Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 DOI: 10.13102/sociobiology.v60i4.429-435Sociobiology 60(4): 429-435 (2013) The Odorant-Binding Protein obp11 Gene Shows Different Spatiotemporal Roles in the Olfactory System of Apis mellifera ligustica and Apis cerana cerana HX Zhao1,2, YX Luo2, JH Lee3, XF Zhang2, Q Liang3, XN Zeng1 Introduction For social insects such as the honeybee, olfactory language plays a critical role in colony life, with important functional roles for work within and outside of the hive. Ac- cording to honeybee biology, newly emerged bees are de- velopmentally immature (Calderone, 1998), and they join a caste that mainly cleans cells while awaiting functional ma- turity (Winston, 1987). From the ages of 4-12 days (Seeley, 1982), the nursing caste feeds larvae or the queen for about 1 week (Seeley, 1979). Middle-aged bees (12-21 days old) build and maintain the nest, and receive and process nectar (Johnson, 2003, 2008a). After 21 days, workers initiate tasks outside of the nest (foraging nectar and pollen, scouting, de- fending) (Seeley, 1995; Seeley & Visscher, 2004; Beekman et al., 2006; Visscher, 2007). These behaviors are correlated Abstract Odorant-binding proteins participate in the olfactory system of the honeybee. Apis mellifera ligustica and Apis cerana cerana are species of honeybee that have different biological functions. The two species have diversified olfactory systems, with A. cerana displaying sensitive olfactory involvement in collecting nectar and pollen from small plants; and A. mellifera collecting from large nectariferous plants. We hypothesized that, given this difference in biological activity, the obp11 genes of A. mellifera and A. cerana may show different olfactory expression patterns. We cloned and sequenced the obp11 genes from A. mellifera (Amobp11) and A. cerana (Acobp11). Using quantitative real-time PCR, we demonstrated that nurse workers, which have the highest olfactory sensitivity in the A. mellifera hive, have the highest expression of the Amobp11 gene; whereas 1-day- emerged workers, which have the lowest olfactory sensitivity, have cor- respondingly low expression. However, the highest expression of the Acobp11 gene is observed for foragers, which display the highest olfactory sensitivity in the A. cerana po- pulation. The OBP11 protein from the two species is highly conserved, with an apparent molecular weight and predicted extracellular localization that is similar to other OBP proteins. The expression of the obp11 gene in A. mellifera and A. cerana correlates with the different roles of the olfactory system for the two different species. These findings support the critical role of odorant-binding proteins in the honeybee olfactory system. Sociobiology An international journal on social insects 1 - South China Agricultural University, Guangzhou, China. 2 - Guangdong Entomological Institute, Guangzhou , China. 3 - Fujian Agriculture and Forestry University, Fuzhou, China. RESEARCH ARTICLE - BEES Article History Edited by: Yi-Juan Xu, SCAU, China Received 27 March 2013 Initial acceptance 04 June 2013 Final acceptance 01 July 2013 Keywords Apis mellifera ligustica, Apis cerana cerana, Odorant-Binding Proteins, Real- time PCR Corresponding author Xinnian Zeng South China Agricultural University Guangzhou, 510642, China E-Mail: zengxn@scau.edu.cn with the function of the olfactory system, which mediates volatile signals to workers, as opposed to contact perception (Maisonnasse, 2010). Moritz and Crewe suggested that the queen emits volatile pheromones to inhibit new queens or the ovary development of workers (Moritz & Crewe, 1991). Honeybees have been observed as a model for the in- sect olfactory system (Vanesa, 2009). The odorant-binding proteins (OBPs) of honeybees are the main functional pro- teins in the olfactory system. OBPs recognize and distinguish volatile compounds and then transport these compounds to olfactory receptors. Honeybee OBPs are small, water solu- ble molecules, which are expressed as abundant extracellular proteins. The genome of the honeybee (Honeybee Genome Sequencing Consortium, 2006) contains 21 genes encoding OBPs (Forêt & Maleszka, 2006), each gene with a markedly different expression pattern. Most of the OBPs are restricted HX Zhao et al. - Different Spatiotemporal Roles in the Olfactory System of Honey Bees430 to olfactory tissues, with particularly high expression in the antenna, while the obp11 gene is expressed exclusively in the antenna of adult bees (Francesca et al., 2010). Apis cerana cerana Fabricius (A. cerana), is an im- portant