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PubMed2001

Metabolic response of Platynota stultana pupae during and after extended exposure to elevated CO(2) and reduced O(2) atmospheres.

The metabolic response of Platynota stultana pupae to elevated CO(2) and reduced O(2) atmospheres was measured using microcalorimetry. Initial measurements at 20 degrees C immediately upon placement in controlled atmosphere indicated a decrease in metabolic heat rate (MHR) of 27, 45, 56, 56, and 72% in an atmosphere of 5, 10, 20, 40, and 79% CO(2), respectively, and a decrease of 20, 50, 66 and 100% under 6, 2, 1, and 0% O(2). With extended exposure to controlled atmospheres, MHR increased under 5, 10, and 20% CO(2) and 6 and 2% O(2); however, the increase was greater and occurred more rapidly with lower CO(2) and higher O(2) concentration. The MHR at 40 and 79% CO(2) remained at the initial reduced level for 8 and 6 days, respectively, then decreased with longer exposure. The MHR of pupae held under 1 and 0% O(2) remained at the initial reduced level for 22 days. Upon transfer to air, the MHR of pupae increased from the reduced levels and then decreased. When the MHR decreased by no more than 30%, as a result of controlled atmosphere treatment, the pupae still developed into adults. However, when the MHR decreased by more than 50%, the energy supply was insufficient and the pupae died. Pupa mortality was comparable between 5% CO(2) and 6% O(2), and 10% CO(2) and 2% O(2). The MHR was reduced less under 20% CO(2) than under 2 or 1% O(2); however, the pupae were more susceptible to 20% CO(2) than 2 or 1% O(2). These and other data indicate an increased toxicity of high CO(2) over low O(2) atmospheres that may be related to an increase in membrane permeability as a result of CO(2) treatment.

Journal Article↗
PubMed2002

Comparison of the circadian eclosion rhythm between non-diapause and diapause pupae in the onion fly, Delia antiqua.

The influence of pupal diapause on adult eclosion rhythm of Delia antiqua was investigated. When non-diapause and diapause pupae were exposed to various photoperiods at 15, 20 and 25 degrees C, both of them emerged as adults close to the light-on time, but the phase of eclosion varied with photoperiod and temperature. Moreover, there was a significant difference in the eclosion time between non-diapause and diapause pupae; the eclosion peak of diapause pupae was earlier than that of non-diapause pupae. When non-diapause and diapause pupae were transferred to constant darkness (DD) after having experienced LD 12:12 at 15, 20 and 25 degrees C, both showed circadian rhythmicity in eclosion. Although the free-running period (tau) decreased slightly as temperature increased in both non-diapause and diapause pupae, the latter tended to show shorter tau than the former. This observation suggests that the observed difference in eclosion time in LD cycles between non-diapause and diapause pupae is due to differences in tau.

Journal Article↗
PubMed2000

Tolerance of pupae and pharate adults of Callosobruchus subinnotatus Pic (Coleoptera: Bruchidae) to modified atmospheres: a function of metabolic rate.

Three developmental stages (pupae, early pharate and late pharate adults) of Callosobruchus subinnotatus (Pic.) were investigated for their tolerance or susceptibility to four modified atmospheres. Two of these atmospheres were hypercarbic and two were hypoxic. The hypercarbic atmospheres were found to cause mortality earlier than hypoxic atmospheres. Late pharate adults died earlier than pupae or early pharate adults. Late pharate adults that survived the exposure took a longer time to eclose than the pupae or early pharate adult.Using high resolution microrespirometric techniques, it was possible to record the oxygen consumption rate and CO(2) output of different developmental stages in air. The metabolic rate was determined manometrically as the oxygen uptake rate at an ambient temperature of 25 degrees C. The oxygen uptake rate differed significantly between groups of 20 individuals of different stages (p<0.01; t-test). The lowest rate of oxygen uptake (510.6+/-52.2 &mgr;l g(-1) h(-1)) was recorded in pupae. Higher oxygen uptake rates were found in early pharate adults (668.4+/-45.6 &mgr;l g(-1) h(-1)) and late pharate adults (1171.2+/-45.0 &mgr;l g(-1) h(-1)), and adult beetles (1310.4+/-53.4 &mgr;l g(-1) h(-1)). The patterns of CO(2) release were similar to those of oxygen uptake. CO(2) release was highest in eclosed adults and late pharate adults followed by early pharate adults, and lowest in pupae. The mode of CO(2) release ranged from continuous CO(2) release in pupae to discontinuous CO(2) release in late pharate and eclosed adults. Thus, high metabolic rates, and perhaps, in conjunction with discontinuous CO(2) of late pharate adults are responsible for their higher susceptibility to modified atmospheres than pupae and early pharate adults.

