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

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

Elda Vitanović 1,2, Julian M. Lopez 3, Jeffrey R. Aldrich 1,4, Maja JukićŠpika 2,5,* , Kyria Boundy-Mills 3 and Frank G. Zalom 1 1 Department of Entomology and Nematology, University of California, Davis, CA 95616, USA; [email protected] (E.V.); drjeff[email protected] (J.R.A.); [email protected] (F.G.Z.) 2 Institute for Adriatic Crops and Karst Reclamation, Put Duilova 11, 21000 Split, Croatia 3 Department of Food Science and Technology, Phaff Yeast Collection, University of California, Davis, CA 95616, USA; [email protected] (J.M.L.); [email protected] (K.B.-M.) 4 Jeffrey R. Aldrich Consulting LLC, P.O. Box 121, Marcell, MN 56657, USA 5 Ctr Excellence Biodivers & Mol Plant Breeding, Svetošimunska Cesta 25, 1000 Zagreb, Croatia * Correspondence: [email protected]; Tel.: +385-21-434-482

Received: 1 September 2020; Accepted: 30 September 2020; Published: 2 October 2020

Abstract: 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 effective 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 effective 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, Scheffersomyces 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.

Keywords: plant protection; integrated pest management; agricultural entomology; insect behavior; McPhail trap; lures; torula

1. 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 [3–5]. 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

Agronomy 2020, 10, 1501; doi:10.3390/agronomy10101501 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1501 2 of 13 from the order Neuroptera, Coleoptera, and Diptera, and parasitoids (Order Hymenoptera) ([3–6] and references therein). The intensive use of pesticides for B. oleae control has stimulated researchers to seek new methods that will preserve olive orchard biodiversity [6]. Dipteran responses to yeast volatiles have been reported by many researchers ([7–11] and references therein). In addition, interactions between yeast and Drosophila species have been repeatedly investigated and documented ([7,12–17] and references therein). Drosophila use yeasts as food for larvae, nutrients for egg production, and in courtship [18]. Becher et al. [19] pointed out that Drosophila melanogaster is primarily attracted to volatiles associated with yeasts found on host fruit rather than to fruit odor itself. The same authors also stated that volatile compounds emitted by the yeasts are usually found in flowers and nectar and that they are common floral signals and insect attractants. Releases of these volatiles attract many different insect species before the emergence of plant flowering and may contribute to the evolution of pollination by insects. Unlike drosophilid flies, interactions between yeasts and tephritid fruit flies have not been well investigated [20]. Piper et al. [20] suggested that a better understanding of interactions between yeasts and tephritids could lead to the development of new formulations of attractants in “attract and kill” lures, as well as improvement in insect mass rearing insect methodology in Sterile Insect Technique (SIT) control programs [21]. Interactions between B. oleae and gut microbiota, such are fungi [22] and bacteria [23–25], have been investigated for similar reasons. Bactrocera oleae is reportedly also attracted to host plant volatiles [26–29], as well as intra- [30,31] and inter-specific semiochemicals [32]. However, the interaction between B. oleae and yeasts other than torula yeast (Cyberlindnera jadinii; syn. Candida utilis) remains little studied. Over 400 yeasts were isolated from infested olives, adult flies, and potential feeding sites during preliminary studies [33]. Of these yeasts, over 130 insect-associated strains showed promise for attracting B. oleae as well or better than the industry standard torula yeast pellet. The aim of the research reported herein was to find the most attractive yeast strains from among the plethora of promising species for the attraction of the olive fruit fly. Bait pellets consisting of hydrolyzed torula yeast and boric acid were developed in 1971 for monitoring of the Caribbean fruit fly [34]. This pellet lure is now used for monitoring and/or control of many tephritids ([35] and references therein), including B. oleae [36]. Burrack et al. [36] found that commercial torula yeast pellets were more effective for attracting olive fruit flies in the field than other attractants, such as lures based on ammonia or pheromones. In our previous study [33], we found that volatiles produced by active torula yeast formulation in potato dextrose broth (PDB) differed greatly from those produced by an inactive commercial pellet of the same yeast species suspended in water. However, from a practical standpoint, the commercialization of yeast for the attraction of tephritid fruit flies is much more easily accomplished with the dry, inactive form of yeast than for active, living cultures [33]. Therefore, the five most attractive yeast species, as found here through testing, were also tested as water suspensions of their non-living freeze-dried residue as a step toward advancing a more commercially-acceptable form that could be used for B. oleae detection and monitoring. Finally, in the course of the current investigation, many adults of one species of the green lacewing, Chrysoperla comanche (Stephens) (Neuroptera: Chrysopidae), were incidentally caught in traps baited with live yeast cultures [37]. Data comparing the attractiveness of yeast to B. oleae versus C. comanche attraction are also presented.

