Seasonal Prevalence ofEntomophthora muscaeand Introduction ofEntomophthora schizophorae(Zygomycotina: Entomophthorales) inMusca domestica(Diptera: Muscidae) Populations on California Dairies

Two entomopathogenic fungi in theEntomophthora muscaespecies complex that infect house flies were used in this study:E. muscaeFresenius (16–17 nuclei/conidium) occurred naturally at four southern California dairies, whileEntomophthora schizophoraeKeller and Wilding (4–8 nuclei/conidium) did not. During the first year of the study, onset of measurableE. muscaeinfections occurred between September and November but varied among sites. At least 20% of the flies at all four dairies were infected by November, and infection at one site exceeded 70%. During the fall epizootic period, infection levels were inversely related to temperature. Average weekly temperatures higher than 17–20°C and maximum daily temperatures higher than 26–28°C were statistically correlated with low infection levels. In the second year,E. schizophoraewas introduced by releasing diseased flies at two dairies (four times at one dairy and three times at the other).E. schizophoraewas recovered for a brief time in the house fly population after the first two releases at one site but not at the second site.     

Plant host relationships of three lineages of the gall-inducing fly <Emphasis Type="Italic">Fergusonina</Emphasis> Malloch (Diptera: Fergusoninidae) on <Emphasis Type="Italic">Eucalyptus</Emphasis> L’Hérit.

The gall-inducing fly family Fergusoninidae, in association with a mutualist nematode, induces galls on Myrtaceae. Each fly species typically targets a particular site on its host plant, giving rise to a range of gall types, and one plant species may host at least four fly species. While incongruent fly–host evolutionary time scales preclude early cospeciation, it is possible that Fergusoninidae have been diverging with their host plants more recently at correspondingly finer taxonomic levels, such as within host subgenera. To test this possibility, we reduced the scale of our analysis and focussed on a clade of ten Eucalyptus species, sampling intensively and using a phylogenetic approach to compare the relationships between these plant hosts and their associated flies. We also took advantage of the fact that three different gall types, each with its own clade of Fergusonina flies, could be sampled on this focal host clade, in effect giving us three different host/fly association tests on the one set of hosts. The phylogenies of flies from the three different gall types were estimated using Bayesian analysis of mtCOI sequences and compared with an existing phylogeny of the eucalypt host clade. While each gall type showed a different pattern of host relationships, heuristic and quantitative analyses showed that there was little correspondence between plant and fly phylogenies and we conclude that host switching is prevalent in this system. There was more host fidelity in the flower bud gallers on this group of eucalypts, and there was least in the leaf blade gallers, with the shoot bud gallers demonstrating an intermediate level of host fidelity. We discuss the possible factors which may have led to their patterns of host association. This is the first study of Fergusonina to focus on one clade of Eucalyptus L’Hérit. (Myrtaceae) with intensive sampling and shows that each host plant species is commonly used by multiple fergusoninid species. This has provided us with the opportunity to study in detail the host relationships of three separate clades of Fergusonina from different plant tissue types, and has revealed many previously unrecorded host plant/gall site associations.     

The occurrence of Entomophthorales on the carrot fly [Psila rosae F.] in the field during two successive seasons

Three species of Entomophthorales were found on adult carrot flies (Psila rosae F.) during two successive seasons:Entomophthora muscae (C.) Fres.,Condiobolus apiculatus (Thax.) Remaud. & Keller andErynia sp.E. muscae was by far the most common species and caused epizootics in one carrot fly population up to 3 times per year. Flies cuaght in the hedge showed a higher infection level than flies caught in the field close to the hedge. Flies caught 200 m away from the hedge showed the lowest infection level. Apparently the hedge was the site of infection, since carrot flies, killed byE. muscae, were found there attached to the underside of the leaves. Flies caught on yellow sticky traps developed only to a limited extend symptoms and gave little information about the fungus infection levels.     

Aleochara suffusa andA. bilineata (Col.: Staphylinidae) as parasitoids of brassica root flies in northern Norway

A sample ofDelia puparia collected in late autumn from a brassica field at Tromsø, northern Norway, was investigated to study the level of parasitism byAleochara. BothA. suffusa andA. bilineata were reared from puparia of the cabbage root fly,Delia radicum, and the bean seed flies,D. florilega and/orD. platura. Only two specimens ofA. bilineata emerged from puparia of the turnip root fly,D. floralis. BothAleochara species hibernated in the larval state and both pupated inside the host puparium. Most specimens ofA. suffusa emerged from small hosts (D. florilega/D. platura), whereas the majority of A.bilineata emerged from host species of larger size (D. radicum/D. floralis). The time to develop from first instar larva to adult was similar for bothA. suffusa andA. bilineata. Parasitoids developing in large hosts emerged later than those in small hosts, the delay being the same for both species ofAleochara.     

