Honey bee (Apis mellifera) transferrin-gene structure and the role of ecdysteroids in the developmental regulation of its expression

Insect Biochemistry and Molecular Biology 34 (2004) 415–424 www.elsevier.com/locate/ibmb

Honey bee (Apis mellifera) transferrin-gene structure and the role of ecdysteroids in the developmental regulation of its expression Adriana Mendes do Nascimento a,
, Virginie Cuvillier-Hot a, Angel Roberto Barchuk b, Zila´ Luz Paulino Simo˜es a, Klaus Hartfelder a a

Departamento de Biologia, Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Avenida Bandeirantes, 3.900, Ribeirao Preto, 14040-901 SP, Brazil b Departamento de Gene´tica, Faculdade de Medicina de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Ribeirao Preto, SP, Brazil Received 7 October 2003; accepted 12 December 2003

Abstract Social life is prone to invasion by microorganisms, and binding of ferric ions by transferrin is an efficient strategy to restrict their access to iron. In this study, we isolated cDNA and genomic clones encoding an Apis mellifera transferrin (AmTRF) gene. It has an open reading frame (ORF) of 2136 bp spread over nine exons. The deduced protein sequence comprises 686 amino acid residues plus a 26 residues signal sequence, giving a predicted molecular mass of 76 kDa. Comparison of the deduced AmTRF amino acid sequence with known insect transferrins revealed significant similarity extending over the entire sequence. It clusters with monoferric transferrins, with which it shares putative iron-binding residues in the N-terminal lobe. In a functional analysis of AmTRF expression in honey bee development, we monitored its expression profile in the larval and pupal stages. The negative regulation of AmTRF by ecdysteroids deduced from the developmental expression profile was confirmed by experimental treatment of spinning-stage honey bee larvae with 20-hydroxyecdysone, and of fourth instar-larvae with juvenile hormone. A juvenile hormone application to spinning-stage larvae, in contrast, had only a minor effect on AmTRF transcript levels. This is the first study implicating ecdysteroids in the developmental regulation of transferrin expression in an insect species. # 2003 Elsevier Ltd. All rights reserved. Keywords: Iron metabolism; Ecdysone; Juvenile hormone; Honey bee; Metamorphosis; Insect immune response

1. Introduction Social life does not only purvey advantages, such as improved defense against predators, higher survival rates of offspring and buffering against environmental variation, but also confers putatively detrimental properties, such as the rapid spreading of pathogenic microbial diseases at high population densities in a controlled microenvironment. This observation, obviously, holds true for any socially organized animal community, where it is manifest in a continuous arms race between pathogens and their hosts. Since defense mechanisms against pathogens and parasites represent a very basic element in metazoan evolution, one can
Corresponding author. Tel.: +1-55-16-602-3153; fax: +1-55-16633-6482. E-mail address: [email protected] (A.M. do Nascimento).

0965-1748/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2003.12.003

expect to find many common strategies employed by phylogenetically even quite distant taxa. In vertebrates, one of these strategies is the restriction of access for pathogens and parasites to free ferric ions by a controlled iron shuttle system (for recent reviews, see Aisen et al., 2001; Koski and Scott, 2003; Ponka, 2003; Stafford and Belosevic, 2003). Such tight control of free iron is not only advantageous as a defense strategy against bacterial and protozoan infections but also is a necessity in view of the toxicity of free iron in the presence of oxygen. Positioned at the crossroads between energy metabolism and immunity, it comes as no surprise that iron metabolism has been an intensively investigated subject also in insects (for recent reviews, see Law, 2002; Nichol et al., 2002), and both ferritin and transferrin orthologs have been identified and cloned, yet, surprisingly, no homologs to transferrin receptors have been

