The oxidative response of rabbit peritoneal neutrophils to leishmanias and other trypanosomatids

Luminometry has been used to measure the respiratory burst of rabbit peritoneal neutrophils that is elicited by different forms and species of Leishmania and Herpetomonas. Mid-log phase and metacyclic promastigotes of L. major evoked large responses; that due to metacyclics was lower and slower, but they also bound in smaller numbers than mid-log phase cells. Promastigotes of L. mexicana mexicana also stimulated a large respiratory burst whereas amastigotes elicited little or none. Leishmania donovani promastigotes and culture forms of H. muscarum muscarum and H. m. ingenoplastis all evoked large responses by neutrophils. There was, however, very little response to L. mexicana mexicana promastigotes, L. donovani promastigotes or H. muscarum muscarum when they were added in large numbers. This 'inhibition' was not apparent with L. major.     

Purine phosphoribosyltransferases of Leishmania mexicana mexicana and other flagellate protozoa

Amastigotes and cultured promastigotes of Leishmania mexicana mexicana and L. m. amazonensis, cultured promastigotes of L. donovani and L. tarentolae, and the culture forms of Crithidia fasciculata, Herpetomonas muscarum muscarum and H. m. ingenoplastis all possessed four phosphoribosyltransferase (PRTase) activities: adenine PRTase, hypoxanthine PRTase, guanine PRTase and xanthine PRTase. The enzymes of L. m. mexicana required divalent cations for activity; Mn2+ or Co2+ produced maximal activity in most cases. Hypoxanthine PRTase, guanine PRTase and xanthine PRTase from all organisms were sedimentable in part, suggesting that they may occur within glycosomes. The enzymes of L. m. mexicana cultured promastigotes were inhibited by a range of purine analogues.     

A comparative study of the purine- and pyrimidine-metabolising enzymes of a range of trypanosomatids

A range of trypanosomatids (amastigotes and cultured promastigotes of Leishmania mexicana mexicana, cultured promastigotes of L. m. amazonensis, L. donovani and L. tarentolae, culture forms of Crithidia fasciculata, Herpetomonas muscarum muscarum and H. m. ingenoplastis and procyclic trypomastigotes of Trypanosoma brucei brucei) have been surveyed for the presence of purine- and pyrimidine-metabolising enzymes. Several common features were observed, including the presence of nucleosidases, catabolic phosphorylases, phosphoribosyltransferases, kinases and cytidine deaminase and the apparent absence of AMP deaminase, anabolic purine phosphorylase and cytosine deaminase. Significant differences between species were discovered, notably in adenine and adenosine metabolism. Nucleoside phosphotransferase active on inosine was detected in insect trypanosomatids but not in L. m. mexicana.     

The Products and Pathways of Glucose Catabolism in Herpetomonas muscarum ingenoplastis and Herpetomonas muscarum muscarum

ABSTRACT. The products and pathways of glucose catabolism in the insect trypanosomatids Herpetomonas muscarum ingenoplastis and Herpetomonas muscarum muscarum have been studied with the aim of elucidating how both organisms are able to proliferate well under aerobic and anaerobic conditions. When incubated in medium containing glucose as the only exogenous carbon source, catabolism was found to be fermentative in both cases. Acetate was a major product of both organisms while H. m. ingenoplastis produced more ethanol and propionate and less succinate than H. m. muscarum . Ethanol production by H. m. ingenoplastis decreased both under anaerobic conditions and in the presence of elevated CO₂ concentrations, whereas succinate and propionate release by this organism were greater in high CO₂ and anoxia, respectively. Succinate production by H. m. muscarum was greatest under anaerobic conditions in elevated CO₂ whereas propionate was only a minor product. The same four products were released during growth of the organisms in complex medium, but the relative proportions differed suggesting that other substrates were being used. Both organisms contained enzymes of the glycolytic and pentose phosphate pathways, but while all activities of the TCA cycle were present in H. m. muscarum . NAD-linked isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate CoA synthase and succinate dehydrogenase were not detected in H. m. ingenoplastis . Fumarate reductase activity was present in both organisms. The data presented suggest that CO₂-fixation and reverse flux through the TCA cycle may be important factors that enable the organisms to undergo anaerobiosis.     

