©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
cDNA Sequence and Expression of the Ceratotoxin Gene Encoding an Antibacterial Sex-specific Peptide from the Medfly Ceratitis capitata (diptera) (*)

(Received for publication, November 11, 1994)

Daniela Marchini (§) Andrea G. O. Manetti Marco Rosetto Luigi F. Bernini (1) John L. Telford (2) Cosima T. Baldari Romano Dallai

From the  (1)Department of Evolutionary Biology, University of Siena, Via P. A. Mattioli 4, I-53100 Siena, Italy, the Department of Human Genetics, University of Leiden, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands, and the (2)Immunobiology Research Institute Siena, Via Fiorentina 1, I-53100 Siena, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ceratotoxins are antibacterial 3-kDa molecular mass amphiphilic peptides isolated from the female reproductive accessory glands of the medfly Ceratitis capitata. They are physiologically related to bee melittin and show amino acid sequence homology with magainin peptides. In this paper, we report the complete sequence of cDNA coding for ceratotoxin A and the expression of the gene during the life cycle of the insect. Experimental data show that the ceratotoxin is a gene expressed exclusively in the imaginal stages and that it is female-specific, related to sexual maturity, and stimulated by mating. Differently from most antibacterial insect hemolymph peptides, it is not induced by microbial infection. Western blot analysis using an anti-ceratotoxin antibody indicates the female accessory glands as the only site where the production of the ceratotoxin peptide occurs.


INTRODUCTION

A large number of antimicrobial peptides have been isolated from both vertebrates and invertebrates. Most of them can be classified into a few major groups based on common sequences, secondary structure, and mechanism of action (for reviews, see (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) ). Some peptides, such as cecropin, sarcotoxin, magainin, and melittin, are characterized, in addition to a strong basicity, by a molecular mass of 2-4 kDa and an amino acid sequence that allows folding into amphiphilic helixes (11, 12, 13, 14) . These structures disrupt prokaryotic and, in some instances, eukaryotic membranes through the formation of ion channels(15, 16, 17) .

Antibiotic peptides are produced as a barrier against infections and may be constitutively expressed or induced in response to endogenous and/or exogenous stimulations or triggered by microorganisms. Constitutively expressed and neuroendocryn/injury-stimulated peptides such as seminalplasmin and magainins (18, 19) are reported more frequently among vertebrates, although in some cases peptides elicited by traumas have been described in insects(20, 21, 22) , and a constitutively expressed defensin-like peptide from a scorpion has also been described(23) .

Most insect antimicrobial peptides are induced by bacterial infection and accumulate in the hemolymph as effectors of the humoral immune response. These include cecropins, the cysteine-containing defensins, sapecins and royalisin, proline rich apidaecins and drosocin, glycin rich attacin-like proteins attacins, sarcotoxin II, diptericins and coleoptericin, and lysozyme (for reviews, see (8) and (24) ). However, a gene encoding a male-specific, antibacterial peptide from Drosophila melanogaster has been shown to be constitutively expressed in the ejaculatory duct(25) . Moreover, an insect toxin, melittin(26) , recently demonstrated to have an antimicrobial spectrum (27, 28) , is constitutively secreted by the honeybee venom gland, which interestingly is also associated with the reproductive organs(29) .

We have recently purified two 3-kDa antibacterial peptides from the medfly Ceratitis capitata, which we named ceratotoxins A and B(30) . These peptides, isolated from the female reproductory system, appeared also constitutively produced(31) . Amino acid sequence determination revealed primary structure homology with magainins (30) and the possibility of their folding into amphiphilic helixes(30) .

Here we report the sequence of cDNA coding for ceratotoxin A peptide and an analysis of its expression. We show that ceratotoxin is specifically expressed in the accessory glands of sexually mature females and that its production is not induced by microbial infections but is enhanced by mating.


