Intestinal probiotics motivate the foraging decision and promote fecundity and survival of Bactrocera dorsalis (Diptera: Tephritidae)

The 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 showed that intestinal probiotics motivate 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 positive control. Conversely, flies fed sugar diet (negative control) were not able to produce any eggs but lived longer than those from the positive control. shape which


Abstract 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 showed that intestinal probiotics motivate 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 positive control. Conversely, flies fed sugar diet (negative control) were not able to produce any eggs but lived longer than those from the positive control.

Conclusions
These results suggest that intestinal bacteria can shape profitable foraging decision in a way which promotes the reproduction and survival of B. dorsalis. Our data could empower the sterile insect technique (SIT) through the mass rearing program.

Background
Insects are capable of shaping their foraging behavior and food consumption in a way that favors their growth and reproduction [1][2][3]. In this light, the substrate specific chemoreceptors are key factors in the responses of insects to environmental stimuli such as food and whose latency to respond depends on the nutritional status of the insect [4][5][6].
Many insects are associated with diverse extracellular microorganisms that can be found, among other sites on the exoskeleton, in the hemocoel, or in the gut lumen [7], and with intracellular microorganisms that populate specialized tissues or organs such as bacteriocytes [8]. Their relationships with their hosts are often linked to their status as intra or extra-cellular symbionts and range from parasitic to mutualistic [9][10][11]. The intra-cellular symbionts are often considered as obligate ones, for, they cannot live outside the host, so they are transmitted vertically (from mother to progeny) [12]. Both, intra and extra-cellular symbionts play a variety of functions on host biology, survival and foraging activity [13][14][15][16][17][18]. For example, the gut microbiome of the vinegar fly Drosophila melanogaster was shown to indirectly influence the foraging behavior of the host by modulating their immune system, lipid and carbohydrate accumulation [19][20][21] and olfactory sensitivity for the own benefits of bacteria [22]. Gut bacteria was shown to be implicated in the resistance and susceptibility of Callosobruchus maculatus to dichlorvos and essential oil, respectively [23]. Intestinal probiotic Klebsiella oxytoca (member of Enterobacteriaceae family) restored the ecological fitness of irradiated B. dorsalis males by promoting food intake and metabolic activities [24].
Furthermore, some insects possess the ability to cultivate and digest their own gut bacteria as additional protein source to fuel their metabolic functions [25,26]. These evidences somehow show that gut bacteria and the host nutritional status are intimately associated in driving the fitness and foraging behavior of the insect.
Tephritidae fruit flies (Diptera: Tephritidae) harbour bacterial communities dominated by species of Enterobacteriacae [27]. These microbes have been shown in other fruit flies to be involved in host longevity [28,29], Nitrogen fixation [30], reproductive success [31,32], protection from pathogens [33] and detoxification of xenobiotics [27,34,35]. In order to survive and reproduce, these flies should acquire nutrients (carbohydrate and protein) from the environment through their foraging activity. Overall, the presence of gut microbiome in adult flies contributes to their nutrition, by providing essential amino acids missing from their diets. For example, symbiotic olive flies Bactrocera oleae have been able to produce eggs when fed only non-essential amino acids, while aposymbiotic flies have been unable to do so [28,29]. Moreover, bacteria supplemented diets were shown to increase the life expectancy and fecundity of the flies in comparison to normal diets. For instance, female olive flies fed sugar diet inoculated with Pseudomonas putida laid more eggs than those fed sugar diet only [36]. Similarly, Enterobacter agglomerans and Klebsiella pneumoniae improved the dietary outcomes of yeast-based foods that positively affected the longevity and female reproductive capacity of the Mediterranean fly Ceratitis capitata [37].
The oriental fruitfly Bactrocera dorsalis (Diptera: Tephritidae:) is a serious pest which causes considerable loss of cultivated crops worldwide and attacks over 350 host species [38,39]. The bacterial populations inhabiting the gut and reproductive organs of this pest were shown to play important roles in host physiology and behavior [38,[40][41][42].
In the present study, we hypothesized that intestinal probiotics motivate the foraging decision and enhance the reproduction fitness and survival of B. dorsalis. The method consisted in offering to protein starved flies, symbiotic or axenic, a choice between full diets (containing all amino acids, sugar and minerals) or full diets supplemented with individual bacterial isolates (Pantoea dispersa, Enterobacter cloacae, Enterococcus faecalis and Klebsiella oxytoca). In the first experimental setting, we evaluated the effects of the presence or absence of bacteria on the responses of the flies (landing latency, food choice and ingestion) to the diets presented, while in the second experiment, we evaluated the fecundity and longevity of females fed full and probiotic diets, respectively. We predicted that, as deprived of their gut bacteria, axenic flies would consistently chose the most profitable diet to sustain their maintenance and reproduction fitness.

