3.1. Bactrocera Oleae and Green Lacewings Attraction to Three Blends of OFF-Associated Yeast Volatile Compounds in Olive Orchard
During the investigation, from the beginning of July until the end of October 2018, 5961 olive fruit fly adults were caught on YS traps containing three blends of OFF-associated yeast volatile compounds (
Figure 1), among which a significant difference was noted (
p = 0.002).
These results support the hypothesis that
B. oleae responds to volatile compounds produced by yeasts associated with olive fruit flies, in the field. Similar results were obtained by Vitanović et al. [
25]. Their study was focused on olive fruit fly behavior when exposed to yeast volatile compounds associated with olives, olive fruit fly and other insects, both in the laboratory and in the field. The authors also tested olive fruit fly attraction to some blends of yeast volatile compounds in olive orchards. Those blends had different influence on olive fruit flies’ behavior, meaning that some of them were not attractive to flies [
25]. Responses of other tephritid fruit flies to yeast volatile compounds have also been reported [
29,
46].
Yellow sticky traps containing a volatile blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate were the most effective in capturing
B. oleae adults (2517), followed by YS traps that contained volatile blend of isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate (2118), and YS traps containing blend of isoamyl alcohol and 2-phenethyl alcohol (1326) (
Figure 1). These results were not expected because a recent study confirmed that
B. oleae was more attracted to YS traps containing blends of three alcohols than YS traps that contained a binary volatile blend of alcohol and ester [
25]. All tested OFF-associated yeast volatile blends in our research captured significantly greater numbers of olive fruit flies than the control traps (
Figure 1). On the contrary, the results of Vitanović et al. [
25] showed that one of the investigated blends of OFF-associated yeast volatiles (isoamyl alcohol, 2-phenethyl alcohol, 2-phenethyl acetate, isobutyl acetate and isobutanol) was not significantly different compared to control traps. In our study, YS traps containing volatile blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate (1258.50 ± 23.33;
p = 0.001) and YS traps that contained blend of isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate (1032.00 ± 35.36;
p = 0.002) were significantly more attractive to olive fruit flies compared to YS traps containing blend of isoamyl alcohol and 2-phenethyl alcohol (710 ± 98.99;
p = 0.036) or YS traps containing hexane as the control (
Figure 1). Moreover, YS traps that contained a binary blend of isoamyl alcohol and 2-phenethyl alcohol (710 ± 98.99;
p = 0.036) caught a significantly greater number of flies than the control traps. Results also show that YS traps containing volatile blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate (1258.50 ± 23.33) did not significantly differ in
B. oleae captures compared to YS traps that contained blend of isoamyl alcohol, 2-phenethyl alcohol, and 2-phenethyl acetate (1032.00 ± 35.36;
p = 0.059) (
Figure 1). The activity of YS traps containing all tested blends compared to the control was expected because isoamyl alcohol and 2-phenethyl acetate were the most attractive volatile compounds to olive fruit flies in both laboratory and in the field, as well as was isobutyl acetate but only in the field [
25]. Besides that, some researchers have confirmed isoamyl alcohol as an olive plant volatile compound, identifying it in leaves [
47] or in fruits [
1] and, as it is well known, host plants volatiles are attractive to
B. oleae [
1,
17,
18,
19]. Davis et al. [
44] state that isoamyl alcohol was also attractive to many of dipterans that were captured during his investigation. However, it is known that alcohols and esters are emitted by ripe or fermenting fruits [
29,
48] and our results may be related to that fact. Davis and Landolt [
48] also claim that some of the fruit flies, such as Queensland fruit fly (
Bactrocera tryoni, Froggatt) and Mexican fruit fly (
Anastrepha ludens, Loew) respond to alcohols and esters as a food attractant. Nevertheless, all the above facts may be the reason why in our investigation on YS traps containing two blends of alcohols and esters together were significantly more attractive to olive fruit fly than YS traps containing only blends of alcohols.
Figure 1 also presents the results of green lacewings (Neuroptera: Chrysopidae) captured on the same YS traps containing different tested blends of OFF-associated yeast volatiles. During the investigation, 438 green lacewings were caught. The differences in attractiveness between YS traps that contained three blends of OFF-associated yeast volatile compounds to green lacewings were also tested. There was no significant difference in green lacewings attraction between YS traps that contained different tested blends. Furthermore, there was no significant difference in green lacewings catch between YS traps that contained tested blends compared to control YS traps (
p = 0.961) (
Figure 1). Mutualism between yeasts and green lacewings was confirmed long ago [
49], but the relation between them is still not understood [
50]. According to the previous discovery that some of the certain yeast strains associated with olives, olive fruit flies or other insects were attractive to green lacewings [
35,
51], we decided to investigate whether YS traps containing OFF-associated yeast volatile compounds be attractive to them, too. The results of our study are promising because olive fruit flies were highly attracted to YS traps containing different blends of OFF-associated yeast volatile compounds, while green lacewings were not. As is mentioned, many species of the green lacewing are beneficial insects, especially in olive orchards [
36,
37,
38,
40,
52]. The implementation of new attractants that might be highly attractive to target pests and would not disturb their biodiversity, would be of great importance for olive protection.