MATERIALS AND METHODS

Bacterial Strains and Media

Escherichia coli LE 392 was used for the induction experiments and antibacterial assays of ceratotoxin. The culture medium was LB (32) supplemented with 0.7% agarose when solid medium was required.

Insects

Ceratitiscapitata flies were reared in standard laboratory conditions at 23 °C, 70% relative humidity and a 14:10 light-dark regime(33) .

RNA Preparation and Analysis

RNA was extracted from both whole flies and dissected reproductive accessory glands of sexually mature females. The material, immediately frozen in liquid nitrogen and stored at -80 °C, was homogenized using a Polytron homogenizer (Kinematica AG) and extracted as described by Chomczynski and Sacchi(34) . Poly(A) RNA was purified on oligo(dT) cellulose (Boehringer Mannheim).

Construction and Screening of Medfly cDNA Libraries

1 µg of poly(A) RNA from female reproductive accessory glands was used for cDNA synthesis using the cDNA synthesis system kit (Amersham Corp.). cDNA was ligated with gt11 vector using the gt11 cDNA cloning system (Amersham Corp.), and packaged with the in vitro packaging kit (Amersham Corp.). The probe for cDNA library screening was prepared by polymerase chain reaction with 100 ng of first-strand cDNA from whole female flies. Degenerate primers were designed by backtranslation of the amino acid sequence of ceratotoxin A (30) . The nucleotide sequence for the 5` primer was deduced by backtranslation from Pro (11) to Ile (16) of the mature peptide for the 3` primer from Ala (27) to Pro(22) . The primer sequences were 5`-CCAGAATTCCC(G/A/T/C)GT (G/A/T/C)GC(G/A/T/C)AA(G/A)AA(G/A)AT-3` and 5`-GACAAGCTTGC(G/A/T/C)GC(C/T)TT(G/A/T/C)GC(G/A/T)AT(G/A/T/C)GG-3` for 5` and 3` primer, respectively. 35 cycles of polymerase chain reaction were performed with 1 min at 94 °C, 1 min at 48 °C, and 1 min at 72 °C with 4 µM primers and 1 mM MgCl(2). The resulting 63-base pair fragment was cloned into the Bluescript plasmid vector (Promega), and its nucleotide sequence was determined by the dideoxynucleotide chain termination method using the Sequenase kit (U. S. Biochemical Corp.). It was then reamplified using perfectly matching primers, end-labeled with [P]ATP, and used to screen the cDNA library as described(32) . The probe used for the Northern blot analysis was a ceratotoxin cDNA clone, P-labeled by random priming using the Prime-a-gene kit (Promega). Hybridization was performed overnight at 60 °C in 1 times Denhardt's solution, 2 times SSC, 50 µg/ml salmon sperm DNA.

Nucleotide Sequence Determination and Analysis

Positive recombinant phages were plaque-purified, and DNA was extracted according to Sambrook et al.(32) . cDNA inserts were separated by 1.2% low temperature melting agarose gel electrophoresis after digestion with EcoRI, ligated to the Bluescript vector, and sequenced on both strands. The nucleotide sequence was determined by the chain termination method (35) using the Sequenase kit from U. S. Biochemical Corp. DNA sequence analysis was done on a VAX computer with the GCG Package from the University of Wisconsin(36) .

Primer Extension

For the primer extension reaction, we used 200 ng of [P]ATP end-labeled oligonucleotide (5`-GCAGCAGGTTCGGCTACTACGCATT-3`) corresponding to amino acids 27-19 of ceratotoxin cDNA and 2 µg of poly(A) RNA. The sample was dried in a Speedvacuum centrifuge (Savant), resuspended in 1 times avian myeloblastosis virus buffer, and then annealed for 1 min at 90 °C and for 45 min at 45 °C. The sample, with added dNTPs (up to 400 µM final concentration) and avian myeloblastosis virus reverse transcriptase (Promega), was incubated for 1 h at 45 °C. After stopping the reaction with 95% formamide, 20 mM EDTA, the sample was denatured for 3 min at 95 °C and loaded on a 6% polyacrylamide gel containing 7 M urea.

