Micro-CT visualization of a promastigote secretory gel (PSG) and parasite plug in the digestive tract of the sand fly Lutzomyia longipalpis infected with Leishmania mexicana

Leishmaniasis is a debilitating disease of the tropics, subtropics and southern Europe caused by Leishmania parasites that are transmitted during blood feeding by phlebotomine sand flies (Diptera: Psychodidae). Using non-invasive micro-computed tomography, we were able to visualize the impact of the laboratory model infection of Lutzomyia longipalpis with Leishmania mexicana and its response to a second blood meal. For the first time we were able to show in 3D the plug of promastigote secretory gel (PSG) and parasites in the distended midgut of whole infected sand flies and measure its volume in relation to that of the midgut. We were also able to measure the degree of opening of the stomodeal valve and demonstrate the extension of the PSG and parasites into the pharynx. Although our pilot study could only examine a few flies, it supports the hypothesis that a second, non-infected, blood meal enhances parasite transmission as we showed that the thoracic PSG-parasite plug in infected flies after a second blood meal was, on average, more than twice the volume of the plug in infected flies that did not have a second blood meal.     

Illuminating the Prevalence of Trypanosoma brucei s.l. in Glossina Using LAMP as a Tool for Xenomonitoring

Background

As the reality of eliminating human African trypanosomiasis (HAT) by 2020 draws closer, the need to detect and identify the remaining areas of transmission increases. Here, we have explored the feasibility of using commercially available LAMP kits, designed to detect the Trypanozoon group of trypanosomes, as a xenomonitoring tool to screen tsetse flies for trypanosomes to be used in future epidemiological surveys.

Methods and Findings

The DNA extraction method was simplified and worked with the LAMP kits to detect a single positive fly when pooled with 19 negative flies, and the absolute lowest limit of detection that the kits were able to work at was the equivalent of 0.1 trypanosome per ml. The DNA from Trypanosoma brucei brucei could be detected six days after the fly had taken a blood meal containing dead trypanosomes, and when confronted with a range of non-target species, from both laboratory-reared flies and wild-caught flies, the kits showed no evidence of cross-reacting.

Conclusion

We have shown that it is possible to use a simplified DNA extraction method in conjunction with the pooling of tsetse flies to decrease the time it would take to screen large numbers of flies for the presence of Trypanozoon trypanosomes. The use of commercially-available LAMP kits provides a reliable and highly sensitive tool for xenomonitoring and identifying potential sleeping sickness transmission sites.     

Effect of host packed cell volume on the bloodmeal size of male tsetse flies, Glossina pallidipes

aematin contents of engorged, male tsetse flies, Glossina pallidipes Austen, were compared with the packed cell volumes of oxen on which they had fed. Haematin contents icnreased with packed cell volume up to packed cell volumes of approximately 30%. Haematin contents appeared to level off or decline with further increase in packed cell volume. These results support a model of blood-feeding in tsetse flies in which the rate of blood consumption decreases as packed cell volume increases, because of increase in blood viscosity, and tsetse are unable to compensate for the decrease in consumption rate by feeding for a longer time. After allowing for the effects of packed cell volume, bloodmeal sizes of tsetse increased with ox body temperature.     

Increased longevity and resistance to heat shock in Drosophila melanogaster flies exposed to hypergravity

In recent years, attempts have been made to increase longevity in animal models (caloric restriction in rodents or overexpression of catalase and superoxide dismutase in transgenic flies, for instance). We report here that flies submitted to hypergravity (3 or 5 g), for 1 or 4 weeks starting from the second day of imaginai life and transferred after that time to 1 g, have a higher resistance to heat shock than flies living continuously at 1 g. Furthermore, male flies that had lived for 2 weeks from the second day of life at 3 or 5 g, lived longer than those living all the time at 1 g; no longevity increase was observed in females. As far as we know, this is the first example in flies showing that a mild stress at a young age not only increases resistance to an acute stress but also increases longevity. A hypothesis to explain these results could be that heat-shock proteins, which are induced by various stress factors, are synthesized in conditions of hypergravity.     

