Yeasts Associated with the Olive Fruit Fly Bactrocera oleae (Rossi) (Diptera: Tephritidae) Lead to New Attractants

: The olive fruit ﬂy ( Bactrocera oleae Rossi) is the primary insect pest in all olive-growing regions worldwide. New integrated pest management (IPM) techniques are needed for B. oleae to mitigate reliance on pesticides used for its control which can result in negative environmental impacts. More e ﬀ ective lures for monitoring olive ﬂies would help to know when and where direct chemical applications are required. The aim of this research was to ﬁnd new, more e ﬀ ective methods for B. oleae detection and monitoring. Twelve insect-associated yeasts were selected and tested as living cultures in McPhail traps for the attraction of olive ﬂies. Certain yeasts were more attractive than others to B. oleae ; speciﬁcally, Kuraishia capsulata , Lachancea thermotolerans , Peterozyma xylosa , Sche ﬀ ersomyces ergatensis , and Nakazawae ernobii, than the industry-standard dried torula yeast ( Cyberlindnera jadinii ; syn. Candida utilis ). The attractiveness of dry, inactive (i.e., non-living) formulations of these ﬁve yeasts was also tested in the ﬁeld. Inactive formulations of K. capsulata , P. xylosa , N. ernobii , and L. thermotolerans were signiﬁcantly more attractive to B. oleae than commercially available torula yeast. Green lacewing, Chrysoperla comanche (Stephens) (Neuroptera: Chrysopidae), adults were incidentally caught in traps baited with the live yeast cultures. This is the ﬁrst ﬁeld study that compares olive ﬂy attraction to yeast species other than torula yeast. Commercialization of yeasts that are more attractive than the torula standard would improve monitoring and associated control of the olive fruit ﬂy.


Introduction
The olive fruit fly (Bactrocera oleae Rossi) is the most damaging olive pest in the world. Olive losses from this tephritid routinely range up to 15% [1], but in years and at sites with high fly populations, total yield loss can occur [2]. These losses affect table olive production more than olive oil production since damaged fruit cannot be processed as table olives. In olive oil production, B. oleae directly affects the amount and quality of the oil. Historically, olive fly control has been based solely on insecticides [3][4][5]. However, chemical control negatively affects biodiversity in the olive canopy by reducing the number of beneficial organisms that include predatory arthropods, such as spiders, insects All active yeast formulations were placed in duplicate in plastic McPhail traps (Great Lakes IPM, Inc., Vestaburg, MI, USA) in an organic olive orchard at UC Davis (coordinates: 38.535861, −121.795023), beginning on 29 October 2015. Traps were placed randomly in every third row with a distance of four trees between traps in the row at 1.5-2 m high in the shade of the olive canopy for 10 days. Positive control traps containing torula yeast pellet solution (5 g of torula in 200 mL deionized water) (ISCA Technologies Inc., Riverside, CA, USA), and negative control traps containing 200 mL of deionized water, or 200 mL of PBD solution were also included in duplicate. Because PDB supports microbial growth in this non-aseptic environment, it was necessary for the PDB-baited traps to be replaced every two days. Two traps for each treatment (yeast) were placed in the olive orchard, and the total number of olive fruit flies in each of them was counted daily by removing all insects with a strainer, and then returning the yeast cultures to the respective trap.

Preparation of the Five Most Attractive Insect-Associated Yeasts
The five most attractive yeast strains from the fall 2015 test were selected for spring 2016 field testing. These were K. capsulata, P. xylosa, N. ernobii, L. thermotolerans, and S. ergatensis. These yeasts were revived and grown in the same manner as described above, as was the commercially available formulation of torula yeast (Cyberlindnera jadinii; syn. Candida utilis).

Preparation of Dry Yeast
Freeze-dried preparations of the five yeast strains that attracted the highest number of olive fruit flies in the fall 2015 trial were prepared for the spring 2016 field trial ( Figure 1).
Agronomy 2020, 10, x 4 of 14 The five most attractive yeast strains from the fall 2015 test were selected for spring 2016 field testing. These were K. capsulata, P. xylosa, N. ernobii, L. thermotolerans, and S. ergatensis. These yeasts were revived and grown in the same manner as described above, as was the commercially available formulation of torula yeast (Cyberlindnera jadinii; syn. Candida utilis).

Preparation of Dry Yeast
Freeze-dried preparations of the five yeast strains that attracted the highest number of olive fruit flies in the fall 2015 trial were prepared for the spring 2016 field trial ( Figure 1).