Herpetomonas megaseliae, Crithidia fasciculata, and Leptomonas collosoma (Kinetoplastida, Trypanosomatidae) in Feces of Lizards Fed Culture Forms of the Flagellates

ABSTRACT. Herpetomonas megaseliae, Crithidia fasciculata , and Leptomonas collosoma from culture survived gut passage in Anolis carolinensis following their ingestion by this lizard. Maximum persistence of H. megaseliae in lizards, as detected by fecal culture, was seven days. No invasion of tissues by H. megaseliae could be detected by means of sectioned material, stained impression slides, or cultures inoculated with material from organs. Crithidia fasciculata was evident in cloacal fluid for up to three days in wet mount preparations. Leptomonas collosoma was observed in feces 24 h after the organisms were fed to lizards. Both C. fasciculata and L. collosoma were cultured from feces of lizards fed the parasites 24 h earlier. Herpetomonas megaseliae was differentiated in lizard feces, with greater than 40% of the forms observed being paramastigotes or opisthomastigotes. Truncate, semispherical forms resembling choanomastigotes were seen, but the kinetoplast was posterior to the nucleus in some of these. Many forms showed extensive coiling of the axoneme within the body of the flagellate. Choanomastigotes and spheromastigotes of C. fasciculata and promastigotes, sphero-mastigotes and amastigotes of L. collosoma were also observed in the feces.     

Comparative real-time kinetic analysis of human complement killing of Leishmania infantum promastigotes derived from axenic culture or from Phlebotomus perniciosus

Although Leishmania metacyclic promastigotes are generally considered resistant to human complement, studies of in vitro-cultured axenic stationary promastigotes using serum concentrations that approximate physiological plasma conditions indicate complement sensitivity. Natural Leishmania infection is caused by sand fly-inoculated promastigotes, whose complement resistance has not been analyzed systematically. We compared Leishmania susceptibility to human complement in L. infantum promastigotes derived from in vitro cultures and from sand flies. Phlebotomus perniciosus sand flies were fed with axenic promastigotes, L. infantum-infected U-937 cells, or spleen cells from L. infantum-infected hamsters. On selected days post-feeding, flies were dissected and promastigotes isolated; in addition, axenic promastigotes were obtained from culture at equivalent days of growth. In near-physiological serum concentration and temperature conditions, measurement of real-time kinetics of propidium iodide uptake showed that approximately 90% of axenic- and sand fly-derived promastigotes were rapidly killed by complement. We found no substantial differences between promastigotes from axenic culture, those isolated from flies on different post-feeding days, or those generated in flies fed with distinct inocula. The results indicate that Leishmania susceptibility to human complement is independent of promastigote developmental stage in the sand fly mid-gut and in axenic culture.     

Herpetomonas mariadeanei sp. n. (Protozoa,Trypanosomatidae) from Muscina stabulans (Falléen, 1816) (Diptera,Muscidae)*

SYNOPSIS. A trypanosomatid flagellate was isolated from the musciod fly Muscina stabulans (Fallén). Cloned cultures of this organism contained promastigotes, opisthomastigotes, and froms containing a long flagellum doubled or coiled within the cell but not protruding outside. These latter forms we are designating endomastigotes. The presence of these hitherto underscribed endomastigotes along with other morphologic and growth characteristics suggest that this is a new species for which the name Herpetomonas mariadeanei is proposed.     

Varying Kinetoplast Ultrastructure in Two Subspecies of Herpetomonas muscarum (Leidy)*

SYNOPSIS. The kinetoplast nucleoid of Herpetomonas muscarum muscarum is a disk-shaped mass of filaments with sharply delimited anterior and posterior margins. The kinetoplast nucleoid of Herpetomonas muscarum ingenoplastis is elongate, tapered, and made up of long, helically arranged filaments. Possible explanations of these structures on the basis of work by others on isolated kinetoplast DNA are given. The double flagellum of Herpetomonas is seen by electron microscope to be two complete flagella in close contact but with no visible connection to each other.     

Conditioning odor-shock associations in the black blowfly,Phormia regina

Discriminative classical conditioning of an olfactory avoidance response was demonstrated in the blowfly, Phormia regina.Learning indices were calculated as the fraction of flies avoiding the CS+ (conditioned stimulus paired with electric shock reinforcement) minus the fraction of flies avoiding the CS-(conditioned stimulus not paired with electric shock), averaged over two different groups of flies, in which reciprocal odors were used as the CS+. Avoidance responses to both odors presented simultaneously at a T-maze choice point yielded mean learning indices of zero for naive flies or pseudoconditioned (shock alone) or sensitized (odors alone) controls. In contrast, pairing an odor with electric shock produced a mean learning index significantly greater than zero. These results are similar to those reported for Drosophila melanogaster.Blow flies, however, showed much lower levels of associative learning than fruit flies.     

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.     

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 o?cyte 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 o?cyte 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.     

