Identification of a novel <Emphasis Type="Italic">Drosophila</Emphasis> gene, <Emphasis Type="Italic">beltless</Emphasis>, using injectable embryonic and adult RNA interference (RNAi)

Background

RNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype.

Results

Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts.

Conclusions

We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system.

     

Observations on the oviposition behaviour ofGoniozus sensorius [Hymenoptera: Bethylidae] a parasite ofDiaphania indica [Lepidoptera: Pyralidae]

Goniozus sensorius Gordh (Hymenoptera: Bethylidae) was recorded as a major parasite of the pumpkin caterpillar,Diaphania indica (Saunders) (Lepidoptera: Pyralidae). This paper provides detailed information on the oviposition behaviour ofG. sensorius. Prior to oviposition, the parasite temporarily paralyses the host larva. The paralysed condition lasts for about 2 h. The egg laying process on each larva requires 30 to 60 min. The maximum number of eggs are laid on the 6^th and 7^th segment, and none in the terminal segments. Generally, oviposition is restricted to 1 segment of the host larva and occasionally on 3 segments. The maximum number of eggs are laid on the 2^nd and 3^rd day after emergence and the mean number of eggs laid on each host larva was 7.1.     

Biology of the flea beetle,Altica carduorum [Col.: Chrysomelidae] on Canada thistle (Cirsium arvense) in South Dakota

A laboratory colony of 50 adults ofAltica carduorum Guérin-Méneville was established at South Dakota State University. Beetles reared in this colony had an average preoviposition period of 7 days when exposed to a regular cycle of 16 hr of light (24°C) and 8 hr of darkness (12.7°C). The laboratory reared females, whose longevity averaged 100 days, laid an average of 259.3±9.7 eggs. High temperatures and/or low RH are limiting factors to beetle survival. In the field eggs were laid throughout June on the underside of leaves along the edges of the veins. Adults fed throughout the summer and then over-wintered in the soil. Some of these beetles emerged the following spring and laid viable eggs. Factors limiting establishment in South Dakota, however, were high temperatures, low humidities, and the predators,Lebia viridis Say andHarpalus pennsylvanicus Degeer.A. carduorum originates from the Swiss Rhône valley where there is a relatively “continental” climate, but is limited to special habitats where comparatively high humidity occurs. South Dakota climate is characterized by cold winters, and hot, dry summers, therefore establishment would be most difficult.     

DNA synthesis in developing two-cell mouse embryos

Mated CF1 (Carworth) female mice were sacrificed at 2 hr intervals between 29 and 43 hr after human chorionic gonadotrophin (HCG) administration. One- and two-cell eggs were incubated in [³H]thymidine for 1 hr. Labeled two-cell embryos were first observed at 31 hr and reached a maximum number at 35 hr. The S period is approximately 6 hr in duration. Although both blastomeres were labeled in most cases, embryos with only one labeled blastomere were more numerous at later times. In vitro labeling was corroborated by injecting [³H]thymidine directly into the isthmic portion of the oviduct. Embryos usually complete the second cleavage division 18–20 hr after onset of DNA synthesis. The cell cycle at the two-cell stage is thus characterized by a G₁ of close to 1 hr, a 6 hr S, and a G₂ of about 12 hr.Embryos developing in vitro frequently fail to progress beyond the two-cell stage. The block is not due to absence of DNA synthesis since these embryos were found to incorporate [³H]thymidine.     

Oviposition Site Selection by the Regal Fritillary, Speyeria idalia, as Affected by Proximity of Violet Host Plants

Selection of oviposition sites by female regal fritillary butterflies, Speyeria idalia (Drury), in relation to the location and abundance of their larval food plant, Viola pedatifida G. Don, was studied in Kansas tallgrass prairie. To identify potential cues that females use to select oviposition microsites, we compared plots in which eggs were laid with paired control plots in terms of violet abundance, distance from plot center to the nearest violet plant, plant species composition and relative abundance, plant diversity (Shannon's index), and average plant canopy height. No significant differences occurred between control and oviposition sites for any of these variables (P > 0.15). Violets were present in only 30% of the 1-m-diameter oviposition plots, compared with 24% of the corresponding controls. Furthermore, eggs were never laid directly on violets even when they were present nearby. These data imply that S. idalia females do not preferentially orient to or oviposit on their larval food plant. Nevertheless, female S. idalia did not oviposit randomly, for they invariably chose microsites near the ground that were shaded by the grass/forb overstory and usually deposited eggs on the underside of dead vegetation. We speculate that environmental stresses such as high temperatures and intense solar insolation during the egg stage or harsh overwintering conditions experienced by first-instar larvae impact survival and influence choice of oviposition sites. High fecundity (more than 2000 eggs in some individuals) may allow this species to use a conditional, sweepstakes strategy, i.e., producing and spreading many eggs over prairie that harbors host plants to compensate for low egg and larval survivorship.     

