Ceratotoxins: female-specific X-linked genes from the medfly, Ceratitis capitata.

In this paper, we report the chromosomal localization of ceratotoxins, a gene family encoding antibacterial female-specific peptides from the mediterranean fruit fly Ceratitis capitata. The analysis of both polytene and mitotic chromosomes by in situ hybridization shows that ceratotoxins are the first case of female-specific X-linked genes from the medfly C. capitata. Southern blot analysis reveals that the ceratotoxin gene family is not specifically amplified in the female reproductive accessory glands of C. capitata.     

The ceratotoxin gene family in the medfly Ceratitis capitata and the Natal fruit fly Ceratitis rosa (Diptera: Tephritidae)

Ceratotoxins (Ctxs) are a family of antibacterial sex-specific peptides expressed in the female reproductive accessory glands of the Mediterranean fruit fly Ceratitis capitata. As a first step in the study of molecular evolution of Ctx genes in Ceratitis, partial genomic sequences encoding four distinct Ctx precursors have been determined. In addition, anti-Escherichia coli activity very similar to that of the accessory gland secretion from C. capitata was found in the accessory gland secretion from Ceratitis (Pterandrus) rosa. SDS-PAGE analysis of the female reproductive accessory glands from C. rosa showed a band with a molecular mass (3 kDa) compatible with that of Ctx peptides, also slightly reacting with an anti-Ctx serum. Four nucleotide sequences encoding Ctx-like precursors in C. rosa were determined. Sequence and phylogenetic analyses show that Ctxs from C. rosa fall into different groups as C. capitata Ctxs. Our results suggest that the evolution of the ceratotoxin gene family might be viewed as a combination of duplication events that occurred prior to and following the split between C. capitata and C. rosa. Genomic hybridization demonstrated the presence of multiple Ctx-like sequences in C. rosa, but low-stringency Southern blot analyses failed to recover members of this gene family in other tephritid flies.     

CD and NMR structural characterization of ceratotoxins,natural peptides with antimicrobial activity

Antibacterial properties of the secretion from the female reproductive accessory glands of medfly Ceratitis capitata are mostly ascribed to the presence of two peptides, ceratotoxin A and B, which exhibit a strong activity against gram-positive and gram-negative bacterial strains, and show sequence and function homology with cecropins, melittin, and magainins. CD experiments performed in different solvents indicate the presence of a significant content of helical structures in organic solvent. Two-dimensional nmr results for ceratotoxin A in methanol show a helical behavior for the 8–25 region of the peptide. A Ramachandran classification of each residue for the structures obtained from distance geometry calculations lead to the definition of four structural families in which the central segment 10–19 is always helical and differences refer to residues 8–9 and 19–23. A sequence analysis of the two ceratotoxins and a systematic search on the protein data bank revealed the occurrence of a KX-hydrophobic-hydrophobic-P motif that seems to be important for helix stabilization. © 1996 John Wiley & Sons, Inc.     

Juvenile hormone regulates the expression of the gene encoding ceratotoxin a,an antibacterial peptide from the female reproductive accessory glands of the medfly Ceratitis capitata

Ceratotoxin A is an antibacterial peptide produced by the reproductive female accessory glands of the medfly Ceratitis capitata. To investigate whether ceratotoxin A gene expression was affected by juvenile hormone, which has gonadotropic functions in adult insects, newly emerged female medflies were treated with precocene II, an antiallotropin compound capable of inhibiting juvenile hormone biosynthesis. Daily treatment of newly emerged flies with precocene II blocked ceratotoxin A gene expression in a dose-dependent manner. Ceratotoxin A gene expression could be recovered after withdrawl of precocene II treatment. Moreover, the effect of precocene II on ceratotoxin A gene expression could be countered by simultaneous treatment with methoprene, a juvenile hormone analogue. The effects of precocene II and methoprene treatments on the growth of both ovaries and accessory glands was also investigated. Our data suggest that ceratotoxin A gene expression is modulated by juvenile hormone.     