Abnormal egg-laying behaviour of female carrot flies (Psila rosae) induced by the fungus Entomophthora muscae

Female carrot flies (Psila rosae F.) infected with Entomophthora muscae (C.) Fres. on days 1 to 4 of adult life were unable to lay their eggs near the food plant. Flies infected on day 5 of adult life showed an egg-laying behaviour similar to that of uninfected flies. E. muscae-infected female carrot flies caught in the field showed an egg-laying behaviour comparable to that of the flies infected in the laboratory during the first day of their live.Eggs laid by infected females were equally as fertile as eggs laid by uninfected females, and developed into normal flies.It is concluded that female carrot flies infected with E. muscae in the field do not in general contribute to the development of the carrot fly population, due to their abnormal egg-laying behaviour.

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Zusammenfassung Wenn Weibchen der echten Möhrenfliegen (Psila rosae) zwischen den ersten und der vierten Tag in dem Laboratorium mit E. muscae infiziert worden waren, könnten sie ihre Eier an den Futterpflanze nicht ablegen. E. muscae-infizierte Weibchen, die ins Feld gefangen worden waren, zeigten auch ein abweichendes Eiablageverhalten. Ihr Behalten war mit dem Verhalten von Fliegen vergleichbar, die an ihrem ersten Lebenstag im Laboratorium infiziert worden waren.Von infizierten Weibchen gelegte Eier waren ebenso fruchtbar wie die von nicht infizierten Weibchen gelegten Eiern. Es wird konkludiert, dass E. muscae-infizierten Weibchen der echten Möhrenfliegen der Entwicklung der Population keinen Beitrag leisten.

     

Laboratory transmission and in vivo incubation of Entomophthora muscae (Entomophthorales: Entomophthoracae) in the onion fly,Delia antiqua (Diptera: Anthomyiidae)

A high-moisture infection chamber was used for the in vivo transmission of Entomophthora muscae within laboratory populations of the onion fly, Delia antiqua. This cadaver-to-fly transmission procedure provided an average experimental infection rate well above 95%. Laboratory infection and temperature-dependent incubation rates of E. muscae were further examined in adult populations of D. antiqua. The time from initial exposure until host death and pathogen sporulation was accurately predicted using a second-order function of the incubation temperature. A developmental base temperature of approximately 5°C was estimated, with 105 degree-days being the average number of heat units required between host infection and death. E. muscae transmission between D. antiqua and D. platura, two insect pests typically associated with Michigan onion production, was verified under laboratory conditions.     

Logistic growth of the host‐specific obligate insect pathogenic fungus Entomophthora muscae in house flies (Musca domestica)

Insect pathogenic fungi (IPF) need to overcome the host immune system in order to sporulate and ensure transmission to new hosts. Some IPF produce immunosuppressive toxins, whereas others rely on rapid fungal proliferation to kill the host by sheer fungal mass, resulting in a trade‐off between allocating resources to toxin production and fungal proliferation. The obligate entomopathogenic fungus, Entomophthora muscae sensu stricto, is host specific to the common house fly, Musca domestica. E. muscae grows as protoplast cells without cell walls and is not known to produce toxins. Here, we assessed the growth of E. muscae, in vivo, using real‐time PCR to measure the amount of a single‐copy actin gene. We find that E. muscae exhibits S‐shaped logistic growth between time post‐exposure and the number of fungal nuclei. The results show that E. muscae initially grows exponentially inside the host until depletion of available nutrient sources signifies the ‘limiting capacity’ where after the host is killed. This growth pattern differs markedly from toxin‐producing IPF species of Metarhizium and Beauveria in which maximal (plateau) growth and sporulation do not occur until well after the death of the host.     

Parasitism of Heterotylenchus autumnalis Nickle (Nematoda: Sphaerulariidae) to the Face Fly,Musca autumnalis De Geer (Diptera: Muscidae)

New information is reported on the parasitism of Heterotylenchus autumnalis upon its principal known host, Musca autumnalis. Black to brown spots are produced on the cuticle of all infected host larvae where the nematode penetrated. The principal damage to the host is castration of the female. In laboratory tests nematode larvae were not infective and did not leave the hosts before the female fly was 1 1 days old. Nematode larvae removed from infected male flies infected other hosts, but it is believed that in nature these larvae are unable to leave the host.     

