Ecological determinants of prevalence of the male-killing bacterium Arsenophonus nasoniae.

Male-killing bacteria are found in a broad range of arthropods. Arsenophonus nasoniae is a male-killing bacterium, causing a 80% reduction of the male progeny in infected Nasonia vitripennis wasps. Although the discovery of A. nasoniae dates from the early 80's, knowledge about the biology and ecology of this endosymbiont is still scarce. One of these poorly studied features is the ecological factors underlying A. nasoniae incidence on its Nasonia spp. hosts in different geographical locations. Here, we studied the prevalence of A. nasoniae in Iberian wild populations of its host N. vitripennis. This wasp species is a common parasitoid of the blowfly Protocalliphora azurea pupae, which in turn is a parasite of hole-nesting birds, such as the blue tit (Cyanistes caeruleus). We also examined the effects of bird rearing conditions on the prevalence of A. nasoniae through a brood size manipulation experiment (creating enlarged, control and reduced broods). Both the wasp and bacterium presence were tested through PCR assays in blowfly pupae. We found A. nasoniae in almost half (47%) of nests containing blowflies parasitized by N. vitripennis. The prevalence of A. nasoniae was similar in the two geographical areas examined (central Portugal and southeastern Spain) and the probability of infection by A. nasoniae was independent of the number of blowfly pupae in the nest. Experimental manipulation of brood size did not affect the prevalence of A. nasoniae nor the prevalence of its host, N. vitripennis. These results suggest that the incidence of A. nasoniae in natural populations of N. vitripennis is high in the Iberian Peninsula, and the infestation frequency of nests by N. vitripennis carrying A. nasoniae is spatially stable in this geographical region independently of bird rearing conditions.

Effects of ivermectin on development of Calliphora vicina, Robineau-Desvoidy 1830 (Diptera, Calliphoridae).

Ivermectin is one of the most widely used drugs for parasite control. Previous studies have shown a reduction in the abundance and diversity of "non-target" coprophilous organisms due to the presence of ivermectin (IVM) in bovine faecal matter (FM). Due to its breadth of behavioural habits, Calliphora vicina is a suitable dipteran species to evaluate the effects of IVM in FM. The aim of this work was to evaluate the effect of five concentrations of IVM in FM (3000, 300, 100, 30, and 3 ng/g) on the development of C. vicina. The following endpoints were evaluated: survival (between the first larval stage and emergence of new adults), larval development times to pupation and pupation times to adult, and adult emergence (% sex) and LC50. Sampling was performed from larval hatching at 60 and 120 min and at 3, 4, 5, and 12 h, and every 24 h specimens were weighed until pupae were observed. Data were analysed by ANOVA using a non-parametric Kruskal-Wallis test and as a function of elapsed development time and accumulated degree hours (ADH). Mortality at 3000 and 300 ng/g was 100% and 97%, respectively. There were statistically significant delays in adult emergence time (p = 0.0216) and in the ADH (p = 0.0431) between the control group (C) and 100 ng/g. The LC50 was determined at 5.6 ng/g. These results demonstrate the lethal and sub-lethal effects of IVM on C. vicina, while highlighting the usefulness of this species as a bioindicator for ecotoxicological studies.

Does tail docking prevent Cochliomyia hominivorax myiasis in sheep? A six-year retrospective cohort study.

Tail docking is a husbandry practice widely incorporated in sheep farms around the world. It is an irreversible mutilation that impairs animal welfare, both immediately and in the longer term. The defence of tail docking as a practice is centred around the perception that doing so contributes to the promotion of local hygiene, allowing the use of the wool, facilitating reproductive management and reducing the chances of myiasis, a disease caused by the invasion of blowfly larvae in the tissues of warm-blooded animals. However, current understanding of farm animal welfare questions the need to maintain practices such as tail docking. Thus, the aim of this study was to evaluate the effect of tail docking on the incidence of Cochliomyia hominivorax myiasis in sheep in an experimental flock in Brazil during a six-year retrospective cohort study. Relative risk, odds ratio and incidence rate ratio were the association measures adopted. A total of 4,318 data-points were collected and supplied the analytical model. Tail docking did not decrease the risk and, on the contrary, was found to increase the chances of sheep being affected by myiasis. The results support the hypothesis that tail docking is not a protective factor against the occurrence of myiasis and further fuel calls for a rethink of tail docking being deployed as a blanket measure in the prevention of myiasis in sheep.

Random attention can explain apparent object choice behavior in free-walking blowflies.

