Oviposition Efficacy of Drosophila suzukii (Diptera: Drosophilidae) on Different Cultivars of Blueberry

Drosophila suzukii (Matsumura) is an important pest of thin-skinned fruits including blueberry, raspberry, strawberry, and cherry. Blueberry was introduced into Japan in the 1950s, and severe economic losses attributable to D. suzukii were first reported in 2002. The objective of this study was to elucidate whether oviposition behavior varies among blueberry cultivars having different firmness of fruit. Fruit firmness in 12 cultivars of highbush blueberry (Vaccinium corymbosum L.) and rabbiteye blueberry (Vaccinium virgatum Aiton) was determined using a rheometer. More eggs tended to be laid in berries of cultivars possessing softer fruits than in those having firmer fruits. Choice tests, where one female was allowed to oviposit on blueberry fruits with different firmness, showed that softer fruits were more vulnerable to D. suzukii females than firmer fruits.

Drosophila suzukii (Matsumura) is one of the most serious pests of thin-skinned fruits including blueberry, raspberry, cherry, grape, and strawberry (Kanzawa 1939; Sasaki and Sato 1995; Lee et al. 2011a,b; Walsh et al. 2011; Bellamy et al. 2013). D. suzukii females possess a serrated ovipositor and lay eggs into fresh fruits before harvest (Kanzawa 1939, Sasaki and Abe 1993, Walsh et al. 2011). This insect originated in eastern Asian countries, including Japan. It has invaded Europe and North America as a soft fruits pest since 2008, and the economic damage has been substantial, with estimated crop losses of up to 50% (Goodhue et al. 2011, Walsh et al. 2011, Cini et al. 2012). However, farmers' losses can reach 100% if D. suzukii larvae are detected in the fruits, because in such a case the complete fruit load will be rejected by an inspector.

Blueberry was introduced into Japan in the 1950s. Its cultivation has become widespread since the late 1990s, and severe economic losses attributable to D. suzukii were first reported in 2002 (Shimizu 2006, Kawase et al. 2007). Highbush blueberry (HB; Vaccinium corymbosum L.) can receive significant damage from D. suzukii, but rabbiteye blueberry (RE; Vaccinium virgatum Aiton) suffers little damage in Tokyo (J. Shimada, T. Shimizu, and T. Motobayashi, personal communication). However, the reasons for this difference in damage are not clear. The objective of this study was to elucidate whether female D. suzukii has a preference for oviposition in particular cultivars of blueberry. According to several previous studies, the firmness of the fruits may affect the efficacy of oviposition by D. suzukii females (Kawase and Uchino 2005, Lee et al. 2011a). Seven HB cultivars and five RE cultivars were tested. In this study, the firmness of the fruits was measured using a rheometer, and the number of eggs laid by individual females was examined for each cultivar and correlated with fruit firmness. Finally, choice tests of different cultivars with different fruit firmness were performed to elucidate the factor(s) associated with oviposition preference of D. suzukii on blueberry fruits.

Materials and Methods

Insects and Fruits.

A population of D. suzukii originally from Yamagata Prefecture was obtained from Tokyo Metropolitan University and reared at 25°C in glass tubes (25 mm in diameter by 90 mm in length) with artificial diet, as used for rearing Drosophila melanogaster (Meigen). The medium contains the following: glucose 100 g, cornmeal 90 g, yeast 40 g, agar 7 g, propionic acid 3 ml, and Bokinin solution 10 ml. Bokinin solution is 10% butyl 4-hydroxybenzoate in 70% ethanol. All insect rearing and experimentation was conducted under a photoperiod of 16:8 (L:D) h and 60% relative humidity (RH).

HB and RE were collected from the farm in Tokyo University of Agriculture and Technology, Fuchu, Tokyo. Mature fruits were identified by the dark blue color of their skin and a dark blue ring along the stalk, according to the method of Ismail and Kender (1969). The HB cultivars used were ‘Darrow,’ ‘Meader,’ ‘Coville,’ ‘Brigitta,’ ‘Dixi,’ ‘Herbert,’ ‘Georgiagem’; RE included ‘Tifblue,’ ‘Woodard,’ ‘Delite’; and one cultivar was unassigned (B-26 strain). The fruits were harvested on 15 and 22 July, 3 August, and 1 September in 2010.

Firmness of Blueberry Fruits in Different Cultivars.

