Influence of Supplemental Protein on the Life Expectancy and Reproduction of the Chinese Citrus Fruit Fly, Bactrocera minax (Enderlein) (Tetradacus minax) (Diptera: Tephritidae)

Influence of Supplemental Protein on the Life Expectancy and Reproduction of the Chinese Citrus... Bactrocera minax (Enderlein) (Diptera: T ephritidae) is a major citrus pest in China, whose artificial rearing technology of the adult is not well documented to date. In this study, we tried to determine if supplementing proteins to the adult diet could result in the enhancement of some fitness parameters of B. minax. Four feeds with varying protein source were provided as F0 (water), F1 (sucrose), F2 (sucrose + yeast), and F3 (sucrose + peptone). F0 and F1 being the control, F2 and F3 were protein food types. The results showed that adults fed by F2 and F3 lived longer with 40.1 d and 32.8 d, respectively, had reduced death rates (death peaks were delayed for 5.6 d and 4.1 d, respectively), increased mating frequencies (8.1 and 5.3 per females, 4.7 and 7 .3 per males, respectively), and longer mating durations (with 42 d and 34 d). In addition, females recorded an increased adult ovary development, more egg load (with 94.8 and 77 .3 brood eggs per ovary) and to greater oviposition rates of 63.2 eggs/female and 19.3 eggs/female. Based on our results, protein supplements enhanced B. minax survival, mating, and fecundity. This study does not only provide basic knowledge to implement artificial rearing of B. minax, but also deepens our understanding on its physiology that could be used to enhance the management of the pest. Key words: Bactrocera minax, protein compositions, survival, mating, fecundity Fruit flies (Diptera: Tephritidae) are among the most destructive agri- occurs in other Asian countries such as Bhutan, Nepal, and India cultural pests known to occur worldwide (White and Elson-Harris (EPPO/CABI 1997, Wang et  al. 2014). In China, B.  minax mainly 1992, Bhattacharya et al. 2013). Fruit flies damage fruits and vege- occurs in temperate provinces such as Guangxi, Guizhou, Hubei, tables by laying their eggs inside the fruits and cause them to rot and Hunan, Jiangsu, Jiangxi, Shaanxi, Sichuan, and Yunnan (EPPO/ drop, thus inducing significant losses in production (Hollingsworth CABI 1997, Wang et al. 2014). The pest exhibits about 6 mo’s pupal et al. 1997, Bhattacharya et al. 2013). Besides causing direct losses diapause from November to May as a way to fight against harsh to a wide range of fruits and vegetables, they limit the development environmental conditions (Chen et al. 2016). Such long pupal dia- of agriculture in many countries because of strict trade quarantines pause is a barrier for laboratory rearing and development of control imposed to prevent their spread. Bactrocera minax (Enderlein) strategies against this pest (Chen et  al. 2016). Damage caused by (Tetradacus minax) (Diptera: Tephritidae) is one of the most impor- larval feeding on the fruit pulp results in fruit segments partially tant quarantinable pests which attacks citrus fruits in China. It also or completely turning into paste. The losses are estimated to range © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/25/4924853 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Journal of Insect Science, 2018, Vol. 18, No. 2 between 5 and 20% and can reach up to 50% (Wang et al. 1995, Materials and Methods 2009). In a global agricultural system that promotes food security Acquisition of Adult Fruit Flies and sustainability, there is a necessity to produce nutritional comple- Mature larvae of B. minax were collected from Orange orchards in ments in quality and quantity to meet the rising demands in fruits Songzi, Hubei, China, between 20 October and 10 November 2015. and vegetables of the population. B.  minax, therefore, represents a The collected larvae were taken to the Yangtze university insectary significant threat to the completion of these challenges unless it is where they were moved into plastic container (21 cm in diameter) well controlled and managed. containing 15% water content sand (sterilized sand by constant tem- Nutrition plays a vital role in the development of tephritid fruit perature 60°C for 24 h to kill Nematodes) and kept indoor at room flies. Life span, maturity development, and reproductive capacity are temperature (4.8–22.6°C, 75% RH ± 5%) till adult emergence. The mostly influenced by the quality of the food (Good and Tatar 2001, plastic container containing fourth stage pupae were placed into big Fontana et  al. 2010). Reproduction and longevity are mutually cage (baby mosquito net) to prevent the emerging adult to escape. dependent in sexually reproducing organisms, with an increase in Adults emerged in late April and early May 2016 and ended on 27 the energy used for reproduction resulting in a subsequent decrease May 2016. in longevity (Partridge and Andrews 1985). Hence, the nutritional conditions in an environment influence the trade-off between current Diet Preparation reproductive effort and life span because of competition for nutrients In this study, a solid diet was used to feed the flies, the diet was between somatic maintenance and gamete production (Kirkwood measured in grams; sucrose 4  g (Tianjin Beilian Fine Chemicals and Rose 1991). Lee and Lee (2005) reported on the benefit of arti- Development Co. Ltd.), sucrose + yeast extract (3  g + 1  g, mak- ficial diet on predator’s development. Also, the reproductive ability ing 3:1) (yeast extract and sucrose + peptone (3 g and 1 g, making in many Tephritids is strongly influenced by diet (Aluja et al. 2001; 3:1)(Guangdong Huankai Microbial Sci & Tech. Co. Ltd). After Carey et  al. 2002, 2008; Harwood et  al. 2013; Liedo et  al. 2013; measurements, the solid food was mixed evenly and placed on Petri Pereira et al. 2013). dishes ready to be provided to flies. Yeast extract was used as source Protein is one of the most important nutrients required for ovar- of protein, as it contains vitamin content and protein which pro- ian development and vitellogenesis in insects (Raikhel and Dhadialla vide excellent growth conditions for more microbes. Peptone is the 1992, Aluja et  al. 2001, Taylor et  al. 2013). In general, fruit flies microbiological culture media prepared by enzymatic digestion of fed with protein supplements, for example Anastrepha ludens selected animal tissues, widely used to suit a range of nutritional (Loew), Ceratitis capitata (Wiedemann), and Bactrocera cucurbitae requirements. Peptone was used as source of protein as it supports (Coquillett), had their fecundity enhanced while given protein sup- excellent growth of various microorganisms. It also contains short plements (Mangan 2003, Harwood et  al. 2013). Also, protein-en- chains peptides and broad spectrum amino acids required for the riched foods were shown to promote the longevity of the fruit fly, microbial growth. Anastrepha serpentina (Wiedemann) (Jácome et  al. 1999). In add- ition, the protein level of the food has a high influence on egg pro- Experimental Design duction (Mangan 2003). For example, while evaluating the effect of The obtained adults were separated in three cohorts. To assess the larval diet on the development and reproductive rates of male and effects of protein supplementation on the survival of B.  minax, a female Mediterranean fruit fly, Kaspi et al. (2002) found that pro- total of 60 newly emerged flies comprising 30 males and 30 females tein-fed larvae were large in size, grew fast, and had more nutritional were introduced in a wire mesh cage (dimensions: 30 cm × 30 cm × reserves upon emergence. 