Female Gland Extracts Research Articles

Salivary gland anticoagulants in culicine and anopheline mosquitoes (Diptera:Culicidae).

Female salivary gland extracts from 9 mosquito species representing the 2 hematophagous subfamilies delayed coagulation of human plasma. All extracts significantly delayed recalcification time, prothrombin time, and activated partial thromboplastin time, indicating an effect on the common coagulation pathway. Chromogenic assays to determine the coagulation inhibition site were performed for coagulation factor Xa (FXa) and thrombin, the major components of the common pathway. Although variation existed in the degree of inhibition, all anophelines have thrombin-directed anticoagulants, and culicine mosquitoes have FXa-directed anticoagulants. Differences in the site of action of the anticoagulants most likely reflected the long period of independent adaptation of the 2 subfamilies to the challenges presented by vertebrate hemostasis.

Identification and synthesis of the sex pheromone of Phtheochroa cranaodes (Lepidoptera: Tortricidae)

The pheromone of Phtheochroa cranaodes (Lepidoptera: Tortricidae) was identified as (3 E,5 Z)-3,5-dodecadienyl acetate (5) by GC-MS analysis of female gland extracts and field trapping of males with synthetic compound.

A Factor Xa-Directed Anticoagulant from the Salivary Glands of the Yellow Fever Mosquito Aedes aegypti

Salivary gland extracts from the yellow fever mosquito Aedes aegypti contain a factor Xa-specific anticoagulant detectable in vitro by clotting assays. The anticoagulant is found only in female salivary glands, which is consistent with its role in bloodfeeding. Extracts of adult female glands significantly delay both the prothrombin time and the activated partial thromboplastin rime, but have no effect on the thrombin clotting time. The anticoagulant has been shown to result from the specific inhibition of coagulation factor Xa based on a FXa clotting assay and the inhibition of FXa-directed cleavage of the synthetic chromogenic substrate, chromozym X. The inhibition of FXa by female salivary gland extracts exhibits noncovalent, noncompetitive inhibition kinetics and is reversible. The anticoagulant is destroyed by boiling for 10 min or heating at 56°C for 30 min, has a 4 mM calcium optimum with no magnesium requirement, and has a pH optimum of 8.0. The anticoagulant activity has an apparent molecular weight of 35.5 kDa, as determined by molecular sieve. We propose that the A. aegypti anticoagulant is a novel, proteinaceous serine protease inhibitor specific for FXa.

Lack of correlation of distribution of prolactin (PRL) charge isoforms with induction of PRL storage.

GH4C1 cells, rat pituitary tumor cells, produce PRL, but store little relative to normal lactotrophs; treating cells with estradiol, insulin, and epidermal growth factor increases both PRL storage and accumulation of dense core granules. Normal lactotrophs contain several differently charged forms of PRL. We investigated whether inducing PRL storage in GH4C1 cells altered the production of these forms. Four forms of PRL that differed by charge were found by immunoblots of two-dimensional gels of extracts of female rat pituitary glands and of secretory granules isolated from the glands. Four forms were also secreted by GH4C1 cells. The relative abundance of the four forms in the medium of GH4C1 cells, determined by [35S]amino acid incorporation for 24h, was 1.2 +/- 0.58%, 91.3 +/- 1.09%, 6.3 +/- 0.72%, and 1.2 +/- 0.39% of total PRL (mean +/- SEM), from the most basic to the most acidic, respectively. Treatment with 1 nM estradiol, 300 nM insulin, and 10 nM epidermal growth factor did not significantly change the relative abundance of the forms. All four forms also were found in GH4C1 cells after 2 h of incubation with [35S]amino acids, although no incorporation of 32PO4 was detectable over the same incubation time. We conclude that GH4C1 cells produce four forms of PRL that differ by charge, as normal lactotrophs do. The increase in storage of PRL caused by insulin, estrogen, and epidermal growth factor does not result in or is caused by increased secretion of a specific form.

Polymorphism of human pituitary FSH: analysis of immunoreactivity and in vitro bioactivity of different molecular species.

