Structural Differences in the Digestive Tract Between Females and Males Could Modulate Regurgitation Behavior in Anastrepha ludens (Diptera: Tephritidae).

With the aim of understanding the mechanisms involved in the regurgitation behavior of tephritid flies, we performed a structural study of the digestive system of the economically important fruit-fly pest, Anastrepha ludens (Loew) using optical, scanning electronic microscopy (SEM) and transmission electron microscopy (TEM), plus a feeding assay. Most structures studied are similar to those previously reported in other adult dipterans, but, importantly, we found sexual differences in some structures that apparently affect regurgitation. We report for the first time sexual differences in the crop duct nerve and large numbers of dense core vesicles within the nerve bundle. Male nerve bundles are bigger and have more secretory vesicles than female ones. The close proximity to the muscles of both the crop lobes and duct suggest that these vesicles (i.e., possibly neurosecretions) might help modulate the muscles regulating regurgitation. The salivary glands are connected to the crop via tracheae, however, SEM/TEM studies failed to find any direct structural connection. Results of the feeding assay indicate that, independently of food type (sucrose or protein) and age, males regurgitate significantly more than females. Regurgitation behavior may also play an important role in capturing bacteria in the environment, and possibly help adults eliminate ingested toxicants such as insecticides. Our findings shed light on an interesting phenomenon that has important practical implications.

Effect of Diet on Adult House Fly (Diptera: Muscidae) Injected With the Salivary Gland Hypertrophy Virus (MdSGHV).

Research to date on the salivary gland hypertrophy virus (SGHV) in three species of flies has focused on adult flies having access to and taking a proteinaceous diet. Since many studies have shown that diet affects viral infection in numerous organisms, this study examined the effect of a protein-free diet on the effect of the SGHV virus in adult house flies, Musca domestica. L. Adults infected with the virus, and maintained on a sugar diet only, showed salivary glands with a blue rather than a grayish color and mild hypertrophy compared with protein-fed flies. It was possible to retrieve the virus from these glands and successfully infect noninfected flies. When injected at various ages, female flies fed only sugar showed that regardless of age, sugar-fed flies still became infected and showed the pathology of the glands. In addition, electron microscope studies revealed at the ultrastructural level that there was no difference between viral replication in cells from salivary glands of adults fed a proteinaceous-free diet and those feeding on protein.

Serotonergic Innervation of the Salivary Glands and Central Nervous System of Adult Glossina pallidipes Austen (Diptera: Glossinidae), and the Impact of the Salivary Gland Hypertrophy Virus (GpSGHV) on the Host.

Using a serotonin antibody and confocal microscopy, this study reports for the first time direct serotonergic innervation of the muscle sheath covering the secretory region of the salivary glands of adult tsetse fly, Glossina pallidipes Austen. Reports to date, however, note that up until this finding, dipteran species previously studied lack a muscle sheath covering of the secretory region of the salivary glands. Direct innervation of the salivary gland muscle sheath of tsetse would facilitate rapid deployment of saliva into the host, thus delaying a host response. Our results also suggest that the neuronal and abnormal pattern seen in viral infected glands by the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is due to a compensatory increased branching of the neurons of the salivary glands, which is associated with the increased size of the salivary glands in viral infected flies. This study shows for the first time serotonin in the cell bodies of the brain and thoracico-abdominal ganglion in adult tsetse, G. pallidipes Austen (Diptera: Glossinidae). A hypothesis is proposed as to whether innervation of the muscle sheath covering of the secretory region of the salivary glands is present in brachyceran compared with nematoceran dipterans; and, a plea is made that more research is needed to develop a blood feeding model, similar to that in the blow flies, for elucidating the various mechanisms involved in production and deployment of saliva.

Effect of Crop Volume on Contraction Rate in Adult House Fly.