Northern Blot Analysis

RNA was subjected to electrophoresis in a denaturing formaldehyde gel system(32) , blotted overnight on Bioblot-NC (Costar) nitrocellulose filters, and hybridized overnight at 65 °C in 1 times Denhardt's, 2 times SSC, 50 µg/ml salmon sperm DNA, with the P-labeled ceratotoxin cDNA as a probe.

Protein Assay

The protein assay was performed according to Bradford(37) , using as standard bovine serum albumin.

Immunization of Rabbits with Ceratotoxin A

1 ml of phosphate-buffered saline containing 0.5 mg of ceratotoxin A (synthesized by Multiple Peptide System, San Diego, California) was thoroughly mixed with 1 ml of complete Freund's adjuvant. The emulsion was injected into six subcutaneous sites of the axial lymph node regions. The immunization was repeated 4 weeks later with 250 µg of peptide in incomplete Freund's adjuvant. Rabbits were bled 5 weeks after the first injection. Following the initial immunization, the animals were boosted with 250 µg of peptide at 5-week intervals, and bled 1 week after the last injection. Sera were tested for antipeptide activity by a standard two-step enzyme-linked immunoassay, using the preimmune serum as control.

Electrophoresis, Western Blot Analysis, and Antibacterial Assay

SDS-polyacrylamide gel electrophoresis was carried out on a 20% polyacrylamide gel according to Laemmli(38) . Samples were homogenized using a 100-µl glass homogenizer in dissociation buffer (38) , boiled for 5 min, and spun at 10,000 times g for 10 min to remove the debris. Low molecular weight markers were purchased from Promega. Synthetic ceratotoxin A (synthesized by Multiple Peptide System, San Diego, California) was also used as marker. Proteins were transferred onto a nitrocellulose filter (Bioblot-NC, Costar) according to Towbin et al.(39) . After Ponceau staining and incubation with phosphate-buffered saline containing 3% bovine serum albumin and 0.1% Triton X-100 (PBSAT) for 30 min, the filters were incubated overnight with a rabbit anti-ceratotoxin antiserum, diluted 1:200 in PBSAT. After three washes with PBSAT, the second antibody (goat anti-rabbit IgG, 1:1000 dilution, horseradish peroxidase-conjugated, (Cappel) was added and incubated for 1 h at room temperature. The color reaction was developed by using 4-chloro-1-naphthol (Merck) in 50 mM Tris-HCl at pH 6.8 and stopped by the addition of H(2)O. Antibacterial activity of the gel-separated proteins was assayed by overlaying the gel slab with an E. coli culture (10^5 cell/ml) in LB-agarose according to Hultmark et al.(40) after renaturation of the gel as reported by Saul et al.(41) .

Insect Treatment and Immunization

We set up a new protocol to obtain flies with a reduced bacterial flora for the immunization assays. Based on the assumption that the accessory gland secretion, containing the ceratotoxin, is spread over the eggs during oviposition and is found on their surface after laying, (^1)the eggs should be protected against bacteria. Nevertheless, in the laboratory rearings, contamination with the fecal content occurs during the collection of the eggs so that the antimicrobial substance on the eggs is not effective enough to be bactericidal. We decided to use such antimicrobial substance after a heat treatment in order to obtain at the same time a partial purification of the heat stable ceratotoxin and a killing of most of the bacteria present on the egg surface. After 3 h of precollection, the eggs were transferred to a tube and added with 0.1 M sodium phosphate buffer (PB) at pH 6.8 (250 mg of eggs/ml) and gently stirred by hand for 3 min at room temperature to solubilize the egg surface substance. After decanting of the eggs, the supernatant was boiled for 5 min and centrifuged at 10,000 times g for 10 min. The supernatant (270 µg/ml total protein), containing the ceratotoxin, was used to treat for 20 min 125 mg of eggs, which had been washed 3 times with 15 ml of sterile PB. The eggs were seeded on Petri dishes containing autoclaved larval food (yeast/sugar/agar). Although control experiments carried out spreading eggs on a LB-agar Petri dish showed no bacterial colony forming units after overnight incubation at 37 °C, we failed to obtain axenical adults. However, the bacterial flora was sensibly reduced with respect to the adults from nontreated eggs. To induce an immune response, sexually mature adults from eggs treated as above were cold anesthetized and punched with a needle dipped into an overnight culture of E. coli. At different times after the injection, the flies were frozen in liquid nitrogen and the RNA was extracted for the Northern blot analysis. A batch of noninjected flies was used as control.

