated diterpene synthesis ability probably evolved not only in the most
derived termites but also more representatives of earlier-branching lineages. Further
studies on the isolation and expression of GGPP synthase genes in soldiers with no frontal
gland (Mastotermitidae, Termopsidae, Hodotermitidae, and Kalotermitidae) or in
soldiers with no diterpene in their frontal gland secretions (e.g. Macrotermitinae,
Coptotermitinae and Rhinotermitinae) should help to elucidate the evolutionary
mechanisms of diterpene chemical defense in termites.
ACKNOWLEDGMENTS
We are grateful to Dr. Nathan Lo who gave us valuable comments on the manuscript
and corrected the English. Thanks are also due to Keisuke Shimada, Kyoko Ishitani,
Ikkei Shirasaki, Iwao Itai, and Satoshi Nakamura for their help during field and
laboratory work. This study was supported by Research Fellowships for Young
Scientists to M. H., and in part by a Grant-in-Aid for Young Scientists (Nos. 19770012
and 21770079 to K. M.) and for Scientific Research on Innovative Areas
(No. 20200059 to K. M.) from the Japan Society for the Promotion of Science.
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expressions, level, speratus, isopterarhinotermitidae, geranylgeranyl, high, synthase, frontal, diphosphate, soldiers, reticulitermes, gland, genes
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