Flies are often observed to approach dark objects. To a naive observer they seem to pay selective attention to one out of several objects although previous research identified as a possible underlying mechanism a reflex-like fixation behavior integrating responses to all objects. In a combination of behavioral experiments and computational modelling, we investigated the choice behavior of flies freely walking towards an arrangement of two objects placed at a variable distance from each other. The walking trajectories were oriented towards one of the objects much earlier than predicted by a simple reactive model. We show that object choice can be explained by a continuous control scheme in combination with a mechanism randomly responding to the position of each object according to a stochastic process. This may be viewed as a special form of an implicit attention-like mechanism, for which the model does not require an explicit decision mechanism or a memory for the drawn decision.

Love at first flight: wing interference patterns are species-specific and sexually dimorphic in blowflies (Diptera: Calliphoridae).

Wing interference patterns (WIPs) are stable structural colours displayed on insect wings which are only visible at specific viewing geometries and against certain backgrounds. These patterns are widespread among flies and wasps, and growing evidence suggests that they may function as species- and sex-specific mating cues in a range of taxa. As such, it is expected that WIPs should differ between species and show clear sexual dimorphisms. However, the true extent to which WIPs vary between species, sexes and individuals is currently unclear, as previous studies have only taken a qualitative approach, without considering how WIPs might be perceived by the insect. Here, we perform the first quantitative analysis of inter- and intra-specific variation in WIPs across seven Australian species of the blowfly genus Chrysomya. Using multispectral digital imaging and a tentative model of blowfly colour vision, we provide quantitative evidence that WIPs are species-specific, highlight that the extent of divergence is greater in males than in females and demonstrate sexual dimorphisms in several species. These data suggest that WIPs have diversified substantially in blowflies as a result of either sexual or ecological selection.

Bacterial association observations in Lucilia sericata and Lucilia cuprina organs through 16S rRNA gene sequencing.

Blowfly (Diptera: Calliphoridae) species Lucilia sericata (Meigen) and related species Lucilia cuprina (Wiedmann) are important agricultural pests, assist in forensic fields and also have a therapeutic role in medicine. Both species (though predominantly L. sericata) are utilised in a clinical setting for maggot debridement therapy (MDT) where the larvae ingest necrotic tissue and bacteria from non-healing wounds. Conversely, larvae of L. cuprina feed invasively, as major initiators of sheep myiasis in Australia, New Zealand, and the UK, among other regions. Both species exhibit larval and adult interactions with bacterially rich environments, but the significance of this in the composition of their microbiome has yet to be considered. This study utilised dissected samples of digestive and reproductive organs from both disinfected and non-disinfected adults and larvae of both species for bacterial DNA extraction, followed by 16S rRNA gene sequencing. Sequencing data indicated unsurprisingly that digestive tracts of both genders and female salivary glands from all non-disinfected samples carry the most concentrated amounts of bacteria. Genera Pseudomonas and Corynebacterium were also highly represented within all organs and species analysed. Comparison of bait lures to sample sequence read output of insect specimens showed no correlation with genera such as Pseudomonas present in insects, while absent from wild bait, and in reduced amounts from fleece bait profiles. With this information, future work can focus on key organs such as the spermathecae and salivary glands, while also providing the potential to identify the role these bacteria may play in the blowfly life cycle. KEY POINTS: Genera Pseudomonas appears consistently in the microbiome of Lucilia species. Female spermathecae and salivary glands show the highest microbial diversity. Bacterial profiles of L. sericata and L. cuprina have similar composition.

A serine protease-associated lectin in the cytolytic system of blowfly (Chrysomya megacephala) larvae: Evidence and characterization.

Cytolytic activity against invading microorganisms is one of the innate forms of immunity in invertebrates. A serine protease-associated sialic acid-specific cytolytic lectin was purified using glutaraldehyde-fixed ox erythrocytes from the larval extract of blowfly (Chrysomya megacephala). The purified lectin lysed vertebrate erythrocytes with effective haemolysis of ox red blood cells (RBCs) in an isotonic medium. The degree of haemolytic (HL) activity of the purified cytolytic lectin depended on its concentration, pH, temperature, and calcium ions. It was sensitive to ethylenediaminetetraacetic acid. The native molecular mass of the C-type lectin was 260 ± 26 kDa, comprising four different polypeptide subunits of 75 kDa (pI ~8), 69 kDa (pI ~7.0), 61 kDa (pI ~5.3), and 55 kDa (pI ~4.6). The association between the C-type lectin and serine protease was confirmed by MALDI-TOF-MS analysis that revealed its homology in the same spectral peak as well as the proteases and phenylmethylsulphonyl fluoride inhibition of HL activity. Haemolysis inhibition by N-acetylneuraminic acid and other sugars revealed the properties of the lectin. The purified lectin distorted the integrity of ox RBCs and Paenalcaligenes hermetiae. This in vitro study documents the presence of a cytolytic system in blowfly (C. megacephala) larvae for the clearance of invading microbial pathogens in their feeding niche.

