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Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)
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Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)
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Keisuke Shimada 1, Kiyoto Maekawa ⇑ Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama, Toyama 930-8555, Japan
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Article history: Received 4 March 2014 Received in revised form 18 April 2014 Accepted 1 May 2014 Available online xxxx
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Keywords: Beta-glucosidase Gene expression Cellulose digestion Social communication Termites Cockroaches
a b s t r a c t Beta-glucosidase (BG) is known as a multifunctional enzyme for social maintenance in terms of both cellulose digestion and social communication in termites. However, the expression profiles of each BG gene and their evolutionary history are not well understood. First, we cloned two types of BG homologs (RsBGI and RsBGII) from the termite Reticulitermes speratus (Kolbe). Gene expression analyses showed that RsBGI expression levels of primary queens and kings from 30 to 100 days after colony foundation were high, but those of reproductives dropped after day 400. Extremely low gene expression levels of RsBGI were observed in eggs, whereas workers had significantly higher expression levels than those of soldiers and other colony members. Consequently, RsBGI gene expression levels changed among each developmental stage, and RsBGI was shown to be involved in cellulose digestion. On the other hand, the RsBGII gene was consistently expressed in all castes and developmental stages examined, and notable expression changes were not observed among them, including in eggs. It was indicated that RsBGII is a main component involved in social communication, for example, the egg-recognition pheromone shown in this species previously. Finally, we obtained partial gene homologs from other termite and cockroach species, including the woodroach (genus Cryptocercus), which is the sister group to termites, and performed molecular phylogenetic analyses. The results showed that the origin of the BG gene homologs preceded the divergence of termites and cockroaches, suggesting that the acquisition of multifunctionality of the BG gene also occurred in cockroach lineages. Ó 2014 Elsevier Ltd. All rights reserved.
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1. Introduction
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In eusocial termites, primary reproductives (alates, queens and kings) and neotenic reproductives (i.e. supplementary queens and kings such as nymphoids and ergatoids) are devoted to reproduction (Korb and Hartfelder, 2008). Workers forage for food and care for their siblings, whereas soldiers defend the colony (Wilson, Q3 1971). Molecular mechanisms to regulate the division of labor among these castes and their sociality have developed during the course of termite evolution, and common mechanisms should be shared by all extant termites (reviewed in Miura and Scharf, 2011). Several common genes responsible for social maintenance and genes showing caste-specific expression have been identified in several termite species (e.g. encoding hexamerines and vitello-
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⇑ Corresponding author. Tel.: +81 76 445 6629; fax: +81 76 445 6641. E-mail address: [email protected] (K. Maekawa). 1 Present address: Ishikawa Museum of Natural History, Ri-441 Choshi-machi, Kanazawa, Ishikawa 920-1147, Japan.
genins) (Scharf et al., 2005a, 2007). Endogenous termite cellulase is also a good example of such a gene. Cellulolytic protists in the gut of lower termites are well-known mutualistic symbionts (Cleveland, 1923), but termites also secrete cellulases by themselves (Watanabe et al., 1998). Endo-beta-1,4glucanase (EG; EC 3.2.1.4) belonging to the glycosyl hydrolase family (GHF) 9 and beta-glucosidase (BG; EC 3.2.1.21) affiliated with GHF1 are known as termite-derived cellulases (reviewed in Lo et al., 2011). EGs and BGs are known as common cellulase components in bacteria, fungi, protists, plants and animals. Cellulose chains are hydrolyzed to cellobiose and cellotriose by EGs, and short-chain sugars are converted into glucose by BGs (Ni et al., 2005; Zhang et al., 2009). In termites and their relatives (i.e. cockroaches), EG genes are found in many species (Lo et al., 2000; Shimada and Maekawa, 2008), but BG genes have only be identified in some termite species, including Neotermes koshunensis (Tokuda et al., 2002), Coptotermes formosanus (Zhang et al., 2010, 2012a), Reticulitermes flavipes (Scharf et al., 2010), Macrotermes barneyi (Wu et al., 2012) and Nasutitermes takasagoensis (Tokuda
http://dx.doi.org/10.1016/j.jinsphys.2014.05.006 0022-1910/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Shimada, K., Maekawa, K. Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae). Journal of Insect Physiology (2014), http://dx.doi.org/10.1016/j.jinsphys.2014.05.006
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et al., 2009). In higher termites, EGs and BGs are mainly produced in the midgut (Tokuda et al., 2004, 2009; Wu et al., 2012). However, salivary glands are the major expression sites of EG and BG genes in lower termites and cockroaches (Tokuda et al., 2004; Shimada and Maekawa, 2008; Scharf et al., 2010; Zhang et al., 2012b). A previous study showed that the EG gene is highly expressed in workers, but soldiers had low expression levels in R. flavipes (Scharf et al., 2005b). Similar EG gene expression patterns were also observed in Hodotermopsis sjostedti (Fujita and Miura, 2008). Furthermore, Zhang et al. (2012a) showed that workers had higher expression levels of BG (clone Glu1B beta-glucosidase) than those of soldiers in C. formosanus. It was also shown that the activity of BG enzyme is higher in workers than soldiers (Sugio et al., 2006; Fujita and Miura, 2008). These differences in gene expression and enzyme activity of cellulases are thought to reflect donor-recipient relationships in trophallactic interactions. Because the task of workers is wood digestion and brood care, they are recognized as a ‘donor’. On the other hand, soldiers are a ‘recipient’ because their mandibles are too specialized to ingest wood by themselves (Machida et al., 2001). Similar tendencies are also observed in the non-termite insect group. In the subsocial wood-feeding cockroach Salganea esakii, EG gene expression levels of first-instar nymphs were extremely low compared with those of adults, whereas first-instar nymphs of the gregarious wood-feeding cockroach Panesthia angustipennis had high EG gene expression levels (Shimada and Maekawa, 2008). The adults of S. esakii showed parental care via stomodeal trophallaxis to their offspring, but the basic social unit of P. angustipennis observed in the field did not appear to be the family (Maekawa et al., 2008a; Nalepa et al., 2008; Shimada and Maekawa, 2011). EG gene expression levels were also shown to vary among different developmental stages in the same caste of termites. Shimada and Maekawa (2010) showed that EG gene expression levels were up-regulated in primary queens and kings of R. speratus between 30 and 100 days after colony foundation, when there were low numbers of workers in the colony. On the other hand, EG gene expression levels of primary reproductives decreased at 400 days after colony foundation (when more than one hundred workers were present), and neotenic reproductives (nymphoids) obtained from a mature field colony had extremely low EG gene expression levels. These results suggested that endogenous cellulose metabolic pathways are differentially regulated among each caste, and those in reproductives are regulated during the course of colony development. Expression level changes of other termite-derived cellulases (i.e. BG) would also be related to those of EG. However, BG is known to be a multifunctional enzyme, and the gene expression patterns among caste and developmental stages are not well understood. Recently, some studies reported that BG and related proteins have a role other than wood digestion. Matsuura et al. (2009) indicated that BG was one of the main components of egg-recognition pheromone in R. speratus. In the drywood termite Cryptotermes secundus, genes specifically expressed in female neotenic reproductives were identified, and Neofem2 similar to BG (belonging to GHF1) was shown to have a function in reproductive suppression (Weil et al., 2007, 2009; Korb et al., 2009). Moreover, male-specific beta-glycosidase protein Lma-p72 (affiliated with GHF1) putatively involved in pheromonal communication was also identified from Madeira cockroach Rhyparobia (=Leucophaea) maderae (Cornette et al., 2003). It would be interesting to understand the evolutionary history of multifunctional BG genes in cockroaches and termites, but comprehensive analyses have not yet been performed. In this study, we cloned two BG gene homologs from R. speratus (Kolbe), and performed expression analyses among castes and developmental stages, especially focusing on the reproductives at
different colony stages. Then, we obtained orthologous genes from other termite and cockroach species, including the woodroach (genus Cryptocercus), which is a sister group to termites, and performed molecular phylogenetic analyses. Based on the results obtained, we discuss the evolution of the multifunctional roles of BG genes.