30 cm) covered with a white cloth at the bottom. Flies were provided A source of sugar is an important nutrient for fruit flies with four types of food designated as F0 (water alone), F1 (sucrose because it provides energy for survival and reproduction. Hagen alone), F2 (sucrose + yeast extract, mass ratio 3:1), and F3 (sucrose + (1953) reported that both sexes of the western cherry fruit fly, peptone, mass ratio 3:1). There were five replicates for each food Rhagoletis indifferens, Curran, require sources of sugar for sur- type, 30 males and 30 females were provided each food type result- vival. Fontellas and Zucoloto (1999) also found that West Indian ing into a total of 60 flies per replicate. After preparation, 4 g solid fruit fly, Anastrepha obliqua, Macquart, did not survive more than food were provided to the colony into the Petri dish (diameter: 9 cm) 3 d without a sugar (sucrose) source. However, Sacchetti et  al. and replaced every 24 h. Also, the flies were provided with 7 ml of (2014) reported that olive fruit fly, Bactrocera oleae, did not sur - water twice a day (morning and evening) on a cotton bud (diameter: vive longer when fed with sugar diet. Similarly, Gavriel et al. (2011) 0.5 cm) that was replaced on a weekly basis. Male and female flies’ reported that male Mediterranean fruit fly had a short life span and mortality was recorded daily. the ability of the female to receive male were not inhibited when To assess the effect of protein supplementation on mating and fed with granulated sugar only. This implies that fruit flies depend egg production, 30 males and 30 females virgin B. minax (0 to 24 h upon these two nutrients for both survival and egg production. To after emergency) were provided each food source (F0, F1, F2, and the best of our knowledge, none of the previous studies about the F3) resulting into a total of 60 flies per replicate. There were five artificial rearing techniques of B. minax has been successful due to replicates for each food type. Fresh orange fruit was provided for egg the high adult mortality rates observed. This unfortunate situation laying as they are adapted to oviposition through the thick skin of hindered a comprehensive investigation of its biological character- oranges (CABI, 2017), and the orange was replaced every three days. istics and the development of effective prevention measures. For The frequency of mating (mating times) was observed and recorded this reason, the understanding of the nutritional factors that could every half an hour from 8:00 a.m. to 6:00 p.m. reduce mortality of B. minax and enhance its reproduction capac- Finally, 800 females were kept in four separate cages (200 ities is of prime importance for the development of an artificial females/cage) where they were provided with four food sources as rearing program. Thus, the present study aimed at evaluating the described above. Twenty insects were dissected every 5 d for a dur- feeding response of adults to different protein supplements through ation of 20 d to assess the ovarian development and egg load (Chou the assessment of their effects on the survival and reproduction of et al. 2012). the Chinese citrus fruit fly, B. minax. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/25/4924853 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 3 swelling. and big. Subsidiary silks diminish or lose. Primary for- Demographic Analysis mation of ovary and egg yolk precipitation. “Third stage”: ovary Adult life expectancy was obtained by the following expression; is growing bigger, and harder egg yolk deposition to form egg. “Life expectancy”  =  (∑Survival days per insect)/Total number of Eggs can be seen in the oviducts. Mature Eggs in ovary, before adult tested. On evaluating the effect of protein on flies, cumulative and after the onset of oviposition (Fig.  1) (Fletcher et  al. 1978; mortality ratio (CMR) and feeding days were compared. First, we Kendra et  al. 2006; Chou et  al. 2012). To calculate the ratio of calculated the mortality ratio (MR %) by MR % = daily mortality/ different ovary stages the following formula was used: “Ratio of total lifetime mortality * 100, then CMR % obtained by the follow- specific ovary stage (%)” = (number of flies with the specific ovary ing formula; CMR (%) = MR + MR + MR + … + MR . Where; n 1 2 3 n stage)/(total number of flies dissected) * 100. MR is the mortality ratio; CMR is the cumulative mortality ratio, n Data on the effect of diet on adult life expectancy, mortal- is the feeding days when food was supplied to the flies. ity, age-specific mortality, mating frequency per female, mating The adult cumulative mortality in all four types of food (F0, F1, F2, and F3) tested were transformed to probability units and ana- lyzed using the logistic regression (y = a/(1 + Exp (b – k x))) where x is the feed days in each food and y is the cumulative mortality in probability units. Time–mortality data were fitted using probability analysis to estimate the lethal time for 50% (LT ) and 90% (LT ) 50 90 cumulative mortality in each food treatment tested. To obtain the age-specific cumulative mortality, cumulative mortality data were subjected Arcsine transformation (arcsine square root transforma- tion) (Sokal and Rohlf 1995, Warton and Hui 2011) and then were subjected to analysis of variance (ANOVA one-way) and means were separated by Tukey’s test at P = 0.05. To access mating dynamic the following expression were used; “Lifetime Mating frequency per female” = ∑(daily number of mat- ing times/daily number of surviving females). “Lifetime Mating fre- quency per male” = ∑(daily number of mating times/daily number of surviving males). The cumulative mating ratio (CMR) for each diet was calculated using the following expression: first, we calculated the mating ratio (MR %) by the following formula, MR% = (life- time mating times)/(total lifetime mating times) * 100. The MR obtained was used to calculate the CMR by using the following for- mula; CMR (%)  =  MR + MR + MR + … + MR . Where; MR n 1 2 3 n is the daily mating ratio; CMR is the cumulative mating ratio; n is the feeding days (days of food supply to the flies, until death of the last fly). The adult cumulative mating in four food sources (F0, F1, F2, and F3) tested was transformed to probability units and analyzed using the logistic regression (y = a/(1 + Exp (b – k x))) where x is the feeding days in each food and y is the cumulative mating in proba- bility units. Mating time data were fitted using probability analysis to estimate the time for 16% (mating onset), 50% (mating peaks), and 84% (mating ends) (Gong et  al. 2012) cumulative mating in each food treatment tested. Mating peaks are the 50% cumulative mating. To obtain the age-specific cumulative mating ratio, cumula- tive mating data were subjected to Arcsine transformation (arcsine square root transformation) (Sokal and Rohlf 1995, Warton and Hui 2011) and then were subjected to ANOVA (one-way) and means were separated by Tukey’s test at P = 0.05. To access the female oviposition, the orange provided for ovi- position was removed from the cage and cut through the oviposi- tion patch or hole. The eggs found were counted by naked eyes and recorded. The following expression was used to estimate the female egg production per female. Oviposition per female = ∑(total weekly fecundity/weekly average number of females surviving). To access the female fecundity of B.  minax, egg load was determined by counting the number of mature oocytes in the egg chambers under stereomicroscope. The estimated standard stages of ovarian development were assigned by relating to particular Fig. 1. Grading of the ovarian in female adult of B. minax. Stages of ovarian oogenesis stages. Hence, the ovarian development in B.  minax development in adult B.  minax, Stages 1–2 represent sexually immature observed was “first stage”: ovary is original, very small, nonde- females, and refer to steps in the ovary maturation phase. Stage 3 represents veloped, nonshaped, and with subsidiary silks. Its diameter is just mature ovaries, before and after the onset of oviposition. (a) means ovarian a little greater than the oviduct. “Second stage”: ovary is growing, stage 1, (b) means ovarian stage 2, (c) means ovarian stage 3. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/25/4924853 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Journal of Insect Science, 2018, Vol. 18, No. 2 frequency per male, age-specific mating frequency, ovarian egg load, F2 (43.8 d) and F3 (31.8 d). Nonprotein diet (F0 and F1) had short and female oviposition were subjected to ANOVA (one-way), and LT90 of 7.68 d and 16.16 d, respectively (Table 3). means were separated by Tukey’s test at P = 0.05. Statistical analyses were performed using SPSS 17.0 (SPSS Inc. 2008). Effect of Protein Supplements on B. minax Mating Behavior Mating frequency Results The food type influenced the mating frequency of B.  minax (F  = 25.617, df = 3, 19, P = 0.000; F  = 12.696, df = 3, 19, P = 0.000) Effect of Protein Supplements on Adult B. minax ♀ ♂ (Table  1). Higher mating frequency was observed when flies were Survival fed with F2. No mating was observed when flies were fed on diet Life expectancy F0. Mating frequency was observed to be the lowest with 0.38 Diet influences life expectancy (F  = 89.055, df = 3, 19, P = 0.000) per female and 0.31 per male in the lifetime when adults B. minax presented in Table 1. Protein supplementation resulted in significant were fed on F1 diet. (F1 vs F2, F3 P  = 0.000, 0.002; P   =  0.030, differences in adult life expectancies. The longest adult life expect- ♀ ♂ 0.001) (Table 1). There was a significant increase in mating frequen- ancy was observed when flies were fed the diet F2 (40.10  ±  1.79 cies when adults’ B.  minax were fed on diet F2 or F3 (P  <  0.05). d) followed by the diet F3 (32.76 ± 2.66 d) (P < 0. 000), the life Hence, the mean values were 8.07 (P = 0.000) for a female and 4.69 expectancy on nonprotein diet (F1) observed to be (19.62 ± 0.09 (P = 0.030) for a male when B.  minax was fed on the diet F2 and d; P = 0.024). 