Disruption of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata Parker (Diptera: Sarcophagidae), by venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

The action of venom from the ectoparasitic wasp, Nasonia vitripennis, was monitored by examining alterations in patterned muscular movements characteristic of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata. Venom injected into larvae prior to pupariation caused a dose-dependent delay in pupariation. Eventually, such larvae did pupariate, but puparia were abnormally formed. Barographic records revealed that all elements of pupariation behavior were present in venom-injected larvae, but pupariation behavior was not well synchronized with tanning, thus implying that the venom caused disruption in the temporal organization of central motor programs. When larvae were ligated and injected with venom posterior to the ligature, no response was evident in the posterior region, suggesting that the venom does not directly stimulate muscles or neuromuscular junctions. Injection of exogenous ecdysteroid into venom-injected larvae restored some elements of pupariation behavior, consistent with ecdysone's role in stimulating the release of anterior retraction factor and puparium tanning factor, two factors that are released from the CNS to regulate pupariation. When the venom was injected into newly emerged imagoes, the duration of extrication behavior was shortened, whereas all phases of post-eclosion behavior were lengthened. These observations imply that the venom affects CNS centers that regulate the muscular systems engaged in extrication and post-eclosion behavior.     

Comparison of the effects of pyrokinins and related peptides identified from arthropods on pupariation behaviour in flesh fly (Sarcophaga bullata) larvae (Diptera: Sarcophagidae)

Peptides from the pyrokinin/PBAN family and some structurally related compounds identified in various arthropods were tested for acceleration of puparial contraction in flesh fly larvae. Modifications of behavioural patterns of pupariation were further studied for the active compounds using a behavioural analysis based on the recording of changes in tension of the cuticle. Nine peptides belonging to the pyrokinin/PBAN family (Lem-PK, Pea-PK-5, Lom-PK II, Hez-PBAN, Bom-DH-I), identified in five different insect species, two pyrokinin peptides derived from the genome of Drosophila melanogaster (capa-3, and hugin), and two pyrokinins identified from the white shrimp Penaeus vannamei were very active in the pupariation assay, with threshold doses within the range of 0.1-5.0 pmol larva(-1). High activity was also detected for a related peptide ETH1 from Drosophila. All of these peptides share a C-terminal PRLamide, which is essential and sufficient for the activity. Interestingly, two other structurally related peptides from Drosophila--ETH2 and capa-1--which feature conservative changes (Ile and Val, respectively) at the C-terminal Leu position, were inactive within a physiological range of concentrations. It is clear that the receptor mediating the acceleration of puparial contraction behaviour is sensitive to the introduction of greater steric bulk at the C-terminal Leu position. The peptides that accelerated pupariation showed very similar patterns of muscular and cuticular activity.     

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, [L9]Neb-PVK-2, [I9]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 [I9]Neb-PVK-2 proved inactive, whereas [L9]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 congruent to 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.     

Wandering behaviour and pupariation in tsetse larvae

Abstract .Following parturition, the third instar larva of Glossina morsitans morsitans West begins a wandering period in which it crawls to the site of pupariation. The duration of wandering can be drastically shortened by pinching or by denying the larva physical contact with the substrate. Contact with water increases the wandering period. Duration of subsequent activities appears to be rigidly fixed. At the end of the wandering period, the larva quickly progresses through a stereotypic sequence of behaviours that include immobilization and excretion of a liquid from the anus, retraction of the anterior segments, cuticular shrinkage, and tanning. Muscular activity and mechanical changes in the cuticle are reflected in changes of haemocoelic pressure. Muscular contractions produce pressure pulses that gradually increase in frequency and intensity, reaching a peak during retraction of the anterior segments. Changes in the mechanical properties of the cuticle cause a more gradual elevation of baseline pressure as the cuticle shrinks and loses its plasticity. As tanning begins, muscular activity ceases and haemocoelic pressure gradually decreases. In spite of its unusual early development within the confines of the female's uterus, the free-living larva shows the full behavioural repertoire observed in other cyclor-rhaphous Diptera at pupariation.     

Fraenkel's pupariation factor identified at last

Thirty-five years ago, Zdarek and Fraenkel demonstrated that nervous tissue extracts influenced development by accelerating pupariation in the grey flesh fly, Neobellieria bullata. We have now identified this pupariation factor as SVQFKPRLamide, designated Neb-pyrokinin-2 (Neb-PK-2). To achieve this, the central nervous system of N. bullata wandering stage larvae, that is, preceding pupariation, were dissected and extracted before HPLC separation. Chromatographic fractions were screened with a bioassay for pupariation accelerating activity. Only one fraction showed huge pupariation activity. Mass spectrometry revealed the presence of a pyrokinin, whose primary sequence could not be unequivocally determined by tandem mass spectrometry. However, this Neb-pyrokinin appeared to be very prominent in the ring gland from which it was subsequently purified and identified. Synthetic Neb-PK-2 accelerates pupariation with a threshold dose of only 0.2 pmol and therefore, Neb-pyrokinin is considered to be the genuine pupariation factor. The immunohistochemical distribution pattern of Neb-PK-2 is very similar to that of Drosophila pyrokinin-2, from which it differs by only one amino acid residue. Hence, the recently identified G-protein coupled receptors (CG8784, CG8795) for Drosophila pyrokinin-2 might play an important role in puparium formation.     