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2. Materials and methods
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2.1. Insects
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Several mature colonies of R. speratus were collected from rotten wood in laurel forests in Toyama and Ishikawa Prefectures, Japan, in 2008–2010. Pieces of nest wood were brought back to the laboratory and kept in plastic cases in constant darkness. Primary kings, nymphoids (neotenic reproductives differentiated from nymphs) with functional reproductive organs, workers (6th or 7th instars; Maekawa et al., 2008b), soldiers, late-stage nymphs, larvae and eggs were collected from mature field colonies. Ergatoids (neotenic reproductives differentiated from workers) with functional reproductive organs were obtained from other colonies maintained under laboratory conditions. Old instar larvae (pseudoergates) of Zootermopsis nevadensis, adults of subsocial cockroach S. esakii and gregarious cockroach P. angustipennis were collected from rotten wood in laurel forests in Japan in 2006 and 2007 (Hyogo, Nagasaki and Ishikawa Pref., respectively). We maintained the live insects in the dark at room temperature prior to use. The adults of subsocial cockroach Cryptocercus punctulatus were provided by Dr. Nalepa (North Carolina State University).
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2.2. Termite colony foundation and sample collection
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Incipient colonies of R. speratus were set up as described by Maekawa et al. (2010), and primary queens and kings were sampled at 30, 50, 100, and 400 days after colony foundation. The details of colony members of each period are shown in Shimada et al. (2013). For RNA extraction (see below), abdomens of reproductives including symbiotic protozoa and bacteria were removed, then heads and thoraxes were immersed in liquid nitrogen immediately. They were stored at 80 °C until use.
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2.3. cDNA preparation
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Total RNA was extracted from heads and thoraxes (including salivary glands) of termites and salivary glands of cockroaches stored at 80 °C using a FastPure RNA kit (Takara Bio, Shiga, Japan). For the analyses of primary queens and kings, different individuals (2–4 individuals) were used for each RNA sample and at least three different RNA samples were prepared for each stage. More than 10 individuals of each caste (workers, soldiers, nymphs and ergaoids), 60 larvae and 100 eggs were used for each RNA sample for the comparison among other colony members. After DNase (Takara) treatment, 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 mRNA (60 ng) was transcribed using SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen, USA) as instructed by the manufacturer.
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2.4. Cloning and sequencing of BG genes
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Specific cDNAs were amplified by PCR using a thermal cycler GeneAmp 2400 (Applied Biosystems, USA) or MJ-Mini (Bio-Rad,
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Please cite this article in press as: Shimada, K., Maekawa, K. Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae). Journal of Insect Physiology (2014), http://dx.doi.org/10.1016/j.jinsphys.2014.05.006
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USA) with degenerate primers. Degenerate PCR primers for BG (insectBGfw1: CAY TGG GAH YTR CCD CAR and insectBGrv1: AAG TTG TCM ATB AGA SWC CA) were newly designed from conserved sequences among insects. PCR products were purified by using a MagExtractor Kit (Toyobo, Japan). Purified fragments were cloned into a pT7Blue-2 T-Vector (Novagen, USA), and the inserts were amplified from a single bacterial colony using U19mer/SP6 primers. At least three randomly chosen clones were picked, and determined using a DYEnamic ET Terminator Kit (Amersham, USA) and an automatic sequencer model 373S (Applied Biosystems).
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2.5. Gene expression analyses by real-time quantitative PCR
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For real-time quantitative PCR analyses, to select the most suitable reference gene, we examined the stability of three housekeeping genes consisting of beta-actin (Accession No. AB520714), NADH-dehydrogenase (NADH-dh) (AB602837) and elongation factor-1-alpha (EF1-alpha) (AB602838) using appropriate software geNorm (Vandesompele et al., 2002) and Normfinder (Andersen et al., 2004). Primer sequences were obtained from Maekawa et al. (2010) and Hojo et al. (2011). These analyses revealed EF1alpha and beta-actin to be the most stable genes in comparisons among different stages of queens and kings, respectively. NADHdh was shown to be the most stable in a comparison among other colony members. Primers for BG genes were designed using Primer Express software (Applied Biosystems) based on the obtained sequence (RsBGIfw: GCC TCA GAC ACT CCA A, RsBGIrv: TCT CGG AGT ATT TTG CCA ATT CT, RsBGIIfw: GCC ACA GCC TCT ACA A and RsBGIIrv: CCT CGA AGT AGT TTG CTA TAA CA). The relative quantification of cDNAs was performed using a SYBR Green I chemistry and a MiniOpticon Real-Time System detection system (Bio-Rad, CA, USA). Each reaction mixture (total volume 20 ll) consisted of 10 ll SYBR Green I master mix (Bio-Rad), 0.4 ll each of the forward and reverse primers (200 nM), 7.2 ll RNase free water, and 2 ll of the cDNA template. The temperature profile for amplifying the target gene and reference gene fragments were 95 °C for 3 min, followed by 40 cycles of 95 °C for 20 s, 60 °C (53 °C and 51 °C in BGI and BGII, respectively) for 20 s, and 72 °C for 30 s. The production of single products was confirmed by dissociation curve analysis conducted using the MiniOpticon system. Quantification data analyses were performed using CFX manager software version 1.5 (Bio-Rad) in accordance with the manufacturer’s instructions. The relative expression levels were calibrated using the mean expression level of workers (6th or 7th instars) collected from a mature field colony as 1.0. For the analyses of primary queens and kings, each biological sample (total 3–5 samples per each stage) was used for the gene expression analyses. For the comparison among other colony members, expression levels were calculated using technical triplicates of the same cDNA sample. These analyses were performed with reference to the MIQE guidelines (Minimum Information for Publication of Quantitative Real-time PCR Experiments) (Bustin et al., 2009). A statistical test was performed by the usual methods for multiple comparisons (Tukey’s test) using Ekuseru-Toukei 2010 (Social Survey Research Information Co., Ltd., Tokyo, Japan). P values less than 0.05 were considered significant.
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2.6. Molecular phylogenetic analyses
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Obtained nucleotide sequences were translated into amino acid sequences by using MacClade 4.08 (Maddison and Maddison, 2005). Nucleotide sequences were subject to a BLASTX search (National Center for Biotechnology Information, USA). Phylogenetic analyses were then performed using PAUP⁄4.0b10
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(Swofford, 2000) with neighbor joining (NJ) and maximum parsimony (MP) criteria, using already published BG sequences from other termites. In NJ analyses, genetic distances were corrected using Kimura’s two-parameter method (Kimura, 1980), and a bootstrap analysis of 1000 replications was performed using PAUP⁄. In MP analysis, all characters were weighted equally, gaps were treated as missing, and a bootstrap analysis of 1000 replications was performed.