Follicle-stimulating hormone is known to be highly heterogeneous in serum and in the pituitary. In the present study, we have partially separated different molecular species of human pituitary FSH and characterized their immunoreactivity and in vitro bioactivity. Pooled extracts of male (n = 15) and female (n = 9) human pituitary glands were chromatographed on a column of Sephacryl S-200 and FSH-containing fractions were fractionated by chromatofocusing in the pH range 4-6. FSH was measured in the individual fractions by an in vitro bioassay, based on the FSH-dependent aromatase activity of immature rat Sertoli cells, and by the following methods based on commercial kits: radioimmunoassay (RIA), immunofluorimetric assay (IFMA), immunoradiometric assay (IRMA), immunoenzymometric assay (IEMA). In each assay, the kit standard, calibrated against the 2nd International Reference Preparation (IRP) 78/549, and the International Standard (IS) 83/575 were run in parallel. The relative potencies of the kit standards in terms of IS 83/575 were: IFMA 3.08, IRMA 1.62, RIA 2.42, IEMA 1.45 and bioassay 1.14. After chromatofocusing, pituitary FSH eluted mostly in fractions with pH approximately 4.5, without sex-related differences. In both sexes approximately 25% of bioactive material showed a pI < 4 and eluted with 1 M NaCl. Although the same IS 83/575 was used in the various assays, the profiles of immunoreactive FSH were significantly different. The highest intermethod variability was observed in the case of male pituitary FSH. The relative biopotency of the different molecular species of FSH did not appear to change according to their pI but, rather, varied with the assay method and the standard.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological and behavioral responses of turnip moth males,Agrotis segetum to fluorinated pheromone analogs

The electrophysiological and behavioral responses of maleAgrotis segetum to fluorinated analogs of (Z)-5-decenyl acetate (Z5-10∶OAc) were investigated. The single sensillum recordings showed that 4,4-difluoro-(Z)-5-decenyl acetate (4,4-F2), 7,7-difluoro-(Z)-5-decenyl acetate (7,7-F2), 10,10,10-trifluoro-(Z)-5-decenyl acetate (10,10,10-F3) and 7,7,8,8-tetrafluoro-(Z)-5-decenyl acetate (7,7,8,8-F4) were each 100-fold less active than the natural Z5-10 ∶ OAc, whereas the 7,7,8,8,9,9,10,10,10-nonafluoro-(Z)-5-decenyl acetate (F9) analog was essentially inactive. A mixture of Z5-10 ∶ OAc, Z7-12 ∶ OAc, and Z9-14 ∶ OAc on a filter paper dispenser was as attractive as female gland extracts when tested in a flight tunnel. With Z5-10∶OAc omitted, the two-component mixture elicited a significantly lower male response. Four analogs, 7,7-F2, 10,10,10-F3, 7,7,8,8-F4, and F9, were added separately to the two-component mixture to replace Z5-10∶OAc. The responses elicited by the mixtures containing the 7,7-F2, 10,10,10-F3, and 7,7,8,8-F4 analogs did not differ significantly from that of the natural three-component mixture and the two-component mixture, whereas the mixture containing F9 elicited a significantly lower male response, as low as the response to the two-component mixture. In a field test the mixtures containing 10,10,10-F3 and 7,7,8,8-F4 were significantly more active than the two-component mixture, but still less active than the natural three-component mixture. It appears that field tests provided greater discrimination among pheromone analogs in assessing their behavioral activity than the flight-tunnel test did. Structure-activity analyses demonstrate the importance of the lipophilic interaction between the terminal alkyl chain and the receptor site for the activity of the stimulus. The lipophobicity of the fluorinated analogs impedes a productive receptor interaction.

Odor-modulated upwind flight of the sphinx moth, Manduca sexta L.

1. Male and female Manduca sexta flew upwind in response to the odor of female sex-pheromone gland extract or fresh tobacco leaf respectively, and generated very similar zigzagging tracks along the odor plume. 2. After loss of odor during flight, males and females alike: (1) first flew slower and steered their flight more across the wind, then (2) stopped moving upwind, and finally (3) regressed downwind. 3. Males flying upwind in a pheromone plume in wind of different velocities maintained their ground speed near a relatively constant 'preferred' value by increasing their air speed as the velocity of the wind increased, and also maintained the average angle of their resultant flight tracks with respect to the wind at a preferred value by steering a course more precisely due upwind. 4. The inter-turn duration and turn rate, two measures of the temporal aspects of the flight track, were maintained, on average, with remarkable consistency across all wind velocities and in both sexes. The inter-turn durations also decreased significantly as moths approached the odor source, suggesting modulation of the temporal pattern of turning by some feature of the odor plume. This temporal regularity of turning appears to be one of the most stereotyped features of odor-modulated flight in M. sexta.

