Molecular basis for the reproductive division of labour in a lower termite
© Weil et al; licensee BioMed Central Ltd. 2007
Received: 17 October 2006
Accepted: 28 June 2007
Published: 28 June 2007
Polyphenism, the expression of different phenotypes with the same genetic background, is well known for social insects. The substantial physiological and morphological differences among the castes generally are the result of differential gene expression. In lower termites, workers are developmentally flexible to become neotenic replacement reproductives via a single moult after the death of the founding reproductives. Thus, both castes (neotenics and workers) are expected to differ mainly in the expression of genes linked to reproductive division of labour, which constitutes the fundamental basis of insect societies.
Representational difference analysis of cDNAs was used to study differential gene expression between neotenics and workers in the drywood termite Cryptotermes secundus (Kalotermitidae). We identified and, at least partially cloned five novel genes that were highly expressed in female neotenics. Quantitative real-time PCR analysis of all five genes in different castes (neotenics, founding reproductives, winged sexuals and workers of both sexes) confirmed the differential expression patterns. In addition, the relative expression of these genes was determined in three body parts of female neotenics (head, thorax, and abdomen) using quantitative real-time PCR.
The identified genes could be involved in the control and regulation of reproductive division of labour. Interestingly, this study revealed an expression pattern partly similar to social Hymenoptera indicating both common and species-specific regulatory mechanisms in hemimetabolous and holometabolous social insects.
Social insects (termites and social Hymenoptera, such as ants, some bees, and wasps) are the exemplars of social life. They are characterized by a reproductive division of labour in which only a few individuals within a colony reproduce (queen/s, and king/s in termites), while the large majority helps in raising offspring (workers, in termites additionally soldiers). This caste system is a result of phenotypic plasticity; i.e. different castes generally arise from environmentally induced differential gene expression [1–3].
We specifically addressed the question of what characterizes a queen by comparing gene expression profiles between workers and female reproductives in the drywood termite Cryptotermes secundus. In termites, neotenic replacement reproductives are especially suited for this purpose because they differ from workers only by traits linked to reproduction, while confounding traits that are developed by winged sexuals for the dispersal process (e.g. compound eyes, wings) are not expressed. Our analysis revealed a number of interesting genes that are primarily expressed in neotenic replacement reproductives and may be involved in processes controlling or maintaining the reproductive division of labour.
Identification of caste-specific transcripts in female neotenics
No. of clones
Identity match by BLASTX [species]
Local identity (%)
PREDICTED: similar to CG4382-PA [Tribolium castaneum]
PREDICTED: similar to CG4382-PA [Apis mellifera]
juvenile hormone esterase [Aedes aegypti]
beta-glucosidase [Neotermes koshunensis]
male-specific beta-glycosidase [Leucophaea maderae]
PREDICTED: similar to CG9701-PA [Tribolium castaneum]
Vitellogenin 1 precursor (Vg-1) [Periplaneta americana]
Vitellogenin [Athalia rosae]
Vitellogenin 2 precursor (Vg-2) [Periplaneta americana]
family 4 Cytochrome P450 [Coptotermes acinaciformis]
Cytochrome P450 4C1 (CYPIVC1) [Blaberus discoidalis]
Cytochrome P450 [Aedes aegypti]
PREDICTED: similar to guanylate cyclase OlGC-R2 [Danio rerio]
Quantitative expression analysis of the Neofem 1–5 genes
The ability of Cryptotermes secundus workers to develop into neotenic replacement reproductives after a single moult offers the unique possibility to study differential gene expression during caste differentiation. In this study, we compared the transcriptomes from female neotenics and workers of both gender using RDA to identify novel neotenics-specific transcripts.
We were able to identify five genes that were highly overrepresented in female neotenics of the drywood termite C. secundus. Four of these five genes were overexpressed in the head. Expression of the genes Neofem1 and Neofem2 of C. secundus was highly specific for female reproductives. Both genes are predicted to encode secretory proteins that are specifically expressed in the heads of female neotenics. The open reading frame of Neofem1 encodes a putative esterase-lipase which shows the highest similarity to yet uncharacterized proteins of the red flour beetle Tribolium castaneum and the honey bee Apis mellifera which are putative orthologs of the Drosophila protein CG4382-PA. In addition, two juvenile hormone esterases (JHE) of the mosquito Aedes aegypti are closely related to the Neofem1 protein sequence. Most interestingly, the Apis mellifera homolog (GB16889, [GenBank: XP_393293]) was found in the brain of adult female worker honey bees  and is closely related to a moth integumental carboxyl/cholinesterase which is implicated in pheromone processing [17, 18].