Figure 2 shows the population dynamic (a) and FTD (b) of
B. oleae for each of the tested YS traps containing blends of OFF-associated yeast volatile compounds during research, where adult flights of three generations of olive fruit fly are clearly seen.
In this part of the Mediterranean region,
B. oleae usually develops three generations, but in years with favorable climatic conditions for its development, it can develop four generations [
53,
54]. During the investigation, for each olive fruit fly generation, significant differences in flies’ attraction to YS traps containing tested blends were found (
Figure 2a). The most differences were noticed during the 2nd generation of
B. oleae adult flight. Yellow sticky traps containing volatile blend of isoamyl alcohol, 2-phenethyl acetate, and isobutyl acetate (321 ± 35.36) and YS traps that contained blend of isoamyl alcohol, 2-phenethyl alcohol, and 2-phenethyl acetate (314.5 ± 28.99) caught significantly more olive fruit flies compared to YS traps that contained a blend of isoamyl alcohol and 2-phenethyl alcohol (142.5 ± 95.46;
p = 0.045 and
p = 0.049, respectively) and control traps. These results may be related with temperatures because during the flight of 2nd generation, the values of the mean temperature were in the optimal range (24.2 °C–27.8 °C) for olive fruit fly reproduction, flying, and development [
45] (
Table 1). Results in
Figure 2a show that between YS traps that contained a blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate (321 ± 35.36) and YS traps that contained blend of isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate (314.5 ± 28.99), no difference in
B. oleae attraction was found (
p = 0.939), as well as between YS traps containing blend of isoamyl alcohol and 2-phenethyl alcohol and YS traps that contained hexane (142.50 ± 95.46 and 118.50 ± 19.09, respectively;
p = 0.764). Observing population dynamics of the 1st and 3rd generation, YS traps containing isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate were significantly more attractive to
B. oleae compared to control traps (
p = 0.034 and
p = 0.028 respectively). The other two tested blends did not differ in olive fruit fly catch compared to control traps. Results in the same figure also show that during the two mentioned generations, YS traps containing different blends of OFF-associated yeast volatile compounds did not significantly differ in
B. oleae captures.
Figure 2b shows the FDT during the investigation. The rate of population change was stable within each of the generations but varied greatly between them for all the tested traps. In general, FTD values for each of the tested YS traps decreased over generations, reaching their highest value in the 3rd generation, for each of the tested blends as well as the hexane (
Figure 2b). Values of FTD for each of the tested YS traps decreased with increasing temperature and with decreasing relative humidity in the olive orchard (
Figure 2b,
Table 1).
3.2. Influence of Climatic Parameters on the Abundance of Bactrocera Oleae and Green Lacewings Caught on YS Traps Containing Different Blends of OFF-Associated Yeast Volatile Compounds
The attractiveness of YS traps containing tested blends to both,
B. oleae and green lacewings, was influenced by climatic parameters (
Table 2). Climatic parameters differed among different generations of olive fruit fly (
Table 1).
Bactrocera oleae is sensitive to high temperature, meaning that temperature above 31 °C causes mortality of all developmental pest stages [
45]. Additionally, olive fruit fly activity decreases below 23 °C and completely stops at 17 °C. Observing the average values of all climatic parameters between three generations of
B. oleae, it was noticed that the lowest and the highest values were recorded during the 1st or 3rd olive fruit fly generation (
Table 1). Climatic conditions with the highest temperatures (up to 31.6 °C) and the lowest relative humidity values (up to 34%) were noticed during July and the beginning of August when the flight of 1st generation was recorded. Contrarily, during the flight of the 3rd generation in September and October, climatic conditions with the lowest temperatures (up to 14.5 °C) and the highest relative humidity values (up to 88%) were found. In both cases, the values of temperature and relative humidity were not favorable for
B. oleae flying [
55,
56]. During the investigation, drought-marked climatic conditions were noticed (
Table 1). The highest amount of rainfall (67.10 mm) was recorded at the end of October during flight of 3rd
B. oleae generation.
The abundance of
B. oleae caught on YS traps containing blend of isoamyl alcohol and 2-phenethyl alcohol (r = −0.739;
p = 0.001) was significantly negatively correlated with temperature (
Table 2). The abundance of
B. oleae caught on the yellow sticky traps containing other tested blends was not correlated with temperature (
Table 2). In addition to isoamyl alcohol, these blends also contained esters, of which blend 3 contained two esters, 2-phenethyl acetate, and isobutyl acetate. Yellow sticky traps that contained blend of alcohol and two esters were significantly more attractive to
B. oleae compared to YS traps containing two alcohols during each of three
B. oleae generations (
Figure 2a,b).
