Sarcophaga Bullata Research Articles

Diapause hormone in the corn earworm, Helicoverpa zea: Optimum temperature for activity, structure–activity relationships, and efficacy in accelerating flesh fly pupariation

Diapause hormone (DH) effectively terminated pupal diapause in Helicoverpa zea. This effect was temperature-dependent, with an optimum of 21 °C. The dose–response curve indicated an ED 50 of DH for diapause termination of approximately 100 pmol. The core sequence and essential amino acids were determined by bioassays using modified and truncated DH analogs. A C-terminal hepta-peptide, LWFGPRLa, was the core sequence required for diapause termination. Activity was lost when Alanine was substituted for any of the amino acids in the hepta-peptide, with the exception of Glycine. A fragment series of analogs suggested that the amide and Arginine were the most important components needed for terminating diapause. Leucine, Tryptophan, and Phenylalanine at the N-terminus of the hepta-peptide were also critical for activity. The C-terminal Leucine was less important: deletion resulted in decreased activity, although it could not be substituted by Alanine. The fact that a portion of the DH sequence is similar to the pyrokinin that accelerates fly pupariation prompted us to also evaluate the capability of DH to accelerate development in the flesh fly, Sarcophaga bullata. The threshold dose of DH essential to accelerate fly pupariation was 5 pmol for immobilization/retraction and longitudinal contraction and 10 pmol for tanning, approximately one or two orders of magnitude lower than the effective dose required for diapause termination in H. zea. Tensiometric measurements revealed that DH affected neuromuscular patterns of pupariation behavior and associated cuticular changes in a manner similar to that of the fly pyrokinins and their analogs.

Mapping the peptide and protein immune response in the larvae of the fleshfly Sarcophaga bullata

We chose the larvae of fleshfly Sarcophaga bullata to map the peptide and protein immune response. The hemolymph of the third-instar larvae of S. bullata was used for isolation. The larvae were injected with bacterial suspension to induce an antimicrobial response. The hemolymph was separated into crude fractions, which were subdivided by RP-HPLC, gel electrophoresis, and free-flow electrophoresis. In several fractions, we determined significant antimicrobial activities against the pathogenic bacteria Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa. Among antimicrobially active compounds we identified dipeptide beta-alanyl-L-tyrosine, protein transferrin, and two variants of peptide sapecin. We also partially characterized two novel antimicrobially active polypeptides; odorant-binding protein 99b, and a peptide which remains unidentified.

Insect Pest Management Agents: Hormonogen Esters (Juvenogens)

The chemical part of this investigation focused on designing structures and synthesizing a series of six new esters (juvenogens), derived from biologically active insect juvenile hormone bioanalogues (juvenoids, JHAs) and unsaturated short-chain linear and branched fatty acids for possible application as biochemically targeted insect hormonogen agents. The structures of the new compounds were assigned on the basis of a detailed NMR analysis of their (1)H and (13)C NMR spectra. The biological part of this investigation focused on introductory biological screening tests with these compounds against the red firebug (Pyrrhocoris apterus), termites (Reticulitermes santonensis and Prorhinotermes simplex), and the blowfly (Neobellieria bullata). The biological activity of the juvenogens was studied in relation to the fatty acid functionality in the structures. Notable biological activity in topical tests and medium activity in peroral tests was found for the juvenogens 3 and 7 with P. apterus. The compounds 6 and 8 showed the lowest activity in both topical and oral assays with P. apterus. Considerable effect of all tested juvenogens was observed in P. simplex; however, the juvenogens 5 and 6 (derivatives of the only branched short-chain fatty acid) showed no activity against R. santonensis. The effect of the compounds 3-8 on larval hatching of N. bullata was only moderate (larval hatching 80-90%); however, the proliferation effect caused by 5, 6, and 8 is more pronounced than the effect caused by 3, 4, and 7.

