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(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
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
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, 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].
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 6199-6204
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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
. 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 
Denhardt's solution, 2
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 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 Denhardt's, 2
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 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 HO.
Antibacterial activity of the gel-separated proteins was assayed by
overlaying the gel slab with an E. coli culture (10
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, (
)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 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
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.
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 10
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.
,
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.
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).
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.
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).
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 
-tubulin
()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).
-helix secondary structure, also demonstrated for ceratotoxin by
circular dichroism spectra ()in addition to the previous
theoretical predictions(30) .
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.
)))
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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