local species in China. A. cerana has a keen olfactory ability, foraging small and dispersed nectariferous plants, de- fending strongly against ectoparasitic V. destructor and chalk- brood disease, and providing tolerance for low environmen- tal temperatures (Sarah et al., 2010). On the other hand, Apis mellifera ligustica Spinola (A. mellifera) belongs to a species of honeybee that mainly forages large nectariferous plant and produces a number of bee products (royal jelly, pollen, propo- lis, etc). A. mellifera is easily infected with Varroa destructor and chalkbrood disease. While A. cerana and A. mellifera are two important honeybee species that exhibit long-term evolu- tionary divergence (Qiu et al., 2012), the colonies of A. cerana suffer less damage than those of A. mellifera from parasites and chalkbrood disease. Because of its sensitive olfactory system, A. cerana recognizes and distinguishes dummy larvae more efficiently than A. mellifera. However, to our knowledge, there have been few direct comparisons between the molecular me- chanisms of olfactory sensing of the two species. Therefore, in this study, experimentation was performed to compare the obp11 genes of A. cerana and A. mellifera at the molecular le- vel. We examined whether the obp11 gene expression pattern correlates with the different roles of the olfactory system in A. mellifera and A. cerana, thus providing a molecular basis for the differences in behavior of the two species. Based on our findings, we propose that the obp11 gene expression patterns vary according to the divergent evolutionary behavior of two species. Material and Methods Samples collection A. mellifera and A. cerana were fed in an experimental apiary of bee science at the College of Bee Science of Fujian Agriculture and Forestry University during the spring of 2011- 2012. Individual insects were collected within 24h of emergen- ce, marked with enamel paint on the thorax to identify them by age, and then introduced into the same healthy colony. When appropriate, antennae were harvested from A. mellifera and A. cerana. The samples were first collected into Trizol re- agent and then stored at -70oC until use. Total RNA extraction and cDNA synthesis Total RNA was extracted from the antennae of sampled bees using Trizol reagent according to the manual’s protocol (Invitrogen, USA). Subsequently, cDNA was synthesized using the Promega RT-PCR System according to the manual’s protocol(Promega, USA). Obp11 gene cloning and expression The primers used for amplifying the obp11 gene from A. mellifera and A. cerana were designed using Primer Pri- mer 5.0 (Table 1). PCR was performed using the following program: denaturation at 94ºC for 1 min; 35 cycles of 94ºC for 40 s, annealing at 57ºC for 50 s, and extension at 72ºC for 50 min; and a final extension at 72ºC for 7 min. PCR products were cloned into pGEM-T vector (Promega, USA) and trans- formed into E. coli DH5α. Positive colonies were selected by identification with the restriction endonucleases EcoR I and Xhol I, and then the obp11 gene fragments were purified using a Gel Extraction Kit (Sangon, Shanghai) and cloned into pET- 28a vector, which was digested with the same restriction en- donucleases, to construct recombinant expression plasmids pET-28a-Amobp11 and pET-28a-Acobp11. We used IPTG to induce expression of recombinant proteins in E. coli. Primer Sense and antisense sequences (5′–3′) Purpose Aobp11 GAATTCATGAAAGCAGCAGAAATTTG / CTCGAGTCACGGAGCAATAAACGCTA cDNA isolation (reverse transcriptase PCR) Bobp11 TCTCGTTTATGGGGAAATCAGCGAT / TCCGTATTCCGTAGCTTCGACATCC Expression analysis (Real-time PCR) β-Actin TGCCAACACTGTCCTTTCTG / AGAATTGACCCACCAATCCA Internal control Table 1. Oligonucleotide primers used for isolation and expression analysis of odorant-binding proteins of Apis mellifera ligustica and Apis cerana cerana Real-time PCR (RT-PCR) Antennae were separately collected at 1, 4, 10, 15, 20, 25 and 30 days of age, and total RNA was extracted and reverse transcribed to synthesize cDNA, in accordance with the Promega manual. Samples were stored at -70ºC until use. Real-time PCR was performed using the Applied Biosystems StepOnePlus Real-Time PCR System with SYBR Green dye (Promega) in 96-well plates (ABI, USA). Relative quantifi- cation analysis was performed according to cycle threshold values (CT) generated from the Promega GoTaq 2-Step RT- qPCR system (Promega, USA). Standard curves were pre- pared using a purified PCR product for the obp11 and β-actin genes. For each experiment, the endogenous β-actin g gene was analyzed in triplicate (internal