Shirlee M. Meola

Inhibition of Cellular Proliferation of Imaginal Epidermal Cells by Diflubenzuron in Pupae of the Stable Fly

A second mode of action has been found for the inhibition of chitin synthesis by diflubenzuron. This compound blocks synthesis of the imaginal cuticle by preventing formation of the adult epidermis in the pupal stage of the stable fly (Stomoxys calcitrans L.).

Inhibition of DNA synthesis by diflubenzuron in pupae of the stable fly Stomoxys calcitrans (L.)

Abstract Biochemical assays of stable fly pupae treated with diflubenzuron at the white prepupal stage and then injected with either [ 3 H]thymidine or [ 14 C]thymidine showed no differences in uptake of the thymidine at 0–4, 0–24, 20–21, or 22–24 hr after injection of the radiolabeled thymidine. However, at 32–36 hr the diflubenzuron pupae incorporated only 10–11% of the amount of labeled thymidine incorporated by the untreated pupae. Autoradiographs taken from diflubenzuron-treated pupae at 22–24 and 32–36 hr after injection of the labeled thymidine showed that a reduction in DNA synthesis had occurred in cells originating from the imaginal epidermal histoblasts. The reduction in DNA synthesis at 22–24 hr was not detectable by the biochemical assay since the number of proliferating epidermal cells was too small a proportion of the total number of cells undergoing histogenesis at this time period. Thus, the insect growth regulator, diflubenzuron, appears to be cell specific in this species of fly in that the reduction in DNA synthesis is observed only in cells originating from the imaginal epidermal histoblasts. However, it is not known at this time whether this effect is primary or secondary.

The oviduct musculature of the horsefly, Tabanus sulcifrons, and its response to 5‐hydroxytryptamine and proctolin

ABSTRACT. A delicate lace‐like membrane covers the ovaries of Tabanus sulcifrons. These membranes were found to contain muscle fibres that provide the organs with motile properties. The lateral oviducts consist of a single layer of longitudinal muscles that form a structural syncytium by means of an extensive anastomosis of the fibres comprising it. The common oviduct is composed of two muscle layers, an outer sheath of circular muscle and an inner substratum of longitudinal muscle. Both of these layers showed evidence of a structural syncytium. When isolated in saline, the oviduct was spontaneously active and gave a simple phasic pattern of contraction. Such muscle preparations were sensitive to both 5‐hydroxytryptamine (5HT) and the insect myotropic peptide, proctolin. Excitation was generally indicated by a rise in muscle tonus or an increase in the frequency and amplitude of individual phasic contractions, or all three characteristics. The threshold for activation with 5HT was variable, ranging from as low as 4 × 10‐9M to 1 × 10‐7M. Proctolin evoked a noticeable increase in the tonus of most oviducts at 10‐10M. However, several preparations responded to as little as 3 × 10‐11M proctolin.

Isolation and Immunocytochemical Characterization of Three Tachykinin-Related Peptides from the Mosquito, Culex salinarius

Three myotropic peptides belonging to the Arg-amide insect tachykinin family were isolated from whole-body extracts of the mosquito, Culex salinarius. The peptides, APSGFMGMR-NH2, APYGFTGMR-NH2 and APSGFFGMR-NH2 (designated culetachykinin I, II, and III) were isolated and purified on the basis of their ability to stimulate muscle contractions of isolated Leucophaea maderae hindgut. Biologically inactive methionine sulfoxides of two of the three peptides were isolated using an ELISA system based upon antiserum raised against APYGFTGMR-NH2 and identified with mass spectrometry. Immunocytochemistry localized these peptides in cells in the brain, antennae, subesophageal, thoracic and abdominal ganglion, proventriculus and midgut. Nerve tracts containing these peptides were found in the median nerve of the brain, central body, nervi corpus cardiaci, cervical nerve, antennal lobe and on the surface of the midgut.

Characterization of an antiserum against an achetakinin I-analog and its use for the localization of Culekinin Depolarizing Peptide II in the mosquito, Culex salinarius

ELISA experiments revealed that an antiserum raised against an achetakinin-analog could specifically detect the recently isolated Culekinin Depolarizing Peptide (CDP)-II from the mosquito, Culex salinarius. The characterization indicated that two different epitopes in the C-terminal region of achetakinin I and CDP-II are recognized. One epitope is the -F-Y-region, the other is the -P-W-region. Among the peptides isolated from C. salinarius, the antiserum reacts only with CDP-II. Pre-absorption tests of the antiserum with CDP-II in immunohistological stainings abolished the reaction, while tests with pre-immune sera did not cause any immunopositive reactions. In the mosquito head ganglia, immunoreactive neurons were detected in the pars lateralis, the optic lobe and the suboesophageal ganglion. Although some immunopositive axons extended into the nervi corporis cardiacii II, no immunoreactivity was observed in the retrocerebral complex. In the thoracic ganglia, immunoreactive neurons were found in the pro-, meso- and metathoracic neuromeres. No immunoreactivity was found elsewhere. With this study we demonstrate that CDP-II, isolated from a whole body extract, is truly a neuropeptide, and the data suggest that its function is neuromodulating or neurotransmitting rather than neurohormonal.

