Aposymbiotic tsetse flies, Glossina morsitans morsitans obtained by feeding on rabbits immunized specifically with symbionts

Tsetse flies fed on rabbits which previously had been immunized with whole flies without symbionts showed an increase in mortality and only a small decrease in fecundity. Flies fed on rabbits immunized with symbionts only became aposymbiotic. Their fecundity decreased drastically while their longevity was not affected. The antibodies found in the flies were specifically attached to the tissues which had been used as antigens.     

The obligate mutualist Wigglesworthia glossinidia influences reproduction, digestion, and immunity processes of its host, the tsetse fly

Tsetse flies (Diptera: Glossinidae) are vectors for trypanosome parasites, the agents of the deadly sleeping sickness disease in Africa. Tsetse also harbor two maternally transmitted enteric mutualist endosymbionts: the primary intracellular obligate Wigglesworthia glossinidia and the secondary commensal Sodalis glossinidius. Both endosymbionts are transmitted to the intrauterine progeny through the milk gland secretions of the viviparous female. We administered various antibiotics either continuously by per os supplementation of the host blood meal diet or discretely by hemocoelic injections into fertile females in an effort to selectively eliminate the symbionts to study their individual functions. A symbiont-specific PCR amplification assay and fluorescence in situ hybridization analysis were used to evaluate symbiont infection outcomes. Tetracycline and rifampin treatments eliminated all tsetse symbionts but reduced the fecundity of the treated females. Ampicillin treatments did not affect the intracellular Wigglesworthia localized in the bacteriome organ and retained female fecundity. The resulting progeny of ampicillin-treated females, however, lacked Wigglesworthia but still harbored the commensal Sodalis. Our results confirm the presence of two physiologically distinct Wigglesworthia populations: the bacteriome-localized Wigglesworthia involved with nutritional symbiosis and free-living Wigglesworthia in the milk gland organ responsible for maternal transmission to the progeny. We evaluated the reproductive fitness, longevity, digestion, and vectorial competence of flies that were devoid of Wigglesworthia. The absence of Wigglesworthia completely abolished the fertility of females but not that of males. Both the male and female Wigglesworthia-free adult progeny displayed longevity costs and were significantly compromised in their blood meal digestion ability. Finally, while the vectorial competence of the young newly hatched adults without Wigglesworthia was comparable to that of their wild-type counterparts, older flies displayed higher susceptibility to trypanosome infections, indicating a role for the mutualistic symbiosis in host immunobiology. The ability to rear adult tsetse that lack the obligate Wigglesworthia endosymbionts will now enable functional investigations into this ancient symbiosis.     

Effects of dietary folic acid level and symbiotic folate production on fitness and development in the fruit fly Drosophila melanogaster

Folic acid is a vitamin for probably all animals. When converted to folate forms, it is used in DNA synthesis and amino acid metabolism. Literature suggests insects must consume folates, folates do not affect others, is a toxin for some, and that a few insects synthesize it. It has been reported that Drosophila melanogaster does not consistently need dietary folate because it can synthesize it. This seems unlikely since animals generally lack this ability. More likely, folates thought to have been made by the fly came from microbial symbionts. We aimed to clarify how dietary folic acid affects fitness and development in fruit flies and whether flies may receive folates from microbial symbionts. We found larvae were more viable and developed faster with increasing dietary folic acid, with the surprising exception that larvae fed nearly-zero folic acid developed faster. Their body folate levels did not significantly differ from those that consumed up to 600 times more folic acid. However, these flies fed little folate only achieved normal body folate levels and development times when antibiotics were excluded from the diet. When flies consumed near-zero folates with antibiotics, their body folate levels decreased and development was prolonged. An assay for the endosymbiont Wolbachia in flies used to generate the experimental flies did not show presence of these bacteria. Our data suggest D. melanogaster can harbor unknown bacterial symbiont(s) that provide essential folates to their host when it is scarce in the diet, allowing the fruit fly to maintain growth and development.     

