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The effect of changing the gaseous diffusion coefficient on the mass loss pattern of Hyalophora cecropia pupae.

The importance of gas phase diffusion in insect gas exchange remains unclear. The role of diffusion in gas exchange of developing Hyalophora cecropia pupae was examined by altering the gaseous diffusion coefficient in the breathing mixture. Gaseous diffusion coefficients were manipulated by substituting helium or sulfur hexafluoride for the nitrogen usually present in air. Sensitive mass loss recordings were employed to monitor gas exchange activity. Mass loss recordings showed a two-phase cycle, open and closed-flutter. Mass loss rates during the open and closed-flutter periods were not altered in proportion to the changes induced in the rate of diffusion. Open-phase duration was inversely and proportionally related to the diffusion coefficient. These results are consistent with changes in spiracle resistance or convective flow during the open period in response to a change in the diffusion coefficient. In addition, they indicate a significant gas phase diffusive resistance within the pupal tracheal system. This previously unreported gas phase resistance appears to be a major determinant of the duration of the open period and thus of overall water loss rates in these pupae.

Animals↗

Releases of Spalangia nigroaenea and Muscidifurax zaraptor (Hymenoptera: Pteromalidae) increase rates of parasitism and total mortality of stable fly and house fly (Diptera: Muscidae) pupae in Illinois cattle feedlots.

Weekly releases of Spalangia nigroaenea Curtis and Muscidifurax zaraptor Kogan & Legner from May through August of 1991-1993 at small, owner-operated cattle feedlots in Illinois provided weekly emergence of 100-300 parasitoids of each species per feedlot animal. In assessments based on fly and parasitoid emergence from > 47,000 stable fly and house fly puparia collected during the 3-yr period, total stable fly mortality was greater in lots where releases were made (60.7%) than in paired, untreated control lots (51.7%) (P = 0.04; paired t-test); parasitism of stable fly pupae by S. nigroaenea averaged 11.6% where releases were made and 6.4% in paired control lots (P = 0.0016). In lots where releases were made, total mortality of house fly pupae was greater (68.7 versus 56.1%; P = 0.0001); unexplained mortality was greater (55.5 versus 46.1%; P = 0.0018); and parasitism by Muscidifurax spp. was greater (2.4 versus 1.4%; P = 0.07) than in paired control lots. Parasitism, unexplained mortality, and total mortality of both fly species varied significantly from 1991 to 1992 in lots that received the same treatment each year, presumably due primarily to weather. Over the 3-yr period, releasing these species, particularly S. nigroaenea, significantly reduced production of stable fly and house fly adults in cattle feedlots. The potential value of such reductions is likely to vary as a result of feedlot conditions and weather.

Animals↗

Immune response is energetically costly in white cabbage butterfly pupae.

Parasite-driven coevolution has led hosts to develop a complicated and potentially costly defence machinery, consisting mainly of the immune system. Despite the evidence for the trade-offs between immune function and life-history traits, it is still obscure how the costs of using and maintaining the immune function are paid. We tested whether immune challenge is energetically costly for white cabbage butterfly (Pieris brassicae L.) diapausing pupa. Individuals challenged with nylon implant raised their standard metabolic rate nearly 8% compared to the controls. Hence, costs of activation of immune system in insect pupa can be expressed in energetic currency.

Analysis of Variance↗

Parasitism of house fly (Musca domestica) pupae by four species of Pteromalidae (Hymenoptera): effects of host--parasitoid densities and host distribution.

Parasitoid-induced mortality of house fly, Musca domestica L., pupae and parasitoid progeny emergence by four species of pteromalid parasitoids, Muscidifurax raptor Girault & Sanders, M.zaraptor Kogan & Legner, Spalangia cameroni Perkins and S.endius Walker, were determined for a 24 h exposure period using parasitoid: host ratios ranging from 1:2 to 1:50. When the number of parasitoids was held constant (n = 5) and the numbers of hosts varied, and when the number of hosts was held constant (n = 100) and the number of parasitoids varied, both the number of pupae killed per parasitoid and the number of parasitoid progeny per parasitoid increased with increasing parasitoid:host ratios to reach an upper limit asymptotically. Maximum values were, respectively: M.raptor (14.7, 11.1), M.zaraptor (12.3, 9.3), S.cameroni (16.9, 5.5), S.endius (14.8, 9.7) with no consistent effects attributed to parasitoid interference. For M.raptor and S.cameroni at parasitoid:host ratios of 1:10, the pupal mortality and progeny emergence were determined for a 24 h exposure period when hosts were distributed in poultry manure at four levels of aggregation ranging from clumped to uniform. Pupal mortality was least in clumped distributions, while parasitoid progeny emergence was not significantly different.

