Roger A. Leopold

The biosynthesis of the male housefly accessory secretion and its fate in the mated female

Abstract Autoradiographic studies of ejaculatory ducts from male houseflies, Musca domestica , previously injected with either tritiated arginine, lysine, or histidine showed that only arginine and lysine were incorporated into the stored accessory secretion. Scintillation spectrophotometry indicated that the amount of [ 3 H] arginine incorporated into the duct correlated with the number of times the male had previously copulated. Cycloheximide and actinomycin D were effective in inhibiting the uptake of [ 3 H] arginine in the ejaculatory ducts of newly emerged males, but only cycloheximide was an effective inhibitor in older males that had mated repeatedly. Autoradiography and electron microscopy showed that during mating the accessory secretion was transferred to the vaginal pouches of the female and that within 10 min after mating began, it was penetrating the intimal lining of the pouches. Cytolysis of many of the cells of these pouches was correlated to the transfer of the secretion during mating. This transfer of the secretion apparently terminated about 40 min after copulation began, although mating usually continued for an additional 10 to 20 min. The amount of 3 H-labelled material in the haemolymph of the females increased until the completion of mating and then decreased about 60 per cent after 8 hr.

Cytological and cytochemical studies on the ejaculatory duct and accessory secretion in Musca domestica.

Abstract The accessory secretion produced in the ejaculatory duct of male adults of Musca domestica was found to be an arginine-rich proteinaceous substance. The synthesis and extracellular storage of the secretion occurs in the anterior region of the duct and apparently coincides with the expression of sexual maturity. Three, and sometimes four, successive matings were required to deplete totally the secretion from the storage area. Renewed synthesis was usually observed within the secretory cells after two successive copulations and continued for more than 24 hr in those ducts which had been depleted by repeated matings. The structure and possible function the accessory secretion serves in the induction of mating refusal behaviour in female flies is discussed.

Ultrastructure of sperm penetration of house fly eggs

Acrosomes of house fly sperm in the micropyle of eggs were examined and compared with those of sperm in the spermathecae and of sperm in the anterior fertilization chamber of females from which the accessory reproductive glands had been removed. The acrosomal membranes of sperm within the spermatheca and those of sperm in the anterior fertilization chamber of glandless females were intact, and the granular material of the acrosomal cavity was present; those of sperm within the egg micropyle of normal females were lysed and largely devoid of granular acrosomal material. Also, numerous clusters of tubular aggregates were identified within the micropylar cap substance of ovarian eggs and of eggs from glandless females but were greatly disassociated and less abundant in eggs that sperm had penetrated. The various enveloping layers that surround the ooplasm and the immediate consequences of sperm entry of the egg were revealed and documented.

Using electroporation and a slot cuvette to deliver plasmid DNA to insect embryos.

Microinjection is the method used almost exclusively to deliver DNA constructs to insect embryos while electroporation is commonly used for DNA delivery to bacteria, cell cultures and certain plant tissues. This communication describes a method using an easily constructed slot cuvette and the electroporation technique for transfer of DNA to insect embryos for possible use in developing methods for germline transformation. This method eliminates time-consuming individual embryo manipulation and thus far has been found to be adaptable for use on several types of insect embryos. Using this method, we show successful transfer of plasmid DNA to embryos of the corn earworm moth, Helicoverpa zea, and the house fly, Musca domestica.

Development and Reproduction of the Egg Parasitoid, Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae), as a Function of Temperature

Abstract The development, fecundity, and life table parameters of Gonatocerus ashmeadi Girault, an egg parasitoid of the glassy-winged sharpshooter, Homalodisca coagulata (Say), were studied in the laboratory at six constant temperatures between 12 and 32°C. At 12°C, the parasitoid failed to develop beyond the third instar, and durations of the egg stage and the first and second instars were prolonged. Development from the egg stage to adult emergence varied from 27.1 d at 16°C to 9.5 d at 28°C. Temperature thresholds for development ranged from 3.8°C for first-instar larvae to 12.8°C for the pupal stage. The thermal constants were lowest for the second-instar larvae (26.7 DD) and highest for the pupae (75.4 DD). Nearly 207 DD were required above the lower temperature threshold of 8.5°C to complete development from egg to adult. The optimum temperature for egg to adult development was 29.2°C. Survival from egg to adult was 67.4% at 16°C and ranged from 83.4 to 86.7% between 20 and 32°C. At 16–32°C, the population had a type I survivorship pattern. At 16°C, longevity of adult females and males averaged 27.1 and 19.0 d, respectively, but declined to 6.4 and 6.9 d at 32°C. At 20–32°C, peak adult emergence occurred on the first day of emergence, but at 16°C, it was greatest on the second day. When exposed to temperatures ranging from 16 to 32°C, the female:male sex ratio was similar, ranging from 3.4 to 5.6. Lifetime fecundity was greatest at 24°C and lowest at 32°C, with the maximum net reproduction also occurring at 24°C. Greatest intrinsic and finite rates of increase, shortest population doubling time, and mean generation time occurred when G. ashmeadi was held at 28°C. The parameters defined in this study can influence geographical distribution and are important for the mass-rearing this wasp as biological control agent for the glassy-winged sharpshooter.

Electrophoresis of the male accessory secretion and its fate in the mated female

Polyacrylamide disk gel electrophoresis of homogenates of the ejaculatory ducts of male houseflies, Musca domestica, yielded 20 stainable protein fractions; 12 proteins (ranging from 650 to 4150 in molecular weight) could be definitely attributed to the ducts, and the remaining 8 were presumed by virtue of the relative mobilities to result from contamination with haemolymph. Studies of the incorporation of 14C-labelled amino acids into the ducts showed that relatively more arginine and asparagine were taken up followed, in order, by lysine, phenylalanine, methionine, glycine, cysteine, tyrosine, valine, threonine, leucine, isoleucine, histidine, and serine. Only trace amounts of proline, tryptophan, aspartic acid, alanine, glutamic acid, and glutamine were found. Since the components of the ejaculatory duct transferred to the female via copulation were selectively absorbed by the head, thorax, and abdomen of the female, we have support for the hypothesis of a multi-functional role of the male accessory secretion in the regulation of female physiology.