Firmness of blueberry fruits was determined using a rheometer (Fudoh Rheometer, RT-3005D, Rheotech, Tokyo, Japan) within 24 h of harvesting. Measurement was performed with a column plunger (diameter of 2 mm) at an insertion speed of 60 mm/min. When the plunger was pushed into a fruit, the maximum resistance value occurred at the moment the plunger penetrated the fruit, and was defined as the firmness of the fruit. Ten berries were examined for each cultivar.

Preference of D. suzukii for Different Blueberry Cultivars.

Oviposition by D. suzukii females on blueberry fruits was examined in the laboratory for berries harvested within the preceding 24 h, using different berries from the same harvested batches of fruits used for testing firmness as described above. Equivalent masses of fruits (5–6 g) were transferred to transparent plastic cups (4.5 cm in diameter by 3 cm in height), and then one 2- to 4-d-old female was placed in each cup to oviposit for 24 h at 25°C. Each cultivar was replicated six times, using females of the same age. The flies were then removed and the fruits kept for 1–2 d at 25°C to allow the eggs to hatch. The fruits were soaked in carbonated water (soda water, Suntory Foods Ltd., Tokyo, Japan) at room temperature, and were shaken for 15–20 min and kept at 25°C for 24 h. Carbonated water allows live fly larvae to emerge from the fruits (Shimizu 2006). The fruits were dissected under a dissecting microscope, and remaining unhatched eggs and dead larvae were counted. The total number of progeny (fly larvae spontaneously emerging into the carbonated water, unhatched eggs, and dead larvae) per female fly was recorded as “estimated oviposition.”

Choice Test for Female Flies Between Firmer and Softer Fruits.

Choice tests were conducted using three cultivars—Darrow (HB), Herbert (HB), and Tifblue (RE). Darrow and Herbert fruits were harvested on 15 July and Tifblue on 22 July of 2010. Until the experiments were conducted on 22 July, fruits of Darrow and Herbert were kept at 4°C, and the firmness of fruits was examined just before the choice test. Combinations of 1) Herbert and Tifblue, and 2) Darrow and Tifblue fruits were placed in plastic cups (two fruits per cultivar in each cup, 4.5 cm in diameter by 3 cm in height) and exposed to a single female D. suzukii for 24 h. After removal of female flies, the fruits were individually transferred to a half-ounce cup (4 cm in diameter by 2 cm in height) and incubated at 25°C for 1–2 d to allow hatching. The fruits were then treated with carbonated water, as described above, to count larvae and residual eggs. Six females were examined in each choice test.

Data Analysis.

Parameters recorded were analyzed with the statistical software JMP 10 (SAS Institute, Cary, NC). The results of fruit firmness were analyzed by one-way analysis of variance (ANOVA). For comparison of the firmness of fruits in two groups (different harvesting dates; HB vs RE; freshly harvested fruits vs 1 wk later), Welch's t-test was conducted. “Estimated oviposition” was analyzed by the Kruskal–Wallis test, because the variables were not normally distributed. The correlation between firmness of fruits and log-transformed number of progeny was examined by Pearson's correlation analysis. The data for estimated oviposition between two cultivars (HB vs RE) were log-transformed and analyzed by Welch's t-test, because the data were normally distributed.

Results

Firmness of Blueberry Fruits in Different Cultivars.

Firmness measurements for blueberry fruit cultivars are shown in Table 1. There were significant differences in the firmness among all blueberry cultivars (F = 20.7; df = 16, 134; P < 0.0001). Two RE cultivars—Delite and Tifblue—were significantly firmer than most of the HB cultivars. However, the HB cultivar Herbert was significantly softer than all other cultivars, both HB and RE, examined. Firmness of fruits was significantly higher for RE than for HB examined (t = 2.39; df = 1, 12.5; P = 0.03). The firmness of Darrow, Woodard, Delite, and Tifblue fruits, which were harvested on different dates, was examined, but the timing of harvesting did not significantly affect the firmness of fruits (F = 3.48; df = 2, 10.5; P = 0.072 for Darrow; t = 0.89; df = 1, 14.8; P = 0.39 for Woodard; t = 0.61, df = 1, 17.5; P = 0.55 for Delite) except Tifblue (t = 2.69; df = 1, 17.5; P = 0.015).

Preference of D. suzukii for Different Blueberry Cultivars.