Electron Microscope Analysis

Females of C. capitata of different ages (newly emerged up to 40 days old) were dissected, and the accessory glands were removed in 0.1 M PB at pH 7.2 as described previously(42) . The material was fixed in 3% glutaraldehyde in PB added with 3% sucrose for 1 h at 4 °C and postfixed with 1% OsO(4) in the same buffer for 1 h and 30 min at 4 °C. After careful rinsing in PB plus 3% sucrose, the glands were dehydrated in a graded ethanol series and embedded in TAAB 812 resin (TAAB Laboratories Equipment Ltd). Thin sections were obtained with a LKB ultramicrotome, stained with uranyl acetate and lead citrate, and observed with a Philips CM 10 transmission electron microscope.


RESULTS

Cloning and Analysis of Ceratotoxin cDNA

We constructed a probe for ceratotoxin gene sequences by polymerase chain reaction using a mixture of primers corresponding to all possible code degenerations for amino acids 11-16 and 22-27, respectively, of ceratotoxins(30) . Reverse transcriptase polymerase chain reaction on poly(A) RNA extracted from adult females resulted in a 63-base pair fragment that was identified by sequencing as the expected cDNA fragment encoding amino acids 11-27 of ceratotoxin A. The polymerase chain reaction product was reamplified using perfectly matching primers and used as probe to screen a medfly gt11 cDNA library constructed from poly(A) RNA extracted from adult female accessory glands. Screening of about 8 times 10^3 plaques allowed us to identify 13 positive clones and to determine the nucleotide sequence of 11 of them. The nucleotide sequence of ceratotoxin A cDNA and its deduced amino acid sequence are presented in Fig. 1A. All sequenced clones lack 5` untranslated sequences. However, as shown in Fig. 1B, primer extension analysis of the mRNA identifies the transcription start site 12 bases upstream of the open reading frame. The sequence contained a single long open reading frame capable of coding for a precursor protein of 71 amino acids. Amino acids 36-64 of the precursor were identical to the previously published sequence of ceratotoxin A. The longest cDNA clones contained 93 base pairs after the stop codon followed by a series of thymidines, which presumably represents the poly(A) tract from the message RNA.


Figure 1: A, nucleotide sequence of a ceratotoxin cDNA clone from the medfly Ceratitis capitata. The deduced amino acid sequence of the open reading frame is shown below the nucleotide sequence. A polyadenylation signal is underlined. The arrows indicate the N and the C termini of the mature peptide, respectively. The arrowhead indicates the putative cleavage site by a signal peptidase (sp). B, determination of the transcription start site of the ceratotoxin gene by primer extension (arrow). The size of the extension product is 67 bases. A, 2 µg of poly (A) RNA. Asterisk indicates the 5` end of the ceratotoxin cDNA clone. C, deduced amino acid sequence of the ceratotoxin A (ctxA). The variations are marked below the sequence. The amino acid sequence of the putative ceratotoxin B (ctxb) derived from the truncated cDNA clone is also shown. Only amino acids differing from those encoded by ceratotoxin A cDNA are shown. Underlined amino acid residues indicate nucleotide substitution at the third base of the codon. Asterisk indicates amino acid residue deletion at position 47. Dashes indicate nonsequenced regions.