Genomic organization of four novel nondisulfide-bridged peptides from scorpion Mesobuthus martensii Karsch: gaining insight into evolutionary mechanism

At least 25 nondisulfide-bridged peptides (NDBPs) have been identified and characterized from scorpions. However, the genomic organization of the genes that encode these peptides have not been reported yet. BmKa1, BmKa2 and BmKb1 are three novel genes that code for NDBPs identified by our group from Mesobuthus martensii Karsch. Based on their cDNA sequences, the genomic DNA sequences encoding these peptides were obtained using the PCR method. Sequence analysis showed that three distinct genomic structural patterns are used to encode these three peptides. The BmKa1 gene is not interrupted by any introns. However, the BmKa2 gene is composed of two exons, interrupted by a 67 bp intron that is located in the DNA region encoding the mature peptide. Two genomic homologues of the BmKb1 cDNA sequence, named BmKb1′ and BmKb2, respectively, were obtained. The BmKb1′ gene contains one intron of 593 bp, inserted into the DNA region that encodes the signal peptide. Similarly, the BmKb2 gene also contains an intron that interrupts the exon that encodes the NDBP signal peptide. The amino acid sequences deduced for BmKb2 and BmKb1′ differ only at one position. The data suggest that the genomic organizational pattern of NDBPs displays more divergence than that exhibited by the genes that encode disulfide-bridged peptides from scorpions.     

Short-term, postcopulatory changes in receptive and proceptive behavior in the female syrian hamster (Mesocricetus auratus)

The interactions between male and female hamsters were recorded in terms of proximity (approaching, leaving, and following by each member of the pair). The time spent sniffing the partner was also recorded. Mated and unmated females were tested three times on the day of estrus and once on the day following estrus. As expected, receptivity declined rapidly in mated but not unmated females. None of the proximity measures changed during estrus in the unmated females. However, female approaching (Ap% ≈ female approaches as percentage of all approaches) decreased 1–3 hr after mating and was associated with a decline in a number of measures of female proceptive behavior. Female leaving (L% = female leaves as percentage of all leaves) did not change. In unmated females Ap% has decreased and L% has increased by the day after estrus (Day 2 of the estrus cycle). In mated females the decrease in Ap% is accelerated (occurring within the 3 hr after mating) and precedes a change in L% which has occurred by the day after estrus (Day 1 of pregnancy). Thus, mating brings about not only a decrease in receptivity, as has been shown for several species, but also a change in female proceptive behavior and in her stimulus value to the male.     

Male age and strain affect ejaculate quality in the Mexican fruit fly

Aging in all organisms is inevitable. Male age can have profound effects on mating success and female reproduction, yet relatively little is known on the effects of male age on different components of the ejaculate. Furthermore, in mass‐reared insects used for the Sterile Insect Technique, there are often behavioral differences between mass‐reared and wild males, while differences in the ejaculate have been less studied. The ejaculate in insects is composed mainly of sperm and accessory gland proteins. Here, we studied how male age and strain affected (i) protein quantity of testes and accessory glands, (ii) the biological activity of accessory gland products injected into females, (iii) sperm viability, and (iv) sperm quantity stored by females in wild and mass‐reared Anastrepha ludens (Diptera: Tephritidae). We found lower protein content in testes of old wild males and lower sperm viability in females mated with old wild males. Females stored more sperm when mated to young wild males than with young mass‐reared males. Accessory gland injections of old or young males did not inhibit female remating. Knowledge of how male age affects different ejaculate components will aid our understanding on investment of the ejaculate and possible postcopulatory consequences on female behavior.     

Antibacterial activity and pore-forming properties of ceratotoxins: a mechanism of action based on the barrel stave model

Ceratotoxins are alpha-helical cationic peptides isolated from the medfly Ceratitis capitata. These amphipathic peptides were found to display strong antibacterial activity and weak hemolytic activity. When reconstituted into planar lipid bilayers, ceratotoxins developed highly asymmetric I/V curves under voltage ramps and formed, in single-channel experiments, well-defined voltage-dependent ion channels according to the barrel stave model. The antibacterial activity and pore-forming properties of these molecules were well correlated. Similar experiments performed with synthesized truncated fragments showed that the C-terminal domain of ceratotoxins is strongly implicated in the formation of helical bundles in the membrane whereas the largely cationic N-terminal region is likely to anchor ceratotoxins on the lipid surface.     

