John G. Stoffolano

Innervation of dromyosuppressin (DMS) immunoreactive processes and effect of DMS and benzethonium chloride on the Phormia regina (Meigen) crop

Antibody to the dipteran myosuppressin peptide, dromyosuppressin, TDVDHVFLRFamide, stained cells and fibers in the brain, optic lobes, subesophageal ganglion, and thoracico‐abdominal ganglion of the blow fly, Phormia regina (Meigen). Dromyosuppressin‐like immunoreactive fibers were detected in the cardiac recurrent nerve, hypocerebral ganglion/corpora cardiaca complex, crop duct, and crop. In order to explore the mechanisms involved in regulating crop movement, we established an in vitro bioassay. The basal rate of crop movement was 50.8 ± 1.5 contractions per minute. Application of 1 μl of saline to the crop did not significantly affect the rate of movement compared to the basal rate (46.1 ± 1.1 contractions per minute, P < 0.05). Application of 1 μl 10‐6 M dromyosuppressin or 1 μl 10‐3 M benzethonium chloride to the crop slowed the rate to 2.2 ± 0.2 and 6.1 ± 0.7 contractions per minute, respectively. Although other data have previously been interpreted to suggest that dipteran crop contractions do not include a neural component, the neuropeptide dromyosuppressin affected P. regina crop motility. Innervation of the crop and crop duct by dromyosuppressin immunoreactive processes that originated in the central nervous system and the effect of dromyosuppressin on crop muscle contractions suggest that dromyosuppressin is released locally to modulate crop contractions and that crop motility is under neural regulation. Myosuppressins isolated from numerous insects have a high degree of structure identity and reduce spontaneous muscle contractions of the hindgut, oviduct, and heart. Benzethonium chloride, previously identified as a myosuppressin agonist on the cockroach hindgut and locust oviduct, mimicked the effect of dromyosuppressin on the crop. This suggests that structural requirements for myosuppressin receptor binding in the cockroach hindgut, locust oviduct, and fruit fly crop are similar. J. Comp. Neurol. 421:136–142, 2000.

The adult Dipteran crop: a unique and overlooked organ.

The diverticulated crop is a unique and overlooked foregut organ in the Diptera that affects many physiological and behavioral functions. Historically, the crop was viewed simply as a reservoir for excess nutrients. The crop lobes and crop duct form an elaborate sphincter and pump system that moves stored nutrients to the crop lobes, oral cavity, and the midgut. The storage capacity of the crop lobes is significant when filled maximally and supplies sufficient carbohydrates to sustain prolonged activity and flight, and adequate protein and lipids to facilitate reproductive events. Crop emptying is under complex neuroendocrine and neural control and may be influenced by multiple neuromessengers, such as serotonin and dromyosuppressin. The crop lobes also serve as a site for the initial mixing of enzymes from the salivary glands and antimicrobials from the labellar glands with ingested food. These food-processing functions are associated with behaviors unique to dipterans, such as regurgitation (or bubbling), nuptial gift giving, and substrate droplet deposition or trap-lining.

Heterotylenchus autumnalis: Hemocytic reactions and capsule formation in the host, Musca domestica

Abstract Failure of the nematode H. autumnalis to develop in M. domestica is attributed to the defense reaction of host larvae to the infective, gamogenetic stage of the parasite. The defense reaction involves melanization and encapsulation of the parasite, and is manifested as changes in the hemocyte picture of the host. Differential hemocyte counts suggest that the initial reaction includes the aggregation and fusion of oenocytoids forming a pigmented layer or sheath capsule that adheres to the cuticle of the parasite. This reaction initiates the aggregation and fusion of other hemocytes forming a homogeneous mass or syncytium (cellular capsule) that completely surrounds the melanized parasite. The total number of hemocytes in parasitized larvae appears to be greater than in nonparasitized larvae of comparable age. However, a comparative study of mitotic indices failed to show any significant difference in the percentage of dividing hemocytes of parasitized and nonparasitized larvae.

