Dopamine Dynamics and Signaling in Drosophila: An Overview of Genes,Drugs and Behavioral Paradigms

Changes in dopamine (DA) signaling have been implicated in a number of human neurologic and psychiatric disorders. Similarly, defects in DA signaling in the fruit fly, Drosophila melanogaster, have also been associated with several behavioral defects. As most genes involved in DA synthesis, transport, secretion, and signaling are conserved between species, Drosophila is a powerful genetic model organism to study the regulation of DA signaling in vivo. In this review, we will provide an overview of the genes and drugs that regulate DA biology in Drosophila. Furthermore, we will discuss the behavioral paradigms that are regulated by DA signaling in flies. By analyzing the genes and neuronal circuits that govern such behaviors using sophisticated genetic, pharmacologic, electrophysiologic, and imaging approaches in Drosophila, we will likely gain a better understanding about how this neuromodulator regulates motor tasks and cognition in humans.     

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.     

Gut bacterial communities in the Mediterranean fruit fly (Ceratitis capitata) and their impact on host longevity

Fruit flies (Diptera: Tephritidae) harbor stable bacterial communities in their digestive system, composed mainly of members of the Enterobacteriaceae. However, the Enterobacteriaceae are not the sole community in this habitat. We examined the hypothesis that Pseudomonas spp. form a cryptic community in the gut of Ceratitis capitata, the Mediterranean fruit fly (‘medfly’). Suicide polymerase restriction PCR (SuPER PCR), a novel culture-independent technique, revealed that Pseudomonas spp. form minor, common and stable communities within the medfly's gut. These include P. aeruginosa, a known pathogen of arthropods. Experimental inoculations with high levels of P. aeruginosa reduced the medfly's longevity while inoculations with members of the Enterobacteriaceae extended the fly's life.Accordingly, we suggest that in addition to their possible contribution to the fly's nitrogen and carbon metabolism, development and copulatory success (as shown in previous studies), the Enterobacteriaceae community within the medfly's gut may also have an indirect contribution to host fitness by preventing the establishment or proliferation of pathogenic bacteria.     

Expression of dengue virus NS3 protein in Drosophila alters its susceptibility to infection

We developed a Drosophila model in which the dengue virus NS3 protein is expressed in a tissue specific and inducible manner. Dengue virus NS3 is a multifunctional protein playing a major role during viral replication. Both protease and helicase domains of NS3 are interacting with human and insect host proteins including innate immune components of the host machinery. We characterized the NS3 transgenic flies showing that NS3 expression did not affect fly development. To further study the links between NS3 and the innate immune response, we challenge the flies with gram-positive and gram-negative bacteria. Interestingly, the Drosophila transgenic flies expressing NS3 were more susceptible to bacterial infections than control flies. However ubiquitous or immune-specific NS3 expression affected neither the life span nor the response to a non-infectious stress of the flies. In conclusion, we generated a new in vivo system to study the functional impact of DENV NS3 protein on the innate immune response.     

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.     

The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis

The gut microorganisms in some animals are reported to include a core microbiota of consistently associated bacteria that is ecologically distinctive and may have coevolved with the host. The core microbiota is promoted by positive interactions among bacteria, favoring shared persistence; its retention over evolutionary timescales is evident as congruence between host phylogeny and bacterial community composition. This study applied multiple analyses to investigate variation in the composition of gut microbiota in drosophilid flies. First, the prevalence of five previously described gut bacteria (Acetobacter and Lactobacillus species) in individual flies of 21 strains (10 Drosophila species) were determined. Most bacteria were not present in all individuals of most strains, and bacterial species pairs co-occurred in individual flies less frequently than predicted by chance, contrary to expectations of a core microbiota. A complementary pyrosequencing analysis of 16S rRNA gene amplicons from the gut microbiota of 11 Drosophila species identified 209 bacterial operational taxonomic units (OTUs), with near-saturating sampling of sequences, but none of the OTUs was common to all host species. Furthermore, in both of two independent sets of Drosophila species, the gut bacterial community composition was not congruent with host phylogeny. The final analysis identified no common OTUs across three wild and four laboratory samples of D. melanogaster. Our results yielded no consistent evidence for a core microbiota in Drosophila. We conclude that the taxonomic composition of gut microbiota varies widely within and among Drosophila populations and species. This is reminiscent of the patterns of bacterial composition in guts of some other animals, including humans.     