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detected in the Drosophila genome (Adams et al., 2000). This could mean that insects use a different element in the shuttle from transferrin-bound iron in the hemolymph into iron requiring cells. Furthermore, and in distinction to vertebrates, insect ferritin is predominantly a secreted rather than a cytosolic protein (Locke and Nichol, 1992). These observations have led to hypothesize that in insects the genuine hemolymph iron transporter may be ferritin, leaving transferrin to serve primarily as an antibiotic factor in defense reactions. Its role would thus be similar to mammalian lactoferrin (Yoshiga et al., 1997; Fallon and Sun, 2001). This functional switch from a transport protein to a component of the insect immune response system makes transferrin one of the candidate determinants of longevity. Intraspecific differences in longevity are particularly pronounced in the castes of highly social Hymenoptera, such as honey bees, where a queen can live up to 4 years. In contrast, worker bees usually do not live much longer than 45 days, except for winter bees in temperate climates, which can have their longevity extended to several months, permitting colonies to survive harsh winters without spending precious energy in short-lived offspring. Such lifespan differences have recently been incorporated into models on honey bee colony organization (Page and Peng, 2001; Amdam and Omholt, 2002), the latter pointing out vitellogenin titer and juvenile hormone as critical variables. Amdam and Omholt (2002) consider that vitellogenin, in addition to its primary role as an egg yolk protein, may also have a function in the honey bee immune defense via its deduced zinc-binding properties, and thus may promote longevity. When taken in this context, transferrin could be a second major candidate factor for lifespan modulation in social insects. In addition, it could even act in conjunction with vitellogenin, since uptake of radioactive iron and transferrin as well as the presence of transferrin mRNA has been demonstrated in fly oocytes (Kurama et al., 1995). Transferrin has also been detected in oocytes of Riptortus clavatus, but there is no evidence for transferrin expression in ovaries (Hirai et al., 2000). Transferrin sequences are available for several insect taxa, the holometabolans Manduca sexta (Bartfeld and Law, 1990), Aedes aegypti (Yoshiga et al., 1997), Bombyx mori (Yun et al., 1999), Drosophila melanogaster and Drosophila silvestris (Yoshiga et al., 1999), Sarcophaga peregrina (Kurama et al., 1995), and the hemimetabolans Riptortus clavatus (Hirai et al., 2000), Blaberus discoidalis (Jamroz et al., 1993) and Mastotermes darwiniensis (Thompson et al., 2003). The latter is the first published sequence of a social insect transferrin. Similar to most insect transferrins, its expression is up regulated in response to infection (Thompson et al., 2003). In a

parallel approach to this study, a complete honey bee transferrin cDNA sequence (AmTRF) has recently been deposited in GenBank by Kucharski and Maleszka (2003). These authors obtained the AmTRF cDNA sequence by transcriptional profiling using a honey bee microarray (Whitfield et al., 2002), and analyzed its tissue-specific expression, primarily in brain and eye development and in the context of bacterial defense of adult worker bees. In distinction to most studies on insect transferrins, we included larval and pupal stages in our study, primarily because these are not only highly susceptible to infection, but they are also the critical stages for caste development, and thus for the configuration and installation of differential longevity programs of queens versus workers. Based on previous information on juvenile hormone (JH) effects on transferrin expression in B. discoidalis (Jamroz et al., 1993) and R. clavatus (Hirai et al., 2000), and also on AmTRF (Kucharski and Maleszka, 2003), the caste-specifically modulated JH titer in honey bee development and reproduction (for reviews, see Hartfelder and Engels, 1998; Bloch et al., 2002) was a natural candidate as a regulator for transferrin expression also in the preimaginal stages. Yet interestingly, and not reported so far for any of the other insect species, ecdysteroids turned out to be the prime modulator of transferrin expression in honey bee larvae and pupae.

2. Materials and methods 2.1. Bees Larvae, pupae and adults were collected from Apis mellifera colonies (Africanized hybrids) maintained at the Experimental Apiary of the University of Sa˜o Paulo at Ribeira˜o Preto, Brazil. The developmental stages of workers were classified according to the criteria proposed by Rachinsky et al. (1990) and Michelette and Soares (1993). 2.2. Bacterial strains and culture conditions Escherichia coli DH5aF used as host for all plasmidsubcloning experiments was grown in LB medium. E. coli LE392, used as host for propagation of the kZAP bacteriophage, was grown in NZY medium. v Bacterial strains were grown at 37 C. 2.3. DNA and RNA isolation For DNA extraction, 10 unpigmented pupae were snap-frozen in liquid nitrogen and ground to fine powder before being transferred to 2 ml polypropylene tubes containing 300 ll of extraction buffer (0.1 M