Discussion
The relationships between the flies and their gut microbiome are interlaced in complex webs in which gut bacteria provide essential nutrients to the host and enhance its reproductive capacity and survival [1,36]. The alteration of gut bacteria generally results in the disruption of physiological functions of the host fly. In order to survive and reproduce under such condition, the flies optimally forage on diets which offer higher profitability in terms of nutrient intakes [1,3]. In a similar experimental setting using symbiotic and aposymbiotic flies, the suppression of gut microbiome by antibiotics treatment disrupted the foraging behavior of the oriental fruit fly B. dorsalis and constrained the fly to consume many food droplets at the cost of extending the foraging duration [18]. In our experiment, by creating different feeding environments, we demonstrated how the supplementation of gut bacteria isolates affected the foraging behavior, diet ingestion and fitness parameters of B. dorsalis in a significant manner. Axenic flies (males and females) showed significant preference for the probiotic meal to which they responded faster (compared to full diet) and maximized the diet ingestion to ensure their maintenance and reproductive fitness. Previously, gut bacterial isolates (Enterococcus cloacae, Citrobacter freundii Bacillus cereus, Enterobacter, Klebsiella etc.) were demonstrated to produce chemical substances which attracted Bactrocera dorsalis andBactrocera cucurbitae, toward available food source [43][44][45]. In addition to being highly attracted toward the probiotic diets, axenic flies were compelled to ingest as many food drops as possible to become satiated (Fig. 3 A & B). This finding suggests that bacterial isolates may facilitate the access and assimilation of the available nutrients from the diets [32], either by increasing the food palatability or positively modulating digestive enzymes [29]. Previous studies also demonstrated that alterations of gut microbiota may result in the change of feeding behavior and may constraint the host fly to make rational decision toward diets with higher rewards [13,18] and the use of intestinal bacteria as dietary supplements may help to restore the initial fitness of the host. For example, commensal bacterial isolates Klebsiella oxytoca (BD177) was able to reinstate the ecological and foraging fitness of B. dorsalis irradiated lines by improving diet ingestion [18] and increasing food metabolism (haemolymph sugar and amino acid contents), respectively [24].
Supplementing the full diet with P. dispersa and E. cloacae resulted in an improvement of female fecundity compared to positive control ( Fig. 4 A & B). The quality of diets and bacteria were shown to interact together in modulating the fecundity of many fruit flies [36,46,47]. The host fly can either use nutrients from the full diets directly to improve its reproduction fitness [28,29], or simultaneously the bacterial isolates can use amino acids from the diets to support their own proliferation, afterward they are digested by the host fly and used as additional source of nutrients for the eggs production [25,26]. Conversely, when the diets were supplemented with E. faecalis and K. oxytoca, the number of eggs laid was reduced by more than 60% in comparison with the positive control and no eggs were recorded from the negative control in which flies were fed white sugar only ( Fig. 4 A & B). Two implications can be attributed to these observations. First, E. faecalis and K. oxytoca were deleterious by negatively affecting B. dorsalis sexual maturity and oogenesis and the little eggs produced were solely sustained by the full diet. Second, the absence of eggs in sugar fed flies indicates that amino acids residues are primary precursors of eggs production in B. dorsalis. Taken together, the association between gut microbiome and diet quality has a nutritional and life history basis [21,36,48,49].
The nutrient content of diets has significant impacts on adult longevity [50,51]. For example, the variation of the concentrations of carbohydrate, protein and a phenolic compound (resveratrol) extended the lifespan of Drosophila melanogaster [52]. When flies forage in an environment with varying protein availability, they generally make compromises between some fitness parameters based on life history tradeoffs. As such, either they sacrifice the reproduction and prolong their lifespan or maximize energy for reproduction and shorten their life expectancy. Irrespective of the compromises made along this process, the gut microbiome may come into play to facilitate peptide synthesis or protein metabolism to sustain the host development and survival [21,48,49].
The presence of E. faecalis and K. oxytoca in adult diets extended the B. dorsalis lifespan in comparison with the positive control ( Fig. 5 A & B). The possible reason could be the ability of these bacterial isolates to reduce biomarkers of physiological and oxidative stresses, and inflammation which are considered as the main cause of early death in flies [53].
The larval diet consisted of all the above ingredients that were mixed, added with 250 g/L of wheat bran and autoclaved before use [18]. Twenty (20) adult flies aged 8-10 days were removed from the laboratory culture and anesthetized at -20 °C for 5 min prior to dissection and isolation of individual guts. A culture-dependent technique was employed to isolate gut bacteria (from anesthetized flies) from which, four isolates were later used in bioassays.