Estimated oviposition, as defined above, is shown in Table 1. Delite, the firmest cultivar, received the fewest eggs, an average of 1.2–1.5 eggs oviposited per female. The Kruskal–Wallis test indicated a significant difference among the cultivars (H = 34.2; df = 16; P = 0.0051).

A linear regression plot of mean estimated oviposition by D. suzukii females on fruits of each cultivar and the firmness of the fruits is shown in Fig. 1. Among 12 different cultivars, estimated oviposition was negatively correlated with fruit firmness (r² = 0.32; df = 1, 15; P = 0.018).

Choice Tests for Female Flies Using RE and HB Fruits.

Results from choice tests in which one female was presented with two cultivars having different fruit firmness are shown in Fig. 2. Fruits of Darrow (HB), with a firmness of 127 mg, received significantly more oviposition by female D. suzukii than those of Tifblue (RE), with a firmness of 150 mg (t = 3.1; df = 1, 10; P = 0.01). Similarly, fruits of Herbert (HB; firmness of 82 mg) received significantly more oviposition by female D. suzukii than did Tifblue (RE) (t = 4.2; df = 1, 10; P = 0.002). The firmness values of fruits for Herbert, Darrow, and Tifblue were 81.9 ± 6.2, 126.6 ± 4.9, and 149.5 ± 5.3 mg, respectively, and were significantly different from one another (F = 34.9; df = 2, 17; P < 0.01). These results also indicated that more eggs were laid in softer fruits than in firmer fruits.

In this experiment, Darrow and Herbert fruits harvested 1 wk previously were compared with Tifblue harvested on the experiment day. Although the harvest timing of fruits used in the choice test was different, the test needed to be performed on a single day. To determine whether preservation at 4°C for a week might affect the firmness of the fruits, firmness was measured again just before the choice tests were conducted. The firmness of Darrow fruits preserved for a week (126.6 ± 5.7 mg) was not significantly different from that of fruits just harvested (t = 1.35; df = 1, 16.4; P = 0.197). Similarly, Herbert fruits stored for a week (81.9 ± 4.6 mg) were not significantly different from those just harvested (t = 2.25; df = 1, 8.2; P = 0.053).

Discussion

Firmness of blueberry fruits varied significantly among cultivars, but not between the two blueberry species—HB and RE—tested in this study (Table 1). The estimated oviposition by female D. suzukii during 24 h on fruits of different cultivars also varied, but was not significantly different among cultivars. Large standard errors of estimated oviposition observed in this experiment may be attributable to various factors. For example, 2- to 4-d-old females were used in this experiment, but the variation in their ages may have affected their oviposition behavior. Kanzawa (1934) showed that female D. suzukii started to oviposit between 24 and 116 h, with an average of 47 h, after adult emergence.

Estimated oviposition and fruit firmness were negatively correlated (Fig. 1). Female D. suzukii clearly tend to oviposit more eggs in softer fruits than in firmer fruits. Similarly, the level of D. suzukii infestation was weakly or moderately correlated with firmness of cultivars of HB blueberry from California and Oregon (Lee et al. 2011a), as well as with pH and Brix (sugar content). However, Lee et al. (2011a) measured the firmness of fruits using a different method from ours, namely as the force needed to squeeze the fruit by 1 mm. The squeezing force may be affected not only by skin firmness but also by other factors including the elasticity, intercellular space, water content, and size and shape of fruits. In the current study, unlike that of Lee et al. (2011a), firmness of fruits was measured by penetration of a plunger, and should thus correlate with the firmness of the fruits' skin. Using a procedure similar to ours, Hoshino et al. (2010) estimated the firmness of blueberry fruit skin from the difference between the firmness of the entire fruit and the firmness of the flesh of the fruit (i.e., estimated firmness of skin = firmness of [entire fruit−flesh of fruit]), and found that the firmness of an entire fruit is positively correlated with the firmness of the fruit's skin. This suggests that firmer- or thicker-skinned fruits are attacked less by D. suzukii females than are thinner-skinned fruits.