The functional aspects of the adult house fly crop have not been studied even though various human and domestic animal pathogens have been discovered within the crop lumen. The average volume consumed (midgut and crop) by flies starved for 24 h was 3.88 µl by feeding both sexes on a sucrose phosphate glutamate buffer. In addition, various volumes of a solution (0.125 M sucrose plus Amaranth dye) were fed to 3-d-old adult female house flies to quantify the crop contraction rate as affected by crop volume. As crop volume increased, the contraction rate increased until it reached a peak at 2 µl, after which it declined. It is hypothesized that the high contraction rate of the crop, which in house fly is almost twice the rate of three other fly species, is one of the factors that makes house fly an excellent vector. The mechanism for such a high contraction rate needs to be investigated.

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.

Efficacy and residual toxicity of chitosan for Rhagoletis pomonella (Diptera: Tephritidae).

Chitosan is a naturally derived polymer that has significant potential for use as a bioinsecticide. Despite this, there is a lack of research as to the efficacy of chitosan for many insect pest species. The apple maggot fly, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), is one such pest for which chitosan toxicity has not been explored. In this study, the toxicity of chitosan for R. pomonella adults was tested via no-choice feeding assays. An aging trial was further used to test the mortality of flies provided dried chitosan-sucrose treatments (CST), which were aged for 0 or 3 days in greenhouse conditions. This study found that the CST is toxic for R. pomonella adults when ingested, leading to a significant increase in the rate of mortality compared to control groups. The use of dried chitosan, however, did not change the mortality of flies, suggesting this will not be an effective delivery mechanism. Effective biopesticide delivery systems have not been defined for chitosan use outside of a laboratory, indicating the need for further research testing delivery mechanisms. It is suggested that an effective method of delivery can be as a food-based bait in attract-and-kill traps, as chitosan must be ingested to kill flies. Forming a viscous solution, chitosan may be best suited to these lure systems.

Ultrastructure of the salivary glands of non-infected and infected glands in Glossina pallidipes by the salivary glands hypertrophy virus.

Light, scanning electron, and transmission electron microscopy analyses were conducted to examine the morphology and ultrastructure of the salivary glands of Glossina pallidipes. Three distinct regions, each with a characteristic composition and organization of tissues and cells, were identified: secretory, reabsorptive and proximal. When infected with the salivary gland hypertrophy (SGH) virus, glands showed a severe hypertrophy, accompanied by profound changes in their morphology and ultrastructure. In addition, the muscular fibers surrounding the secretory region of the glands were disrupted. The morphological alterations in the muscular tissue, caused by viral infection, could be an important aspect of the pathology and may shed light on the mode of action of the SGH virus. Results were discussed with regard to the potential effect of viral infection on normal salivation and on the ability of infected tsetse flies to transmit a trypanosome parasite.

Effect of channel blockers on the smooth muscle of the adult crop of the queen blowfly, Phormia regina.

Few studies have examined the various factors affecting the rate of contraction of the supercontractile muscles of the crop lobes of adult Phormia regina Meigen (Diptera: Calliphoridae). Using an in situ bioassay of the crop organ, various ion channel blockers were tested and it was demonstrated that in all cases the blockers (i.e., against the following conductances: Cl⁻, Ca²âº, Na⁺, and a FMRF-amide action) significantly reduced the contraction rates of the crop lobes, which were filled with 4.5 µL of 1.0 M sucrose containing 10 mM of the dye amaranth. Benzyltrimethylammonium chloride, never before reported for its effect on insect muscle, was as effective in suppressing crop muscle contraction as benzethonium chloride, which is a reported agonist of dromyosuppressin.

Rescuing the Inhibitory Effect of the Salivary Gland Hypertrophy Virus of Musca domestica on Mating Behavior.

Infection with salivary gland hypertrophy virus (MdSGHV) of Musca domestica prevents female flies from accepting copulation attempts by healthy or virus-infected males. This study focused on supplemental hormonal rescue therapy for mating behavior in virus-infected female house flies. The inhibitory effect of the virus on mating behavior in females injected with MdSGHV was reversed by hormonal therapy in the form of octopamine injections, topical application of methoprene, or both therapies combined along with 20-hydroxyecdysone. Infected females whose mating responsiveness had been restored continued to have other viral pathologies associated with infection such as hypertrophy of the salivary glands and a lack of ovarian development.

