Puncturing apple fruits increases survival of Grapholita molesta (Lepidoptera: Tortricidae) in laboratory rearing.

Grapholita molesta (Busck) is a major pest in orchards of apple, peach, and plum. For better rearing in the laboratory, we compared the life history characteristics of G. molesta by providing larvae with either punctured or unpunctured apple fruits. The development time of immatures and the fecundity of adult females were similar between punctured and unpunctured apples. However, the overall survival rate of G. molesta (larvae to adult emergence) was 1.7 times higher on punctured apples than unpunctured ones, resulting in a higher intrinsic rate of population increase. Therefore, punctured apples would be a better food source for rearing of G. molesta.

Introduction

Oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), is a major pest of important tree fruits, including peaches, apples, and pears, belong to Rosaceae family [1-5]. Grapholita molesta is widely distributed throughout Asia, Europe, the Americas, Africa, and Australia’s temperate and subtropical regions [1,4]. It is also a quarantine pest that affects shipment of fruit between countries [6]. Depending on the temperature and location, the pest can have three to six generations per year [7-11].

According to a survey (1992–2005) from Korea, fruit damage by G. molesta at harvest varied from 0.02 to 1.64%, and the orchard infested with G. molesta was 13 to 71% [12]. In some Brazilian orchards (in 1985), G. molesta was reported to have damaged up to 90% of all apples [13]. Since its introduction into the United States in the early twentieth century, G. molesta has become a serious pest of tree fruits in that country [14].

Organophosphate, carbamate, and synthetic pyrethroid pesticides are commonly used to control G. molesta [15-18], but insecticide resistance poses a serious threat to the fruit industry [17], and G. molesta has developed resistance to 14 insecticides, including 10 organophosphates [19]. To address this problem, new IPM programs must be developed and implemented. Preliminary trials in Bulgaria have shown that population densities of G. molesta on peaches were reduced during a pilot program that combined classic sterile insect release (SIR) with F1 male sterility [20]. Grapholita molesta can also be potentially controlled by using natural enemies [21,22].

To provide insects for bioassay and for inexpensive production of natural enemies, an efficient diet and rearing system for G. molesta is needed. Green immature apples have been shown to be a good food source for G. molesta larvae [23,24]. To rear G. molesta, Vetter et al. [25] compared an artificial diet to a punctured-apple diet and found that the punctured-apple diet produced more pupae than the artificial diet. However, many important details, such as the number of punctures, larval development period, fecundity, and female longevity were not reported in that study. Also, no comparison of insect survival has been made of punctured versus unpunctured apples as rearing diets for G. molesta. Therefore, in this study, we hypothesized that punctured apple fruit may increase fitness of G. molesta by enhancing development and reproduction. We compared biological and life table parameters of G. molesta reared on between punctured and non-punctured apple fruits.

Materials and methods

Insect rearing

Larvae were reared on apple fruits described by Sarker and Lim [26]. Naturally infested apples were collected from Bioresource Research Institute (Andong, Republic of Korea) in 2015 and kept in ventilated plastic containers (24.0 L × 17.0 W × 8.0 H cm) in a growth chamber (DS-11BPL, Dasol Scientific Co. Ltd, Hwaseong, Republic of Korea) held at 24.9 ± 0.1°C, 50.2 ± 1.3% RH, and a 16:8 h (L:D) photoperiod. After about 20 d of collection, larvae reached the fifth instar and emerged from apples to build their cocoons. To support pupation, a paper towel was placed in boxes. Pupae were then collected about 10 d and held in different breeding dishes (10.0 D × 4.0 H cm, 310102, SPL, Pocheon, Republic of Korea) for adult emergence. After adult moths emerged, 10–15 pairs of adult moths were transferred into ventilated acrylic oviposition cylinders (25.5 H × 8.5 D cm), and a piece of cotton soaked with a 10% sugar solution was provided as an adult food source in each cage. These acrylic cylinders were kept at 25.6 ± 0.1°C and 91.2 ± 0.1% RH in a growth chamber. These oviposition cylinders were examined and changed daily to collect 1-day-old eggs starting when moths first began to lay eggs on the wall. The acrylic cylinders with eggs on the walls were kept in a separate growth chamber at 25.1 ± 1.5°C and 94.3 ± 5.4% RH until the eggs hatched, after which the first instar larvae were collected for use in experiments, or were returned to the mass rearing. OFM was reared for about six generations while experiments were carried out. Wild males were field collected and added to the mass rearing 2–3 times in a year to reduce inbreeding depression.

Plant materials

Pruned fruits of green fresh apple [Malus domestica Borkh. Variety “Fuji” (strain ‘Busa’)] as an experimental unit were collected from unsprayed apple orchards in Gilan County, Andong City, the Republic of Korea, in 2017. Apples were measured to be 6.2 ± 0.2 cm diameters and sealed in plastic zipper bags held at 4°C in a refrigerator before being used in our experiment.