5.31 (P = 0.002) for female and 7.30 (P = 0.001) for a male when the diet F3 was used. However, the mating frequency showed no Mortality dynamic statistical difference between the diets F3 and F2 for both females Adult mortality was observed to vary with the nutrient type sup- and males (P  = 0.094; P  = 0.283). ♀ ♂ plemented. The mortality rate was highest when B. minax was fed with F0 and F1 diets (Table  2). F2 and F3 diets had a different effect on their mortality rates; 100% adult mortality was observed Mating dynamic at age 65 d and 75 d when flies were fed with F2 and F3, respec- No mating behavior was observed for adults fed with diet F0 only tively; 100% adult mortality was observed at 20 and 30 d on flies (Fig.  3) By contrast, adults fed with diets F1, F2, and F3 started fed with F0 and F1food types, respectively. The flies fed on diet F2 mating at 12, 10, and 9 feeding days, respectively. The mating was and F3 showed a slow cumulative mortality (Fig.  2).The LT of observed to end at 18 d for flies fed with diet F1, 51 d for F2, and 42 adults fed with the diet F0 was the shortest (5.32 d), whereas LT d for F3. The flies fed with diet F2 mating behavior lasted for 42 d, was longer when flies were fed with diet F2 (14.6 d) followed by followed by flies fed with F3 diet lasted for 34 d (Table 4). Among diet F3 (LT  = 13.1 d) and diet F1 (LT  = 9.0 d). The use of pro- the diets tested, F2 and F3 showed similar mating peaks (50% cumu- 50 50 tein-enriched foods extended the peak of adult mortality (Fig. 2). In lative mating ratio) (Table 5). addition, when flies were fed with diets F2 and F3, their mortality The mating peaks (50% mating ratio) for the fruit flies fed with F2 and peaks were delayed by 5.6 d and 4.1 d than that of adults fed with F3 diets were observed at 24.6 and 21.7 d old, respectively, whereas for diet F1. LT90 were observed to be longer when flies were fed with flies fed with diet F1 the peak occurred only at 14.9 d (Fig. 3 and Table 5). Table 1. Life expectancy, mating frequency, ovarian egg load, and female egg production data for B. minax maintained at different food sources (protein diet and non-protein diets) Food sources Life expectancy Mating frequency Ovarian egg load Female oviposition ♀ ♂ F0 5.7 ± 0.1d 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0c F1 19.6 ± 0.1c 0.38 ± 0.2b 0.31 ± 0.1b 15.0 ± 7.6b 0.0 ± 0.0c F2 40.1 ± 1.8a 8.07 ± 0.3a 4.69 ± 0.4a 94.79 ± 6.3a 63.2 ± 8.7a F3 32.8 ± 2.7b 5.31 ± 1.5a 7.3 ± 1.9a 77.29 ± 7.0a 19.3 ± 2.5b The mean ± standard deviation (life expectancy, mating frequency/male or female, number of eggs/ovary, and egg production per female) are represented. F0– Diet containing Water alone; F1–Diet containing Sucrose alone; F2–Diet containing Sucrose and Yeast; F3–Diet containing Sucrose and Peptone. Different letters between food types are statistically different after Tukey’s test at P = 0.05. Table 2. Age-specific cumulative mortality ratio Food sources Age-specific cumulative mortality ratio (%) 5 d 10 d 30 d 50 d 65 d 75 d F 37.0 ± 4.1a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a F 36.3 ± 12.3a 66.0 ± 8.3b 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a F 23.3 ± 2.1a 49.7 ± 0.6b 77.0 ± 3.1b 87.7 ± 2.3b 98.7 ± 0.6b 100.0 ± 0.0a F 25.3 ± 0.6a 49.3 ± 1.5b 82.7 ± 4.4b 96.7 ± 2.1a 100.0 ± 0.0a 100.0 ± 0.0a Arcsine transformation (arcsine square root transformation used to transform cumulative mortality data and then one-way ANOVA, means were separated by Tukey’s test at P = 0.05. Different letters between food types are statistically different after Tukey’s test at P = 0.05. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/25/4924853 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 5 No egg was laid when females were fed with diets F0 and F1. The Effect of protein supplements on adult B. minax mean number of eggs laid was of 63.2  ±  8.7 eggs/individual in fecundity females fed with diet F2 and 19.3 ± 2.5 eggs/individual in females Ovarian development fed with diet F3. The results showed a significant difference in The timing of ovary development varied in relation to the type of diet the amount of eggs laid by females fed with diet F2 and diet F3 the flies were fed with (Fig. 4). When fed with diets F2 and F3, the ovary (P = 0.000). of the adult B. minax developed faster than that of F1 diet. The ovaries of female fed with diets F2 and F3 developed to stage 2 on the 5th day, to stage 3 on the 10th day and were found to have stage 2 and stage 3 Discussion ovaries 5 d earlier than those female fed with diet F1. For females fed with diets F3, F2, and F1, the disappearance of stage 1 ovaries occurred Sugar and other nutritional components like protein are necessary at 20 d, 15 d, and 10 d, respectively. The disappearance time in females dietary requirement for energy production and development that fed with diets F3 and F2 was shorter than for females fed with diet F1. greatly affect the life history traits in adults of many insect species (Bateman 1972, Jácome et al. 1999, May et al. 2015). In the present Ovarian egg load study, it was observed that provision of water or sugar alone resulted The ovarian egg load of B.  minax appeared to be different depend- in high mortality rates of the adults’ B. minax. However, provision ing on the food type provided to adult flies (F   =  14.979, df  =  3, 46, of sugar and protein enhanced the survival and reproduction of P  =  0.000) (Table  1). For females fed with water only, no egg was adult B. minax. Adult mortality of 100% were observed at age 30 d observed in the ovary. For females fed with diet F1, brood eggs per on the flies fed with nonprotein diet, while at age 75 d 100% mor - ovary were 15.00 ± 7.57 (n = 3), significantly lower than in females fed tality was observed for protein-fed adult. These results congruent with diets F2 (94.79 ± 6.28; n = 24; P = 0.000) and F3 (77.29 ± 6.97; with Teal et  al. (2004) who found that Anastrepha suspensa pro- n = 45; P = 0.006). The results, however, showed there was no difference vided with a diet of sugar and protein lived longer than those fed between brood eggs of females fed with diet F2 and diet F3 (P = 0.233). with sugar alone. Sucrose diet facilitates metabolism of insect where the energy obtained extend insect life span (Lardies et al. 2004, Naya et  al. 2007). Protein is an important nutritional element for insect Female oviposition reproduction as it contains amino acids necessary for insect ovipo- There were significant differences in egg production when differ - sition (Lardies et al. 2004, Nash et al. 2014). Alamzeb et al. (2006) ent foods were fed (F = 43.677, df = 3, 19, P = 0.000) (Table  1). reported that protein is necessary for female oocytes maturation to reach vitellogenic stage. Harwood et  al. (2013) also reported that protein supplement following eclosion period led to higher survival and reproductive abilities in the Mediterranean fruit fly, C. capitata Wiedemann and melon fly, B. cucurbitae Coquillett suggesting that provisioning protein-enriched foods extend fruit flies life expectancy and reproduction. Chinese citrus fruit fly showed different mating frequencies fol- lowing the food type provided. Protein diet (yeast and peptone) 40 showed increase in mating frequencies. Mating started at early age F0 around nine feeding days after eclosion on protein-fed adult, mat- F1 ing duration on protein-fed flies lasted for 42 d.  Shelly et al. (2002) 20 F2 reported that C. capitata male mating was more achieved when sub- F3 jected to protein diet than nonprotein diet. On females, they observed that the females were sighted more near protein-fed males than non- protein fed males. Joachim-Bravo et  al. (2009) investigated on the role of protein on sexual behavior of C. capitata and, observed that Feeding time (Days) male fed with high protein diet showed greater number of copu- Fig. 2. Effect of different food types on cumulative mortality of adult B. minax. lation. In addition, young males and males fed with high protein Table 3. Curve equation for regression between cumulative mortality and feeding days of B. minax adults fed with different food types Food types Regression equation R R R Feeding days 0.05 0.01 LT LT 50 90 F0 Y = 102.057/(1 + Exp(4.665 − 0.869X)) 1.000 0.666 0.798 5.3 7.7 F1 Y = 99.255/(1 + Exp(2.817 − 0.315X)) 0.994 0.388 0.496 9.0 16.2 F2 Y = 93.472/(1 + Exp(1.364 − 0.103X)) 0.967 0.413 0.526 14.6 44.9 F3 Y = 96.843/(1 + Exp(1.612 − 0.128X)) 0.982 0.707 0.834 13.1 32.7 Y: represents the cumulative mortality of B. minax; X: feeding days (days of food supply to the flies, until death of the last fly) of B. minax; LT50: (Lethal Time) is the time required to record 50% adult mortality. LT90: the maximum lethal time required to record 90% adult mortality; R: coefficient of determination; R : 0.05 coefficient of determination at 95%, R : coefficient of determination at 99%. (If R-value is greater than 0.05 and 0.01 are statistically significant and R-value less 0.01 than 0.05 and 0.01 are not statistically significant). The adult cumulative mortality was transformed to probability units and analyzed using the logistic regression (y = a/(1 + Exp (b − k x))) where x is the feed days in each food and y is the cumulative mortality in probability units. Time–mortality data was fitted using prob- ability analysis to estimate the lethal time for 50% (LT 50) and 90% (LT 90) cumulative mortality in each food treatment tested. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/25/4924853 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Cumulative mortality ratio (%) 6 Journal of Insect Science, 2018, Vol. 18, No. 2 showed greater participation and called more often than older For most of the studied biological characteristics of B.  minax, males and males fed with nonprotein diet (Roriz and Joachim-Bravo our results showed that protein source diet in B. minax had the high- 2013). Egg production increased proportionally with higher mating est effect on egg production and survival compared with nonpro- frequencies. This trade-off interaction resulted in the reduction of tein sources (F0 and F1) where flies died shortly. The results are in female life expectancy when fed with protein diet as earlier reported agreement with previous studies on physiology of fruit flies (Nash by Papanastasiou et al. (2013). The ovary growth was faster when et al. 2014, Shinwari et al. 2015). Between the two protein sources adults B. minax were fed F2 and F3 diet than F1. This indicates that tested, addition of yeast extract as source of protein had the highest protein supplementation resulted in a faster ovary maturation and effect compared with the addition of peptone, yeast extract showed female egg production. For example, Jácome et al. (1999) reported positive response on flies survival and reproduction. The results are that female A. serpentina requires access to protein-enriched diets in in agreement with previous studies on physiology of fruit flies (Nash order to produce significant number of eggs. et al. 2014, Shinwari et al. 2015). Zajitschek et al. (2012) reported that yeast diet had a strong influence on survival and female repro- ductive fitness. The higher effects of yeast extract can be attributed to its composition (nitrogenous compounds, carbon, sulfur, trace nutrients, vitamin B complex) and also the fact that it’s made up of microorganisms from fungi groups. Apart from amino acids, other substances such as B-complex vitamins and mineral were incorpo- rated into the yeast diets. These components play important catalytic roles in the physiology of many insect species (Fraenkel and Blewett 1943, Nash et al. 2014, Zhou et al. 2016) and are known to enhance the fecundity in fruit flies (Ben-Yosef et al. 2010). F0 Loss of reproductive ability (reproduction senescence) for F1 B.  minax was observed when flies were subjected to nonprotein F2 diet. This explains the significance of protein on flies’ reproduction. F3 Delaying host access and protein sources result to reduced repro- duction known as reproduction senescence (Harwood et al. 2013). Bonduriansky et al. (2008) reported that trade-off between survival and reproduction is influenced by time and duration of poor repro- Feeding time (Days) ductive conditions may decrease the overall fecundity of an organ- ism because of senescence unless the reproductive ability can be Fig.  3. Cumulative mating of adult B.  minax fed on different food sources (50% represent adult mating peaks). extended to advanced ages. Table 4. Age-specific cumulative mating ratio Food sources Age-specific cumulative mating ratio (%) 5 d 15 d 18 d 30 d 42 d 51 d F0 — — — — — — F1 0.0 ± 0.0a 43.3 ± 23.3a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a F2 0.0 ± 0.0a 7.1 ± 1.7a 21.4 ± 4.8b 88.5 ± 4.8b 95.1 ± 1.4b 100.0 ± 0.0a F3 0.0 ± 0.0a 14.0 ± 2.7a 34.4 ± 7.7b 74.1 ± 7.9a 100.0 ± 0.0a 100.0 ± 0.0a Arcsine transformation (arcsine square root transformation used to transform mating frequency data and then one-way ANOVA, means were separated by Tukey’s test at P = 0.05. Different letters between food types are statistically different after Tukey’s test at P = 0.05. Table 5. Curve equation from regression analysis between cumulative mating ratio and feeding days for B. minax adults fed on different food sources Food types Regression equation R R R Feeding days 0.05 0.01 Mating Mating Mating onset (16%) peak (50%) end (84%) F0 — — — — — — — F1 Y = 106.933/(1 + Exp(16.30 − 1.087X)) 0.972 0.775 0.875 13.4 14.9 16.2 F2 Y = 97.131/(1 + Exp(5.425 − 0.223X)) 0.997 0.304 0.393 17.1 24.6 32.7 F3 Y = 99.740/(1 + Exp(5.407 − 0.249X)) 0.994 0.339 0.436 15.1 21.8 28.4 Y: represents the cumulative mating ratio of B. minax; X: represents the feeding time (days of food supply to the flies, until death of the last fly) of B. minax; —: represent unmated; R: coefficient of determination; R : coefficient of determination at 95%, R : coefficient of determination at 99%. (If R-value is Greater 0.05 0.01 than 0.05 and 0.01 are statistically significant and R-value Less than 0.05 and 0.01 are not statistically significant). The adult cumulative mating ratio in four food sources (F0, F1, F2, F3) tested was transformed to probability units and analyzed using the logistic regression (y = a/(1 + Exp (b − k x))) where x is the feeding days in each food, y is the cumulative mating ratio of B. minax in probability units. Mating time data were fitted using probability analysis to estimate the time for 16% (mating onset), 50% (mating peaks), and 84% (mating ends) cumulative mating in each food treatment tested. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/25/4924853 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Cumulative mating ratio (%) Journal of Insect Science, 2018, Vol. 18, No. 2 7 stage1 stage2 stage3 ( and above ) 0d 5d 10d 15d 20d 0d 5d 10d 15d 20d 0d 5d 10d 15d 20d F1 F2 F3 Fig. 4. Ovarian development for female B. minax flies fed with different food types. d: indicates feeding days when food was supplied to the flies. CABI. 2017. Bactrocera minax. In Invasive Species Compendium. CAB During this study, both sugar and protein appeared to be essen- International, Wallingford, UK. https://www.cabi.org/isc/datasheet/8726. tial components for the survival and reproduction of B. minax. The Carey, J. R., P. Liedo, L. Harshman, Y. Zhang, H. G. Müller, L. Partridge, and J. deprivation of protein and sugar from adult diets resulted in high L. Wang. 2002. Life history response of Mediterranean fruit flies to dietary mortality rates. However, the use of yeast extract as source protein restriction. Aging Cell. 1: 140–148. enhanced adult life span, mating frequency, fecundity, and ovarian Carey, J. R., L. G. Harshman, P. Liedo, H. G. Müller, J. L. Wang, and Z. Zhang. development in B. minax. Same results were obtained by Lee et al. 2008. Longevity-fertility trade-offs in the tephritid fruit fly, Anastrepha (2008), Fanson et al. (2009), and Fanson and Taylor (2012) as diet ludens, across dietary-restriction gradients. Aging Cell. 7: 470–477. had influence on life span and egg production on Queensland fruit Chen, Z., Y. Dong, Y. Wang, A. A. Andongma, M. A. Rashid, P. Krutmuang, fly and D. melanogaster. These results suggest that yeast extract is a and C. Niu. 2016. Pupal diapauses termination in Bactrocera minax: an suitable source of protein in mass rearing of B. minax. Nevertheless, insight on 20-hydroxyecdysone induced phenotypic and genotypic expres- sions. Scientific Reports, 6: 27440. more studies are needed to unveil other factors required for a suc- Chou, M. Y., R. F.  Mau, E. B.  Jang, R. I.  Vargas, and J. C.  Piñero. 2012. cessful artificial rearing of this devastative citrus fly so as to imple- Morphological features of the ovaries during oogenesis of the Oriental fruit fly, ment an effective management strategy of their populations. Bactrocera dorsalis, in relation to the physiological state. J. Insect Sci. 12: 1–12. EPPO/CABI. 1997. Data Sheets on Quarantine Pests, Bactrocera minax [J]. Acknowledgments http://www.eppo.org/QUARANTINE/listA1.htm. Fanson, B. G., and P. W. Taylor. 2012. Protein:carbohydrate ratios explain life This work was supported by the National Science Foundation of China span patterns found in Queensland fruit fly on diets varying in yeast:sugar (31572010). We thank Dr. Mazarin Akami from Huazhong Agricultural ratios. Age (Dordr). 34: 1361–1368. University for reviewing the final version of our manuscript. Fanson, B. G., C. W. Weldon, D. Pérez-Staples, S. J. Simpson, and P. W. Taylor. 2009. 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Influence of Supplemental Protein on the Life Expectancy and Reproduction of the Chinese Citrus Fruit Fly, Bactrocera minax (Enderlein) (Tetradacus minax) (Diptera: Tephritidae)