Comparative real-time kinetic analysis of human complement killing of Leishmania infantum promastigotes derived from axenic culture or from Phlebotomus perniciosus

Although Leishmania metacyclic promastigotes are generally considered resistant to human complement, studies of in vitro-cultured axenic stationary promastigotes using serum concentrations that approximate physiological plasma conditions indicate complement sensitivity. Natural Leishmania infection is caused by sand fly-inoculated promastigotes, whose complement resistance has not been analyzed systematically. We compared Leishmania susceptibility to human complement in L. infantum promastigotes derived from in vitro cultures and from sand flies. Phlebotomus perniciosus sand flies were fed with axenic promastigotes, L. infantum-infected U-937 cells, or spleen cells from L. infantum-infected hamsters. On selected days post-feeding, flies were dissected and promastigotes isolated; in addition, axenic promastigotes were obtained from culture at equivalent days of growth. In near-physiological serum concentration and temperature conditions, measurement of real-time kinetics of propidium iodide uptake showed that 90% of axenic- and sand fly-derived promastigotes were rapidly killed by complement. We found no substantial differences between promastigotes from axenic culture, those isolated from flies on different post-feeding days, or those generated in flies fed with distinct inocula. The results indicate that Leishmania susceptibility to human complement is independent of promastigote developmental stage in the sand fly mid-gut and in axenic culture.     

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.     

Effects of substituted phenylethylamines on blowfly feeding behavior

The pharmacology of adult Phormia regina (Meigen) feeding behavior was explored by injecting candidate drugs into starved blowflies and then determining their responsiveness to aqueous sucrose, via the proboscis extension reflex. d-Amphetamine caused responsiveness to fall dramatically, while related drugs and biogenic amines had varying effects. When d-amphetamine-treated flies were fed 1 M sucrose, they consumed significantly more than control flies. Electrophysiological studies demonstrated that the responses of tarsal sugar receptor neurons to aqueous sucrose were not significantly altered by d-amphetamine. These observations are compatible with our hypothesis that octopamine positively modulates blowfly feeding behavior and suggest that other aromatic biogenic amines affect feeding behavior in this insect.     

Growth, Fine Structure and Cyst Formation of a Microbial Mat Ciliate: Pseudocohnilembus pusillus (Ciliophora, Scuticociliatida)

ABSTRACT Pseudocohnilembus species exhibit a polymorphic life cycle consisting of trophic cells, theronts, and cysts. Pseudocohnilembus pusillus isolated from the intertidal mats of Laguna Figueroa, Baja California Norte, Mexico, forms desiccation-resistant cysts in response to bacterial food depletion. This isolate is a euryhaline organism, able to grow at salinities from freshwater to 96 ppt total salinity and from pH 6–9. Electron micrographs show that oral and somatic cilia and kinetids are retained inside young cysts. Cyst walls are composed of a single layer (0.1 μm) of granular material. Under all conditions, as bacterial food was depleted, P. pusillus cells formed cysts, except for a small proportion (1–5%) that continued to swim. Changes in pH and salinity did not directly induce cyst formation. Salinity did greatly affect growth rate. Doubling times were shortest at 16 ppt salinity and at pH 7–8. Cyst formation occurred later in the growth cycle as more food bacteria were added. Additionally, ciliates grown in small culture volumes (10 ml) formed cysts sooner than cultures in larger volumes (100 ml), suggesting that crowding may influence cyst formation. Mature cysts may survive desiccation at least as long as one month at 37° C and for as long as one year at 20 ± 3° C. Although trophic cells did not survive desiccation or anoxia, encysted ciliates from liquid stationary phase cultures kept in anoxic seawater for one month excysted into swimming cells within 2.5 h after exposure to air. The adaptability of P. pusillus to extremes of salinity, pH, desiccation, and anoxia permits survival in its environmentally variable, microbial mat habitat.     

The fly factor phenomenon is mediated by interkingdom signaling between bacterial symbionts and their blow fly hosts

We tested the recent hypothesis that the"fly factor"phenomenon(food cur-rently or previously fed on by flies attracts more flies than the same type of food kept inccessible to flies)is mediated by bacterial symbionts deposited with feees or regur-gitated by feeding flies.We allowed laboratory-reared black blow flies,Phormia regina(Meigen),to feed and de fecate on bacterial Luria-Bertani medium solidified with agar,and isolated seven morphologically distinct bacterial colonies.We identified these us-ing matrix-assisted laser desorption/ionization mass spectrometry and sequencing of the 165 rRNA gene.In two-choice laboratory experiments,traps baited with cultures of Pro-teus mirabilis Hauser,Morganella morganii subsp.sibonii Jensen,or Serratia marcescens Bizio,captured significantly more flies than corresponding control jars baited with tryptic soy agar only.A mixture of seven bacterial strains as a trap bait was more attractive to flies than a single bacterial isolate(M.m.siboni).In a field experiment,traps baited with agar cultures of P:mirabilis and M.m siboni in combination captured significantly more flies than lraps baited with either bacterial isolate alone or the agar control.As evident by gas chromatography-mass spectrometry,the odor profiles of bacterial isolates differ,which may explain the additive effect of bacteria to the attractiveness of bacterial trap baits.As"generalist bacteria,"P mirabilis and M.m.sibonii growing on animal protein(beef liver)or plant protein(tofu)are similarly effective in attracting flies.Bacteria-derived airborne semiochemicals appear to mediate foraging by flies and to inform their feeding and oviposition decisions.     