Field Studies of Entomophthora (Zygomycetes: Entomophthorales)—Induced Behavioral Fever in Musca domestica (Diptera: Muscidae) in Denmark

House flies were collected over 3 days (three to five times per day) from specific sites on a dairy farm with a range of high to low temperatures. Flies were held individually to determine whether the distribution of fungus-infected (Entomophthora muscae and E. schizophorae) house flies differed according to the stage of infection and temperature. All but 2 of 396 infected flies (99.5%) had E. muscae. More E. muscae-infected flies collected from cool areas were in later stages of infection (i.e., dying 0–2 days after capture), whereas flies collected on sun-exposed surfaces tended to be in earlier stages of infection (i.e., dying 6–8 days after capture). Most flies died 3–5 days after capture and were consequently in the middle stages of infection. A mark and release experiment was conducted to determine whether E. schizophorae-inoculated flies frequented surfaces with higher temperatures than did uninfected control flies. About 3000 yellow-marked house flies inoculated with E. schizophorae and 3000 blue-marked control flies were released in an enclosed swine farrowing barn. Significantly more inoculated flies were recorded on the heat lamps than flies in the control group. The results suggest that behavioral fever occurs in the field for flies infected with both E. muscae and E. schizophorae and that flies can cure themselves of infection through the use of artificial heat sources.     

Infection with Haemoproteus iwa affects vector movement in a hippoboscid fly—frigatebird system

Haemosporidian parasites, which require both a vertebrate and invertebrate host, are most commonly studied in the life stages occurring in the vertebrate. However, aspects of the vector's behaviour and biology can have profound effects on parasite dynamics. We explored the effects of a haemosporidian parasite, Haemoproteus iwa, on a hippoboscid fly vector, Olfersia spinifera. Olfersia spinifera is an obligate ectoparasite of the great frigatebird, Fregata minor, living among bird feathers for all of its adult life. This study examined the movements of O. spinifera between great frigatebird hosts. Movement, or host switching, was inferred by identifying host (frigatebird) microsatellite genotypes from fly bloodmeals that did not match the host from which the fly was collected. Such host switches were analysed using a logistic regression model, and the best‐fit model included the H. iwa infection status of the fly and the bird host sex. Uninfected flies were more likely to have a bird genotype in their bloodmeal that was different from their current host's genotype (i.e. to have switched hosts) than infected flies. Flies collected from female birds were more likely to have switched hosts than those collected on males. Reduced movement of infected flies suggests that there may be a cost of parasitism for the fly. The effect of host sex is probably driven by differences in the sex ratio of bird hosts available to moving flies.     

The culture ofEntomophthora muscae (C) Fres. in carrot flies (Psila rosae F.) and the effect of temperature on the pathology of the fungus

A method for maintaining anin vivo culture ofEntomophthora muscae (C) Fres. on its original host, adult carrot flies (Psila rosae F.), is described. The lethal time for adult carrot flies was greatly influenced by temperature, both for infected and for uninfected flies. In the range 8.2°C–20.2°C the LT₅₀ for infected flies was about 5.4 times shorter than the estimated average life-span for uninfected flies. The discharge of primary spores was also strongly dependent on temperature. The total number of primary spores discharged per fly at 100% RH and in darkness ranged between 1.2×10⁴ and 9.6×10⁴ with a mean of 5.1×10⁴.      

Female-derived sex pheromone mediates courtship behaviour in the parasitoid Lariophagus distinguendus

Brassiceye^®traps baited with ethylisothiocyanate were modified and used to collect live adults of Delia radicum(L.) and Delia floralis(Fallén) (Diptera: Anthomyiidae) from the field to observe the prevalence of Entomophthora muscae(Cohn) Fresenius and Strongwellsea castransBatko & Weiser. The traps were highly effective and selective for D. radicumand D. floralis. Of the flies identified, 98.4% in 1996 and 93.7% in 1997 were either D. radicumor D. floralis. In 1997 the maximum mean catch was as high as 82 flies per trap per day, and more than 80% of these were females.During both seasons E. muscaecaused relatively high levels of mortality in adult populations of D. radicumand D. floralis. The fungus caused a total infection level of 17.9% in 1996 and 47.7% in 1997 with infection peaks of 82.4% in 1996 and 87.5% in 1997. Both years, a significant positive correlation was found between E. muscaeprevalence and temperature. One infection peak was observed for S. castransin 1996, and during that season the total S. castransinfection level was 18.0%. In 1997, the total S. castransinfection level was as low as 8.1%. There is no strong indication that the prevalence of E. muscaeor S. castransdiffers between either the fly species or sexes within species.     

