Effect of thermal acclimation on the tolerance of the peach fruit fly (Bactrocera zonata: Tephritidae) to heat and cold stress.

Understanding the thermal biology of insects is of increasing importance for predicting their geographic distribution, particularly in light of current and future global temperature increases. Within the limits set by genetic makeup, thermal tolerance is affected by the physiological conditioning of individuals (e.g., through acclimation). Considering this phenotypic plasticity may add to accurately estimating changes to the distribution of insects under a changing climate. We studied the effect of thermal acclimation on cold and heat tolerance of the peach fruit fly (Bactrocera zonata) - an invasive, polyphagous pest that is currently expanding through Africa and the Middle East. Females and males were acclimated at 20, 25 and 30 °C for up to 19 days following adult emergence. The critical thermal minimum (CTmin) and maximum (CTmax) were subsequently recorded as well adult survival following acute exposure to chilling (0 or -3 °C for 2 h). Additionally, we determined the survival of pupae subjected for 2 h to temperatures ranging from -12 °C to 5 °C. We demonstrate that acclimation at 30 °C resulted in significantly higher CTmax and CTmin values (higher heat resistance and lower cold resistance, respectively). Additionally, adult recovery following exposure to -3 °C was significantly reduced following acclimation at 30 °C, and this effect was significantly higher for females. Pupal mortality increased with the decrease in temperature, reaching LT50 and LT95 values following exposure to -0.32 °C and -6.88 °C, respectively. Finally, we found that the survival of pupae subjected to 0 and 2 °C steadily increased with pupal age. Our findings substantiate a physiological foundation for understanding the current geographic range of B. zonata. We assume that acclimation at 30 °C affected the thermal tolerance of the flies partly through modulating feeding and metabolism. Tolerance to chilling during the pupal stage probably changed according to temperature-sensitive processes occurring during metamorphosis, rendering younger pupae more sensitive to chilling.

Comparative Proteomics of Coxiella like Endosymbionts (CLEs) in the Symbiotic Organs of Rhipicephalus sanguineus Ticks.

Maternally transmitted obligatory endosymbionts are found in the female gonads as well as in somatic tissue and are expected to provide missing metabolite to their hosts. These deficiencies are presumably complemented through specific symbiotic microorganisms such as Coxiella-like endosymbionts (CLEs) of Rhipicephalus ticks. CLEs are localized in specialized host tissue cells within the Malpighian tubules (Mt) and the ovaries (Ov) from which they are maternally transmitted to developing oocytes. These two organs differ in function and cell types, but the role of CLEs in these tissues is unknown. To probe possible functions of CLEs, comparative proteomics was performed between Mt and Ov of R. sanguineus ticks. Altogether, a total of 580 and 614 CLE proteins were identified in Mt and Ov, respectively. Of these, 276 CLE proteins were more abundant in Mt, of which 12 were significantly differentially abundant. In Ov, 290 CLE proteins were more abundant, of which 16 were significantly differentially abundant. Gene Ontology analysis revealed that most of the proteins enriched in Mt are related to cellular metabolic functions and stress responses, whereas in Ov, the majority were related to cell proliferation suggesting CLEs function differentially and interdependently with host requirements specific to each organ. The results suggest Mt CLEs provide essential nutrients to its host and Ov CLEs promote proliferation and vertical transmission to tick progeny. IMPORTANCE Here we compare the Coxiella-like endosymbionts (CLEs) proteomes from Malpighian tubule (Mt) and the ovaries (Ov) of the brown dog tick Rhipicephalus sanguineus. Our results support the hypothesis that CLEs function interdependently with host requirements in each of the organs. The different functional specificity of CLE in the same host suggest that metabolic capabilities evolved according to the constrains imposed by the specific organ function and requirements. Our findings provide specific CLE protein targets that can be useful for future studies of CLE biology with a focus on tick population control.

Effects of Thermal Acclimation on the Tolerance of Bactrocera zonata (Diptera: Tephritidae) to Hydric Stress.