Interspecific Transfer of Bacterial Endosymbionts between Tsetse Fly Species: Infection Establishment and Effect on Host Fitness

Tsetse flies (Glossina spp.) can harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis, are transmitted maternally to tsetse flies' intrauterine larvae. The third symbiont, from the genus Wolbachia, parasitizes developing oocytes. In this study, we determined that Sodalis isolates from several tsetse fly species are virtually identical based on a phylogenetic analysis of their ftsZ gene sequences. Furthermore, restriction fragment-length polymorphism analysis revealed little variation in the genomes of Sodalis isolates from tsetse fly species within different subgenera (Glossina fuscipes fuscipes and Glossina morsitans morsitans). We also examined the impact on host fitness of transinfecting G. fuscipes fuscipes and G. morsitans morsitans flies with reciprocal Sodalis strains. Tsetse flies cleared of their native Sodalis symbionts were successfully repopulated with the Sodalis species isolated from a different tsetse fly species. These transinfected flies effectively transmitted the novel symbionts to their offspring and experienced no detrimental fitness effects compared to their wild-type counterparts, as measured by longevity and fecundity. Quantitative PCR analysis revealed that transinfected flies maintained their Sodalis populations at densities comparable to those in flies harboring native symbionts. Our ability to transinfect tsetse flies is indicative of Sodalis ' recent evolutionary history with its tsetse fly host and demonstrates that this procedure may be used as a means of streamlining future paratransgenesis experiments.     

An Investigation into the Protein Composition of the Teneral Glossina morsitans morsitans Peritrophic Matrix

Background

Tsetse flies serve as biological vectors for several species of African trypanosomes. In order to survive, proliferate and establish a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM), which is an acellular gut lining surrounding the blood meal. Crossing of this multi-layered structure occurs at least twice during parasite migration and development, but the mechanism of how trypanosomes do so is not understood. In order to better comprehend the molecular events surrounding trypanosome penetration of the tsetse PM, a mass spectrometry-based approach was applied to investigate the PM protein composition using Glossina morsitans morsitans as a model organism.

Methods

PMs from male teneral (young, unfed) flies were dissected, solubilised in urea/SDS buffer and the proteins precipitated with cold acetone/TCA. The PM proteins were either subjected to an in-solution tryptic digestion or fractionated on 1D SDS-PAGE, and the resulting bands digested using trypsin. The tryptic fragments from both preparations were purified and analysed by LC-MS/MS.

Results

Overall, nearly 300 proteins were identified from both analyses, several of those containing signature Chitin Binding Domains (CBD), including novel peritrophins and peritrophin-like glycoproteins, which are essential in maintaining PM architecture and may act as trypanosome adhesins. Furthermore, 27 proteins from the tsetse secondary endosymbiont, Sodalis glossinidius, were also identified, suggesting this bacterium is probably in close association with the tsetse PM.

Conclusion

To our knowledge this is the first report on the protein composition of teneral G. m. morsitans, an important vector of African trypanosomes. Further functional analyses of these proteins will lead to a better understanding of the tsetse physiology and may help identify potential molecular targets to block trypanosome development within the tsetse.     

Effect of antibiotic treatment and gamma-irradiation on cuticular hydrocarbon profiles and mate choice in tsetse flies (Glossina m. morsitans)

Background

Symbiotic microbes represent a driving force of evolutionary innovation by conferring novel ecological traits to their hosts. Many insects are associated with microbial symbionts that contribute to their host’s nutrition, digestion, detoxification, reproduction, immune homeostasis, and defense. In addition, recent studies suggest a microbial involvement in chemical communication and mating behavior, which can ultimately impact reproductive isolation and, hence, speciation. Here we investigated whether a disruption of the microbiota through antibiotic treatment or irradiation affects cuticular hydrocarbon profiles, and possibly mate choice behavior in the tsetse fly, Glossina morsitans morsitans. Four independent experiments that differentially knock down the multiple bacterial symbionts of tsetse flies were conducted by subjecting tsetse flies to ampicillin, tetracycline, or gamma-irradiation and analyzing their cuticular hydrocarbon profiles in comparison to untreated controls by gas chromatography – mass spectrometry. In two of the antibiotic experiments, flies were mass-reared, while individual rearing was done for the third experiment to avoid possible chemical cross-contamination between individual flies.