Production of experimental flies Symbiotic flies
Symbiotic flies were collected from the laboratory established colony (as described above). A total of 690 newly emerged flies (1 day old) were fed sugar diet and water for seven days prior to bioassays (to starve them of protein source) using 9 cm Petri dishes presented in cotton wool. One hundred fifty flies were used for foraging tests (75 males and 75 females) (Experiment 1). Three hundred sixty males collected from the lab culture were used to fertilize the 180 females assigned for fecundity and longevity assays (Experiment 2). For Experiment 1, the flies were divided by sex (75 males and 75 females) and separately held in 45x30x30cm cages. Then, individual fly was transferred to a 15x15x15cm cages for bioassays. For Experiment 2, 180 females were separately held in 6 cages of 30 flies each. Then, 60 males (same age) from the lab culture were added in each cage (from the fourth sugar treatment day) to mate with the experimental females. Mating couples (duration ≥ 10 min) were retrieved and held in a separate cage and later used for bioassays.

Axenic flies
Axenic flies were obtained from sterilized eggs (embryos) collected from our lab culture and grown on sterile diets as previously described [56,57]. Briefly, 300 collected eggs (aged 4 hours) were individually surface sterilized twice in ethanol 70% and then rinsed twice in deionized distilled water (ddw), before being immersed in phosphate-buffered saline (PBS) solution for 5 min. The resulting embryos were dechorionated in 2.7% sodium hypochlorite solution for 2 min, and then washed twice in sterile ddw, before being transferred to autoclaved larval diet and allowed to develop for about nine days (to reach the third instar larvae). The third instar larvae were allowed to pupate and eclose in sterile sands under lab conditions (12:12 L: D; 25±3ºC, and 67±5% RH). The axenic state of disinfected embryos was validated by PCR of the 16S rRNA gene on ten individual egg homogenates using universal primers (27F/1459R) and by culturing technique on ten individual egg homogenates using dilution plating of eggs homogenate on agar plate-medium, respectively. The PCR reactions were performed in a programmed thermal cycler under the following conditions: Initial denaturation at 95°C for 5 min, followed by 30 cycles at 94°C for 1 min, annealing at 53°C, 54°C, 55°C or 58 °C for 1 min 30 sec, 72°C for 1 min and a final extension at 72°C for 5 min. Any disinfected sample containing grown colonies or agarose bands were discarded and repeated until no bacterial colonies or DNA bands were seen. The repartition of the number of axenic flies and procedures in both experimental settings (1 and 2) is similar to that of the symbiotic flies as described in the previous section, with the only difference that we used axenic flies here.

Insect dissection
The 20 flies previously anesthetized (see section 1 above) were individual washed in 70% ethanol for 2 min and rinsed 3 times in sterile distilled water before dissection. The dissection was carried out aseptically with two pairs of sterilized forceps on a sterilized glass slide spotted with 50 µL of sterile distilled water using a stereomicroscope. The intact guts were individually and separately transferred into Eppendorf tubes containing 750 μL TE buffer (10 mM Tris-Cl, pH 8; 1 mM EDTA, pH 8) and manually crushed with adapted pestle. Homogenized gut suspensions were serially diluted by 10 −4 -10 −8 , 50 μL of which were plated onto Luria Bertani (LB) agar media (Table 1) and incubated at 37°C for 24-48 h. After the incubation, the representative bacteria colonies were randomly pooled based on the size, color, opacity and morphology of each colony. The pre-selected colonies were purified through repeated sub-culturing before being used for DNA extraction and sequencing or preserved in glycerol at -80°C in 50% glycerol (v/v) for future use. The whole dissection procedures were performed in a laminar flow hood to avoid aerial contamination.