Animals↗

Temperature-dependent development and parasitism rates of four species of Pteromalidae (Hymenoptera) parasitoids of house fly (Musca domestica) pupae.

Parasitoid development, parasitoid-induced host mortality and parasitoid progeny emergence were determined at five constant temperatures for Muscidifurax raptor Girault and Sanders, Muscidifurax zaraptor Kogan and Legner, Spalangia cameroni Perkins and Spalangia endius Walker using pupae of the house fly, Musca domestica L., as hosts. At temperatures of 20, 25, 30 and 35 degrees C the median development times (days from oviposition to adult emergence), respectively, were M. raptor (28.4, 20.7, 14.3, 14.5), M. zaraptor (30.6, 22.8, 14.1, 14.2), S. cameroni (55.6, 35.2, 21.8, 25.0) and S. endius (52.4, 31.5, 16.3, 14.6). All species failed to emerge at 15 degrees C. Using densities of five parasitoids and 100 hosts and a 24 h exposure period, Muscidifurax species oviposited at a greater rate over a wider range of temperatures than Spalangia species. At 15, 20, 25, 30 and 35 degrees C the mean number of pupae killed per parasitoid were, respectively, M. raptor (1.4, 7.4, 10.5, 13.7, 14.1), M. zaraptor (0.0, 3.3, 8.9, 14.4, 15.0), S.cameroni (0.0, 7.8, 11.0, 11.9, 7.4), S.endius (0.6, 4.0, 7.5, 12.0, 11.7), and means of the number of parasitoid progeny per parasitoid were, respectively, M.raptor (0.2, 5.2, 7.9, 11.8, 11.6), M.zaraptor (1.3, 4.4, 8.2, 13.0, 13.7), S.cameroni (0.0, 2.4, 4.7, 5.1, 1.0), S.endius (0.0, 0.9, 3.4, 7.5, 4.9). Development and ovipositional activity in S.cameroni was strongly inhibited at 35 degrees C. The model by Sharpe & DeMichele (1977) was used to describe temperature-dependent development and the number of parasitoid progeny produced per parasitoid at temperatures of 15-30 degrees C in all species.

Animals↗

RNA polymerase III from Drosophila hydei pupae. Purification and partial characterization.

DNA-dependent RNA polymerase III, which is usually highly resistant to alpha-amanitin, has been purified from Drosophila hydei pupae. The enzyme is insensitive to alpha-amanitin concentrations up to 0.1 microM; at 100 microM alpha-amanitin the enzyme activity is inhibited by approximately 10%. The enzyme was extracted at low ionic strength and purified to homogeneity by six purification steps; 0.1--0.2 mg enzyme/kg pupae could be obtained. The subunit composition of the enzyme was determined after sucrose-gradient centrifugation by sodium dodecyl sulphate electrophoresis in polyacrylamide gels. The enzyme was resolved into putative subunits of molecular weight 154 000, 135 000, 62 000, 58 000, 38 000, 32 000, 31 000, 27 200, 26 500, 21 500 and 17 500. This subunit composition is in general accord with that of eucaryotic class III enzymes. The catalytic properties (salt-activation profile, ratio of activity with denatured DNA to that with native DNA) and the order of elution of the enzyme from DEAE-cellulose with respect to RNA polymerase II agree with the classification of the isolated enzyme as an RAN polymerase III.

Amanitins↗

CecC, a cecropin gene expressed during metamorphosis in Drosophila pupae.

Cecropins are antibacterial peptides, induced in insects in response to bacterial infections. In Drosophila, three cecropin genes have previously been characterized, CecA1, CecA2, and CecB, in a dense cluster at 99E on the third chromosome. From the same locus, we now describe a fourth member of the cecropin gene family, CecC, which is mainly expressed at the early pupal stage. In situ hybridization to immunized pupae show that CecC is induced in the anterior end of the larval hindgut and in other larval tissues that are undergoing histolysis. Within these other tissues it is often expressed in distinct foci that may correspond to hemocytes. A similar pattern of expression in the metamorphosing pupa is also observed for the CecA and CecB genes. Comparing the DNA sequences of the cecropin genes, a conserved region is observed about 30 bp upstream of the TATA box. It consists of three shorter motifs, two of which are reminiscent of a putative promoter element in immune protein genes from the cecropia moth.