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3. Results
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3.1. Molecular cloning and sequencing BG genes of R. speratus
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We analyzed seven clones, and two different partial BG cDNA sequences were obtained [RsBGI: 906 bp (two clones) and RsBGII: 900 bp (five clones)]. These two sequences were defined as BGI and BGII based on the inferred phylogenetic relationships (see below). Nucleotide sequence data of RsBGI and RsBGII are available in the DDBJ/EMBL/GenBank databases (AB915865 and AB915866, respectively). Translated amino acid sequences of these genes did not include a terminator codon and were well conserved compared with other published amino acid sequences of GHF1 members. Conserved motifs putatively involved in substrate binding and catalysis were also observed in both sequences (Fig. 1). BLASTX showed that nucleotide sequences of some termite BG genes published previously were retrieved with the high similarity. RsBGI gene was 91% identical to R. flavipes beta-glucosidase gene (HM152540), 86% identical to C. formosanus clone Glu1B betaglucosidase gene (GQ911585) and Coptotermes gestroibeta-1,4-glucosidase gene (KC891004). RsBGII gene was 81% identical to the C. formosanus clone Glu1C beta-glucosidase gene (JN565079) and 77% identical to the Odontotermes formosanus beta-glucosidase gene (GU591172).
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3.2. Changes in BG gene expression levels of queens and kings during colony development
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For real-time quantitative PCR for each gene, a single peak on the melting curve was observed, showing that the target PCR products were amplified selectively and that primer dimers and other products were not formed. The results showed that the expression patterns were completely different between RsBGI and RsBGII. The expression levels of the RsBGI gene changed with colony development, but expression patterns were similar in both queens and kings (Fig. 2). Female and male alates had low expression levels (1.4% and 0.4% of worker levels, respectively), whereas the expression levels in queens and kings from days 30 to 100 were relatively high (from 12% to 98% of worker levels). However, at day 400, the expression levels dropped again in queens and kings (0.6% and 1.3% of worker levels, respectively). The expression levels in physogastric nymphoids were lower than those of queens at day 400 (Fig. 2). These results were consistent with the expression patterns of the R. speratus EG gene shown in the previous report (Shimada and Maekawa, 2010). On the other hand, RsBGII gene expression levels in both queens and kings did not change during colony development (Fig. 2). Female alates had low expression levels (11.7% of worker levels). However, the expression levels at day 400 were not low in primary queens (24.6% of worker level), and there were no significant differences of expression levels among other primary queens (from 22.9% to 31.5% of worker levels). High expression levels were observed in physogastric nymphoids (45.7% of worker levels). Male alates also had low expression levels (4.4% of worker levels), but those of the kings in days 100 and 400 were significantly higher (24.6% and 24.1% of worker levels, respectively) (Fig. 2).
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Please cite this article in press as: Shimada, K., Maekawa, K. Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae). Journal of Insect Physiology (2014), http://dx.doi.org/10.1016/j.jinsphys.2014.05.006
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Fig. 1. Multiple alignment of amino acid sequences (302 aa) of BG genes of R. speratus (RsBGI and RsBGII) and other termite species (RfBG: R. flavipes BG, HM152540; CfBG Glu1B: C. formosanus BG clone Glu1B, GQ911585; CfBG Glu1C: C. formosanus BG clone Glu1C, JN565079; OfBG: O. formosanus BG, GU591172). All amino acid residues matching with the first taxon are shaded. Conserved amino acid residues putatively involved in substrate binding and catalysis are indicated by asterisks. An N-glycosylation site is indicated by black arrowheads, and putative proton donor and nucleophile sites are shown by white and gray arrowheads, respectively.
Fig. 2. Relative expression levels (mean ± S.D. values) of RsBG genes in queens (A) and kings (B) of R. speratus. NP indicates female nymphoids obtained from a mature field colony. Black columns show RsBGI, and white columns show RsBGII. The expression levels were calibrated using the mean expression level of workers (6th or 7th instars collected from the field colony) as 1.0. The biological replicate numbers of analyses are shown in parentheses. Different letters over the bars denote significant differences (Tukey’s test, p < 0.05).
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3.3. BG gene expression in eggs and other colony members
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Gene expression patterns among other colony members were also completely different between RsBGI and RsBGII (Fig. 3). Eggs had extremely low levels of RsBGI gene expression relative to workers (0.07%). The expression levels of RsBGI in workers were higher than those of soldiers (0.9% of worker levels). These results were consistent with previous studies on EG and BG gene expression levels in other termite species (Scharf et al., 2005b; Fujita and Miura, 2008; Zhang et al., 2012a). Moreover, low expression levels were also observed in nymphs (0.3%), ergatoids (1.4%) and larvae (0.1%). On the other hand, RsBGII gene expression levels ranged from 8% (nymphs) to 101% (ergatoids) of worker levels among other colony members. The expression levels in eggs were
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relatively high, and were not significantly different from those in larvae. Thus, differences in gene expression levels in RsBGI were much larger than those observed in RsBGII.
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3.4. Molecular phylogeny of BG genes in termites and cockroaches
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Using degenerate PCR primers, we obtained the partial cDNA sequences (906 bp) of BGI genes from Z. nevadensis (ZnBGI), C. punctulatus (CpBGI), S. esakii (SeBGI) and P. angustipennis (PaBGI). Using the same PCR primers, BGII genes (900 bp) were obtained from Z. nevadensis (ZnBGII) and S. esakii (SeBGI). All of the translated amino acid sequences from these cDNAs showed high similarity (60–76% identical) to other BGs from termites obtained
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Please cite this article in press as: Shimada, K., Maekawa, K. Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae). Journal of Insect Physiology (2014), http://dx.doi.org/10.1016/j.jinsphys.2014.05.006
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Fig. 3. Relative expression levels (mean ± S.D. values) of RsBG genes in workers (W), soldiers (S), nymphs (N), ergatoids (Er), larvae (L) and eggs of R. speratus. Black columns show RsBGI, and white columns show RsBGII. The expression levels were calibrated using the mean expression level of workers (6th or 7th instars) as 1.0. Three real-time quantitative PCR analyses were performed using the same cDNA sample (10–100 different individuals were used; see the Section 2.3) of each developmental stage. Different letters over the bars denote significant differences (Tukey’s test, p < 0.05).
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previously. These nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases (AB915867–72). Molecular phylogenetic analyses revealed that all obtained sequences were shown to form monophyletic groups with known BG sequences of termite species (NJ bootstrap value/MP bootstrap value: 97%/96%) (Fig. 4). Topologies were essentially the same in both NJ and MP analyses. All obtained sequences of BGI (RsBGI, ZnBGI, CpBGI, SeBGI and PaBGI) were shown to be included in a monophyletic group with most of other termite BGs (NJ/MP: 94%/65%). CpBGI was included in the termite BGI group (NJ/MP: 100%/99%), whereas other cockroach BGIs (SeBGI and PaBGI) clustered with each other (NJ/MP: 100%/100%). These relationships inferred from BGI were consistent with the currently accepted phylogeny of termites and cockroaches (Lo et al., 2000; Klass and
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Meier, 2006; Inward et al., 2007). Similarly, all obtained sequences of BGII (RsBGII, ZnBGII and SeBGII) were shown to be included in a monophyletic group with C. formosanus clone Glu1C betaglucosidase (JN565079) and O. formosanus BG (GU591172) (NJ/MP: 100%/82%). Termite BGIIs formed monophyletic groups with one another with high bootstrap values (NJ/MP: 100%/99%).
