Pupal Extract Research Articles

The Expression of the Drosophila awd Gene during Normal Development and in Neoplastic Brain Tumors Caused by lgl Mutations

The abnormal wing discs (awd) gene of Drosophila is homologous to the nm23 gene of mammals, a gene whose expression is altered in metastatic tumors. Both awd and nm23 encode nucleoside diphosphate kinases (NDP kinases). We have examined the accumulation of AWD/NDP kinase during normal development by assaying enzyme activity in extracts. There is a nearly constant level of activity throughout larval and pupal development. We have examined the tissue-specific transcription of the awd gene by RNA in situ hybridization and by reporter gene expression. In imaginal discs and brains there is no detectable awd gene expression until the beginning of the third larval instar, despite the constant level of enzyme activity measured in extracts of larvae and pupae. The most intense awd gene expression in imaginal discs and brains occurs after the end of larval development. We have also examined awd gene expression in neoplastic brain tumors caused by mutations in the lethal giant larvae (lgl) gene. In lgl mutant brains, as in normal brains, awd gene expression begins during the third larval instar. No tumors form in brains from lgl-;awd- double mutant larva, so awd gene expression is required for tumor formation and/or proliferation. There is more accumulation of AWD/NDP kinase in lgl- mutant brains than there is in normal brains. Using an awd reporter gene, we show that this is a consequence of an increased proportion of awd gene-expressing cells in mutant brains. Using the same awd reporter gene as a marker of donor cells, we have confirmed the invasiveness of lgl-induced neuroblastomas.

Morphogenetic and growth related changes in lipids of Drosophila ananassae (Doleschall).

The lipid component of Drosophila ananassae have been identified qualitatively. Neutral and phospholipid fractions are specific for pupal, larval or adult extracts. Some of these lipid components are identical with those obtained from the temperate D. melanogaster and other species. This demonstrates biochemical convergence between geographically isolated species groups.

Opioid detection in pupal extract of the Chinese oak silkwormAntheraea pernyi guerin

Opioid detection in pupal extract of the Chinese oak silkwormAntheraea pernyi guerin

A fluorimetric assay of general esterase activity in individual larva, pupa and adult mosquito

The general esterase activity in larvae, pupae and adult mosquitoes of Aedes togoi, Aedes aegypti (ROCK), Aedes albopictus, Culex quinquefasciatus (Kembangan) and Armigeres subalbatus was determined by a continuous fluorimetric assay which employs 4-methylumbelliferylacetate (4-MUA) as the substrate. The specific activities of the fourth-instar larva and adult of C. quinquefasciatus (Kembangan) were significantly higher than the corresponding activities of the other species of mosquitoes. When measured in the fourth-instar larvae, the total esterase activity per larva was significantly higher in C. quinquefasciatus (Kembangan) and Armigeres subalbatus compared to the other three species examined. This was correlated with correspondingly higher LD 50 values, measured with malathion, for these two species. 1-Naphthylacetate (1-NA) showed inhibition of 4-MUA-esterase activity. A comparison of the total esterase activity measured in parallel with 4-MUA and 1-NA as substrates in 11 extracts of adults and pupae of different species of mosquitoes showed a good correlation ( r = 0.96). The procedure was also modified as a quantitive end-point assay suitable for rapid screening of multiple samples. For investigation of field populations, the fluorescence of the released 4-MU could be observed under illumination of a 96-well plate with a hand-held u.v. lamp.

Immunochemical and biological analysis of pheromone biosynthesis activating neuropeptide in Heliothis peltigera.

This study describes the preparation and characterization of a highly specific antiserum to Helicoverpa zea pheromone biosynthesis activating neuropeptide (Hez-PBAN), and the use of this antiserum, in an enzyme linked immunosorbent assay (ELISA), to determine: a) the content of endogenous PBAN in head extracts of male and female Heliothis peltigera; b) the level of PBAN at different developmental stages; and c) the content of PBAN in four different moth species. Cross-reactivity studies revealed that the antiserum is directed mainly toward the N-terminal region of the neuropeptide, and that it exhibits similar binding affinities toward the oxidized and reduced forms of PBAN. Analysis of PBAN content in head extracts of male and female H. peltigera, at scotophase, revealed the presence of 4.97 and 4.58 pmol, respectively, in 3-day-old moths, and 5.33 and 4.78 pmol, respectively, in 7-day-old moths. The similarity in the content of PBAN at both ages and sexes was in accordance with the amount of pheromonotropic activity in these extracts which stimulated pheromone biosynthesis to a similar level. Analysis of PBAN-like immunoreactivity (IR) in head extracts of H. peltigera larvae and pupae demonstrated the existence of the neuropeptide in the 4th larval instar and continued to increase as a function of development. No IR could be detected in the first three larval instars. The larval and pupal extracts also exerted pheromonotropic activity which followed a similar pattern. The activity in these extracts, however, was considerably lower than that found in adult male and female heads. IR was also detected in head extracts of three other Noctuidae moths: Helicoverpa armigera, Cornutiplusia circumflexa and Spodoptera littoralis, indicating a high degree of chemical and structural similarity of PBAN in these moths.

