The effect of methyl eugenol exposure on subsequent mating performance of sterile males of Bactrocera dorsalis
Qinge Ji (corresponding author), Fujian Agriculture and Forestry University, Fuzhou 350002, China. E-mail: firstname.lastname@example.org
Methyl eugenol (ME) is ingested and used by several fruit fly species of the genus Bactrocera as a precursor of the sexual pheromone produced by adult males. This can result in an increase in male sexual activity and performance, which is important to the development of an effective sterile insect technique programme in China. The effect of ME on mating competitiveness, multiple mating, and the incidence of female remating were studied in sterile males from a genetic sexing strain of Bactrocera dorsalis (Hendel) irradiated with 100 Gy of 137Cs. The results obtained in field cages showed that feeding on ME did not increase the frequency of multiple mating by sterile males, but significantly increased the mating competitiveness of sterile males against that of semi-wild males. At the same time, sterile males fed ME did not significantly affect the remating of semi-wild females 5 days after the initial mating with a semi-wild male, but the ME-fed males increased the remating frequency of females 10 and 15 days after the initial mating.
The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera/Tephritidae), is a serious pest of fruits in southern China. It attacks almost all fruiting plants in tropical, subtropical and temperate regions, reducing fruit quantity and quality, and increasing quarantine costs in 14 provinces of China (Ministry of Agriculture of the P.R.C., unpublished data).
The traditional method of controlling this pest is the application of insecticide cover sprays. However, this strategy causes undesirable environmental and health problems (Hua and Jiang 2000; Zhang 2004). The sterile insect technique (SIT), although in its infancy in China, applied as part of an environmental friendly area-wide integrated approach (Hendrichs et al. 2007), has been successful in controlling fruit fly pests in many countries around the world (Krafsur 1998; Dyck et al. 2005).
Implementing and improving the effectiveness of the SIT are currently a high priority in China. At Fruit Fly Control Center in Fuzhou, Fujian, China, a genetic sexing strain of B. dorsalis based on pupal colour (McCombs and Saul 1995) is maintained under mass rearing, producing 6 million males per week. Quality control tests for adult emergence, flight ability, mating performance and longevity of mass-reared males, pre- and post-irradiation, are conducted according to the International Quality Control Manual (FAO/IAEA/USDA 2003) to evaluate the quality of the insects released in the field. The mating competitiveness, which is one of the most important factors, i.e. the mating performance of sterile males with wild females when competing with wild males in the field, ultimately determines the success of the SIT.
The parapheromone methyl eugenol (ME) is a known powerful attractant of B. dorsalis males (Steiner 1952; Steiner et al. 1965). It has been shown to function as a precursor for the male sex pheromone in B. dorsalis and to increase the attractiveness of the pheromone to females. (Tan and Nishida 1996; Shelly et al. 2000; Shelly 2001). However, there is a lack of knowledge concerning ME’s effect on the frequency of multiple matings (remating) by both sterile males and wild females. Studying remating is important because if the level of remating is significant and if the wild female tends to choose to remate with wild males instead of sterile males, there will be a corresponding negative impact on the effectiveness of the SIT programme. This study examined the effect of ME on the overall level of mating by sterile males and remating by wild mated females to be able to improve the effectiveness of the SIT.
Materials and Methods
The strain in production at Fruit Fly Control Center in Fuzhou, Fujian Agriculture and Forestry University, Fujian, China, is the genetic sexing strain of B. dorsalis originated from a pupal colour sexing strain developed in Hawaii (McCombs and Saul 1995). It was established in 2007 in Fujian, China, by hybridizing and backcrossing males of the Hawaiian strain with wild Chinese B. dorsalis females (Ji et al. 2007). It was reared on artificial diet for ca. 40 generations under laboratory conditions (25°C, 65 ± 5% RH and 12 : 12 L/D).
Male pupae from the mass-reared colony were dyed with a fluorescent dye (ca. 4 g/L) and irradiated at the standard sterilizing dose for B. dorsalis of 100 Gy (Shelly et al. 2010) using the 137Cs source at the Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang ca. 48 h before peak emergence. After emergence, flies were maintained in cages (30 × 30 × 30 cm, stainless steel frame with nylon mesh gauze). Water and food (3 : 1 sugar/hydrolysed yeast) were provided until they reached sexual maturation (10–12 days old) and were ready for experimentation.