Journal Article↗
PubMed2004

Epicuticular wax of large and small white butterflies, Pieris brassicae and P. rapae crucivora: qualitative and quantitative comparison between diapause and non-diapause pupae.

We compared the quantity and quality of the epicuticular wax of diapause and non-diapause pupae in two closely related Pieris species, P. brassicae and P. rapae crucivora. Main components of their epicuticular wax were identified as hydrocarbons. In P. brassicae, more than 95% of hydrocarbons were saturated regardless of whether the pupae were in diapause or not. In P. rapae crucivora, 93% of hydrocarbons were saturated in non-diapause pupae whereas in diapause pupae 41% were saturated and 59% unsaturated. From measurements of body surface area by nuclear magnetic resonance microimaging, we calculated the average thickness of the wax layer. The thickness in diapause and non-diapause pupae of P. brassicae was 800 and 160 nm, respectively. In P. rapae crucivora, the thickness was 195 nm in diapause and 11 nm in non-diapause. This is the first report to clarify the compositional difference in epicuticular wax between diapause and non-diapause pupae.

Animals↗
PubMed1990

The fate of Bacillus thuringiensis var. israelensis in B. thuringiensis var. israelensis-killed pupae of Aedes aegypti.

Carcasses of mosquito larvae killed by Bacillus thuringiensis var. israelensis allow its complete growth cycle (germination, vegetative growth, and sporulation), thus becoming toxic themselves to scavenging larvae. In this study, we demonstrate that the bacterium is capable of inducing death of Aedes aegypti pupae and of recycling in the resulting carcasses. B. thuringiensis var. israelensis-killed pupae were obtained by treating 40-hr-old synchronized fourth instar larvae with a low dose of spores (8000/ml). The fraction of dead pupae was reduced by higher or lower spore concentrations as well as by treating younger or older larval populations (both fourth instar): Increased proportions of dead larvae were obtained at higher concentration or by earlier treatment, whereas lower concentrations or later treatment resulted in more living pupae. Multiplication of B. thuringiensis var. israelensis is shown to occur in the carcasses of dead pupae. The number of spores in each pupal carcass followed a similar kinetic as in larval carcasses, but the final yield was about 10-fold higher, apparently reflecting the difference in dry weight between the two mosquito developmental stages (426 micrograms vs 83 micrograms, respectively). The specific larvicidal activity in a homogenized dead pupa was similar to that of B. thuringiensis var. israelensis powder, LC50 of about 600 spores/ml.

Aedes↗
PubMed2001

The energetic costs of diving in Aedes aegypti and Aedes albopictus pupae.

Undisturbed mosquito pupae rest at the water surface and respond to passing shadows or vibrations by diving. Pupae do not feed and rely solely on energy stored from the larval stage. The ability of a newly emerged adult mosquito to survive, and therefore to transmit disease, depends on these energy reserves. Earlier studies of diving behavior in Aedes aegypti, Ae. albopictus, and Ae. triseriatus pupae provided evidence that pupae sense their state of buoyancy and modify their diving behavior accordingly. With strong stimulation pupae tend to dive to a depth where they become neutrally or negatively buoyant and commonly rest on the bottom. This behavior, as well as the tendency to rest when not disturbed, may logically be viewed as energy-conserving. The results of these studies also generated the hypothesis that the diving behavior displayed by these container-breeding mosquitoes helps them avoid being washed from their container by overflowing water during rainfall. Rainfall stimulates diving and logically, prolonged, heavy rainfall stimulates excessive diving, a likely drain on energy reserves. Our objectives were to determine, in Ae. aegypti and Ae. albopictus, the energetic costs associated with resting behavior, with frequent diving, and with buoyancy reduction. Using survival rate, mean survival after adult emergence, and measurement of total calories, we found a clear energetic cost associated with frequent diving. In contrast, relative to diving, essentially no energy cost was associated with buoyancy reduction, that is, pupae behave in response to variations in buoyancy in a way that does not impact significantly on energy reserves.