Immunocytochemical localization of leucomyosuppressin-like peptides in the CNS of the cockroach,Leucophaea maderae

Immunocytochemistry was used to determine sites of synthesis and pathways for the transport of the neuropeptide, Leucomyosuppressin (pQDVDHVFLRFamide) in the cockroach,Leucophaea maderae. This study led to identification of neurons in the brain and thoracic ganglia reactive to polyclonal antibodies raised against this peptide. No immunoreactive cells were found in the subsophageal or abdominal ganglia. Although the corpus cardiacum contained no intrinsic cells immunoreactive to LMS antibodies, the periphery of this organ and that of the nervi corporis allati contain an abundance of LMS-reactive terminals.

The egg fertilization site within the house fly, Musca domestica (L.) (diptera: Muscidae)

Abstract The surface morphology of a dome-shaped genital chamber in the female Musca domestica L., where the sperm and egg meet following ovulation, was examined to determine its role in fertilization. The inner surface of the chamber was found to be lined with 3 types of nonarticulated cuticular spines. Examination of eggs removed from the chamber indicated that the distinctly robust spines at the apex were involved with the removal of a mucoid secretion which occludes the micropyle opening. The spines lining the rest of the chamber were more slender and flexible than those at the apex, and may function in ensuring that sperm remain at the fertilization site when the egg is placed into the chamber.

The pupal instar of Stomoxys calcitrans: Cuticle deposition and chitin synthesis

Abstract Incorporation of radioactive N -acetyl- d -glucosamine peaked at 1 and also at 4 days post-pupation. Histological studies indicated that these peaks were related to the production of the ecdysial membrane and underlying imaginal cuticulin layer on the first day and the production of imaginal cuticle on the fourth day. The formation and/or secretion of the larval exocuticle, pre-pupal cuticle and ecdysial membranes were studied.

Heart structure and beat in the stable fly, Stomoxys calcitrans

ABSTRACT. The heart of the adult stable fly, Stomoxys calcitrans (L.), is suspended from the dorsal sclerites of the abdomen by strands of connective tissue, and supported from below by alary muscles that insert into a central band of longitudinal muscle just beneath the aorta. Valved openings occur in three of the heart segments. The central band of muscle beneath the heart is innervated but there is no well‐defined lateral cardiac nervous system. The myocardium consists of a single layer of circular muscle composed of a series of muscle fibres that are joined dorsally and ventrally by intercalated discs in the midline of the insect. T‐system tubules are closely associated with the sarcoplasmic reticulum, forming dyads. The heart rate of intact stable flies varied from short intervals of almost no activity to periods with a very rapid beat (126–294 pulses/min), and when the connections to the central nervous system were severed the heart beat became very regular (258 pulses/min). Slight pressure applied to the dorsal septum stopped myocardial contractions in that segment. The myocardium was insensitive to perfusion with 10‐3M acetylcholine, l‐aspartic acid, l‐glutamic acid, Λ‐aminobutyric acid, 5‐hydroxytryptamine, octopamine, tyramine and proctolin 10‐5m. However, Mn++ caused either an intermittent beat at lower concentrations (0.5 min) or near arrest at higher concentrations (2 mM).

Ultrastructural Analysis of Neurosecretory Cells in the Antennae of the Mosquito, Culex Salinarius (Diptera: Culicidae)

An antiserum raised against the peptide, culetachykinin II, immunocytochemically detected a group of neurosecretory cells in the first flagellar segment of the antennae of both males and females of the mosquito, Culex salinarius. This is the first insect species in which neurosecretory cells have been found in the antennae. The ultrastructure of these antennal neurosecretory cells (ANC) is described, as well as their relationship to other neurons in the antennae and antennal lobe of the mosquito. These tachykinin-reactive cells contain relatively small (140–220 nm) elementary neurosecretory granules. Not only do the ANC have axons that terminate on specific glomeruli of the deutocerebrum, but these neurons also have collaterals that form neurohemal terminals in the receptor lymph channels of the dendrites of the sensory neurons. Thus, the ANC not only influence higher centers of the brain that interpret signals from the antennal sensillae, but also modulate the response of the sensory receptors. To our knowledge, this is the first report of neurosecretory cells directly affecting the signal reception of sensory neurons.