Potential of a fly gut microbiota incorporated gel-based larval diet for rearing Bactrocera dorsalis (Hendel).
ABSTRACT: BACKGROUND:The Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is an important polyphagous pest of horticultural produce. The sterile insect technique (SIT) is a proven control method against many insect pests, including fruit flies, under area-wide pest management programs. High quality mass-rearing process and the cost-effective production of sterile target species are important for SIT. Irradiation is reported to cause severe damage to the symbiotic community structure in the mid gut of fruit fly species, impairing SIT success. However, studies have found that target-specific manipulation of insect gut bacteria can positively impact the overall fitness of SIT-specific insects. RESULTS:Twelve bacterial genera were isolated and identified from B. dorsalis eggs, third instars larval gut and adults gut. The bacterial genera were Acinetobacter, Alcaligenes, Citrobacter, Pseudomonas, Proteus, and Stenotrophomonas, belonging to the Enterobacteriaceae family. Larval diet enrichment with the selected bacterial isolate, Proteus sp. was found to improve adult emergence, percentage of male, and survival under stress. However, no significant changes were recorded in B. dorsalis egg hatching, pupal yield, pupal weight, duration of the larval stage, or flight ability. CONCLUSIONS:These findings support the hypothesis that gut bacterial isolates can be used in conjunction with SIT. The newly developed gel-based larval diet incorporated with Proteus sp. isolates can be used for large-scale mass rearing of B. dorsalis in the SIT program.
Project description:The Mediterranean fruit fly (medfly), Ceratitis capitata, is a pest of worldwide substantial economic importance, as well as a Tephritidae model for sterile insect technique (SIT) applications. The latter is partially due to the development and utilization of genetic sexing strains (GSS) for this species, such as the Vienna 8 strain, which is currently used in mass rearing facilities worldwide. Improving the performance of such a strain both in mass rearing facilities and in the field could significantly enhance the efficacy of SIT and reduce operational costs. Recent studies have suggested that the manipulation of gut symbionts can have a significant positive effect on the overall fitness of insect strains. We used culture-based approaches to isolate and characterize gut-associated bacterial species of the Vienna 8 strain under mass rearing conditions. We also exploited one of the isolated bacterial species, Enterobacter sp., as dietary supplement (probiotic) to the larval diet, and we assessed its effects on fitness parameters under the standard operating procedures used in SIT operational programs. Probiotic application of Enterobacter sp. resulted in improvement of both pupal and adult productivity, as well as reduced rearing duration, particularly for males, without affecting pupal weight, sex ratio, male mating competitiveness, flight ability and longevity under starvation.
Project description:<h4>Background</h4>The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera, Tephritidae) is the most significant insect pest of Australian horticulture. Bactrocera tryoni is controlled using a range of tools including the Sterile Insect Technique (SIT). Mass-rearing and irradiation of pupae in SIT can reduce the fitness and quality of the released sterile insects. Studies have also showed reduced microbial gut diversity in domesticated versus wild tephritids.<h4>Results</h4>Transmission electron microscopy confirmed the presence of the bacterial isolates in the mid-gut of mass-reared larvae, and plate counts from individual larval guts showed increased numbers of bacteria in supplemented larvae. Several developmental and fitness parameters were tested including larval development time (egg-hatch to pupation), pupal weight, emergence, flight ability, sex-ratio, and time to adult eclosion (egg-hatch to adult eclosion). Enterobacter sp. and Asaia sp. shortened larval development time, while this was delayed by Lactobacillus sp., Leuconostoc sp. and a blend of all four bacteria. The mean time from egg hatch to adult eclosion was significantly reduced by Leuconostoc sp. and the blend for males and females, indicating that the individual bacterium and consortium affect flies differently depending on the life stage (larval or pupal). There was no impact of bacterial supplemented larvae on pupal weight, emergence, flight ability, or sex ratio.<h4>Conclusions</h4>Our findings show that bacteria fed to the larval stage of B. tryoni can impart fitness advantages, but the selection of probiotic strains (individual or a consortium) is key, as each have varying effects on the host. Bacteria added to the larval diet particularly Leuconostoc sp. and the blend have the capacity to reduce costs and increase the number of flies produced in mass-rearing facilities by reducing time to adult eclosion by 1.3 and 0.8 mean days for males, and 1.2 and 0.8 mean days for females.