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NaCl, 1 mM EDTA, 1.5% SDS, 50 mM Tris–HCl, adjusted to pH 7.5) plus proteinase K (0.5 mg ml1). v After incubation at 55 C for 2 h, the homogenate was centrifuged (12; 000g for 10 min) and DNA was extracted from the supernatant by repeated phenol– chloroform liquid-phase separation, followed by isopropanol precipitation. Total RNA was extracted from whole-body samples using a TRIzol (Invitrogen) protocol. When used for RT-PCR assays, the RNA extracts were incubated in the presence of 3 units of DNAse I (Promega) for v 30 min at 37 C to eliminate contaminating DNA. 2.4. Sequencing of honey bee transferrin genomic and cDNA In order to identify a transferrin-encoding gene from A. mellifera, we designed degenerated primers for use in a reverse transcription PCR (RT-PCR) protocol. The primers were based on consensus sequences in conserved regions of insect transferrin cDNAs (Yoshiga et al., 1997). The following primers gave positive results: TRF1 (50 -GAMCCYAAGGAYATGTAYGTRGC-30 ) and TRF2 (50 -YCWCKYTCWATIACITCYKTGTA30 ). First-strand cDNA was synthesized by reverse transcription (SuperScript II; Invitrogen) from 1 lg of total RNA. Aliquots of first-strand cDNA products were employed in PCR reactions using Invitrogen Taq polymerase. The thermal cycling program consisted of 2 min v v at 94 C followed by 30 cycles of 30 s at 94 C, 45 s at v v v 52 C, 1 min at 72 C and a final extension step at 72 C for 10 min. The amplification products were analyzed by electrophoresis in 1% agarose gels. Fragments of about 550 bp were purified and subcloned into the EcoRI site of pCR 2.1-TOPO plasmid (TOPO TA Cloning kit, Invitrogen). Insert-containing plasmids were subjected to sequencing reactions using the M13reverse and M13-forward universal primers. The genomic sequence of AmTRF was determined by using two specific primers TRF3 (50 -GTTCGTC GCGAATTTTTCATC-30 ) and TRF4 (50 -GTTAAATA ATTCACGACGAGC-30 ) that were synthesized based on the cDNA sequence and were employed in subsequent PCR amplification with genomic DNA as template. A resulting 3.1 kb PCR fragment was cloned into pCR 2.1-TOPO plasmid and sequenced. Comparison of the genomic sequence with the cDNA sequence was performed with Sequencher 3.1 software. 2.5. Screening of the kZAP cDNA library Recombinant plaques of a brain kZAP cDNA library of A. mellifera were immobilized on nylon membranes (Biodyne B, Invitrogen), and were probed with a PCR-generated 550 bp AmTRF fragment that was labeled with [a-32P]-dCTP using a Random-Primer

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DNA labeling system (Invitrogen). Hybridization was v carried out overnight at 65 C in 5 SSC supplemented with Denhardt’s solution in the presence of 0.1% SDS. Stringency washes were performed once in v 3 SSC (0.1% SDS) for 15 min at 65 C and once in v 1 SSC (0.1% SDS) at 65 C. Positive plaques were selected for further screenings. Subsequent to a tertiary screening, the eluted phage of a positive plaque was used as template for PCR amplification with the M13reverse and M13-forward universal primers that match the phage sequences flanking the cDNA insert. After electrophoresis in a 1% agarose gel, a PCR fragment of about 2 kb was purified and subcloned into a pCR 2.1TOPO plasmid. The complete sequence of the insert was obtained by primer walking. 2.6. Sequencing and bioinformatics tools DNA sequencing was performed by the dideoxy sequencing method, using a Big-dye terminator v3.0 Cycle Sequencing Ready Reaction for an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Contigs were assembled using Sequencher 3.1 software. Sequence homology searches were performed by BLAST (blastx algorithm), and putative orthologs were aligned by means of ClustalW. For a molecular phylogeny analysis of insect transferrins, the ClustalW results were transformed to Mega 2.1 format (Kumar et al., 2001). A pairwise distance matrix was constructed by Poisson correction on 526 sites. The Neighbor Joining procedure was used for the tree construction and tree branch significance was evaluated by 500 bootstrap repetitions. 2.7. Northern blot analysis Total RNA (10 lg for developmental stages, 20 lg for hormone-treated larvae) was isolated from whole bodies and run on denaturing 1.2% agarose gels containing formaldehyde. The RNAs were transferred to nylon membranes (Biodyne B Membrane, Invitrogen) by capillary blotting in the presence of 20 SSC for v 16 h. The membranes were prehybridized at 65 C for at least 1 h in 5 SSC containing 0.1% SDS, 5% dextran and 5% liquid block (Amersham, RPN 3540). A fluorescein-labeled 550 bp AmTRF fragment prepared according to the protocol of Gene Images2 Random Prime Labelling Module (Amersham) was added to the prehybridization solution for overnight v hybridization at 65 C. Stringency washes and fluorescent signal detection followed the manufacturer’s protocol of Gene Images. For normalization, membranes were stripped and rehybridized with a 28S subunit rDNA probe.