Insects, similarly to other small terrestrial invertebrates, are particularly susceptible to climatic stress. Physiological adjustments to cope with the environment (i.e., acclimation) together with genetic makeup eventually determine the tolerance of a species to climatic extremes, and constrain its distribution. Temperature and desiccation resistance in insects are both conditioned by acclimation and may be interconnected, particularly for species inhabiting xeric environments. We determined the effect of temperature acclimation on desiccation resistance of the peach fruit fly (Bactrocera zonata, Tephritidae) - an invasive, polyphagous pest, currently spreading through both xeric and mesic environments in Africa and the Eurasian continent. Following acclimation at three constant temperatures (20, 25, and 30°C), the survival of adult flies deprived of food and water was monitored in extreme dry and humid conditions (<10 and >90% relative humidity, respectively). We found that flies acclimated at higher temperatures were significantly heavier, and contained more lipids and protein. Acclimation temperature significantly and similarly affected the survival of males and females at both high and low humidity conditions. In both cases, flies maintained at 30°C survived longer compared to 20 and 25°C - habituated counterparts. Regardless of the effect of acclimation temperature on survival, overall life expectancy was significantly shortened when flies were assayed under desiccating conditions. Additionally, our experiments indicate no significant difference in survival patterns between males and females, and that acclimation temperature had similar effects after both short (5-10 days) and long (11-20 days) acclimation periods. We conclude that acclimation at 30°C prolongs the survival of B. zonata, regardless of ambient humidity levels. Temperature probably affected survival through modulating feeding and metabolism, allowing for accumulation of larger energetic reserves, which in turn, promoted a greater ability to resist starvation, and possibly desiccation as well. Our study set the grounds for understanding the phenotypic plasticity of B. zonata from the hydric perspective, and for further evaluating the invasion potential of this pest.

Shifting microbiomes complement life stage transitions and diet of the bird parasite Philornis downsi from the Galapagos Islands.

Domestication disconnects an animal from its natural environment and diet, imposing changes in the attendant microbial community. We examine these changes in Philornis downsi (Muscidae), an invasive parasitic fly of land birds in the Galapagos Islands. Using a 16S rDNA profiling approach we studied the microbiome of larvae and adults of wild and laboratory-reared populations. These populations diverged in their microbiomes, significantly more so in larval than in adult flies. In field-collected second-instar larvae, Klebsiella (70.3%) was the most abundant taxon, while in the laboratory Ignatzschineria and Providencia made up 89.2% of the community. In adults, Gilliamella and Dysgonomonas were key members of the core microbiome of field-derived females and males but had no or very low representation in the laboratory. Adult flies harbour sex-specific microbial consortia in their gut, as male core microbiomes were significantly dominated by Klebsiella. Thus, P. downsi microbiomes are dynamic and shift correspondingly with life cycle and diet. Sex-specific foraging behaviour of adult flies and nest conditions, which are absent in the laboratory, may contribute to shaping distinct larval, and adult male and female microbiomes. We discuss these findings in the context of microbe-host co-evolution and the implications for control measures.

Coxiella-Like Endosymbiont of Rhipicephalus sanguineus Is Required for Physiological Processes During Ontogeny.