2. Materials and Methods

2.1. Experiment in 2015

2.1.1. Preparation of 12 Insect-Associated Yeasts Twelve yeast strains from a preliminary screening of over 130 insect-associated yeast strains that showed promise for attracting B. oleae [33] were selected for field testing (Table1). All yeast cultures were prepared in duplicate. All yeast strains were revived from cryopreserved stocks onto potato dextrose agar (PDA) and incubated at room temperature for five days. Yeast seed Agronomy 2020, 10, 1501 3 of 13 cultures were prepared by inoculating 10 mL of potato dextrose broth (PDB; DifcoTM, Sparks, NJ, USA) with about 1 µL of cells from each fresh plate, and incubating at room temperature in a roller drum [38]. After three days, 5 mL of each yeast culture was added to flasks containing 200 mL PDB and incubated four days with shaking at 200 rpm and 30 ◦C. After four days, flask yeast cultures were diluted with PDB so that all had equivalent OD 600 [39], and then 200 mL of each live yeast culture was transferred into a sterile 250 mL bottle. Bottles with active yeast formulations were chilled then transported to the olive orchard.

Table 1. Yeast strains from the Phaﬀ Yeast Culture Collection (UCDFST).

Yeast Strain and UCDFST ID Number Geographic Source Habitat Source Kuraishia capsulata UCDFST 68-897.1 British Columbia, Canada Beetle frass in Pinus contorta Peterozyma xylosa UCDFST 52-162 California, USA Drosophila miranda Frass of bark beetle Ips oregoni Ogataea pini UCDFST 52-128 California, USA in Pinus contorta Bactrocera oleae (olive fruit fly) Solicoccozyma terrea UCDFST 05-757 California, USA adult male Caeca of larva of Ernobius Nakazawaea ernobii UCDFST 51-28 Unknown mollis beetle Bactrocera oleae (olive fruit fly) Lachancea thermotolerans UCDFST 04-833 California, USA adult male Bactrocera oleae (olive fruit fly) Vishniacozyma cf. foliicola UCDFST 05-508 California, USA adult female Scheffersomyces ergatensis UCDFST 80-53 Spain Ergastes faber beetle Bactrocera oleae (olive fruit fly) Filobasidium oeirensis UCDFST 05-501 California, USA adult male Wickerhamomyces subpelliculosus UCDFST 76-85 USA Fermenting cucumbers Bark beetle frass in Abies Jianyunia aff. sakaguchii UCDFST 73-1015 California, USA magnifica Cyberlindnera jadinii (syn. Candida utilis, USA Torula yeast cultivation tank Torula yeast) UCDFST 75-33

2.1.2. Field Testing 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.

2.2. Experiment in 2016

2.2.1. 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 Agronomy 2020, 10, x 4 of 14

The five most attractive yeast strains from the fall 2015 test were selected for spring 2016 field Agronomy 2020, 10, 1501 4 of 13 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 formulationwere of revived torula and yeast grown (Cyberlindnera in the same manner jadinii as; describedsyn. Candida above, utilis as was). the commercially available formulation of torula yeast (Cyberlindnera jadinii; syn. Candida utilis).