Results

All three antibiotic experiments yielded significant effects of antibiotic treatment (particularly tetracycline) on cuticular hydrocarbon profiles in both female and male G. m. morsitans, while irradiation itself had no effect on the CHC profiles. Importantly, tetracycline treatment reduced relative amounts of 15,19,23-trimethyl-heptatriacontane, a known compound of the female contact sex pheromone, in two of the three experiments, suggesting a possible implication of microbiota disturbance on mate choice decisions. Concordantly, both female and male flies preferred non-treated over tetracycline-treated flies in direct choice assays.

Conclusions

While we cannot exclude the possibility that antibiotic treatment had a directly detrimental effect on fly vigor as we are unable to recolonize antibiotic treated flies with individual symbiont taxa, our results are consistent with an effect of the microbiota, particularly the obligate nutritional endosymbiont Wigglesworthia, on CHC profiles and mate choice behavior. These findings highlight the importance of considering host-microbiota interactions when studying chemical communication and mate choice in insects.

     

Interspecific transfer of bacterial endosymbionts between tsetse fly species: infection establishment and effect on host fitness

Tsetse flies (Glossina spp.) can harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis, are transmitted maternally to tsetse flies' intrauterine larvae. The third symbiont, from the genus Wolbachia, parasitizes developing oocytes. In this study, we determined that Sodalis isolates from several tsetse fly species are virtually identical based on a phylogenetic analysis of their ftsZ gene sequences. Furthermore, restriction fragment-length polymorphism analysis revealed little variation in the genomes of Sodalis isolates from tsetse fly species within different subgenera (Glossina fuscipes fuscipes and Glossina morsitans morsitans). We also examined the impact on host fitness of transinfecting G. fuscipes fuscipes and G. morsitans morsitans flies with reciprocal Sodalis strains. Tsetse flies cleared of their native Sodalis symbionts were successfully repopulated with the Sodalis species isolated from a different tsetse fly species. These transinfected flies effectively transmitted the novel symbionts to their offspring and experienced no detrimental fitness effects compared to their wild-type counterparts, as measured by longevity and fecundity. Quantitative PCR analysis revealed that transinfected flies maintained their Sodalis populations at densities comparable to those in flies harboring native symbionts. Our ability to transinfect tsetse flies is indicative of Sodalis ' recent evolutionary history with its tsetse fly host and demonstrates that this procedure may be used as a means of streamlining future paratransgenesis experiments.     

Diversity in symbiont consortia in the pea aphid complex is associated with large phenotypic variation in the insect host

Virtually all eukaryotes host microbial symbionts that influence their phenotype in many ways. In a host population, individuals may differ in their symbiotic complement in terms of symbiont species and strains. Hence, the combined expression of symbiont and host genotypes may generate a range of phenotypic diversity on which selection can operate and influence host population ecology and evolution. Here, we used the pea aphid to examine how the infection with various symbiotic complements contributes to phenotypic diversity of this insect species. The pea aphid hosts an obligate symbiont (Buchnera aphidicola) and several secondary symbionts among which is Hamiltonella defensa. This secondary symbiont confers a protection against parasitoids but can also reduce the host’s longevity and fecundity. These phenotypic effects of H. defensa infection have been described for a small fraction of the pea aphid complex which encompasses multiple plant-specialized biotypes. In this study, we examined phenotypic differences in four pea aphid biotypes where H. defensa occurs at high frequency and sometimes associated with other secondary symbionts. For each biotype, we measured the fecundity, lifespan and level of parasitoid protection in several aphid lineages differing in their symbiotic complement. Our results showed little variation in longevity and fecundity among lineages but strong differences in their protection level. These differences in protective levels largely resulted from the strain type of H. defensa and the symbiotic consortium in the host. This study highlights the important role of symbiotic complement in the emergence of phenotypic divergence among host populations of the same 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.     