Neofem2 showed highest similarity to a digestive β-glycosidase from the salivary glands of the termite Neotermes koshuensis . Insect glycosidases are known to include, amongst others, digestive and pheromone degrading enzymes [12, 13]. However, the lack of expression in males suggests a sex specific function. Thus Neofem2 is presumably not a digestive enzyme. Rather the close match to Lma-p72 protein of the Madeiran cockroach , which is sex specifically expressed in the abdominal glands of male cockroaches to attract females, may indicate a pheromonal function.
The C. secundus Neofem4 protein is closely related to family 4 cytochrome P450 enzymes (CYP4) from arthropods, with highest similarities to an uncharacterized termite CYP4 from Coptotermes acinaciformes and to CYPIVC1 from Blaberus discoidalis (Blattodea; ). Cytochrome P450 enzymes of insects are generally associated with the metabolism of endogenous substrates or hormones, and with detoxification (summarized by Feyereisen ). In termites and social Hymenoptera, some cytochrome P450 enzymes are expressed in a caste specific manner [3, 6, 21–23]. Contrary to these studies on Hymenoptera and on Coptotermes acinaciformes that all revealed highest expression levels in non-reproducing castes, Neofem4 of C. secundus was overexpressed specifically in female neotenics. In termites, cytochrome P450 enzymes are involved in metabolic pathways (C. acinaciformis) or insecticide resistance (Mastotermes darwinensis) [6, 24]. However, the specific expression of Neofem4 in the head of female neotenics suggests that Neofem4 is involved in the metabolism of endogenous substrates like ecdysteroids or JH rather than insecticide resistance.
The gene Neofem3 is the only gene that is distributed almost equally in all body parts of female neotenics. It showed highest similarities to insect vitellogenins (Vgs), specifically to Vg1 of the American cockroach Periplaneta americana and a Vg of the turnip sawfly Athalia rosae. In most insect species vitellogenins are synthesized extraovarially in female fat body cells as large precursor proteins of vitellin (the major yolk protein of insects). Vgs are secreted into the haemolymph and then incorporated into developing oocytes [25, 26]. High expression levels of Vg in female reproductives (primaries and neotenics) were expected because of their ovarian activity. The elevated Vg expression in male reproductives may be explained by the function of Vgs as storage proteins . Recently it was shown that functionally sterile nursing honey bee workers utilize vitellogenin to produce royal jelly to feed larvae . The above findings suggest that an ancestral reproductive protein, Vg, was repeatedly co-opted in different social species to serve different functions in different castes. Thus, Vg seems to function as an important developmental protein.
We isolated and characterized five genes that were up-regulated in female replacement reproductives compared to non-reproducing workers of the drywood termite Cryptotermes secundus (Kalotermitidae). Interestingly, potential homologues of some of these genes appear to be expressed in different insect species, hemimetabolous as well as holometabolous, in a caste- and species-specific manner. Especially, pheromone-processing genes and Vg emerge as major players that were repeatedly exploited in social evolution of insect societies.
All chemical reagents used were purchased from Sigma-Aldrich (Taufkirchen, Germany) unless otherwise noted. Oligonucleotides were synthesized either by Metabion international AG (Martinsried, Germany) or by Carl Roth GmbH (Karlsruhe, Germany). Sequences of all Oligonucleotides are given in Additional file 1.
Complete termite colonies (Cryptotermes secundus) were collected in mangroves around Darwin (NT, Australia) and held in climate chambers at 27°C and a relative humidity of 70% (for details see Korb and Schmidinger ). Primary reproductive and alates were taken from these colonies.
To obtain neotenic reproductives, big colonies were split and groups of at least 15 workers were placed together in new Pinus radiata wood blocks (16 × 4 × 4 cm3). After about two weeks neotenic reproductives developed which were removed together with two workers. The sex of the neotenics was determined by their sex-specific morphology as described by Grassé .
Total RNA from different castes and developmental stages was prepared using the RNAwiz™ solution (Ambion). Poly(A)mRNA was enriched using the MicroPoly(A)Purist™ Kit (Ambion) according to the manufacturer's recommendations. RNA purity and integrity were checked by agarose gel electrophoresis and by UV/Vis spectrometry.