The metabolism of 3H-moulting hormone in Calliphora erythrocephala during larval development

The activity in whole insects for converting ³H-α-ecdysone to ³H-β-ecdysone after injection is low (half-maximal) in young last instar larvae, maximal in mature larvae, and minimal (fourth-maximal) at the white puparial stage. Because moulting hormone titre is low throughout the last larval instar and increases at the formation of the puparium it appears that hydroxylation at C-20 is not a key step in regulating β-ecdysone biosynthesis during larval development.The activity for catabolizing ³H-β-ecdysone is maximal in second instar larvae, about thirdmaximal throughout most of the third instar, and minimal at pupariation (thirtieth-maximal). Thus inactivation may play a rôle in regulating moulting hormone titre during larval development.     

Calcium transport from Malpighian tubules to puparial cuticle ofMusca autumnalis

Summary The transport of calcium from mineralized granules stored in the Malpighian tubules to the puparium of the face fly,Musca autumnalis De Geer, was studied. Calcium was transported directly from the tubules to the cuticle via the hemolymph. Little, if any, calcium entered the hindgut or other tissues during or prior to transport. A total of approximately 0.8 mg of calcium per larva was transported, beginning at the wandering stage; peak hemolymph concentrations occurred at anterior retraction. Hemolymph calcium levels subsequently decreased as puparial calcium increased. Puparial mineralization utilized most of the minerals stored during the larval stage, with lesser amounts of minerals being recovered in the adult or excreted. Deposition of mineral salts in the cuticle was accompanied by an increase in cuticular pH from 7.0 to 8.4. The house fly,Musca domestica L., which contains much lower concentrations of minerals in the puparial cuticle, exhibited no increase in cuticular pH during pupariation. Biomineralization of the face fly puparial cuticle appears to occur, in part, as a result of ionic equilibria involving calcium and magnesium phosphates and carbonates, which have relatively low solubility products at alkaline pH.Contribution No. 87-237-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas. Cooperative investigation between Agricultural Research Service, U.S. Department of Agriculture and the Kansas Agricultural Experiment Station. K.J.K. is a research chemist and adjunct professor at U.S. Grain Marketing Research Laboratory and Kansas State University, respectively. Mention of a proprietary product in this paper does not imply its approval by the USDA to the exclusion of other products that may also be suitable. Reprint requests to A.B. Broce     

The calcified puparium of the face fly, Musca autumnalis (Diptera: Muscidae)

The general composition of the calcified puparium of Musca autumnalis was determined. Ash-weight analyses show that 62% of the puparium is inorganic material. The major components of the puparium are calcium, magnesium, phosphate and carbonate. Calcium and magnesium phosphate are the predominant salts in the puparium, with a lesser contribution by their respective carbonates. Scanning electron microscopy revealed that the exocuticle was the site of calcification in the puparium. Inorganic analyses of the insoluble salt isolated from face fly larvae showed it to be compositionally identical with the puparial salt. Major organic components of the puparium are lipid, chitin and protein. The presence of carbonic anhydrase and alkaline phosphatase activity in post-feeding larvae was confirmed.     

X-ray investigation of gas bubble formation, and water loss in tsetse fly pupae

ABSTRACT. Using a microfocal X-ray apparatus, a gas bubble was detected within the puparium of Glossina morsitans. The bubble appeared between 6 and 15 h after pupariation and was associated with one of the longitudinal tracheal trunks of the third instar larva. The bubble grew and achieved maximum size approximately 96 h after pupariation. It then disappeared at the time of eversion of the pupal appendages. There was a close correlation between bubble size and the weight of water lost since the time of pupariation. At the time of eversion of the pupal appendages the gas bubble apparently passed out through the longitudinal tracheal trunk and posterior spiracle to occupy the space between larval (puparial) and pupal cuticle. It is suggested that the bubble plays a vital role in the separation of these cuticular layers and that to this end water loss from the puparium is essential.     

Effect of x-irradiation on the formation of the puparium in the fleshfly, Sarcophaga bullata

Post-feeding, pre-critical stage larvae (36 hr before pupariation) of Sarcophaga bullata were exposed to X-rays and the effects on pupariation observed. With doses ranging from 1250 to 10,000 R the prepuparial period was prolonged and the duration of this delay increased with higher doses. Doses above 2500 R inhibited the retraction of anterior segments, longitudinal contraction and cuticular shrinkage resulting in larval-like tanned puparia. With the anterior part of the body shielded during irradiation, normal puparia were formed, but after a delay proportional to the area irradiated. Injection of β-ecdysone counteracted this delay. With the doses used, irradiation had no effect on post-critical stage larvae. This suggested that the CNS has a special mechanism which controls the neuromuscular processes of pupariation, and when this mechanism is damaged by irradiation larval-like puparia are formed. The pupariation delay was attributed to a temporary block in the synthesis, or release, or both of α-ecdysone (in whole-body or anteriorly only irradiated larvae) and its final conversion to β-ecdysone (in posteriorly only irradiated larvae). The fact that post-critical stage larvae are insensitive to irradiation suggests that the neuromuscular and neurosecretory processes which are affected by irradiation are already completed at that stage.