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4. Discussion
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4.1. The functions of BGI and BGII in termites
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Our study showed that at least two types of BG gene homologs were shared by extant termites and cockroaches, and BGII is thought to have a different function from BGI. In C. formosanus, clone Glu1B beta-glucosidase (i.e. CfBGI) (GQ911585) was shown to act as a digestive enzyme hydrolyzing cellobiose or cellotriose, and glucose was generated as the end product (Zhang et al., 2010, 2012b). The digestive activity of BG enzyme RfBGluc-1 (i.e. RfBGI) was also confirmed in R. flavipes (Scharf et al., 2010). Zhang et al. (2012a) showed that the gene expression levels of clone Glu1B beta-glucosidase were higher in donors (workers) than recipients (soldiers), which was consistent with previous studies (Sugio et al., 2006; Fujita and Miura, 2008). Our results (Figs. 2 and 3) also suggested that the function of RsBGI would be cellulose digestion because their gene expression patterns were similar to those of RsEG and C. formosanus clone Glu1B beta-glucosidase (Shimada and Maekawa, 2010; Zhang et al., 2012a). Matsuura et al. (2009) showed that the egg-recognition pheromone of R. speratus was composed of BG and lysozyme (EC 3.2.1.17), and that BG activity was confirmed in eggs using a fluorescent probe. Our results showed that extremely low levels of gene expression in eggs were observed for RsBGI but not RsBGII (Fig. 3). Thus, RsBGII is a candidate for the main component functioning as an egg-recognition pheromone in R. speratus. However, BGs produced by P. angustipennis and egg-mimicking fungus (termite balls)
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Fig. 4. Phylogenetic tree inferred from BG cDNA sequences of termites and cockroaches based on the Neighbor-Joining (NJ) method. Bootstrap values (greater than 50%) from NJ and Most Parsimonious analyses are shown above and below branches to indicate the level of support for each node, respectively.
Please cite this article in press as: Shimada, K., Maekawa, K. Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae). Journal of Insect Physiology (2014), http://dx.doi.org/10.1016/j.jinsphys.2014.05.006
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showed strong egg-recognition pheromone activity (Matsuura et al., 2009), suggesting that the relatively slight differences between RsBGI and II amino acid sequences were not completely related to the specificity of egg-recognition pheromone. In termite egg-recognition, other minor chemical compounds might be involved (Matsuura et al., 2009). The RsBGII gene was expressed in other colony members (Figs. 2 and 3), and BGII genes were also present in Z. nevadensis, Co. formosanus (JN565079) and O. formosanus (GU591172) (Fig. 4). Although the precise function of BGII in a termite colony except in egg-recognition is unknown, it might be involved in pheromonal communication (e.g. nestmaterecognition) in all extant termites. We also obtained a BGII gene from the wood-feeding subsocial cockroach S. esakii (Fig. 4). Because the functions of BGII in cockroaches are completely unknown, further analyses are needed to confirm whether there are any functional differences between BG gene homologs of cockroaches and termites. For example, determinations of fulllength cDNA sequences and their gene expression sites should be performed in these taxa. 4.2. BG gene expression levels in reproductives and other colony members
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The gene expression levels of RsBGIs in primary reproductives changed substantially with colony development (Fig. 2), and their gene expression patterns were similar to those of EG in the same species (Shimada and Maekawa, 2010). Primary queens and kings showing high RsBGI gene expression levels at days 30–100 might be devoted to parental provisioning of young offspring as donors because these colonies were short of workers. Indeed, only 2.2 ± 1.6 young workers were observed at day 50, and 13.9 ± 3.9 workers at day 100 in R. speratus (Shimada et al., 2013). However, at day 400, the RsBGI gene expression levels of primary queens and kings dropped significantly (Fig. 2), by which time worker number had increased (105.3 ± 27.4) (Shimada et al., 2013). Moreover, queens and kings in this period might be able to receive trophallactic food from workers and they could be relieved of the cost of brood care, resulting in specialization for reproduction. The numbers of cellulolytic protists in the gut of primary reproductives were shown to be high at days 30–100, but they had been lost at day 400 (Shimada et al., 2013). Together with the low EG expression levels (Shimada and Maekawa, 2010) and absence of symbiotic protists (Shimada et al., 2013), it was demonstrated that cellulose metabolism in mature reproductives was suppressed entirely. Other recipients of trophallaxis (i.e. larvae, soldiers, nymphs, ergatoids and nymphoids) also had low RsBGI gene expression levels (Figs. 2 and 3). These results suggested that RsBGI gene expression levels are related to roles in trophallactic interactions, with RsBGI highly expressed in donors of trophallaxis. Because the gene expression patterns were completely different between RsBGI and RsBGII, the regulatory mechanisms for these genes should not be the same. To confirm this hypothesis, genetic factors related to regulation of these genes should be investigated.
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4.3. Molecular evolution of BG genes in termites and cockroaches
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Phylogenetic analyses showed that each BGI and BGII gene homolog comprised monophyletic groups, respectively. BGI genes (encoding digestive enzymes) formed a monophyletic group with BGII genes (encoding proteins probably involved in pheromonal communication) (Fig. 4). These phylogenetic relationships observed in BG genes were only partly in agreement with those of EG genes in termites and cockroaches (Shimada and Maekawa, 2008). For example, in Salganea and Panesthia, each homologous gene obtained from the same species formed a monophyletic group with the homologous genes (orthologs) of another species (i.e.
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SeEGI formed a monophyletic group with PaEGI but not SeEGII). However, most EG gene homologs obtained from the same species clustered with one another rather than those of other species (Shimada and Maekawa, 2008). Such a clustering pattern was only seen in the BG genes of N. takasagoensis (Fig. 4). Therefore, it was indicated that the molecular evolutionary histories are quite different between BG and EG genes, in spite of their functional similarities. In the case of EG, the common ancestor of cockroaches and termites was thought to have a single EG gene, and it was duplicated later in more recently evolved lineages (Lo et al., 2011). However, the present results strongly support that the evolutionary event (i.e. gene duplication) of BGI and BGII genes occurred before the divergence of cockroaches and termites (Fig. 4). Consequently, the existence of BGII genes might be common in all extant cockroaches including termites. As known in a wider range of other taxa, BGs are a common cellulase component (Lo et al., 2011). Thus, the most reasonable scenario is that, after gene duplication, in addition to the original functions (cellulose digestion by BGI), another function (probably pheromonal communication by BGII) evolved in the common ancestor of cockroaches and termites. We suggest that the acquisition of the multifunctionality of BG genes might be a preadaptation of termite social evolution. To clarify this hypothesis, functional analyses (e.g. RNA interference of BG gene homologs identified in this study) will be needed both in termites and cockroaches.