2.2.2. Preparation2.2.2. Preparation of Dry of Yeast Dry Yeast Freeze-driedFreeze-dried preparations preparations of the of the five five yeast yeast strains strains that that attracted attracted the highestthe highest number number of olive of fruit olive fruit flies in theflies fall in the2015 fall trial 2015 were trial were prepared prepared for for the the spring 2016 2016 field field trial trial (Figure (Figure1). 1).

Figure 1.Figure Photomicrographs 1. Photomicrographs of active of active cultures cultures of of fivefive insect-associatedinsect-associated yeast yeast strains strains selected selected for the for the spring 2016 field testing, grown in 3 or 5 yeast extract/malt extract (YM) broth as described. Photos 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, were takenphase with contrast. 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 Liquid(Marcor yeast Development extract/malt Corporation, extract (YM) KODA broth Distribution media (10 Group, g/L dextrose, Carlstadt, NJ,3 g/L USA), yeast 3 gextract/L malt (Marcor extract (Home Brew Stuff, Inc., Idaho, USA), 5 g/L Bacto peptone (Thermo Fisher Scientific, Waltham, Development Corporation, KODA Distribution Group, Carlstadt, NJ, USA), 3 g/L malt extract (Home MA, USA), and 10 g/L glucose (Bulkfoods-Natural Food Inc., Toledo, OH, USA) was used to cultivate Brew Stuff,yeasts Inc., for Idaho, attraction USA), studies. 5 g/L For Bacto most yeastpepton strains,e (Thermo yeast extract Fisher/malt Scientif extractic, media Waltham, (YM) broth MA, USA), and 10 g/Lwas glucose prepared (Bulkfoods-Natural at five times (5 ) the standard Food Inc., concentration Toledo, ofOH, all ingredientsUSA) was toused boost to yeast cultivate biomass yeasts for × productivity, except for the yeasts S. ergatensis and N. enobii for which three times (3 ) the normal attraction studies. For most yeast strains, yeast extract/malt extract media (YM) ×broth was prepared at five timesconcentration (5×) the of standard YM was used concentration instead because of it all resulted ingredients in higher cellto boost biomass. yeast Each biomass yeast strain productivity, was revived on PDA plates and incubated for three days at room temperature. A loopful of cells was added except for the yeasts S. ergatensis and N. enobii for which three times (3×) the normal concentration of to 10 mL either 3 YM (S. ergatensis) or 5 YM (remaining yeasts) broth in triplicate in a 50-mL conical × × YM wasbio-reaction used instead tube (Cat.#because 229475, it resulted BR tubes, in Celltreat, higher Shirley, cell biomass. MA, USA) Each and incubated yeast strain 24 h at was 200 rpmrevived 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 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

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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.

2.2.3. 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.

2.3. 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).

3. Results

3.1. 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 (Table1). A significant di fference 2 in fly attraction was noted (χ = 317.64; p0.05 = 23.68) (Figure2). 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 (Figure2). Results in Figure2 show that there were significant di fferences in the attraction of the sexes (χ2 = 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. Agronomy 2020, 10, x 6 of 14 Agronomy 2020, 10, 1501 6 of 13