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Abstract

Bactrocera minax (Enderlein) (Diptera: T ephritidae) is a major citrus pest in China, whose artificial rearing technology of the adult is not well documented to date. In this study, we tried to determine if supplementing proteins to the adult diet could result in the enhancement of some fitness parameters of B. minax. Four feeds with varying protein source were provided as F0 (water), F1 (sucrose), F2 (sucrose + yeast), and F3 (sucrose + peptone). F0 and F1 being the control, F2 and F3 were protein food types. The results showed that adults fed by F2 and F3 lived longer with 40.1 d and 32.8 d, respectively, had reduced death rates (death peaks were delayed for 5.6 d and 4.1 d, respectively), increased mating frequencies (8.1 and 5.3 per females, 4.7 and 7 .3 per males, respectively), and longer mating durations (with 42 d and 34 d). In addition, females recorded an increased adult ovary development, more egg load (with 94.8 and 77 .3 brood eggs per ovary) and to greater oviposition rates of 63.2 eggs/female and 19.3 eggs/female. Based on our results, protein supplements enhanced B. minax survival, mating, and fecundity. This study does not only provide basic knowledge to implement artificial rearing of B. minax, but also deepens our understanding on its physiology that could be used to enhance the management of the pest. Key words: Bactrocera minax, protein compositions, survival, mating, fecundity Fruit flies (Diptera: Tephritidae) are among the most destructive agri- occurs in other Asian countries such as Bhutan, Nepal, and India cultural pests known to occur worldwide (White and Elson-Harris (EPPO/CABI 1997, Wang et  al. 2014). In China, B.  minax mainly 1992, Bhattacharya et al. 2013). Fruit flies damage fruits and vege- occurs in temperate provinces such as Guangxi, Guizhou, Hubei, tables by laying their eggs inside the fruits and cause them to rot and Hunan, Jiangsu, Jiangxi, Shaanxi, Sichuan, and Yunnan (EPPO/ drop, thus inducing significant losses in production (Hollingsworth CABI 1997, Wang et al. 2014). The pest exhibits about 6 mo’s pupal et al. 1997, Bhattacharya et al. 2013). Besides causing direct losses diapause from November to May as a way to fight against harsh to a wide range of fruits and vegetables, they limit the development environmental conditions (Chen et al. 2016). Such long pupal dia- of agriculture in many countries because of strict trade quarantines pause is a barrier for laboratory rearing and development of control imposed to prevent their spread. Bactrocera minax (Enderlein) strategies against this pest (Chen et  al. 2016). Damage caused by (Tetradacus minax) (Diptera: Tephritidae) is one of the most impor- larval feeding on the fruit pulp results in fruit segments partially tant quarantinable pests which attacks citrus fruits in China. It also or completely turning into paste. The losses are estimated to range © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/25/4924853 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Journal of Insect Science, 2018, Vol. 18, No. 2 between 5 and 20% and can reach up to 50% (Wang et al. 1995, Materials and Methods 2009). In a global agricultural system that promotes food security Acquisition of Adult Fruit Flies and sustainability, there is a necessity to produce nutritional comple- Mature larvae of B. minax were collected from Orange orchards in ments in quality and quantity to meet the rising demands in fruits Songzi, Hubei, China, between 20 October and 10 November 2015. and vegetables of the population. B.  minax, therefore, represents a The collected larvae were taken to the Yangtze university insectary significant threat to the completion of these challenges unless it is where they were moved into plastic container (21 cm in diameter) well controlled and managed. containing 15% water content sand (sterilized sand by constant tem- Nutrition plays a vital role in the development of tephritid fruit perature 60°C for 24 h to kill Nematodes) and kept indoor at room flies. Life span, maturity development, and reproductive capacity are temperature (4.8–22.6°C, 75% RH ± 5%) till adult emergence. The mostly influenced by the quality of the food (Good and Tatar 2001, plastic container containing fourth stage pupae were placed into big Fontana et  al. 2010). Reproduction and longevity are mutually cage (baby mosquito net) to prevent the emerging adult to escape. dependent in sexually reproducing organisms, with an increase in Adults emerged in late April and early May 2016 and ended on 27 the energy used for reproduction resulting in a subsequent decrease May 2016. in longevity (Partridge and Andrews 1985). Hence, the nutritional conditions in an environment influence the trade-off between current Diet Preparation reproductive effort and life span because of competition for nutrients In this study, a solid diet was used to feed the flies, the diet was between somatic maintenance and gamete production (Kirkwood measured in grams; sucrose 4  g (Tianjin Beilian Fine Chemicals and Rose 1991). Lee and Lee (2005) reported on the benefit of arti- Development Co. Ltd.), sucrose + yeast extract (3  g + 1  g, mak- ficial diet on predator’s development. Also, the reproductive ability ing 3:1) (yeast extract and sucrose + peptone (3 g and 1 g, making in many Tephritids is strongly influenced by diet (Aluja et al. 2001; 3:1)(Guangdong Huankai Microbial Sci & Tech. Co. Ltd). After Carey et  al. 2002, 2008; Harwood et  al. 2013; Liedo et  al. 2013; measurements, the solid food was mixed evenly and placed on Petri Pereira et al. 2013). dishes ready to be provided to flies. Yeast extract was used as source Protein is one of the most important nutrients required for ovar- of protein, as it contains vitamin content and protein which pro- ian development and vitellogenesis in insects (Raikhel and Dhadialla vide excellent growth conditions for more microbes. Peptone is the 1992, Aluja et  al. 2001, Taylor et  al. 2013). In general, fruit flies microbiological culture media prepared by enzymatic digestion of fed with protein supplements, for example Anastrepha ludens selected animal tissues, widely used to suit a range of nutritional (Loew), Ceratitis capitata (Wiedemann), and Bactrocera cucurbitae requirements. Peptone was used as source of protein as it supports (Coquillett), had their fecundity enhanced while given protein sup- excellent growth of various microorganisms. It also contains short plements (Mangan 2003, Harwood et  al. 2013). Also, protein-en- chains peptides and broad spectrum amino acids required for the riched foods were shown to promote the longevity of the fruit fly, microbial growth. Anastrepha serpentina (Wiedemann) (Jácome et  al. 1999). In add- ition, the protein level of the food has a high influence on egg pro- Experimental Design duction (Mangan 2003). For example, while evaluating the effect of The obtained adults were separated in three cohorts. To assess the larval diet on the development and reproductive rates of male and effects of protein supplementation on the survival of B.  minax, a female Mediterranean fruit fly, Kaspi et al. (2002) found that pro- total of 60 newly emerged flies comprising 30 males and 30 females tein-fed larvae were large in size, grew fast, and had more nutritional were introduced in a wire mesh cage (dimensions: 30 cm × 30 cm × reserves upon emergence. 