Some Like It Hot: Evolution and Ecology of Novel Endosymbionts in Bat Flies of Cave-Roosting Bats (Hippoboscoidea,Nycterophiliinae)

We investigated previously unknown associations between bacterial endosymbionts and bat flies of the subfamily Nycterophiliinae (Diptera, Streblidae). Molecular analyses revealed a novel clade of Gammaproteobacteria in Nycterophilia bat flies. This clade was not closely related to Arsenophonus-like microbes found in its sister genus Phalconomus and other bat flies. High population infection rates in Nycterophilia across a wide geographic area, the presence of the symbionts in pupae, the general codivergence between hosts and symbionts, and high AT composition bias in symbiont genes together suggest that this host-symbiont association is obligate in nature and ancient in origin. Some Nycterophilia samples (14.8%) also contained Wolbachia supergroup F (Alphaproteobacteria), suggesting a facultative symbiosis. Likelihood-based ancestral character mapping revealed that, initially, obligate symbionts exhibited association with host-specific Nycterophilia bat flies that use a broad temperature range of cave environments for pupal development. As this mutualism evolved, the temperature range of bat flies narrowed to an exclusive use of hot caves, which was followed by a secondary broadening of the bat flies'' host associations. These results suggest that the symbiosis has influenced the environmental tolerance of parasite life history stages. Furthermore, the contingent change to an expanded host range of Nycterophilia bat flies upon narrowing the ecological niche of their developmental stages suggests that altered environmental tolerance across life history stages may be a crucial factor in shaping parasite-host relationships.     

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies.     

The Bacteriome of Bat Flies (Nycteribiidae) from the Malagasy Region: a Community Shaped by Host Ecology,Bacterial Transmission Mode,and Host-Vector Specificity

The Nycteribiidae are obligate blood-sucking Diptera (Hippoboscoidea) flies that parasitize bats. Depending on species, these wingless flies exhibit either high specialism or generalism toward their hosts, which may in turn have important consequences in terms of their associated microbial community structure. Bats have been hypothesized to be reservoirs of numerous infectious agents, some of which have recently emerged in human populations. Thus, bat flies may be important in the epidemiology and transmission of some of these bat-borne infectious diseases, acting either directly as arthropod vectors or indirectly by shaping pathogen communities among bat populations. In addition, bat flies commonly have associations with heritable bacterial endosymbionts that inhabit insect cells and depend on maternal transmission through egg cytoplasm to ensure their transmission. Some of these heritable bacteria are likely obligate mutualists required to support bat fly development, but others are facultative symbionts with unknown effects. Here, we present bacterial community profiles that were obtained from seven bat fly species, representing five genera, parasitizing bats from the Malagasy region. The observed bacterial diversity includes Rickettsia, Wolbachia, and several Arsenophonus-like organisms, as well as other members of the Enterobacteriales and a widespread association of Bartonella bacteria from bat flies of all five genera. Using the well-described host specificity of these flies and data on community structure from selected bacterial taxa with either vertical or horizontal transmission, we show that host/vector specificity and transmission mode are important drivers of bacterial community structure.     

Culture of Entomophthora muscae in vivo and its infectivity for six species of muscoid flies

A method for the continuous culture of Entomophthora muscae (Entomophthoraceae) in adult house flies (Musca domestica) is described. Using this method we have maintained the host-pathogen system in the laboratory for more than one year. The ability of this isolate to cause fatal infections in virtually all of the house flies at risk has remained constant. The face fly (Musca autumnalis), the onion fly (Hylemya antiqua), and the seed-corn fly (H. platura) are susceptible to this isolate. The stable fly (Stomoxys calcitrans), the black blow fly (Phormia regina), and the false stable fly (Muscina stabulans) are not.     