The rôle of juvenile hormone in housefly ovarian follicle morphogenesis

Oögenesis in the housefly, Musca domestica, was divided into a series of 10 stages where stage 1 was the germarium, stage 4 was the beginning of yolk deposition, stage 7 was characterized by maximal nurse cell development, stage 9 by the degeneration of the nurse cells and chorion formation, and stage 10 was the mature egg. It required 69 hr from eclosion at 27°C to develop mature eggs. This represented an oöcyte volume increase of 3700-fold, a seventeenfold increase in follicle length, and a sevenfold increase in weight. The application of 2 μg of isopropyl (E,E)-11-methoxy-3,7,11-trimethyldodeca-2,4-dienoate (ZR-515) to allatectomized (-CA) flies stimulated egg development, which progressed at the same rate as the controls. The -CA flies did not develop eggs past stage 4, which represented a cessation of development at a volume of 1·4 per cent that of a mature egg and an ovarian dry weight of 11 per cent that of a mature ovary. The follicle cells from -CA flies did not differentiate into the squamous condition over the nurse chamber, did not become columnar over the oöcyte, did not produce the chorion or vitelline membrane, and did not decrease in number as they did on the stage 10 follicles. Endomitosis in the nurse cell nuclei of -CA flies stopped development at 290 c, but maximum development of 2400 c occurred in stage 7 follicles from controls, and then the nurse cells began to disintegrate.     

Quantification of Food Intake in Drosophila

Measurement of food intake in the fruit fly Drosophila melanogaster is often necessary for studies of behaviour, nutrition and drug administration. There is no reliable and agreed method for measuring food intake of flies in undisturbed, steady state, and normal culture conditions. We report such a method, based on measurement of feeding frequency by proboscis-extension, validated by short-term measurements of food dye intake. We used the method to demonstrate that (a) female flies feed more frequently than males, (b) flies feed more often when housed in larger groups and (c) fly feeding varies at different times of the day. We also show that alterations in food intake are not induced by dietary restriction or by a null mutation of the fly insulin receptor substrate chico. In contrast, mutation of takeout increases food intake by increasing feeding frequency while mutation of ovo^D increases food intake by increasing the volume of food consumed per proboscis-extension. This approach provides a practical and reliable method for quantification of food intake in Drosophila under normal, undisturbed culture conditions.     

Failure of Sterne- and Pasteur-Like Strains of Bacillus anthracis to Replicate and Survive in the Urban Bluebottle Blow Fly Calliphora vicina under Laboratory Conditions

This study aimed to elucidate the bacteriological events occurring within the gut of Calliphora vicina, selected as the European representative of blow flies held responsible for the spread of anthrax during epidemics in certain parts of the world. Green-fluorescent-protein-carrying derivatives of Bacillus anthracis were used. These lacked either one of the virulence plasmids pXO1 and pXO2 and were infected, or not infected, with a worm intestine phage (Wip4) known to influence the phenotype and survival of the pathogen. Blood meals were prepared for the flies by inoculation of sheep blood with germinated and, in case of pXO2+ strains, encapsulated cells of the four B. anthracis strains. After being fed for 4 h an initial 10 flies were externally disinfected with peracetic acid to ensure subsequent quantitation representing ingested B. anthracis only. Following neutralization, they were crushed in sterile saline. Over each of the ensuing 7 to 10 days, 10 flies were removed and processed the same way. In the absence of Wip4, strains showed steady declines to undetectable in the total B. anthracis counts, within 7–9 days. With the phage infected strains, the falls in viable counts were significantly more rapid than in their uninfected counterparts. Spores were detectable in flies for longer periods than vegetative bacteria. In line with the findings in both biting and non-biting flies of early workers our results indicate that B. anthracis does not multiply in the guts of blow flies and survival is limited to a matter of days.     

Dynamics of follicle development and lipid storage in the tsetse fly Glossina palpalis palpalis fed on serum albumin deficient diets

Females of Glossina p. palpalis were fed on a mixture of bovine erythrocytes and a serum substitute which had been lyophilized, for a period of 30 days. The rate of ovarian development and lipid incorporation and the fatty acid composition of whole flies were examined at five-day intervals. Results showed that in flies fed serum albumin-deficient diets, ovarian development was seriously impaired and ovulation did not take place. Flies which were fed on a serum albumin-containing diet ovulated and produced larvae, though at a lower rate than control flies. Compared with controls, lipid incorporation was reduced in experimental flies, but more in the albumin-deficient group than in the albumin-containing group. The diet composition had no marked influence on the relative composition of fatty acids in any one group. It is concluded that, though an essential nutritional element for cow blood fed G. p. palpalis, serum albumin was not the only missing essential factor in the diets that were used in this study.     