Feeding of the Nematode Acrobeloides nanus on Bacteria

Information on the effect of bacteria-feeding nematodes on bacterial populations in the soil is sparse. We have isolated, cultured, and microscopically examined bacteria and nematodes coexisting within an agricultural soil and have studied their feeding relationship. The bacterium Pseudomonas corrugata isolate 2140R is a biocontrol agent against the pathogenic fungus Gaeumannomyces graminis var. tritici. The nematode Acrobeloides nanus is a cosmopolitan, bacteria-feeding organism widespread in agricultural and arid soils throughout Australia. Using light and electron microscopy, we observed the ingestion and breakdown of P. corrugata in the pharynx of A. nanus and bacterial passage through the nematode intestine as well as the accumulation of fluorescent compounds from ingested and broken P. fluorescens in the lumen of the nematode''s intestine. We also observed A. nanus feeding, growing, and reproducing on the Gram-positive bacterium Clavibacter toxicus, the causative agent of the disease annual ryegrass toxicity, and detected crushed bacteria in the nematode''s intestine.     

The host range of the male-killing symbiont Arsenophonus nasoniae in filth fly parasitioids

The Son-killer bacterium, Arsenophonus nasoniae, infects Nasonia vitripennis (Hymenoptera: Pteromalidae), a parasitic wasp that attacks filth flies. This gammaproteobacterium kills a substantial amount of male embryos produced by an infected female. Aside from male death, the bacterium does not measurably affect the host, and how it is maintained in the host population is unknown. Interestingly, this bacterial symbiont can be transmitted both vertically (from mother to offspring) and horizontally (to unrelated Nasonia wasps developing in the same fly host). This latter mode may allow the bacterium to spread throughout the ecological community of filth flies and their parasitoids, and to colonize novel species, as well as permit its long-term persistence.We tested 11 species of filth flies and 25 species of their associated parasitoids (representing 28 populations from 16 countries) using diagnostic PCR to assess the bacterium’s actual host range. In addition to 16S rRNA, two loci were targeted: the housekeeping gene infB, and a sequence with high homology to a DNA polymerase gene from a lysogenic phage previously identified from other insect symbionts. We identified infections of A. nasoniae in four species of parasitoids, representing three taxonomic families. Highly similar phage sequences were also identified in three of the four species. These results identify the symbiont as a generalist, rather than a specialist restricted solely to species of Nasonia, and also that horizontal transmission may play an important role in its maintenance.     

Bacterial farming by the fungus Morchella crassipes

The interactions between bacteria and fungi, the main actors of the soil microbiome, remain poorly studied. Here, we show that the saprotrophic and ectomycorrhizal soil fungus Morchella crassipes acts as a bacterial farmer of Pseudomonas putida, which serves as a model soil bacterium. Farming by M. crassipes consists of bacterial dispersal, bacterial rearing with fungal exudates, as well as harvesting and translocation of bacterial carbon. The different phases were confirmed experimentally using cell counting and ¹³C probing. Common criteria met by other non-human farming systems are also valid for M. crassipes farming, including habitual planting, cultivation and harvesting. Specific traits include delocalization of food production and consumption and separation of roles in the colony (source versus sink areas), which are also found in human agriculture. Our study evidences a hitherto unknown mutualistic association in which bacteria gain through dispersal and rearing, while the fungus gains through the harvesting of an additional carbon source and increased stress resistance of the mycelium. This type of interaction between fungi and bacteria may play a key role in soils.     

Persistence of antibiotic-resistant and -sensitive Proteus mirabilis strains in the digestive tract of the housefly (Musca domestica) and green bottle flies (Calliphoridae)

Synanthropic flies have been implicated in the rapid dissemination of antibiotic-resistant bacteria and resistance determinants in the biosphere. These flies stably harbor a considerable number of bacteria that exhibit resistance to various antibiotics, but the mechanisms underlying this phenomenon remain unclear. In this study, we investigated the persistence of antibiotic-resistant bacteria in the digestive tract of houseflies and green bottle flies, using Proteus mirabilis as a model microorganism. One resistant strain carried the blaTEM and aphA1 genes, and another carried a plasmid containing qnrD gene. Quantitative PCR and 454 pyrosequencing were used to monitor the relative abundance of the Proteus strains, as well as potential changes in the overall structure of the whole bacterial community incurred by the artificial induction of Proteus cultures. Both antibiotic-resistant and -sensitive P. mirabilis strains persisted in the fly digestive tract for at least 3 days, and there was no significant difference in the relative abundance of resistant and sensitive strains despite the lower growth rate of resistant strains when cultured in vitro. Therefore, conditions in the fly digestive tract may allow resistant strains to survive the competition with sensitive strains in the absence of antibiotic selective pressure. The composition of the fly-associated bacterial community changed over time, but the contribution of the artificially introduced P. mirabilis strains to these changes was not clear. In order to explain these changes, it will be necessary to obtain more information about bacterial interspecies antagonism in the fly digestive tract.     