New Insights on Antennal Sensilla of Anastrepha ludens (Diptera: Tephritidae) Using Advanced Microscopy Techniques.

Using light, transmission, scanning electron, and confocal microscopy, we carried out a morphological study of antennal sensilla and their ultrastructures of the Mexican Fruit Fly Anastrepha ludens (Loew), an economically important species that is a pest of mangos and citrus in Mexico and Central America. Our goal was to update the known information on the various sensilla in the antennae of A. ludens, involved in the perception of odors, temperature, humidity, and movement. Based on their external shape, size, cuticle-thickness, and presence of pores, we identified six types of sensilla with 16 subtypes (one chaetica in the pedicel, four clavate, two trichoid, four basiconic, one styloconic, and one campaniform-like in the flagellum, and three additional ones in the two chambers of the sensory pit (pit-basiconic I and II, and pit-styloconic)), some of them described for the first time in A. ludens. We also report, for the first time, two types of pores in the sensilla (hourglass and wedge shapes) that helped classify the sensilla. Additionally, we report a campaniform-like sensillum only observed by transmission electronic microscopy on the flagellum, styloconic and basiconic variants inside the sensory pit, and an "hourglass-shaped" pore in six sensilla types. We discuss and suggest the possible function of each sensillum according to their characteristics and unify previously used criteria in the only previous study on the topic.

Synanthropic Flies-A Review Including How They Obtain Nutrients, along with Pathogens, Store Them in the Crop and Mechanisms of Transmission.

An attempt has been made to provide a broad review of synanthropic flies and, not just a survey of their involvement in human pathogen transmission. It also emphasizes that the crop organ of calliphorids, sarcophagids, and muscids was an evolutionary development and has served and assisted non-blood feeding flies in obtaining food, as well as pathogens, prior to the origin of humans. Insects are believed to be present on earth about 400 million years ago (MYA). Thus, prior to the origin of primates, there was adequate time for these flies to become associated with various animals and to serve as important transmitters of pathogens associated with them prior to the advent of early hominids and modern humans. Through the process of fly crop regurgitation, numerous pathogens are still readily being made available to primates and other animals. Several studies using invertebrate-derived DNA = iDNA meta-techniques have been able to identify, not only the source the fly had fed on, but also if it had fed on their feces or the animal's body fluids. Since these flies are known to feed on both vertebrate fluids (i.e., from wounds, saliva, mucus, or tears), as well as those of other animals, and their feces, identification of the reservoir host, amplification hosts, and associated pathogens is essential in identifying emerging infectious diseases. New molecular tools, along with a focus on the crop, and what is in it, should provide a better understanding and development of whether these flies are involved in emerging infectious diseases. If so, epidemiological models in the future might be better at predicting future epidemics or pandemics.

Involvement of Follicular Patency in the Ovarian Developmental Block in Virus-infected, MdSGHV, House Flies, Musca domestica (Diptera: Muscidae).

The Musca domestica salivary gland hypertrophy virus (MdSGHV) is known to have marked effects on the female Musca domestica L. (or common house fly) reproductive system, particularly regarding the size and functionality of the ovaries. Examination of the terminal ovarian follicles can help determine if and how MdSGHV mechanistically causes the block in ovarian development. In this study, terminal ovarian follicle lengths were measured and monitored for patency using Trypan blue dye staining. We examined the effect of MdSGHV infection on female house fly ovarian follicles and attempted to rescue the diminished ovarian follicles in MdSGHV-infected house flies through the application of a hormonal treatment (i.e., methoprene). Comparison of patency in control saline-injected females, virus-injected females with no methoprene application, and virus-injected females with topical methoprene application revealed that none of the virus-infected flies showed an increase in terminal follicular length beyond stage 3 follicles (staging according to Adams 1974). Additionally, none showed evidence of patency. In control, saline-injected females, we found the threshold length of the terminal follicles for the onset of patency to be 600 µm. When examined at 48, 72, and 96 h post-eclosion, average follicle length for infected females seldom reached 250 µm and they also failed to display patency. Thus, the virus is somehow involved in shutting down the mechanism involved in follicular patency. The lack of patency in infected follicles may also be one of the determining factors preventing vertical transmission of the pathogen.