Microheterogeneity was observed in the cDNA sequences, and a second form of the precursor protein could be deduced, which had a phenylalanine at position 18 and an isoleucine at position 31 of the prepro region of the precursor (Fig. 1C). We also found a single truncated cDNA that could code for the ceratotoxin B, which differs from ceratotoxin A at 2 amino acids. The cDNA also varied at three other positions in the prepro region of the precursor and lacked a valine at position 47 (Fig. 1C).

Ceratotoxin Gene Expression during the Life Cycle of C. capitata

Ceratotoxin was first identified as an antibacterial activity, which could be purified from the accessory glands of the female reproductory apparatus from 5-20-day-old sexually mature females(31, 30) . However, it was not clear from these data whether production of the peptide was restricted to this site. To answer this question, we analyzed the spatial and temporal expression of ceratotoxin using a polyclonal antibody raised against synthetic ceratotoxin A. The antiserum was shown to specifically recognize the synthetic ceratotoxin itself, which comigrates with the biologically active, antibacterial peptide extracted from sexually mature female accessory glands (Fig. 2). In spite of the structural and sequence homology with cecropin peptides (for review, see (24) ) including cecropins from C. capitata(43) , no cross-reaction could be detected with Drosophila cecropin A (44) (Fig. 2).


Figure 2: A, protein staining (Ponceau) and (B) immunoblot of the same filter using anti-ceratotoxin antiserum from a SDS-polyacrylamide gel electrophoresis loaded with 2.8 µg of synthetic D. melanogaster cecropin A (cec) and synthetic ceratotoxin A (ctx). The anticeratotoxin antibody recognizes exclusively the ceratotoxin peptide. C, SDS-polyacrylamide gel electrophoresis overlaid with viable E. coli LE 392 to detect antibacterial activity. G, whole accessory glands; GS, gland secretion; BGS, boiled gland secretion (supernatant). The material loaded on each lane was from 8 accessory glands (four insects). The inhibition growing zone is compatible with the molecular size of the ceratotoxin.



Fig. 3A shows a Western blot analysis of proteins extracted from the reproductive apparatus of females and males at different stages of their adult life using this antibody. No ceratotoxin could be detected in males at any stage examined and in just emerged females, whereas it was abundantly present both in 10-day and 40-day adult females. A lower expression could be observed in ageing females with respect to young females. The antibody weakly recognized also a protein of about 26 kDa (Fig. 3), showing no antibacterial activity (see Fig. 2C), which will be investigated elsewhere.


Figure 3: A, Western blot analysis of ceratotoxin in the reproductive apparatuses of C. capitata. Each lane was loaded with material extracted from five flies. M and F indicate males and females, respectively. 1, 10, and 40 indicate the age of the flies (in days). Ovaries were removed from reproductive apparatus of 10- and 40-day-old females in order to avoid overloading of material on the gel. ctx, 2.8 µg of synthetic ceratotoxin A. The position of molecular size markers expressed in kDa is indicated. B, Western blot analysis of ceratotoxin in accessory glands (G), vaginae plus spermathecae (VS), and ovaries (OV) from 10-day-old females (five flies). Y, whole reproductive apparatus from 25 just emerged females.



The sex specificity and the restriction of ceratotoxin expression to the female adult sexually mature stages was confirmed by Northern blot analysis of total RNA extracted from whole female and male flies at different stages of the adult life, extending the experiment also to the preimaginal stages (embryos, larvae, and pupae). As shown in Fig. 4, ceratotoxin gene expression was not detectable at any stage of the male adult life cycle, the preimaginal stages, or in just emerged females. However, a high level of ceratotoxin gene expression was observed in sexually mature females. As in the Western blot analysis, expression levels were shown to decrease in ageing females (Fig. 4).