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2.8. Hormone treatments S1-stage worker larvae (early cocoon-spinning stage according to Rachinsky et al., 1990) were removed from their brood cells and injected with 20-hydroxyecdysone (20E) (Sigma), diluted to 1 mg/ml in a honey bee saline (Rachinsky and Hartfelder, 1998). A final dose of 1 lg 20E was injected into the hemocoel through a lateral protrusion of the abdominal body wall. Strong muscle contraction occurring in this protrusion after injection prevented backflow of hemolymph in most animals. Specimens that exhibited hemolymph extrusion were discarded. Worker larvae injected with 1 ll honey bee saline served as sham and untreated larvae as general control. To test for effects of JH on AmTRF expression, S1-stage larvae received a topical application of 5 lg juvenile hormone III (Fluka, diluted in acetone to a stock solution of 2.5 lg/ll). Larvae were maintained in an incubator at v 34 C and 70% relative humidity for 6 h. After incubation, the bees were homogenized in Trizol reagent v (Invitrogen) and frozen at 80 C until RNA extraction. In an additional experiment, we topically applied 5 lg JH III on fourth-instar worker larvae while still in their brood cells. After JH application, the brood frames v were kept at 34 C for 1 h for solvent evaporation before returning them to low-rejection colonies, that is to honey bee colonies showing a low degree of hygienic behavior (Gramacho et al., 1999). The JH-treated larvae were then allowed to develop until they reached the late spinning stages (S2/S3). For RNA extraction, they were thus retrieved from the frames 3 days after JH treatment. The rationale for this protocol was to check whether the response observed in the 20E injection experiments indeed represented a physiological effect of 20E. The JH dose used in this experiment elicits the physiological queen differentiation response characterized by an elevated ecdysteroid titer in the late spinning stages (Rachinsky et al., 1990; Rachinsky and Engels, 1995). 3. Results 3.1. Cloning and characterization of the honey bee transferrin gene To identify a transferrin-encoding gene in A. mellifera, we performed reverse transcription experiments on total RNA of non-pigmented pupae (Pw) using degenerate oligonucleotide primers corresponding to conserved transferrin motifs from other insects. Agarose-gel electrophoresis of PCR products generated by the primer combination TRF1 and TRF2 revealed a strong band of about 550 bp which was subcloned and sequenced. The 550 bp PCR fragment generated by this primer combination was also used as probe to screen a brain

kZAP cDNA library of A. mellifera. From about 60,000 recombinant phage plaques, more than 10 positive clones were identified and purified. Purified bacteriophage DNA of one of these clones was used as template for a new PCR with M13-forward and M13reverse universal primers that annealed to the bacteriophage sequence in the opposite insert flanking regions. A single PCR product of about 2.4 kb was purified, subcloned into a TOPO plasmid and used for sequence analysis by primer walking. In silico translation of the 2346 bp nucleotide contig obtained by assembly of the sequencing runs revealed the presence of an open reading frame (ORF) of 2136 bp. To determine the structural organization of the AmTRF gene, we identified the corresponding genomic sequence and compared it to the cDNA sequence. For this purpose, two specific primers, TRF3 and TRF4, were designed based on the cDNA sequence and these were used for subsequent PCR amplification with honey bee genomic DNA as template. The amplification resulted in a 3.1 kb fragment, which was subcloned and sequenced. Analysis by overlapping the genomic and cDNA sequences indicated that AmTRF consists of nine exons (Fig. 1) flanked by GT/AG exon/intron splicing sites typical for eukaryotes (Breathnach and Chambon, 1981). Genomic and cDNA nucleotide sequences for AmTRF from cloned fragments were deposited in a public database (GenBank accession numbers AY336529 and AY336528, respectively). The conceptual translation product of the AmTRF gene is a 686 amino acid polypeptide with an estimated molecular mass of 76 kDa and a pI value of 6.59 (calculated by using the Compute pI/Mw tool, http:// www.expasy.org/tools/pi_tools.html). It is preceded by a putative secretory signal peptide of 26 amino acids, as predicted by the SignalP www server (http:// www.cbs.dtu.dk/services/SignalP/). Blastp comparison of the deduced amino acid sequence of AmTRF obtained in the present study revealed a 100% match with the previously deposited AmTRF sequence (Kucharski and Maleszka, 2003). Since the latter sequence has been obtained by an independent approach that used a different cloning strategy, it is highly probable that transferrin is represented by a single transcript in the honey bee transcriptome. Blastx analysis of the AmTRF cDNA sequence revealed significant similarity to the transferrins of B. discoidalis (48%), M. darwiniensis (47%), R. clavatus (43%), M. sexta (42%), B. mori (42%), D. melanogaster (33%), and also to human transferrin (30%). Fig. 2 shows the respective sequence alignments indicating the presence of three conserved iron-binding domains located in the N-terminal lobe. The C-lobe does not contain such domains suggesting that AmTRF belongs to the monoferric group.