Aedes↗
PubMed2000

Locomotory modes in the larva and pupa of Chironomus plumosus (Diptera, Chironomidae).

The locomotory kinematics of Chironomus plumosus larvae and pupae were investigated in order to determine how different locomotory techniques may be related to (a) possible underlying patterns of muscle activation and (b) the particular lifestyles and behaviours of these juvenile stages. Larvae display three independent modes of motile activity: swimming, crawling and whole-body respiratory undulation. Swimming and respiratory undulation involve the use of metachronal waves of body bending which travel in a head-to-tail direction. Whereas swimming is produced by side-to-side flexures of the whole body, respiratory undulation employs a sinusoidal wave. Crawling appears to result from an independent programme of muscle activation. Instead of a longitudinally transmitting metachronal wave of body flexure, a simultaneous arching of the body, combined with the alternating use of the abdominal and prothoracic pseudopods as anchorage points, produces a form of locomotion analogous to caterpillar-looping. Larval swimming has a set speed and rhythm and is an 'all-or-nothing' locomotory manoeuvre, but the neural programme controlling larval crawling is adaptable; switching from a less to a more slippery substrate resulted in a shorter, faster stepping pattern. The pupa displays two swimming modes, somersaulting and eel-like whole-body undulation, the former being principally a brief, escape manoeuvre, the latter being a faster form of locomotion employed to deliver the pupa to the surface prior to adult emergence. Comparison with the pupa of the culicid Culex pipiens shows that this insect also uses the somersault mechanism but at a higher cycle frequency which produces a faster swimming speed. This appears to be related to differences in lifestyle; the surface-living culicid pupa is exposed to greater predator threat than the bottom-dwelling chironomid pupa, and consequently needs a faster escape.

Journal Article↗
PubMed2001

Evidence of intracolony transmission of Thelohania solenopsae (Microsporidia: Thelohaniidae) in red imported fire ants (Hymenoptera: Formicidae) and the first report of spores from pupae.

Red imported fire ant, Solenopsis invicta, colonies were infected horizontally by introducing live brood (mainly larvae and pupae) infected with Thelohania solenopsae. Live, infected brood introduced into uninfected colonies were adopted and raised to adulthood instead of being executed by the recipient colony. Introductions of infected larvae with uninfected pupae, which eclose into adult worker caste fire ants, resulted in an 80% infection rate of the inoculated colonies. Infections from introductions of infected pupae with uninfected larvae resulted in a 37.5% infection of inoculated colonies. Infections were also detected in 11.6 and 3.7% of the adult worker caste ants that eclosed from uninfected large larvae and pupae, respectively, that were held with infected adult workers. Microscopic examination of infected brood revealed sporoblasts and large numbers of spores of T. solenopsae in S. invicta pupae.

Animals↗
PubMed1980

Ultrastructure of the prothoracic gland of variously aged female pupae of Xyleborus ferrugineus and associated ecdysteroid titers.