30 cm) covered with a white cloth at the bottom. Flies were provided A source of sugar is an important nutrient for fruit flies with four types of food designated as F0 (water alone), F1 (sucrose because it provides energy for survival and reproduction. Hagen alone), F2 (sucrose + yeast extract, mass ratio 3:1), and F3 (sucrose + (1953) reported that both sexes of the western cherry fruit fly, peptone, mass ratio 3:1). There were five replicates for each food Rhagoletis indifferens, Curran, require sources of sugar for sur- type, 30 males and 30 females were provided each food type result- vival. Fontellas and Zucoloto (1999) also found that West Indian ing into a total of 60 flies per replicate. After preparation, 4 g solid fruit fly, Anastrepha obliqua, Macquart, did not survive more than food were provided to the colony into the Petri dish (diameter: 9 cm) 3 d without a sugar (sucrose) source. However, Sacchetti et  al. and replaced every 24 h. Also, the flies were provided with 7 ml of (2014) reported that olive fruit fly, Bactrocera oleae, did not sur - water twice a day (morning and evening) on a cotton bud (diameter: vive longer when fed with sugar diet. Similarly, Gavriel et al. (2011) 0.5 cm) that was replaced on a weekly basis. Male and female flies’ reported that male Mediterranean fruit fly had a short life span and mortality was recorded daily. the ability of the female to receive male were not inhibited when To assess the effect of protein supplementation on mating and fed with granulated sugar only. This implies that fruit flies depend egg production, 30 males and 30 females virgin B. minax (0 to 24 h upon these two nutrients for both survival and egg production. To after emergency) were provided each food source (F0, F1, F2, and the best of our knowledge, none of the previous studies about the F3) resulting into a total of 60 flies per replicate. There were five artificial rearing techniques of B. minax has been successful due to replicates for each food type. Fresh orange fruit was provided for egg the high adult mortality rates observed. This unfortunate situation laying as they are adapted to oviposition through the thick skin of hindered a comprehensive investigation of its biological character- oranges (CABI, 2017), and the orange was replaced every three days. istics and the development of effective prevention measures. For The frequency of mating (mating times) was observed and recorded this reason, the understanding of the nutritional factors that could every half an hour from 8:00 a.m. to 6:00 p.m. reduce mortality of B. minax and enhance its reproduction capac- Finally, 800 females were kept in four separate cages (200 ities is of prime importance for the development of an artificial females/cage) where they were provided with four food sources as rearing program. Thus, the present study aimed at evaluating the described above. Twenty insects were dissected every 5 d for a dur- feeding response of adults to different protein supplements through ation of 20 d to assess the ovarian development and egg load (Chou the assessment of their effects on the survival and reproduction of et al. 2012). the Chinese citrus fruit fly, B. minax. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/25/4924853 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 3 swelling. and big. Subsidiary silks diminish or lose. Primary for- Demographic Analysis mation of ovary and egg yolk precipitation. “Third stage”: ovary Adult life expectancy was obtained by the following expression; is growing bigger, and harder egg yolk deposition to form egg. “Life expectancy”  =  (∑Survival days per insect)/Total number of Eggs can be seen in the oviducts. Mature Eggs in ovary, before adult tested. On evaluating the effect of protein on flies, cumulative and after the onset of oviposition (Fig.  1) (Fletcher et  al. 1978; mortality ratio (CMR) and feeding days were compared. First, we Kendra et  al. 2006; Chou et  al. 2012). To calculate the ratio of calculated the mortality ratio (MR %) by MR % = daily mortality/ different ovary stages the following formula was used: “Ratio of total lifetime mortality * 100, then CMR % obtained by the follow- specific ovary stage (%)” = (number of flies with the specific ovary ing formula; CMR (%) = MR + MR + MR + … + MR . Where; n 1 2 3 n stage)/(total number of flies dissected) * 100. MR is the mortality ratio; CMR is the cumulative mortality ratio, n Data on the effect of diet on adult life expectancy, mortal- is the feeding days when food was supplied to the flies. ity, age-specific mortality, mating frequency per female, mating The adult cumulative mortality in all four types of food (F0, F1, F2, and F3) tested were transformed to probability units and ana- lyzed using the logistic regression (y = a/(1 + Exp (b – k x))) where x is the feed days in each food and y is the cumulative mortality in probability units. Time–mortality data were fitted using probability analysis to estimate the lethal time for 50% (LT ) and 90% (LT ) 50 90 cumulative mortality in each food treatment tested. To obtain the age-specific cumulative mortality, cumulative mortality data were subjected Arcsine transformation (arcsine square root transforma- tion) (Sokal and Rohlf 1995, Warton and Hui 2011) and then were subjected to analysis of variance (ANOVA one-way) and means were separated by Tukey’s test at P = 0.05. To access mating dynamic the following expression were used; “Lifetime Mating frequency per female” = ∑(daily number of mat- ing times/daily number of surviving females). “Lifetime Mating fre- quency per male” = ∑(daily number of mating times/daily number of surviving males). The cumulative mating ratio (CMR) for each diet was calculated using the following expression: first, we calculated the mating ratio (MR %) by the following formula, MR% = (life- time mating times)/(total lifetime mating times) * 100. The MR obtained was used to calculate the CMR by using the following for- mula; CMR (%)  =  MR + MR + MR + … + MR . Where; MR n 1 2 3 n is the daily mating ratio; CMR is the cumulative mating ratio; n is the feeding days (days of food supply to the flies, until death of the last fly). The adult cumulative mating in four food sources (F0, F1, F2, and F3) tested was transformed to probability units and analyzed using the logistic regression (y = a/(1 + Exp (b – k x))) where x is the feeding days in each food and y is the cumulative mating in proba- bility units. Mating time data were fitted using probability analysis to estimate the time for 16% (mating onset), 50% (mating peaks), and 84% (mating ends) (Gong et  al. 2012) cumulative mating in each food treatment tested. Mating peaks are the 50% cumulative mating. To obtain the age-specific cumulative mating ratio, cumula- tive mating data were subjected to Arcsine transformation (arcsine square root transformation) (Sokal and Rohlf 1995, Warton and Hui 2011) and then were subjected to ANOVA (one-way) and means were separated by Tukey’s test at P = 0.05. To access the female oviposition, the orange provided for ovi- position was removed from the cage and cut through the oviposi- tion patch or hole. The eggs found were counted by naked eyes and recorded. The following expression was used to estimate the female egg production per female. Oviposition per female = ∑(total weekly fecundity/weekly average number of females surviving). To access the female fecundity of B.  minax, egg load was determined by counting the number of mature oocytes in the egg chambers under stereomicroscope. The estimated standard stages of ovarian development were assigned by relating to particular Fig. 1. Grading of the ovarian in female adult of B. minax. Stages of ovarian oogenesis stages. Hence, the ovarian development in B.  minax development in adult B.  minax, Stages 1–2 represent sexually immature observed was “first stage”: ovary is original, very small, nonde- females, and refer to steps in the ovary maturation phase. Stage 3 represents veloped, nonshaped, and with subsidiary silks. Its diameter is just mature ovaries, before and after the onset of oviposition. (a) means ovarian a little greater than the oviduct. “Second stage”: ovary is growing, stage 1, (b) means ovarian stage 2, (c) means ovarian stage 3. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/25/4924853 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Journal of Insect Science, 2018, Vol. 18, No. 2 frequency per male, age-specific mating frequency, ovarian egg load, F2 (43.8 d) and F3 (31.8 d). Nonprotein diet (F0 and F1) had short and female oviposition were subjected to ANOVA (one-way), and LT90 of 7.68 d and 16.16 d, respectively (Table 3). means were separated by Tukey’s test at P = 0.05. Statistical analyses were performed using SPSS 17.0 (SPSS Inc. 2008). Effect of Protein Supplements on B. minax Mating Behavior Mating frequency Results The food type influenced the mating frequency of B.  minax (F  = 25.617, df = 3, 19, P = 0.000; F  = 12.696, df = 3, 19, P = 0.000) Effect of Protein Supplements on Adult B. minax ♀ ♂ (Table  1). Higher mating frequency was observed when flies were Survival fed with F2. No mating was observed when flies were fed on diet Life expectancy F0. Mating frequency was observed to be the lowest with 0.38 Diet influences life expectancy (F  = 89.055, df = 3, 19, P = 0.000) per female and 0.31 per male in the lifetime when adults B. minax presented in Table 1. Protein supplementation resulted in significant were fed on F1 diet. (F1 vs F2, F3 P  = 0.000, 0.002; P   =  0.030, differences in adult life expectancies. The longest adult life expect- ♀ ♂ 0.001) (Table 1). There was a significant increase in mating frequen- ancy was observed when flies were fed the diet F2 (40.10  ±  1.79 cies when adults’ B.  minax were fed on diet F2 or F3 (P  <  0.05). d) followed by the diet F3 (32.76 ± 2.66 d) (P < 0. 000), the life Hence, the mean values were 8.07 (P = 0.000) for a female and 4.69 expectancy on nonprotein diet (F1) observed to be (19.62 ± 0.09 (P = 0.030) for a male when B.  minax was fed on the diet F2 and d; P = 0.024). 