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transferrins contain two iron-binding sites, and thus form a distinct group of insect transferrins. 3.3. Transferrin expression during larval and pupal development

Fig. 1. Schematic representation of an Apis mellifera transferrin gene (AmTRF) and its deduced transcript. (A) Genomic architecture of the regions encoding honey bee AmTRF. A white reversed triangle denotes an initiation codon, and a black one indicates a termination codon. Exons are indicated by boxes and the positions of their respective initial and final nucleotides are shown below the exons. The direction of transcription is indicated by an arrow. (B) Deduced amino acid sequence. A bold line beneath the amino acid sequence represents the open reading frame of each exon. Protein, cDNA and genomic sequences of AmTRF are deposited in the GenBank database under accession numbers AAQ02339.1, AY336528 and AY336529, respectively.

3.2. Molecular phylogeny of insect transferrins To run a molecular phylogeny analysis using Mega 2.1, we included as outgroup an arthropod pacifastin heavy-chain precursor, a transferrin-like protein from the crustacean Pacifastacus leniusculus (Malacostraca) (Liang et al., 1997). The retrieved tree (Fig. 3) is based on 526 sites obtained by Neighbor Joining, and its significance was tested by 500 bootstrap repetitions. The two drosophilids plus S. peregrina group together as cyclorraphan dipterans and form a well-supported branch with the nematoceran A. aegypti. The closest phylogenetic relationship emerged for the lepidopteran transferrins (M. sexta and B. mori). The cockroach B. discoidalis and the termite M. darwiniensis constitute a third well-defined group. The other branches within this tree, which are less well supported, group the lepidopteran transferrins with AmTRF. Inclusion of the hemipteran R. clavatus within the holometabolan branch of this tree can be explained by the fact that the hemipteran transferrin is monoferric like the holometabolan transferrins, whereas cockroach and termite

The developmental profile of AmTRF expression in the larval and pupal stages was assessed by Northern blot analyses, starting with the second larval instar, and covering all the critical stages of the fifth larval instar and of pupal development. The Northern blots (Fig. 4) revealed a distinct pattern of AmTRF expression in the fifth larval instar with a maximum expression level right at the transition from the larval-feeding to the cocoonspinning phase. This is a developmentally decisive time point, as nurse bees will thereafter start to seal the brood cells. Subsequently, the expression level showed a marked decline towards the end of the spinning phase and appeared strongly diminished during the entire prepupal phase. AmTRF mRNA made a brief and distinct reappearance right after pupation, in the white-eyed pupae, before dropping again to basal levels for the rest of the pupal phase. When superposing the published ecdysteroid titer curves for honey bee workers on this expression profile (Fig. 4, redrawn from Feldlaufer et al., 1985; Rachinsky et al., 1990; Pinto et al., 2002), a downregulation of transferrin expression by ecdysteroids was strongly suggested. 3.4. Effect of experimental ecdysteroid titer manipulation on transferrin expression To confirm the putative down-regulation of AmTRF by circulating ecdysteroids, we performed two sets of experiments. In the first one, we directly injected 1 lg of 20E into the hemocoel of S1-stage larvae and analyzed the ecdysone response on transferrin expression 6 h later. The S1 stage was chosen because ecdysteroid titers in worker larvae are still low during this period, but soon thereafter will build up to form the prepupal peak, which marks its full presence approximately 36 h later (Rachinsky et al., 1990). Furthermore, S1 larvae are competent to respond to ecdysteroids, as previously shown by protein synthesis and gene expression studies (Hartfelder et al., 1995; Hepperle and Hartfelder, 2001). 20E-injected larvae showed a marked decrease in AmTRF expression when compared to untreated larvae and to larvae injected with honey bee saline (Fig. 5), providing evidence that the proposed negativeregulator hypothesis should indeed be correct. The 20E dose commonly used in such an experimental protocol, however, by far exceeds the endogenous ecdysteroid levels, even those found during the very high pupal peak. In strict pharmacological terms, this experiment thus only confirms an acute response in AmTRF expression to high exogenous hormone doses.

