Virgin Flies Research Articles

Courtship of Drosophila Melanogaster in Large Observation Chambers: the Influence of Female Reproductive State

Abstract1. Mixed sex groups of D. melanogaster were observed in large observation chambers in which food was available. The ratio of mated to virgin females was 3:1. 2. Fertilised females spent more time feeding than virgins and virgin flies spent more time in locomotor activity. 3. Most courtship behaviour took place on the food and consequently mated females received more courtships. 4. To test whether fertilised females were excluding virgin females from the food, groups of females without males were observed. The groups were composed either of virgin, or of a mixture of virgin and mated females. The results showed that even in the absence of fertilised females virgins fed less and were more active. There was no evidence that mated females excluded the virgins. 5. The adaptive significance of the observations is discussed in relation to dispersal from the site of eclosion, oviposition and remating.

The Structure of Courtship in the Drosophila Melanogaster Species Sub-Group

[A quantitative comparison and sequence analysis of courtship behaviour of four-day old virgin flies from seven species of the Drosophila melanogaster species subgroup is described. In three species (D. melanogaster, D. simulans and D. mauritiana) courtship was observed until copulation. In four species (D. yakuba, D. teissieri, D. erecta and D. orena) courtships were truncated after a standard period. Wing rowing by the male was seen in four species (simulans, mauritiana, yakuba and teissierz). In both mauritiana and simulans the proportion of time spent in rowing tended to increase as courtship proceeded. The four species showing the greatest proportion of wing vibration in courtship (melanogaster, yakuba, erecta and orena) also showed an increase in the proportion of time spent vibrating as courtship proceeded. The amount of wing vibration and wing extension in yakuba is greater than in any other species in the subgroup. The proportion of time spent in wing extension in yakuba declined whilst vibration increased during courtship. In six species the proportion of courtship time spent licking increased during courtship. The mean lick duration was longer in yakuba than in any other species of the subgroup. When males were following actively moving females the time they spent performing courtship behaviours and the mean bout lengths were shorter than in courtship as a whole. D. simulans males expressed scissoring at a low level when courting moving females. Females of all species preened more than the males, but preening during courtship appeared to have no excitatory or inhibitory effect on the male. The behavioural sequence characteristic of the species subgroup is orientation→wing display→licking→ copulation. In five species wing vibration was the only element of wing display immediately to precede licking, but in simulans licking could also be preceded by scissoring and in yakuba by wing extension. The transition vibration→licking appears to be a characteristic of the subgroup., A quantitative comparison and sequence analysis of courtship behaviour of four-day old virgin flies from seven species of the Drosophila melanogaster species subgroup is described. In three species (D. melanogaster, D. simulans and D. mauritiana) courtship was observed until copulation. In four species (D. yakuba, D. teissieri, D. erecta and D. orena) courtships were truncated after a standard period. Wing rowing by the male was seen in four species (simulans, mauritiana, yakuba and teissierz). In both mauritiana and simulans the proportion of time spent in rowing tended to increase as courtship proceeded. The four species showing the greatest proportion of wing vibration in courtship (melanogaster, yakuba, erecta and orena) also showed an increase in the proportion of time spent vibrating as courtship proceeded. The amount of wing vibration and wing extension in yakuba is greater than in any other species in the subgroup. The proportion of time spent in wing extension in yakuba declined whilst vibration increased during courtship. In six species the proportion of courtship time spent licking increased during courtship. The mean lick duration was longer in yakuba than in any other species of the subgroup. When males were following actively moving females the time they spent performing courtship behaviours and the mean bout lengths were shorter than in courtship as a whole. D. simulans males expressed scissoring at a low level when courting moving females. Females of all species preened more than the males, but preening during courtship appeared to have no excitatory or inhibitory effect on the male. The behavioural sequence characteristic of the species subgroup is orientation→wing display→licking→ copulation. In five species wing vibration was the only element of wing display immediately to precede licking, but in simulans licking could also be preceded by scissoring and in yakuba by wing extension. The transition vibration→licking appears to be a characteristic of the subgroup.]