Occupational sensitivity to Tenebrio molitor linnaeus (yellow mealworm)

Tenebrio molitor is an abundant stored-grain pest in the northern United States. We evaluated an individual with work-related symptoms of rhinoconjunctivitis on exposure to this insect. Prick skin tests with extracts prepared from the larval, pupal, and adult-life stages were positive for the patient and for another individual with allergy to a closely related species of beetle, Alphitobius diaperinus. Specific IgE antibodies to the extracts were demonstrated by RAST. RAST inhibition demonstrated immunologic cross-reactivity between the life stages of T. molitor and also between T. molitor and A. diaperinus, as well as slight cross-reactivity with blowfly. The proteins in the extracts of each life stage were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 15 protein bands were detected in each of the extracts, although the patterns of separation were different for each life stage. After immunoblotting and autoradiography, six different IgE-binding proteins were identified in the larval extract, five in the pupal extract, and seven in the adult extract, with similar IgE-binding patterns noted for the larval and adult extracts. We conclude that this patient developed IgE-mediated sensitivity to T. molitor antigens as the result of occupational exposure. This study confirms the fact that beetles of the Tenebrionid family are potentially significant allergens for workers exposed to grains or grain products.

Effect of Experience on the Responses of the Parasitoid Brachymeria intermedia (Hymenoptera: Chalcididae) to Its Host, Lymantria dispar (Lepidoptera: Lymantriidae), and to Kairomone

In a choice olfactometer, female Brachymeria intermedia (Nees) walk upwind to odor from host pupae, Lymantria dispar L., and pupal extracts. However, females with oviposition experience on a host pupa were more likely than naive females to walk upwind in kairomone-laden air and did so more rapidly. After arrival at a kairomone-impregnated patch, experienced females were more likely to probe with their ovipositors than naive females. In female B. intermedia , previous experience with a host appears to be an important feature in orientation to and recognition of its host by kairomone.

Inhibitory action of phosphine on acetylcholinesterase of Ephestia cautella (Lepidoptera: Pyralidae)

The present study aims to investigate the inhibitory effects of phosphine gas on the acetylcholinesterase enzyme of Ephestia cautella. The results showed that the activity of this enzyme is relatively high at the larval and adult stages. Significant reductions in enzyme activity were observed in dead or live adults produced from 3–4-day old pupae exposed to various concentrations of phosphine. Treatment of cholinesterase preparation from pupal extract showed in vitro inhibitory action of phosphine on this enzyme.

Occupational sensitivity to Atphitabius diaperinus (Panzer) (lesser mealworm)

Alphitobius diaperinus is an important beetle in the grain and poultry industries. We evaluated three individuals with work-related symptoms of asthma, rhinitis, conjunctivitis, urticaria, and angioedema on exposure to the insect. Prick skin tests with extracts prepared from the larval, pupal, and adult life stages were positive in all three patients. Specific IgE antibodies to these extracts were demonstrated by RAST or radioimmunoassay. RAST and radioimmunoassay inhibition confirmed the specificity of IgE binding and further demonstrated immunologic cross-reactivity between the three life stages. Peripheral blood leukocytes from two of the individuals demonstrated significant histamine release when they were compared with cells from nonexposed atopie and normal control subjects. The proteins in the extracts of each life stage were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. More than 30 protein bands were detected in each of the extracts; however, the patterns of separation were different for each life stage. After immunoblotting and autoradiography, IgE-binding proteins were recognized by sera from all three individuals in the larval extract at 90 kilodaltons (kd), in the pupal extract at 90, 64, and 38 kd, and in the adult extract at 84 kd. Additionally, several other proteins were identified as being allergenic in some of the patients. We conclude that these three patients developed IgE-mediated sensitivity to A. diaperinus antigens as the result of occupational exposure. To our knowledge, this is the first description of sensitivity to this grain beetle.