Obligatory hematophagous arthropods such as lice, bugs, flies, and ticks harbor bacterial endosymbionts that are expected to complement missing essential nutrients in their diet. Genomic and some experimental evidence support this expectation. Hard ticks (Acari: Ixodidae) are associated with several lineages of bacterial symbionts, and very few were experimentally shown to be essential to some aspects of tick's fitness. In order to pinpoint the nature of interactions between hard ticks and their symbionts, we tested the effect of massive elimination of Coxiella-like endosymbionts (CLE) by antibiotics on the development and fitness of the brown dog tick (Rhipicephalus sanguineus). Administration of ofloxacin to engorged (blood fed) nymphs resulted in significant and acute reduction of their CLE loads - an effect that also persisted in subsequent life stages (aposymbiotic ticks). As a result, the post-feeding development of aposymbiotic female (but not male) nymphs was delayed. Additionally, aposymbiotic adult females needed a significantly prolonged feeding period in order to replete (detach from host), and had reduced engorgement weight and a lower capacity to produce eggs. Consequently, their fecundity and fertility were significantly reduced. Eggs produced by aposymbiotic females were free of CLE, and the resulting aposymbiotic larvae were unable to feed successfully. Our findings demonstrate that the observed fitness effects are due to CLE reduction and not due to antibiotic administration. Additionally, we suggest that the contribution of CLE is not mandatory for oocyte development and embryogenesis, but is required during feeding in females, when blood meal processing and tissue buildup are taking place. Presumably, under these extreme physiological demands, CLE contribute to R. sanguineus through supplementing essential micro- and macronutrients. Further nutrient complementary studies are required to support this hypothesis.

Behavioral Responses of the Invasive Fly Philornis downsi to Stimuli from Bacteria and Yeast in the Laboratory and the Field in the Galapagos Islands.

Philornis downsi Dodge and Aitken (Diptera: Muscidae) is an avian parasitic fly that has invaded the Galapagos archipelago and exerts an onerous burden on populations of endemic land birds. As part of an ongoing effort to develop tools for the integrated management of this fly, our objective was to determine its long- and short-range responses to bacterial and fungal cues associated with adult P. downsi. We hypothesized that the bacterial and fungal communities would elicit attraction at distance through volatiles, and appetitive responses upon contact. Accordingly, we amplified bacteria from guts of adult field-caught flies and from bird feces, and yeasts from fermenting papaya juice (a known attractant of P. downsi), on selective growth media, and assayed the response of flies to these microbes or their exudates. In the field, we baited traps with bacteria or yeast and monitored adult fly attraction. In the laboratory, we used the proboscis extension response (PER) to determine the sensitivity of males and females to tarsal contact with bacteria or yeast. Long range trapping efforts yielded two female flies over 112 trap-nights (attracted by bacteria from bird feces and from the gut of adult flies). In the laboratory, tarsal contact with stimuli from gut bacteria elicited significantly more responses than did yeast stimuli. We discuss the significance of these findings in context with other studies in the field and identify targets for future work.

The host fruit amplifies mutualistic interaction between Ceratitis capitata larvae and associated bacteria.

BACKGROUND: The Mediterranean fruit fly Ceratitis capitata is a major pest in horticulture. The development of fly larvae is mediated by bacterial decay in the fruit tissue. Despite the importance of bacteria on larval development, very little is known about the interaction between bacteria and larvae in their true ecological context. Understanding their relationship and inter-dependence in the host fruit is important for the development of new pest control interfaces to deal with this pest. RESULTS: We find no negative effects on egg hatch or larval development brought about by the bacterial isolates tested. The various symbionts inhabiting the fly's digestive system differ in their degree of contribution to the development of fly larvae depending on the given host and their sensitivity to induced inhibition caused by female produced antimicrobial peptides. These differences were observed not only at the genus or species level but also between isolates of the same species. We demonstrate how the microbiota from the mother's gut supports the development of larvae in the fruit host and show that larvae play a major role in spreading the bacterial contagion in the infected fruit itself. In addition, we present (for the first time) evidence for horizontal transfer of bacteria between larvae of different maternal origin that develop together in the same fruit. CONCLUSIONS: Larvae play a major role in the spread and shaping of the microbial population in the fruit. The transfer of bacteria between different individuals developing in the same fruit suggests that the infested fruit serves as a microbial hub for the amplification and spread of bacterial strains between individuals.

Symbiotic bacteria affect oviposition behavior in the olive fruit fly Bactrocera oleae.