The prothoracic glands of female pupae of Xyleborus ferrugineus at three ages (0-h, 48-h, and 72-h-old) were examined for ultrastructural changes that correlate with high titers of ecdysteroids in the entire pupa. In all three ages, the prothoracic gland cells appear compact with a prominent nucleus. Lysosome-like structures with concentrically oriented internal membrane are observed in both 0-h and 72-h-old gland cells. Mitochondria are abundant in the glands of both ages, and rough endoplasmic reticulum is relatively sparse. Numerous microtubules are present in the prothoracic gland cells of 48-h-old female pupae that contain relatively high titers of ecdysteroids. Rough endoplasmic reticulum also is especially abundant at this age of the pupa. Ecdysone titers, estimated by the radioimmunoassay method (RIA), were 161.04 +/- 26.80 pg/mg, 704.25 +/- 69.02pg/mg and 298.72 +/- 26.80pg/mg body weight in 0-h, 48-h and 72-h-old female pupae, respectively.

Animals↗
PubMed2006

Expression, purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor.

To date, many recombinant proteins have been expressed in Bombyx mori cells or silkworm larvae, apart from in pupae. Silkworm pupae may be more suitable for the expression of heterologous proteins as a bioreactor. If maintained at an appropriate temperature, silkworm pupae could be inoculated with recombinant baculovirus for the expression of a protein of interest. In this study, human granulocyte-macrophage colony-stimulating factor was successfully expressed in silkworm pupae using B. mori nucleopolyhedrovirus, purified and characterized with respect to its physico-chemical properties. The target protein expressed had an apparent molecular mass of 29 kDa and an isoelectric point of 5.1. The protein was purified using three chromatographic steps with a final recovery of 10.3%. Finally, approximately 3.5mg of the protein was obtained with a biological activity of up to 8.4 x 10(6) cfu mg(-1). The results of this study suggest that silkworm pupae represent a convenient and low-cost bioreactor for the expression of heterologous proteins.

Animals↗
PubMed2005

Temporal expression patterns of diapause-associated genes in flesh fly pupae from the onset of diapause through post-diapause quiescence.

Distinct differences in the temporal expression patterns of genes associated with pupal diapause were noted in the flesh fly, Sarcophaga crassipalpis. The first change observed was a decline in expression of the gene encoding heat shock protein 90 (hsp90) 2 days after pupariation (1 day before the pupa reaches the phanerocephalic stage characteristic of diapause). In contrast, hsp23 and hsp70 transcripts were undetectable in nondiapause samples and d1-d4 diapause-programmed pupae, but were up-regulated just after the start of diapause, 5 days after pupariation. An increase of glycerol content in diapausing pupae was also noted at the start of diapause. The gene encoding proliferating cell nuclear antigen (pcna) was diapause down-regulated, and this occurred in two phases, with the first decline in expression 7 days after pupariation and a second decline in the level of expression on day 14. For pupae held at 20 degrees C for 20 days and transferred to 10 degrees C, diapause ended after 90-100 days at the lower temperature. However, pupae remained in a state of post-diapause quiescence (d100-d150) and sustained diapause-like hsp and pcna expression patterns until adult development was initiated. Glycerol concentrations and survival declined during the post-diapause phase. This study suggests a distinct sequence in the pattern of gene expression at the onset of diapause, but the genes we have monitored do not contribute to the switch to covert developmental potential at the transition from diapause to post-diapause quiescence.

Animals↗
PubMed2005

Antibacterial and antifungal lysozyme-type activity in Cameraria ohridella pupae.

Lysozyme-type antibacterial and antifungal activity in pupae of Cameraria ohridella was studied. Activity against Micrococcus luteus and Bacillus megaterium was detected in pupae extract. Also antifungal activity from C. ohridella pupae extract directed against Saccharomyces cerevisiae strain W 303 was shown. During immunoblotting two bands in pupae extract, with molecular mass of about 15 and 28 kDa were recognized by antibodies directed against HEWL. After acid electrophoresis followed by bioautography of the extract, two lytic zones showing lysozyme-type activity against M. luteus were observed. Two bacteria: Gram-positive Aerococcus viridans and Gram-negative Aeromonas salmonicida ssp. masoucida were isolated from pupae of C. ohridella. Their activity against M. luteus, B. megaterium, and S. cerevisiae W303 was detected. After immunoblotting with antibodies against HEWL, also two proteins from bacterial suspensions of A. viridans and A. salmonicida were detected, about 15 and 28 kDa.

