In vivo uptake and metabolism of [3H]ecdysone in the vitellogenic follicles of Sarcophaga bullata (Diptera) and its localisation by autoradiography

Within 4 h after injection of [³H]ecdysone, almost all tritiated material has disappeared from the haemolymph, indicating that the uptake by the tissues is very fast. After only 15 min, 19% of the label was found in the ecdysterone fraction and 4% in the highly polar products (HPP) fraction. The uptake of [³H]ecdysone by the ovary (mid-vitellogenic) is almost complete within 1 h after injection. The pattern of [³H]ecdysteroids in the ovaries follows a well ordered sequence: firstly, [³H]ecdysone is the major component of the [³ H]ecdysteroids but it disappears within 2 h, next a peak value of [³H]ecdysterone was found at 1 h, whereafter this also disappeared, and from 2 h on, there was a considerable increase in HPP. The HPP consisted of 3 fractions (A, B and C). Glusulase treatment revealed that apparently only fraction B consisted of glucuronide and/or sulphate-conjugates of ecdysteroids. Autoradiographic experiments confirmed that the uptake of [³H]ecdysone was a very rapid process. In ovaries fixed 1 h after injection, the silver grains were abundant in the ooplasm but were also found in the follicle cell cytoplasm and in trophocytes. In follicles examined 16 h after injection, only a few silver grains were observed in the trophocytes and follicle cells. However, the cytoplasm of the oocyte was labelled. The border cells also accumulated label.

The major results indicate that all cell types of the follicle seem to be able to absorb ecdysone from the haemolymph and that there seems to be a rather selective uptake of ecdysone. In the ooplasm, ecdysone is converted to highly polar conjugates.     

Mapping of serotonin-immunoreactive neurons in the larval nervous system of the flies Calliphora erythrocephala and Sarcophaga bullata

Summary Serotonin-immunoreactive (5-HTi) neurons were mapped in the larval central nervous system (CNS) of the dipterous flies Calliphora erythrocephala and Sarcophaga bullata. Immunocytochemistry was performed on cryostat sections, paraffin sections, and on the entire CNS (whole mounts).The CNS of larvae displays 96–98 5-HTi cell bodies. The location of the cell bodies within the segmental cerebral and ventral ganglia is consistent among individuals. The pattern of immunoreactive fibers in tracts and within neuropil regions of the CNS was resolved in detail. Some 5-HTi neurons in the CNS possess axons that run through peripheral nerves (antenno-labro-frontal nerves).The suboesophagealand thoracico-abdominal ganglia of the adult blowflies were studied for a comparison with the larval ventral ganglia. In the thoracico-abdominal ganglia of adults the same number of 5-HTi cell bodies was found as in the larvae except in the metathoracic ganglion, which in the adult contains two cell bodies less than in the larva. The immunoreactive processes within the neuropil of the adult thoracico-abdominal ganglia form more elaborate patterns than those of the larvae, but the basic organization of major fiber tracts was similar in larval and adult ganglia. Some aspects of postembryonic development are discussed in relation to the transformation of the distribution of 5-HTi neurons and their processes into the adult pattern.     

Immunohistochemical localisation of the hypertrehalosaemic hormone II (Cam-HrTH-II) and related peptides in the nervous system of Carausius morosus and Sarcophaga bullata

Summary A polyclonal antiserum was prepared against an N-terminal modified Cam-HrTH-II (Leu-Asn-Phe-...), one of the members of the large AKH/RPCH peptide family, first isolated from Carausius morosus. The localisation of this peptide was performed by means of immunocytochemical methods in the brain and corpora cardiaca-corpora allata complex of the stick insect, Carausius morosus and the grey fleshfly, Sarcophaga bullata. The distribution patterns of molecules reactive to the Cam-HrTH-II and the LomAKH-I antisera in both insect species were compared. In Carausius, both antisera reacted in the same cell bodies. In Sarcophaga, some neurons were stained by both, others only by one of the two antisera. By combining two different antisera, we demonstrated that there are no Lom-AKH-I-like molecules present in Carausius and that there must occur at least three different AKH-like molecules in the brain of Sarcophaga. One is similar to Cam-HrTH-II, the second to Lom-AKH-I and the third is an AKH/RPCH-like peptide, different from Lom-AKH-I and Cam-HrTH-II.     