Symbiotic bacteria associated with gut symbiotic organs and female genital accessory organs of the leaf beetle <Emphasis Type="Italic">Bromius obscurus</Emphasis> (Coleoptera: Chrysomelidae)

Leaf beetles (Coleoptera: Chrysomelidae) constitute one of the most species-rich insect families, and live exclusively on leaves or other plant parts. Early histological works described the presence of symbiotic bacteria in gut-associated symbiotic organs of some chrysomelid species, but their microbiological nature has been poorly characterized except for those associated with reed beetles of the subfamily Donaciinae. Here we investigated symbiotic bacteria of the leaf beetle Bromius obscurus (L.) belonging to the subfamily Eumolpinae. Specific bacterial 16S ribosomal RNA and gyrB gene sequences were consistently obtained from the symbiotic organs, which radially surround the foregut-midgut junction, of all adult males and females examined. In adult females, the same sequences were also obtained from a pair of genital accessory organs, which are presumably for vertical symbiont transmission. Whole mount in situ hybridization specifically detected the symbiont in the gut symbiotic organs endocellularly and also in the female genital accessory organs extracellularly. In the gut symbiotic organs, the endocellular symbiont cells were small and rosette-like or aggregated and granule-like, whereas in the female genital organs the extracellular symbiont cells were of a condensed form. Molecular phylogenetic analysis showed that the symbiont of B. obscurus constitutes a distinct lineage in the Gammaproteobacteria. Molecular evolutionary analysis has identified significantly accelerated molecular evolution and a highly adenine–thymine-biased nucleotide composition of the symbiont genes, presumably reflecting reductive evolution of the symbiont genome. These results suggest an intimate and stable host-symbiont association in B. obscurus, in which the symbiont may play some important, though hitherto unknown, biological roles in its herbivorous insect host.     

Expression of allatostatins in the Mediterranean field cricket, Gryllus bimaculatus de Geer (Ensifera, Gryllidae)

The allatostatin (AST) type A gene of the cricket Gryllus bimaculatus encodes a hormone precursor including at least 14 putative peptides with a common C-terminus Y/FXFGL/Iamide. By RT-PCR we have analyzed the expression of the allatostatin precursor in various tissues of 0-21 days old adult virgin and mated females. In 3-day-old virgin females, the gene is strongly expressed in the brain (oesophageal ganglion), the suboesophageal ganglion and the caecum, but to a lower extent in other parts of the digestive tract (ileum, midgut, colon), and in various other tissues such as the fat body, ovaries and female accessory reproductive glands. In the brain and ovaries of virgin females, the AST expression is rather constant throughout adult life, whereas in brains of mated animals, expression is low until day 7, but increases sharply from day 8 onwards to reach values triple those before day 7. In ovaries of mated animals AST gene expression is also age-dependent, with high expression rates during the first 4 days after imaginable moult, a second but smaller peak from day 15 to 21, and very low values in between. In the fat body of virgin crickets allatostatin expression is high during the first 9 days after ecdysis and declines thereafter, whereas in mated animals two peak values, day 1 and day 6, are observed, and a third peak in older animals.     

Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedusa hypochondrialis

Amphibian antimicrobial peptides have been known for many decades and several of them have been isolated from anuran species. Dermaseptins are among the most studied antimicrobial peptides and are found in the skin secretion of tree frogs from the Phyllomedusinae subfamily. These peptides exert a lytic action on bacteria, protozoa, yeast, and filamentous fungi at micromolar concentrations, but unlike polylysines, present little hemolytic activity. In this work, two antimicrobial peptides were isolated from the crude skin secretion of Phyllomedusa hypochondrialis and tested against Gram-positive and Gram-negative bacteria, presenting no hemolytic activity at the tested concentrations. One of them was identified with the recently reported peptide PS-7 belonging to the phylloseptin family, and another was a novel peptide, named DPh-1, which was fully purified, sequenced by ‘de novo’ mass spectrometry and grouped into Dermaseptins (DPh-1).     

