The Effect of the Hypertrophy Virus (MdSGHV) on the Ultrastructure of the Salivary Glands of Musca domestica (Diptera: Muscidae).

The salivary glands of insects play a key role in the replication cycle and vectoring of viral pathogens. Consequently, Musca domestica (L.) (Diptera: Muscidae) and the Salivary Gland Hypertrophy Virus (MdSGHV) serve as a model to study insect vectoring of viruses. A better understanding of the structural changes of the salivary glands by the virus will help obtain a better picture of the pathological impact the virus has on adult flies. The salivary glands are a primary route for viruses to enter a new host. As such, studying the viral effect on the salivary glands is particularly important and can provide insights for the development of strategies to control the transmission of vector-borne diseases, such as dengue, malaria, Zika, and chikungunya virus. Using scanning and transmission electron microscopic techniques, researchers have shown the effects of infection by MdSGHV on the salivary glands; however, the exact location where the infection was found is unclear. For this reason, this study did a close examination of the effects of the hypertrophy virus on the salivary glands to locate the specific sites of infection. Here, we report that hypertrophy is present mainly in the secretory region, while other regions appeared unaffected. Moreover, there is a disruption of the cuticular, chitinous lining that separates the secretory cells from the lumen of the internal duct, and the disturbance of this lining makes it possible for the virus to enter the lumen. Thus, we report that the chitinous lining acts as an exit barrier of the salivary gland.

Vibrio cholerae laboratory infection of the adult house fly Musca domestica.

The present study was designed to test the hypothesis that house flies may be capable of specifically harbouring ingested Vibrio cholerae in their digestive tracts. Flies were continuously fed green fluorescent protein (GFP)-labelled, non-O1/non-O139 environmental strains of V. cholerae. Bacterial burdens were quantitatively measured using plate counts and localization was directly observed using confocal microscopy. Vibrio cholerae were present in the fly alimentary canal after just 4 h, and reached a plateau of ∼107 colony-forming units (CFU)/fly after 5 days in those flies most tolerant of the pathogen. However, individual flies were resistant to the pathogen: one or more flies were found to carry < 180 V. cholerae CFU at each time-point examined. In flies carrying V. cholerae, the pathogen was predominantly localized to the midgut rather than the rectal space or crop. The proportion of house flies carrying V. cholerae in the midgut was dose-dependent: the continuous ingestion of a concentrated, freshly prepared dose of V. cholerae increased the likelihood that fluorescent cells would be observed. However, V. cholerae may be a transient inhabitant of the house fly. This work represents the first demonstration that V. cholerae can inhabit the house fly midgut, and provides a platform for future studies of host, pathogen and environmental mediators of the successful colonization of this disease vector.

Disruption of the salivary gland muscle in tsetse, Glossina pallidipes Austen, as a result of salivary gland hypertrophy virus infection.

The secretory region of the salivary glands in Glossina pallidipes Austen (Diptera: Glossinidae) is characterized by an external muscle layer. Scanning electron microscopy and transmission electron microscopy investigations provide a detailed description of the longitudinal muscle fibres and a comparison of their structure when affected by salivary gland hypertrophy virus. The virus is responsible for hypertrophy of the salivary glands in symptomatic flies, specifically of the muscle fibres, the cytoarchitecture of which is completely altered. Although observations did not reveal viral particles in the muscle cells of either asymptomatic or symptomatic flies, muscle fibres were enlarged and detached from one another and their associated basement membrane only in symptomatic flies. A decrease in type IV collagen labelling in the basement membrane of the muscles in symptomatic flies is reported and is considered a potential cause of the salivary gland muscle alteration and, possibly, myopathy. The maintenance of an organized muscular layer is essential for the normal secretion of saliva and hence its pathology in symptomatic tsetse flies could affect the normal transmission of the trypanosome that develops inside the salivary gland epithelium. Therefore, a better understanding of the possible role of the virus is essential in order to elucidate its impact on salivary deployment in symptomatic flies.

Effect of serotonin and calcium on the supercontractile muscles of the adult blowfly crop.

Bioassays and electrophysiological recordings were conducted to determine the role of serotonin and calcium on the supercontractile pump muscles of the diverticulated crop of adult blowflies. Using in situ crop preparations, serotonin was found to significantly increase the rates of contractions of a specific pump in the crop wall, pump P4. The addition of the serotonin antagonist, mianserin, or calcium free saline, both significantly reduced the contraction rates of this pump. Recordings, using suction electrodes from pump P4, confirm the in situ bioassay data and show that serotonin promotes muscle activity in empty crops in which no pump activity is normally observed. Moreover, our data indicate the crucial role of extracellular calcium ions in crop pump contractile activity. These results provide new information on how the crop of adult dipterans is modulated and suggest that serotonin, possibly supplied by neurons in the thoracico-abdominal neural plexus, may be involved in modulating the pumping of crop contents into the midgut for digestion or triggering antiperistalsis from the foregut in the process known as regurgitation or 'bubbling'.