The quantitative relationship between life span, food ingestion, egg production, mating, and flight-activity of protein-fed blowfly Phormia terrae novae females

The age-dependent variations in the amount of food ingestion, egg production, and flight activity of protein-fed female Phormia terrae novae were analyzed. Also the influence of mating and forced physical performance on the time course of these parameters and on life span were tested. In virgin females, the life span with the investigated factors is generally delayed, together with mean and maximum life span. In comparison to mated females virgin females exhibit a 40% prolongation of mean life span and ingest more sugar with a later intake maximum. Their egg production starts 5 days later (Day 10) but reaches only half the amount occurring in mated females. Flight performance is generally higher in virgin females, reaching its maximum 2 days later (Day 14), with a more gradual decline instead of the sudden drop in flight performance seen in mated flies. Females receiving their food by flight only ("forced fliers") show a shorter mean life span (which is more pronounced in virgin flies). The maximum of sugar intake shifts forward to an earlier point after eclosion, and the amount is reduced by about 30% over the entire life span. Similarly, the egg production rate reaches lower values and tends to have an earlier maximum in comparison to corresponding groups that had access to sugar on the cage floor. The results further support the hypothesis that Phormia exhibits a strictly programmed aging pattern. Its time course depends on reproduction sufficiency and physical performance. Both factors act to shorten life expectation.

Male Esterase 6 Catalyzes the Synthesis of a Sex Pheromone in Drosophila melanogaster Females

Esterase 6, a component of the seminal fluid of Drosophila melanogaster males, hydrolyzes cis-vaccenyl acetate, a lipid made only by males, to cis-vaccenyl alcohol. This reaction occurs in the female reproductive tract and is virtually complete within 6 hours after copulation. Both the alcohol and the acetate decrease the number of matings among pairs of virgin flies in which the female is treated topically with these substances. Although females tested 10 minutes after copulation elicit less courtship than virgin females, females tested 6 hours after copulation stimulate even less courtship if they received active esterase 6 in the seminal fluid of their respective mates. Either the alcohol or a derivative appears to be an antiaphrodisiac that decreases courtship elicited by inseminated females and thus reduces the probability of further mating. Thus the activity of the pheromone depends on a final reaction which occurs in the female, using both substrate and enzyme provided by the male.

In vitro activation of Drosophila eggs

Mature ovarian eggs of Drosophila can be activated by treatment with hypotonic buffers. Well-fed, 4-day-old virgin flies contain large numbers of partially dehydrated mature eggs. When these eggs are transferred to a hypotonic culture medium, the ovarian eggs swell immediately and within minutes up to 70% become impermeable. The following cellular events ensue: meiosis, which had been arrested at metaphase I, is completed and the “polar body” nuclei fuse; cortical multivesicular bodies with acid phosphatase activity appear within minutes; polar granules fragment, dissociate from mitochondria, and become associated with polysomes; finally, monosomal ribosomes move to the polysomal region of a sucrose gradient. Each of these events corresponds to the normal in vivo effects of ovulation of oocytes, whether or not they are fertilized. When ovarian eggs of a parthenogenetic strain of D. mercatorum were activated by hypotonic treatment, some eggs developed into normal embryos. The presence of high potassium, low pH, and polyethylene glycol enhanced the frequency of normal development. Thus, we suggest that the rehydration of mature oocytes, as they move from the ovary to the uterus, activates the maternal program of the oocyte.

Mating Behavior of Wild Anastrepha fraterculus (Diptera: Tephritidae) on a Caged Host Tree

The mating behavior of the South American fruit fly, Anastrepha fraterculus (Wiedemann) is described, following release of wild virgin flies onto a field-caged host tree (guava). Sexual encounters and attempted copulations occurred almost exclusively from 0700-0900 hours and were confined to leaf nodes or the bottom surface of leaves, where males stationed themselves, often forming leks, and appeared to emit a sex pheromone which was attractive to virgin females. There were no male visitations or sexual encounters on green guava fruit, even though females, after mating, frequently oviposited there.