Molecular cloning and biological characterization of novel antimicrobial peptides, pilosulin 3 and pilosulin 4, from a species of the Australian ant genus Myrmecia

Venom of an Australian ant species of the Myrmecia pilosula species complex (mss. name Myrmecia banksi Taylor) contains two major allergenic peptides, pilosulin 1 and pilosulin 2. To obtain novel cDNA clones that encode the pilosulin-related bioactive peptides, mRNA of another Myrmecia species was subjected to RT-PCR in which the forward primer corresponds to a nucleotide sequence in the leader sequences of pilosulin 1 and pilosulin 2. As a result, we isolated cDNA clones encoding the novel antimicrobial peptides pilosulin 3 and pilosulin 4. The nucleotide and the amino acid sequences of all four pilosulins have high homology except for the mature peptide coding regions. Synthetic pilosulin 3 and pilosulin 4 peptides displayed antimicrobial activity with histamine-releasing and low hemolytic activities.     

Fixation of mutations in the viral genome during an outbreak of foot-and-mouth disease: heterogeneity and rate variations

Rates of fixation of mutations during the evolution of the foot-and-mouth disease virus (FMDV) C₁ in nature have been estimated by hybridization of viral RNA to cloned cDNAs representing defined FMDV genome segments, and comparison of the selected RNAs by T₁ RNase oligonucleotide fingerprinting. Values ranged from <0.04 × 10⁻² to 4.5 × 10⁻² substitutions per nucleotide per year (s/nt/yr), depending on the time period and the genomic segment considered. Rates for viral structural protein genes were up to sixfold higher than for nonstructural protein genes. Values in excess of 10⁻² s/nt/yr have been measured for the RNA region that encodes VP1–VP3. The nucleotide sequences of the major immunogenic region of capsid protein VP1 have been determined for six new FMDV C₁ isolates, and they are compared with the two previously known sequences of FMDV C₁ (C-S8 and C₁-O). Both oligonucleotide fingerprinting of selected RNA fragments and direct nucleotide sequencing demonstrate that genetic heterogeneity exists among three viruses isolated on the same day, introducing a significant indetermination in the evaluation of fixation rates of mutations. During the FMDV C₁ outbreak, amino acid substitutions did occur that are known to affect the immunological properties of the virus. The proportion of mutations between two viral RNAs does not increase significantly with the time elapsed between the two isolations, suggesting a cocirculation of multiple, related, nonidentical FMDVs (‘evolving quasispecies’) as the mode of evolution of this agent.     

Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes

The nucleotide (nt) sequences of the Sc3 and Sc4 genes of the filamentous fungus Schizophyllum commune, and the deduced amino acid (aa) sequences, were determined; moreover, the previously published sequence for the ScI gene [Dons et al., EMBO J. 3 (1984) 2101–2106] was corrected. All three independently isolated genes were found to have similar structures and nt sequences of their coding regions. At the aa level the homology is 43–62% (63–69% in the C-terminal parts of the proteins), the hydrophobic aa predominate and the hydrophobicity patterns are similar. All three proteins contain leader sequences and eight cysteines among about 110 aa, conserved at the same positions. Yet these genes are differentially regulated: Sc1 and Sc4 are only expressed at high levels in fruiting dikaryons, whereas Sc3 is highly expressed in both monokaryons and dikaryons, independent from fruiting.     