Semi-wild males and females were obtained from a strain recently adapted to laboratory conditions (six generations) that originated from infested guava fruit (Psidium guajava L.) collected from Zhangzhou, Fujian. The colony was maintained in the laboratory on guava fruit. Pupae were collected, and flies separated by sex before reaching 2 days of age and kept in different rooms to ensure they remained unmated and to avoid exposing the flies to male pheromone prior to testing. Water and food (same as above) were provided until they reached sexual maturation (10–12 days old) and ready for experimentation. Flies of both the genetic sexing strain and the semi-wild strain were kept under laboratory conditions (25°C, 65 ± 5% RH and 12 : 12 D/L).
Methyl eugenol feeding
An amount of 0.5 ml of ME (obtained from Changzhou Hefeng Institute of Biochemistry, Jintan, Jiangsu, China) was dispensed on a piece of filter paper (7 cm diameter) placed inside a Petri dish. After the ME spread for ca. 2 min, the dish was placed into a cage (same as above) for ca. 800 sterile, sexually mature 10-day-old males to freely feed on for 2 h to ensure that all of the males fed to satiation on the ME.
Effect of ME feeding on competitiveness of sterile males
Two hours before dusk on a test day (about 16: 30), 50 semi-wild males and 50 sterile males, non-ME-fed, were placed into the canopy of a pruned guava tree, ca. 2-m tall, inside a field cage (3 × 3 × 2.3 m, aluminium frame with 100 mesh nylon gauze, (treatment A), while at the same time, 50 semi-wild males and 50 sterile males, ME-fed, were placed into another similar field cage (treatment B). Fifteen minutes later, 50 virgin semi-wild females were placed into each field cage. Tests were conducted according to the quality control mating performance field cage test procedure (FAO/IAEA/USDA 2003). Mating pairs were collected in 20-ml plastic vials and later scored for the presence or absence of fluorescent dye in the ptilina of all males. The total number of pairs per male type was recorded. Five replications were conducted.
Effect of ME feeding on consecutive remating of sterile males
Four field cages were marked A or A’ (for non-ME-fed males) and B or B’ (for ME-fed males). Two hours before dusk on the test day, 50 non-ME-fed sterile males were placed in field cage A and 50 ME-fed sterile males were placed in field cage B. Fifteen minutes later, 50 virgin semi-wild females were placed into field cages A and B. Mating pairs collected in 20-ml plastic vials were gently introduced into field cages A’ and B’, respectively, until there were no more mating pairs in field cages A and B (ca. 2 h). The total number of mating pairs collected from each field cage (A and B) was recorded. On the next morning, the mated females in field cages A’ and B’ were removed, while leaving the males. New virgin semi-wild females equal to the number of once-mated males in field cages A’ and B’ were introduced into these cages separately 2 h before sunset of the same day. Mated pairs were transferred to empty field cages (new A’ and B’), and the total number of pairs was counted at the end of the test, as before. The above procedures were repeated on successive days until no more sterile males mated in either field cage A’ or B’. The total numbers of sterile males mating once, twice, three times, etc. were obtained by counting the numbers of males remaining in cages A and B before each next round and recording these numbers for both non-ME-fed and ME-fed flies. Three replications of the entire procedure were carried out.
Effect of ME feeding by males on remating of semi-wild females
Two hours before dusk on the test day, about 500 pairs of mature semi-wild males and females were placed into a field cage A. Mating pairs from this cage A were gently introduced into another field cage B using 20-ml plastic vials until there were no more mating pairs in field cage A (several hours). The next morning, males were removed from field cage B. Remaining females were allowed to oviposit ad libitum into plastic containers (500 ml, cylindrical plastic bottles with lids and a sponge soaked with diluted orange juice) each day. Additional field cages were prepared (C and D) that were used on the 5th, 10th and 15th days after the first mating. Once again, 2 h before dusk on the test day, 50 sterile males, non-ME-fed, were placed into field cage C, while, at the same time, 50 ME-fed sterile males were placed into field cage D. Fifteen minutes later, 50 semi-wild females from field cage B above which had mated once with semi-wild males were placed into each field cage C and D. Mating pairs from these field cages were removed until there were no more mating pairs in either field cage, then the total numbers of mating pairs were recorded for each cage. Three replications were performed.