Isolation and characterization of antimicrobial peptides from larvae of flesh fly Neobellieria bullata

Different types of antimicrobial peptides have been identified in seeds from different plant species. The aim of this study was to isolate and characterize peptides present in chilli pepper seeds (Capsicum annuum L.) and evaluate their toxic activities against some yeast species. Initially, proteins from seed flour were extracted in phosphate buffer, pH 5.4, for 3 h at 4 °C and the pellet obtained at 90% saturation with ammonium sulfate was heated at 80 °C for 15 min. The resulting suspension was clarified by centrifugation and the supernatant was extensively dialyzed against water; the peptide-rich extract was then named F/0-90. Cation-exchange chromatography was performed to separate low molecular mass proteins. One of the resulting fractions, named F3, enriched with basic proteins of 6–16 kDa, was submitted to reverse-phase chromatography in a C2/C18 column by HPLC, resulting in four fractions denominated RP1, RP2, RP3 and RP4. When these fractions were submitted to N-terminal sequencing, the comparative analysis in databanks revealed homology for two of these peptides, isolated from fractions RP3 and RP4, with sequences of proteinase inhibitors and 2S albumins, respectively. The F3 fraction, rich in peptides, inhibited the growth of yeasts Saccharomyces cerevisiae, Candida albicans, Candida parapsilosis, Candida tropicalis, Pichia membranifaciens, Kluyveromyces marxiannus and Candida guilliermondii. The RP3 and RP4 fractions showed high inhibitory activity against the growth of the yeast S. cerevisiae. The F3 fraction was also able to inhibit glucose-stimulated acidification of the medium by yeast cells of S. cerevisiae and to cause several morphological changes in different yeasts, such as cell wall disorganization, bud formation as well as the formation of pseudohyphae.

Effects of Male Age and Cervical Proprioceptors on Sexual Aerial Pursuit by Male Flesh Flies, Neobellieria bullata (Diptera: Sarcophagidae)

Information from multiple sensory systems is likely combined to provide guidance for male muscoid flies engaged in aerial pursuit of females. To specify the female’s position to the thoracic flight motor, head-centered visual information should be integrated with propriosensory information about head position relative to the thorax because the flies’ heads are flexible around the neck. Head position is encoded by a proprioceptive organ in the ventral neck membrane. We determined in the flesh fly Neobellieria bullata (Sarcophagidae) that accurate propriosensory information is necessary for aerial capture of females by shaving mechanosensory hairs from the organ in male flies and competing them against their sham operated brothers in pursuits of virgin females. We also determined that normal male flies are not successful at capturing females until the second day post-eclosion.

Assortative Mating in the Jewel Wasp: 1. Female Matching of Eye-Color Genotype, Not Host-Feeding Phenotype

ABSTRACT An experiment was performed to test the influence of two factors on assortative mating in the Jewel wasp (Nasonia vitripennis): (1) inherited eye-color genotype, and (2) conditioned foraging phenotype. To determine the female choice of sires, genetically marked wasps were used of two recessive mutant eye-color genotypes: white or red. Each female (of independently crossed eye-color and juvenile host) was given a choice of two males, one of each eye-color, counterbalanced for juvenile host: blowfly (Sarcophaga bullata) or housefly (Musca domestica) pupae. Blowfly-reared females produced more total offspring, indicating higher fertility. Red-eyed females produced more offspring sired by red-eyed males, indicating assortative mating by eye color. Blowfly-reared females, however, did not produce more offspring sired by blowfly-reared males, indicating no assortative mating by juvenile host.

Morphology and physiology of the prosternal chordotonal organ of the sarcophagid fly Sarcophaga bullata (Parker)

The anatomy and the physiology of the prosternal chordotonal organ (pCO) within the prothorax of Sarcophaga bullata is analysed. Neuroanatomical studies illustrate that the approximately 35 sensory axons terminate within the median ventral association centre of the different neuromeres of the thoracico-abdominal ganglion. At the single-cell level two classes of receptor cells can be discriminated physiologically and morphologically: receptor cells with dorso-lateral branches in the mesothoracic neuromere are insensitive to frequencies below approximately 1 kHz. Receptor cells without such branches respond most sensitive at lower frequencies. Absolute thresholds vary between 0.2 and 8 m/s 2 for different frequencies. The sensory information is transmitted to the brain via ascending interneurons. Functional analyses reveal a mechanical transmission of forced head rotations and of foreleg vibrations to the attachment site of the pCO. In summed action potential recordings a physiological correlate was found to stimuli with parameters of leg vibrations, rather than to those of head rotation. The data represent a first physiological study of a putative predecessor organ of an insect ear.