Figure 2. Total number of Bactrocera oleae captured in traps that contained active culture formulations Figure 2. Total number of Bactrocera oleae captured in traps that contained active culture formulations of 12 yeast strains and controls field-tested in fall 2015. Bars associated with different lowercase letters of 12 yeast strains and controls field-tested in fall 2015. Bars associated with different lowercase letters are significantly different at p 0.05 by the Chi-square test. Asterisks represent significant differences are significantly different at p≤ ≤ 0.05 by the Chi-square test. Asterisks represent significant differences in the attraction of the sexes within the same yeast strain, while different uppercase letters indicate in the attraction of the sexes within the same yeast strain, while different uppercase letters indicate differences in the attraction of the sexes in all tested traps (p 0.05 by Chi-square test). differences in the attraction of the sexes in all tested traps (p≤ ≤ 0.05 by Chi-square test). 3.2. Experiments in 2016 Results in Figure 2 show that there were significant differences in the attraction of the sexes (χ2 3.2.1.= 10.48, Attraction df = 1, p of= 0.0012)B. oleae withto the males Five Mostexhibiting Active 23 and percent Inactive greater Yeasts attraction to B. oleae than females overall.During However, May 2016, not 394all olivetested fruit yeast flies strains were collected showed in differences McPhail traps in the containing attraction active of orthe inactive sexes. formulationsIndividually representingonly S. terrea, the J. aff. five sakaguchii most attractive, and N. yeast ernobii strains were from more the attractive 2015 field to trial. males Traps than containing females. activeTraps yeastthat contained formulations F. oeirensis accounted caught for 112 the of same the flies number caught, of andboth they sexes. were significantly less attractive 2 to B. oleae then traps containing inactive yeast formulations (χ = 73.35; p0.05 = 3.84). The attractiveness 3.2. Experiments in 2016 of the single yeast strain and their relationship to the control samples is shown in Figure3. The attraction of B. oleae to the active and inactive formulations of K. capsulata, P.xylosa, and N. ernobii 3.2.1. Attraction of B. oleae to the Five Most Active and Inactive Yeasts is presented in Figure3A–C, and a significant di fference in adult captures among these yeast strains was foundDuring ( χMay2 = 184.95; 2016, 394χ2 =olive267.76; fruitχ 2flies= 408.28; were collectedp = 12.59). in McPhail Mean traps ( SD) containingB. oleae captures active inor 0.05 ± McPhailinactive formulations traps containing representing inactive formulations the five most ofattracK. capsulatative yeast(14.75 strains from4.78), theP.xylosa 2015 field(20.00 trial.12.02), Traps ± ± andcontainingN. ernobii active(22.5 yeast16.82) formulations were significantly accounted greater for 112 when of the comparedflies caught, to and traps they that were contained significantly active ± formulationsless attractive ofto theB. oleae same then yeast traps strains containing and to inactive all control yeast traps formulations (torula yeast, (χ2 = water, 73.35; PDB,p0.05 = media3.84). The 3 × andattractiveness media 5 ).of There the single were noyeast di ffstrainerences and in thei attractivenessr relationship to B. to oleae the betweencontrol samples active formulations is shown in × ofFigureK. capsulata 3. , 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 (Figure3D–E). There was no significant di fference 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

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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 (χ2 = 6.03, df = 1, p = 0.0140; 69 males: 43 females) 2 andAgronomy inactive 2020 yeast, 10, x strain formulations (χ = 22.69, df = 1, p 0.0001; 181 males: 101 females). 7 of 14 ≤

Figure 3. Bactrocera oleae collected in McPhail traps containing active and inactive formulations of Figure 3. Bactrocera oleae collected in McPhail traps containing active and inactive formulations of (A) (A) Kuraishia capsulata,(B) Peterozyma xylosa,(C) Nakazawaea ernobii, (D) Lachancea thermotolerans, Kuraishia capsulata, (B) Peterozyma xylosa, (C) Nakazawaea ernobii, (D) Lachancea thermotolerans, and (E) E Sche ersomyces ergatensis andScheffersomyces ( ) ff ergatensis as attractantsas attractants placed in placed olive orchard. in olive Results orchard. presented Results presentedare mean values are mean of valuesfour measurements of four measurements (duplicate (duplicate traps in trapstwo independen in two independentt repetitions). repetitions). Bars labeled Bars by labeled different by di lettersfferent letters are significantly different at p 0.05 by the Chi-square test. Error bars indicate SE. Control traps are significantly different at p ≤ 0.05≤ by the Chi-square test. Error bars indicate SE. Control traps were were torula yeast, water, PDB, media 3 , and media 5 (see also Table1). torula yeast, water, PDB, media 3×, and× media 5× (see× also Table 1). 3.2.2. Attraction of a green lacewing to the five most active and inactive yeasts The attraction of B. oleae to the active and inactive formulations of K. capsulata, P. xylosa, and N. ernobiiIn a is previous presented study, in Figure we noted 3A–C, that and one a speciessignificant of green difference lacewing, in adultC. comanche captures, wasamong highly these attracted yeast tostrains active was formulations found (χ2 of= 184.95; different χ2 yeast= 267.76; strains χ2 =that 408.28; included p0.05 = 12.59). two of Mean the five (±SD) yeasts, B. oleaeL. thermotolerans captures in andMcPhailC. terreus traps, that containing were deployed inactive informulations the present of study K. capsulata [37]. (14.75 ± 4.78), P. xylosa (20.00 ± 12.02), andMcPhail N. ernobii traps (22.5 containing ± 16.82) were active significantly formulations greater of insect-associated when compared yeastto traps strains that placedcontained inan active olive 2 orchardformulations in spring of the 2016 same showed yeast significant strains and di tofferences all control (χ =traps258, (torulap0.05 = yeast,14.07) water, in attraction PDB, media to C. comanche 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.