Figure 4: Northern blot analysis of ceratotoxin gene expression during the life cycle of the medfly. E, embryos at 12 and 24 h; L, first, second, and third instar larvae; P, 5 and 10-day-old pupae; A, just emerged (je) and sexually mature 6 and 40-day-old males (M) and females (F). 30 µg of total RNA were used for each sample. The arrow indicates 18 S RNA.



We tested different organs of the reproductive apparatus of Ceratitis sexually mature females to understand whether accessory glands are the only site of ceratotoxin production. Proteins extracted from spermathecae, vagina, and ovaries in addition to the accessory glands were analyzed by Western blot for the presence of ceratotoxin. As shown in Fig. 3B, the accessory glands appear to be the only site of ceratotoxin production.

Morphological Analysis of the Accessory Gland Maturation

The expression pattern of ceratotoxin in the accessory glands of C. capitata was compared with the secretion cycle of the accessory glands at different stages of the female sexual maturity. Fig. 5shows the ultrastructure of the accessory glands in just emerged (A), 3-day-old (B), and 10-day-old (C) females. The epithelium of the glands is formed by two types of cells as in the insect type 3 ectodermal glands(45) . Large secretory cells, provided with an extracellular central cavity lined by microvilli, are in continuity with the gland lumen through an efferent duct produced by flat duct-forming cells, which are disposed beneath the cuticle surrounding the gland lumen(46) . In newly emerged females, the accessory glands are small and devoid of luminal secretion. The cytoplasm of secretory cells is reduced, and only a few cisterns of endoplasmic reticulum and Golgi apparatus are visible. No sign of secretory activity is evident and in the central cavity, devoid of secretion, only the fibrous material forming the end apparatus is visible (Fig. 5A). In 3-4-day-old females, the accessory glands enlarge, but no large amounts of secretion are yet accumulated. However, the secretory cells begin to be active, and small drops of electron-dense material can be detected in the central cavity (Fig. 5B). In 5-10-day-old females, the accessory glands reach their maximal size. A dense, transparent secretion has accumulated in the gland lumen, and it can easily flow out when glands are dissected. The secretory cells are enlarged, and their cytoplasm is rich in rough endoplasmic reticulum and Golgi systems. The central cavity is very expanded and filled with electron-dense homogenous material that passes through the efferent duct and flows into the gland lumen (Fig. 5C). In 10-40-day-old females, the accessory glands do not change substantially in size. However cells at different stages of secretion and some degenerated cells are visible within the epithelium (not shown). Thus the pattern of expression of ceratotoxin at different stages of the female adult life parallels the pattern of maturation of the accessory glands.


Figure 5: Electron microscope analysis of female medfly accessory glands in just emerged (A), 3-day-old (B), and 10-day-old (C) females. Note the lack of dense secretion in (A) and different amount of content (B and C) in the extracellular central cavity (ex) of the secretory cell at different stages of maturation. mv, microvilli; ed, efferent duct; L, gland lumen. Bar = 1 µm.



Ceratotoxin Gene Expression Is Not Induced by Bacterial Infection

With the exception of andropin (25) and possibly melittin(26) , all known insect antibacterial peptides are induced by bacterial infection(8) . Although ceratotoxin appears constitutively produced in adult females, we could not rule out a modulation of its expression by bacterial infection. To answer this question, we injected sexually mature axenically grown females with an E. coli LE 392 suspension and collected them 6 and 12 h after infection. Noninfected flies from the same batch were used as control. The result of a Northern blot analysis of ceratotoxin gene expression is presented in Fig. 6. No difference could be detected between noninfected and infected flies (Fig. 6A). On the other hand, as expected, cecropin expression, analyzed on the same blot using C. capitata cecropin 1 (43) as a probe, was barely detectable in noninfected flies but was strongly induced at 6 h and began to decrease at 12 h (Fig. 6B).


Figure 6: A, analysis of ceratotoxin mRNA in infected flies (I). Uninfected flies (U) were frozen at the same time as the flies frozen at 6 h after injection. All of the flies were sexually mature (6-day-old) females. Flies were injected with a bacterial suspension and collected 6 and 12 h after injection. 50 µg of total RNA were used for each hybridization. B, the same blot was probed with a C. capitata cecropin 1 cDNA clone(43) .