Sex pheromone perception in male pine sawflies, Neodiprion sertifer (Hymenoptera; Diprionidae)

Electroantennographic and single sensillum recordings were performed on male pine sawfly, Neodiprion sertifer, antennae. Responses to the sex pheromone component (2S, 3S, 7S)- 3,7-dimethyl-2-pentadecenyl (diprionyl) acetate (SSS:OAc), to the behavioral inhibitor (2S, 3R, 7R)-diprionyl acetate (SRR:OAc), to the six other enantiomers of diprionyl acetate, and to the biosynthetic precursor diprionol were recorded. Responses to trans-perillenal, a monoterpene identified in female gland extracts and to (2S, 3S, 7S)-diprionyl propionate (SSS:OPr), a field attractant for N. sertifer and some related sawfly species were also recorded. EAG recordings demonstrated a high antennal sensitivity to SSS:OAc and to SSS:OPr. A somewhat lower response was elicited by SRR:OAc. Single sensillum recordings revealed 8-12 different cells firing in each sensillum, corresponding to the number of cells observed in earlier morphological investigations. Out of these cells all, except one, responded to SSS:OAc and to SSS:OPr. No differences in the response to the two components could be observed. The largest amplitude cell in each sensillum was specifically tuned to the behavioral antagonist, SRR:OAc. The pheromone perception system encountered in male pine sawflies thus differs clearly from that observed in moths.

Identification of sex pheromone of tomato pinworm,Keiferia lycopersicella (Wals.)

A sex pheromone produced by femaleKeiferia lycopersicella (Walsingham) was isolated and identified as (E)-4-tridecenyl acetate, based on chemical analyses, electroantennogram assays, and field trapping in California and Florida. Males were captured equally well in traps baited with (E)-4-tridecenyl acetate alone or a variety of (Z)- and (E)-4-tridecenyl acetate blends, although theZ isomer was not detected in extracts of female glands.

Identification of sex pheromone of bristly cutworm,Lacinipolia renigera (Stephens)

The sex pheromone of the bristly cutworm moth,Lacinipolia renigera was identified as a blend of (Z)-9-tetradecenyl acetate (itZ9-14): Ac and (Z, E)-9,12-tetradecadienyl acetate (ZE-9,12-14: Ac). Extracts of female glands were analyzed by capillary gas chromatography on three columns of different polarities. In each analysis, peaks with retention times identical to Z9-14:Ac andZE-9, 12-14: Ac were observed. GC-MS analysis of gland extracts supported the identification of these two compounds. Volatiles emitted from female sex pheromone glands during 10-min collection periods contained 7.8 ±2.01 ng ofZ9- 14: Ac. On average the blend contained 3.8 ± 1.43%ZE-9,12-14: Ac. Blends ranging from 1% to 10%ZE- 9,12-14: Ac in Z9-14: Ac (1 mg) were effective in capturing males in the field. The number of males captured in traps baited with a 3 % blend ofZE- 9,12-14: Ac in Z9-14: Ac was not significantly different than the number caught in traps containing two virgin females.

Does the imported cabbageworm,Pieris rapae, use an oviposition deterring pheromone?

Eggs ofPieris rapae (L.) contain a water- and methanol-soluble oviposition-deterring pheromone (ODP) that is avoided by ovipositing females offered treated and untreated cabbage leaves in a choice situation. Water extracts of female accessory glands also deter oviposition. Egg washes and gland extracts stimulate a contact chemoreceptor in sensilla on the fore tarsi. Electroantennogram (EAG) recordings show that antennal receptors respond to volatiles from conspecific eggs, but not to volatiles from eggs ofMamestra brassicae. No differences were detected between North American and European insects with regard to production and perception of ODP. These results differ from conclusions in the literature based upon field observations. It is concluded that this discrepancy in due to methodological differences.

Western blot analysis of Amblyomma americanum-derived stage-specific and shared antigens using serum from guinea pigs expressing resistance

Serum from guinea pigs expressing resistance to larval, nymphal and adult Amblyomma americanum ticks was used in Western blot analyses to identify potential antigens from egg, larval and nymphal, and female salivary gland extract preparations. The results demonstrate multiple antigens unique to each life stage, as well as several shared proteins between the three life stages. However, it appears as if two particular proteins of 25 and 38 kDa may be more important than others, based upon their prevalence and intensity of recognition in this assay relative to other polypeptides.