Inhibitory effect of an in vivo activated soft analogue of A-phenyl-B-triazolium metyrapone during pupariation in Sarcophaga bullata

Experiments with the ovoviviparous fleshfly Sarcophaga bullata (Parker) (Dipt., Sarcophagidae), showed that the compound 4-(dodecanoyloxymethyl)-1-(2-methyl-1-oxo-1-phenyl-2-propyl)-(1,2,4-triazolium) chloride (NKI-42002) was an in vivo inhibitor of the cytochrome P-450-dependent monooxygenase system (MFO) during pupariation. The hard analogue of A-phenyl-B-triazolium metyrapone and the soft analogue of A-phenyl-B-imidazolium metyrapone showed no specific activity. Our results suggest that the soft quaternary group may help the transport of A-phenyl-B-triazolium metyrapone to the MFO system.A dose of 0.8 nmole/spec. of NKI-42002 had both a specific (delay in pupariation:1.64 ratio) and toxic (perc. of mortality:56) effect. The effects of the 0.4 nmole/spec. NKI-42002 can be eliminated by the simultaneous injection of 0.4 pmole/spec. of 20-OH ecdysone. These reversal studies support the hypothesis that NKI-42002 interferes with the biosynthesis of 20-OH ecdysone.

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Résumé Au cours d'études sur la mouche ovovivipare Sarcophaga bullata, on a constaté que le composé 4-(dodécanoyloxyméthyl)-1-(2-méthyl-1-oxo-1-phenyl-2-propyl)-(1,2,4-triazolium) chlorure (NKI-42002) était un inhibiteur in vivo du système cyt. P-450 dépendant mono-oxygénase (MFO) lors de la formation des pupes. Les analogues, solide du A-phényl-B-triazolium métyrapone et fluide du A-phényl-B-imidazolium métyrapone, n'ont présenté aucune activité spéciale. Ces résultats suggèrent que le groupe quaternaire fluide peut faciliter le transfert du A-phényl-B-triazolium métyrapone au système MFO.La dose de 0,8 nmole/ind. de NKI-42002 a présenté une action tant spécifique (retard à la pupaison; ratio 1,64), que toxique (mortalité 56%). On peut éliminer l'influence d'une dose de 0,4 nmole/ind. de NKI-42002 par injection simultanée d'une dose de 0,4 pmole/ind. de 20-OH ecdysone. Ces résultats confortent l'hypothèse suivant laquelle le NKI-42002 interfère avec la biosynthèse de la 20-OH ecdysone.

     

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.     

First description of the female of Sarcophaga (Sarcorohdendorfia) gracilior (Chen, 1975) (Diptera,Sarcophagidae)

Sarcophaga (Sarcorohdendorfia) gracilior (Chen, 1975) is documented from specimens collected in Hubei Province, China, using morphological characters and wing interference patterns (WIPs). The female of S. (S.) gracilior is described for the first time, the male is redescribed, and both sexes are photographed. The distribution of the species is updated.     

Interneurons of the subesophageal ganglion of Sarcophaga bullata responding to gustatory and mechanosensory stimuli

Summary Intracellular recordings were made from interneurons in the subesophageal ganglion (SEG) of Sarcophaga bullata while stimulating the labellar lobes with solutions of sucrose, NaCl and with distilled water. Neurons that responded to sucrose did not respond to NaCl and vice versa, while sucrose-sensitive neurons often responded weakly to water. Several of the recorded neurons were filled with Lucifer Yellow, and their morphology was reconstructed. Most showed extensive arborizations within the SEG, suggesting that they were local interneurons involved in the early stages of gustatory processing. Some of the filled neurons had extensive projections to the brain, in addition to arborizations in the SEG. This is the first published record of gustatory interneurons in the higher flies.Abbreviations LY lucifer yellow - SEG subesophageal ganglion     

Traumatic Myiasis Caused by an Association of Sarcophaga tibialis (Diptera: Sarcophagidae) and Lucilia sericata (Diptera: Calliphoridae) in a Domestic Cat in Italy

We describe here a rare case of traumatic myiasis occurred in August 2014, caused by an association of 2 Diptera species, Sarcophaga tibialis Macquart (Diptera: Sarcophagidae) and Lucilia sericata (Meigen) (Diptera: Calliphoridae), in a domestic cat in northern Italy. Species identification was based on adult male morphology. The present case is the first report of S. tibialis as an agent of myiasis in Italy, and also the first ever report of myiasis caused by an association of S. tibialis and L. sericata. The cat developed an extensive traumatic myiasis in a large wound on the rump, which was treated pharmacologically and surgically. The biology, ecology, and distribution of S. tibialis and L. sericata are also discussed. A literature review is provided on cases of myiasis caused by S. tibialis, and cases of myiasis by L. sericata involving cats worldwide and humans and animals in Italy.     