Effects of diluted Concord grape juice laced with sodium chloride and selected boron-containing compounds on attraction, consumption, crop muscle contractions, and mortality of adult Drosophila suzukii Matsumura (Diptera: Drosophilidae).

BACKGROUND: In recent years, there has been interest in low-cost, reduced-risk materials that could be used for attract-and-kill of the invasive pest, spotted-wing Drosophila, Drosophila suzukii. This pest causes heavy economic damage to soft-skinned fruits in many countries. In this study, we evaluated physiological and behavioral effects of adding either borax, boric acid, or sodium chloride to diluted Concord grape juice (DGJ), a material that is attractive to adult D. suzukii. RESULTS: Results showed that the addition of borax, sodium chloride and boric acid did not significantly affect the response of adult D. suzukii, relative to DGJ alone. Increases in concentrations (to 5% and 10%) of borax, sodium chloride and boric acid were correlated with decreased ingestion of materials. Mortality of males and females was almost 100% with lower concentrations (1% and 5%) of borax and boric acid within 72 h. The higher concentrations of sodium chloride (5% and 10%) resulted in 100% mortality of both sexes within 72 h. There was no significant effect of chemicals on the number of crop contractions of flies when fed for 4 h. CONCLUSIONS: This study suggests that some substances such as boric acid and borax may act as toxicants without influencing the behavioral response of D. suzukii. © 2021 Society of Chemical Industry.

The Effect of the Musca domestica Salivary Gland Hypertrophy Virus on Food Consumption in Its Adult Host, the Common House Fly (Diptera: Muscidae).

The Musca domestica salivary gland hypertrophy virus (MdSGHV) substantially enlarges the house fly's salivary glands and prevents or delays ovarian development in its adult host, but the effect that MdSGHV has on the house fly's food consumption is currently unknown. Using house flies from a laboratory-reared colony, we evaluated the effect of MdSGHV infection on food consumption over a 7-d period. Both treatment (virus-infected) and control (saline-injected) flies were provided with a choice of 8% sucrose solution and 4% powdered milk solution to determine food preferences. Quantities of each solution consumed were measured every 24 h for each fly to measure food consumptions. Infected house flies were shown to consume less overall of both solutions than house flies injected with saline. The largest consumption discrepancy was seen between female house flies. Healthy female flies with developing ovaries continued to consume a sugar and protein diet, whereas infected female flies fed predominantly on a sugar diet. Additionally, infected male and female flies consumed significantly lower quantities of protein and sucrose than control flies. This suggests that MdSGHV has a negative consumption effect (e.g., hunger, starvation) on its host. Thus, differences in food consumption of infected and control flies probably represent differences in the nutritional requirements of flies resulting from viral infection.

Persistence and Significance of Chlamydia trachomatis in the Housefly, Musca domestica L.

Trachoma is the leading cause of infectious blindness worldwide. Ocular infection by the obligate intracellular pathogen, Chlamydia trachomatis, causes the eyelashes to turn in and scratch the cornea, leading to blindness if left untreated. The disease is most prevalent in poor, rural communities that lack the infrastructure for basic hygiene, clean water, and proper sanitation. Infection is often spread through infected clothes, contaminated hands, and face seeking flies. The goal of this research was to understand the biological role of Musca domestica flies in the transmission of C. trachomatis. PCR, tissue culture, and immunofluorescence microscopy were used to determine the presence, viability, and the anatomical location of C. trachomatis within the digestive tract of M. domestica. Flies were fed with C. trachomatis and then harvested at various time intervals after feeding. The data confirmed the presence of C. trachomatis DNA and viable elementary bodies (EBs) in fly crops, up to 24 h postfeeding. C. trachomatis DNA was also isolated from the upper portions of the alimentary tract of flies up to 48 h postfeeding. In addition, DNA was isolated from the regurgitation material from fly crops up to 12 h postfeeding. The viability of isolated C. trachomatis EBs was repeatedly confirmed between 12 and 48 h and up to 7 days in ex vivo crops stored at room temperature. Our data suggest that eye-seeking flies such as M. domestica can ingest C. trachomatis during regular feeding. Because M. sorbens does not occur in continental United States, we did not use it in any of our studies. These data also confirm, for the first time, that ingested chlamydia remains viable inside the flies for 24-48 h postfeeding. We further show that these flies can regurgitate and transmit the trachoma agent at their next feeding. We believe that these findings reveal an opportunity for efficient intervention strategies through fly vector control, especially as we near new target date for global elimination of trachoma.