Nutritional requirements for reproduction and survival in the blowfly Lucilia sericata.

Insects with access to finite energy resources must allocate these between maintenance and reproduction in a way that maximizes fitness. This will be influenced by a range of life-history characteristics and the environment in which any particular insect species lives. In the present study, females of the blowfly Lucilia sericata (Diptera: Calliphoridae) were fed diets differing in protein and carbohydrate (sucrose) content and the allocation of lipid to reproduction was quantified using a spectrophotometric method of analysis. Immediately after adult emergence, total body lipid, scaled for differences in body size, showed an initial decline as it was utilized to meet the metabolic demands of cuticle deposition, muscle maturation and then flight. When flies were denied access to sucrose, stored lipid then continued to decrease until flies died, usually within 4 days of emergence. However, flies given access to sucrose were able to increase body lipid content, demonstrating that carbohydrate is essential for homeostasis and that it can be used to synthesize lipid. Nevertheless, female flies fed sucrose only were unable to synthesize egg yolk. Only flies provided with protein were able to mature eggs. However, the rate of egg maturation and number and size of eggs matured were greater for female flies given liver compared with flies provided with pure whey protein powder. The results demonstrate the importance of different dietary components for different elements of the life-history of L. sericata, namely survival and reproduction.

Variation in parasitoidism of Protocalliphora azurea (Diptera: Calliphoridae) by Nasonia vitripennis (Hymenoptera: Pteromalidae) in Spain.

Parasitoid wasps may act as hyperparasites and sometimes regulate the populations of their hosts by a top-down dynamic. Nasonia vitripennis (Walker, 1836) is a generalist gregarious parasitoid that parasitizes several host flies, including the blowfly Protocalliphora Hough, 1899 (Diptera, Calliphoridae), which in turn parasitizes bird nestlings. Nonetheless, the ecological factors underlying N. vitripennis prevalence and parasitoidism intensity on its hosts in natural populations are poorly understood. We have studied the prevalence of N. vitripennis in Protocalliphora azurea (Fallén, 1817) puparia parasitizing wild populations of pied flycatcher (Ficedula hypoleuca) and blue tit (Cyanistes caeruleus) birds in two Mediterranean areas in central and southern Spain. We found some evidence that the prevalence of N. vitripennis was higher in moist habitats in southern Spain. A host-dependent effect was found, since the greater the number of P. azurea puparia, the greater the probability and rate of parasitoidism by the wasp. Our results also suggest that N. vitripennis parasitizes more P. azurea puparia in blue tit nests than in pied flycatcher nests as a consequence of a higher load of these flies in the former. Based on the high prevalence of N. vitripennis in P. azurea puparia in nature, we propose that this wasp may regulate blowfly populations, with possible positive effects on the reproduction of both bird species.

Spatial and temporal habitat partitioning by calliphorid blowflies.

Calliphorid blowflies perform an essential ecosystem service in the consumption, recycling and dispersion of carrion nutrients and are considered amongst the most important functional groups in an ecosystem. Some species are of economic importance as facultative agents of livestock myiasis. The interspecific ecological differences that facilitate coexistence within the blowfly community are not fully understood. The aim of this work was to quantify differences in habitat use by calliphorid species. Thirty traps were distributed among three habitats at two sites in southwest England for collections made during March-August 2016. A total of 17 246 specimens were caught, of which 2427 were Lucilia sericata, 51 Lucilia richardsi, 6580 Lucilia caesar, 307 Lucilia ampullacea, 4881 Calliphora vicina and 2959 Calliphora vomitoria (all: Diptera: Calliphoridae). Lucilia sericata was the dominant species in open habitats, whereas L. caesar was the most abundant species in shaded habitats. Calliphora specimens were more abundant in the cooler months. These findings suggest that Calliphora and Lucilia species show strong temporal segregation mediated by temperature, and that species of the genus Lucilia show differences in the use of habitats that are likely to be driven by differences in humidity tolerance and light intensity. These factors in combination result in effective niche partitioning.