Hormonal regulation of trehalose metabolism in the blowfly, Phormia regina: interaction between hypertrehalosemic and hypotrehalosemic hormones

Hypertrehalosemia occurs two days after cardiacectomy of adult male Phormia regina with no attendant change in fat body glycogen. In spite of this, cardiacectomized flies caused to fly for 10 min show a lower rate of haemolymph trehalose turnover, and seem to have a decreased capability for synthesizing trehalose from haemolymph glucose. Phormia brain is shown to contain a hypotrehalosemic hormone whose release depends on the integrity of the stomatogastric nervous system. It is possible that the hypertrehalosemic condition in cardiacectomized flies is a result of the absence of this hormone from the blood.     

A New Chamber for Studying the Behavior of Drosophila

Methods available for quickly and objectively quantifying the behavioral phenotypes of the fruit fly, Drosophila melanogaster, lag behind in sophistication the tools developed for manipulating their genotypes. We have developed a simple, easy-to-replicate, general-purpose experimental chamber for studying the ground-based behaviors of fruit flies. The major innovative feature of our design is that it restricts flies to a shallow volume of space, forcing all behavioral interactions to take place within a monolayer of individuals. The design lessens the frequency that flies occlude or obscure each other, limits the variability in their appearance, and promotes a greater number of flies to move throughout the center of the chamber, thereby increasing the frequency of their interactions. The new chamber design improves the quality of data collected by digital video and was conceived and designed to complement automated machine vision methodologies for studying behavior. Novel and improved methodologies for better quantifying the complex behavioral phenotypes of Drosophila will facilitate studies related to human disease and fundamental questions of behavioral neuroscience.     

Influence of the White Locus on the Courtship Behavior of Drosophila Males

Since its discovery by Morgan, the Drosophila white gene has become one of the most intensely studied genes and has been widely used as a genetic marker. Earlier reports that over- and misexpression of White protein in Drosophila males leads to male-male courtship implicated white in courtship control. While previous studies suggested that it is the mislocalization of White protein within cells that causes the courtship phenotype, we demonstrate here that also the lack of extra-retinal White can cause very similar behavioral changes. Moreover, we provide evidence that the lack of White function increases the sexual arousal of males in general, of which the enhanced male-male courtship might be an indirect effect. We further show that white mutant flies are not only optomotor blind but also dazzled by the over-flow of light in daylight. Implications of these findings for the proper interpretation of behavioral studies with white mutant flies are discussed.     

The effects of dietary sodium and potassium on rapid diuresis in the tsetse fly Glossina morsitans

A self-supporting colony of Glossina morsitans can be maintained by feeding flies through artificial membranes but only if a diet of defibrinated pig blood is used. The potassium concentration of pig serum was found to be higher than that of cow or rabbit serum. However, this did not affect the rate of diuresis which was lower after in vitro diets than after feeding on a live host.After feeding in vivo on rabbits, and in vitro on pig blood and cow blood, the concentrations of sodium and potassium in the haemolymph remained constant. After feeding on salt solutions and distilled water, on the other hand, it was found that the more the composition of the meal varied from that of blood serum the lower was the rate of diuresis and the more the concentrations of sodium and potassium in the haemolymph were displaced from their normal values. Large fluctuations in the concentrations of these ions resulted in the paralysis and death of the flies.The results demonstrate that the tsetse fly is able to reabsorb sodium from the primary urine produced by the Malpighian tubules during diuresis and it is suggested that this reabsorption of ions may lead to an increased circulation of water through the excretory system.     