Larval diapause in the mosquito aedes triseriatus: effects of diet and temperature on photoperiodic induction

Larval diapause was induced in both a northern (Ohio) and a southern (Alabama) strain of Aedes triseriatus by photoperiodic treatment of the larval stage itself. The effect of a short-day (10 hr) photoperiod during larval development was cumulative and resulted in failure of the fourth instar to pupate. The larvae, which had been obtained from long-day (16 hr) eggs, underwent diapause when their development was slowed by a low-quantity diet or low temperature (16°C) for a sufficient number of days to allow the short-day photoperiod to exert a visible effect.     

Effect of hydration,photoperiod and temperature on diapause termination in eggs ofPetrobia (Tetranychina) harti (Acari: Tetranychidae)

Petrobia harti (Ewing) displays a facultative summer diapause in the egg stage. An adult female will lay only either diapause or non-diapause eggs throughout her life. In the laboratory, diapause eggs are laid by females which develop on detachedOxalis articulata leaves under long-day photoperiods and a relatively low temperature of 19±1°C.Diapause occurs in a stage of advanced embryonic development, in which the embryo appears U-shaped when observed from the egg's ventral side. Embryonic development ceased at this stage, and no further growth occurred when the eggs were kept under a relative humidity of about 70% in various photoperiod and temperature conditions. However, when the eggs were hydrated by placing them on wet cotton wool, development in some embryos (apparently in those which had completed their diapause development) proceeded beyond the U-stage at a rate similar to that in non-diapause embryos and the eggs hatched.Under LD 168 and 19±1°C or 26±1°C, the later from oviposition the period of egg hydration started, the higher the percentage of diapause termination. Under LD 168 and 26±1°C, diapause termination occurred mostly during the first week of hydration, while at 19±1°C mostly during the second and third week.At 26±1°C, in eggs hydrated 15 days but not 30 days from oviposition, the percentage of diapause termination was higher under a long-day than under a short-day photoperiod.Under LD 168, when the eggs were hydrated continuously from oviposition or starting 15, 30 and 45 days from it, the percentage of diapause termination was higher at 26±1°C than at 19±1°C.The percentage of diapause-laying adult females and the intensity of egg diapause were higher when the pre-imaginal mites grew at LD 1212 and 19±1°C, than when they grew at LD 168 and 26±1°C. This maternal effect on egg diapause intensity was expressed when the eggs were maintained at LD 1212 and 19±1°C but not at LD 168 and 26±1°C.     

Temporally uncontrolled expression of linearized plasmid DNA which carries bacterial chloramphenicol acetyltransferase gene withXenopus cardiacα-actin promoter after injection intoXenopus fertilized eggs

Summary Circular and linearized plasmid DNA which contained bacterial chloramphenicol acetyltransferase (CAT) gene connected toXenopus cardiac-actin promoter was injected intoXenopus fertilized eggs to study their expression in the course of early embryonic development. While circular DNA was slightly replicated and expressed only after embryos reached neurula stage, linearized DNA formed a large amount of concatemers, and was expressed as early as at blastula stage, or about 14 hr earlier than the time of circular DNA expression. Similarly earlier expression of linearized DNA occurred slightly more strongly when the DNA was injected into presumptive dorsal than in ventral blastomeres at 4-cell stage, and the expression was not affected when embryos were dissociated at blastula stage and their cells were cultured under reaggregating or nonreaggregating conditions. These results show that although circular actin-CAT fusion gene is expressed during development according to endogenous temporal control, the expression of linearized DNA deviates from such developmental control even though it contains intact promoter of-actin gene. It is then recommended that study of the control of the expression of exogenously-introduced DNA inXenopus fertilized eggs should be carried out with circular but not linearized plasmids.     