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

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

Hosts use altered macronutrient intake to circumvent parasite-induced reduction in fecundity

Explanations for the evolution of pathogen-induced fecundity reduction usually rely on a common principle: the trade-off between host longevity and reproduction. Recent advances in nutritional research have, however, challenged this assumption and shown that longevity and reproduction are not inextricably linked. In this study, we showed that beetles infected by cysticercoids of the tapeworm Hymenolepis diminuta increased their total food intake and, more particularly, their carbohydrate consumption compared with uninfected insects. This increased intake was only pronounced during the first 12 days p.i., when the parasite grows and develops into a mature metacestode. Despite consuming more nutrients, infected individuals sustained lower levels of body lipid and were less efficient at converting ingested protein to body protein. However they demonstrated a capacity to compose a diet that sustained high levels of reproductive output unless confined to foods that were nutritionally dilute. We did not find any indication that macronutrient intakes had an effect on host pro-phenoloxidase activity; however, phenoloxidase activity was significantly affected by protein intake. Our results showed that when offered nutritionally complementary diets, infected hosts do not systematically suffer a reduction in fecundity. Thus, in our view, the assumption that a reduction in host reproduction represents an adaptive response by the host or the parasite to divert resources away from reproduction toward other traits should be reassessed.     

The parasitoid of a fruit moth caterpillar utilizes fruit components as nutrient source to increase its longevity and fertility

Longevity and fecundity of female wasps are two decisive factors for the effectiveness of parasitoid species as biological control agents. Accessibility and suitability of nutrient sources determine parasitoid survival and reproduction. Host, nectar and honeydew feeding are frequent adult parasitoid behaviors to cover nutritional needs. Here we postulate that especially parasitoid species of endophytic herbivores might use plant tissue as a nutrient source that becomes accessible upon herbivory. We investigated the influence of plant consumption and host feeding on longevity and fecundity of Hyssopus pallidus, a gregarious ecto-parasitoid of caterpillars of the codling moth that feed inside apple fruits. Longevity of unmated and mated ovipositing female parasitoids was highest in treatments with fruit pulp. While longevity in this treatment was not significantly different from that with honey, it was significantly higher than in treatments without food, with water or with a host alone.Reproduction was significantly increased by these sugar-rich nutrient sources compared to the control with a host alone. In contrast, host feeding did not yield any significant contribution to longevity and fecundity in a series of bioassays with different host–parasitoid ratios and with differently aged and sized hosts, compared to controls without food.We conclude that in this synovigenic species host feeding does not contribute to longevity and fecundity, but females can increase survival and reproduction in the field relying solely on the plant tissue damaged by their host caterpillar.     

Only helpful when required: a longevity cost of harbouring defensive symbionts

Maternally transmitted symbionts can spread in host populations if they provide a fitness benefit to their hosts. Hamiltonella defensa, a bacterial endosymbiont of aphids, protects hosts against parasitoids but only occurs at moderate frequencies in most aphid populations. This suggests that harbouring this symbiont is also associated with costs, yet the nature of these costs has remained elusive. Here, we demonstrate an important and clearly defined cost: reduced longevity. Experimental infections with six different isolates of H. defensa caused strongly reduced lifespans in two different clones of the black bean aphid, Aphis fabae, resulting in a significantly lower lifetime reproduction. However, the two aphid clones were unequally affected by the presence of H. defensa, and the magnitude of the longevity cost was further determined by genotype × genotype interactions between host and symbiont, which has important consequences for their coevolution.     