Student’s t-tests were used for paired comparisons of means. Following an analysis of variance (anova), Fisher’s least significant difference (LSD) tests were used for multiple comparisons of means (SPSS 15.0 for windows). The raw data generated values of the relative sterility index (RSI) (McInnis et al. 1996; FAO/IAEA/USDA 2003) and the sterile male competitiveness index, C (RSI), for each field cage (Paranhos et al. 2013). The RSI simply measures the proportion of wild females mated to sterile males and is expressed as a decimal value between 0 and 1. When equal numbers of sterile and wild males are released into a cage, the expected RSI for sterile males would be 0.5 if sterile males are equally competitive with wild males. The index, C (RSI) = RSI/(1 − RSI), compares the proportion of treated sterile male matings to the proportion of untreated sterile male matings. As the numbers of treated and semi-wild males for each test was the same (50), the expected RSI = 0.5 and C(RSI) = 1.0 if ME-treated or non-treated sterile male competitiveness equals that of semi-wild males.
Effect of ME feeding on competitiveness of sterile males
The results for the tests of sterile male competitiveness treated with ME or not are shown in table 1. In the ME-treated field cages, a mean of 22.20 ± 1.24 sterile males compared with a mean of 23.20 ± 1.07 semi-wild males mated with semi-wild females. In the control field cages, with non-ME-treated males, there was a mean of 11.60 ± 0.87 sterile males and 30.60 ± 1.72 semi-wild males mating with semi-wild females, respectively. The mean RSI values of 0.49 for ME-treated males and 0.28 for non-ME-fed males were significantly different from each other (t = 6.781, d.f. = 8, P = 0.0001), where an RSI value of 0.5 indicates equal competitiveness of wild and sterile males. The mean sterile male competitiveness values, C (RSI), for ME-treated (0.97 ± 0.09) and control (0.39 ± 0.04) sterile males were also significantly different from each other (t = 6.083, d.f. = 8, P = 0.0029). The effect of ME on competitiveness of sterile males can also be noted in the ratio of the C (RSI) (treated/control) values. These values ranged between 1.76 and 3.59 over the five replications, and the mean value (2.63 ± 0.32) was substantially different from 1.0, signifying higher competitiveness of ME-treated sterile males (t = 5.095, d.f. = 4, P = 0.007). All together, the above results indicate that feeding ME significantly improved the mating competitiveness of sterile males.
Table 1. Mating competitiveness of methyl eugenol (ME)-treated or non-treated (control) sterile males, compared with semi-wild males when competing for virgin semi-wild females
|Mean ± SE||22.20 ± 1.24||23.20 ± 1.07||0.49 ± 0.02||0.97 ± 0.09||11.60 ± 0.87||30.60 ± 1.72||0.28 ± 0.02||0.39 ± 0.04||2.63 ± 0.32|
Effect of ME feeding on consecutive remating of sterile males
Sterile males mated repeatedly over consecutive days, with some mating up to nine times (table 2). As expected, over the nine successive days, the proportions of males mating additional times decreased rapidly. For example, 32.67% of males were able to mate four times, while <10% mated seven times. There were no significant differences between control (non-ME-fed) and ME-fed sterile males for both the proportions mating each consecutive time and the mean total number of matings per male (t-tests, P > 0.05).
Table 2. Remating ability of methyl eugenol (ME)-treated or non-treated (control) sterile males over nine consecutive matings with virgin semi-wild females
|1||42.67 ± 0.88||41.33 ± 1.45||85.33 ± 1.76||82.67 ± 2.91||0.477|
|2||35.67 ± 1.86||34.00 ± 1.53||71.33 ± 3.71||68.00 ± 3.06||0.526|
|3||21.00 ± 2.08||21.33 ± 1.20||42.00 ± 4.16||42.67 ± 2.40||0.896|
|4||16.33 ± 0.33||16.33 ± 1.76||32.67 ± 0.67||32.67 ± 3.53||1.000|
|5||11.00 ± 1.15||10.00 ± 1.53||22.00 ± 2.31||20.00 ± 3.06||0.629|
|6||7.33 ± 2.03||5.00 ± 0.58||14.67 ± 4.06||10.00 ± 1.15||0.330|
|7||4.00 ± 1.15||2.00 ± 0.58||8.00 ± 2.31||4.00 ± 1.15||0.196|
|8||1.33 ± 0.88||0.33 ± 0.33||2.67 ± 1.76||0.67 ± 0.67||0.349|
|9||–||0.33 ± 0.33||–||0.67 ± 0.67||0.374|
| Y ||139.33 ± 3.93||130.67 ± 6.36||–||–||0.311|
| Z ||2.79 ± 0.08||2.61 ± 0.13||–||–||0.311|
Effect of ME feeding by males on remating of semi-wild females
A higher percentage of once-mated semi-wild females remated with ME-treated sterile males compared with the non-treated control males at each of the 5th, 10th and 15th days after the initial mating with a semi-wild male (table 3). While the difference was not statistically significant for the 5th day after the initial mating (t-test, P = 0.069), there were significant differences at the 10th and 15th days after the initial mating (P = 0.022 and 0.023, respectively). Following an analysis of variance, tests of multiple mean comparisons indicated that there was a significant difference between the 5th day and both the 10th and 15th days after the initial mating for both ME-treated and non-treated control males, with the percentage of remating females declining sharply between day 5 and day 10 (P < 0.001) There was no significant difference between the 10th and the 15th days after the initial mating for both ME-treated and control males (P > 0.05).