For YS traps containing a blend of isoamyl alcohol and 2-phenethyl alcohol, besides the temperature, significant positive correlation with rainfall amount (r = 0.572;
p = 0.041) was also observed (
Table 2). In the same table, significant negative correlation between temperature and abundance of
B. oleae caught on YS traps containing only hexane was also noticed (r = −0.632;
p = 0.009). However, temperature as a climatic parameter was negatively correlated with the abundance of olive fruit fly in both cases, regardless of whether it was a control trap or YS trap containing isoamyl alcohol and 2-phenethyl alcohol. On the contrary, the abundance of green lacewings caught on YS traps that contained other tested blends was significantly positively correlated with temperature, as follows: blend of isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate (r = 0.729;
p = 0.002) and blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate (r = 0.618;
p = 0.014) (
Table 2). In general, the abundance of green lacewings decreased with decreasing temperature, especially when the temperature dropped below 20 °C (
Table 1,
Supplementary Figure S1). The significant negative correlation was noticed between the abundance of green lacewings caught on YS traps containing isoamyl alcohol, 2-phenethyl alcohol, and 2-phenethyl acetate (r = −0.557,
p = 0.048) and rainfall amounts (
Table 2).
Relative humidity as climatic parameter did not show correlations with the abundance of
B. oleae neither with green lacewings (
Table 2).
Observing the population dynamic of both insects, we can conclude that the abundance of
B. oleae increased with a temperature drop and relative humidity decreasing in the end of summer with the most significant peak in the beginning of October, while the abundance of green lacewings decreased, despite the YS traps difference (
Figure 2a and
Supplementary Figure S1). At the same time, YS traps containing blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate were the most attractive to olive fruit fly among all tested traps with significance in relation to control traps (
Figure 2a). These results are promising because October is olive harvest time, so the use of pesticides for
B. oleae controlling is not allowed and at the same time the abundance of the olive fruit fly in the olive orchard is high.
3.3. Daily Volatile Release of Investigated Blends in the Field
Daily monitoring of the individual evaporation of each of the tested OFF-associated yeast volatile compounds was carried out in October 2021. As it is shown in
Figure 3, a total of five volatile compounds (two alcohols, two esters and one alkane) were tested and identified using the HS-SPME–GC/FID method.
Samples were measured daily, for seven days, and analyzed using HS-SPME–GC/FID yielded peak area results, reflecting the amount of each sample present in the vial. Peak area of the measured samples was compared, and they reflected their proportion in the vials exposed in olive orchard. Hexane showed the largest decrease in peak area (89.29%), as did esters, especially isobutyl acetate (54.91%), while 2-phenethyl acetate decreased to a much lesser extent (25.81%). In contrast, by measuring alcohols in dispensers, isoamyl alcohol and 2-phenethyl alcohol showed an increase of 8.21 and 11.32%, respectively (
Figure 3). During all tested days, as well as the last day of testing, the presence of all tested compounds was confirmed (
Supplementary Figure S2) after exposure to outdoor field conditions.
Vitanović et al. [
25] previously stated that these tested OFF-associated volatile compounds individually were attractive to
B. oleae in the field, thus it was crucial to test their different blends in the olive orchard and to determine the release rate for each of them individually. Our results showed, by comparing the levels of OFF-associated yeast volatile components, that all of them were present in dispensers during their presence in the olive orchard (
Figure 3). However, to our knowledge, the release of tested OFF-associated yeast volatile compounds has not been studied, both under controlled conditions and in the field. Important factors influencing the release of a chemical compound include temperature, airflow, wind, sunlight exposure, precipitation and relative humidity. In addition to these factors, the release rate is also affected by the characteristics of the compound itself and by the type of the used dispenser [
57]. The vapor pressure of a compound directly affects its volatility, so a compound that has a higher vapor pressure, at a certain temperature evaporates faster than a compound that has a lower vapor pressure [
28]. This explains the faster evaporation of isobutyl acetate release rate for OFF-associated yeast volatile compounds. In the case of dispensers placed in the field, variations in the release rate can lead to an increase in volatility of the chemical, changes in their composition, as well as degradation of the material of the dispenser in which it is applied [
58].