Formula omitted] of dopamine and tyramine by mosquito pupae ( Aedes togoi)

The N- acetyltransferase (NAT) activity of mosquito pupae was measured by a radioenzymatic assay, using [ 14C]-, [ 3H]dopamine, [ 14C]tyramine or [ 14C]acetyl-CoA. The pupal extract could also generate acetyl-CoA from ATP, acetate and CoA for this acetylation reaction. Both the dopamine- and tyramine-NAT reactions proceeded linearly up to 20 min at an optimum pH of 8.4. It is possible that the same enzyme is involved in the acetylation of both biogenic amines as shown by the competitive inhibition kinetics obtained, and the similarities of the NAT reaction with both amines, in the presence of metal chelators, metal ions, SH reagents and MAO inhibitors. Mn 2+ stimulated and Zn 2+ inhibited the reaction. The specific activity of NAT in individual pupae measured soon after pupation showed no significant difference between the male and female pupae: the values obtained were, respectively, 893 ± 57 and 861 ± 30 pmol [ 14C]NAcT formed/min per mg protein and 21.9 ± 1.2 and 22.0 ± 1.4 pmol [ 3H]NADA formed/min per mg protein.

Juvenile hormone in Drosophila melanogaster: Identification and titer determination during development

Juvenile hormone (JH) III was identified in whole-body extracts of eggs, larvae, pupae, pharate adults and adults of Drosophila melanogaster. Titers of the hormone varied according to stage of development. Highest levels were found in post-feeding (wandering) larvae and adults; only low levels were found in feeding last-stadium larvae and in pupae. None of the other known JHs were detected (limit of detection ≈0.01 ng/g). These results are discussed in the light of the known physiological roles of JH in the development of Drosophila and other insects.

Phenolsulphotransferase activity in the developing mosquito ( Aedes togoi)

Phenolsulphotransferase (PST) activity was measured with N-acetyldopamine (NADA) and harmol as substrates in the larvae, pupae and adult mosquito ( Aedes togoi). Only the newly emerged pupae showed high PST activity 1–4 hr after pupation. PST activity could also be measured in each individual pupa, with the female exhibiting significantly higher specific activity (30 ± 3.7 pmol NADA [ 35S]sulphate/min per mg protein) than the males (13.6 ± 2.9 pmol NADA [ 35S]sulphate/min per mg protein). The optimum pH for the PST reaction was 9.0. The K m values for [ 35S]PAP were 0.55 and 2.5 μM when measured with NADA and harmol as acceptors, respectively; the corresponding K m values for these two substrates were 2.61 and 16.1 μM. Studies with 2,6-dichloro-4-nitrophenol showed a dose-dependent inhibition of PST. Sulphate conjugation of NADA from ATP and sodium [ 35S]sulphate was also demonstrated with pupal extracts, with pH optimum between 8.6 and 9.0. The specific activity of this overall sulphate conjugation, measured in the female pupal extract was 5.08 pmol NADA [ 35S]sulphate/min per mg protein and 1.68 pmol harmol [ 35S]sulphate/min per mg protein. The importance and possible function of sulphate conjugation of NADA in insects is discussed.

Biosynthesis of makisterone A and 20‐hydroxyecdysone from labeled sterols by the honey bee, Apis mellifera

AbstractIn an effort to determine the sterol precursor(s) of the 28‐carbon ecdysteroid, makisterone A, honey bee pupae (13 days post‐oviposition) were injected with radiolabeled sterols and subsequently examined for labeled ecdysteroids. High performance liquid chromatography of the pupal extracts revealed that [3H]campesterol was converted to a compound that behaved chromatographically identical to authentic makisterone A, and [14C]cholesterol was incorporated into a compound chromatographically like 20‐hydroxyecdysone. No incorporation of either 24‐[3H]methylenecholesterol or [14C]sitosterol into an ecdysteroid was observed. The neutral sterols of uninjected honey bee pupae contained 49.8% 24‐methylenecholesterol on a relative percent basis and, with three other C28 and C29 sterols, accounted for over 99% of the total sterols present.