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Fig. 2. Alignment of AmTRF (AY336528) with Blaberus discoidalis (A47275), Manduca sexta (A36500), Drosophila melanogaster (AAC67389.1) and human transferrin (P02788) amino acid sequences. The colons and asterisks represent conserved residues and cysteins, respectively. Putative iron-binding residues are indicated by bold dots below the gray crossbars. Amino acids are numbered on the right margin.

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Fig. 2 (continued )

The second experiment was, therefore, specifically designed to discriminate possible pharmacological effects in our titer manipulation experiments from truly physiological effects of endogenous ecdysteroids. In this experiment, we applied JH to fourth instar worker larvae where it acts as a mediator and manipulator of prothoracic gland activity during the larval spinning stage. The application of JH to fourth instar larvae is an established protocol to elicit queen differentiation in honey bee larvae (for review, see Hartfelder and

Fig. 3. Molecular phylogeny of insect transferrins. ClustalW alignment of AmTRF cDNA sequence (AY336528) with transferrins sequenced for other insect species: Aedes aegypti (AAB87414.1), Blaberus discoidalis (A47275), Bombyx mori (AAD02419.1), Drosophila melanogaster (AAC67389.1), Drosophila silvestris (AAC77913.1), Manduca sexta (A36500), Mastotermes darwiniensis (AAN03488.1), Riptortus clavatus (AAD02419.1), and Sarcophaga peregrina (S68986). The pacifastin heavy-chain sequence, a transferrin-like protein from the crustacean Pacifastacus leniusculus (U81825) was used as outgroup to root the tree. Tree topology was calculated by using Mega 2.1 (Kumar et al., 2001), based on a pairwise distance matrix for Neighbor Joining. Tree branch significance was evaluated by 500 bootstrap repetitions.

Engels, 1998). An important mode of action of JH in this context appears to be its effect on ecdysteroid secretion by the prothoracic gland, leading to a queenlike ecdysteroid titer profile in these larvae (Rachinsky and Engels, 1995). In JH-treated larvae, the ecdysteroid titer forming the prepupal peak is already increasing in the late spinning stages (S2/3S3) similar to what is observed in natural queens, whereas in control worker larvae, it is still at basal levels during the S2/S3 stages. This JH application protocol thus manipulates the endogenous ecdysteroid titer within a physiological range, and based on JH metabolism calculations (Mane and Rembold, 1977), the experimentally increased JH titer during the fourth larval instar should have returned to normal levels until the late spinning stages. Interestingly, the results obtained in this experiment gave an even clearer picture, because the postu-

Fig. 4. Expression profile of AmTRF analyzed by Northern blot of total RNA (10 lg) from larvae and pupae. Total RNA of each developmental stage was probed with a AmTRF cDNA probe (550 bp fragment) and with a 28S-rDNA subunit probe for normalization. The ecdysteroid titer profile was redrawn based on data from Feldlaufer et al. (1985), Rachinsky et al. (1990) and Pinto et al. (2002) to fit the sequence of developmental stages presented in the Northern blots. L2, L3 and L4 represent second, third and fourth instar larvae, respectively; the fifth instar is subdivided into nine stages, with F1 to F3 being feeding stages, S1 to S3 the spinning stages and PP1 to PP3 the prepupal stages; the pupal phase is subdivided into seven stages according to progressive eye pigmentation (Pw to Pd) and subsequently by the degree of cuticle tanning (Pdl to Pdd); Pdd is the last pupal stage before ecdysis of the adult worker bee.