Wolbachia interactions that determine Drosophila melanogaster survival

Abstract.— We have recently described a mutualistic symbiosis in which Wolbachia bacteria were shown to improve the fitness of some Drosophila melanogaster stocks. Wolbachia did not extend longevity in all Drosophila genotypes, even though 16s rDNA sequences indicated that our Drosophila stocks were infected with the same Wolbachia strain. Here, we use reciprocal hybrid crosses between two Drosophila strains, one that lived longer with Wolbachia (Z53) and one that did not (Z2), to investigate the inheritance of the survival phenotype and its dependence on the host genotype, sex, and mating conditions. Wolbachia's positive effects were more apparent in hybrid flies than in parental flies, ruling out exclusive maternal inheritance or the dependence of the survival phenotype on Wolbachia strain differences. The Wolbachia survival effects were more apparent in single-sex cages, where courtship and mating were not permitted. In these cages, nearly all flies with Wolbachia lived longer than uninfected flies, even though strain Z2 showed no Wolbachia effect in mixed-sex mating cages. We used comparisons between single- and mixed-sex cages to estimate the cost of reproduction for both sexes. Our data suggest that Wolbachia infection may increase the inferred cost of reproduction, particularly in males. Wolbachia can even produce a positive survival effect almost as large as the negative survival effect associated with reproduction. We discuss the implications of our experiments for the study of insect symbioses.     

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

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

Several biological properties of E. coli SK cells]

Escherichia coli SK cells are not colicinogenic, but possess multiple resistance to antibiotics (tetracycline, kanamycin, penicillin, polymyxin, ampicillin). Numerous variations, sensitive to one or several of the antibiotics listed were obtained by cloning the initial culture. Experiments with acridine orange failed to eliminate completely the resistance of E. coli cells to all five of the antibiotics under study. Partial modifications in the spectrum of antibiotic resistance did not influence the host specificity system present in the cells SK. The capacity of restriction and the activity of the methylating enzymes in all the clones under study in the initial strain proved to be the same.     

The effect of host odour on the landing responses of tsetse flies (Glossina morsitans morsitans) in a wind tunnel with and without visual targets

Abstract The effect of artificial host odour on the landing responses of males of Glossina m.morsitans West, and on their reaction to visual targets has been investigated in a wind tunnel. Landing was induced in flies that traversed steep odour gradients as they flew upwind and downwind across the edge of an odour plume, irrespective of whether visual targets were present or not; the landing response could be elicited over a wide range of odourconcentrations. When targets were present such odour gradients also tended to increase the proportion of landing flies which alighted on or near the targets; and the bigger the target, or the hungrier the flies, the greater was the propensity for target landing. In air which was more uniformly permeated with odour, the propensity to land on targets was increased only at high odour concentration.     

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.     

Characterization of the gut bacterial community in Manduca sexta and effect of antibiotics on bacterial diversity and nematode reproduction

The tobacco hornworm, Manduca sexta , is a model lepidopteran insect used to study the pathogenic and mutualistic phases of entomopathogenic nematodes (EPNs) and their bacterial symbionts. While intestinal microbial communities could potentially compete with the EPN and its bacterial partner for nutrient resources of the insect, the microbial gut community had not been characterized previously. Here, we show that the midgut of M. sexta raised on an artificial diet contained mostly Gram-positive cocci and coryneforms including Staphylococcus, Pediococcus, Micrococcus and Corynebacterium . Major perturbation in the gut community was observed on addition of antibiotics to the diet. Paenibacillus and several Proteobacteria such as Methylobacterium, Sphingomonas and Acinetobacter were primary genera identified under these conditions. Furthermore, the reproduction of the nematode Steinernema carpocapsae was less efficient, and the level of nematode colonization by its symbiont Xenorhabdus nematophila reduced, in insects reared on a diet containing antibiotics. The effect of antibiotics and perturbation of gut microbiota on nematode reproduction is discussed.