Representational difference analysis
Double-stranded cDNA was prepared by reverse transcription of 2 μg poly(A) mRNA using the Universal Riboclone® cDNA Synthesis System (Promega). RDA was performed essentially as described by Heinz et al. . Briefly, the driver representation consisted of cDNA generated from the pooled mRNA of 25 Cryptotermes secundus workers. This representation was subtracted from tester cDNA representation of the mRNA repertoire of 11 C. secundus female replacement reproductives. After three rounds of subtraction (driver excess: 50 ×, 400 × and 10.000 × in successive rounds) and amplification, the entire third difference product was gel-extracted and "shotgun"-cloned into the BamH I restriction site of the pZErO-2 vector (Invitrogen) according to the manufacturer's instructions. To check for specificity of the difference product, inserts of randomly picked clones were PCR-amplified from single bacterial colonies utilizing vector-specific primers. The PCR products were denatured with 3 M NaOH for 30 min at room temperature and blotted in duplicates on two separate nylon membranes (Magna NT, 0.22 μm; MSI) in 20 × SSC using a vacuum dot blot manifold (Schleicher und Schuell). After UV-cross-linking, one blot was hybridized to driver (worker), the other blot to tester (female neotenics) cDNA representation, which had been labelled radioactively with Klenow fragment (Roche Biochemicals) according to standard protocols. After stringent washing, membranes were exposed to a Molecular Dynamics Storage Phosphor Screen overnight and scanned on a Typhoon 9200 Variable Mode Imager (Amersham Pharmacia). An additional RDA was performed starting with the first difference product of the first RDA. The procedure was modified by adding Dpn II fragments of three genes obtained from the first round to achieve additional Dpn II fragments. Here two additional rounds of subtraction (driver excess: 400 × and 5.000 × in successive rounds) and amplification were performed. Products were cloned and analysed as above.
RNA ligase-mediated 5'- and 3'-Rapid Amplification of c DNA Ends (RACE)-PCR
To obtain complete cDNAs of genes corresponding to the identified RDA fragments, 5'- and 3'-end RACE-PCRs and inter-fragment PCRs were performed. One μg of total RNA from female neotenics was used for cDNA synthesis with the FirstChoice™ RLM-RACE Kit (Ambion). The outer and inner primers for nested PCRs of the genes Neofem 1–5 and the putative transferrin were derived from gene-specific PCR fragments obtained during the RDA (sequences are given in Additonal file 1). They were used to amplify 5'- and/or 3'-cDNA fragments. PCR products were cloned into pCR2.1-TOPO vector (TOPO Cloning Kit, Invitrogen) and inserts from several individual plasmid-containing bacterial colonies were sequenced (by GENEART, Regensburg, Germany). Oligonucleotide primers for full-length cDNA amplification were designed according to sequence alignments. PCR products were cloned into pCR2.1-TOPO (TOPO Cloning Kit, Invitrogen) and subsequently sequenced.
Quantitative real-time PCR
Total RNA (1 μg) was reverse transcribed using Superscript II RT (Invitrogen) and Random Decamers (Ambion). qRT-PCR was performed on a Mastercycler® ep realplex (Eppendorf) using the QuantiTect SYBR green PCR Kit (Qiagen) according to the manufacturer's instructions. Primers are given in Additional file 1. Melting curves were analyzed to control for specificity of the PCR reactions. Expression data for genes were normalized for expression of the 18S rRNA. The relative units were calculated from a standard curve plotting 3 different concentrations of log dilutions against the PCR cycle number (CP) at which the measured fluorescence intensity reached a fixed value. Values represent mean +SD of three independent experiments.
Alignments were performed using the software Gene Runner Version 3.05 (Hastings Software Inc.) and BioEdit Version 7.0.1 (Tom Hall Isis Pharmaceuticals, Inc.). BLAST-X database  searches were conducted to establish cDNA clone identity.
The authors would like to thank Jürgen Heinze and Reinhard Andreesen for providing resources. We express appreciation to Lucia Pfeilschifter-Schwarzfischer for technical assistance. We thank Estelle Roux and Alexander Fuchs for sampling colonies. This work was supported by a Deutsche Forschungsgemeinschaft (DFG) grant to JK and MR.