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Fig. 5. Down-regulation of AmTRF message in response to ecdysteroids. Relative quantification of AmTRF expression by Northern blot (with 20 lg of total RNA) and normalization to 28S rDNA expression levels for untreated S1-stage larvae (untreated), sham-control larvae that received a honey bee saline injection in S1 (ecdysone control), larvae that received an injection of 1 lg 20E in saline into the hemocoel in the S1-stage (20E injected in S1), larvae that received a JH application in the S1 stage (JH applied on S1), and larvae that had received a topical JH application (5 lg) in the fourth instar (JH applied in L4) to manipulate the endogenous ecdysteroid titer in S1. The strongest reduction in AmTRF expression in the S1-stage was observed when the endogenous ecdysteroid titer was increased in consequence of the JH treatment in the fourth instar.

lated queen-like (increased) ecdysteroid titer in the S2/S3 stage completely blocked transferrin transcription thus confirming the hypothesis that the hemolymph ecdysteroid titer de facto controls transferrin expression in honey bee postembryonic development. As a further countercheck to this experiment, we applied JH to S1-stage larvae to evaluate the effect of JH itself on AmTRF expression after a 6-h incubation. Even at the high concentrations of exogenous JH, AmTRF expression exhibited only a slight reduction when compared to untreated larvae. Thus, the elevated JH titer by treatment of S1-larvae cannot explain the observed down-regulation of AmTRF expression occurring in consequence of the JH application to fourth larval instar, and JH, therefore, seems to exert its effect primarily via an interendocrine modulation of the ecdysteroid titer.

4. Discussion 4.1. Structure of the honey bee transferrin gene and its position in a molecular phylogeny A PCR amplification approach using degenerate primers derived from conserved domains of insect transferrins permitted cloning of the complete coding sequence of A. mellifera transferrin. The cDNA sequence corresponds to the honey bee sequence recently annotated as honey bee transferrin by Kucharski and Maleszka (2003). Blastx comparisons and ClustalW alignment with other insect transferrins further confirmed this annotation, which is also supported by the protein molecular mass and isoelectric point calculations. In silico analysis of protein domains characterized AmTRF as one of the monoferric transferrins typical of holometabolan insects, with which it also grouped in

the deduced molecular phylogeny for the nine insect species for which transferrin sequences have been published. Even though this molecular tree is exclusively based on transferrin cDNA sequence information and also includes only nine insect species, the species clusters are congruent with general insect phylogeny. In the tree, AmTRF grouped with the lepidopteran transferrins, although the bootstrap value supporting this branch was fairly low. The Apis-Lepidoptera branch connects to the dipteran branch that also joins with the hemipteran R. clavatus. Obviously, the rather uncertain deep-level tree morphology (except for the clear separation of the lepidopterans, dipterans and the Dictyoptera/Isoptera branch) has to be attributed the low number of insect transferrin sequences available for the comparison. Molecular phylogenies that include the honey bee and that are based on single proteins or protein families have been presented for honey bee opsins (Mardulyn and Cameron, 1999), elongation factor-alpha (Danforth and Ji, 1998), the nicotinic acetylcholine receptor alpha 3 (Thany et al., 2003), vitellogenin (Piulachs et al., 2003), and in the most extensive form for the tetraspanin superfamily (Todres et al., 2000). All of these single protein or protein family trees did not produce congruent molecular phylogenies, yet rather reflect the evolutionary history of the individual proteins or protein family members. This factor is also well represented in the transferrin tree which shows a clear separation of the diferric transferrins (Dictyoptera and Isoptera), versus the transferrins of the other insect orders which, so far, are all described as monoferric. 4.2. Hormonal regulation of AmTRF expression and its putative role in postembryonic development When superposing the developmental profile of transferrin expression on the hemolymph ecdysteroid titer of honey bee worker larvae and pupae (Feldlaufer et al., 1985; Rachinsky et al., 1990; Pinto et al., 2002), a negative regulation of AmTRF expression by ecdysteroids became immediately apparent. We confirmed this hypothesis experimentally by two types of experiments where we manipulated the ecdysteroid titer during the stages of maximal AmTRF expression. Since the response obtained after 20E injection into spinningstage larvae could be attributed to pharmacological effects, we adopted a protocol that indirectly manipulates the ecdysteroid titer via a JH application. In this case, hormone intervention and sampling were separated by at least 6 days, permitting that the JH titer returns to physiological levels, yet exerting its effects on the ecdysteroid titer via an interendocrine pathway (Rachinsky and Engels, 1995). This clear evidence for an ecdysteroid control of AmTRF expression in the preimaginal stages of the