Figure 1.
Photomicrographs of active cultures of five insect-associated yeast strains selected for the spring 2016 field testing, grown in 3× or 5× yeast extract/malt extract (YM) broth as described. Photos were taken with a Carl Zeiss Axio Imager.A1, Jena, Germany with a 100X oil immersion objective, phase contrast.
Liquid yeast extract/malt extract (YM) broth media (10 g/L dextrose, 3 g/L yeast extract (Marcor Development Corporation, KODA Distribution Group, Carlstadt, NJ, USA), 3 g/L malt extract (Home Brew Stuff, Inc., Idaho, USA), 5 g/L Bacto peptone (Thermo Fisher Scientific, Waltham, MA, USA), and 10 g/L glucose (Bulkfoods-Natural Food Inc., Toledo, OH, USA) was used to cultivate yeasts for attraction studies. For most yeast strains, yeast extract/malt extract media (YM) broth was prepared at five times (5×) the standard concentration of all ingredients to boost yeast biomass productivity, except for the yeasts S. ergatensis and N. enobii for which three times (3×) the normal concentration of YM was used instead because it resulted in higher cell biomass. Each yeast strain was revived on PDA plates and incubated for three days at room temperature. A loopful of cells was added to 10 mL either 3× YM (S. ergatensis) or 5× YM (remaining yeasts) broth in triplicate in a 50-mL conical bioreaction tube (Cat.# 229475, BR tubes, Celltreat, Shirley, MA, USA) and incubated 24 h at 200 rpm 30 °C. This seed culture was then inoculated into 790 mL of the same media in 2.8 L Fernbach flasks and incubated 96 h at 200 rpm at 30 °C, except S. ergatensis, which was grown at 25 °C. Cells were washed Liquid yeast extract/malt extract (YM) broth media (10 g/L dextrose, 3 g/L yeast extract (Marcor Development Corporation, KODA Distribution Group, Carlstadt, NJ, USA), 3 g/L malt extract (Home Brew Stuff, Inc., Idaho, USA), 5 g/L Bacto peptone (Thermo Fisher Scientific, Waltham, MA, USA), and 10 g/L glucose (Bulkfoods-Natural Food Inc., Toledo, OH, USA) was used to cultivate yeasts for attraction studies. For most yeast strains, yeast extract/malt extract media (YM) broth was prepared at five times (5×) the standard concentration of all ingredients to boost yeast biomass productivity, except for the yeasts S. ergatensis and N. enobii for which three times (3×) the normal concentration of YM was used instead because it resulted in higher cell biomass. Each yeast strain was revived on PDA plates and incubated for three days at room temperature. A loopful of cells was added to 10 mL either 3× YM (S. ergatensis) or 5× YM (remaining yeasts) broth in triplicate in a 50-mL conical bio-reaction tube (Cat.# 229475, BR tubes, Celltreat, Shirley, MA, USA) and incubated 24 h at 200 rpm Agronomy 2020, 10, 1501 5 of 13 30 • C. This seed culture was then inoculated into 790 mL of the same media in 2.8 L Fernbach flasks and incubated 96 h at 200 rpm at 30 • C, except S. ergatensis, which was grown at 25 • C. Cells were washed three times by centrifuging at 4000 rpm for 10 min at room temperature, then resuspending cells in 150 mL deionized water. The cell pellets were frozen 24 h at −80 • C, then freeze-dried for 48 h below −47 • C, vacuum >0.12 Pa in a Labconco Freeze Zone 4.5 freeze dryer. Freeze-dried cell mass was transferred to a pre-weighed plastic bag, weighed, then stored at −80 • C.
For field bioassays, 5 g of each yeast strain was suspended in 200 mL of deionized water in 250 mL bottles.

Field Testing
McPhail traps baited with the various active and inactive yeast-strain formulations were deployed as described for 2015 in 2 sets (10 days each), beginning on May 11, 2016. In addition to commercial torula yeast pellets as a positive control and deionized water as a negative control, as described previously, 5× and 3× YM media were also included as negative controls. The 5× and 3× YM media in traps were replaced every two days. The total number of olive fruit flies per trap was counted daily by removing all insects with a strainer, and then returning the yeast cultures of each respective trap.
In the course of the spring 2016 investigation, we noticed that traps baited with all yeast formulations captured many green lacewings (Neuroptera: Chrysopidae) as well, and these were counted too.

Statistical Analysis
Data were subjected to non-parametric χ2 goodness of fit test to compare the attractiveness of active and inactive formulations of yeasts to olive fruit fly and to green lacewings. For the frequencies (e.g., less than 10 on average), V-square statistics as a Chi-square corrected for sample size [40,41] were used. The correction of the significance value was performed by the Bonferroni test [42,43] for controlling the effect of multiple testing. Analyses were performed by Statistica software version 12.0 (StatSoft, Inc., Tulsa, OK, USA, 2013).