Project description:Larval diets used for artificial rearing can have a significant effect on insect biology. The Queensland fruit fly (aka "Qfly"), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), is one of the greatest challenges for fruit growers in Australia. The sterile insect technique (SIT) is being developed to manage outbreaks in regions that remain free of Qfly and to reduce populations in regions where this species is endemic. Factory scale rearing is essential for SIT; however, artificial larval diets are known to affect the microbiome of Qfly, which may then affect fly performance. In this study, high-throughput Illumina sequencing was used to assess the Qfly microbiome in colonies reared, for five generations from nature, on two common artificial diets (carrot and gel). At generation five (G5), the microbiome was assessed in larvae, pupae, adult males and adult females and standard fly quality control parameters were assessed together with additional performance measures of mating propensity and survival under nutritional stress. At the genus level, bacterial communities were significantly different between the colonies reared on the two larval diets. However, communities converged at Phyla to family taxonomic levels. Bacterial genera of Morganella, Citrobacter, Providencia, and Burkholderia were highly abundant in all developmental stages of Qfly reared on the gel diet, when compared to the carrot diet. Despite abundance of these genera, a greater percentage of egg hatching, heavier pupal weight and a higher percentage of fliers were found in the Qfly reared on the gel diet. Mating propensity and survival under nutritional stress was similar for adult Qfly that had been reared on the two larval diets. Overall, our findings demonstrate that the artificial larval diet strongly influences the microbiome and quality control measures of Qfly, with likely downstream effects on performance of flies released in SIT programs.
Project description:<h4>Background</h4>Insect pests belonging to genus Bactrocera sp. (Diptera: Tephritidae) pose major biotic stress on various fruits and vegetable crops around the world. Zeugodacus and Bactrocera sp. are associated with diverse bacterial communities which play an important role in the fitness of sterile insects. The wild populations of melon fly, Zeugodacus cucurbitae (Coquillett) and Oriental fruit fly, Bactrocera dorsalis (Hendel) were collected from pumpkin and mango fields, respectively. The laboratory populations of Z. cucurbitae and B. dorsalis were mass-reared on bottle gourd and sweet banana, respectively. Bacterial communities present in the gut of wild and mass-reared mature (~?12?days old) and newly emerged (<?1?h after emergence) male and female adults of Z. cucurbitae and B. dorsalis were assessed. We used Illumina HiSeq next-generation sequencing of 16S rRNA gene to profile the gut bacterial communities of wild and mass-reared mature and newly emerged Z. cucurbitae and B. dorsalis adults.<h4>Results</h4>We found diverse bacterial composition in the gut of wild and mass-reared Z. cucurbitae (ZC) and B. dorsalis (BD) with varied relative abundance. Few taxonomic groups were common to both the species. The most dominant phyla in all samples of Z. cucurbitae and B. dorsalis adults were Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. The phylum Proteobacteria occurred more in wild Z. cucurbitae (~?87.72%) and B. dorsalis (~?83.87%) as compared to mass-reared Z. cucurbitae (64.15%) and B. dorsalis (~?80.96%). Higher relative abundance of Phylum Firmicutes was observed in mass-reared fruit fly than wild adults. Cyanobacteria/Chloroplast and Actinobacteria were also present with very low relative abundance in both wild as well as mass-reared melon fly and Oriental fruit fly. Enterobacteriaceae (61.21%) was dominant family in the gut of both wild and mass-reared adults. Providencia and Lactococcus were dominant genera with varied relative abundance in wild as well as in mass-reared mature and newly emerged fruit fly adults of both species. Some of the genera like Morganella and Serratia were only detected in mass-reared mature and newly emerged Z. cucurbitae and B. dorsalis adults. Principal Coordinate Analysis (PCoA) showed that fruit fly adult samples were grouped based on species and age of the adults while no grouping was observed on the basis of sex of the adult fruit fly.<h4>Conclusions</h4>The gut bacterial communities associated with wild and mass-reared mature and newly emerged adults of Z. cucurbitae and B. dorsalis showed variation that depends on species and age of the insects. Understanding the gut microbiota of wild and mass-reared Z. cucurbitae and B. dorsalis using high throughput technology will help to illustrate microbial diversity and this information could be used to develop efficient mass-rearing protocols for successful implementation of sterile insect technique (SIT).