Amino Acid Sequence↗

Gloverin, an antibacterial protein from the immune hemolymph of Hyalophora pupae.

Gloverin is an inducible antibacterial insect protein isolated from pupae of the giant silk moth Hyalophora. It is a basic (pI 8.5) protein with a molecular mass of 13.8 kDa, containing a large number of glycine residues (18.5%) but no cysteine, and has an amino acid sequence that reveals no strong degree of identity with any known proteins. Gloverin inhibits the growth of Escherichia coli at a minimal concentration of 1-3 microM, i.e. less than 5% of the concentration of gloverin in the hemolymph of infected pupae. The prime effect of gloverin, following its interaction with lipopolysaccharide (LPS) in the bacterial envelope, is a specific inhibition of the synthesis of vital outer membrane proteins, leading to an increased permeability of the outer membrane. The activity of gloverin is not affected by heating (100 degrees C, 10 min) but is inhibited by Mg2+ and by free LPS. The gloverin molecule will undergo conformational transitions from a monomeric random coil to an alpha-helix upon transfer from an aqueous to a hydrophobic environment, a property likely to be of importance for its interaction with cell-bound LPS. The activity of gloverin is in many respects similar to that of attacin, another antibacterial protein, originally found in Hyalophora [for a review see Boman, H. G., Faye, I., Gudmundsson, G. H., Lee, J.-Y. & Lindholm, D. A. (1991) Eur J. Biochem. 201, 23-31].

Amino Acid Sequence↗

Evidence for two immune inhibitors from Bacillus thuringiensis interfering with the humoral defense system of saturniid pupae.

Mutants of Bacillus thuringiensis lacking either beta-exotoxin or gamma-endotoxin were compared for their virulence using pupae of a giant silk moth. Known doses of viable log-phase bacteria were injected, and the response was followed as the number of viable bacteria in the hemolymph. The results obtained imply that, in the system used, neither the beta-exotoxin nor the gamma-endotoxin and the sporeforming ability are of importance for virulence. Results with sterile culture filtrate from B. thuringiensis have given evidence for the production of two inhibitors, A and B, which interfere with the humoral defense system in pupae of Hyalophora cecropia. Inhibitor A, which blocked the lysis of Escherichia coli,was precipitated by trichloroacetic acid and sensitive to heating. Inhibitor B, which blocked the killing of Bacillus cereus, was soluble in trichloroacetic acid and resistant to 90 degrees C for 5 min. Both inhibitors are believed to contribute to the insecticidal nature of B. thuringiensis.

Animals↗

Insect immunity. III. Purification and partial characterization of immune protein P5 from hemolymph of Hyalophora cecropia pupae.

We previously showed that in pupae of Hyalophora cecropia, eight hemolymph proteins (P1 through P8) were selectively synthetized after immunization (Faye et al., Infect, Immun. 12:1426-1438, 1975). We also showed that a gross fractionation was obtained by a series of ammonium sulfate precipitations (designed A through D) and that protein P5 was enriched in fraction A. Starting from fraction A, we have now purified protein P5 by using dialysis, isoelectric focusing, and hydroxylapatite chromatography. The final product gave a single band in both gradient gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using the latter method, proteins P5 and P8 were found to be enriched in fraction A, but they were absent in fraction Ac prepared from nonimmunized pupae. Protein P5 was found to have a pI of 6.6 and a molecular weight of 24,000 in sodium dodecyl sulfate and 96,000 in tris (hydroxymethyl) aminomethane-borate, pH 9.0. These data suggest a structure for P5 composed of four subunits of equal size. Protein P5 stimulated the killing of Escherichia coli by hemolymph fractions B and D, but it had neither killing nor phenol oxidase activity of its own.

Animals↗

Sex differences in gene expression for pupa weight in long term selected lines of Tribolium.

Full-sib matings in two populations of Tribolium which had been selected for increased pupa weight for more than 85 generations resulted in a significant inbreeding depression in male progeny but showed no effect in the female progeny. An analysis of variance of a population produced by backcrossing the selected populations to the inbred lines originally used to establish the select populations (Design-III) indicated some genes were still segregating which produce dominance effects in males but not in females. The data support the hypothesis that a class of genes exists, associated with the autosomes, that differ in their dominance effects of pupa weight in the two sexes.