Moulting hormone,juvenile hormone and the ultrastructure of the fat body of adult Sarcophaga bullata (Diptera)

Summary In the ovoviviparous fly, Sarcophaga bullata, vitellogenesis is cyclic; a process reflected in ultrastructural changes in the fat body cells and oenocytes. At eclosion the larval fat body has not yet completely disappeared. During vitellogenesis the fat body cells are specialized for intensive protein synthesis showing a very extensive RER and numerous invaginations of the plasma membrane. These features disappear when the eggs descend into the oviducts to complete embryogenesis. The predominant feature of the oenocytes is their very prominent SER. The fat body cells of the males are never as specialized for protein synthesis as those of the females. Feeding of ecdysterone to males for 3 or more days induces a rather extensive subcellular apparatus for protein synthesis, i.e., invaginations of the plasma membrane and an extensive RER. Juvenile hormone is completely ineffective in this respect. Both ecdysterone and juvenile hormone have pronounced but different effects on the oenocytes of males.     

Induction and decay of thermosensitivity in the flesh fly,Sarcophaga crassipalpis

When pharate adults of the flesh fly Sarcophaga crassipalpis are exposed to 40°C for 4 h they become more tolerant of high temperatures that are normally lethal (thermotolerance). In contrast, a 1-h exposure to 45°C decreases tolerance to a subsequent high temperature challenge (thermosensitivity). While control flies experience little mortality when held at 35°C for 24–48 h the thermosensitized flies die when exposed to 35°C. Sensitivity to a second thermal challenge slowly decays over a 72-h period. The acquisition of thermotolerance prevents the development of thermosensitivity. Brains from thermosensitized flies cultured at 43°C express the 72-kDa heat-shock protein and normal protein synthesis is inhibited. This implies that development of thermosensitivity is not associated with a loss in the capacity to express the 72-kDa heat-shock protein.Abbreviations ICN ICN Biomedicals, Inc. PO Box 19536, Irvine, CA 92713-9921 - LD light dark cycle - LT₅₀ time required to kill 50% of the test animals - SDS sodium dodecyl sulfate - TRIS Tris(hydroxymethyl)aminomethane     

Sarcophaga (Liosarcophaga) dux (Diptera: Sarcophagidae): A flesh fly species of medical importance

Background

Although tropical climate of Thailand is suitably endowed with biodiversity of insects, flies of medical importance is not well investigated. Using information from literature search, fly survey approach and specialist’s experience, we review database of Sarcophaga (Liosarcophaga) dux Thomson (Diptera: Sarcophagidae), one of the priorities flesh fly species of medical importance in Thailand.

Results

This review deals with morphology, bionomics and medical involvement. Important morphological characteristics of egg, larva, puparia and adult were highlighted with illustration and/or micrographs. Search pertaining to molecular analysis used for fly identification and developmental rate of larvae were included. Medical involvement of larvae was not only myiasis-producing agent in humans and animals, but associated with human death investigations.

Conclusions

This information will enable us to accurate identify this species and to emphasis the increase medically important scene in Thailand.     

Comparative micromorphology of third instar larvae and the breeding biology of some Afrotropical Sarcophaga (Diptera: Sarcophagidae)

Four sympatric species of Sarcophaga, viz S. cruentata Meigen, S. exuberans Pandelle, S. nodosa Engel and S. tibialis Macquart, which occur in the Transvaal, South Africa, showed oviparity under optimum laboratory breeding conditions. Details of the life cycle duration under these conditions are discussed. Rearing and colonizing methods were developed. Scanning electron microscopy of third instar larvae provided useful data in distinguishing between the four species. The characters which were examined were the spinulation of the body segments and the rim surrounding the spiracular atrium of the posterior spiracles, the anterior spiracles and the spiracular hairs of the posterior spiracles.     

Photoperiodic sensitivity and diapause induction during ovarian,embryonic and larval development of the flesh fly,Sarcophaga argyrostoma

Sensitivity to the daily photoperiod, particularly with respect to pupal diapause induction, was studied during ovarian, embryonic, and larval development of the flesh flySarcophaga argyrostoma. Large flies were shown to have a greater number of primary follicles in their ovaries and to be capable of limited ovarian maturation in the absence of exogenous protein (autogeny). Such ovarian development occurred independently of photoperiod. However, long days experienced during embryogenesis caused more rapid development, and earlier larviposition, than short days. Short days during embryonic and subsequent larval development also induced pupal diapause, whereas long days led to continuous or non-diapause development of the pupae. Pupal diapause could not be induced by photoperiods during the vitellogenic phase of ovarian development. InSarcophaga argyrostoma, a maternal effect preventing pupal diapause among the progeny of files with a diapause history was not observed.     