Ceratotoxin Expression Is Enhanced by Mating

To establish whether ceratotoxin, like the male-specific peptide andropin in Drosophila(25) , was induced by mating, we isolated 60 female flies immediately after eclosion and kept them separated from males until they reached sexual maturity. 40 of them were then singly mated with males of the same batch and collected at 3 and 12 h after mating. The results of a Northern blot analysis of ceratotoxin gene expression in the mated and nonmated females are presented in Fig. 7A. Ceratotoxin expression increased 3 h after mating and then showed a slight decrease at 12 h. Probing the same filter with a probe specific for C. capitata beta-tubulin(^2)confirmed that equivalent amounts of RNA were present in each lane. In addition, no difference was detected among the samples when the blot was probed for C. capitata cecropin 1 mRNA (43) (data not shown).


Figure 7: A, Northern blot analysis of the induction of ceratotoxin mRNA in response to mating. VR, sexually mature (6-day-old) virgin females; MT, females 3 and 12 h after mating. B, Western blot analysis of ceratotoxin expression in response to mating. VR, MT, 7-day-old virgin, and mated females, respectively; ctx, synthetic ceratotoxin A (2.8 µg).



The induction of ceratotoxin expression in response to mating was confirmed by Western blot analysis of accessory glands from virgin and mated 7-day-old females, where significantly more ceratotoxin was found in mated compared with virgin flies (Fig. 7B).


DISCUSSION

In this paper we report the cDNA sequence and the expression of the ceratotoxin A gene encoding an antibacterial peptide from the medfly C. capitata.

The single long open reading frame is capable of coding for a 71-amino acid polypeptide containing the mature ceratotoxin A sequence between amino acids 36 and 64. The mature peptide starts after a putative proteolytic cleavage site immediately preceding the lysine-arginine dipeptide at position 34-35 as reported for the processing of the precursors of magainins (47) and other bioactive peptides including hormones(48) . Moreover the preprosequence shows a potential cleavage site of the signal peptide after position 23, according to von Heijne (49) . Starting from this site, the liberation of the mature ceratotoxin from the prosequence could also occur via cleavage of dipeptides by a dipeptidylaminopeptidase, in agreement with the experimental data on the enzymatic processing of melittin precursor(50) , which has a similar length to ceratotoxin. A motif of 5 amino acids (EPAAE) at position 24-28 recalls similar sequences (EPXAE) in the propeptides of the apidaecins (51) and melittin(50) . Moreover, the EP dipeptide is also present in the prosequence of cecropins A and B from Hyalophora cecropia(52, 7) which, however, is substantially different in length from that of ceratotoxin. Since the 7-amino acid hydrophobic tail is present in the translational product of the cDNA of ceratotoxin but not in the purified peptide, a post-translational enzymatic processing at C terminus can be hypothesized, as occurs for the removal of the tetrapeptide of the attacins (53) and the dipeptide of cecropin D from Hyalophora cecropia(7) . However, as no protease inhibitors were used during the preparation of the material for the purification of the ceratotoxin (30) , we cannot exclude carboxyl-terminal loss of amino acids, as has been reported by Thompson et al.(54) during the purification of the pardaxins defence peptides from the sole Pardachirus pavoninus.

We are working, at the present, on the genomic organization of the ceratotoxin gene in order to explain whether the microheterogeneity observed in the cDNA sequences reflects genetic polymorphism or whether the ceratotoxin genes are repeated. However, most antimicrobial peptides are coded for by multigene families, and it has been suggested that the respective peptides have evolved through a series of gene duplications(7) . Multigene families have been described for cecropins (44, 55, 56) , attacins(53) , apidaecins(51) , and caerulein precursor (57) .