Inhibition of PK/PBAN-mediated functions in insects: Discovery of selective and non-selective inhibitors

The antagonistic properties of a few linear and backbone cyclic (BBC) conformationally constraint peptide libraries and their analogs, were tested for the ability to inhibit pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) mediated functions: sex pheromone biosynthesis in Heliothis peltigera female moths, cuticular melanization in Spodoptera littoralis larvae, pupariation in the fleshfly Neobellieria bullata and hindgut contraction in Leucophaea maderae, elicited by exogenously injected PBAN, pheromonotropin (PT), leucopyrokinin (LPK), myotropin (MT) or by the endogenous peptides. The data revealed differential inhibitory patterns within the same assay with different elicitors (in both the pheromonotropic and melanotropic assays) and among the different functions and disclosed selective antagonists, hinting at the possibility that the receptors that mediate those functions may differ from one another structurally.

Venom from the Ectoparasitic Wasp <I>Melittobia digitata</I> (Hymenoptera: Eulophidae) Induces Paralysis and Developmental Delay in Natural and Factitious Hosts

Melittobia digitata Dahms (Hymenoptera: Eulophidae) is a gregarious ectoparasitoid that attacks the pupae of solitary bees and wasps. Although this wasp has been intensively studied in terms of behavior and life history, almost nothing is known about its venom. Moreover, the use of venom in the host–parasite relationship has been disputed despite observations that parasitized hosts seem to be halted in development and enter a state of paralysis. In this study, we examined the role of venom from M. digitata during parasitism by comparing the developmental fate of natural (alfalfa leafcutting bee, Megachile rotundata F.) and factitious [yellow mealworm, Tenebrio molitor L. and flesh fly, Sarcophaga bullata (Parker)] hosts after either natural envenomation or artificial venom injection. Isolated crude venom collected either by “milking” adult females or by dissections of venom glands was nearly equally effective in halting the development of M. rotundata, T. molitor, and S. bullata. Host responses to venom injection were dependent on the developmental stage of the host tested and concentration of venom injected. For all three hosts, venom seemed to induce paralysis, but the onset of paralysis was not the same in each host. T. molitor and M. rotundata entered a paralyzed state several hours (2–4) earlier than larvae of S. bullata. Likewise, the type of paralysis induced by venom from M. digitata was not the same for the three insects tested as evidenced by limited mobility and some progression of development in venom-injected M. rotundata and T. molitor, and a flaccid paralysis characterized by complete immobilization in larvae of S. bullata. Ultimately, all venom-injected hosts that became paralyzed eventually died. Our data suggest two potential modes of action for M. digitata venom.

Activation kinetics of cetylpyridinium chloride on the prophenol oxidase from pupae of blowfly ( Sarcophaga bullata)

Prophenol oxidase (PPO) (EC 1.14.18.1) is isolated from pupae of blowfly ( Sarcophaga bullata) and purified by employing ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose, and gel filtration through Sephadex G-100 column chromatography. The enzyme exists in a latent or inactive state. Cetylpyridinium chloride (CPC), a cationic detergent, is found to activate the PPO activity. The activation of the enzyme by CPC has first been studied by using the kinetic method of the substrate reaction described by Tsou. The results show that the enzyme is activated by a complexing scheme that has not been previously identified. The enzyme first reversibly and quickly binds CPC and then undergoes a slow reversible active course. The activation reaction is a single molecule reaction and the apparent activation rate constant is dependent on the CPC concentration with the function relationship fit with a hyperbola. The micro rate constants of activation and the association constant are determined from the measurements. Substrate binding does not affect the micro rate constants of activation by CPC.