control, Shu et al., 2011) with a non-template reaction (negative control) and a water only reaction (blank control). Relative quantification analy- sis was performed using the comparative standard method (Schefe et al., 2006). Statistical analysis Quantitative data are presented as mean ± standard de- viation (SD) for real-time PCR experiments. One-way ANOVA (SPSS 17.0 Statistical software) was used to analyze the dif- ferent expression pattern of each gene at different ages. Sociobiology 60(4): 429-435 (2013) 431 Fig. 1 Analysis of the sequences of the Amobp11 and Acobp11 genes. A-Alignment of the Amobp11 and Acobp11 nucleic acid sequences. Divergent nucleic acid sequences are circled. B-Alignment of the deduced AmOBP11 and AcOBP11 protein sequences. Divergent amino acid sequences are circled. C-Alignment the CDS protein sequences of 12OBPs (1-11 and 13) and AmOBP11 and AcOBP11. The conserved cysteine residues in this alignment are shaded in dark. Results Sequence analysis of the Amobp11 and Acobp11 genes To characterize the Amobp11 and Acobp11 genes, a 432 bp open reading frame was amplified and sequenced. The Amobp11 and Acobp11 genes have many similar characteristics with about 99.31% identity (Fig. 1A). The Amobp11 gene encodes a 144 amino acid protein that contains a 23 amino acid hydrophobic signal peptide at the N-terminus. The software SignalP 4.0 (Petersen et al., 2011) predicted a molecular weight of 16.6 kDa and a pI of 5.06. The Acobp11 gene encodes a 138 amino acid protein that has a pre- dicted molecular weight of 16.2 kDa and a predicted pI of 4.95 (http://web.expasy.org/cgi-bin/protparam/protparam). The de- duced amino acid sequences of AmOBP11 and AcOBP11 were aligned using DNAMAN software (Fig.1B). The alignment shows that the two sequences vary in sequence at the C-termi- nus, which is truncated by 6 cysteine residues for AcOBP11. Using TMHMM2.0 posterior probabilities for sequences out- side the transmembrane, AmOBP11 and AcOBP11, like the other OBPs, are predicted to be extracellular proteins. Ac- cording to the Kyte and Doolittle method, AmOBP11 and AcOBP11 are hydrophilic in property; however, these pro- teins have some aliphatic amino acids, with an aliphatic in- dex of 81.88 for AcOBP11 and 83.15 for AmOBP11, as well as four region of lipophilicity (http://web.expasy.org/prot- param/). Compared with 12 OBPs (1-11and 13), AmOBP11 and AcOBP11 have six conserved cysteine residues, which are shaded in dark (Fig. 1C). Heterologous expression of the Amobp5 and Acobp5 gene We cloned the Amobp11 and Acobp11 genes into plas- mids, as verified by digestion with the restriction endonucleases EcoR I and Xhol I. Subsequently, cohesive termini of the tar- get gene were inserted into the expression vector pET-28a and transformed into E. coli BL21/Rosetta competent cells. Finally, we used 1 mM IPTG to induce Amobp11 and Acobp11 gene expression. The electrophoretic bands corresponding to recombinant OBP11 protein from pET-28a-Amobp11-E.coli Rosetta (AmOBP11) and pET-28a-Amobp11-E.coli Rosetta (AcOBP11) were verified to be approximately 16 kDa on a 12% SDS-PAGE gel (Fig.2). We used ultrasonic energy to release AmOBP11 and AcOBP11 protein, which formed an insoluble inclusion body. Expression profiling of Amobp11 and Acobp11 genes by real- time PCR To further characterize the functions of the respective obp11 genes in each species of honeybee, we used real-time PCR to quantitatively assess expression levels. The PCR am- plification efficiency for the obp11 gene was 94.8% (slope = -3.453). The efficiency for the β-actin gene was 96.4% (slope HX Zhao et al. - Different Spatiotemporal Roles in the Olfactory System of Honey Bees432 Fig. 2 SDS-PAGE analysis of recombinant AmOBP11 (A) and AcOBP11 (B). A: Lane M: Protein molecular weight marker; Lane 1: pET-28a+E.coli Rosetta; Lane 2: pET-28a-Acobp11+E.coli Rosetta without IPTG to induce expression; Lanes 3-5: pET-28a-Acobp11+E. coli Rosetta induced with IPTG; Lane 6-7: pET-28a-Amobp11 +E.coli Rosetta induced with IPTG; Lane 8: pET-28a-Amobp11+E. coli Rosetta without IPTG; Lane 9: E.coli Rosetta; Lane 10: pET-28- a+E.coli Rosetta. Arrows show the OBP11 expression proteins. Fig.3 Amobp11 gene expression levels in Apis mellifera ligustica an- tennae across seven ages determined by qRT-PCR Expression levels of the Amobp11 gene were calculated relative to the control ß-actin gene using the standard curve method. Bars on each column represent SD for three independent experiments. The Amobp11 gene expres- sion levels in 10-day-old and 15-day-old workers were significantly higher than others ages (F=5.269, P<0.01, n=3), and expression of the Amobp11 gene in 1-day-old workers was the lowest. There were no significant differences between expression in 10-day-old and 15- day-old workers, or others days workers (Duncan’ ANOVA test). = -3.412). This indicates that the relative standard curve method for real-time PCR analysis with SYBR Green dye is experi- mentally suitable. The transcript abundance was calculated based on the difference in threshold cycle (Ct) values between the obp11 and β-actin gene transcripts. The Amobp11 gene demonstrated the highest expres- sion in the antennae of 10-day-old and 15-day-old workers, with nearly 200-fold increase. Other days presented relatively lower expression (Fig.3). In contrast, the Acobp11 gene demonstrated expression that was low at 1 day with a slight increase up to 20 days. The highest expression was observed in 25-day-old and 30-day-old workers, which demonstrated approximately 9- to 12-fold increase in expression as compared to 1-day-old workers (Fig.4). Fig.4 Acobp11 gene expression levels in Apis cerana cerana anten- nae across seven ages determined by qRT-PCR expression levels of the Acobp11 gene were calculated relative to the control ß-actin gene using the standard curve method. Bars on each column represent SD for three independent experiments. The Acobp11 gene expres- sion levels in 25-day-old and 30-day-old workers were significantly higher than others ages (F=6.507, P<0.01, n=3), and expression of the Acobp11 gene in 1-day-old workers was the lowest. There were no significant differences between expression in 25-day-old and 30- day-old workers, or between expression in 30-day-old and 10-day- old workers (Duncan’ ANOVA test). Discussion In this study, we cloned and identified the Acobp11 and Amobp11 genes. Based on our sequence analysis, we concluded that the obp11 genes of the two species have high similarity. The predicted protein sequences of AmOBP11 and AcOBP11 are conserved throughout the molecule, with the exception of one internal residue and six residues at the c- terminus. Furthermore, hydrophobicity analysis suggests that, similar to other honeybee OBPs, AmOBP11 and AcOBP11 are extracellular proteins. We heterologously expressed the Amobp11 and Acobp11 genes in E. coli and demonstrated an apparent molecular mass that agrees with the predicted values for the native honeybee proteins. As a social insect, the foragers of A. mellifera seek large nectariferous plants, while the A. cerana foragers seek small and dispersed nectariferous plants. Therefore, the diver- gent behavior of A. mellifera and A. cerana foragers suggests a diversity of olfactory function. Based on this diversity, we proposed that the expression of the obp11 gene may differ for A. mellifera and A. cerana. Previously, Forêt and Maleszka (2006) indicated that the obp11 gene is expressed exclusive- ly in the antennae of adult bees; thus, we collected different aged workers’ antennae to analyze developmental variations in obp11 gene expression. Varying expression patterns of the obp11 gene in the life cycle of each species could lead to dif- ferent behaviors within and outside of the colony. Both Amobp11 and Acobp11 genes showed the lowest expression levels in 1-day-old workers. Winston (1987) and Calderone (1998) reported that newly emerged bees cannot fly or sting, and thus are developmentally immature (Winston, Sociobiology 60(4): 429-435 (2013) 433 1987; Calderone, 1998). Therefore, the low expression of the obp11 gene in the 1-day-old workers of both species correlates with reduced olfactory function. Olfactory sensing for the two species is closely correlated with the function of the obp11 gene. Our results demonstrate that the developmental and temporal expression profile of the Amobp11 and Acobp11 genes differs in the time and extent of induction after the first day of emergence. For A. mellifera, the obp11 gene expres- sion peaks at 10 to 15 days of age in workers, with nearly 200-fold higher expression than at others ages; whereas for A. cerana, the obp11 gene, expression rises gradually with the highest expression in the 25-day-old workers. The different peaks of expression for the two species correlate with diffe- rent behavioral functions in the life cycle of the honeybee. For most honeybee species, 10-day-old workers feed larvae, while 12- to 21-day-old workers build and store and process food. At the peak of Amobp11 gene expression (10 and 15 days old workers), the honeybees perform nurse-tasks, begin to secrete beeswax, and work to clean within the hive. While performing these in-hive tasks, the 