Evidence for haemoglobins as common allergenic determinants in IgE‐mediated hypersensitivity to chironomids (non‐biting midges)

Chironomids (non-biting midges) are known to cause IgE-mediated hypersensitivity in man. This study compares the cross-reactivity between the chironomid midge Cladotanytarsus lewisi ('green nimitti'), a widespread cause of allergy in the Sudan and Chironomus riparius (= thummi, CTT) where larvae are used as pet fish food and where haemoglobins were previously shown to be major allergens. As with C. riparius, immature forms of C. lewisi also contain allergenic material since skin test responses to larval, pupal and adult extracts were obtained in Sudanese individuals. Crossed radioimmunoelectrophoresis of the C. lewisi larval and pupal extracts indicate that they contain a higher proportion of the allergenic fractions than adults. Further evidence of common allergen determinants between C. lewisi and C. riparius were obtained by the demonstration of positive skin-prick tests, in Sudanese patients, to extracts of larval, adult and isolated haemoglobin extracts of C. riparius. Cross-reactivity between C. lewisi and C. riparius was also demonstrable by RAST inhibition studies. A dose-dependent inhibition was observed using both the C. lewisi adult midge RAST and the C. riparius haemoglobin RAST, the two respective antigens, and sera from individuals hypersensitive to either C. lewisi or C. riparius. Due to the immunological cross-reactivity found between these distantly related species, we conclude that chironomids should be seen as significant environmental and occupational allergens.

Suitability of Lymantria dispar (Lepidoptera: Lymantriidae) as a Host for the Entomogeneous Nematode, Steinernema feltiae (Rhabditida: Steinernematidae)

Larvae, pupae, and adults of the gypsy moth, Lymantria dispar (L.), were susceptible to the entomogeneous nematode, Steinernema feltiae Filipjev (=Neoaplectana carpocapsae Weiser). The original inoculum, obtained from the wax moth, Galleria mellonella (L.), was infective at a dosage as low as 10 infective-stage nematodes per ml (LC50 = 38 nematodes per ml). After two passages through gypsy moth larvae, the pathogenicity increased (LC50 = 15 nematodes per ml), and remained stable thereafter. Nematodes reproduced within gypsy moth cadavers and progeny emerged 5 to 7 days after initial challenge. Nematode progeny increased from 35 per host (neonate larvae); 1,475 per second-instar larvae; 3,910 per third-instar larvae; to 11,106 per fourth-instar larva. Nematode progeny production in fifth-instar male larvae remained unchanged at 10,700; decreased in pupae to 8,900; and increased in adults to 13,500. Nematode production reached its peak among fifth-instar female larvae (43,000 per host), but decreased dramatically among sixth-instar female larvae (3,200 per host), and continued to decline in pupae (200 per host) and adult females (50 per host). Tissue extracts (10%) obtained from neonates, fourth- and fifth-instar larvae had little effect or inhibited nematode progeny production, while extracts from second- and third-instar larvae appeared to be stimulatory. The addition of sixth-instar (female) larval, prepupal, pupal, and adult female extracts increased nematode production slightly but was still below that of the standard (H2O). On the other hand, the addition of male prepupal, pupal, and especially adult male extracts resulted in greater numbers of nematodes than the standard.

Pupal and larval cuticle proteins of Drosophila melanogaster.

Proteins, soluble in 7 M urea, were extracted from third-instar larval and pupal cuticles of Drosophila melanogaster. Both extracts contain a limited number of polypeptides resolved by one- or two-dimensional electrophoresis. The five major larval proteins have low molecular weights (less than 20000) and are not glycosylated. The major pupal cuticle proteins fall into two size classes: two with apparent molecular weights of 56K and 82K and four with molecular weights between 15K and 25K. The proteins with high apparent molecular weights are glycosylated. In nondenaturing gels, no components of the larval and pupal cuticle extracts comigrate. One-dimensional "fingerprints" indicate that cuticle proteins from these two stages have unique primary structures. Immunological results indicate that the major low molecular weight larval and pupal cuticle proteins are comprised of two families of proteins that share antigenic determinants. The high molecular weight pupal cuticle proteins are immunologically unrelated to the low molecular weight components. We conclude that the pupal and larval proteins are encoded in part by multigene families that have arisen by gene duplication and evolutionary divergence.