We also conducted choice tests between softer and firmer fruits using individual D. suzukii females (Fig. 2). In both experiments, the estimated oviposition was higher in softer fruits than in firmer fruits. Two choice tests in this study showed that the HB fruits were attacked more than RE by D. suzukii females: the flies preferred Darrow (HB) to Tifblue (RE), whose fruits were significantly firmer than those of Darrow. Herbert (HB) was more strongly preferred by D. suzukii females to Tifblue (RE), a pair in which fruit firmness was more markedly different. Lee et al. (2011a) suggested that the firmness of fruits is a factor in the oviposition preference displayed by D. suzukii females. Other factors including the sugar and acid content of the fruits, which may affect fruit odor, should also be examined to compare the vulnerability of RE and HB cultivars. Choice tests using HB cultivars from Oregon and California were also performed by Lee et al. (2011a), but the number of eggs laid did not differ among them. This inconsistency may be because RE and HB were used in this study whereas only HB cultivars, among which fruit firmness and other factors might vary to a lesser extent, were used by Lee et al. (2011a). Silva et al. (2005) examined the physicochemical, carbohydrate, and sensory characteristics of HB and RE blueberry cultivars, and reported that the firmness of the HB cultivars examined (Bluecrop and Jersey) was significantly lower than that of the RE cultivars examined (Climax, Premier, and Tifblue).

The Japanese name of D. suzukii is “outou-shoujoubae,” which is derived from sweet cherry (outou) and fruit fly (shoujoubae). Initially, D. suzukii was an important pest of sweet cherry (Prunus avium L.) in Japan (Kanzawa 1934), but blueberry became an additional host plant of D. suzukii after it had spread in Japan, because blueberry is well suited to D. suzukii as a host plant. Sasaki and Sato (1995) found that the host plants of D. suzukii in Fukushima Prefecture were, in order of fruit ripening, cherry Prunus yedoensis Matsumura (late May to early June), sweet cherry (late May to early July), Elaeagnus multiflora Thunb. (late June), mulberry Morus bombycis Koidz. (late June to early July), blueberry (late June to October), blackberry and raspberry (middle July to October), Rubus parvifolius L. (early to late August), peach Prunus persica (L.) Batsch. (early August), Prunus buergeriana Miq. (middle to late September), and American pokeweed, Phytolacca americana L. (early October to late November). Thus, D. suzukii seems to switch among host plants when ripe fruits are available. The fly population may build up after repeated occurrence on different host plants, because D. suzukii was estimated to undergo >10 generations per year in a laboratory study (Kanzawa 1939). Because all the host plants examined are sufficiently soft to allow D. suzukii females to attack, identifying potential host fruits that ripen before blueberry and controlling the population before blueberry is attacked may suppress outbreaks of this pest on fruit crops.

Recently, blueberry breeding has been focusing on the firmness of the fruits (Proctor and Miesle 1991, Silva et al. 2005). Firmer fruits are preferred by fresh fruit consumers and are preserved longer than softer-skinned fruits. Breeding of firmer fruits may also be beneficial from the point of view of reducing damage by D. suzukii. Our data suggest that a firmness of 140 mg is the level that the fruit would need to attain for effective resistance against penetration by D. suzukii females, and this could be a target for fruit-breeding programs.

Furthermore, laboratory studies indicate that cultivars or species producing firmer fruits are less vulnerable to D. suzukii females than those producing softer fruits (Lee et al. 2011a, Burrack et al. 2013, this study). Burrack et al. (2013) recently addressed host selection by D. suzukii in the field and laboratory, and showed that fruit firmness may be one driver of this host selection. However, field infestation was not simply correlated with the firmness of fruits. If softer fruits can act as a trap crop around more resistant crops, the preference trait of D. suzukii could be used to establish a cultural control strategy to manage D. suzukii in the field, as mentioned by Burrack et al. (2013). To connect this laboratory observation and field management, further practical field studies are needed to establish a successful cultural control strategy against D. suzukii.

Acknowledgments

We sincerely thank Isao Ogiwara, Takashi Motobayashi, Hidenobu Tsujimura, Jun Shimada, Tomoe Shimizu, Satoshi Noma, and Mika Mashimo, all of Tokyo University of Agriculture and Technology; Koichiro Tamura of Tokyo Metropolitan University; and Ian Smith of Nara Institute of Science and Technology. This project was partially supported by a special research fund of MEXT, Japan: “Research and development of security and safe crop production to reconstruct agricultural lands in Fukushima Prefecture based on novel techniques to remove radioactive compounds using advanced bio-fertilizer and plant protection strategies.”

References Cited