Clonidine, octopaminergic receptor agonist, reduces protein feeding in the blow fly, Phormia regina (Meigen).

Results in this study are consistent with those of Murdock and his colleagues who clearly demonstrated that clonidine, an agonist of octopaminergic receptors in some insects, significantly increases sucrose feeding. Their studies, however, did not examine the effect of clonidine on protein feeding. Injection of a 20 microg/microl/fly dose of clonidine significantly reduces protein feeding in both sexes of Phormia regina, instead of stimulating feeding as is observed with carbohydrate feeding. The manner in which the flies are fed prior to starvation and the method of testing influences the amounts of diet consumed. It is proposed that the biogenic amines influence the state of hunger (i.e., protein versus carbohydrates) while other chemicals and neural mechanisms (i.e., such as sulfakinins and stretch receptors, respectively) affect satiety.

Insect satiety: sulfakinin localization and the effect of drosulfakinin on protein and carbohydrate ingestion in the blow fly, Phormia regina (Diptera: Calliphoridae).

Sulfakinins, which are satiety factors in invertebrates, have previously been shown to inhibit feeding in the German cockroach and desert locust. This study examines the occurrence of sulfakinin immunoreactivity and the role of sulfakinin as a feeding satiety factor in the black blow fly, Phormia regina. Specifically, this study examines the effect of sulfakinin on two of the blow fly's nutrient requirements (i.e., carbohydrates and proteins). We observed sulfakinin immunoreactive cells in the brains of both male and female flies. We found that drosulfakinin I (DrmSKI, FDDY[SO(3)H]GHMRFa) significantly inhibited carbohydrate feeding by 44% at the most effective dose (10 nmol) in female flies. Statistically, there was no significant effect on males; however, injections of 10 nmol DrmSKI reduced carbohydrate feeding by 34% compared to the sham. Drosulfakinin had no effect on protein feeding and no significant inhibition was detected in females or males. The results of this study lend further support to the idea that carbohydrate and protein feeding are regulated by separate control mechanisms, especially in Calliphoridae.

Allatotropic activity in the brain of female Phormia regina (Diptera: Calliphoridae).

In Phormia regina, the rate of juvenile hormone (JH) synthesis rises rapidly after the ingestion of an adequate protein meal. In a previous publication we have localized the neurons containing Manduca sexta allatotropin (Mas-AT)-like substances in the brain of P. regina and demonstrated the allatotropic effect of synthetic Mas-AT in sugar-fed flies in vitro. In this current study, we examined the possible role of the brain of P. regina after the fly received a protein meal. In vitro studies showed that the brain releases, at 8 h after a protein meal, a factor(s) with a strong allatotropic effect. This factor(s) stimulates the corpus allatum (CA) to produce 6.9 times more juvenile hormones (JHs) than the control CA. Time course studies showed that the release of this allatotropic factor(s) is temporally controlled. Only the brains collected from flies at 6 and 8 h after the onset of a liver meal release allatotropic factor(s). Injection of anti-Mas-AT antiserum partially suppressed the fly follicle development in vivo. Presence of anti-Mas-AT antiserum decreased the allatotropic effect of the brain released allatotropic factor(s) in vitro. In addition to a Mas-AT-like substance, it is possible that the brains of liver-fed P. regina females may synthesize other allatotropic factors that are chemically unrelated or partially related to Mas-AT, which cannot be recognized/neutralized by our anti-Mas-AT antiserum.

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 microl 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 microl 10(-6) M dromyosuppressin or 1 microl 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.

Detection of cryptic species in the Tabanus nigrovittatus (Diptera: Tabanidae) complex in Massachusetts by morphometric and cuticular hydrocarbon analysis.

Greenhead flies of the Tabanus nigrovittatus complex from Massachusetts salt marshes were identified as T. nigrovittatus Macquart and T. conterminus Walker using the morphometric model developed by Sofield et al. Four body measurements from a total of 5,983 female flies collected over 2 consecutive years yielded canonical scores producing a unimodal rather than the expected bimodal distribution. The lack of bimodality indicated that both species were not present at the study site. This was substantiated by cuticular hydrocarbon (CHC) analysis of a subsample of these specimens. Fifteen, female flies of the Tabanus nigrovittatus complex from the same site were identified to species using the Sofield et al. morphometric model and validated using CHC analysis. Two individuals of the T. nigrovittatus complex were identified incorrectly as T. conterminus by the morphometric model. The tendency of this model to incorrectly classify some individuals of T. nigrovittatus as T. conterminus brings into question the identity of the Walker syntypes of T. conterminus. Based on CHC analysis, our study shows that both species coexist within our study area.