In addition to the strong homology displayed by the ceratotoxin mature peptide to one of the caerulein precursor fragment peptides from Xenopus laevis(19, 30) , the putative ceratotoxin precursor shows a considerable amino acid sequence homology (70% similarity and 46.66% identity in 32 amino acids) with dermaseptin, another antimicrobial peptide secreted by the skin of the amphibian Phyllomedusa sauvagei(58) (Table 1). Moreover, similarities among these peptides are found in the alpha-helix secondary structure, also demonstrated for ceratotoxin by circular dichroism spectra (^3)in addition to the previous theoretical predictions(30) .



Analysis of ceratotoxin expression during the life cycle of the medfly both at the transcriptional and translational levels confirms that this protein is sex-specific and is localized exclusively in the accessory glands of the reproductive apparatus of sexually mature females. Moreover, the expression decreases with the age of the females.

Ceratotoxin is the first female-specific gene demonstrated to be related to the reproductive apparatus of insects. Interestingly, the honeybee venom peptide, melittin, also has antibacterial activity and is produced in the venom gland, which is a modified reproductive accessory gland(29) . The male-specific antibacterial peptide andropin in Drosophila may share a common function with ceratotoxin in protection of the reproductive tract from bacterial invasion in males or females, respectively. This may be important if the presence of bacteria could indirectly interfere with sperm motility and fertilization. However, as ceratotoxin was also found on the surface of laid eggs,^1 we can speculate on a possible role of ceratotoxin in creating a microbiologically controlled oviposition environment that favors early larval development. Work is in progress to verify this hypothesis, which could have importance for the biological control of the medfly, which represents a serious agricultural pest.

Our investigations on possible mechanisms of induction of ceratotoxin expression demonstrated that the ceratotoxin gene is not induced by bacterial infection and/or injury, as most insect antibacterial peptides including cecropins from C. capitata (present work). Since ceratotoxin is a sex-specific peptide, we investigated mating as a possible mechanism of induction of ceratotoxin expression. Our results indicate that ceratotoxin mRNA production, also detectable in virgin females, is increased by mating. The analysis of the ceratotoxin peptide in virgin and mated females at the beginning of sexual maturity also showed an increased production of peptide in the accessory glands of mated females. The increase in expression of ceratotoxin on mating may be due to the transfer of a factor to the female from the male. It is known that during mating males transfer factors to the females inhibiting receptivity and/or inducing egg laying(59, 60) . Factors of this nature could be responsible for ceratotoxin gene regulation directly or by enhancing juvenile hormone production. Moreover, direct transfer of male accessory gland juvenile hormone to females by mating has been suggested in Aedesaegypti(61) . Although this gonadotropic hormone seems unable to induce the male-specic andropin gene(25) , our data suggest an involvement of juvenile hormone in the ceratotoxin expression, since it parallels accessory gland and ovary maturation (present work and (46) ).


FOOTNOTES

*
This work was supported by the National Research Council of Italy (CNR), special project RAISA, subproject 2, paper 2051. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L34403[GenBank].

§
To whom correspondence should be addressed. Tel.: 39-577-298918; Fax: 39-577-298898; MARCHINID{at}unisi.it.

(^1)
D. Marchini, A. G. O. Manetti, M. Rosetto, L. F. Bernini, J. L. Telford, C. T. Baldari, and R. Dallai, unpublished results.

(^2)
A cDNA clone from a medfly library.

(^3)
L. Ragona, H. Molinari, L. Zetta, R. Longhi, D. Marchini, R. Dallai, L. F. Bernini, L. Lozzi, M. Scarselli, and N. Niccolai, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Dan Hultmark for the helpful discussion and for the gift of the cecropin A-amide from Drosophila, chemically synthesized by Åke Engström, Department of Immunology, University of Uppsala. We also thank Dr. Laura Marri, Department of Molecular Biology, University of Siena, for the suggestion of the protocol for the SDS-polyacrylamide gel electrophoresis renaturation.


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