Subunit composition of vitellin, and concentration profiles of vitellogenin, and vitellin in Phormia regina (Meig.) following a protein meal

1. 1. The vitellin (Vt) of Phormia regina eggs was isolated by extraction at low ionic strength and purified by TEAE-cellulose chromatography. SDS polyacrylamide gel electrophoresis and staining with horseradish peroxidase-concanavalin A showed that the Vt contains four subunits of Mr 42,000, 43,000, 44,000 and 45,500, all of which are glycosylated. 2. 2. Murine polyclonal antibodies to the Vt, employed in conjunction with radioimmunoblotting, recognized all four subunits, and revealed that Vt and/or vitellogenin (Vg) are present exclusively in the hemolymph, fat bodies and ovaries of liver-fed females but not those fed sugar. 3. 3. Using these antibodies in an ELISA, concentrations of Vg in the hemolymph and Vt in the ovaries were determined periodically following liver feeding. At 28°C, the hemolyph Vg titer reaches a maximum 28 hr post-feeding and then decreases rapidly as ovaries begin to sequester Vt, whose concentration in that organ peaks about 48 hr post-feeding.

The influence of age and diet on respiration in adult male and female blowflies, Phormia regina

Abstract Female Phormia regina , in general, were found to utilize more oxygen than males especially after approximately the sixth day of adulthood on both a μl/specimen and μl/mg basis. The females exhibited cyclic respiratory rates which correlated with the gonadotrophic cycles. Following its final gonadotrophic cycle the respiratory rates of females progressively declined. The male exhibited its highest respiratory rates during the fifth and tenth days after emergence; thereafter, there was a general decline in respiratory rates. Females have a significantly higher fresh body weight than males when both sexes are fed on a protein diet. Females fed on a protein-deficient diet exhibited significantly higher respiratory rates on a μl/mg basis than females fed on a protein diet. This extremely high respiratory rate was directly correlated with the occurrence of a protein hunger drive. Males fed on a protein-deficient diet also exhibited higher respiratory rates on a μl/mg basis than males fed on protein diet. However, the differences were not as great between the males as compared to the female comparison.

The diverticulated crop of adult Phormia regina.

The crop of adult Phormia regina consists of a duct that diverges from the esophagus, just in front of the cardia, and extends ventrally and posteriorly into the thorax and abdomen where it forms a bilobed sac. Flattened epithelial cells produce the cuticular lining of the crop. When empty, or partially full, the epithelial cells and cuticular lining form folds extending into the lumen, thus providing for expansion as the crop sac fills. Covering the sac on the hemolymph side is a layer of anastomosed, intrinsic muscles connected to one another by intercellular cytoplasmic bridges. Mitochondria are located at the periphery of the sarcomere. Also inside the sarcomere are glycogen, sarcoplasmic reticula, and transverse tubular systems (T-system). I, A, and Z-bands are present and the Z-bands are not in register making the muscle-type supercontractile. Important structures, not previously researched and associated with the crop muscles, are the crop nerves. Coming off the corpora cardiaca, and running down each side of the crop duct, is a pair of nerves, each housing several axons. These nerves extend to and branch over the crop sac. Here they penetrate the muscle mass and form neuromuscular junctions where electron-dense droplets of neurosecretion are released. Based on the literature, and research in our laboratory, it has now been shown that these nerves carry adipokinetic hormone, Drosophila insulin-like peptide, and a dromyosuppressin-like neuropeptide.

Role of Phote-HrTH (Phormia terraenovae hypertrehalosemic hormone) in modulating the supercontractile muscles of the crop of adult Phormia regina Meigen.

Phote-HrTH (Phormia terraenovae hypertrehalosemic hormone) has been demonstrated in the Diptera to be involved in flight metabolism, reproduction, and diapause. Each of these events needs the hormones action and requirement for carbohydrates is the common denominator. In Diptera, carbohydrates are taken up during feeding by action of the cibarial pump and are then stored in the crop. Using adult Phormia regina, both a bioassay and electrophysiological recordings show that Phote-HrTH slows down or inhibits the crop lobe muscles (P5) and, at the same time, stimulates the muscles of the pump 4 (P4) involved in pushing fluids out of the crop and up into the midgut for digestion. The EC50 for P4 was in the nanomolar range while the IC50 for P5 was 1.4-75.1 pM. The effect of Phote-HrTH on P4/5 suggests that the peptide is important in coordinating the two pumps, which are involved in moving carbohydrates up into the midgut for digestion. The adult crop organ is an essential storage organ for carbohydrates and now should be considered an important structure capable of delivering nutrients to the midgut for digestion.