Effects of chitosan and erythritol on labellar taste neuron activity, proboscis extension reflex, daily food intake, and mortality of male and female spotted-winged drosophila, Drosophila suzukii.

In recent years, there has been interest in reduced-risk materials with insecticidal properties for the invasive pest spotted-wing drosophila, Drosophila suzukii. Here, we compared the peripheral sensitivity (via the tip-recording technique, used to monitor the neural activity of gustatory receptor neurons [GRNs]) and palatability (via the Proboscis Extension Reflex [PER]) of chitosan, a polysaccharide derived from chitin, with that of erythritol, a sugar alcohol, to male and female D. suzukii. Because in some insect species it has previously been shown that chitosan has some insecticidal properties, then treatment effects on mortality rates of male and female D. suzukii were quantified. Physiological recordings from the l-type labellar sensilla showed that erythritol evoked responses from one GRN, while chitosan elicited spiked activity from a second one. The first PER bioassay revealed that the level of response to erythritol increased significantly for males and females as the concentrations increased, and the effect of fly sex was non-significant. The second PER bioassay compared the male and female response to chitosan and erythritol each at 0.125, 0.25, 0.5, 1, and 2% concentrations. The overall female PER to erythritol was significantly greater than that exhibited by males, and no differences were noted between sexes when chitosan was evaluated. These results indicate that chitosan alone can elicit PER responses in adult D. suzukii. In the third experiment, chitosan was toxic to D. suzukii. When combined with sucrose (2%), chitosan elicited high levels (80-100%) of mortality of adult D. suzukii within 3 days, particularly in males. The presence of erythritol did not seem to increase the toxic effect of chitosan.

Effect of chitosan on adult longevity when fed, in no-choice experiments, to Musca domestica L., Tabanus nigrovittatus Macquart, and Phormia regina (Meigen) adults and its consumption in adult Musca domestica L.

BACKGROUND: The literature reports that more research needs to be done on using chitosan as an environmentally friendly bioinsecticide, especially against dipterans. Thus, we examined the effect of chitosan when fed, in no-choice experiments, to adult Musca domestica, Tabanus nigrovittatus, and Phormia regina. RESULTS: A 2% chitosan solution was fed, in no-choice experiments, to adults of Musca domestica, Tabanus nigrovittatus, and Phormia regina to study the effects on survivorship. In all species, the uptake of chitosan caused a significant decrease in survivorship. Examination of the digestive tract of house flies showed a thick material within the midgut, plus a shriveling of the midgut only in chitosan-treated flies. A survivorship curve of adult house flies fed a 10% sucrose and 2% w/v ascorbic acid-only solution showed that the effect of the chitosan solution was due to the chitosan and not the ascorbic acid. Intake experiments revealed that by day 2, chitosan treated house flies consumed significantly less of the diet compared to controls. CONCLUSION: Chitosan, a biopesticide, when fed in no-choice experiments to three species of adult flies produced severe mortality within 4-6 days of ingestion. A working hypothesis suggests that by day 2 of the chitosan diet there appears to be a malfunction of the digestive tract and possibly the midgut microbiome. © 2020 Society of Chemical Industry.

Fly foregut and transmission of microbes.