一株棕尾别麻蝇胚胎细胞系的建立及其特性分析

双翅目昆虫细胞系广泛应用于遗传学、发育生物学、分子生物学、人和动物体病原学以及昆虫抗微生物肽的研究。本研究建立了一株新的棕尾别麻蝇Sarcophaga peregrina胚胎细胞系。该细胞系的原代培养始于2008年11月17日, 取材于棕尾别麻蝇晚期胚胎组织, 在Shields & Sang M3昆虫培养基中于28℃恒温培养, 在第26天进行第1次传代, 至今已历时21个月, 传代72次, 生长状态稳定, 被命名为Sp-E-HNU11。该细胞系的细胞形态主要呈梭形和近圆形, 杂以少量巨型细胞, 紧密贴壁生长。细胞群体倍增时间为42 h。染色体数目一般为10条或12条, 为二倍体或亚二倍体细胞系; 除一对颗粒状微型染色体外, 其他染色体呈短杆状。细胞系的β-萘酯酶和谷草转氨酶同工酶谱上分别显示出1条和3条酶带。随机引物扩增多态性 (random amplified polymorphic DNA, RAPD) 分析结果显示, 该细胞系与小菜蛾细胞系Px-E-HNU12、草地贪夜蛾细胞系IPLB-Sf-9和家蚕细胞系Bm-21E-HNU5呈现明显不同的带型特征。 Sp-E-HNU11细胞系的建立为昆虫抗微生物肽及其他相关的研究工作增添了新的研究工具和生产载体。     

Morphological markers of anteroposterior and dorsoventral polarity in developing oocytes of the hymenopteranCosmoconus meridionator (Ichneumonidae)

Summary The progressive establishment of anteroposterior and dorsoventral polarity in developing oocytes ofCosmoconus meridionator is described. In fully grown oocytes, the asymmetrical (polar) organization is apparent in the localization of the oocyte nucleus (germinal vesicle) and oosome, and in the uneven (graded) distribution of lipid droplets, yolk spheres and specific organelles termed accessory nuclei (AN). The latter structures occur preferentially within the anteroventral periplasm. The developmental significance of AN is discussed.     

Segmental organisation of the tail region in the embryo of Drosophila melanogaster

Summary The segmental organisation of the tail region in the embryo of Drosophila melanogaster, which is defined here as the epidermal region posterior to the boundary between abdominal segments A7 and A8, has been investigated by means of ultraviolet (UV) laser fate-mapping and phenotypic analysis of embryonic mutants that alter the segmental pattern of the larval cuticle. Wild-type embryos were irradiated in the presumptive tail region with a UV- laser microbeam of 20 m diameter at the blastoderm stage. The ensuing defects were scored in the cuticle pattern of the tail region of the first-instar larva, which is described in detail in this paper. The spatial distribution of defect frequencies was used to construct a blastoderm fate-map of the cuticle structures of the larval tail region. The segmental origin of the larval tail structures was inferred from the phenotypic analysis of segmentation and homoeotic mutants, which revealed pattern repetition throughout the embryonic tail region corresponding to four segment anlagen, A8 to A11, and a non-segmental telson. These data enabled the transformation of the blastoderm fate-map of cuticle structures into a map of tail segment anlagen. The tail anlage occupies about 10% of the egg length (EL), bounded by segment A7 anteriorly at 20% EL and by the proctodaeum posteriorly at 10% EL, as measured from the posterior pole. The anlagen of segments A8 and A9 appear to be narrow dorso-ventral strips of blastoderm cells similar to the anlagen of the trunk segments, whereas the anlagen of A10 and A11 are smaller and produce fewer pattern elements. The telson is represented in the cuticle by the tuft which derives from a very dorsal posterior position. The antero-posterior axis of the entire tail anlage appears curved upward posteriorly. Differences in the mode of development between tail and trunk segments are discussed, as are similarities of larval and imaginal tail development in Drosophila. Comparison with tail development in other insects suggests that, during evolution, the transition from semi-long-germ to long-germ development modified the organisation of the tail region without affecting its primary subdivision into metameric units.