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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 (χ2 = 6.03, df = 1, p = 0.0140; 69 males: 43 females) and inactive yeast strain formulations (χ2 = 22.69, df = 1, p ≤ 0.0001; 181 males: 101 females).

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]. Agronomy 2020, 10, 1501 8 of 13 McPhail traps containing active formulations of insect-associated yeast strains placed in an olive orchard in spring 2016 showed significant differences (χ2 = 258, p0.05 = 14.07) in attraction to C. comanche (Figure 44).All).All testedtested yeast strain strain active active formulations formulations were were significantly signiﬁcantly more more attractive attractive than than were were the controlthe control traps traps (torula (torula yeast, yeast, PDB, PDB, and andwater). water). Mean Mean (±SD) ( capturesSD) captures were weresignificantly signiﬁcantly greater greater in traps in ± baitedtraps baited with L. with thermotoleransL. thermotolerans (30.75(30.75 ± 23.12)23.12) than thanin those in those baited baited with with K. capsulataK. capsulata (21.00(21.00 ± 10.61),10.61), C. ± ± C.silvicola silvicola (19.75(19.75 ± 16.27)16.27) or S. or ergatensisS. ergatensis (18.00(18.00 ± 7.11).7.11). ± ±

Figure 4. ChrysoperlaChrysoperla comanche comanche collectedcollected in in McPhail McPhail traps traps containing containing active active formulations formulations of Peterozyma xylosa,xylosa, ScheScheffersomycesffersomyces ergatensisergatensis,, Nakazawaea Nakazawaea ernobii ernobii,, Kuraishia Kuraishia capsulata capsulata,, and andLachancea Lachancea thermotolerans thermotoleransas asattractants attractants placed placed in in an an olive olive orchard orchard in in spring spring 2016. 2016. Results Results are are mean mean valuesvalues ofof four measurements (duplicate traps in two independentindependent repetitions). Bars Bars labeled labeled by by different different letters are significantlysignificantly different at p 0.05 by the Chi-square test. Error bars indicate SE. different at p ≤ 0.05 by the Chi-square test. Error bars indicate SE. Results of the attractiveness of inactive formulations to C. comanche placed in the same olive Results of the attractiveness of inactive formulations to C. comanche placed in the same olive orchard in spring 2016 were similar to those for the active formulations (Figure5), and as for active Agronomyorchard 2020 in spring, 10, x 2016 were similar to those for the active formulations (Figure 5), and as for active9 of 14 2 formulation, there were significant differences between formulations (χ = 262, p0.05 = 15.51). formulation, there were significant differences between formulations (χ2 = 262, p0.05 = 15.51).