- Hamilton WD: The genetical evolution of social behaviour. I, II. J Theor Biol. 1964, 7: 1-52. 10.1016/0022-5193(64)90038-4.PubMedView ArticleGoogle Scholar
- Crozier RH, Pamilo P: Evolution of social insect colonies: sex allocation and kin selection. 1996, Oxford: Oxford University PressGoogle Scholar
- Evans JD, Wheeler DE: Differential gene expression between developing queens and workers in the honey bee, Apis mellifera. Proc Natl Acad Sci USA. 1999, 96: 5575-5580. 10.1073/pnas.96.10.5575.PubMed CentralPubMedView ArticleGoogle Scholar
- Roisin Y: Diversity and evolution of caste patterns. Termites: Evolution, Sociality, Symbioses, Ecology. pp. 95-120. Edited by: Abe T, Bignell DE, Higashi M. 2000, Dordrecht: Kluwer Academic Publishers, 95-120.View ArticleGoogle Scholar
- Miura T, Kamikouchi A, Sawata M, Takeuchi H, Natori S, Kubo T, Matsumoto T: Soldier caste-specific gene expression in the mandibular glands of Hodotermopsis japonica (Isoptera: Termopsidae). Proc Natl Acad Sci USA. 1999, 96: 13874-13879. 10.1073/pnas.96.24.13874.PubMed CentralPubMedView ArticleGoogle Scholar
- Cornette R, Koshikawa S, Hojo M, Matsumoto T, Miura T: Caste-specific cytochrome P450 in the damp-wood termite Hodotermopsis sjostedti (Isoptera, Termopsidae). Insect Mol Biol. 2006, 15 (2): 235-244. 10.1111/j.1365-2583.2006.00632.x.PubMedView ArticleGoogle Scholar
- Scharf ME, Ratliff CR, Wu-Scharf D, Zhou X, Pittendrigh BR, Bennett GW: Effects of juvenile hormone III on Reticulitermes flavipes : changes in hemolymph protein composition and gene expression. Insect Biochem Mol Biol. 2005, 35: 207-215. 10.1016/j.ibmb.2004.12.001.PubMedView ArticleGoogle Scholar
- Zhou X, Oi FM, Scharf ME: Social exploitation of hexamerin: RNAi reveals a major caste-regulatory factor in termites. Proc Natl Acad Sci USA. 2006, 103: 4499-4504. 10.1073/pnas.0508866103.PubMed CentralPubMedView ArticleGoogle Scholar
- Zhou X, Tarver MR, Bennett GW, Oi FM, Scharf ME: Two hexamerin genes from the termite Reticulitermes flavipes : Sequence, expression, and proposed functions in caste regulation. Gene. 2006, 376: 47-58. 10.1016/j.gene.2006.02.002.PubMedView ArticleGoogle Scholar
- Noirot C, Pasteels JM: Ontogenetic development and evolution of the worker caste in termites. Experientia. 1987, 43: 851-860. 10.1007/BF01951642.View ArticleGoogle Scholar
- Noirot C: Sexual castes and reproductive strategies in termites. An evolutionary approach to castes and reproduction. pp. 5-35. Edited by: Engels W. 1990, Berlin: Springer Verlag, 5-35.Google Scholar
- Tokuda G, Saito H, Watanabe H: A digestive beta-glucosidase from the salivary glands of the termite, Neotermes koshunensis (Shiraki): distribution, characterization and isolation of its precursor cDNA by 5'- and 3'-RACE amplifications with degenerate primers. Insect Biochem Mol Biol. 2002, 32: 1681-1689. 10.1016/S0965-1748(02)00108-X.PubMedView ArticleGoogle Scholar
- Cornette R, Farine JP, Abed-Viellard D, Quennedey B, Brossut R: Molecular characterization of a male-specific glycosyl hydrolase, Lma-p72, secreted on to the abdominal surface of the Madeira cockroach Leucophaea maderae (Blaberidae, Oxyhaloinae). Biochem J. 2003, 372: 535-541. 10.1042/BJ20030025.PubMed CentralPubMedView ArticleGoogle Scholar
- Tufail M, Lee JM, Hatakeyama M, Oishi K, Takeda M: Cloning of vitellogenin cDNA of the American cockroach, Periplaneta americana (Dictyoptera), and its structural and expression analyses. Arch Insect Biochem Physiol. 2000, 45: 37-46. 10.1002/1520-6327(200009)45:1<37::AID-ARCH4>3.0.CO;2-8.PubMedView ArticleGoogle Scholar