Progeny Quality of Gonatocerus ashmeadi (Hymenoptera: Mymaridae) Reared on Stored Eggs of Homalodisca coagulata (Hemiptera: Cicadellidae)

Abstract This study assessed the effects of refrigerated storage on the suitability of eggs of the glassy-winged sharpshooter,Homalodisca coagulata (Say) (Hemiptera: Cicadellidae), as hosts for propagation of the parasitoidGonatocerus ashmeadi Girault (Hymenoptera: Mymaridae). Development of the host eggs was terminated by chilling at 2°C for 5 d before storage was initiated at 10°C for up to 70 d. Parasitism, adult emergence rate, developmental time, and sex ratio were used to gauge the suitability of the eggs as hosts after storage. In addition to these measures, demographic growth parameters also were used to assess the quality of the wasp progeny through the F2 generation. Host eggs stored 20 d remained fully acceptable to the wasps for attack. Although the parasitism rate decreased with storage time, > 80% adult parasitoid emergence was realized from eggs stored 30 d. After 70 d storage, adult emergence rate was decreased by 48%, fecundity decreased by 53%, female production by 19%, developmental time was extended 3 d, and female longevity was shortened 5 d. The emergence pattern of F1 but not F2 adults varied with storage time of the parental and grandparental hosts, respectively. For the F2 generation, emergence rate, development, and sex ratio did not vary with storage time when the F1 parents parasitized fresh host eggs. Demographic parameters for the F1 population showed that net reproductive rate was > 20 although it decreased significantly after their parental host eggs were stored for > 30 d. The intrinsic and finite rates of increase, population doubling time, and mean generation time decreased only after storage for 60 d. Our results show that short-term cold storage could be used for maintaining wasp populations in a mass-rearing program and that the detrimental effects of chilling host eggs in storage for over 30 d do not extend to F2 generation.

The egg fertilization site within the house fly, Musca domestica (L.) (diptera: Muscidae)

Abstract The surface morphology of a dome-shaped genital chamber in the female Musca domestica L., where the sperm and egg meet following ovulation, was examined to determine its role in fertilization. The inner surface of the chamber was found to be lined with 3 types of nonarticulated cuticular spines. Examination of eggs removed from the chamber indicated that the distinctly robust spines at the apex were involved with the removal of a mucoid secretion which occludes the micropyle opening. The spines lining the rest of the chamber were more slender and flexible than those at the apex, and may function in ensuring that sperm remain at the fertilization site when the egg is placed into the chamber.

Internal genitalia of the female house fly, Musca domestica L. (Diptera: Muscidae): Analysis of copulation and oviposition

Abstract The morphology of the internal genitalia of the adult female Musca domestica L. was examined to determine the role of these structures during copulation and oviposition. Three major structures, the dorsal, ventral, and posterior valves, project into the lumina of the posterior common oviduct and anterior vagina in the region of the vaginal pouches. The dorsal valve is composed of 2 folds of epithelial tissue suspended from the dorsal vaginal wall, and houses the posterior openings of the spermathecal and accessory gland ducts. During copulation, the dorsal valve receives the aedeagus and is the site of sperm deposition. The ventral valve lies directly anterior to the dorsal valve and consists of a large, transverse muscle band and a posteriorly directed epithelial projection. Immediately beneath the epithelial projection, the vagina forms a blind, conical pocket, the anterior chamber. Fertilization is accomplished within this structure during oviposition. The posterior valve consists of 2 widely separated folds (or arms) of epithelial tissue arising from the ventral floor of the vagina. These arms, along with those of the dorsal valve, hold the egg in position while sperm penetration occurs.

Composition of the surface hydrocarbons from the vitelline membranes of dipteran embryos.

Hydrocarbons were the major lipid class extracted by hexane from the vitelline membrane surface of dechorionated eggs of the house fly, Musca domestica, the New World screwworm, Cochliomyia hominivorax, the secondary screwworm, Cochliomyia macellaria, the green bottle fly, Phaenicia sericata, the sheep blow fly, Lucilia cuprina and the Mexican fruit fly, Anastrepha ludens. The length of time the embryos must be exposed to hexane with or without a small amount of alcohol in order to attain permeability was species-dependant. Long-chain n-alkanes comprised the major lipid class removed from vitelline membranes of all species except A. ludens where 2-methylalkanes were the major class. The range in size by the total number of carbon atoms in the hydrocarbons was: C23-C49 in C. hominivorax, C27-C33 in C. macellaria, C24-C35 in L. cuprina, C25-C36 in M. domestica, C25-C33 in P. sericata and C21-C51 in A. ludens. The major hydrocarbon component, expressed as percent of the total hydrocarbons, was n-nonacosane (C29) in C. hominivorax (40%), C. macellaria (43%), L. cuprina (38%), M. domestica (39%) and P. sericata (60%). However, in A. ludens, 2-methyloctacosane (32%) was the major hydrocarbon. Unsaturated hydrocarbons, monoenes (16%) and dienes (11%), were abundant only in A. ludens. Since prior studies indicated that the length of time the embryos must be exposed to hexane with or without a small amount of alcohol in order to attain permeability is species dependant, we suggest that the differences in hydrocarbon composition may contribute to this variation in lipid extractability.