Regulation of mating behavior by nutrition and the corpus allatum in both male and female Phormia regina (Meigen).

Both dietary protein and the corpus allatum (CA) were required for normal mating behavior in both male and female black blow fly, Phormia regina (Meigen). Nutrition (protein diet) activated the CA in both sexes. More than 10 mg of dietary liver was required for each male to result in 80% insemination of females, while 20 mg of liver was required for each female to allow 78% of females to become inseminated. Between 10 to 15 mg of protein meal (i.e., liver) was required to activate sexual receptivity in 71% of the females, while between 15 to 20 mg of liver was needed to support full oöcyte development in 70% of the females (Yin, C.-M., Zou, B.-X., Li, M.-F., Stoffolano, J.G., Jr. 1994. Discovery of a midgut peptide hormone which activates the endocrine cascade leading to oogenesis in Phormia regina Meigen. Journal of Insect Physiology, 40, 283-292). Allatectomy suppressed mating behavior more than 2-fold in both sexes. Topical application of 10 &mgr;g of S-methoprene (a juvenile hormone analogue) at 12 h after the onset of liver feeding restored sexual activity of both allatectomized males and females. Incidence of successful insemination increased as the oöcyte development progressed. Ovariectomy suppressed sexual receptivity more than 3-fold in liver-fed females. Thus, in addition to nutrition and the CA, ovaries and their developmentaal status can also affect the sexual receptivity in female P. regina.

Control of pupal fat body disappearance in the female black blow fly, Phormia regina (Meigen) by the brain and the corpus allatum.

Neck-ligation, brain implantation, allatectomy, methoprene treatment, and ovariectomy indicated that the disappearance of pupal fat body cells in newly emerged adult female blow fly, Phormia regina, is controlled by the brain and the corpus allatum (CA). Absence of brain or CA greatly lowered the rate of fat body cell disappearance (i.e. death). Dependency on the CA decreased from 0 to 36h post-emregence, indicating that the CA was active during the earlier part of this timespan. Methoprene treatment enhanced pupal fat body cell disappearance in allatectomized females. Brain implantation restored the rate of pupal fat body cell disappearance in neck-ligated flies. Brains from day 1 sugar-fed flies proved to be more effective than those from day 2 sugar-fed flies, indicating that there may be a window after adult emergence that allows the brain to act directly or indirectly on the death of pupal fat body cells. Ovariectomy did not alter the rate of pupal fat body cell death in test animals. Dying pupal fat body cells were smaller in size, less dense (i.e. did not sink in saline like normal pupal fat body cells), and stickier (i.e. attached to other tissues tighter) than the healthy cells. A possible role played by ecdysteroids is also discussed.

Cuticular hydrocarbons and their role in copulatory behavior in Phormia regina (Meigen).

The cuticular hydrocarbons from adult Phormia regina (Meigen) were characterized by gas chromatography-mass spectrometry. Both sexes had similar components in nearly identical quantities, consisting of complex mixtures of saturated n-, monomethyl- and dimethylalkanes from 23 to 33 total carbons. Although no diet-, age-, or sex-specific differences were observed, cuticular hydrocarbons were shown to be involved in copulatory behavior. Hydrocarbon profiles of wild, compared to laboratory reared flies, showed no major differences. Behaviorally, males responded the same to dead decoys of either sex. Removal of the hydrocarbons, using hexane, from either male or female decoys, did not affect the number of mating strikes, but markedly reduced the number of copulatory attempts and the amount of time males spent mounted on either decoy. House fly, Musca domestica L., males when paired with a female M. domestica decoy produced copulatory attempts: whereas, when P. regina males were placed with M. domestica female decoys, there were no copulatory attempts. It is concluded that the cuticular hydrocarbons of P. regina function as species-specific but not sex-specific mating cues and elicit species-specific copulatory behavior in males.

Comparison between tsetse fly synthetic diet and bovine blood on reproduction and survivorship in Tabanus nigrovittatus (Diptera: Tabanidae).