Animals↗
PubMed2005

Effects of amino acids from selenium-rich silkworm pupas on human hepatoma cells.

Selenium (Se) plays an important role in cancer-prevention. Silkworm pupas have been used as a Chinese traditional medicine since ancient time. In order to find effective carcinostatic agents, Se-rich amino acids were extracted from Ziyang silkworm pupas. The Se content of Ziyang pupas was measured to be 215 times higher than that of Luoyang normal ones, and the majority of Se was stored in proteins. Composition analysis showed that Se-rich amino acids from Ziyang pupas had higher amounts of selenomethionine, methionine, cystine, and tyrosine than normal amino acids from Luoyang pupas which were rich in amino acids containing alkyl side chains. When cultured with human hepatoma cells SMMC-7721, Se-rich amino acids at concentrations of 0.5, 1.5, and 2.5 micromol L(-1) Se significantly and dose-dependently inhibited cell viability, induced changes in cell morphology and cycle, and caused cell apoptosis. On the contrary, normal amino acids did not show any inhibitory effect on SMMC-7721 cells. Sodium selenite or selenomethionine at the same Se concentrations only slightly inhibited the hepatoma cells. Mechanism study showed that selenium-rich amino acids could increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine partially inhibited the increase of ROS. Those results suggested that Se-rich amino acids were effective carcinostatic agents compared with sodium selenite and selenomethionine. The mechanism for their hepatoma-inhibitory effects was the induction of cellular apoptosis through ROS generation.

Amino Acids↗
PubMed2006

Isolation and characterization of neutral lipids of desilked eri silkworm pupae grown on castor and tapioca leaves.

The neutral lipid of desilked eri silkworm pupae (Samia cynthia ricini) grown on two different host plants, castor (Ricinus communis Linn.) and tapioca (Manihot utilizsima Phol.) leaves, was extracted with hexane. The oil content in pupae was estimated to be in the range of 18-20% (dry basis). The pupal oil was found to be enriched with alpha-linolenic acid (ALA) with palmitic acid as the second major fatty acid. The level of ALA in the oil of silkworm pupae was found to be significantly higher (P < 0.001) when grown on tapioca (58.3%) as compared to those grown on castor (42.9%). Other chemical parameters such as percent free fatty acid, peroxide value, phosphorus content, percent unsaponifiable matter, and composition of sterols were also determined in both of the oils and compared. Reversed-phase high-performance liquid chromatography analysis of triacylglycerol molecular species showed that the pupal oil is rich in molecular species with equivalent carbon numbers (ECN) C36, C40, C42, C44, and C48. There was a significantly higher level (P < 0.001) of trilinolenin (C36) in the oil of tapioca-based silkworm as compared to castor-based silkworm pupae. Regiospecific analysis of the oil showed a higher level of ALA at the sn-2 position of silkworm pupae grown on tapioca (60.2%) as compared to those grown on castor (47.3%) oil. Thus, the presence of a large amount of ALA and their predominance at the sn-2 position make the eri pupal oil highly nutritious, provided that the oxidative stability is ensured.

Animals↗
PubMed2003

The cypovirus Diadromus pulchellus RV-2 is sporadically associated with the endoparasitoid wasp D. pulchellus and modulates the defence mechanisms of pupae of the parasitized leek-moth, Acrolepiopsis assectella.

Diadromus pulchellus is a solitary endoparasitoid wasp that parasitizes the pupae of the leek-moth, Acrolepiosis assectella (Lepidoptera). Hitherto, every individual D. pulchellus from France that has been investigated was infected by an orthoreovirus, DpRV-1, and an ascovirus, DpAV-4. Recently, a new strain of D. pulchellus, established from a French field population, was found to be able to develop on leek-moth pupae, but lacked both DpRV-1 and DpAV-4. However, all these wasps were infected with a new cypovirus, DpRV-2. This cypovirus is transmitted to the A. assectella pupae at each wasp oviposition and is replicated mainly in the gut cells of the parasitized pupae. DpRV-2, like the ascovirus DpAV-4, is able to inhibit the defence reaction of A. assectella pupae and so contributes to the parasitic success of D. pulchellus wasps.