Experiment in 2015
Attraction of B. oleae to 12 Insect-Associated Yeasts During November 2015, 657 olive fruit flies (370 males and 287 females) were captured in McPhail traps baited with active cultures of 12 insect-associated yeast strains (Table 1). A significant difference in fly attraction was noted (χ 2 = 317.64; p 0.05 = 23.68) ( Figure 2). Traps baited with L. thermotolerans captured the most olive fruit flies, followed by N. ernobii and P. xylosa. Among the 12 active yeast formulations, six of them (L. thermotolerans, N. ernobii, P. xylosa, S. ergatensis, K. kapsulata, and C. jadinii) captured significantly greater numbers of olive fruit flies than did the PDB, water, or torula pellet control traps. McPhail traps baited with V. cf. foliicola, W. subpelliculosus, O. pini, J. aff. sakaguchii, and S. terrea did not differ in B. oleae captures compared to torula yeast pellets, but they did capture significantly more flies than did the PDB or water control traps. Only the F. oeirensis baited traps captured significantly fewer B. oleae than those baited with torula pellets (Figure 2).
Results in Figure 2 show that there were significant differences in the attraction of the sexes (χ 2 = 10.48, df = 1, p = 0.0012) with males exhibiting 23 percent greater attraction to B. oleae than females overall. However, not all tested yeast strains showed differences in the attraction of the sexes. Individually only S. terrea, J. aff. sakaguchii, and N. ernobii were more attractive to males than females. Traps that contained F. oeirensis caught the same number of both sexes.  Results in Figure 2 show that there were significant differences in the attraction of the sexes (χ 2 = 10.48, df = 1, p = 0.0012) with males exhibiting 23 percent greater attraction to B. oleae than females overall. However, not all tested yeast strains showed differences in the attraction of the sexes. Individually only S. terrea, J. aff. sakaguchii, and N. ernobii were more attractive to males than females. Traps that contained F. oeirensis caught the same number of both sexes.    Figure 3.

Experiments in
The attraction of B. oleae to the active and inactive formulations of K. capsulata, P. xylosa, and N. ernobii is presented in Figure 3A-C, and a significant difference in adult captures among these yeast strains was found (χ 2 = 184.95; χ 2 = 267.76; χ 2 = 408.28; p 0.05 = 12.59). Mean (±SD) B. oleae captures in McPhail traps containing inactive formulations of K. capsulata (14.75 ± 4.78), P. xylosa (20.00 ± 12.02), and N. ernobii (22.5 ± 16.82) were significantly greater when compared to traps that contained active formulations of the same yeast strains and to all control traps (torula yeast, water, PDB, media 3× and media 5×). There were no differences in attractiveness to B. oleae between active formulations of K. capsulata, P. xylosa, and the industry-standard (inactive torula yeast pellet), but active yeast formulations mentioned were more attractive to B. oleae than the other control attractants. The N. ernobii active formulation was significantly less attractive to B. oleae than was torula yeast, and no difference in attractiveness was noted compared to other control attractants.
The differences in attractiveness between active and inactive formulations of L. thermotolerans and S. ergatensis to olive fruit fly were also tested ( Figure 3D-E). There was no significant difference in mean (±SD) B. oleae attraction between McPhail traps containing active (6.75 ± 4.57) and inactive (6.75 ± 3.09) formulations of L. thermotolerans or between traps containing active (8.50 ± 2.38) and inactive (6.50 ± 3.11) formulations of S. ergatensis. The number of flies captured in McPhail traps containing either active or inactive formulations of L. thermotolerans and S. ergatensis was significantly greater than control traps that contained water, PDB, 3× YM broth, or 5× YM broth. However, B. oleae captured in traps containing either the active or inactive L. thermotolerans formulations or the active S. ergatensis were significantly greater compared to captures in traps torula yeast pellets, while the number captured in traps containing the inactive S. ergatensis formulation was not different. When considering the total number of flies captured in McPhail traps, significant differences in attraction were observed between the sexes for both active (χ 2 = 6.03, df = 1, p = 0.0140; 69 males: 43 females) and inactive yeast strain formulations (χ 2 = 22.69, df = 1, p ≤ 0.0001; 181 males: 101 females).  Table 1).
The attraction of B. oleae to the active and inactive formulations of K. capsulata, P. xylosa, and N. ernobii is presented in Figure 3A-C, and a significant difference in adult captures among these yeast strains was found (χ 2 = 184.95; χ 2 = 267.76; χ 2 = 408.28; p0.05 = 12.59). Mean (±SD) B. oleae captures in McPhail traps containing inactive formulations of K. capsulata (14.75 ± 4.78), P. xylosa (20.00 ± 12.02), and N. ernobii (22.5 ± 16.82) were significantly greater when compared to traps that contained active formulations of the same yeast strains and to all control traps (torula yeast, water, PDB, media 3× and media 5×). There were no differences in attractiveness to B. oleae between active formulations of K. capsulata, P. xylosa, and the industry-standard (inactive torula yeast pellet), but active yeast formulations mentioned were more attractive to B. oleae than the other control attractants. The N. ernobii active formulation was significantly less attractive to B. oleae than was torula yeast, and no difference in attractiveness was noted compared to other control attractants.