5.31 (P = 0.002) for female and 7.30 (P = 0.001) for a male when the diet F3 was used. However, the mating frequency showed no Mortality dynamic statistical difference between the diets F3 and F2 for both females Adult mortality was observed to vary with the nutrient type sup- and males (P  = 0.094; P  = 0.283). ♀ ♂ plemented. The mortality rate was highest when B. minax was fed with F0 and F1 diets (Table  2). F2 and F3 diets had a different effect on their mortality rates; 100% adult mortality was observed Mating dynamic at age 65 d and 75 d when flies were fed with F2 and F3, respec- No mating behavior was observed for adults fed with diet F0 only tively; 100% adult mortality was observed at 20 and 30 d on flies (Fig.  3) By contrast, adults fed with diets F1, F2, and F3 started fed with F0 and F1food types, respectively. The flies fed on diet F2 mating at 12, 10, and 9 feeding days, respectively. The mating was and F3 showed a slow cumulative mortality (Fig.  2).The LT of observed to end at 18 d for flies fed with diet F1, 51 d for F2, and 42 adults fed with the diet F0 was the shortest (5.32 d), whereas LT d for F3. The flies fed with diet F2 mating behavior lasted for 42 d, was longer when flies were fed with diet F2 (14.6 d) followed by followed by flies fed with F3 diet lasted for 34 d (Table 4). Among diet F3 (LT  = 13.1 d) and diet F1 (LT  = 9.0 d). The use of pro- the diets tested, F2 and F3 showed similar mating peaks (50% cumu- 50 50 tein-enriched foods extended the peak of adult mortality (Fig. 2). In lative mating ratio) (Table 5). addition, when flies were fed with diets F2 and F3, their mortality The mating peaks (50% mating ratio) for the fruit flies fed with F2 and peaks were delayed by 5.6 d and 4.1 d than that of adults fed with F3 diets were observed at 24.6 and 21.7 d old, respectively, whereas for diet F1. LT90 were observed to be longer when flies were fed with flies fed with diet F1 the peak occurred only at 14.9 d (Fig. 3 and Table 5). Table 1. Life expectancy, mating frequency, ovarian egg load, and female egg production data for B. minax maintained at different food sources (protein diet and non-protein diets) Food sources Life expectancy Mating frequency Ovarian egg load Female oviposition ♀ ♂ F0 5.7 ± 0.1d 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0c F1 19.6 ± 0.1c 0.38 ± 0.2b 0.31 ± 0.1b 15.0 ± 7.6b 0.0 ± 0.0c F2 40.1 ± 1.8a 8.07 ± 0.3a 4.69 ± 0.4a 94.79 ± 6.3a 63.2 ± 8.7a F3 32.8 ± 2.7b 5.31 ± 1.5a 7.3 ± 1.9a 77.29 ± 7.0a 19.3 ± 2.5b The mean ± standard deviation (life expectancy, mating frequency/male or female, number of eggs/ovary, and egg production per female) are represented. F0– Diet containing Water alone; F1–Diet containing Sucrose alone; F2–Diet containing Sucrose and Yeast; F3–Diet containing Sucrose and Peptone. Different letters between food types are statistically different after Tukey’s test at P = 0.05. Table 2. Age-specific cumulative mortality ratio Food sources Age-specific cumulative mortality ratio (%) 5 d 10 d 30 d 50 d 65 d 75 d F 37.0 ± 4.1a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a F 36.3 ± 12.3a 66.0 ± 8.3b 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a F 23.3 ± 2.1a 49.7 ± 0.6b 77.0 ± 3.1b 87.7 ± 2.3b 98.7 ± 0.6b 100.0 ± 0.0a F 25.3 ± 0.6a 49.3 ± 1.5b 82.7 ± 4.4b 96.7 ± 2.1a 100.0 ± 0.0a 100.0 ± 0.0a Arcsine transformation (arcsine square root transformation used to transform cumulative mortality data and then one-way ANOVA, means were separated by Tukey’s test at P = 0.05. Different letters between food types are statistically different after Tukey’s test at P = 0.05. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/25/4924853 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 5 No egg was laid when females were fed with diets F0 and F1. The Effect of protein supplements on adult B. minax mean number of eggs laid was of 63.2  ±  8.7 eggs/individual in fecundity females fed with diet F2 and 19.3 ± 2.5 eggs/individual in females Ovarian development fed with diet F3. The results showed a significant difference in The timing of ovary development varied in relation to the type of diet the amount of eggs laid by females fed with diet F2 and diet F3 the flies were fed with (Fig. 4). When fed with diets F2 and F3, the ovary (P = 0.000). of the adult B. minax developed faster than that of F1 diet. The ovaries of female fed with diets F2 and F3 developed to stage 2 on the 5th day, to stage 3 on the 10th day and were found to have stage 2 and stage 3 Discussion ovaries 5 d earlier than those female fed with diet F1. For females fed with diets F3, F2, and F1, the disappearance of stage 1 ovaries occurred Sugar and other nutritional components like protein are necessary at 20 d, 15 d, and 10 d, respectively. The disappearance time in females dietary requirement for energy production and development that fed with diets F3 and F2 was shorter than for females fed with diet F1. greatly affect the life history traits in adults of many insect species (Bateman 1972, Jácome et al. 1999, May et al. 2015). In the present Ovarian egg load study, it was observed that provision of water or sugar alone resulted The ovarian egg load of B.  minax appeared to be different depend- in high mortality rates of the adults’ B. minax. However, provision ing on the food type provided to adult flies (F   =  14.979, df  =  3, 46, of sugar and protein enhanced the survival and reproduction of P  =  0.000) (Table  1). For females fed with water only, no egg was adult B. minax. Adult mortality of 100% were observed at age 30 d observed in the ovary. For females fed with diet F1, brood eggs per on the flies fed with nonprotein diet, while at age 75 d 100% mor - ovary were 15.00 ± 7.57 (n = 3), significantly lower than in females fed tality was observed for protein-fed adult. These results congruent with diets F2 (94.79 ± 6.28; n = 24; P = 0.000) and F3 (77.29 ± 6.97; with Teal et  al. (2004) who found that Anastrepha suspensa pro- n = 45; P = 0.006). The results, however, showed there was no difference vided with a diet of sugar and protein lived longer than those fed between brood eggs of females fed with diet F2 and diet F3 (P = 0.233). with sugar alone. Sucrose diet facilitates metabolism of insect where the energy obtained extend insect life span (Lardies et al. 2004, Naya et  al. 2007). Protein is an important nutritional element for insect Female oviposition reproduction as it contains amino acids necessary for insect ovipo- There were significant differences in egg production when differ - sition (Lardies et al. 2004, Nash et al. 2014). Alamzeb et al. (2006) ent foods were fed (F = 43.677, df = 3, 19, P = 0.000) (Table  1). reported that protein is necessary for female oocytes maturation to reach vitellogenic stage. Harwood et  al. (2013) also reported that protein supplement following eclosion period led to higher survival and reproductive abilities in the Mediterranean fruit fly, C. capitata Wiedemann and melon fly, B. cucurbitae Coquillett suggesting that provisioning protein-enriched foods extend fruit flies life expectancy and reproduction. Chinese citrus fruit fly showed different mating frequencies fol- lowing the food type provided. Protein diet (yeast and peptone) 40 showed increase in mating frequencies. Mating started at early age F0 around nine feeding days after eclosion on protein-fed adult, mat- F1 ing duration on protein-fed flies lasted for 42 d.  Shelly et al. (2002) 20 F2 reported that C. capitata male mating was more achieved when sub- F3 jected to protein diet than nonprotein diet. On females, they observed that the females were sighted more near protein-fed males than non- protein fed males. Joachim-Bravo et  al. (2009) investigated on the role of protein on sexual behavior of C. capitata and, observed that Feeding time (Days) male fed with high protein diet showed greater number of copu- Fig. 2. Effect of different food types on cumulative mortality of adult B. minax. lation. In addition, young males and males fed with high protein Table 3. Curve equation for regression between cumulative mortality and feeding days of B. minax adults fed with different food types Food types Regression equation R R R Feeding days 0.05 0.01 LT LT 50 90 F0 Y = 102.057/(1 + Exp(4.665 − 0.869X)) 1.000 0.666 0.798 5.3 7.7 F1 Y = 99.255/(1 + Exp(2.817 − 0.315X)) 0.994 0.388 0.496 9.0 16.2 F2 Y = 93.472/(1 + Exp(1.364 − 0.103X)) 0.967 0.413 0.526 14.6 44.9 F3 Y = 96.843/(1 + Exp(1.612 − 0.128X)) 0.982 0.707 0.834 13.1 32.7 Y: represents the cumulative mortality of B. minax; X: feeding days (days of food supply to the flies, until death of the last fly) of B. minax; LT50: (Lethal Time) is the time required to record 50% adult mortality. LT90: the maximum lethal time required to record 90% adult mortality; R: coefficient of determination; R : 0.05 coefficient of determination at 95%, R : coefficient of determination at 99%. (If R-value is greater than 0.05 and 0.01 are statistically significant and R-value less 0.01 than 0.05 and 0.01 are not statistically significant). The adult cumulative mortality was transformed to probability units and analyzed using the logistic regression (y = a/(1 + Exp (b − k x))) where x is the feed days in each food and y is the cumulative mortality in probability units. Time–mortality data was fitted using prob- ability analysis to estimate the lethal time for 50% (LT 50) and 90% (LT 90) cumulative mortality in each food treatment tested. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/25/4924853 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Cumulative mortality ratio (%) 6 Journal of Insect Science, 2018, Vol. 18, No. 2 showed greater participation and called more often than older For most of the studied biological characteristics of B.  minax, males and males fed with nonprotein diet (Roriz and Joachim-Bravo our results showed that protein source diet in B. minax had the high- 2013). Egg production increased proportionally with higher mating est effect on egg production and survival compared with nonpro- frequencies. This trade-off interaction resulted in the reduction of tein sources (F0 and F1) where flies died shortly. The results are in female life expectancy when fed with protein diet as earlier reported agreement with previous studies on physiology of fruit flies (Nash by Papanastasiou et al. (2013). The ovary growth was faster when et al. 2014, Shinwari et al. 2015). Between the two protein sources adults B. minax were fed F2 and F3 diet than F1. This indicates that tested, addition of yeast extract as source of protein had the highest protein supplementation resulted in a faster ovary maturation and effect compared with the addition of peptone, yeast extract showed female egg production. For example, Jácome et al. (1999) reported positive response on flies survival and reproduction. The results are that female A. serpentina requires access to protein-enriched diets in in agreement with previous studies on physiology of fruit flies (Nash order to produce significant number of eggs. et al. 2014, Shinwari et al. 2015). Zajitschek et al. (2012) reported that yeast diet had a strong influence on survival and female repro- ductive fitness. The higher effects of yeast extract can be attributed to its composition (nitrogenous compounds, carbon, sulfur, trace nutrients, vitamin B complex) and also the fact that it’s made up of microorganisms from fungi groups. Apart from amino acids, other substances such as B-complex vitamins and mineral were incorpo- rated into the yeast diets. These components play important catalytic roles in the physiology of many insect species (Fraenkel and Blewett 1943, Nash et al. 2014, Zhou et al. 2016) and are known to enhance the fecundity in fruit flies (Ben-Yosef et al. 2010). F0 Loss of reproductive ability (reproduction senescence) for F1 B.  minax was observed when flies were subjected to nonprotein F2 diet. This explains the significance of protein on flies’ reproduction. F3 Delaying host access and protein sources result to reduced repro- duction known as reproduction senescence (Harwood et al. 2013). Bonduriansky et al. (2008) reported that trade-off between survival and reproduction is influenced by time and duration of poor repro- Feeding time (Days) ductive conditions may decrease the overall fecundity of an organ- ism because of senescence unless the reproductive ability can be Fig.  3. Cumulative mating of adult B.  minax fed on different food sources (50% represent adult mating peaks). extended to advanced ages. Table 4. Age-specific cumulative mating ratio Food sources Age-specific cumulative mating ratio (%) 5 d 15 d 18 d 30 d 42 d 51 d F0 — — — — — — F1 0.0 ± 0.0a 43.3 ± 23.3a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a F2 0.0 ± 0.0a 7.1 ± 1.7a 21.4 ± 4.8b 88.5 ± 4.8b 95.1 ± 1.4b 100.0 ± 0.0a F3 0.0 ± 0.0a 14.0 ± 2.7a 34.4 ± 7.7b 74.1 ± 7.9a 100.0 ± 0.0a 100.0 ± 0.0a Arcsine transformation (arcsine square root transformation used to transform mating frequency data and then one-way ANOVA, means were separated by Tukey’s test at P = 0.05. Different letters between food types are statistically different after Tukey’s test at P = 0.05. Table 5. Curve equation from regression analysis between cumulative mating ratio and feeding days for B. minax adults fed on different food sources Food types Regression equation R R R Feeding days 0.05 0.01 Mating Mating Mating onset (16%) peak (50%) end (84%) F0 — — — — — — — F1 Y = 106.933/(1 + Exp(16.30 − 1.087X)) 0.972 0.775 0.875 13.4 14.9 16.2 F2 Y = 97.131/(1 + Exp(5.425 − 0.223X)) 0.997 0.304 0.393 17.1 24.6 32.7 F3 Y = 99.740/(1 + Exp(5.407 − 0.249X)) 0.994 0.339 0.436 15.1 21.8 28.4 Y: represents the cumulative mating ratio of B. minax; X: represents the feeding time (days of food supply to the flies, until death of the last fly) of B. minax; —: represent unmated; R: coefficient of determination; R : coefficient of determination at 95%, R : coefficient of determination at 99%. (If R-value is Greater 0.05 0.01 than 0.05 and 0.01 are statistically significant and R-value Less than 0.05 and 0.01 are not statistically significant). The adult cumulative mating ratio in four food sources (F0, F1, F2, F3) tested was transformed to probability units and analyzed using the logistic regression (y = a/(1 + Exp (b − k x))) where x is the feeding days in each food, y is the cumulative mating ratio of B. minax in probability units. Mating time data were fitted using probability analysis to estimate the time for 16% (mating onset), 50% (mating peaks), and 84% (mating ends) cumulative mating in each food treatment tested. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/25/4924853 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Cumulative mating ratio (%) Journal of Insect Science, 2018, Vol. 18, No. 2 7 stage1 stage2 stage3 ( and above ) 0d 5d 10d 15d 20d 0d 5d 10d 15d 20d 0d 5d 10d 15d 20d F1 F2 F3 Fig. 4. Ovarian development for female B. minax flies fed with different food types. d: indicates feeding days when food was supplied to the flies. CABI. 2017. Bactrocera minax. In Invasive Species Compendium. CAB During this study, both sugar and protein appeared to be essen- International, Wallingford, UK. https://www.cabi.org/isc/datasheet/8726. tial components for the survival and reproduction of B. minax. The Carey, J. R., P. Liedo, L. Harshman, Y. Zhang, H. G. Müller, L. Partridge, and J. deprivation of protein and sugar from adult diets resulted in high L. Wang. 2002. Life history response of Mediterranean fruit flies to dietary mortality rates. However, the use of yeast extract as source protein restriction. Aging Cell. 1: 140–148. enhanced adult life span, mating frequency, fecundity, and ovarian Carey, J. R., L. G. Harshman, P. Liedo, H. G. Müller, J. L. Wang, and Z. Zhang. development in B. minax. Same results were obtained by Lee et al. 2008. Longevity-fertility trade-offs in the tephritid fruit fly, Anastrepha (2008), Fanson et al. (2009), and Fanson and Taylor (2012) as diet ludens, across dietary-restriction gradients. Aging Cell. 7: 470–477. had influence on life span and egg production on Queensland fruit Chen, Z., Y. Dong, Y. Wang, A. A. Andongma, M. A. Rashid, P. Krutmuang, fly and D. melanogaster. These results suggest that yeast extract is a and C. Niu. 2016. Pupal diapauses termination in Bactrocera minax: an suitable source of protein in mass rearing of B. minax. Nevertheless, insight on 20-hydroxyecdysone induced phenotypic and genotypic expres- sions. Scientific Reports, 6: 27440. more studies are needed to unveil other factors required for a suc- Chou, M. Y., R. F.  Mau, E. B.  Jang, R. I.  Vargas, and J. C.  Piñero. 2012. cessful artificial rearing of this devastative citrus fly so as to imple- Morphological features of the ovaries during oogenesis of the Oriental fruit fly, ment an effective management strategy of their populations. Bactrocera dorsalis, in relation to the physiological state. J. Insect Sci. 12: 1–12. EPPO/CABI. 1997. Data Sheets on Quarantine Pests, Bactrocera minax [J]. Acknowledgments http://www.eppo.org/QUARANTINE/listA1.htm. Fanson, B. G., and P. W. Taylor. 2012. Protein:carbohydrate ratios explain life This work was supported by the National Science Foundation of China span patterns found in Queensland fruit fly on diets varying in yeast:sugar (31572010). We thank Dr. Mazarin Akami from Huazhong Agricultural ratios. Age (Dordr). 34: 1361–1368. University for reviewing the final version of our manuscript. Fanson, B. G., C. W. Weldon, D. Pérez-Staples, S. J. Simpson, and P. W. Taylor. 2009. 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