Monitoring long-term evolutionary changes following Wolbachia introduction into a novel host: the Wolbachia popcorn infection in Drosophila simulans

Wolbachia may act as a biological control agent for pest management; in particular, the Wolbachia variant wMelPop (popcorn) shortens host longevity and may be useful for dengue suppression. However, long-term changes in the host and Wolbachia genomes can alter Wolbachia spread and/or host effects that suppress disease. Here, we investigate the phenotypic effects of wMelPop in a non-native host, Drosophila simulans, following artificial transinfection approximately 200 generations ago. Long-term rearing and maintenance of the bacteria were at 19°C in the original I-102 genetic background that was transinfected with the popcorn strain. The bacteria were then introgressed into three massbred backgrounds, and tetracycline was used to create uninfected sublines. The effect of wMelPop on longevity in this species appears to have changed; longevity was no longer reduced at 25°C in some nuclear backgrounds, reflecting different geographical origin, selection or drift, although the reduction was still evident for flies held at 30°C. Wolbachia influenced productivity and viability, and development time in some host backgrounds. These findings suggest that long-term attenuation of Wolbachia effects may compromise the effectiveness of this bacterium in pest control. They also emphasize the importance of host nuclear background on Wolbachia phenotypic effects.     

A strain of the bacterial symbiont Regiella insecticola protects aphids against parasitoids

Aphids commonly harbour facultative bacterial endosymbionts and may benefit from their presence through increased resistance to parasitoids. This has been demonstrated for Hamiltonella defensa and Serratia symbiotica, while a third common endosymbiont, Regiella insecticola, did not provide such protection. However, this symbiont was recently detected in a highly resistant clone of the peach-potato aphid, Myzus persicae, from Australia. To test if resistance was indeed conferred by the endosymbiont, we eliminated it from this clone with antibiotics, and we transferred it to two other clones of the same and one clone of a different aphid species (Aphis fabae). Exposing these lines to the parasitoid Aphidius colemani showed clearly that unlike other strains of this bacterium, this specific isolate of R. insecticola provides strong protection against parasitic wasps, suggesting that the ability to protect their host against natural enemies may evolve readily in multiple species of endosymbiotic bacteria.     

Signaling-mediated cross-talk modulates swarming and biofilm formation in a coral pathogen Serratia marcescens

Interactions within microbial communities associated with marine holobionts contribute importantly to the health of these symbiotic organisms formed by invertebrates, dinoflagellates and bacteria. However, mechanisms that control invertebrate-associated microbiota are not yet fully understood. Hydrophobic compounds that were isolated from surfaces of asymptomatic corals inhibited biofilm formation by the white pox pathogen Serratia marcescens PDL100, indicating that signals capable of affecting the associated microbiota are produced in situ. However, neither the origin nor structures of these signals are currently known. A functional survey of bacteria recovered from coral mucus and from cultures of the dinoflagellate Symbiodinium spp. revealed that they could alter swarming and biofilm formation in S. marcescens. As swarming and biofilm formation are inversely regulated, the ability of some native α-proteobacteria to affect both behaviors suggests that the α-proteobacterial signal(s) target a global regulatory switch controlling the behaviors in the pathogen. Isolates of Marinobacter sp. inhibited both biofilm formation and swarming in S. marcescens PDL100, without affecting growth of the coral pathogen, indicative of the production of multiple inhibitors, likely targeting lower level regulatory genes or functions. A multi-species cocktail containing these strains inhibited progression of a disease caused by S. marcescens in a model polyp Aiptasia pallida. An α-proteobacterial isolate 44B9 had a similar effect. Even though ∼4% of native holobiont-associated bacteria produced compounds capable of triggering responses in well-characterized N-acyl homoserine lactone (AHL) biosensors, there was no strong correlation between the production of AHL-like signals and disruption of biofilms or swarming in S. marcescens.     

Use of Arabidopsis thaliana and Pseudomonas syringae in the Study of Plant Disease Resistance and Tolerance

The interaction between Arabidopsis thaliana and the bacterium Pseudomonas syringae is being developed as a model experimental system for plant pathology research. Race-specific ("gene-for-gene") resistance has been demonstrated for this interaction, and pathogen genes that determine avirulence have been isolated and characterized. Because certain lines of both Arabidopsis and soybean are resistant to bacteria carrying the avirulence genes avrRpt2 and avrB, extremely similar pathogen recognition mechanisms are apparently present in these two plant species. Isogenic bacterial strains that differ by the presence of single avirulence genes are being used to analyze plant resistance. Plant resistance genes have been identified in crosses between resistant and susceptible lines. The extensive map-based cloning tools available in Arabidopsis are being used to isolate these resistance genes. In a related project, ethylene-insensitive Arabidopsis mutants are being used to examine the role of ethylene in disease development. Ethylene apparently mediates symptom formation in susceptible plants and is not required for resistance, suggesting possible strategies for enhancement of disease tolerance in crops.     