Some effects of prolonged virginity on feeding and milk-gland activity in females of the tsetse Glossina austeni (Newst.)

The effects of virginity on feeding and milk-gland (MG) activity were investigated in 4-week-old virgin females of Glossina austeni. The flies take in smaller but constant amount of blood roughly equivalent to their own weight during each feed. This syndrome of feeding to a constant weight with time leads to fatbody hypertrophy largely because of decreased metabolic demands due to the absence of a larva. It is suggested that the small blood-meals of virgin females are probably related to the space limitations in their abdomens, and are probably hormone-mediated, possibly operating via the cuticle. The MG of virgin flies is well-differentiated but the cycles of synthetic activity which the distal MG tubules exhibit in mated flies during a pregnancy cycle are not so apparent in virgins. The secretions of this gland are used to nourish the developing larva and if the MG were very active the secretions produced would be lost. It is concluded that in virgin females the blood-meal is reduced in size, probably as a result, at least partly, of a reduction or absence of neurosecretory out-flow related to mating and ovulation, and the products of their blood-meal apparently are diverted not to the MG but to the fatbody for storage. These effects of virginity on blood-meal size, fatbody and MG activity can be reversed at any time by mating.

Long‐term effects of diet on the neuroendocrine system of the sheep blowfly, Lucilia sericata

ABSTRACT. The medial neurosecretory cells (MNC), corpus allatum (CA) and ovaries of Lucilia sericata Mg. are quiescent in mature flies fed on a diet of sugar and water, but these tissues rapidly respond to the ingestion of a protein meal. The present study revealed that after oogenesis, virgin flies synthesized and stored neurosecretory material, but this activity declined as they grew older. At the same time, the CA, following a decrease in size during the latter stages of oogenesis, became enlarged again. This is interpreted as denoting synthesis but not release of CA hormone. In meat‐fed mated flies, cycles of oviposition and oogenesis were reflected in the rise and fall in activity of the MNC and CA. The MNC and CA of male flies also responded to the ingestion of a protein meal, and the activity of these tissues persisted for several days before it declined. The similar response of the MNC and CA in both males and females demonstrates the importance of a meat meal to reproduction in this species: in females it is required for oogenesis, but its ingestion by both sexes is necessary for overall reproductive success.

Protein synthesis inhibition alters Drosophila mating behavior

Fruit fly Drosophila pseudoobscura virgin AR females mate preferentially with AR males when given a choice between AR and or males. However, AR females which mated with or males when young show a significant change in sexual selection in favor of or over AR males in subsequent simultaneous choice tests. Ingestion of food moistened with 4 ml cycloheximide (400 μg/ml) produces 75% protein synthesis inhibition in the female flies. Females exposed to cycloheximide (CXM) immediately after their initial copulation with or males resemble virgin flies in their choice of mates, and mate preferentially with AR males. Females exposed to CXM before their initial copulatory experience with or males resemble or-experienced but untreated (no CXM) flies in their choice of mates, and mate preferentially with or males. The change in sexual preference shown by females with prior copulatory experience resembles learning in that it is subject to disruption by CXM in ways analogous to those reported in the literature.

Aging and frequency-dependent mating in Drosophila.

Frequency-dependent mating behavior has been observed in Drosophila pseudoobcura, but most experiments have used only 4-day-old virgin flies. One report has indicated that rare-male behavior may be affected by age. Males and females of the Arrowhead strain along with rare orange-eyed males were used in the present study to confirm that frequency-dependent mating behavior is operating with 4-day-old females but is absent by 11 days. The same situation is also observed when Chricahua males and females are used along with rare Arrowhead males.

