(Received for publication, November 11, 1994)
From the
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.
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.
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).
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.
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.
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) .
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).
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
-helix secondary structure, also demonstrated for ceratotoxin by
circular dichroism spectra (
)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, 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) ).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L34403[GenBank].