- Lu KH, Bradfield JY, Keeley LL: Juvenile hormone inhibition of gene expression for cytochrome P4504C1 in adult females of the cockroach, Blaberus discoidalis. Insect Biochem Mol Biol. 1999, 29: 667-673. 10.1016/S0965-1748(99)00034-X.PubMedView ArticleGoogle Scholar
- Pfaffl MW, Tichopad A, Progmet C, Neuvians TP: Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004, 26: 509-515. 10.1023/B:BILE.0000019559.84305.47.PubMedView ArticleGoogle Scholar
- Claudianos C, Ranson H, Johnson RM, Biswas S, Schuler MA, Berenbaum MR, Feyereisen R, Oakeshott JG: A deficit of toxification enzymes: pesticide sensitivity and enviornmental response in the honeybee. Insect Mol Biol. 2006, 15: 615-636. 10.1111/j.1365-2583.2006.00672.x.PubMed CentralPubMedView ArticleGoogle Scholar
- Ishida Y, Leal WS: Cloning of putative odorant-degrading enzyme and integumental esterase cDNAs from the wild silkmoth, Antheraea polyphemus . Insect Biochem Mol Biol. 2002, 32: 1775-1780. 10.1016/S0965-1748(02)00136-4.PubMedView ArticleGoogle Scholar
- Bradfield JY, Lee Y-H, Keeley LL: Cytochrome P450 family 4 in a cockroach: Molecular cloning and regulation by hypertrehalosemic hormone. Proc Natl Acad Sci USA. 1991, 88: 4558-4562. 10.1073/pnas.88.10.4558.PubMed CentralPubMedView ArticleGoogle Scholar
- Feyereisen R: Insect P450 enzymes. Annu Rev Entomol. 1999, 44: 507-533. 10.1146/annurev.ento.44.1.507.PubMedView ArticleGoogle Scholar
- Evans JD, Wheeler DE: Expression profiles during honeybee caste determination. Genome Biol. 2000, 2: 1-10.1186/gb-2000-2-1-research0001.View ArticleGoogle Scholar
- Liu N, Zhang L: CYP4AB1, CYP4AB2, and Gp-9 gene overexpression associated with workers of the red imported fire ant, Solenopsis invicta Buren. Gene. 2004, 327: 81-87. 10.1016/j.gene.2003.11.002.PubMedView ArticleGoogle Scholar
- Judice CC, Carazzole MF, Festa F, Sogayar MC, Hartfelder K, Pereira GAG: Gene expression profile underling alternative caste phenotypes in a highly eusocial bee, Melipona quadrifasciata . Insect Mol Biol. 2006, 15: 33-44. 10.1111/j.1365-2583.2005.00605.x.PubMedView ArticleGoogle Scholar
- Falckh PHJ, Balcombe W, Haritos VS: Isolation and identification of a cytochrome P450 sequence in an Australian termite, Mastotermes darwiniensis . Biochem Biophys Res Commun. 1997, 241: 579-583. 10.1006/bbrc.1997.7856.PubMedView ArticleGoogle Scholar
- Wheeler D: The role of nourishment in oogenesis. Annu Rev Entomol. 1996, 41: 407-431. 10.1146/annurev.en.41.010196.002203.PubMedView ArticleGoogle Scholar
- Amdam GV, Norberg K, Hagen A, Omholt SW: Social exploitation of vitellogenin. Proc Natl Acad Sci USA. 2003, 100: 1799-1802. 10.1073/pnas.0333979100.PubMed CentralPubMedView ArticleGoogle Scholar
- Korb J, Schmidinger S: Help or disperse? Cooperation in termites influenced by food conditions. Behav Ecol Sociobiol. 2004, 56: 89-95. 10.1007/s00265-004-0757-x.View ArticleGoogle Scholar
- Grasse PP: Termitologia: anatomie-physiologie-biologie-systematique des termites. Anatomie-physiologie-reproduction. 1982, Paris: MassonGoogle Scholar
- Heinz S, Krause SW, Gabrielli F, Wagner HM, Andreesen R, Rehli M: Genomic organization of the human gene HEP27: alternative promoter usage in HepG2 cells and monocyte-derived dendritic cells. Genomics. 2002, 79: 608-615. 10.1006/geno.2002.6743.PubMedView ArticleGoogle Scholar
- ULR: [http://www.ncbi.nlm.nih.gov/BLAST/]
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