Electrical properties of the Drosophila egg membrane

Electrical properties of the egg membrane of Drosophila melanogaster were examined using intracellular microelectrodes. Unfertilized eggs and fertilized eggs for the period up to the syncytial blastoderm stage were used. Among Na, K, and Cl, K was most permeant to the membrane. The K permeability, however, did not completely determine the membrane potential. The resting potential in standard solution was −63.5 ± 8.0 mV (mean ± SD) in unfertilized eggs collected within 2–3 days after virgin flies started to lay eggs. The resting potential in fertilized eggs was −27.0 ± 8.4 mV within 20 min after egg deposition, while it was −55.1 ± 6.5 mV at the syncytial blastoderm stage. These changes at different developmental stages were associated with changes in the K-dependence of the membrane. The larger amplitude of the resting potential was suggested to be due to increased K permeability but not to decreased nonspecific leakage. The current-voltage relation was linear throughout the stages examined. Action potentials, such as those in eggs of other animals, were not observed. High Ca media significantly increased the amplitude of the resting potential associated with increase in the membrane resistance. A remarkable nonlineality in the I–V relation appeared in high Ca media, which caused continuously increasing hyperpolarization during sustained inward current. Eggs of temperature-sensitive mutants, shi ts1 and para ts1 showed properties similar to those in wild-type eggs with transient temperature changes.

Effects of Parasitization by the Nematode, Heterotylenchus autumnalis, on Mating and Oviposition in the Host, Musca autumnalis

Male face flies parasitized by the nematode Heterotylenchus autumnalis are capable of mating and inseminating females. However, less than 50% of the flies inseminated by infected males oviposit. Female hosts are castrated by the nematodes which penetrate and destroy the ovaries. Although the testes are not destroyed by the nematode, acessory glands are lacking in many heavily infected males 9 to 12 days old. The fate of the nematode in male hosts appears to be a dead end, since there is no natural means for the parasite to exit from male flies. Mating appears to be necessary for infected females to deposit nematodes. Heterotylenchus autumnalis is a nematode parasite of the face fly, Musca autumnalis, one of the leading pests of dairy and beef cattle in the United States and Canada. The life cycle of the parasite involves an alternation of gamogenetic and parthenogenetic stages, with infection of host larvae occurring in bovine feces, the breeding site of the fly (Stoffolano and Nickle, 1966; Nickle, 1967). Female hosts are castrated by the larval nematodes which penetrate and destroy the ovaries of the flies. Infected female flies go through a mock and deposit larval nematodes, instead of fly eggs, into manure. Heterotylenchus autumnalis may prove to be an effective biological control agent against M. autumnalis since the nematode sterilizes female flies and is naturally disseminated by the host. There is also some evidence to suggest the parasite is host-specific (Jones and Perdue, 1967; Stoffolano and Streams, 1971; Nappi and Stoffolano, 1971, 1972; Stoffolano, 1973), but additional experimental evidence is needed to determine the relationships of the nematode with other dung-breeding Diptera. Information is also needed concerning the fate of the nematode in male flies, and the effects of parasitization on the reproductive biology of the host. The present investigation was undertaken to determine whether nematode-infected male face flies could successfully mate and inseminate female flies, and if mating stimuli were necessary for parasitized flies to deposit nematodes. Since the ovipositor of the female is Received for publication 16 July 1973. positioned within the genital atrium of the male during copulation, the possibility of a transfer of infection was also considered. MATERIALS AND METHODS Fa e flies used in this study were maintained at 27 ? 2 C, 16 hr photophase, and 50 to 55% relative humidity. The adult flies were fed powdered milk, sugar, and water. Fresh bovine feces was used as the oviposition and larval rearing medium. The nematodes were obtained from a laboratory colony of infected face flies maintained at the Entomology Research Division, USDA, Lincoln, Nebraska. To propagate the nematodes the abdomens of 12-day-old infected adult flies were dissected and the parasites spread over the surface of manure containing face fly larvae. Infected and noninfected flies were reared separately. To obtain virgin flies of known age, fly pupae were individually placed into glass vials and covered. Following eclosion the flies were sexed, removed from the vials, and transferred to separate wire-screen cages. Mating experiments were performed with 7-day-old females and infected males 5 to 12 days old. Each pair of flies was placed within a screen cage for 24 hr to allow for mating. No individual was used more than once. After 24 hr the males were dissected to determine whether they were parasitized. Female flies that had been paired with infected males were each reared in isolation and provided with fresh bovine feces twice a week for a period of 2 weeks. The surface of the manure was examined to determine the presence of eggs. Females that did not oviposit and produce offspring were dissected or examined histologically for the presence of spermatozoa in the spermathecae. For histological examinations the flies were fixed in either Kahle's or Mahdissan's solution (8 hr), cleared in 2 changes of dioxane (6 to 8 hr each), and vacuum infiltrated in a paraffin oilParaplast series for 1 to 2 days. Longitudinal