Lethal stage of female embryos in crosses between two local populations of the spider mite,Tetranychus quercivorus Ehara et Gotoh (Acari: Tetranychidae)

Unidirectional incompatibility has been known in the spider mite, Tetranychus quercivorus Ehara et Gotoh. In a cross between females from Tsukuba (36°N) and males from Sapporo (43°N), most eggs obtained hatched and reached adulthood with a female-biased sex ratio, whereas in the reciprocal cross, almost all fertilized eggs died and an extremely male-biased sex ration occurred. Embryonic development was examined to determine the lethal stage of hybrid eggs obtained from the incompatible cross (Sapporo X Tsukuba ). Development of normal embryos was divided into 18 stages, which were described and illustrated. In the incompatible cross, most embryos attained the eye-spot stage without showing any abnormality, but the majority of them stopped their growth then and died at this stage.     

γ-Glutamyltranspeptidase in echinoderm eggs and larvae: fertilization-dependent and developmentally-induced changes in specific activity

• 1.1. γ-Glutamyltranspeptidase is present in echinoderm eggs and larvae: in homogenates the level of activity is comparable to that of rat cerebral cortex.
• 2.2. In eggs of Lytechinus pictus, fertilization induces an early rapid and sustained (5 min–6 hr) 37% increase in the activity of γ-glutamyltranspeptidase in homogenate fractions.
• 3.3. Relative to these homogenate levels, the specific activity of γ-glutamyltranspeptidase are ≈60% lower in 40,000 g supernatant fractions and 2.7-fold higher in 40,000 g particulate fractions in both unfertilized and 15 min post-fertilized Lytechinus pictus eggs.
• 4.4. The subcellular distribution of γ-glutamyltranspeptidase is the same in both unfertilized and 15-min post-fertilized Lytechinus pictus eggs: 78% in 40,000 g particulate fractions, 22% in 40,000 g soluble fractions.
• 5.5. In both unfertilized and 15 min post-fertilized eggs of Lytechinus pictus the enzyme responds to heat (50 vs 37°C) by activation in a similar manner: 1.72- and 1.68-fold homogenates; 2.6- and 3.0-fold in supernatants; 1.97- and 1.90-fold in particulate fractions.
• 6.6. In homogenates of Pisaster ochraceous larvae, γ-glutamyltranspeptidase activity increases steadily during the course of larval development: relative to the low activity at day 5, activities exhibit an increase of 1.2-, 2.0-, 3.1- and 5.4-fold at days 10, 16, 22 and 28, respectively.

     

Reproduction and development in some species of the weakly electric genus <Emphasis Type="Italic">Campylomormyrus</Emphasis> (Mormyridae,Teleostei)

Reproduction in captivity of four species of the mormyrid genus Campylomormyrus was investigated. Cyclical reproduction was provoked by changing water conductivity (C) alone: decreasing C led to gonadal recrudescence, an increase induced gonad regression. Data on the reproduction and development of three species are presented. All three species are indeterminate fractional spawners. Spawning intervals ranged from 6 to 66 days in C. rhynchophorus, 10–75 days in C. tshokwe, and 18 days in C. compressirostris (calculated values). Fecundities (eggs per fractional spawning) ranged from 70 to 1570 eggs in C. rhynchophorus, 100–1192 in C. tshokwe, and 38–246 in C. compressirostris. Spawnings/ovipositions occurred during the second half of the night; no parental care was observed; no special spawning substrates were necessary. C. compressirostris successfully spawned in breeding groups, C. rhynchophorus as pair. Agonistic behavior in the C. tshokwe pair forced us to divide the breeding tank; therefore, only ovipositions occurred. However, injection of an artificial GnRH hormone allowed us to obtain ripe eggs and sperm and to perform successful artificial reproduction. All three species produce yolky, slightly sticky eggs. Egg diameter ranges from 2.3–3.0 mm. Hatching occurred on day 3, feeding started on day 11. Transition from larval to juvenile stage occurred at around 20 mm total length (TL). At this size C. rhynchophorus developed a higher body than the two other species and differences between the species in the melanin pigmentation of the unpaired fins occurred. Between 32 and 35 mm TL the upper and lower jaws started to elongate.     