Laboratory transmission of an Asian strain of Leishmania tropica by the bite of the southern European sand fly Phlebotomus perniciosus

Imported cases of anthroponotic cutaneous leishmaniasis due to Leishmania tropica are increasingly documented in Europe. We investigated the ability of Phlebotomus perniciosus, a competent vector of Leishmania infantum widespread in southwestern Europe, to support the growth and transmissibility of an Asian strain of L. tropica recently isolated from a refugee. Parasite growth behavior was investigated in laboratory-reared sand flies fed artificially with promastigotes as well as in sand flies infected after biting on footpad lesions induced in hamsters by promastigote inoculation. The evolution of infection was checked by gut microscopy and quantitative real-time PCR, and it was found to be similar between promastigote- and amastigote-initiated infections. In 80% of infected sand flies, despite survival and flourishing growth of promastigotes after blood digestion and defecation, either the parasites died, or failed to migrate to the foregut and/or to mature into infective forms. However, in the remaining 20% L. tropica developed into abundant metacyclic promastigotes. The quantitative real-time PCR assay detected variable loads of gut promastigotes irrespective of morphological evidence of viability or progressive/final death. Parasite transmissibility was investigated by exposing naive hamsters to P. perniciosus previously infected on chronic lesions induced in hamsters which survived to take a second blood meal. Two months post exposure, lesions developed in skin sites bitten by sand flies confirmed to harbor metacyclic promastigotes; in the following months, the presence of viable and transmissible L. tropica parasites in lesions was demonstrated by xenodiagnosis assays. Our findings support the hypothesis that, in particular epidemiological situations, P. perniciosus may play the role of an occasional L. tropica vector.     

Post-feed buzzing in the tsetse, Glossina morsitans morsitans, is an endothermic mechanism

ABSTRACT. Post-feed buzzing in Glossina morsitans morsitans Westw. causes a rise in thoracic temperature relative to the length of the buzz. As lift is proportional to the square of wing-beat frequency, which increases with temperature up to 32°C, buzzing results in an increase in the lift which the fly can produce. Heat generated by buzzing, in combination with the heat received from the host at the time of feeding, may well allow the fly to maximize lift generated in the immediate post-feeding period. Buzzing flies excrete excess water from the meal more rapidly than non-buzzing flies. It is argued that this is due to a rise in abdominal temperature. Maximized lift in the immediate post-feeding period and the rapid elimination of water from the very large blood meals taken by these flies may be expected to have strong selective advantages for the flies.     

Ethanol extract of Cassia siamea L. increases life span in Drosophila melanogaster

The stem of Cassia siamea L. (Fabaceae) has been used in traditional Thai medicine as a longevity remedy. The objective of this study was to investigate the effect of ethanolic stem extract of C. siamea (CSE) on the life span of Drosophila melanogaster. The results showed that a diet containing 10 mg/mL CSE could significantly extend the mean life span of D. melanogaster by 14% compared with the control diet (P < 0.01). The maximum life span was 74, 78, and 84 days in control, CSE (5 mg/mL) and CSE (10 mg/mL) groups, respectively. Supplementation of CSE at 10 mg/mL also significantly increases the activity of superoxide dismutase (SOD) and catalase (CAT) at days 25 and 40 compared with the control diet. Treatment of CSE at 5 and 10 mg/mL significantly increased the climbing ability of D. melanogaster both on days 25 and 40 compared with the control flies. Paraquat and H₂O₂ challenge test showed that flies fed with CSE at 10 mg/mL had a longer survival time than the control flies (P < 0.01). This study provides supportive evidence that supplementation with CSE prolonged life span and reduced oxidative stress in D. melanogaster.     

The molybdoenzyme system of Drosophila melanogaster. I. Sulfite oxidase: Identification and properties. Expression of the enzyme in maroon-like (mal), low-xanthine dehydrogenase (lxd), and cinnamon (cin) flies

Sulfite oxidase (sulfite: ferricytochrome c oxidoreductase; EC 1.8.2.1) has been detected in Drosophila melanogaster and some of its properties have been studied. In most respects this enzyme resembles the mammalian sulfite oxidases except for its molecular weight (148,000), which is somewhat higher than that of rat sulfite oxidase (116,000). Cytochrome c, potassium-ferricyanide, and oxygen can serve as electron acceptors in the oxidation of sulfite by the enzyme. Although definite evidence can be obtained only through the analysis of the pure enzyme, experiments involving tungstate feeding suggest that Drosophila sulfite oxidase is most probably a molybdoenzyme. Extracts of mal flies show normal levels of sulfite oxidase, whereas lxd flies have only 5–10% of the activity of wild type, and in cin flies the enzyme is apparently absent. While it is possible that the lxd and cin mutations are at some level responsible for the defective synthesis of a molybdenum-containing cofactor (supposed to be present in most molybdoenzymes), the evidence accumulated so far by several authors and the results of the present investigation argue against the involvement of a Mo cofactor in the multiple enzyme deficiencies observed in mal flies.     