Host Tissues and Metabolic Products as Ultraviolet Screens for the Gypsy Moth (Lepidoptera: Lymantriidae) Nucleopolyhedrosis Virus 1

The addition of tissue extracts from first to fourth stage Lymantria dispar (L.) larvae resulted in a 2-fold increase in virus-caused mortality, after exposure to ultraviolet (UV) radiation. The addition of extracts from fifth-stage male or fifth- and sixth-stage female larvae had little effect upon virus stability. Both male and female prepupal extracts acted as UV screens. Male and female pupal extracts had some protective activity, but were not as effective as those from prepupae. The addition of adult female extract was ineffective, while the adult male extract was the most effective UV screen. The addition of fecal filtrates enhanced viral stability to radiation and the effect was concentration dependent. For example, a 1% filtrate enhanced stability by a factor of 1.23, and a 5% filtrate by a factor of 2.45. The addition of a 20% filtrate enhanced persistence by a factor of 3.66 (=70% viruscaused larval mortality). Various compounds involved in purine catabolism were examined as UV screens. At a 1% concentration, hypoxanthine, urea, and xanthine were ineffective, while allantoin and uric acid had some activity. Adenine and guanine were effective protectants of the virus. In a subsequent test, uric acid was effective at concentrations ranging from 1 to 10%. The addition of 10% uric acid to the gypsy moth nucleopolyhedrosis virus (NPV) enhanced viral stability by 3.58-fold (=ca. 81% NPV-caused mortality).

Characterization and culture of virus replicating continuous insect cell lines from the bollworm,Heliothis zea (boddie)

A series of five discrete virus replicating insect cell lines were isolated from the ovarian and fat body tissues ofHeliothis zea pupae. Two of these cell lines (IPLB-HZ-1075 and-HZ-1079) were studied in depth as to their growth and virus replication responses to specific nutrients (acetyl-β-methylcholine, fresh glutamine) in a number of media. The same two cell lines were identified to species by serological (microimmunodiffusion) and isozyme (phosphoglucoisomerase and peptidase:glycyl-leucine) techniques. Distinguishing comparisons were made with other cell lines that have been confused with the present lines in the literature and with cell line and host pupal extracts from the same and other lepidopteran species studied concurrently in this laboratory. Sterility culture tests were negative for mycoplasmas. The present fiveH. zea lines were the first insect cell lines to replicate polyhedra from a unicapsid multiple embedded nuclear polyhedrosis virus (Baculovirus Group A), in this case the homologous virus obtained from larvae ofH. zea.

DNA polymerase activity in developmental stages of Drosophila melanogaster

AbstractAn assay procedure was developed that allowed the first reproducible measurement of DNA polymerase activity in all developmental stages of Drosophila melanogaster. Evidence is presented that the same enzymatic species is present in extracts of embryos, pupae, and adults of both sexes and that this activity has many properties similar to vertebrate α‐polymerases. Polymerase activity per individual is low in embryos and rises steadily through larval instars, reaches a peak in early pupae, declines through the late pupal period, and remains low in newly eclosed adults of both sexes. A dramatic increase is observed in adult females as mature oocytes are formed. This pattern of enzyme activity is completely coincident with changes in DNA levels during development, and suggests that the Drosophila enzyme, like vertebrate α‐polymerases, functions in cellular DNA replication. Two mutagen‐sensitive mutants, deficient in both replication on undamaged templates and postreplication repair, were found to have normal levels of this α‐polymerase activity. Our results suggest that a single enzymatic species of α‐polymerase holoenzyme exists in Drosophila and is common to all developmental stages of this organism.

The cuticular proteins of Tenebrio molitor: I. Electrophoretic banding patterns during postembryonic development

Buffer-soluble cuticular proteins of the abdomen of the yellow mealworm, Tenebrio molitor, were analyzed by SDS-polyacrylamide gel electrophoresis. Since the abdominal epidermis of Tenebrio persists throughout the insect's life, these cuticular proteins reflect the secretory history of a continuous line of cells during its entire metamorphic developmental program. Twenty-two to thirty-eight bands were detected in extracts of larval cuticle, 11 to 35 in pupal cuticular extracts, and 30 to 41 in extracts from adults. No population polymorphism was apparent, nor was there any sexual dimorphism, in these cuticular proteins. At each metamorphic stage, the cuticular proteins formed a unique banding pattern. Bands unique to the larval and to the adult exocuticular extracts were observed. Extracts from cuticles of freshly ecdysed animals (exocuticle) differed from extracts from animals in which sclerotization and postecdysial (endocuticle) deposition had occurred, both in number of hands and in their molecular weight distributions. Some proteins became less soluble during sclerotization. The majority of the exocuticular bands from all three stages had molecular weights below 25,000; higher-molecular-weight proteins were extracted from postecdysial animals of each stage.