Preliminary observations on change of sex by the coral-inhabiting snails Coralliophila violacea (Lamarck) (Gastropoda: Coralliophilidae)

The coral-inhabiting snails, Coralliophila violacea (Lamarck), are widely distributed in the Indo–Pacific region. It has been inferred from field data that sex changes have occurred and might be influenced by neighbors. In this study, we designed a field experiment with 2 treatments, i.e. ‘single males' and ‘male–female pairings' to test the hypothesis of sex changes as well as the role of neighbors on sex changes. Snails were collected by SCUBA diving from the surface of massive coral Porites lobata in shallow waters (<6 m) in southern Taiwan. A total of 73 males and 32 females were marked and returned to the surface of a colony of the massive coral, P. lobata. After 4–5 months, a total of 25 marked males and eight marked females were recaptured. In the treatment of ‘single males', eight of 17 males had changed their sex. None of eight recaptured males in the treatment of ‘male–female pairings' had changed their sex. The occurrence of sex changes is dependent on the presence or absence of a female neighbor (P<0.05, Fisher's exact test). In this study, we have provided direct evidence of socially controlled sex changes in Coralliophila violacea.     

Synthetic peptides from C-reactive protein containing tuftsin-related sequences

Peptides containing Lys-Pro-Arg or Thr-Lys-Arg segments corresponding to various regions of human C-reactive protein were synthesized. The peptides prepared were composed of amino acid residues, 37–58, 51–58, 173–187 and 181–187 of C-reactive protein. The relationship between C-reactive protein, its synthetic fragments and tuftsin (Thr-Lys-Pro-Arg) was investigated in binding studies, enhancement of phagocytosis and change in cyclic nucleotide levels of mouse macrophages. The peptides AA 51–58 and 181–187 did enhance macrophage phagocytosis capacity to a similar extent to that of tuftsin. They showed however only negligible binding to the cells. The effect of C-reactive protein and the synthetic peptides on metabolic activity of neutrophils was also investigated. It was shown that the peptides inhibited to some degree superoxide production, lysozyme release and Vitamin B₁₂ binding protein release from neutrophils in the absence and presence of the stimulants, PMA or Con A. Comparable activity with tuftsin was not found.     

Possible recognition of the GnRH receptor by an antiserum against a peptide encoded by nucleotide sequence complementary to mRNA of a GnRH precursor peptide

Two peptides, rHRnG and hproHRnG, which were encoded by the nucleotide sequences complementary to mRNA of rat hypothalamic gonadotropin-releasing hormone (GnRH) and human placental proGnRH(−3–13), respectively, were synthesized. A remarkable hydropathic anti-complementarity was observed in the N-terminal region between hproHRnG and human proGnRH(−3–13). Neither hproHRnG nor rHRnG bound GnRH in ELISA unless exremely high concentrations of peptides were used. ¹²⁵I-GnRH failed to bind with either rHRnG or hproHRnG previously coated polypropylene tubes. Antisera against these peptides were generated in rabbits. All the rabbits produced antibodies with high titer as tested by ELISA. One rabbit immunized with hproHRnG showed markedly reduced serum testosterone levels as compared with those of other rabbits. Intravenous administration of 1 ml serum from this rabbit, antiserum R281, into ovariectomized rats significantly decreased plasma LH. Using antiserum R281, about 10% of female rat pituitary cells were stained by immunohistochemistry. The staining was specific to hproHRnG since it was abolished by preabsorption of the antiserum with hproHRnG, but not with rHRnG, GnRH, LH nor any other peptide tested. This particular antiserum may have recognized the GnRH receptor, and thereby interfered with the action of endogenous GnRH. These results appear to be in agreement with the view that there is a structural similarity between the receptor for a peptide and the so-called complementary peptide.     

The Novel Antibacterial Peptide Ceratotoxin A Alters Permeability of the Inner and Outer Membrane of Escherichia coli K-12

Ceratotoxins are antibacterial 3-kDa amphiphilic peptides isolated from the female reproductive apparatus of the medfly Ceratitis capitata. The antibacterial activity of a chemically synthesized ceratotoxin A (ctx A) has been investigated. Ctx A was mainly active against Gram-negative organisms, and it had a lytic effect on nongrowing Escherichia coli K-12. Data showed that ctx A alters both the outer and the inner membrane of E. coli K-12 cells. Received: 4 August 1995 / Accepted: 2 January 1996