Comparative study of the antennal sensilla of five species of root maggots: Delia radicum L., D. floralis F., D. antiqua Mg., D. platura MG. (Diptera : Anthomyiidae) and Psila rosae F. (Diptera : Psilidae)

A comparative study of the antennal sensilla of Delia radicum L., D. floralis F., D. antiqua Mg., D. platura Mg. (Diptera : Anthomyiidae) and Psila rosae F. Diptera Psilidae) is undertaken. For both sexes of each species, the type, distribution, and density of sensilla are determined. All 5 species have trichoid (olfactory) and grooved (olfactory) sensilla. Basiconica I (blunt) sensilla (olfactory) are found on each of the species examined, except D. platura. Basiconica II (tapered) (olfactory) and clavate (olfactory) sensilla are found only on Delia species. Also, only Delia species have single-chambered, dorsal pits, and these contain basiconic II pit sensilla (olfactory). Common to all 5 species is a multi-chambered ventral pit (olfactory). In the ventral pit, all 5 species have grooved pit sensilla (olfactory). In addition to this type of sensillum the Delia species have smooth-walled conical pit sensilla (hygro-/thermosensitive) and P. rosae has granular pit sensilla (hygro-/thermosensitive). Smooth-walled tapered pit sensilla (hygro-/thermosensitive) are found in D. radicum. Similarities and differences in the density of surface sensilla between dorsal and ventral funicular surfaces, male and female flies, and oligophagous (D. antiqua, D. radicum, D. floralis and P. rosae) and polyphagous (D. platura) species are compared. Several differences in sensillum density between the dorsal and ventral funicular surfaces are observed, but these do not fit into a consistent trend. Except for D. radicum, there are differences in sensillum density between male and female flies. For the oligophagous species, females have a greater sensillum density, whilst for the polyphagous D. platura males have a greater sensillum density. Comparisons between species show the greatest differences between the Delia species and P. rosae, and within the 4 Delia species, differences in sensillum density do not correlate with host range or body size.     

Comparison of the sensitivity of four Delia species to host and non-host plant compounds

Abstract. The behavioural facilitation hypothesis, tested in the present study, suggests that evolution of host-plant shifts by phytophagous insects is based on the preadaptation of insects to the chemistry of potentially novel plant hosts. Thus, closely-related insects should have similar sensitivities to compounds that are shared by different host plants. The chemoreception is investigated for four phytophagous flies, Delia radicum, Delia floralis, Delia antiqua and Delia platura (Diptera, Calyptratae: Anthomyiidae), belonging to the same genus but developing mainly on different plant families, with particular secondary plant compound profiles. In addition, the carrot fly, Psila rosae, an acalyptrate Diptera, is included as an unrelated species that is associated with completely different host plants. For the comparison, the known oviposition stimulants of the cabbage root fly (glucobrassicin, sinalbin, sinigrin and a thia-triaza-fluorene compound; CIF-1) present on the cabbage leaf surface were chosen. Responses from prothoracic tarsal sensilla are recorded to contact stimulation in a dose-dependent manner. Among the different flies tested, only D. radicum responds to all the compounds. By contrast, D. floralis is only sensitive to CIF-1, and not specifically on the C₅ sensillum, a finding that is in conflict with previously published results. This discrepancy is possibly an indication of the variability among flies originating from different cultures or habitats. With the exception of sinigrin at high concentration, the various compounds tested do not stimulate D. antiqua or D. platura. However, the carrot fly appears to be completely insensitive to sinigrin even at the highest tested concentration of 10⁻¹ M. The responses of the contact-chemoreceptor neurones to the selected compounds therefore provide little evidence of common sensitivities that would explain host shift in Delia species and specialization at the physiological level. The wide divergence within closely-related species and rearing cultures appears to indicate that the sensitivity and distribution of sensory receptor neurones is very variable on an evolutionary scale.     

Community of bat flies (Streblidae and Nycteribiidae) on bats in the Cerrado of Central-West Brazil: hosts,aggregation, prevalence,infestation intensity,and infracommunities

Streblidae and Nycteribiidae are families of bloodsucking flies that parasitize bats exclusively. We studied the community of these flies in a Cerrado area in the Central-West Brazil. We captured 708 bats over 17 nights from October 2012 to March 2013. Forty-five per cent of the hosts were parasitized by 836 specimens of bat flies of 22 species. The most abundant flies were Trichobius joblingi on Carollia perspicillata, followed by Megistopoda aranea on Artibeus planirostris, and Strebla guajiro on C. perspicillata. All bat flies showed a high level of specificity for their hosts. Trichobius joblingi was the bat fly with the highest prevalence (80%) and mean intensity of infestation (3.5) on hosts with a representative sample size (n > 20). This result is likely related to the type of roosting (cavity) used by C. perspicillata, primary host of this fly species. Anoura caudifer hosted the largest infracommunities (n = 7). However, most bats were parasitized by a single fly species, suggesting a pattern in infestations. The aggregation index was high, indicating an unequal occurrence in parasite infestations. The majority of hosts were infested by few or no flies and few hosts were highly infested, showing a negative binomial distribution.