The Larval Development of the Chiton <Emphasis Type="Italic">Deshayesiella curvata</Emphasis> (Carpenter in Dall, 1879) (Mollusca: Polyplacophora)

Chronological study of the larval development of the chiton Deshayesiella curvata with sketching of the first stages of egg division and embryos up to the seven-valved shell stage is presented for the first time. D. curvata exhibits pelagic development with lecithotrophic larvae. Larval eyes form at the age of 3 days; at the age of 6 days, a Schwabe organ appears near the eyes in the form of two pigmented spots. Chitons at the stage of seven-valved shell have larval eyes, as well as the Schwabe organ.     

Expression of the zygotic genome in blastoderm stage embryos ofDrosophila: Analysis of a specific protein

Summary The synthesis of a protein which has been detected in blastoderm cells but not in pole cells (Gutzeit and Gehring 1979) has been studied further by means of two-dimensional gel electrophoresis. This protein could not be detected at the nuclear multiplication stage. The protein is translated from mRNA which is transcribed at the blastoderm stage since it is not synthesized in detectable amounts when embryos are injected with -amanitin prior to the blastoderm stage. Also the protein could not be detected when RNA from freshly laid eggs was translated in vitro. Embryos from females which are homozygous for the mutationmat (3) 1 form pole cells but no blastoderm cells (Rice and Garen 1975). Thesemat (3) 1 embryos, as we will call them in this report, express the protein if aged for a period of time sufficient for completion of blastoderm cell formation in control wild-type embryos.mat (3) 1 embryos and embryos injected with -amanitin show the same syndrome of visible developmental anomalies; however, the studied protein could only be detected inmat (3) 1 embryos but not in -amanitin injected embryos.Supported by the DFG, SFB 46     

Germ-line chimera by lower-part blastoderm transplantation between diploid goldfish and triploid crucian carp

Germ-line chimerism was successfully induced by blastoderm transplantation from donor triploid crucian carp, which reproduces gynogenetically, to recipient diploid goldfish, which reproduces bisexually. Lower part of donor blastoderm including primordial germ cells (PGCs) was sandwiched between recipient blastoderm at the mid- to late-blastula stage. When donor grafts were prepared from intact embryos or ventralized ones by removing vegetal yolk hemisphere at the 1- to 2-cell stage, malformations including double axes were observed in the resultant chimeras transplanted with grafts from intact embryos at the hatching stage, while a few malformations in those from ventralized embryos. PGCs originated from donor grafts were observed around the gonadal anlage at 10 days post-fertilization in chimeras. When ploidy of erythrocytes and epidermal cells in chimeric fish was examined by flow-cytometry, no triploid cells were detected at 1- and 5-year-old chimeras. Three-year-old chimeric fish (n=5) laid eggs originated from the donor together with those from the recipient. The frequency of eggs from the donor crucian carp blastoderm varied from 3.1 to 89.3% between chimeras.     

The effects of juvenile hormone analogues on the embryogenesis of Drosophila melanogaster

Two juvenile hormone analogues (JHA), 6,7-Epoxy-3-methyl-7-ethyl-1-(3,4-(methyl-enedioxy)phenoxy)-2-cis/trans-octene (Hoffman-LaRoche RO 20-3600) and 6,7-Epoxy-3,7-dimethyl-1-(p-ethyphenoxy)-2-cis/trans-octene (Stauffer R-20458), produced embryonic and some first larval instar lethality when topically applied to freshly laid Drosophila eggs at concentrations above 0·1μg RO-20-3600/μl acetone or 1·0 μg R-20458/μl acetone. With progressively later JHA treatments during embryonic development, lethality decreased while the proportion of larvae dying at the time of hatching increased. No delayed postembryonic lethality was observed after the first larval instar. JHA applications to freshly laid eggs produced abnormal head and tracheal formation in some embryos treated with RO-20-3600 and caused developmental arrest during the beginning of posterior gut formation in most eggs treated with R-20458. Treatment with either JHA after the beginning of blastoderm formation resulted in normal looking first instar larvae which failed to hatch.     