Feeding and breeding aspects of Stomoxys calcitrans (Diptera: Muscidae) under laboratory conditions

Bionomic aspects of Stomoxys calcitrans (Linnaeus, 1758) (Diptera: Muscidae) were studied under laboratory conditions. For this reason, laboratory-rearing techniques were optimized at the National Veterinary School of Toulouse. The colony was maintained at 25 ± 2 °C, 50 ± 10% RH under a 12-hour light cycle and observed daily. The size of each adult cage is 30 x 30 x 30 cm and designed to house about 500-1,000 flies. The average cycle from egg to adult was 19.2 ± 1.7 days. The mean longevity of imagos was 9.3 ± 5.8 days and not significantly different between sexes. Stable flies were split into two groups; the first was fed with blood, honey and water, and the second was fed only with honey and water. The mean weight of a blood meal was 11.1 ± 3.8 mg with no significant differences between males and females. The mean longevity of non-blood fed flies was found to be significantly higher (10.4 ± 3.9 days) than those fed with blood. The maximum lifespan was shorter for non-blood fed males (17 days) and females (18 days) than for those fed with blood (females: 24 days, males: 23 days). Under these laboratory conditions, S. calcitrans rearing was successfully established. In the end, the number of expected generations of S. calcitrans and the net reproduction rate were estimated to be 11.8 generations/year and 16.2 living females per female respectively.     

‘Hunger’ in the tsetse fly: The nutritional correlates of behaviour

The spontaneous activity and visual responsiveness of starved tsetse flies, Glossina morsitans, increase exponentially for about 5 days after feeding until a pre-moribund decline sets in. Evidently the depletion of food reserves affects the behaviour of the fly. The causal relationship in this interaction was sought in two ways: (a) by looking for changes in nutritional parameters that follow the same trend and time course as the behavioural change, (b) by calculating the correlations between behaviour and these various parameters from concurrent data on individual flies. None of the three factors traditionally measured in tsetse (per cent hydration, lipid content, and residual blood meal) changed along a course similar to that followed by behaviour, nor did any expression of them correlate as well with behaviour (r = 0·75, 0·74, and 0·71, respectively) as did the duration of starvation alone (r = 0·79). On the other hand, abdominal weight and total body weight both changed in parallel with behaviour and correlated substantially better with it than did starvation time (r = 0·85 and 0·89, respectively); they also correlated very strongly with each other (r = 0·98). It is suggested that the mean levels of the fly's behavioural thresholds are modulated on the basis of information about its overall nutritional state derived from monitoring its weight or abdominal volume, or perhaps some third variable with which these two are closely correlated.     

Influence of diet on synthesis and utilisation of lipids for reproduction by the tsetse fly Glossina morsitans

Fatty acid composition of lipids from adult Glossina morsitans was unaffected by an in vitro fed diet of cow blood which induces production of under-sized offspring compared to that of flies fed on a superior diet of pig blood. The commonest fatty acids were C_16:0, C_16:1 and C_18:1 and only small differences in proportions were detected between virgin and pregnant females. Rate of lipid accumulation by males and females was the same and was unaffected by diet, but males achieved a maximum of 2.5 mg on day 9 while both virgin and fertilised females reached a maximum of 5,0 mg on day 14 of adult life. Lipid content of pregnant flies then fell to 3.0 mg on the day of larviposition and accumulation began again. A cow blood diet reduced the extent to which the lipids were utilised for larval growth and this was reflected in an altered secretory activity cycle in the female uterine gland. However, no effect on the growth of adult fat body was detectable in such flies. Mating and fertilisation, which influence reproductive events through activity of the endrocine system do not seem to affect the acquisition of lipid reserves by female Glossina. However, they clearly exert considerable influence over distribution of such reserves between fat body and uterine gland, which distribution is also affected by diet.