Attraction of a green lacewing to the five most active and inactive yeasts
In a previous study, we noted that one species of green lacewing, C. comanche, was highly attracted to active formulations of different yeast strains that included two of the five yeasts, L. thermotolerans and C. terreus, that were deployed in the present study [37].
Agronomy 2020, 10, x; doi: www.mdpi.com/journal/agronomy 3.2.2. Attraction of a green lacewing to the five most active and inactive yeasts In a previous study, we noted that one species of green lacewing, C. comanche, was highly attracted to active formulations of different yeast strains that included two of the five yeasts, L. thermotolerans and C. terreus, that were deployed in the present study [37].
Chi-square analysis revealed that significantly more C. comanche were attracted to traps baited with active yeast formations than inactive formulations (χ 2 = 24.90, df = 1, p ≤ 0.0001). Of the 689 total C. comanche captured in traps baited with the yeast strains evaluated, 410 were recorded in traps baited with active formulations and 279 with inactive formulations (Figures 4 and 5).

Discussion
This is the first in-depth study of yeast strains other than the commercially-available Cyberlindnera jadinii; syn. Candida utilis) (torula yeast) dry pellet vis-à-vis olive fruit fly attraction. Commercialization of yeast strains that are more attractive than the industry standard torula yeast pellet would improve its monitoring and potentially its control, too.
In an earlier field study [33], we evaluated 12 yeast strains as active yeasts formulations for B. oleae attraction and found that certain yeast strains were more attractive than others. Five of them were significantly more attractive to B. oleae than the torula yeast pellets (Figure 2). We also felt that evaluating inactive formulations of these yeasts (which is similar in concept to that of torula yeast pellet) might afford more uniform release of volatiles over time than the active formulations that had been previously evaluated and would provide better shelf life making them more attractive and usable as commercial baits.
The effectiveness of inactive and active formulations of five yeasts, K. capsulata, L. thermotolerans, P. xylosa, S. ergatensis, and N. ernobii, were compared to one another and commercial torula yeast pellets in a California olive orchard. Inactive formulations of K. capsulata, P. xylosa, N. ernobii, and L. thermotolerans yeasts were significantly more attractive to olive fruit flies when compared to commercial torula yeast pellets, while the inactive formulation of S. ergatensis was not. Of these, the inactive formulation of N. ernobii was the most attractive to B. oleae among the yeasts and formulations evaluated, resulting in 2.6-fold more flies being captured in plastic McPhail traps relative to torula yeast pellets. Bactrocera oleae were also attracted to active formulations of N. ernobii, P. xylosa, K. capsulata, and L. thermotolerans, although trap captures were lower in all cases relative to inactive formulations of the same yeasts. As anticipated, B. oleae trap captures proved somewhat inconsistent between years when using the active formulations of these yeasts. For example, the N. ernobii active formulation was second most attractive among the yeast strains tested in fall 2015, yet no B. oleae were captured in the spring 2016 study. Bactrocera oleae were also more attracted to active formulations of P. xylosa and K. capsulate in 2015 relative to 2016, while active formulations of L. thermotolerans were relatively attractive in both years. The reasons for this inconsistency in attraction for the active formulations of these yeasts are not known, but the cooler conditions present at the time of the fall 2015 field study relative to the spring 2016 trial could have been a factor influencing yeast fermentation during these studies or could affect fly populations. Consistency of active formulations seasonally and under different ambient field conditions could perhaps be improved with additional research.
The study results suggest that inactive formulations of N. ernobii, P. xylosa, K. capsulate, or L. thermotolerans could be used as attractants for B. oleae detection, monitoring, and/or control, and that attraction to these yeasts could prove more effective than to torula yeast. These four yeast strains are more attractive to olive fruit fly than torula yeast, and this could be due to their host associations in nature. The original sources from which these yeast strains were collected (insects) differs from that of torula yeast (C. jadinii; syn. C. utilis), which is associated with plants. The L. thermotolerans strain used in this study was first isolated from a male olive fruit fly trapped in a plastic McPhail trap [33]. The P. xylosa used in this study was first isolated from a Drosophilidae species, Drosophila miranda, while the K. capsulata and N. ernobii were first isolated from beetles (Table 1) [33]. Our results are also consistent with the numerous studies of interactions between other Diptera species and yeasts (e.g., [8][9][10][11][14][15][16] and references therein).