Microbial associations are widespread across the insects. In the olive fruit fly Bactrocera oleae (Diptera: Tephritidae), vertically transmitted gut symbionts contribute to larval development inside the olive host, and to adult nutrition. Nevertheless, their effect on behavioural decisions of adults is unknown. In this study, we show that symbiotic bacteria affect oviposition behaviour in B. oleae. We studied the effect of different fruits as hosts and different gut-bacteria as gut-symbionts on oviposition attempts and fly development in B. oleae. Untreated flies that had native gut-symbionts attempted oviposition significantly more times than axenic flies as well as flies treated with medfly-associated Pantoea or Klebsiella bacteria. Axenic flies provided with a diet containing the homogenized gut of symbiotic flies recovered the same number of oviposition attempts as their symbiotic counterparts. As for as the different hosts, green olives (unripe) and grapes were preferred while black olives (ripe) elicited the least number of oviposition attempts, with an interactive effect of host and bacterial treatments. It appears that both the host attributes and the native gut-symbionts drive oviposition preference towards green olives in B. oleae. Moreover, both bacterial treatments and hosts significantly affected the development of B. oleae larvae. Though grapes elicited as many oviposition attempts as green olives, they yielded no pupae. Taken together, our results suggest that the intimate association between B. oleae and their gut-microbes, extends beyond nutritional support to behaviour.

Host-specific associations affect the microbiome of Philornis downsi, an introduced parasite to the Galápagos Islands.

The composition and diversity of bacteria forming the microbiome of parasitic organisms have implications for differential host pathogenicity and host-parasite co-evolutionary interactions. The microbiome of pathogens can therefore have consequences that are relevant for managing disease prevalence and impact on affected hosts. Here, we investigate the microbiome of an invasive parasitic fly Philornis downsi, recently introduced to the Galápagos Islands, where it poses extinction threat to Darwin's finches and other land birds. Larvae infest nests of Darwin's finches and consume blood and tissue of developing nestlings, and have severe mortality impacts. Using 16s rRNA sequencing data, we characterize the bacterial microbiota associated with P. downsi adults and larvae sourced from four finch host species, inhabiting two islands and representing two ecologically distinct groups. We show that larval and adult microbiomes are dominated by the phyla Proteobacteria and Firmicutes, which significantly differ between life stages in their distributions. Additionally, bacterial community structure significantly differed between larvae retrieved from strictly insectivorous warbler finches (Certhidea olivacea) and those parasitizing hosts with broader dietary preferences (ground and tree finches, Geospiza and Camarhynchus spp., respectively). Finally, we found no spatial effects on the larval microbiome, as larvae feeding on the same host (ground finches) harboured similar microbiomes across islands. Our results suggest that the microbiome of P. downsi changes during its development, according to dietary composition or nutritional needs, and is significantly affected by host-related factors during the larval stage. Unravelling the ecological significance of bacteria for this parasite will contribute to the development of novel, effective control strategies.

Transcriptomic responses of the olive fruit fly Bactrocera oleae and its symbiont Candidatus Erwinia dacicola to olive feeding.

The olive fruit fly, Bactrocera oleae, is the most destructive pest of olive orchards worldwide. The monophagous larva has the unique capability of feeding on olive mesocarp, coping with high levels of phenolic compounds and utilizing non-hydrolyzed proteins present, particularly in the unripe, green olives. On the molecular level, the interaction between B. oleae and olives has not been investigated as yet. Nevertheless, it has been associated with the gut obligate symbiotic bacterium Candidatus Erwinia dacicola. Here, we used a B.oleae microarray to analyze the gene expression of larvae during their development in artificial diet, unripe (green) and ripe (black) olives. The expression profiles of Ca. E. dacicola were analyzed in parallel, using the Illumina platform. Several genes were found overexpressed in the olive fly larvae when feeding in green olives. Among these, a number of genes encoding detoxification and digestive enzymes, indicating a potential association with the ability of B. oleae to cope with green olives. In addition, a number of biological processes seem to be activated in Ca. E. dacicola during the development of larvae in olives, with the most notable being the activation of amino-acid metabolism.

Draft Genome Sequence of the Bactrocera oleae Symbiont "Candidatus Erwinia dacicola".