Sex pheromone components of three species ofSemiothisa (Geometridae), (Z,Z,Z)-3,6,9-heptadecatriene and two monoepoxydiene analogs

Adult males ofSemiothisa signaria dispuncta (Walker) were attracted to field traps baited with (Z,Z,Z)-3,6,9-octadecatriene and (Z,Z)-6,9-cis-3,4-epoxy-octadecadiene. However, analyses of sex pheromone gland extracts of females of this species by GC-MS and by GC in combination with an electroantennograph detector (GC-EAD) showed the pheromone to be comprised of a mixture of the next lower homologs: (Z,Z,Z)-3,6,9-heptadecatriene and (Z,Z)-6,9-cis-3,4-epoxy-heptadecadiene. Blends of these two C17 compounds were subsequently found to be more attractive to males in the field than the corresponding C18 mixtures. Sex pheromones of two otherSemiothisa species were also found to contain C17 components. (Z,Z,Z)-3,6,9-Heptadecatriene, detected by GC-EAD analysis of a female abdominal tip extract ofS. bicolorata (Fabricius), attracted conspecific males, and this attraction was significantly reduced by additions of (Z,Z)-6,9-cis-3,4-epoxyheptadecadiene, the major pheromone component ofS. signaria dispuncta, to the lure. (Z,Z)-3,9-cis-6,7-Epoxy-heptadecadiene was detected by GC-EAD analysis as the primary male antennal stimulatory component present in abdominal tip extracts ofS. ulsterata (Pearsall), and males of this species were attracted to traps baited with this epoxide. Each of these three C17 compounds constitute previously unknown lepidopteran sex pheromone components. Blends of (Z,Z, Z)-3,6,9-heptadecatriene and (Z,Z)-3,9-cis-6,7-epoxyheptadecadiene attracted males of a fourth species,S. delectata Hulst, but no females of this species were obtained to permit analysis of its sex pheromone. The occurrence of (Z,Z,Z)-3,6,9-heptadecatriene inS. neptaria (Guenee) females was indicated by GC-MS analysis of an abdominal tip extract; however, no males were attracted to any of the fielded mixtures containing this hydrocarbon.

Blackheaded fireworm: Laboratory and field studies of its sex pheromone

Electroantennogram (EAG) responses of maleRhopobota naevana (Hübner), the blackheaded fireworm, to all of the monoene straightchain 12- and 14-carbon alcohols and acetates implicated (Z)-11-tetradecenl-1-ol (Z11-14∶OH) and its acetate (Z11-14∶Ac) as sex pheromone components.Z11-14∶Ac produced the strongest EAG response of all compounds tested. Gas chromatography-mass spectrometry (GC-MS) analysis of extract of female sex pheromone glands (SPG) confirmed the presence ofZ11-14∶OH (125 pg/female) andZ11-14∶Ac (600 pg/female) (all other monoenes had different retention times). In field tests, traps baited withZ11-14∶OH alone captured males, but traps baited withZ11-14∶Ac alone did not. Traps baited with a combination ofZ11-14∶OH andZ11-14∶Ac in various ratios did not produce better trap catches thanZ11-14∶OH alone. (Z)-9-Dodecen-1-ol acetate (Z9-12∶Ac), reported by others to be a field attractant, did not produce trap catch in our tests, but in combination withZ11-14∶ OH (98∶2 in septa corresponding to 95:5 in vapor,Z11-14∶OH toZ9-12∶AC) produced a sevenfold increase in catch overZ11-14∶OH alone. IfZ9-12∶AC had been present in extract of SPG at 2-5% ofZ11-14∶OH, it would not have been detected in our GC-MS experiment.

Sex pheromone ofGrapholita funebrana occurrence ofZ-8- andZ-10-Tetradecenyl acetate as secondary components

Z-8-Dodecenyl acetate (Z8-12∶Ac),E-8-dodecenyl acetate (E8-12∶Ac),Z-8-tetradecenyl acetate (Z8-14∶Ac),Z-10-tetradecenyl acetate (Z10-14∶Ac), andZ-8-dodecen-1-ol (Z8-12∶OH) were identified in the proportions 100∶1∶30∶5∶2 in female sex gland extracts ofGrapholita funebrana, accompanied by saturated acetates from 12 to 20 carbons with tetradecyl acetate predominating.Z10-14∶Ac has not previously been described as a lepidopteran sex pheromone component. Best attraction of males is obtained withZ8-12∶Ac in the presence of a higher proportion ofE8-12∶Ac than in the female. Inclusion of the 14-carbon acetates did not augmentG. funebrana catches but inhibitedG. molesta. On the other hand, addition ofZ8-12∶OH at the level optimal forG. molesta reduced attraction ofG. funebrana.

Pheromones of two arctiid moths (Creatonotos transiens andC. gangis)

The two major components of the female sex pheromones of twoCreatonotos species have been identified as an achiral C21 triene, (Z,Z,Z)-3,6,9-heneicosatriene, and a chiral epoxide, (Z,Z)-2(2,5-octadienyl)-3-undecyloxirane. The ratios of these components in the two species fall into nonoverlapping ranges. Two additional achiral minor components, (Z,Z)-6,9-heneicosadiene and (Z,Z,Z)-3,6,9-tricosatriene, were also identified in the female sex gland extracts. The male pheromone of both species consists of hydroxydanaidal, a chiral dihydropyrrolizine derived from pyrrolizidine alkaloids in the larval diet.Creatonotos transiens was found to convert dietary heliotrine into (R)-(-)-hydroxydanaidal, with inversion at the single asymmetric carbon atom. The possible biological and biosynthetic significance of the chiral pheromone components are discussed, and they are compared with known examples of chiral lepidopteran pheromones.