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honey bee perfectly explains the data presented by Kucharski and Maleszka (2003) who contrasted AmTRF expression in white-eyed pupae with a single late pupal stage. They hypothesized that AmTRF expression may be related to the metamorphic reorganization of the brain in this stage. We could now pinpoint the main player in this process, the pupal ecdysteroid titer, which goes through a minimum exactly during the white-eyed-pupal stage before the large and very broad pupal peak builds up (Feldlaufer et al., 1985; Rachinsky et al., 1990; Pinto et al., 2002). Furthermore, we observed the transient activation of AmTRF expression in white-eyed pupae in whole-body extracts, so this response may not be limited to the brain but rather could reflect a generalized metamorphic regulation of AmTRF expression. In distinction to ecdysteroids, JH has previously been reported to regulate transferrin expression. It stimulates transferrin expression in B. discoidalis and also in R. clavatus (Jamroz et al., 1993; Hirai et al., 2000), whereas in adult workers of the honey bee it appears to have a slightly repressive effect on AmTRF (Kucharski and Maleszka, 2003). For the larval–pupal transition in honey bees, we could detect a similar, slightly repressive mode of action of JH when it was topically applied to spinning-stage larvae. Yet in contrast to ecdysteroids, the effect of on AmTRF transcript levels was clearly only a minor one. Most of its modulatory effect may thus be attributable to an interendocrine effect on the hemolymph ecdysteroid titer. At present, we cannot answer the question whether this negative regulation by ecdysteroids observed in honey bee metamorphosis is an isolated case, as these life stages have not been studied for other insects in this context. The overexpression of honey bee transferrin at the transition from the larval feeding to the cocoon-spinning phase and the subsequent ecdysteroiddependent drop in transferrin expression during the prepupal and pupal stages may, however, represent a general element in insect metamorphosis, possibly related to defense mechanisms. The putative role of transferrin in the insect defense system against pathogens and endoparasites is supported by its upregulation in response to bacterial infection observed in several insect species (Yoshiga et al., 1997, 1999), including the honey bee (Kucharski and Maleszka, 2003). In this respect, the observed high expression levels for transferrin at the end of the larval feeding period and the transition to the spinning phase in A. mellifera larvae (F3/S1 stage) become meaningful. The brood cells are sealed in this developmental stage and the larvae will no longer benefit from grooming by the nurses. Overexpressing proteins with antibiotic roles during this particular time period may thus contribute to guarantee survival of honey bee larvae at the onset and in preparation for metamorphosis.

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The present study provides strong evidence and, in fact, is also the first one to report a negative regulation of transferrin expression by ecdysteroids. This result adds to the already controversial literature on the involvement of ecdysteroids in the insect immune response system. Expression of the diptericin gene, for instance, has been shown to increase in Drosophila larvae fed with an ecdysone-containing diet (Meister and Richards, 1996). This contrasts with the expression pattern of diptericin and other antibacterial peptides in the pupal stages. In Calliphora vicina, their expression turned out to be down-regulated by the elevated pupal 20E levels, and when ecdysone was co-injected with bacteria in diapausing Calliphora pupae, the level of induced antibacterial activity was reduced (Chernysh et al., 1995). The elucidation of the role and mode of action of ecdysteroids in the insect defense system against pathogens and parasites thus will require further efforts. These will mainly have to discern the ecdysone response cascades set in action in each case, since, as suggested by Meister and Richards (1996), the ecdysone effect on immune gene induction may involve a regulatory pathway different from the ecdysone response cascade for normal development.

Acknowledgements We wish to thank Luı´z Roberto Aguiar for technical assistance in the apiary and Anete Pedro Lourenc¸o for providing the cDNA samples used in the initial RT-PCR assays. A brain kZAP cDNA library of Apis mellifera was kindly provided by Dr. Gene Robinson (University of Illinois). V.C.-H. was supported by a fel´ trange`res— lowship from Ministe`re des Affaires E France (Bourse Lavoisier). This research was supported by FAPESP (99/00719-6).

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