Agronomy 2020, 10, x; doi: www.mdpi.com/journal/agronomy

FigureFigure 5.5. ChrysoperlaChrysoperla comanche comanchecollected collected in McPhail in McPhail traps containingtraps contai inactivening inactive formulations formulations of Nakazawae of Nakazawaeernobii, Peterozyma ernobii, xylosaPeterozyma, Scheff ersomycesxylosa, Scheffersomyces ergatensis, Kuraishia ergatensis, capsulate, Kuraishiaand Lanhanceacapsulate, thermotoleransand Lanhanceaas thermotoleransattractants placed as attractants in an olive placed orchard in an in olive spring orchard 2016. Resultsin spring are 2016. mean Results values are of mean four measurementsvalues of four measurements(duplicate traps (duplicate in two independent traps in two repetitions).independent Barsrepetitions). labeled Bars by di labeledfferent lettersby different are significantly letters are different at p 0.05 by the Chi-square test. Error bars indicate SE. Asterisks represent a significant significantly different≤ at p ≤ 0.05 by the Chi-square test. Error bars indicate SE. Asterisks represent a significantdifference difference in mean number in mean ofnumberC. comanche of C. comanchecaptured captured in traps in with traps inactive with inactive compared compared with active with activeformulations formulations (see Figure (see Figure4) of the 4) sameof the yeast same strains. yeast strains. Traps baited with the inactive formulation of L. thermotolerans captured significantly more Traps baited with the inactive formulation of L. thermotolerans captured significantly more C. C. comanche than did the other inactive yeast formulations (29.50 16.27). L. thermotolerans and comanche than did the other inactive yeast formulations (29.50 ± 16.27).± L. thermotolerans and K. K. capsulata (14.75 12.24) baited traps caught significantly more C. comanche than did the control traps capsulata (14.75 ± 12.24)± baited traps caught significantly more C. comanche than did the control traps containing 5 YM broth, 3 YM broth, torula yeast, or water. There was no difference in captures containing 5×× YM broth, 3×× YM broth, torula yeast, or water. There was no difference in captures between McPhail traps that contained inactive formulations of S. ergatensis (10.50 ± 9.40), P. xylosa (7.75 ± 3.68), or N. ernobii (7.50 ± 5.19) (Figure 5). Traps baited with the latter three yeast strains captured significantly more C. comanche than did traps containing torula yeast pellets and water as a control. Chi-square analysis revealed that significantly more C. comanche were attracted to traps baited with active yeast formations than inactive formulations (χ2 = 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).

4. 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

Agronomy 2020, 10, x; doi: www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1501 9 of 13 between McPhail traps that contained inactive formulations of S. ergatensis (10.50 9.40), P. xylosa ± (7.75 3.68), or N. ernobii (7.50 5.19) (Figure5). Traps baited with the latter three yeast strains ± ± captured signiﬁcantly more C. comanche than did traps containing torula yeast pellets and water as a control. Chi-square analysis revealed that signiﬁcantly more C. comanche were attracted to traps baited with active yeast formations than inactive formulations (χ2 = 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 (Figures4 and5).

4. 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 (Figure2). 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 (Table1)[ 33]. Our results are Agronomy 2020, 10, 1501 10 of 13 also consistent with the numerous studies of interactions between other Diptera species and yeasts (e.g., [8–11,14–16] 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 [44–48] 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 [49–51]. 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].

5. 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 Agronomy 2020, 10, 1501 11 of 13 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.

Author Contributions: Conceived and designed the experiments, E.V., J.R.A., K.B.-M., and F.G.Z.; performed the laboratory and field experiments and analyzed the data, E.V., J.M.L., J.R.A., K.B.-M., and F.G.Z.; statistical analyses, M.J.Š.; writing—original draft preparation, E.V., J.M.L., M.J.Š.; writing—review and editing, J.R.A., K.B.-M., and F.G.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the Fulbright Scholar Program Award 68150029 to E.V. in 2015/2016, and the Department of Entomology and Nematology University of California, Davis, CA, USA. In addition, this research was supported by the project KK.01.1.1.04.0002 New methods in olive pests controlling using plant volatiles, Split, Croatia, funded by European Union. Acknowledgments: Funding from the Fulbright Program of the U.S. Department of State is gratefully acknowledged. Personal thanks are extended to N. Nicola and L.A. Garay for technical assistance, D. Flynn (Executive Director of the UC Davis Olive Center) for providing the olive orchard where our field studies were conducted, and S.L. Winterton of the California State Collection of Arthropods, California Department of Food and Agriculture, Sacramento, for green lacewings identification. Conflicts of Interest: The authors declare no conflict of interest.

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