Viability of sandflies (Diptera, Psychodidae, Phlebotominae) under the influence of infection with promastigotes of various species of Leishmania]

Sand flies were infected with different species of promastigotes from reptiles and warm-blooded animals. Optimal doses of promastigotes were used which ensured the adaptation of Protozoa in the host's intestine. The infection with a mixed culture resulted in the death of most Sand flies: the mortality rate was the highest at the simultaneous introduction of two species and was some what lower at the subsequent infection. The survival of Sand flies infected with one species of leishmania decreased to the greatest extent if "incidental" for them strains of promastigotes were introduced: for Ph. papatasi -- cultures isolated from reptiles, for Sergentomyia arpaklensis -- those isolated from L. tropica major. Natural infection rate of the genera Phlebotomus and Sergentomyia with leishmania of different species agrees with laboratory data on the survival of sand flies. Ph. caucasicus and Ph. papatasi are infected, in general, with L. tropica major, S. arpaklensis -- with L. gymnodactyli.     

Distant olfactory response of the onion fly, Delia antiqua, to host-plant odour in the field

ABSTRACT. Distant olfactory orientation of female adult Delia antiqua (Meigen) to the host-plant Allium volatile dipropyl-disulphide (DPDS) was examined in the field using mark-release-recapture experiments and observations of flight behaviour. Onion-reared, post-diapause, virgin females from a laboratory colony dispersed upwind when released in the centre of 25, 50 and 100 m radius circles of eight 50 μl UDPDS baits. Percentage recapture and dispersal directedness did not decrease as a function of increasing distance to baits. In all cases the mean flight direction of recaptured flies closely correlated with mean wind direction. However, modes of the circular distributions of recaptured flies were located further crosswind when odour-baits were more distant. When distance was held constant (25 m) and DPDS concentration serially reduced (500–0.05 μ/bait), flies dispersed randomly in the absence of DPDS, crosswind in response to 0.05 μl baits and upwind in response to all other baits. Percentage recaptures on DPDS-baited traps of all concentrations were significantly greater than unbaited traps. Results from markrecapture studies were corroborated by observations of flight behaviour downwind. Flies located 100 m downwind from 50 μl DPDS baits flew upwind on take off while take-off flights in the absence of DPDS were random. Our data indicate that Allium volatiles like DPDS are involved not only in the acceptance phase of host-selection, but also in the first and probably most important stage when onion flies are initiating search long distances downwind. We conclude that D. antiqua orients to host-plants using olfactory cues from distances that should be classified as long-range ( sensu Kennedy , 1977     

Morphology of the first instar of Calliphora vicina, Phormia regina and Lucilia illustris (Diptera, Calliphoridae)

Scanning electron microscopy documentation of first instar Calliphora vicina Robineau-Desvoidy, Phormia regina (Meigen) and Lucilia illustris (Meigen) (Diptera: Calliphoridae) is presented for the first time, and the following morphological structures are documented: pseudocephalon; antenna; maxillary palpus; facial mask; labial lobe; thoracic and abdominal spinulation; spiracular field; posterior spiracles, and anal pad. Light microscopy documentation and illustrations are provided for the cephaloskeleton in lateral and ventral views. New diagnostic features are revealed in the configuration of the facial mask, cephaloskeleton and posterior spiracles. The first instar morphology of C. vicina, Ph. regina and L. illustris is discussed in the light of existing knowledge about early instars of blowflies.     

Patterns of selective protein ingestion by the blowfly Phormia regina

ABSTRACT. The patterns of protein ingestion by Phormia regina were reexamined with an apparatus more sensitive than that used in Dethier's (1961) original study. During the first vitellogenic cycle, females exhibited two peaks of protein consumption if given access to protein from the time of emergence. The first occurred during days 1–2 post-eclosion; the second during days 3–5, when oocytes were developing. Once the eggs were mature, protein ingestion remained depressed until oviposition; it peaked again for 1–2 days following each oviposition. In the male fly only the first peak of elevated protein ingestion occurred; thereafter, protein ingestion remained low. For both sexes, protein-deprivation, enforced since eclosion, intensified initial protein meals, which thereafter tailed off rapidly.