A synthetic diet, developed for tsetse flies and fed to Tabanus nigrovittatus Macquart before the 2nd gonotrophic cycle, supported complete egg maturation. T. nigrovittatus is autogenous only during the 1st cycle. Overall, 52% of females fed bovine blood and 46% fed the synthetic diet produced mature, stage 10 follicles. Of these, 76% of the blood-fed females and 65% of those fed the synthetic diet laid egg masses, all of which hatched. The median adult survivorship was 9 d for blood-fed and 8 d for those fed the synthetic diet.

Faeces feeding by adult Phormia regina (Diptera: Calliphoridae): impact on reproduction.

Fresh beef liver, sugar, and five different types of faeces were evaluated as supportive diets for egg development in the blowfly Phormia regina. Females on a sugar diet were unable to develop follicles beyond stage 3, whereas liver proved to be the best diet for complete egg maturation. Some faecal diets were unable to support egg maturation when fed upon for a short period of time; however, longer periods of feeding produced complete egg maturation. The necessity to feed for longer periods of time in order to produce eggs on most of these diets was attributed to their low protein content. Males, in a shorter period of time than females, obtained enough protein from faeces to activate the neuroendocrine system involved in mating.

Crop and midgut filling and emptying in a female Phormia regina (Diptera: Calliphoridae) fed a liver diet.

To study the role of the crop in protein storage, crop and midgut filling in female Phormia regina (Meigen) was measured volumetrically and the soluble protein content determined. During a 4-h feeding period on homogenized liver, nonligated females consumed and stored enough protein in the crop and midgut to develop a normal compliment of eggs. Within 26 h from the onset of feeding, 75% of the protein content of the crop and a similar amount from the midgut were emptied. Neck ligation or sham neck ligation 7 h after females fed to repletion slowed but still allowed delivery of sufficient protein for egg development.

Electrophysiological recordings from the taste chemosensilla of Tabanus nigrovittatus (Diptera: Tabanidae) and determination of behavioral tarsal acceptance thresholds.

A modified behavioral test for determining the median tarsal acceptance thresholds of field-collected, female Tabanus nigrovittatus Macquart to sugars showed that flies were sensitive to the three major nectar sugars (fructose, sucrose, and glucose, in that order of decreasing sensitivity) whereas galactose, a nonnectar sugar, was not comparably stimulating. Comparisons of various treatment effects on the median tarsal acceptance thresholds showed that for future studies, flies should be fed 10% sucrose before testing, should be starved for 24 h before testing, and they do not have to be pretested for a water response. Electrophysiological recordings showed that gustatory chemosensilla were located on the labellum, tip of labrum, labral food canal, foretarsi, tibia, and antennae. All chemosensilla responded to sugar and salt, whereas only those chemosensilla at the tip of the labrum and those lining the food canal, which normally contact the blood meal, responded to sera and plasma.

Ovipositor chemosensilla in Tabanus nigrovittatus (Macq.) Chrysops fuliginosus (Wied.), and Rhagoletis pomonella (Walsh).

The possible presence of chemosensilla in the ovipositor region has been investigated in three different pest insect species by means of the standard tip-recording electrophysiological technique. Results showed that hair-like structures responding to chemical stimulation were located at the lower-inner edge of the anal leaflets in Tabanus nigro Macq. as well as in Chrysops fuliginosus Wied, whereas peg-like chemosensilla proved to be located in two longitudinal grooves at the apical portion of the ovipositor in Rhagoletis pomonella Walsh.. At least 3 chemosensory units could be detected in each chemosensillum tested. No hair-or peg-like structure on the ovipositor region other than that we described showed any chemosensory responsiveness.

The labellar and tarsal chemosensilla in Anopheles maculipennis atroparvus Van Thiel: electrophysiological and morphological observation.

The presence of chemosensilla on labella and tarsi of Anopheles m.a. has been demonstrated by means of morphological (S.E.M.) and electro physiological (tip-recording) techniques. Results have shown that: 1) at least 3 chemosensory units are located in both types of chemosensilla; 2) stimulation with NaCl evoked various response patterns depending on NaCl concentration in the labellar chemosensilla and just a single pattern in the tarsal ones.

Electrophysiological activity of labellar and tarsal chemosensilla of Hybomitra lasiophtalma (Macquart) to stimulation with NaCl and various sugars.

The responses from labellar and tarsal chemosensilla of Hybomitra lasiophtalma to stimulation with NaCl or sugars (sucrose, glucose, fructose, galactose) were recorded. Results showed that 4 chemosensory units were located in each chemosensillum. Among these, a water-, a salt-, and a sugar-sensitive unit were detectable. NaCl (higher than 50 mM) as well as sucrose (higher than 100 mM) sensitivity thresholds were determined; sucrose and galactose proved to be more active as stimuli than fructose and glucose.