In a previous study [33], solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS) analysis of active formulations of three of these yeast strains identified five volatile compounds that were attractive to B. oleae in a laboratory Y-tube olfactometer assay. Bactrocera oleae were also highly attracted to these yeasts in our field studies. The same study [33] showed that volatile compounds produced by active torula yeast (C. jadinii; syn. C. utilis) in PDB greatly differed from those produced by inactive commercial torula yeast pellets suspended in water and that inactive torula yeast pellets were much more attractive to B. oleae than active C. jadinii; syn. C. utilis [33]. Identification of the volatile profiles of inactive formulations of K. capsulata, P. xylos, L. thermotolerans, and N. ernobii could lead to the discovery of specific volatile compounds that may be responsible for olive fruit fly attraction that can be used as improved attractants in integrated pest management (IPM) programs.
We observed that plastic McPhail traps baited with inactive and active formulations of K. capsulata, P. xylosa, N. ernobii, L. thermotolerants, or S. ergatensis also captured many different dipterans as well as other insects. This is unsurprising as it is well known that many insects from several orders, including beetles, flies, ants, and bees, interact with yeasts [44][45][46][47][48] In both years of our study, plastic McPhail traps containing L. thermotolerans captured the greatest number and diversity of insects compared to the other yeast strains. Nakazawa ernobii attracted the greatest number of wasps, especially the active yeast formulation (data not shown). Previous researches on eusocial insects extensively describe their associations with yeasts and other microbes [49][50][51].
The discovery that inactive yeasts were more attractive than the corresponding active formulations sheds doubt on the assumption of mutualism between olive fruit fly and yeasts, the nature of which is reconsidered. In contrast, the concomitant data presented showing that many adults of one species of green lacewing, C. comanche, were incidentally caught in traps baited with live yeast cultures, substantiates the existence of a truly mutualistic relationship between this group of aphid predators and yeast. According to Hagen, et al. [52], yeasts are involved in the nutritional ecology of green lacewings, but the relationship between them is still not well-understood [53]. Because of that, our discovery stimulated us to collect them to investigate which of tested yeasts will be more attractive to lacewings than to B. oleae or vice versa. Generally, McPhail traps that contained active yeast formulations caught a greater number of C. comanche compared to the traps containing their inactive formulations, especially K. capsulata, L. thermotolerans, and S. ergatensis. These yeast strains were significantly more attractive to C. comanche than to B. oleae. Volatile compounds emitted by active yeast cultures were likely more attractive to C. comanche than inactive formulations. By contrast, olive fruit flies were more attracted to inactive yeast formulations. Only the inactive formulation of L. thermotolerans was significantly more attractive to C. comanche than to B. oleae. This is important because our results indicate that inactive formulations of K. capsulata, P. xylosa, L. thermotolerans, and N. ernobii have the potential to be used as new attractants for detection, monitoring, and/or control of olive fruit fly. Therefore, the use of L. thermotolerans as a pest management tool could lead to the disruption of C. comanche, which are common natural enemies in agroecosystems but are used in augmentation biological control programs [54].

Conclusions
The behavioral response of olive fruit fly to 12 insect-associated yeast strains was confirmed. Our data indicated that B. oleae were more attractive to some yeast strains than others. Further, plastic McPhail traps containing certain inactive yeast formulations also captured significantly more adult flies than those baited with the commercial standard, torula yeast. Of all yeast strains evaluated, inactive formulations of K. capsulata, P. xylosa, N. ernobii, and L. thermotolerans were significantly more attractive to B. oleae compared with torula yeast pellets. These four yeast strains represent promising candidates as attractants for B. oleae detection, monitoring, and/or control. Results from the present study confirmed those of a prior study that suggested differential attraction of the green lacewing, C. comanche, to different yeast strains also highlight opportunities for future research toward understanding and perhaps utilizing the behavioral response of beneficial insects to yeasts. This is the first study to investigate the interaction between insect-associated yeast strains and B. oleae in an olive orchard. Results set the foundation for using insect-associated yeasts as attractants for detection, monitoring and/or control of olive fruit fly, and have implications for the management of other tephritids as well.