Animals↗
PubMed1986

Cellular changes in the prothoracic glands of diapausing pupae of Manduca sexta.

Prothoracic glands from diapausing pupae of the tobacco hornworm, Manduca sexta, synthesize markedly less ecdysone in vitro in response to prothoracicotropic hormone (PTTH) than do glands from non-diapausing pupae. Impaired steroidogenesis is also observed in glands from diapausing animals exposed to agents that enhance ecdysone synthesis in non-diapausing pupal glands by increasing intracellular levels of cAMP (1-methyl-3-isobutylxanthine, dibutyryl cAMP, and the calcium ionophore A23187). In contrast, prothoracic glands from diapausing pupae synthesize significantly more cAMP in response to PTTH and A23187 than do those from non-diapausing pupae. These observations indicate that the PTTH-refractoriness characteristic of prothoracic glands during diapause results from a lesion in the steroidogenic pathway occurring beyond the level of the PTTH receptor-adenylate cyclase system. The diapause condition of the prothoracic glands (reduced ecdysone synthesis accompanied by enhanced cAMP formation) can be mimicked by extirpation of the brain of a non-diapausing pupa. Thus, cellular changes in the prothoracic glands associated with diapause may arise as a result of the absence of some factor produced by the pupal brain (e.g. PTTH).

1-Methyl-3-isobutylxanthine↗
PubMed1999

Buoyancy and diving behavior in mosquito pupae.

Mosquito pupal diving behavior has been studied mostly in Aedes aegypti and in this species pupal buoyancy varies relative to several factors. The research reported herein addresses the 2 following questions. Does diving behavior vary among different mosquito genera and species? How is diving behavior influenced by variation in buoyancy? Depth and duration of dive, and dive pattern, were compared among Ae. aegypti, Culex pipiens, Anopheles stephensi, Aedes albopictus, and Aedes triseriatus. In response to the stimulation associated with transferring pupae between containers, diving behavior varied dramatically among the different genera studied. Culex pipiens and An. stephensi make short-duration, shallow dives and remain positively buoyant. The 3 aedine species studied make longer-duration dives, typically to a depth at which they become neutrally or negatively buoyant. Buoyancy reduction effects were studied in the 3 aedine species. Normally buoyant pupae tend to dive to greater depths and for longer periods of time than reduced-buoyancy pupae. Aedine pupal diving behavior clearly is closely regulated relative to buoyancy variation. To the earlier hypotheses that pupal behavior may help avoid predation and be energy-conserving, we add the suggestion that the diving behavior displayed by the container-breeding aedine pupae we studied represents an adaptation that helps keep them from being washed from their container habitat by overflowing water during rainfall. We also suggest that the diving behavior of all the species studied may help pupae survive heavy, pelting rainfall by enabling them to avoid the mechanical shock of a direct hit by a raindrop, which could cause disruption of the gas in the ventral air space, thereby causing the loss of hydrostatic balance and drowning.

Aedes↗
PubMed1979

Comparative levels of lysozyme activity in larvae and pupae of Galleria mellonella after particulate and soluble materials injection.

Using an assay system for lysozyme activity determination in which haemolymph was compared in activity to known standards, the quantitative shifts in the lysozyme activity level of larvae and pupae of Galleria mellonella, after particulate and soluble materials injection, were compared. The level of this activity in all injected larvae was higher fro that of pupae, but the immune response of pupae was more effective in all treatments when compared with activity in the non-injected controls. Furthermore, there was a significant higher level of lysozyme activity in pupae subjected to mechanical immunizing agents, except inversed pupae in which alternation in activity was not significantly different from activity in the corresponding nonexposed insects. Although the exact role of lysozyme in insect immunity is still uncertain, it is possible that this lysosomal enzyme may degrade the cell wall of susceptible parasites prior to being phagocytosed, and thereby act as a nonspecific inducible humoral defence molecule.

Animals↗