"Candidatus Erwinia dacicola" is a Gammaproteobacterium that forms a symbiotic association with the agricultural pest Bactrocera oleae Here, we present a 2.1-Mb draft hybrid genome assembly for "Ca. Erwinia dacicola" generated from single-cell and metagenomic data.

Symbiotic bacteria enable olive fly larvae to overcome host defences.

Ripe fruit offer readily available nutrients for many animals, including fruit fly larvae (Diptera: Tephritidae) and their associated rot-inducing bacteria. Yet, during most of their ontogeny, fruit remain chemically defended and effectively suppress herbivores and pathogens by high levels of secondary metabolites. Olive flies (Bactrocera oleae) are uniquely able to develop in unripe olives. Unlike other frugivorous tephritids, the larvae maintain bacteria confined within their midgut caeca. We examined the interaction between larvae, their associated bacteria, and fruit chemical defence, hypothesizing that bacterial contribution to larval development is contingent on the phenology of fruit defensive chemistry. We demonstrate that larvae require their natural complement of bacteria (Candidatus Erwinia dacicola: Enterobacteriaceae) in order to develop in unripe olives. Conversely, when feeding on ripe fruit, larval development proceeds independently of these bacteria. Our experiments suggest that bacteria counteract the inhibitory effect of oleuropein-the principal phenolic glycoside in unripe olives. In light of these results, we suggest that the unique symbiosis in olive flies, compared with other frugivorous tephritids, is understood by considering the relationship between the fly, bacteria and fruit chemistry. When applied in an evolutionary context, this approach may also point out the forces which shaped symbioses across the Tephritidae.

Phylogenetic, metabolic, and taxonomic diversities shape mediterranean fruit fly microbiotas during ontogeny.

The Mediterranean fruit fly (medfly) (Ceratitis capitata) lays eggs in fruits, where larvae subsequently develop, causing large-scale agricultural damage. Within its digestive tract, the fly supports an extended bacterial community that is composed of multiple strains of a variety of enterobacterial species. Most of these bacteria appear to be functionally redundant, with most strains sustaining diazotrophy and/or pectinolysis. At least some of these bacteria were shown to be vertically inherited, but colonization, structural, and metabolic aspects of the community's dynamics have not been investigated. We used fluorescent in situ hybridization, metabolic profiling, plate cultures, and pyrosequencing to show that an initial, egg-borne, diverse community expands throughout the fly's life cycle. While keeping "core" diazotrophic and pectinolytic functions, it also harbors diverse and fluctuating populations that express varied metabolic capabilities. We suggest that the metabolic and compositional plasticity of the fly's microbiota provides potential adaptive advantages to the medfly host and that its acquisition and dynamics are affected by mixed processes that include stochastic effects, host behavior, and molecular barriers.

Give us the tools and we will do the job: symbiotic bacteria affect olive fly fitness in a diet-dependent fashion.

Olive flies (Bactrocera oleae) are intimately associated with bacteria throughout their life cycle, and both larvae and adults are morphologically adapted for housing bacteria in the digestive tract. We tested the hypothesis that these bacteria contribute to the adult fly's fitness in a diet-dependent fashion. We predicted that when dietary protein is superabundant, bacterial contribution will be minimal. Conversely, in the absence of protein, or when only non-essential amino acids are present (as in the fly's natural diet), we predicted that bacterial contribution to fitness will be significant. Accordingly, we manipulated diet and the presence of bacteria in female olive flies, and monitored fecundity--an indirect measure of fitness. Bacteria did not affect fecundity when females were fed a nutritionally poor diet of sucrose, or a protein-rich, nutritionally complete diet. However, when females were fed a diet containing non-essential amino acids as the sole source of amino nitrogen, egg production was significantly enhanced in the presence of bacteria. These results suggest that bacteria were able to compensate for the skewed amino acid composition of the diet and may be indispensable for wild adult olive flies that subsist mainly on nitrogen-poor resources such as honeydew.