Bacterial DNA extraction
The DNA was extracted following the CTAB (Cetyl TrimethylAmmonium Bromide) method. Briefly, single purified colony was cultured in LB broth for ~7 h. 1.5 mL of bacterial suspensions were centrifuged at 10,000 rpm for 10 min and the recovered pellets were re-suspended in 557 µL of TE buffer. 10 µL of lysozyme (5 mg/ml) was added to the suspension and incubated at 37°C for 20 min.
Then, 3 µL proteinases K (20 mg/mL) and 30 µL SDS (10%) were respectively added and incubated again at 37°C for 40 min, afterward 100 µL of NaCl (5mol/l) and 80 µL of CTAB/NaCl were added to the solution and incubated again at 65 °C for 10 min. Then, Phenol/chloroform/ isoamyl alcohol (25:24:1) was finally added to the upper phase solution and centrifuged at 13,400 g for 4 min. Finally, Isopropyl alcohol was added to precipitate the DNA pellets which were later rinsed in 70% ethanol, resuspended in TE buffer and kept at -20°C until use for PCR analysis.

Polymerase chain reactions (PCR)
PCR reactions of the 16S rRNA gene were performed using the bacterial universal primers 27F:5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'. A total volume of 50 µL of PCR reactions containing 1 µL of DNA template, 1 µL of F/R primers, 5 µL of High Fidelity DNA buffer (x10), 4 µL of dNTPs (2.5 mM), 1 µL of Hifi DNA polymerase and 38 µL of deionized distilled water was prepared. The amplification was carried out in a programmed thermal cycler under the following conditions: an initial denaturation step of 95°C for 5 min followed by 34 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, an extension phase of 72°C for 1 min and a final extension at 72°C for 10 min. The PCR amplicons were analyzed by electrophoresis on a 1% agarose gel and visualized under UV light after staining with ethidium bromide. The target band (1.5kb) was purified with a DNA gel extraction kit (Axygen, China). The purified DNA samples were sequenced using Illumina sequencing technology (Novogene, China) and identified by comparison with the most similar sequences from NCBI nucleotide database using the megablast algorithm (http://blast.ncbi.nlm.nih.gov/) ( Table 2).

Preparation of experimental diets Full and sugar diet
A total of two different diets were prepared: a full diet (F) containing all amino acids (essential and non-essential), sucrose, and minerals required for an optimal maintenance and reproductive development of adult flies [29]; a sugar diet consisting of 60% sucrose and minerals used to maintain flies for seven days of protein starvation ( Table 3). The diet compositions and preparation procedures were done as previously described [29] and filtered before use.

Probiotic diets
Four bacterial isolates (Pantoea dispersa, Enterobacter cloacae, Enterococcus faecalis and Klebsiella oxytoca) ( Table 2) were separately grown in LB broth (Table 1) and centrifuged at 10,000 rpm for 5 min. The harvested pellets were washed twice and resuspended in sterile distilled water. The concentration of bacteria in the solvent was adjusted to 0.5 optical density (OD) at 550 nm wavelength using a spectrophotometer (Eppendorf AG, Germany) [32]. 500 µL of each bacterial suspension was added to 50 g of full diets (treatment groups) while 500 µL of sterile distilled water only was respectively added to full and sugar diets (positive and negative controls, respectively). Two Petri dishes seeded with 5 drops of 5 µL volume of full and probiotic diets (randomly displayed in Petri dishes), respectively, were used for the foraging experiment. The flies assigned for fecundity and longevity assays were fed with 1 mL daily of each experimental diet presented in petri dishes seeded with autoclaved cotton wool.

Experimental procedures Experiment 1: Foraging assays
Following the seven day preparatory period during which flies were fed only sugar (as described above), individual fly was transferred to a 15x15x15cm cage and allowed to acclimatize for 20 min before introducing simultaneously a pair of petri dishes containing combinations of Full diet and bacteria supplemented diets at similar volumes and densities ( Figure 1A). Five treatment groups were set up representing the four bacterial isolates and a control group ( Figure 1A). Female and male flies were tested separately and to motivate their foraging responses, they were all starved for 24 hours before experimental trials. Each observation event was replicated 15 times, males and females, symbiotic and axenic flies, representing a total of 300 observation events ( [29]. The female survival was assessed by daily cage inspection and counting of dead flies till their complete death in all treatment groups. The data were pooled and analyzed within and between treatments and symbiotic status.

Statistical analysis
A one-way analysis of variance (ANOVA) was performed to separately test the effects of symbiotic status and diet types on the foraging behavior (landing latency and diet consumption) (male and female), female fecundity and longevity. All data were tested for homogeneity of variances using Levene's tests and only data on the effects of diet types on female fecundity were log transformed.  Note: The preparation of LB broth follows the same procedures but without agar powder.     Number of nutritional drops consumed by symbiotic and axenic Bactrocera dorsalis exposed to two diets patches (containing full diet and full diet enriched with bacteria, respectively).