Animals↗

Respecified larval proleg and body wall muscles circulate hemolymph in developing wings of Manduca sexta pupae.

Most larval external muscles in Manduca sexta degenerate at pupation, with the exception of the accessory planta retractor muscles (APRMs) in proleg-bearing abdominal segment 3 and their homologs in non-proleg-bearing abdominal segment 2. In pupae, these APRMs exhibit a rhythmic 'pupal motor pattern' in which all four muscles contract synchronously at approximately 4 s intervals for long bouts, without externally visible movements. On the basis of indirect evidence, it was proposed previously that APRM contractions during the pupal motor pattern circulate hemolymph in the developing wings and legs. This hypothesis was tested in the present study by making simultaneous electromyographic recordings of APRM activity and contact thermographic recordings of hemolymph flow in pupal wings. APRM contractions and hemolymph flow were strictly correlated during the pupal motor pattern. The proposed circulatory mechanism was further supported by the findings that unilateral ablation of APRMs or mechanical uncoupling of the wings from the abdomen essentially abolished wing hemolymph flow on the manipulated side of the body. Rhythmic contractions of intersegmental muscles, which sometimes accompany the pupal motor pattern, had a negligible effect on hemolymph flow. The conversion of larval proleg and body wall muscles to a circulatory function in pupae represents a particularly dramatic example of functional respecification during metamorphosis.

Animals↗

[Frequency of Aedes aegypti and Aedes albopictus larvae and pupae in traps, Brazil].

OBJECTIVE: To evaluate the monthly frequency of larvae and pupae of Aedes albopictus, Aedes aegypti and other mosquitoe species in tires, and the influence of environmental factors on that. METHODS: The immature stages of mosquitoes were collected monthly from four tires in the municipality of Nova Iguaçu, Brazil, from November 1997 to October 1998. The following variables were measured: rainfall, temperature, water volume, water pH. The tires were arranged in a pyramid, one at the base (tire 1) and 3 others (2,3 e 4) laying over it. RESULTS: Were collected 10,310 larvae and 612 pupae. Aedes albopictus was the most common species in both the larval and pupal stages. Aedes aegypti and Aedes albopictus were collected throughout the year but were more frequent during in the rainy season. The number of Aedes aegypti was significantly correlated with the temperature, rainfall and water volume of the tires. The correlation between water pH and number of larvae was not significant. Aedes albopictus larvae were more frequent in tires left in the shade. CONCLUSIONS: Aedes albopictus was more abundant in tires than Aedes aegypti. Discarded tires seem to be an important source of both Aedes species throughout the year. The favored environmental conditions of the tires, such as water volume and exposure to sunlight differ for Aedes albopictus and Aedes aegypti.

Aedes↗

First occurrence of parasitoid Spalangia endius (Walker) (Hymenoptera: Pteromalidae) in pupae of Zaprionus indianus Gupta (Diptera: Drosophilidae) in Brazil.

This paper reports the first occurrence of the parasitoid Spalangia endius collected in pupae of Zaprionus indianus using traps with fruit bait. The experiment was carried out at the University of Lavras in Lavras, Minas Gerais, Brazil, from November to December, 2001. A total of 4 Spalangia endius specimens were obtained from 105 Zaprionus indianus pupae. The overall prevalence of parasitism was 3.8%.

Animals↗

Electrical properties of the cerebral prothoracicotropic hormone cells in diapausing and non-diapausing pupae of the tobacco hornworm, Manduca sexta.

Prothoracicotropic hormone (PTTH) is an insect brain neuropeptide that is a primary factor regulating an insect development. Curtailment of its release is thought to be responsible for the pupal diapause of tobacco hornworm, Manduca sexta. The cell synthesizing and secreting the PTTH has been identified as a pair of neurosecretory cells in the pars lateralis on each brain hemisphere. Using intracellular recording techniques, we have demonstrated electrical properties of the PTTH cells in different physiological status, i.e., diapausing and developing pupae. In diapausing pupae, they showed threshold value increasing and input resistance decreasing with the progress of diapausing state, indicating that they were getting unexcitable. Spontaneous action potentials and excitatory postsynaptic potentials (EPSPs) were rarely observed in deeply diapausing state. Non-diapausing PTTH cells were almost silent except day-2, showing rather constant values of electrical properties. On day-2, a significant proportion of the cells had spontaneous action potentials, showing less negative membrane potential values than inactive cells. Exclusively inhibitory postsynaptic potentials (IPSPs) were observed in significant numbers of the cells during the period from day-2 to day-5. On the basis of the results obtained, we proposed a working hypothesis that electrical activities of the PTTH cell may be primarily regulated by its membrane properties which are further modulated by the synaptic mediation.