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Acknowledgments
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We thank Dr. C.A. Nalepa (North Carolina State University) for Q4 providing the samples of Cryptocercus punctulatus. Thanks are also due to D. Watanabe, K. Toga, S. Nakamura, R. Saiki, M. Yoshimura, F. Nakayama, Y. Hashimoto, H. Yaguchi and Y. Masuoka for their help during our field and laboratory work. This study was supported in part by Grants in-Aid for JSPS Fellows (No. 10929 to K.S.) and for Young Scientists (Nos. 21770079 and 24570022 to Q5 K.M.) from the Japan Society for the Promotion of Science.
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Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)
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Keisuke Shimada 1, Kiyoto Maekawa ⇑ Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama, Toyama 930-8555, Japan
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Article history: Received 4 March 2014 Received in revised form 18 April 2014 Accepted 1 May 2014 Available online xxxx
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Keywords: Beta-glucosidase Gene expression Cellulose digestion Social communication Termites Cockroaches
a b s t r a c t Beta-glucosidase (BG) is known as a multifunctional enzyme for social maintenance in terms of both cellulose digestion and social communication in termites. However, the expression profiles of each BG gene and their evolutionary history are not well understood. First, we cloned two types of BG homologs (RsBGI and RsBGII) from the termite Reticulitermes speratus (Kolbe). Gene expression analyses showed that RsBGI expression levels of primary queens and kings from 30 to 100 days after colony foundation were high, but those of reproductives dropped after day 400. Extremely low gene expression levels of RsBGI were observed in eggs, whereas workers had significantly higher expression levels than those of soldiers and other colony members. Consequently, RsBGI gene expression levels changed among each developmental stage, and RsBGI was shown to be involved in cellulose digestion. On the other hand, the RsBGII gene was consistently expressed in all castes and developmental stages examined, and notable expression changes were not observed among them, including in eggs. It was indicated that RsBGII is a main component involved in social communication, for example, the egg-recognition pheromone shown in this species previously. Finally, we obtained partial gene homologs from other termite and cockroach species, including the woodroach (genus Cryptocercus), which is the sister group to termites, and performed molecular phylogenetic analyses. The results showed that the origin of the BG gene homologs preceded the divergence of termites and cockroaches, suggesting that the acquisition of multifunctionality of the BG gene also occurred in cockroach lineages. Ó 2014 Elsevier Ltd. All rights reserved.
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1. Introduction
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In eusocial termites, primary reproductives (alates, queens and kings) and neotenic reproductives (i.e. supplementary queens and kings such as nymphoids and ergatoids) are devoted to reproduction (Korb and Hartfelder, 2008). Workers forage for food and care for their siblings, whereas soldiers defend the colony (Wilson, Q3 1971). Molecular mechanisms to regulate the division of labor among these castes and their sociality have developed during the course of termite evolution, and common mechanisms should be shared by all extant termites (reviewed in Miura and Scharf, 2011). Several common genes responsible for social maintenance and genes showing caste-specific expression have been identified in several termite species (e.g. encoding hexamerines and vitello-
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⇑ Corresponding author. Tel.: +81 76 445 6629; fax: +81 76 445 6641. E-mail address: [email protected] (K. Maekawa). 1 Present address: Ishikawa Museum of Natural History, Ri-441 Choshi-machi, Kanazawa, Ishikawa 920-1147, Japan.
genins) (Scharf et al., 2005a, 2007). Endogenous termite cellulase is also a good example of such a gene. Cellulolytic protists in the gut of lower termites are well-known mutualistic symbionts (Cleveland, 1923), but termites also secrete cellulases by themselves (Watanabe et al., 1998). Endo-beta-1,4glucanase (EG; EC 3.2.1.4) belonging to the glycosyl hydrolase family (GHF) 9 and beta-glucosidase (BG; EC 3.2.1.21) affiliated with GHF1 are known as termite-derived cellulases (reviewed in Lo et al., 2011). EGs and BGs are known as common cellulase components in bacteria, fungi, protists, plants and animals. Cellulose chains are hydrolyzed to cellobiose and cellotriose by EGs, and short-chain sugars are converted into glucose by BGs (Ni et al., 2005; Zhang et al., 2009). In termites and their relatives (i.e. cockroaches), EG genes are found in many species (Lo et al., 2000; Shimada and Maekawa, 2008), but BG genes have only be identified in some termite species, including Neotermes koshunensis (Tokuda et al., 2002), Coptotermes formosanus (Zhang et al., 2010, 2012a), Reticulitermes flavipes (Scharf et al., 2010), Macrotermes barneyi (Wu et al., 2012) and Nasutitermes takasagoensis (Tokuda
http://dx.doi.org/10.1016/j.jinsphys.2014.05.006 0022-1910/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Shimada, K., Maekawa, K. Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae). Journal of Insect Physiology (2014), http://dx.doi.org/10.1016/j.jinsphys.2014.05.006
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et al., 2009). In higher termites, EGs and BGs are mainly produced in the midgut (Tokuda et al., 2004, 2009; Wu et al., 2012). However, salivary glands are the major expression sites of EG and BG genes in lower termites and cockroaches (Tokuda et al., 2004; Shimada and Maekawa, 2008; Scharf et al., 2010; Zhang et al., 2012b). A previous study showed that the EG gene is highly expressed in workers, but soldiers had low expression levels in R. flavipes (Scharf et al., 2005b). Similar EG gene expression patterns were also observed in Hodotermopsis sjostedti (Fujita and Miura, 2008). Furthermore, Zhang et al. (2012a) showed that workers had higher expression levels of BG (clone Glu1B beta-glucosidase) than those of soldiers in C. formosanus. It was also shown that the activity of BG enzyme is higher in workers than soldiers (Sugio et al., 2006; Fujita and Miura, 2008). These differences in gene expression and enzyme activity of cellulases are thought to reflect donor-recipient relationships in trophallactic interactions. Because the task of workers is wood digestion and brood care, they are recognized as a ‘donor’. On the other hand, soldiers are a ‘recipient’ because their mandibles are too specialized to ingest wood by themselves (Machida et al., 2001). Similar tendencies are also observed in the non-termite insect group. In the subsocial wood-feeding cockroach Salganea esakii, EG gene expression levels of first-instar nymphs were extremely low compared with those of adults, whereas first-instar nymphs of the gregarious wood-feeding cockroach Panesthia angustipennis had high EG gene expression levels (Shimada and Maekawa, 2008). The adults of S. esakii showed parental care via stomodeal trophallaxis to their offspring, but the basic social unit of P. angustipennis observed in the field did not appear to be the family (Maekawa et al., 2008a; Nalepa et al., 2008; Shimada and Maekawa, 2011). EG gene expression levels were also shown to vary among different developmental stages in the same caste of termites. Shimada and Maekawa (2010) showed that EG gene expression levels were up-regulated in primary queens and kings of R. speratus between 30 and 100 days after colony foundation, when there were low numbers of workers in the colony. On the other hand, EG gene expression levels of primary reproductives decreased at 400 days after colony foundation (when more than one hundred workers were present), and neotenic reproductives (nymphoids) obtained from a mature field colony had extremely low EG gene expression levels. These results suggested that endogenous cellulose metabolic pathways are differentially regulated among each caste, and those in reproductives are regulated during the course of colony development. Expression level changes of other termite-derived cellulases (i.e. BG) would also be related to those of EG. However, BG is known to be a multifunctional enzyme, and the gene expression patterns among caste and developmental stages are not well understood. Recently, some studies reported that BG and related proteins have a role other than wood digestion. Matsuura et al. (2009) indicated that BG was one of the main components of egg-recognition pheromone in R. speratus. In the drywood termite Cryptotermes secundus, genes specifically expressed in female neotenic reproductives were identified, and Neofem2 similar to BG (belonging to GHF1) was shown to have a function in reproductive suppression (Weil et al., 2007, 2009; Korb et al., 2009). Moreover, male-specific beta-glycosidase protein Lma-p72 (affiliated with GHF1) putatively involved in pheromonal communication was also identified from Madeira cockroach Rhyparobia (=Leucophaea) maderae (Cornette et al., 2003). It would be interesting to understand the evolutionary history of multifunctional BG genes in cockroaches and termites, but comprehensive analyses have not yet been performed. In this study, we cloned two BG gene homologs from R. speratus (Kolbe), and performed expression analyses among castes and developmental stages, especially focusing on the reproductives at
different colony stages. Then, we obtained orthologous genes from other termite and cockroach species, including the woodroach (genus Cryptocercus), which is a sister group to termites, and performed molecular phylogenetic analyses. Based on the results obtained, we discuss the evolution of the multifunctional roles of BG genes.