Two areas of research that have greatly increased in attention are: dipterans as vectors and the microbes they are capable of vectoring. Because it is the front-end of the fly that first encounters these microbes, this review focuses on the legs, mouthparts, and foregut, which includes the crop as major structures involved in dipteran vectoring ability. The legs and mouthparts are generally involved in mechanical transmission of microbes. However, the crop is involved in more than just mechanical transmission, for it is within the lumen of the crop that microbes are taken up with the meal of the fly, stored, and it is within the lumen that horizontal transmission of bacterial resistance has been demonstrated. In addition to storage of microbes, the crop is also involved in depositing the microbes via a process known as regurgitation. Various aspects of crop regulation are discussed and specific examples of crop involvement with microorganisms are discussed. The importance of biofilm and biofilm formation are presented, as well as, some physical parameters of the crop that might either facilitate or inhibit biofilm formation. Finally, there is a brief discussion of dipteran model systems for studying crop microbe interactions.

FMRFamide-like immunoreactivity in the central nervous system and alimentary tract of the non-hematophagous blow fly, Phormia regina, and the hematophagous horse fly, Tabanus nigrovittatus.

FMRFamide-related peptides (FaRPs) are a diverse and physiologically important class of neuropepeptides in the metazoa. In insects, FaRPs function as brain-gut neuropeptides and have been immunolocalized throughout the nervous system and alimentary tract where they have been shown to affect feeding behavior. The occurrence of FMRFamide-like immunoreactivity (FLI) was examined in the central nervous system and alimentary tract of non-hematophagous blow fly, Phormia regina Meigen (Diptera: Calliphoridae), and the hematophagous horse fly, Tabanus nigrovittatus Macquart (Diptera:Tabanidae). Although the central nervous system and alimentary anatomy differ between these two dipteran species, many aspects of FLI remain similar. FLI was observed throughout the central and stomatogastric nervous systems, foregut, and midgut in both flies. In the central nervous system, cells and processes with FLI occurred in the brain, subesophageal ganglion, and ventral nerve cord. FLI was associated with neurohemal areas of the brain and ventral nerve cord. A neurohemal plexus of fibers with FLI was present on the dorsal region of the thoracic central nervous system in both species. In the gut, processes with FLI innervated the crop duct, crop and anterior midgut. Endocrine cells with FLI were present in the posterior midgut. The distribution of FLI in these two flies, in spite of their different feeding habits, further supports the role of FaRPs as important components of the braingut neurochemical axis in these insects and implicates FaRPs as regulators of insect feeding physiology among divergent insect taxa.

The imbalance of serotonergic circuitry impairing the crop supercontractile muscle activity and the mitochondrial morphology of PD PINK1B9Drosophila melanogaster are rescued by Mucuna pruriens.

Despite its great potentiality, little attention has been paid to modelling gastrointestinal symptoms of Parkinson's disease (PD) in Drosophila melanogaster (Dm). Our previous studies on standardized Mucuna pruriens extract (Mpe) have shown usefulness in the Drosophila model of PD. In this communication, we provide new information on the effect of Mpe on basal and serotonin treated contractions in the crop (i.e., an important and essential part of the gut) in Drosophila PD mutant for PTEN-induced putative kinase 1 (PINK1B9) gene. The effect of Mpe on PINK1B9 supplied with standard diet to larvae and/or adults, were assayed on 10-15 days old flies. Conversely from what we observed in the wild type flies, recordings demonstrated that exogenous applications of serotonin on crop muscles of untreated PINK1B9 affect neither the frequency nor the amplitude of the crop contraction, while the same muscle parameters are enhanced following brain injections of serotonin, thus suggesting that PINK1B9 mutants may likely have an impairment in the serotonergic pathways. Also, the mitochondrial morphology in the crop muscles is strongly compromised, as demonstrated by the transmission electron microscopy analysis. The Mpe treatment rescued the crop muscle parameters and also the mitochondrial morphology when supplied to both larvae and adults. Overall, this study strengthens the relevance of using PINK1B9 Dm as a translational model to study the gastrointestinal symptoms in PD and also confirms the useful employment of M. pruriens for PD treatment.