Setiferous Plaques on Antennal Pedicels of Muscoid Diptera: Appearance in Various Species and Tests of Function2

The antennal pedicel of the green bottle fly Phoenicia sericata (Meigen) possesses a dozen or so plaques, each surmounted by a bulbous seta and containing a tuft of microsetae. Twenty-nine muscoid species and one drosophilid have been studied with the scanning electron microscope to ascertain the presence or absence of these structures, interspecific differences, and intraspecific variation. Green bottle flies with Vaseline™ -coated pedicels behaved like controls in response to 5% acetic acid, 0.1% trimethylamine, or 0.1% indole in T-tube preference tests. Treated house flies, Musca domestica L., responded like controls to 3 concentrations of ammonia. Virgin, treated green bottle flies of either sex responded like controls when virgin flies of either sex were used as the attractant. These results suggest that the structures on the pedicel do not have an olfactory function.

Effect of blood intake of Glossina austeni Newst. on pupal weights in successive reproductive cycles

Ten fertilised female Glossina austeni Newst. reared from pupae with a weight range of 17–33 mg were maintained singly in small cages, and were offered food six days per week. The mean data for the flies were 151 days life span, 14–5 pupae per female and 25–4 mg pupal weight. One fly showed an abnormal digestive condition, and several failed to larviposit at the end of a cycle. The weight of pupae produced was related to the total amount of blood ingested in each normal cycle. A comparison between fertilised and virgin flies indicated that 50% of the blood ingested by the former was for pupal production. Large flies took larger blood-meals and fed less frequently than small flies.

Effect of gamma-radiation on the tsetse fly Glossina Morsitans Westw

Puparia of Glossina morsitans Westw. were collected in the Zambesi valley and contained developmental stages from final phases of third-instar larvae to pupae near to eclosion. These puparia or the emerging adults were exposed to gamma-radiation from a Co60 source at rates between 54 and 122 rad/min. Effective mortality in the pupal stage increased from 23 per cent, at 2,000 rads to 64 per cent, at 15,000 rads, while those deposited in spring (August-October) were more sensitive to gamma-rays than those collected in winter (May-July).Under described test conditions, 25 pairs of untreated flies produced a mean of 21 pupae in a 28-day test period, insemination averaged 95 per cent., and 56 and 62 per cent, of the males and females survived, respectively. Continuous irradiation reduced reproduction by over 95 per cent, between 8,000 and 15,000 rads wiih males emerging within the first two weeks after treatment; while males emerging during the third post-treatment week were completely sterilised with dosages above 4,000 rads. Treatments given as fractions one and four days apart gave a slightly higher level of residual fertility than that obtained with similar but continuous total dosages. Irradiation of adult males reduced reproduction to an extent comparable with that observed in males emerging from pupae within two weeks of treatment (> 90 per cent, from 8,000 rads). Females treated either as adults or as pupae were completely sterilised with doses of 1,000 to 8,000 rads. Males exposed to 8,000 and 12,000 rads retained their sterility throughout 39- and 45-day test periods, and were fully competitive with untreated males.Survival of virgin flies emerging from treated pupae was reduced both by increasing dosage from 4,000 to 15,000 rads, and decreasing pupal age at the time of treatment. Mortality during the 28-day mating period decreased with 1,000 and 2,000 rads, increased up to 8,000 rads, and apparently decreased with 12,000 and 15,000 rads. When the doses were fractionated, survival was longer than when treatment was continuous, and mortality was reduced to the level expected from the size of the first fraction. Virgin adult males given doses between 4,000 and 18,000 rads, and females given 2,000 and 4,000 rads, lived longer than the corresponding controls.The ability of the males to inseminate females was not affected by any of the treatments, and sperm from irradiated males were mobile and apparently behaved normally. Dominant lethality in the males was expressed by the failure to produce pupae in matings with untreated females, and in the treated female fly by the complete failure of oogenesis.