10- to 15-day-old workers are attracted by brood pheromones. For this reason, we pro- pose that the Amobp11 gene expression in the olfactory sys- tem is correlated with nursing tasks. On the other hand, at the peak of Acobp11 expression (25 days of age) most worker bees begin to forage for nectar and pollen (Johnson, 2008b, 2010). The later peak of Acobp11 expression is similar to that of Acer-ASP2 (antenna special protein), which is expressed more highly in 27-day-old workers than at other ages (Lee et al., 2008). Acer-ASP2 is related to odorant sensors that function in the collection of certain nec- tars and pollens (Danty et al., 1997; Li, et al., 2008). There- fore, we propose that the Acobp11 gene has a similar function in out-hive foraging, and that the difference in the temporal expression of the Amobp11 and Acobp11 genes might suggest different functional outcomes in sensing the intention of the OBP11 proteins for the two species. The differences in the levels of induction of the Amobp11 and Acobp11 genes also may be indicative of diffe-rent func- tions. The Amobp11 gene is induced nearly 200-fold at 10- day-old workers, while the Acobp11 gene is induced approxi- mately 5-fold in 10-day-old workers; and 10- to 12-fold at its peak induction in 25 to 30-day-old workers. Further experiments are needed to assess why obvious expression differences of the Amobp11 and Acobp11 genes, and the obp11 gene show different roles in the olfactory system of A. mellifera and A. cerana. The olfactory stimuli that motivate honeybees may come from outside the hive or from within the hive, and dif- fering exposure to each of these stimuli would undoubtedly have a profound effect, which is consistent with the OBP ex- pression and may determine the behavior of the honeybee. The olfactory system within the hive functions to motivate workers to perform different tasks based on the age of larvae (Le Conte, 2001, 2008; Maisonnasse, 2009). Larvae of dif- ferent ages emit volatile compounds to adjust the behavior of the honeybees. Young larvae emit E-ß-ocimene, a highly volatile pheromone that is dispersed within the colony to ac- celerate forage behavior of in-hive workers by the olfactory system (Maisonnasse, 2010). Brood ester pheromones moti- vate older workers to take care of the brood (Slessor, 2005; Pankiw, 2007; Peters, 2010). Newly emerging bees engage in some nursing behaviors too, whereas the others ages, such as middle-aged and 12-21 days old workers do not engage in nursing behavior (Ben-Shahar et al., 2002; Ben-Shahar, 2005). Meanwhile, E-ß-ocimene is one of the monoterpene volatile organic compounds, which is emited by larvae to engage workers in nursing tasks. During the process of per- forming nursing tasks, OBPs play an important role in the olfactory system of the honeybee. OBPs assist E-ß-ocimene to transfer to the olfactory receptor, which elicits the corre- sponding behavior. The Amobp11 gene belongs to one of the main antennae OBPs, The 10- to 15-day-old workers have the highest expression of Amobp11 gene. Thus, we propose that the high Amobp11 gene expression of 10- to 15-day-old workers is correlated with sensitive olfactory reception of volatile brood compounds, which might encourage them to engage in nursing behaviors (Maisonnasse, 2009). On the other hand, because the peak of Acobp11 gene expression is in the foraging stage, it is likely that the func- tion of this obp11 gene for A. cerana is more highly evolved to respond to a different set of olfactory stimuli, which are encountered outside of the hive. The olfactory system of A. cerana is known to be more sensitive than that of A. mellifera, especially in regards to the collection of honey and pollen. Therefore, it is likely that the OBP11 proteins of these two species might performing in the response to different sources of stimuli to determine the differing behavior of these two species at different stages. We conclude that the spatiotempo- ral expression patterns of the obp11 gene in A. mellifera and A. cerana suggests that this gene plays different roles in the olfactory sensitivity of workers. Conclusions We demonstrated that the nurse workers, which have the highest olfactory sensitivity in the A. mellifera colony, have the highest expression of Amobp11 gene; whereas 1-day-old workers, which have lowest olfactory sensitivity, have corre- spondingly low expression. However, the highest expression of Acobp11 gene is observed for foragers, which display the highest olfactory sensitivity in the A. cerana population. 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