Project description:<h4>Background</h4>Mass-rearing, domestication and gamma irradiation of tephritid fruit flies used in sterile insect technique (SIT) programmes can negatively impact fly quality and performance. Symbiotic bacteria supplied as probiotics to mass-reared fruit flies may help to overcome some of these issues. However, the effects of tephritid ontogeny, sex, diet and irradiation on their microbiota are not well known.<h4>Results</h4>We have used next-generation sequencing to characterise the bacterial community composition and structure within Queensland fruit fly, Bactrocera tryoni (Froggatt), by generating 16S rRNA gene amplicon libraries derived from the guts of 58 individual teneral and mature, female and male, sterile and fertile adult flies reared on artificial larval diets in a laboratory or mass-rearing environment, and fed either a full adult diet (i.e. sugar and yeast hydrolysate) or a sugar only adult diet. Overall, the amplicon sequence read volume in tenerals was low and smaller than in mature adult flies. Operational taxonomic units (OTUs), belonging to the families Enterobacteriaceae (8 OTUs) and Acetobacteraceae (1 OTU) were most prevalent. Enterobacteriaceae dominated laboratory-reared tenerals from a colony fed a carrot-based larval diet, while Acetobacteraceae dominated mass-reared tenerals from a production facility colony fed a lucerne chaff based larval diet. As adult flies matured, Enterobacteriaceae became dominant irrespective of larval origin. The inclusion of yeast in the adult diet strengthened this shift away from Acetobacteraceae towards Enterobacteriaceae. Interestingly, irradiation increased 16S rRNA gene sequence read volume.<h4>Conclusions</h4>Our findings suggest that bacterial populations in fruit flies experience significant bottlenecks during metamorphosis. Gut bacteria in teneral flies were less abundant and less diverse, and impacted by colony origin. In contrast, mature adult flies had selectively increased abundances for some gut bacteria, or acquired these bacteria from the adult diet and environment. Furthermore, irradiation augmented bacterial abundance in mature flies. This implies that either some gut bacteria were compensating for damage caused by irradiation or irradiated flies had lost their ability to regulate bacterial load. Our findings suggest that the adult stage prior to sexual maturity may be ideal to target for probiotic manipulation of fly microbiota to increase fly performance in SIT programmes.
Project description:BACKGROUND:Gut microbiota affects tephritid (Diptera: Tephritidae) fruit fly development, physiology, behavior, and thus the quality of flies mass-reared for the sterile insect technique (SIT), a target-specific, sustainable, environmentally benign form of pest management. The Queensland fruit fly, Bactrocera tryoni (Tephritidae), is a significant horticultural pest in Australia and can be managed with SIT. Little is known about the impacts that laboratory-adaptation (domestication) and mass-rearing have on the tephritid larval gut microbiome. Read lengths of previous fruit fly next-generation sequencing (NGS) studies have limited the resolution of microbiome studies, and the diversity within populations is often overlooked. In this study, we used a new near full-length (>?1300 nt) 16S rRNA gene amplicon NGS approach to characterize gut bacterial communities of individual B. tryoni larvae from two field populations (developing in peaches) and three domesticated populations (mass- or laboratory-reared on artificial diets). RESULTS:Near full-length 16S rRNA gene sequences were obtained for 56 B. tryoni larvae. OTU clustering at 99% similarity revealed that gut bacterial diversity was low and significantly lower in domesticated larvae. Bacteria commonly associated with fruit (Acetobacteraceae, Enterobacteriaceae, and Leuconostocaceae) were detected in wild larvae, but were largely absent from domesticated larvae. However, Asaia, an acetic acid bacterium not frequently detected within adult tephritid species, was detected in larvae of both wild and domesticated populations (55 out of 56 larval gut samples). Larvae from the same single peach shared a similar gut bacterial profile, whereas larvae from different peaches collected from the same tree had different gut bacterial profiles. Clustering of the Asaia near full-length sequences at 100% similarity showed that the wild flies from different locations had different Asaia strains. CONCLUSIONS:Variation in the gut bacterial communities of B. tryoni larvae depends on diet, domestication, and horizontal acquisition. Bacterial variation in wild larvae suggests that more than one bacterial species can perform the same functional role; however, Asaia could be an important gut bacterium in larvae and warrants further study. A greater understanding of the functions of the bacteria detected in larvae could lead to increased fly quality and performance as part of the SIT.
Project description:BACKGROUND:The gut bacteria of tephritid fruit flies play prominent roles in nutrition, reproduction, maintenance and ecological adaptations of the host. Here, we adopted an approach based on direct observation of symbiotic or axenic flies feeding on dishes seeded with drops of full diet (containing all amino acids) or full diet supplemented with bacteria at similar concentrations to explore the effects of intestinal bacteria on foraging decision and fitness of Bactrocera dorsalis. RESULTS:The results show that intestinal probiotics elicit beneficial foraging decision and enhance the female reproduction fitness and survival of B. dorsalis (symbiotic and axenic), yet preferences for probiotic diets were significantly higher in axenic flies to which they responded faster compared to full diet. Moreover, females fed diet supplemented with Pantoea dispersa and Enterobacter cloacae laid more eggs but had shorter lifespan while female fed Enterococcus faecalis and Klebsiella oxytoca enriched diets lived longer but had lower fecundity compared to the positive control. Conversely, flies fed sugar diet (negative control) were not able to produce eggs, but lived longer than those from the positive control. CONCLUSIONS:These results suggest that intestinal bacteria can drive the foraging decision in a way which promotes the reproduction and survival of B. dorsalis. Our data highlight the potentials of gut bacterial isolates to control the foraging behavior of the fly and empower the sterile insect technique (SIT) program through the mass rearing.