Biology of a California rodent bot fly Cuterebra latifrons Coquillett (Diptera: Cuterebridae).

The behavior of Cuterebra Latifrons adults was observed at hilltop aggregation sites in Marin County, California, from June through October with peak periods in late August. Male flies were active only in the morning and were spaced by their territorial behavior. Resighting of marked male fliles indicated that they remained on the hilltops for their lifetime of 10–12 days. Female flies made up 5% of the population, staying at these sites only long enough to mate. Virgin flies did not move to hilltops until they were 5 days old and ready to oviposit. The average fecundity of each female was 1,300 eggs laid in groups of about 7 per site. Oviposition sites were located at woodrat (Neotoma fuscipes) houses, and eggs appeared to survive winter environments. Eggs developed in 1–2 weeks depending on temperature, and infective larvae remained inside the chorion until stimulated to hatch by a slight, Sudden increase in temperature. Seasonal peaks in numbers of infected woodrats appear related to host activities. Infective larvae entered moist natural openings or abraded skin and appeared at dermal developmental sites 6–8 days after infection. First stage larvae cut the warble pores and molted in the ventral cervical region of the host. The larva, inside a warble cyst, molted again about the 19th day and remained in the host 28–38 days. Mature larvae pupated in 12–24 hrs in damp soil and the pupual period lasted from 28 to 178 days depending on temperature and season of the year. Both sexes required equal time in the pupal stage. Adults emerged in the morning and were reluctant to fly on the day of emergence. In the San Francisco Bay area, C. latifrons is probably bivoltine.

The susceptibility of tsetse flies to topical applications of insecticides. III.—The effects of age and pregnancy on the susceptibility of adults of Glossina morsitans Westw

Solutions of dieldrin, γ BHC, p, p′DDT and Baytex were applied topically to pregnant females of Glossina morsitans Westw. and also, in most cases, to fertilised flies over 21 days old and not noticeably pregnant, to virgin flies over 21 days old, and to males over 18 days old. Pregnant flies required about nine times as much dieldrin as young flies for comparable mortalities, and non-pregnant fertilised flies about four times as much. It was not possible to determine proper regression lines for other insecticides and classes of flies but significant increases in tolerance, as compared with that of young flies, were shown by pregnant flies to γ BHC and DDT, by old virgins to dieldrin and DDT but not γ BHC, and by fertilised flies to γ BHC and DDT. Pregnant flies and virgins showed no increased tolerance to Baytex, and old males showed none to dieldrin, γ BHC or DDT.These results are discussed in relation to aerial spraying of tsetse habitats and it is suggested that they explain some unexpected past results.

Non-Disjunction Produced by Carbon Dioxide

THE abnormal mitoses seen in tissue cultures kept under high tensions of carbon dioxide were described in a communication in NATURE of Mar. 17, 1928, p. 420 (see also Brit. J. Exp. Path., 9, 240; 1928). Fragmentation of the chromosomes and irregular migration of the chromatin to the controsomes were seen, exactly similar to the mitotic changes observed many years previously in the eggs of Ascaris megalocephala subjected to radium (Arch. Middx. Hosp., 30, 98 1913). These observations suggested that carbon dioxide might produce hereditary disturbances, when applied to germ cells, analogous to those produced by X-rays and radium. In order to test this, observations on non-disjunction in Drosophila have been made, in which newly hatched virgin red-eyed flies were subjected to either pure carbon dioxide for a few hours, or to varying mixtures of air and carbon dioxide for longer periods of time. They were then crossed with white-eyed males, and allowed to breed.