Electrophysiological studies of gustatory and olfactory responses of the sensilla on the ovipositor of the apple maggot fly, Rhagoletis pomonella Walsh

Abstract Electrophysiologic techniques were used to study the bioelectri‐cal activity of the ovipositor chemosensilla of the apple maggot fly, Rhagoletis pomonella Walsh (Diptera, Tephritidae), in response to contact and volatile stimulation with various chemicals associated with fruits. The spike amplitude peaks evoked by contact stimuli showed that the chemosensilla are sensitive to glucose, malic acid, and quinic acid but not to fructose. There is evidence that ovipositor chemosensilla may provide information allowing glucose to be distinguished from the two acids. In addition, slow compound potentials were recorded in response to various volatiles associated with fruits. The contact stimulation experiments indicate that the ovipositor chemosensilla permit females to distinguish between a sugar and two acids that occur in apples in variable concentrations depending on the stage of ripeness, and that this difference may provide the fly with information about host suitability and/or quality.

F-actin distribution in the ovaries of pre-vitellogenic and vitellogenic black blowflies, Phormia regina (Meigen) (Diptera : Calliphoridae)

Abstract The spatial distribution of F-actin microfilaments in the ovaries of previtellogenic and vitellogenic female black blowflies, Phormia regina (Diptera : Calliphoridae), as the females shift from a sugar to a liver diet, is determined using rhodamine-labelled phalloidin (rh-phalloidin). During the pre-vitellogenic stages of ovarian development (i.e. corresponding to a sugar diet) a single bright fluorescent layer marks the interface between follicle cells and the oocyte. Fluorescence is also most evident at the inner surface of the ring canals of the nurse cells. This is observed in the nurse cells both in the distal part of the germarium, and in the vitellogenic growing oocyte. However, when liver-fed (i.e. necessary for vitellogenesis), 2 bright fluorescent layers are observed at the follicle cell-oocyte interface. In addition, the cytoplasm of the nurse cells during vitellogenesis appears full of fluorescent microfilaments and the actin rings are found to increase in size and thickness. The changing organization of the F-actin microfilaments in the follicles during the process of both egg chamber and oocyte formation is discussed and possible functions considered.

Regulatory mechanisms and the role of calcium and potassium channels controlling supercontractile crop muscles in adult Phormia regina.

Bioassays and electrophysiological recordings were conducted in the adult blowfly Phormia regina to provide new insights into the regulatory mechanisms governing the crop filling and emptying processes of the supercontractile crop muscles. The cibarial pump drives ingestion. Simultaneous multisite extracellular recordings show that crop lobe (P5) distension during ingestion of a 4.7 μl sugar meal does not require muscle activity by any of the other pumps of the system. Conversely, pumping of fluids toward the anterior of the crop system during crop emptying is brought about by active muscle contraction, in the form of a highly coordinated peristaltic wave starting from P5 and progressively propagating to P6, P4 and P3 pumps, with P5 contracting with a frequency about 3.4 times higher than the other pumps. The crop contraction rate is also modulated by hemolymph-borne factors such as sugars, through ligand recognition at a presumptive receptor site rather than by an osmotic effect, as assessed by both behavioural and electrophysiological experiments. In this respect, sugars of equal osmolarity produce different effects, glucose being inhibitory and mannose ineffective for crop muscles, while trehalose enhances crop activity. Finally, voltage and current clamp experiments show that the muscle action potentials (mAPs) at the P4 pump are sustained by a serotonin-sensitive calcium conductance. Serotonin enhances calcium entry into the muscle cells and this could lead, as an indirect modulatory effect, to activation of a Ca(2+)-activated K(+) conductance (IK(Ca)), which sustains the following mAP repolarization phase in such a way that further mAPs can be generated early and the frequency consequently increased.