Table 3. Remating rate of semi-wild females with methyl eugenol (ME)-treated or non-treated (control) sterile males at 5, 10 and 15 days after their first mating with a semi-wild male (N = 3 replications)
|5||48.67 ± 3.53 a||39.33 ± 1.33 a||0.069|
|10||30.67 ± 1.76 b||22.67 ± 1.33 c||0.022|
|15||30.00 ± 2.31 b||21.33 ± 0.67 c||0.023|
Our study showed that pre-release feeding of ME to sterile males significantly improved their relative mating competitiveness in direct field cage comparisons with semi-wild males. These results are in agreement with previous mating performance studies involving the use of ME as a mating stimulant carried out elsewhere on B. dorsalis (Shelly 1994, 1995; Shelly and Dewire 1994; Shelly et al. 2005, 2010). However, feeding ME did not affect, over consecutive days, the total number of matings by a cohort of sterile males, nor the mean number of matings made by single males. The possible reason is that the amount of precursor for the male sex pheromone converged by ME was maintained relatively constant over the life of the males. The tests indicated that remating behaviour was prevalent among sterile males, with no differences between ME-fed and non-ME-fed males. Some sterile males had the capacity to mate up to nine times on nine consecutive days, with a mean of 2.8 and 2.6 times per male for treated and control males, respectively. Nevertheless, it was not assessed whether during these consecutive matings, sterile males, either treated or not with ME, transferred sufficient sperm and accessory gland products to reduce remating in wild females. To compete with wild males and enhance the effect of the SIT, it is recommended to continuing to improve the mass rearing nutrition and quality of sterile males.
Sterile ME-fed males did not affect the remating frequency of semi-wild females that had mated initially with a wild male 5 days prior, but did significantly increase wild female remating 10 or 15 days after the initial mating, compared with control, untreated sterile males. In general, female remating may be related to the age, the amount of oviposition or sperm usage that has transpired, the quality and quantity of stored sperm, and probably, as suggested in this study, by the mating quality of the male involved (Hee and Tan 1998; Shelly and Edu 2008). Males fed on ME appear to have the capacity of inducing more wild females to remate than non-ME-fed males. They seem to become more attractive after feeding on ME, perhaps by releasing more pheromone, and/or more attractive pheromone, or by superior courtship behaviour compared with non-ME-fed males (Hee and Tan 1998).
Additional studies are required mating first virgin wild females with ME-fed, non-ME-fed sterile males or wild males, rather than only with wild males as in our work, and then exposing them for remating over time to wild, ME-fed or non-ME-fed males. In this way, one can corroborate whether there is any impact of sterile male ME-feeding on female remating.
Based on our and previous findings, it will be necessary to further develop existing procedures of large-scale pre-release feeding of ME to maturing sterile males (McInnis et al. 2006; Shelly et al. 2010) to be able to obtain the benefits we have identified in operational SIT programmes in China.
This research was supported by the Food and Agriculture Organization/International Atomic Energy Agency (FAO/IAEA) through research contract No. 14751, by the Ministry of Agriculture of P.R.C through the research programme No. 200903047 and by the Fujian Provincial Department of Science & Technology through the research programme No. 2009N0017.
This article was published online on 10 November 2011. The reference for Paranhos et al. (2013) has now been updated to show the correct citation details for J. Appl. Entomol. Vol. 137, Suppl. 1.