Delta5-3beta-hydroxysteroid dehydrogenase and estradiol-17beta-hydroxysteroid dehydrogenase activity in preimplantation hamster embryos

Preimplantation golden hamster (Mesocricetus auratus) embryos were recovered on days 1 (= day of finding spermatozoa in the vagina) through 4 of pregnancy. Postimplantation embryos were studied in sectioned gestation sacs excised on days 5 and 6. Δ⁵-3β-Hydroxysteroid dehydrogenase (3β-HSD) activity in embryos was determined histochemically. There was no enzyme activity on days 1 and 2. Weak activity was first observed at 08:00–09:00 hr on day 3, the activity then increased, peaked at 01:00–03:00 hr on day 4, considerably declined by 08:00–09:00 hr (day 4), and was absent on days 5 and 6. These results suggest that the preimplantation embryos synthesize steroid hormones. It was previously hypothesized (Dickmann and Dey, 1973, Dickmann and Dey, 1974) that, hormones synthesized by the preimplantation rat embryo participate in the regulation of morula to blastocyst transformation and implantation of the blastocyst. This hypothesis is applicable to the hamster.In addition to 3βHSD, estradiol-17β-hydroxysteroid dehydrogenase activity was observed in day 3 embryos, suggesting that the embryo synthesizes estrogen.     

Exposure of <Emphasis Type="Italic">Eocanthecona furcellata</Emphasis> (Hemiptera: Pentatomidae) nymphs and adults to high temperatures induces an aestivo-hibernal egg diapause: a strategy for surviving hot summers

The predatory stink bug Eocanthecona furcellata (Wolff) (Hemiptera: Pentatomidae), which is widely distributed in the subtropics and tropics, is a potential biological control agent for lepidopteran, coleopteran, and hemipteran pests. We investigated development of eggs oviposited by E. furcellata females subjected to various photoperiods (i.e., 8L:16D, 12L:12D, and 16L:8D) and temperature regimes (i.e., 26:26, 30:28, 32:30, 34:32, 35:33, and 36:34 °C; temperatures at light and dark phases, respectively) at different developmental stages. Exposure of E. furcellata nymphs and adults to different photoperiods and temperature regimes affected the development of eggs they subsequently oviposited. Temperatures greater than 30 °C induced diapause in a portion of the eggs, as evidenced by the delayed appearance of the black circle and the red spot, characteristics of early and late stage of embryonic development, respectively. The proportion of diapausing eggs generally increased with increasing temperature exposure, whereas it decreased with advancing developmental stage/age at which E. furcellata was subjected to the temperature regime. Although viability of diapausing eggs was lower than that of non-diapausing eggs, diapausing eggs survived longer under high temperatures. The trade-off between survivability and viability may be a strategy to survive unpredictability of summer temperatures.     

Two antigenically distinct forms of β-1,3-glucanase in sea urchin embryonic development

The enzyme β-1,3-glucanase is contained in the unfertilized eggs of most species of sea urchin. In some species, including Lytechinus variegatus, there is also substantial activity following gastrulation, and during remaining larval development. To determine if the same form of β-1,3-glucanase is present in both unfertilized eggs and after gut differentiation, an affinity purification procedure was utilized to isolate enzyme from unfertilized Lytechinus eggs. β-1,3-Glucanase is a 70,000-Da protein in this species, similar to the molecular weight of enzyme isolated from Strongylocentrotus purpuratus. Purified enzyme was used to generate an antibody that specifically recognized a 70,000-Da protein in unfertilized eggs by Western blot analysis, and stained the cortical granules of unfertilized eggs by immunofluorescence. The antibody also specifically immunoprecipitated β-1,3-glucanase activity from egg sonicates. The antibody was used to demonstrate that the form of β-1,3-glucanase present following gastrulation is antigenically distinct from the egg form. The 70,000-Da protein recognized by the antibody was no longer present by 24 hr, but embryos of this and later stages contained substantial amounts of activity, indicating the enzyme at these stages differs from the egg-specific form. In addition, the antibody was not capable of immunoprecipitating enzyme activity from pluteus sonicates. β-1,3-Glucanase has been partially purified from pluteus stage embryos, and appears to be a complex of approximately 200,000 Da. The enzyme is specific to endoderm and appears following differentiation of the gut, suggesting that it may function in larval digestion.