Project description:We modeled the cost-effectiveness of rearing Anastrepha ludens, a major fruit fly pest currently mass reared for sterilization and release in pest control programs implementing the sterile insect technique (SIT). An optimization model was generated by combining response surface models of artificial diet cost savings with models of A. ludens pupation, pupal weight, larval development time and adult emergence as a function of mixtures of yeast, a costly ingredient, with corn flour and corncob fractions in the diet. Our model revealed several yeast-reduced mixtures that could be used to prepare diets that were considerably cheaper than a standard diet used for mass rearing. Models predicted a similar production of insects (pupation and adult emergence), with statistically similar pupal weights and larval development times between yeast-reduced diets and the standard mass rearing diet formulation. Annual savings from using the modified diets could be up to 5.9% of the annual cost of yeast, corn flour and corncob fractions used in the standard diet, representing a potential saving of US $27.45 per ton of diet (US $47,496 in the case of the mean annual production of 1,730.29 tons of artificial diet in the Moscafrut mass rearing facility at Metapa, Chiapas, Mexico). Implementation of the yeast-reduced diet on an experimental scale at mass rearing facilities is still required to confirm the suitability of new mixtures of artificial diet for rearing A. ludens for use in SIT. This should include the examination of critical quality control parameters of flies such as adult flight ability, starvation resistance and male sexual competitiveness across various generations. The method used here could be useful for improving the cost-effectiveness of invertebrate or vertebrate mass rearing diets worldwide.
Project description:<h4>Background</h4>Insect species have established sophisticated symbiotic associations with diverse groups of microorganisms including bacteria which have been shown to affect several aspects of their biology, physiology, ecology and evolution. In addition, recent studies have shown that insect symbionts, including those localized in the gastrointestinal tract, can be exploited for the enhancement of sterile insect technique (SIT) applications against major insect pests such as the Mediterranean fruit fly (medfly) Ceratitis capitata. We previously showed that Enterobacter sp. AA26 can be used as probiotic supplement in medfly larval diet improving the productivity and accelerating the development of the VIENNA 8 genetic sexing strain (GSS), which is currently used in large scale operational SIT programs worldwide.<h4>Results</h4>Enterobacter sp. AA26 was an adequate nutritional source for C. capitata larvae, comprising an effective substitute for brewer's yeast. Incorporating inactive bacterial cells in the larval diet conferred a number of substantial beneficial effects on medfly biology. The consumption of bacteria-based diet (either as full or partial yeast replacement) resulted in decreased immature stages mortality, accelerated immature development, increased pupal weight, and elongated the survival under stress conditions. Moreover, neither the partial nor the complete replacement of yeast with Enterobacter sp. AA26 had significant impact on adult sex ratio, females' fecundity, adults' flight ability and males' mating competitiveness. The absence of both yeast and Enterobacter sp. AA26 (deprivation of protein source and possible other important nutrients) from the larval diet detrimentally affected the larval development, survival and elongated the immature developmental duration.<h4>Conclusions</h4>Enterobacter sp. AA26 dry biomass can fully replace the brewer's yeast as a protein source in medfly larval diet without any effect on the productivity and the biological quality of reared medfly of VIENNA 8 GSS as assessed by the FAO/IAEA/USDA standard quality control tests. We discuss this finding in the context of mass-rearing and SIT applications.
Project description:BACKGROUND:Symbiotic interactions between insects and bacteria have been associated with a vast variety of physiological, ecological and evolutionary consequences for the host. A wide range of bacterial communities have been found in association with the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), an important pest of cultivated fruit in most regions of the world. We evaluated the diversity of gut bacteria in B. dorsalis specimens from several populations in Kenya and investigated the roles of individual bacterial isolates in the development of axenic (germ-free) B. dorsalis fly lines and their responses to the entomopathogenic fungus, Metarhizium anisopliae. RESULTS:We sequenced 16S rRNA to evaluate microbiomes and coupled this with bacterial culturing. Bacterial isolates were mono-associated with axenic B. dorsalis embryos. The shortest embryonic development period was recorded in flies with an intact gut microbiome while the longest period was recorded in axenic fly lines. Similarly, larval development was shortest in flies with an intact gut microbiome, in addition to flies inoculated with Providencia alcalifaciens. Adult B. dorsalis flies emerging from embryos that had been mono-associated with a strain of Lactococcus lactis had decreased survival when challenged with a standard dosage of M. anisopliae ICIPE69 conidia. However, there were no differences in survival between the germ-free lines and flies with an intact microbiome. CONCLUSIONS:These findings will contribute to the selection of probiotics used in artificial diets for B. dorsalis rearing and the development of improved integrated pest management strategies based on entomopathogenic fungi.