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2. Materials and methods
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2.1. Insects
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Several mature colonies of R. speratus were collected from rotten wood in laurel forests in Toyama and Ishikawa Prefectures, Japan, in 2008–2010. Pieces of nest wood were brought back to the laboratory and kept in plastic cases in constant darkness. Primary kings, nymphoids (neotenic reproductives differentiated from nymphs) with functional reproductive organs, workers (6th or 7th instars; Maekawa et al., 2008b), soldiers, late-stage nymphs, larvae and eggs were collected from mature field colonies. Ergatoids (neotenic reproductives differentiated from workers) with functional reproductive organs were obtained from other colonies maintained under laboratory conditions. Old instar larvae (pseudoergates) of Zootermopsis nevadensis, adults of subsocial cockroach S. esakii and gregarious cockroach P. angustipennis were collected from rotten wood in laurel forests in Japan in 2006 and 2007 (Hyogo, Nagasaki and Ishikawa Pref., respectively). We maintained the live insects in the dark at room temperature prior to use. The adults of subsocial cockroach Cryptocercus punctulatus were provided by Dr. Nalepa (North Carolina State University).
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2.2. Termite colony foundation and sample collection
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Incipient colonies of R. speratus were set up as described by Maekawa et al. (2010), and primary queens and kings were sampled at 30, 50, 100, and 400 days after colony foundation. The details of colony members of each period are shown in Shimada et al. (2013). For RNA extraction (see below), abdomens of reproductives including symbiotic protozoa and bacteria were removed, then heads and thoraxes were immersed in liquid nitrogen immediately. They were stored at 80 °C until use.
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2.3. cDNA preparation
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Total RNA was extracted from heads and thoraxes (including salivary glands) of termites and salivary glands of cockroaches stored at 80 °C using a FastPure RNA kit (Takara Bio, Shiga, Japan). For the analyses of primary queens and kings, different individuals (2–4 individuals) were used for each RNA sample and at least three different RNA samples were prepared for each stage. More than 10 individuals of each caste (workers, soldiers, nymphs and ergaoids), 60 larvae and 100 eggs were used for each RNA sample for the comparison among other colony members. After DNase (Takara) treatment, 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 mRNA (60 ng) was transcribed using SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen, USA) as instructed by the manufacturer.
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2.4. Cloning and sequencing of BG genes
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Specific cDNAs were amplified by PCR using a thermal cycler GeneAmp 2400 (Applied Biosystems, USA) or MJ-Mini (Bio-Rad,
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USA) with degenerate primers. Degenerate PCR primers for BG (insectBGfw1: CAY TGG GAH YTR CCD CAR and insectBGrv1: AAG TTG TCM ATB AGA SWC CA) were newly designed from conserved sequences among insects. PCR products were purified by using a MagExtractor Kit (Toyobo, Japan). Purified fragments were cloned into a pT7Blue-2 T-Vector (Novagen, USA), and the inserts were amplified from a single bacterial colony using U19mer/SP6 primers. At least three randomly chosen clones were picked, and determined using a DYEnamic ET Terminator Kit (Amersham, USA) and an automatic sequencer model 373S (Applied Biosystems).
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2.5. Gene expression analyses by real-time quantitative PCR
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For real-time quantitative PCR analyses, to select the most suitable reference gene, we examined the stability of three housekeeping genes consisting of beta-actin (Accession No. AB520714), NADH-dehydrogenase (NADH-dh) (AB602837) and elongation factor-1-alpha (EF1-alpha) (AB602838) using appropriate software geNorm (Vandesompele et al., 2002) and Normfinder (Andersen et al., 2004). Primer sequences were obtained from Maekawa et al. (2010) and Hojo et al. (2011). These analyses revealed EF1alpha and beta-actin to be the most stable genes in comparisons among different stages of queens and kings, respectively. NADHdh was shown to be the most stable in a comparison among other colony members. Primers for BG genes were designed using Primer Express software (Applied Biosystems) based on the obtained sequence (RsBGIfw: GCC TCA GAC ACT CCA A, RsBGIrv: TCT CGG AGT ATT TTG CCA ATT CT, RsBGIIfw: GCC ACA GCC TCT ACA A and RsBGIIrv: CCT CGA AGT AGT TTG CTA TAA CA). The relative quantification of cDNAs was performed using a SYBR Green I chemistry and a MiniOpticon Real-Time System detection system (Bio-Rad, CA, USA). Each reaction mixture (total volume 20 ll) consisted of 10 ll SYBR Green I master mix (Bio-Rad), 0.4 ll each of the forward and reverse primers (200 nM), 7.2 ll RNase free water, and 2 ll of the cDNA template. The temperature profile for amplifying the target gene and reference gene fragments were 95 °C for 3 min, followed by 40 cycles of 95 °C for 20 s, 60 °C (53 °C and 51 °C in BGI and BGII, respectively) for 20 s, and 72 °C for 30 s. The production of single products was confirmed by dissociation curve analysis conducted using the MiniOpticon system. Quantification data analyses were performed using CFX manager software version 1.5 (Bio-Rad) in accordance with the manufacturer’s instructions. The relative expression levels were calibrated using the mean expression level of workers (6th or 7th instars) collected from a mature field colony as 1.0. For the analyses of primary queens and kings, each biological sample (total 3–5 samples per each stage) was used for the gene expression analyses. For the comparison among other colony members, expression levels were calculated using technical triplicates of the same cDNA sample. These analyses were performed with reference to the MIQE guidelines (Minimum Information for Publication of Quantitative Real-time PCR Experiments) (Bustin et al., 2009). A statistical test was performed by the usual methods for multiple comparisons (Tukey’s test) using Ekuseru-Toukei 2010 (Social Survey Research Information Co., Ltd., Tokyo, Japan). P values less than 0.05 were considered significant.
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Obtained nucleotide sequences were translated into amino acid sequences by using MacClade 4.08 (Maddison and Maddison, 2005). Nucleotide sequences were subject to a BLASTX search (National Center for Biotechnology Information, USA). Phylogenetic analyses were then performed using PAUP⁄4.0b10
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(Swofford, 2000) with neighbor joining (NJ) and maximum parsimony (MP) criteria, using already published BG sequences from other termites. In NJ analyses, genetic distances were corrected using Kimura’s two-parameter method (Kimura, 1980), and a bootstrap analysis of 1000 replications was performed using PAUP⁄. In MP analysis, all characters were weighted equally, gaps were treated as missing, and a bootstrap analysis of 1000 replications was performed.