Characterization of phenoloxidase activity in venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

Crude venom isolated from the ectoparasitic wasp Nasonia vitripennis was found to possess phenoloxidase (PO) activity. Enzyme activity was detected by using a modified dot blot analysis approach in which venom samples were applied to nylon membranes and incubated with either l-DOPA or dopamine. Dot formation was most intense with dopamine as the substrate and no activators appeared to be necessary to evoke a melanization reaction. No melanization occurred when venom was incubated in Schneider’s insect medium containing 10% fetal bovine serum or when using tyrosine as a substrate, but melanization did occur when larval or pupal plasma from the fly host, Sarcophaga bullata, was exposed to tyrosine. Only fly larval plasma induced an enzyme reaction with the Schneider’s insect medium. The PO inhibitor phenylthiourea (PTU) and serine protease inhibitor phenylmethylsulfonylfluoride (PMSF) abolished PO activity in venom and host plasma samples, but glutathione (reduced) only inhibited venom PO. Elicitors of PO activity (sodium dodecyl sulfate and trypsin) had no or a modest effect (increase) on the ability of venom, or larval and pupal plasma to trigger melanization reactions. SDS–PAGE separation of crude venom followed by in-gel staining using l-DOPA as a substrate revealed two venom proteins with PO activity with estimated molecular weights of 68 and 160 kDa. In vitro assays using BTI-TN-5B1-4 cells were performed to determine the importance of venom PO in triggering cellular changes and evoking cell death. When cell monolayers were pre-treated with 10 mM PTU or PMSF prior to venom exposure, the cells were protected from the effects of venom intoxication as evidenced by no observable cellular morphological changes and over 90% cell viability by 24 h after venom treatment. Simultaneous addition of inhibitors with venom or lower concentrations of PMSF were less effective in affording protection. These observations collectively argue that wasp venom PO is unique from that of the fly hosts, and that the venom enzyme is critical in the intoxication pathway leading to cell death.

A nondiapausing variant of the flesh fly, Sarcophaga bullata, that shows arrhythmic adult eclosion and elevated expression of two circadian clock genes, period and timeless

We describe a variant of the flesh fly, Sarcophaga bullata, which fails to enter pupal diapause in response to short daylength and low temperatures. This fly also has an arrhythmic adult eclosion pattern: rather than eclosing in early photophase, the variant ecloses arrhythmically throughout the photophase and scotophase. The loss of both diapause (photoperiodic response) and the gating of adult eclosion (presumably a circadian response) suggests that the same clock system is involved in these two responses. An examination of the expression patterns of the clock genes period and timeless demonstrates that both genes are present in the nondiapausing variant, but surprisingly, both genes are expressed at higher levels. This abnormality we observe, possibly the consequence of an upstream clock gene malfunction or a malfunction of the autoregulatory loop, results in disruption of a component of the clock system that is apparently needed for both photoperiodism and circadian rhythmicity.

Preparation of tritiated oostatic peptides for study of radioactivity incorporation in flesh fly Neobellieria bullata

A series of insect oostatic peptides containing 3,4-dehydroproline in the C-terminal part or inside of the peptide chain was synthesized and tritiated by addition of (3)H2 to double bond of 3,4-dehydroproline residue. (3)H-label was introduced also into tyrosine residue of oostatic tetra- and pentapeptides by isotopic exchange of benzyl beta-hydrogens. In this way, three types of tritiated peptides were prepared, different in the radiolabeled amino acid position: [(3)H] Tyr-Asp-Pro-Ala-OH, H-Tyr-Asp-[(3)H] Pro-Ala-OH, [(3)H] Tyr-Asp-Pro-Ala-Pro-OH, H-Tyr-Asp-[(3)H] Pro-Ala-Pro-OH, H-Tyr-Asp-Pro-Ala-[(3)H] Pro-OH, H-Tyr-Asp-Pro-Ala-Pro(5)-[(3)H] Pro-OH and H-Asp-[(3)H] Pro-OH. These peptides made possible a highly sensitive comparative study on radioactivity incorporation into head and ovaries of the flesh fly Neobellieria bullata, which revealed this process to proceed differently. The reasons of the found differences are discussed.