Discrimination and production of disparlure enantiomers by the gypsy moth and the nun moth

ABSTRACT. Two odour receptor cells were physiologically identified within male antennal hair sensillae of the gypsy moth, Lymantria dispar L, and the nun moth, L. monacha L. In the gypsy moth, one cell responded to (+)‐disparlure, while a neighbouring cell responded to (‐)‐disparlure. In the nun moth both cells responded to (+)‐disparlure. The lack of sensitivity to (‐)‐disparlure in the nun moth was corroborated by electroantennogram (EAG) recordings, which indicated no affinity to this enantiomer. Single cell responses of male gypsy moth to different concentrations of the synthetic enantiomers of disparlure were then compared to responses elicited by hexane extracts of female glands of both species. The gypsy moth's extracts stimulated almost exclusively the receptor cell specialized for (+)‐disparlure, while both cells were simultaneously stimulated by the extracts of the nun moths. From the response characteristic of the cells it is estimated that pheromone production of the nun moth is about 10% (+) and 90% (‐)‐disparlure, and that of the gypsy moth is almost 100% (+)‐disparlure. Stimulation of the antenna of each species by female gland extracts of both species did not indicate the presence of receptors for other hexane elutable pheromone components in either species.

Characteristics of aminopeptidase activity of female housefly reproductive accessory glands

Proteolytic activity was detected in crude extracts of female reproductive accessory glands and the following characteristics of the principal aminopeptidase activities were determined: substrate specificity, pH optima, molecular weights, and effects of inorganic salts. The greatest aminopeptidase activities were found with the β-naphthylamides of: alanine at pH 7.5 and 9.5, leucine at pH 8.0, and methionine at pH 6.5. The methionine-specific activity in the crude extract was stimulated 3 times by 100 mM MgCl2, CaCl2, NaCl, or KCl. Inhibition was noted, and ID50 was determined for each of the other principal substrates with the following salts: CdCl2, CaCl2, ZnCl2, HgCl2, MgCl2, and MnCl2. Molecular weights, estimated on Sephadex G-200 and on Sepharose-6B, were found to be around 210 000 for each of these principal aminopeptidase activities in the crude extract.

Continuous single sensillum recording as a detection method for moth pheromone components in the effluent of a gas chromatograph

ABSTRACT. Single sensillum recordings were made from male antennal pheromone receptors of Agrotis segetum (Schiff.) (Noctuidae) and Adoxophyes orana (F.v.R.) (Tortricidae). A gas chromatograph (GC) was used to prepare and to deliver the odour stimuli. Samples of female abdominal gland extracts were injected into the column of the GC and the responses of the receptor cells to the effluent were recorded continuously. The receptor cells responded with an increase of their action potential frequency during elution of the major pheromone components. The pheromone receptors of Agrotis showed a much higher sensitivity than those of Adoxophyes. In the extract of female glands from Agrotis, compounds were detected which have not been identified in previous studies of the sex pheromone of this species. It is suggested that the combination of GC techniques with direct single sensillum recording may serve as a valuable supplement to electro‐antennographic techniques.

Sexual dimorphism of SDS peptide patterns from the submaxillary glands of mice.

In mice (Mus musculus) sexual dimorphism is evident in the structure and function of the submaxillary glands, convoluted tubules being much more prominent in males than in females. Several enzymes are more abundant in the male gland. We have examined the submaxillary glands of male and female mice for the amounts of arginine peptidase and have made electrophoretic scans of SDS peptides in extracts of male and female glands during postnatal development and after androgen treatment. Larger peptides are relatively more abundant in female extracts, and smaller peptides are more abundant in the male extracts. The peptide patterns reflect the amount of arginine peptidase present in the extracts. No arginine peptidase could be detected in submaxillary salivary glands of mice from birth until 3 weeks of age. Then enzyme activity gradually increased, apparently induced by androgen hormones. The amounts of SDS peptides of high molecular weight steadily decreased with increasing age of the mouse and with increasing titers of arginine peptidase. Castration of the male led to the appearance of female SDS patterns; male patterns reappeared after treatment with testosterone. Females could be induced to form male patterns of SDS peptides by treatment with androgen hormones.