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3. Results
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3.1. Molecular cloning and sequencing BG genes of R. speratus
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We analyzed seven clones, and two different partial BG cDNA sequences were obtained [RsBGI: 906 bp (two clones) and RsBGII: 900 bp (five clones)]. These two sequences were defined as BGI and BGII based on the inferred phylogenetic relationships (see below). Nucleotide sequence data of RsBGI and RsBGII are available in the DDBJ/EMBL/GenBank databases (AB915865 and AB915866, respectively). Translated amino acid sequences of these genes did not include a terminator codon and were well conserved compared with other published amino acid sequences of GHF1 members. Conserved motifs putatively involved in substrate binding and catalysis were also observed in both sequences (Fig. 1). BLASTX showed that nucleotide sequences of some termite BG genes published previously were retrieved with the high similarity. RsBGI gene was 91% identical to R. flavipes beta-glucosidase gene (HM152540), 86% identical to C. formosanus clone Glu1B betaglucosidase gene (GQ911585) and Coptotermes gestroibeta-1,4-glucosidase gene (KC891004). RsBGII gene was 81% identical to the C. formosanus clone Glu1C beta-glucosidase gene (JN565079) and 77% identical to the Odontotermes formosanus beta-glucosidase gene (GU591172).
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3.2. Changes in BG gene expression levels of queens and kings during colony development
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For real-time quantitative PCR for each gene, a single peak on the melting curve was observed, showing that the target PCR products were amplified selectively and that primer dimers and other products were not formed. The results showed that the expression patterns were completely different between RsBGI and RsBGII. The expression levels of the RsBGI gene changed with colony development, but expression patterns were similar in both queens and kings (Fig. 2). Female and male alates had low expression levels (1.4% and 0.4% of worker levels, respectively), whereas the expression levels in queens and kings from days 30 to 100 were relatively high (from 12% to 98% of worker levels). However, at day 400, the expression levels dropped again in queens and kings (0.6% and 1.3% of worker levels, respectively). The expression levels in physogastric nymphoids were lower than those of queens at day 400 (Fig. 2). These results were consistent with the expression patterns of the R. speratus EG gene shown in the previous report (Shimada and Maekawa, 2010). On the other hand, RsBGII gene expression levels in both queens and kings did not change during colony development (Fig. 2). Female alates had low expression levels (11.7% of worker levels). However, the expression levels at day 400 were not low in primary queens (24.6% of worker level), and there were no significant differences of expression levels among other primary queens (from 22.9% to 31.5% of worker levels). High expression levels were observed in physogastric nymphoids (45.7% of worker levels). Male alates also had low expression levels (4.4% of worker levels), but those of the kings in days 100 and 400 were significantly higher (24.6% and 24.1% of worker levels, respectively) (Fig. 2).
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Fig. 1. Multiple alignment of amino acid sequences (302 aa) of BG genes of R. speratus (RsBGI and RsBGII) and other termite species (RfBG: R. flavipes BG, HM152540; CfBG Glu1B: C. formosanus BG clone Glu1B, GQ911585; CfBG Glu1C: C. formosanus BG clone Glu1C, JN565079; OfBG: O. formosanus BG, GU591172). All amino acid residues matching with the first taxon are shaded. Conserved amino acid residues putatively involved in substrate binding and catalysis are indicated by asterisks. An N-glycosylation site is indicated by black arrowheads, and putative proton donor and nucleophile sites are shown by white and gray arrowheads, respectively.
Fig. 2. Relative expression levels (mean ± S.D. values) of RsBG genes in queens (A) and kings (B) of R. speratus. NP indicates female nymphoids obtained from a mature field colony. Black columns show RsBGI, and white columns show RsBGII. The expression levels were calibrated using the mean expression level of workers (6th or 7th instars collected from the field colony) as 1.0. The biological replicate numbers of analyses are shown in parentheses. Different letters over the bars denote significant differences (Tukey’s test, p < 0.05).
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3.3. BG gene expression in eggs and other colony members
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Gene expression patterns among other colony members were also completely different between RsBGI and RsBGII (Fig. 3). Eggs had extremely low levels of RsBGI gene expression relative to workers (0.07%). The expression levels of RsBGI in workers were higher than those of soldiers (0.9% of worker levels). These results were consistent with previous studies on EG and BG gene expression levels in other termite species (Scharf et al., 2005b; Fujita and Miura, 2008; Zhang et al., 2012a). Moreover, low expression levels were also observed in nymphs (0.3%), ergatoids (1.4%) and larvae (0.1%). On the other hand, RsBGII gene expression levels ranged from 8% (nymphs) to 101% (ergatoids) of worker levels among other colony members. The expression levels in eggs were
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relatively high, and were not significantly different from those in larvae. Thus, differences in gene expression levels in RsBGI were much larger than those observed in RsBGII.
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3.4. Molecular phylogeny of BG genes in termites and cockroaches
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Using degenerate PCR primers, we obtained the partial cDNA sequences (906 bp) of BGI genes from Z. nevadensis (ZnBGI), C. punctulatus (CpBGI), S. esakii (SeBGI) and P. angustipennis (PaBGI). Using the same PCR primers, BGII genes (900 bp) were obtained from Z. nevadensis (ZnBGII) and S. esakii (SeBGI). All of the translated amino acid sequences from these cDNAs showed high similarity (60–76% identical) to other BGs from termites obtained
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Fig. 3. Relative expression levels (mean ± S.D. values) of RsBG genes in workers (W), soldiers (S), nymphs (N), ergatoids (Er), larvae (L) and eggs of R. speratus. Black columns show RsBGI, and white columns show RsBGII. The expression levels were calibrated using the mean expression level of workers (6th or 7th instars) as 1.0. Three real-time quantitative PCR analyses were performed using the same cDNA sample (10–100 different individuals were used; see the Section 2.3) of each developmental stage. Different letters over the bars denote significant differences (Tukey’s test, p < 0.05).
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previously. These nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases (AB915867–72). Molecular phylogenetic analyses revealed that all obtained sequences were shown to form monophyletic groups with known BG sequences of termite species (NJ bootstrap value/MP bootstrap value: 97%/96%) (Fig. 4). Topologies were essentially the same in both NJ and MP analyses. All obtained sequences of BGI (RsBGI, ZnBGI, CpBGI, SeBGI and PaBGI) were shown to be included in a monophyletic group with most of other termite BGs (NJ/MP: 94%/65%). CpBGI was included in the termite BGI group (NJ/MP: 100%/99%), whereas other cockroach BGIs (SeBGI and PaBGI) clustered with each other (NJ/MP: 100%/100%). These relationships inferred from BGI were consistent with the currently accepted phylogeny of termites and cockroaches (Lo et al., 2000; Klass and
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Meier, 2006; Inward et al., 2007). Similarly, all obtained sequences of BGII (RsBGII, ZnBGII and SeBGII) were shown to be included in a monophyletic group with C. formosanus clone Glu1C betaglucosidase (JN565079) and O. formosanus BG (GU591172) (NJ/MP: 100%/82%). Termite BGIIs formed monophyletic groups with one another with high bootstrap values (NJ/MP: 100%/99%).