Mistaken view of taxonomic validity undermines conservation of an evolutionarily distinct mouse: a response to Ramey et al. (2005)

1 Department of Biology, University of Washington, Seattle, WA, USA 2 Department of Fisheries and Wildlife Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA 3 Department of Biology, Valdosta State University, Valdosta, GA, USA 4 New Mexico Museum of Natural History, Albuquerque, NM, USA 5 Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, CO, USA 6 School of Molecular and Life Sciences, University of Limpopo, Sovenga, South Africa

Host Ranges of Six Solitary Filth Fly Parasitoids (Hymenoptera: Pteromalidae, Chalcididae) from Florida, Eurasia, Morocco, and Brazil

Abstract Attack rates, progeny production, sex ratios, and host use efficiency of Muscidufurax raptor Girualt and Sanders, Spalangia cameroni Perkins, S. endius (Walker), S. nigroaenea Curtis, S. gemina Boucek (Hymenoptera: Pteromalidae), and Dirhinus himalayanus (Hymenoptera: Chalcididae) were evaluated in laboratory bioassays with five dipteran hosts: house fly (Musca domestica L.), stable fly (Stomoxys calcitrans L.), horn fly (Hematobia irritans L.), black dump fly [Hydrotaea aenescens (Weidemann)] (Diptera: Muscidae), and a flesh fly (Sarcophaga bullata Parker) (Diptera: Sarcophagidae). M. raptor, S.cameroni, and S. endius readily attacked and produced progeny on all five host species, with substantially lower production from S. bullata than from the muscid hosts. Rates of host attacks by S. nigroaenea and S. gemina were similar on house fly, stable fly, and black dump fly hosts, with lower rates on horn fly; almost no progeny were produced by S. nigroaenea on S. bullata hosts. D. himalayanus, a larg...

Can juvenogens, biochemically targeted hormonogen compounds, assist in environmentally safe insect pest management?

Two different types of juvenogens, biochemically targeted hormonogen compounds were tested for their potency to act as insect pest management agents. In the performed biological screening, wax-like esteric juvenogens ( 3– 10) proved to be convenient agents for controlling blowfly and termites, and displayed species selectivity: cis- N-{2-[4-(2-butanoyloxycyclohexyl)methyl]phenoxy}ethyl carbamate ( 3) was highly active on blowfly ( Neobellieria bullata), while trans- N-{2-[4-(2-hexadecanoyloxycyclohexyl)methyl]-phenoxy}ethyl carbamate ( 6) showed high activity on termite ( Prorhinotermes simplex). Glycosidic juvenogens, isomeric N-{2-{4-{[2-( β-D-galactopyranosyloxy)cyclohexyl]methyl}phenoxy}ethyl carbamates ( 13 and 14), were proved to act as systemic agents, suitable for protecting plants against phytophagous insects (e.g. aphids). Due to the prolonged action of juvenogens, which is connected with the sequential liberating of the biologically active molecule of the insect juvenile hormone bioanalog from the juvenogen molecule by means of enzymic systems of target insects and/or their host plants, more insect individuals can be treated by juvenogens, which are species-targeted structures due to their different physicochemical properties. The results achieved with both types of juvenogens were promising, concerning their final effect on the tested insect species, and the compounds 3–6, 9 ( cis-(9 Z)- N-{2-[4-(2-(octadec-9-enoyl)oxycyclohexyl)methyl]phenoxy}ethyl carbamate), 13 and 14 proved to represent convenient insect pest management agents for potential practical applications against different insect pests.

A comparison of the pupariation acceleration activity of pyrokinin-like peptides native to the flesh fly: Which peptide represents the primary pupariation factor?

Five native pyrokinin-like peptides (Neb-PK-1, Neb-PK-2, Neb-PVK-1, [L 9]Neb-PVK-2, [I 9]Neb-PVK-2) identified in the neuropeptidome of the flesh fly Neobellieria bullata were compared for their quantitative and/or qualitative effects on puparium formation (pupariation). In a standard pupariation bioassay, both Neb-PVK-1 and [I 9]Neb-PVK-2 proved inactive, whereas [L 9]Neb-PVK-2 demonstrated only weak activity. In contrast, both Neb-PK-1 and Neb-PK-2 demonstrated potent threshold doses, with Neb-PK-2 about 10-fold more active than Neb-PK-1. Analysis of neuromuscular activity during pupariation using a tensiometric technique demonstrates that the two native Neb-PKs accelerate the onset of immobilization and cuticular shrinkage more than motor programs associated with retraction of the anterior segments and longitudinal body contraction. It was further determined that the sensitivity of various components of the pupariation process to these peptides decreases in the following order: immobilization > cuticular shrinkage > motor program for anterior retraction > motor program for longitudinal contraction ≅ tanning of cuticle of the newly formed puparium. A paradoxical situation was observed whereby the motor programs of pupariation are temporally dissociated from actual morphogenesis of the puparium. The tensiometric data suggest that the most likely candidate for a primary pupariation factor is Neb-PK-2, rather than Neb-PK-1.