Animals↗

Comparative action of RH-0345 and pyriproxyfen on molting hormone production and protein analysis in mealworm pupae.

RH-0345 (halofenozide) was found to mimic the action of 20-hydroxyecdysone while pyriproxyfen was presented as a juvenile hormone analogue. In this study, RH-0345 (5, 10 micrograms/pupa) and pyriproxyfen (0.10 and 0.20 microgram/pupa), applied topically on newly ecdysed pupae of Tenebrio molitor, were investigated on ecdysteroid titers and hemolymph protein concentrations. During normal pupal development, the ecdysteroid titer measured using an enzyme immunoassay showed a single peak at day 3. The two compounds did not shifted the moment of the pupal ecdysteroid peak. However, RH-0345 at the highest dose (10 micrograms/pupa) increased the ecdysteroid titres recorded from the second day after topical application while pyriproxyfen decreased this hormonal production. In a second series of experiments, the compounds were evaluated on the protein concentrations in the hemolymph. In control pupae, change in protein concentrations showed two peaks. At a 5 micrograms dose, RH-0345 was found to reduce significantly the value of the first peak and at the highest dose, the first peak was not observed and the values recorded following treatment were significantly lower than in controls. Changes in protein concentrations showed that pyriproxyfen suppressed the second peak and the values recorded were either significantly increased at day 2 and 3, either reduced from day 4.

Animals↗

[Effect of white spot syndrome virus envelope protein Vp28 expressed in silkworm (Bombyx mori) pupae on disease resistence in Procambarus clarkii].

The vaccine made of recombinant envelope protein (rVp28) of white spot syndrome virus (WSSV) expressed in silkworm (Bombyx mori) pupae using a baculovirus vector was used to investigate the efficacy of oral administration on WSSV disease resistance of Procambarus clarkii. Vaccine was mixed with diet at a ratio of 2% (w/w), and Procambarus clarkii were orally administered throughout 75 days. Vaccination with rVP28 showed the significantly higher cumulative survival compared with positive and negative control (P < 0.05) following an oral challenge on the 35th day post-vaccination (dpv), with PRP values 54.16% and 59.26%, respectively. rVP28 induced higher resistance via IM (intramuscular) injection challenge with WSSV stock, with PRP value of 46.12% and 49.99%, respectively. The survivors were subsequently re-challenged on the 55th dpv. rVP28 induced the significantly higher resistance to oral re-challenge (P < 0.05), with both PRP values 55.80% and 63.16%, respectively. rVP28 induced higher resistance to IM injection re-challenge, with both PRP values 31.25%. A DIG labeled WSSV DNA probe was used to detect WSSV by in situ hybridization. The positive cells were observed in epithelial cells of stomach, hepatopancreas and gut of the infected control crayfish, while negative reaction were observed in the tissues of survivors-vaccinated. These results indicated that vaccination of crayfish with recombinant protein had significant effect on oral infection, and had higher resistance against intramuscular injection challenge. This suggested the protection against WSSV could be induced in crayfish by recombinant protein rVp28 expressed in silkworm pupae.

Animals↗

Isolation and characterization of NADP+-specific isocitrate dehydrogenase from the pupa of Bombyx mori.

NADP+-specific isocitrate dehydrogenase was found in several tissues of the pupa of the silkworm, Bombyx mori. This enzyme was highly purified from the whole bodies of pupae. This is the first isolation of the enzyme from insect materials. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis. The reaction catalyzed by the purified enzyme was readily reversible. The pH optimum for the forward reaction (reduction of NADP+) was 7.8, and that for the reverse reaction (oxidation of NADPH) was 6.6. The enzyme had a molecular weight of 86,000 and was found to be composed of two identical subunits, which have a molecular weight of 44,000. The activity of the enzyme in the forward reaction was slightly inhibited by citrate, oxaloacetate, alpha-ketoglutarate, and others. Citrate stabilized the activity over a wide pH region.

Animals↗