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4. Discussion
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4.1. The functions of BGI and BGII in termites
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Our study showed that at least two types of BG gene homologs were shared by extant termites and cockroaches, and BGII is thought to have a different function from BGI. In C. formosanus, clone Glu1B beta-glucosidase (i.e. CfBGI) (GQ911585) was shown to act as a digestive enzyme hydrolyzing cellobiose or cellotriose, and glucose was generated as the end product (Zhang et al., 2010, 2012b). The digestive activity of BG enzyme RfBGluc-1 (i.e. RfBGI) was also confirmed in R. flavipes (Scharf et al., 2010). Zhang et al. (2012a) showed that the gene expression levels of clone Glu1B beta-glucosidase were higher in donors (workers) than recipients (soldiers), which was consistent with previous studies (Sugio et al., 2006; Fujita and Miura, 2008). Our results (Figs. 2 and 3) also suggested that the function of RsBGI would be cellulose digestion because their gene expression patterns were similar to those of RsEG and C. formosanus clone Glu1B beta-glucosidase (Shimada and Maekawa, 2010; Zhang et al., 2012a). Matsuura et al. (2009) showed that the egg-recognition pheromone of R. speratus was composed of BG and lysozyme (EC 3.2.1.17), and that BG activity was confirmed in eggs using a fluorescent probe. Our results showed that extremely low levels of gene expression in eggs were observed for RsBGI but not RsBGII (Fig. 3). Thus, RsBGII is a candidate for the main component functioning as an egg-recognition pheromone in R. speratus. However, BGs produced by P. angustipennis and egg-mimicking fungus (termite balls)
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Fig. 4. Phylogenetic tree inferred from BG cDNA sequences of termites and cockroaches based on the Neighbor-Joining (NJ) method. Bootstrap values (greater than 50%) from NJ and Most Parsimonious analyses are shown above and below branches to indicate the level of support for each node, respectively.
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showed strong egg-recognition pheromone activity (Matsuura et al., 2009), suggesting that the relatively slight differences between RsBGI and II amino acid sequences were not completely related to the specificity of egg-recognition pheromone. In termite egg-recognition, other minor chemical compounds might be involved (Matsuura et al., 2009). The RsBGII gene was expressed in other colony members (Figs. 2 and 3), and BGII genes were also present in Z. nevadensis, Co. formosanus (JN565079) and O. formosanus (GU591172) (Fig. 4). Although the precise function of BGII in a termite colony except in egg-recognition is unknown, it might be involved in pheromonal communication (e.g. nestmaterecognition) in all extant termites. We also obtained a BGII gene from the wood-feeding subsocial cockroach S. esakii (Fig. 4). Because the functions of BGII in cockroaches are completely unknown, further analyses are needed to confirm whether there are any functional differences between BG gene homologs of cockroaches and termites. For example, determinations of fulllength cDNA sequences and their gene expression sites should be performed in these taxa. 4.2. BG gene expression levels in reproductives and other colony members
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The gene expression levels of RsBGIs in primary reproductives changed substantially with colony development (Fig. 2), and their gene expression patterns were similar to those of EG in the same species (Shimada and Maekawa, 2010). Primary queens and kings showing high RsBGI gene expression levels at days 30–100 might be devoted to parental provisioning of young offspring as donors because these colonies were short of workers. Indeed, only 2.2 ± 1.6 young workers were observed at day 50, and 13.9 ± 3.9 workers at day 100 in R. speratus (Shimada et al., 2013). However, at day 400, the RsBGI gene expression levels of primary queens and kings dropped significantly (Fig. 2), by which time worker number had increased (105.3 ± 27.4) (Shimada et al., 2013). Moreover, queens and kings in this period might be able to receive trophallactic food from workers and they could be relieved of the cost of brood care, resulting in specialization for reproduction. The numbers of cellulolytic protists in the gut of primary reproductives were shown to be high at days 30–100, but they had been lost at day 400 (Shimada et al., 2013). Together with the low EG expression levels (Shimada and Maekawa, 2010) and absence of symbiotic protists (Shimada et al., 2013), it was demonstrated that cellulose metabolism in mature reproductives was suppressed entirely. Other recipients of trophallaxis (i.e. larvae, soldiers, nymphs, ergatoids and nymphoids) also had low RsBGI gene expression levels (Figs. 2 and 3). These results suggested that RsBGI gene expression levels are related to roles in trophallactic interactions, with RsBGI highly expressed in donors of trophallaxis. Because the gene expression patterns were completely different between RsBGI and RsBGII, the regulatory mechanisms for these genes should not be the same. To confirm this hypothesis, genetic factors related to regulation of these genes should be investigated.
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4.3. Molecular evolution of BG genes in termites and cockroaches
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Phylogenetic analyses showed that each BGI and BGII gene homolog comprised monophyletic groups, respectively. BGI genes (encoding digestive enzymes) formed a monophyletic group with BGII genes (encoding proteins probably involved in pheromonal communication) (Fig. 4). These phylogenetic relationships observed in BG genes were only partly in agreement with those of EG genes in termites and cockroaches (Shimada and Maekawa, 2008). For example, in Salganea and Panesthia, each homologous gene obtained from the same species formed a monophyletic group with the homologous genes (orthologs) of another species (i.e.
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SeEGI formed a monophyletic group with PaEGI but not SeEGII). However, most EG gene homologs obtained from the same species clustered with one another rather than those of other species (Shimada and Maekawa, 2008). Such a clustering pattern was only seen in the BG genes of N. takasagoensis (Fig. 4). Therefore, it was indicated that the molecular evolutionary histories are quite different between BG and EG genes, in spite of their functional similarities. In the case of EG, the common ancestor of cockroaches and termites was thought to have a single EG gene, and it was duplicated later in more recently evolved lineages (Lo et al., 2011). However, the present results strongly support that the evolutionary event (i.e. gene duplication) of BGI and BGII genes occurred before the divergence of cockroaches and termites (Fig. 4). Consequently, the existence of BGII genes might be common in all extant cockroaches including termites. As known in a wider range of other taxa, BGs are a common cellulase component (Lo et al., 2011). Thus, the most reasonable scenario is that, after gene duplication, in addition to the original functions (cellulose digestion by BGI), another function (probably pheromonal communication by BGII) evolved in the common ancestor of cockroaches and termites. We suggest that the acquisition of the multifunctionality of BG genes might be a preadaptation of termite social evolution. To clarify this hypothesis, functional analyses (e.g. RNA interference of BG gene homologs identified in this study) will be needed both in termites and cockroaches.
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Acknowledgments
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We thank Dr. C.A. Nalepa (North Carolina State University) for Q4 providing the samples of Cryptocercus punctulatus. Thanks are also due to D. Watanabe, K. Toga, S. Nakamura, R. Saiki, M. Yoshimura, F. Nakayama, Y. Hashimoto, H. Yaguchi and Y. Masuoka for their help during our field and laboratory work. This study was supported in part by Grants in-Aid for JSPS Fellows (No. 10929 to K.S.) and for Young Scientists (Nos. 21770079 and 24570022 to Q5 K.M.) from the Japan Society for the Promotion of Science.
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Please cite this article in press as: Shimada, K., Maekawa, K. Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae). Journal of Insect Physiology (2014), http://dx.doi.org/10.1016/j.jinsphys.2014.05.006