20-Hydroxyecdysone and juvenile hormone regulate the laminarin-induced nodulation reaction in larvae of the flesh fly, Neobellieria bullata

Nodulation, which is considered the predominant defense reaction to infection in insects, is a complex process influenced by various endogenous factors. However, the precise mechanisms underlying nodulation remain largely unknown. In the present study, we examined the influence of the insect hormones 20-hydroxyecdysone (20E) and juvenile hormone (JH) on the laminarin-induced nodulation reaction in larvae of the flesh fly Neobellieria bullata. Treating third-instar larvae of N. bullata with 20E prior to laminarin injection enhanced the nodulation response in a dose-dependent manner. The ecdysone agonists RH2485, RH5849 and RH0345 similarly enhanced the nodulation reaction, although they were less active than 20E. In contrast to ecdysone stimulation, supplying larvae with JH or the juvenile hormone analogs (JHA), fenoxycarb and pyriproxyfen, significantly impaired their ability to form nodules in response to laminarin. These findings demonstrate for the first time that 20E and JH play an important regulatory role in the nodulation process.

Stress-induced accumulation of glycerol in the flesh fly, Sarcophaga bullata: Evidence indicating anti-desiccant and cryoprotectant functions of this polyol and a role for the brain in coordinating the response

Nondiapausing larvae of the flesh fly, Sarcophaga bullata, responded to several forms of short-term environmental stress (low temperature, anoxia and desiccation) by accumulating glycerol. Elevation of this polyol, regardless of the type of stress that induced accumulation, conferred cold resistance: larvae with high glycerol levels were 3–4 times more tolerant of a 2 h exposure to −10 °C than unstressed larvae. Protection against low temperature injury, as well as dehydration, was also attained by injection of exogenous glycerol into third instar larvae. This artificially induced cold hardiness was only temporary: when glycerol-injected larvae were exposed to −10 °C immediately after injection, survival was high, but none survived if they were injected and then held at 25 °C for 2 days before the −10 °C exposure. Larvae ligated behind the brain immediately after low temperature exposure failed to accumulate glycerol, but glycerol did accumulate in larvae ligated 6–24 h after cold treatment, thus implying a critical role for the brain in initiating glycerol production. Interestingly, a much shorter exposure (2 h) to low temperature was sufficient to reduce the maximum rate of water loss. Collectively, these observations suggest that multiple pathways may be exploited in response to stress: one pathway is most likely associated with rapid cold hardening (RCH) which generates immediate protection, and a second pathway remains activated for a longer period to enhance the initial protection afforded by glycerol.

Mass spectrometric assignment of Leu/Ile in neuropeptides from single neurohemal organ preparations of insects

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF–TOF) tandem mass spectrometry has been applied for the first time on an insect/arthropod target, focusing on PVK/CAP2b neuropeptides in the housefly Musca domestica and flesh fly Neobellieria bullata. The peptidomic analysis of single neurohemal organ preparations allows the unambiguous assignment of internal Leu/Ile positions not distinguishable by previous mass spectrometric techniques. The confirmation of side-chain fragments which allows assignment of Leu/Ile even from samples as small as neurohemal organs will greatly accelerate the identification of novel neuropeptides that are implicated in the regulation of critical physiological processes in insects. The unnatural Ile analog is 4.5 times more active than the native Leu sequence in a housefly Malpighian tubule fluid secretion assay, which reinforces the caveat that potency values in a biological assay cannot be relied upon to predict the native sequence.