Tylenchulus semipenetrans Alters the Microbial Community in the Citrus Rhizosphere

Infection of citrus seedlings by Tylenchulus semipenetrans was shown to reduce subsequent infection of roots by Phytophthora nicotianae and to increase plant growth compared to plants infected by only the fungus. Hypothetical mechanisms by which the nematode suppresses fungal development include nutrient competition, direct antibiosis, or alteration of the microbial community in the rhizosphere to favor microorganisms antagonistic to P. nicotianae. A test of the last hypothesis was conducted via surveys of five sites in each of three citrus orchards infested with both organisms. A total of 180 2-cm-long fibrous root segments, half with a female T. semipenetrans egg mass on the root surface and half without, were obtained from each orchard site. The samples were macerated in water, and fungi and bacteria in the suspensions were isolated, quantified, and identified. No differences were detected in the numbers of microorganism species isolated from nematode-infected and uninfected root segments. However, nematode-infected root segments had significantly more propagules of bacteria at all orchard sites. Bacillus megaterium and Burkholderia cepacia were the dominant bacterial species recovered. Bacteria belonging to the genera Arthrobacter and Stenotrophomonas were encountered less frequently. The fungus community was dominated by Fusarium solani, but Trichoderma, Verticillum, Phythophthora, and Penicillium spp. also were recovered. All isolated bacteria equally inhibited the growth of P. nicotianae in vitro. Experiments using selected bacteria, T. semipenetrans, and P. nicotianae, alone or in combination, were conducted in both the laboratory and greenhouse. Root and stem fresh weights of P. nicotianae-infected plants treated with T. semipenetrans, B. cepacia, or B. megaterium were greater than for plants treated only with the fungus. Phytophthora nicotianae protein in roots of fungus-infected plants was reduced by nematodes (P ≤ 0.001), either alone or in combination with either bacterium. However, treatment with bacteria did not affect P. nicotianae development in roots. The results suggest different mechanisms by which T. semipenetrans, B. cepacia, and B. megaterium may mitigate virulence of P. nicotianae.     

Adverse effects of members of the Enterobacteriaceae family on boar sperm quality

Semen samples collected in 2012 from 1785 boars belonging to five different breeds were recruited from the quality control laboratory of Magapor SL, Spain. These samples came from 43 boar studs and resulted from diluting the ejaculates in commercial semen extenders. Evaluation of the semen sample characteristics (color, smell, pH, osmolality, concentration, motility of sperm cells, agglutination, acrosome integrity, short hypoosmotic swelling test, and abnormal forms) revealed that they met the international standards. The samples were also tested for the presence of aerobic bacterial contamination. In the present study, 14.73% (n = 263) of the semen samples were contaminated above 3 × 10² colony-forming units/mL with at least one type of bacteria. The Enterobacteriaceae family was by far the major contaminant, being present in 40.68% of the contaminated samples (n = 107). Bacterial strains of the Enterobacteriaceae family isolated from boar semen samples were in order of incidence (percentage of the contaminated samples): Serratia marcescens (12.55%), Klebsiella oxytoca (11.79%), Providencia stuartii (9.12%), Morganella morganii (3.80%), Proteus mirabilis (1.90%), and Escherichia coli (1.52%). We have seen that the presence in semen samples of S. marcescens, K. oxytoca, M. morganii, or P. mirabilis, but not P. stuartii or E. coli, was negatively associated with sperm motility (P < 0.05). The mean sperm concentration (P < 0.05), the mean percentage of spermatozoa with curled tails after the short hypoosmotic swelling test (P < 0.01), and the incidence of morphologically normal acrosomes (P < 0.05) were also lower in semen samples infected with M. morganii compared with uninfected ones. Moreover, P. mirabilis was negatively associated with the presence of abnormal forms. Thus, on the basis of the pathological effects that some of these strains may have on boar sperm quality, bacterial contamination should always be examined in semen samples prepared for artificial insemination.     

Swarming Characteristics of Proteus mirabilis Under Anaerobic and Aerobic Conditions

Nutrients have a pronounced effect on the growth and swarming behaviour of Proteus mirabilis 7002. Iron, zinc, amino acids, and dioxygen are important for rapid growth and normal swarming. Anaerobically grown cultures of P. mirabilis 7002 were unable to swarm on anaerobically maintained rich nutrient agar. Upon exposure to aerobic conditions, P. mirabilis 7002 resumed swarming behaviour. Scanning electron microscopy was used to demonstrate the presence of community organization and mature rafts during normal swarming. These results support the importance of dioxygen and redox status in cell differentiation.