Development and survival rate of immature stages

In this experiment, there were two treatments: punctured and unpunctured apple fruits. Punctures were made (2.6–3.2 mm deep) with an insect pin (Insect Pins, Stainless steel No. 6, BioQuip Products, California, USA), with 20 punctures scattered around the apple fruit. Before being used, insect pins were sterilized with an alcohol lamp, followed by dipping in 70% ethanol. After preparation, punctured and unpunctured apples were placed individually in insect breeding dishes (310122, 120 D × 80 H mm, SPL, Pocheon, Republic of Korea). Five larvae (<5 h old) of G. molesta were then placed on the surface of each fruit with the help of camel hair brush, and fruits were then held at 24.9 ± 0.1°C and 51.7 ± 1.6% RH in a growth chamber. In total, in this experiment, there were 30 larvae used in each of the punctured and unpunctured apple treatments with six replications (5 insects/ apple fruit). When a mature larva emerged from a fruit (exiting larva), it was transferred for pupation into another dish (10.0 D × 4.0 H) that was filled with tissue paper. The duration of the larval stage was estimated as the time from the day of egg hatch (= the day fruit were inoculated with neonate larvae) to the day the mature larva exited the fruit. The duration of the prepupal stage was the period from the exit of the mature larva from the fruit to the pupation, and the pupal stage was from the day of pupation to adult emergence.

Longevity and fecundity of adult females

Adults of G. molesta reared from larvae in treatments were transferred into transparent square breeding dishes (7.2 L × 7.2 W × 10.0 H cm SPL, Pocheon, Republic of Korea, each with three 40 mm-mesh screens; one per side) immediately after their emergence and held with in pairs (1M, 1F) per dish. A piece of cotton soaked with a 10% sugar solution was added to each cage as a food source. Freshly laid eggs were deposited on the container’s surface, and eggs were marked and recorded daily until the adult female died. Breeding dishes holding adult pairs were replaced daily with new ones to avoid pathogenic infections. An absence of spermatophores was used to indicate an unmated female, and such moths were excluded from the analysis [5] and a total of 11 females for punctured and 6 for unpunctured were used in this study. If the male died before the female or if there was uneven number of male and female produced from the treatment, a new male was provided from the cohort cage.

Statistical analysis

Differences in developmental time, preoviposition period, oviposition period, lifetime fecundity, the longevity of adult females, and pupal weight of G. molesta for animals reared on punctured versus unpunctured apple fruits were analyzed with t-tests using SAS 9.4 [27]. Survival rate of each stage was analyzed using two proportion Z- tests [28].

The jackknife procedure was performed to test the differences in population parameters, i.e., doubling time (DT), finite rate of increase (λ), intrinsic rate of increase (r), net production rate (R), and mean generation time of (T) [29]. The survival rate (S) (x = age, j = stage) is the probability that a newly laid egg would survive to age x and stage j. Jackknife algorithms for estimating the means and variances and constructing confidence intervals were described only for R (the net contribution of each female to the next generation, expressed as the total female offspring per female over the whole oviposition period) [29]. The same procedures were used to estimate other parameters (r, T, DT, and λ). All fertility data in the life tables were entered in a computer program (LIFETABLE.SAS) [29] and analyzed using SAS 9.4 [27].

Results

Development and survival rates of immature stages

No significant differences were found in durations of egg stage (t = 0.27, df = 58, P = 0.791), larval stage (t = 0.13, df = 33, P = 0.898), prepupal stage (t = 0.57, df = 28, P = 0.576), pupal stage (t = 1.84, df = 28, P = 0.076), or immature stage (t = 1.36, df = 28, P = 0.183) between punctured and unpunctured apple diets (Table 1).

Table 1

Duration (d) (± SE) of each stage of Grapholita molesta (n = 30 for punctured and n = 30 for unpunctured) reared on apple fruits under laboratory conditions.

Treatment	Egg	Larva	Prepupa	Pupa	Immature
Punctured	3.37 ± 0.09 a(n = 30)	15.52 ± 0.48 a(n = 21)	3.95 ± 0.38 a(n = 19)	8.95 ± 0.40 a(n = 19)	28.58 ± 0.54 a(n = 19)
Unpunctured	3.33 ± 0.09 a(n = 30)	15.43 ± 0.54 a(n = 14)	4.36 ± 0.72 a(n = 11)	10.36 ± 0.74 a(n = 11)	30.27 ± 1.35 a(n = 11)

Means within a column with different letters are significantly different (P < 0.05).

The rate larvae exited from fruits (Zc = 1.83, P = 0.067), the pupation rate (Zc = 0.99, P = 0.324), and the adult emergence rate (Zc = 0.00, P = 1.000) were also not different between punctured and unpunctured apples, but the overall survival rate (Zc = 2.07, P = 0.039) was significantly different between punctured and unpunctured apples (Table 2). The overall survival rate of G. molesta larvae in punctured apples was 60%, which was higher than that of larvae reared in unpunctured apples (40%).

Table 2

Survival rate of each stage of Grapholita molesta provided with punctured and unpunctured apple fruits under laboratory conditions.

Treatment	Exiting rate	Pupation rate	Emergence rate	Overall survival rate
Punctured	0.70 (21/30) a	0.90 (19/21) a	1.00 (19/19) a	0.63 (19/30) a
Unpunctured	0.47 (14/30) a	0.79 (11/14) a	1.00 (11/11) a	0.37 (11/30) b

Means within a column with different letters are significantly different (P < 0.05).

The survival rates of G. molesta larvae reared on punctured and unpunctured apples were highest on 23rd and 21st day, respectively (Fig 1). We found that no fungal development occurred within three weeks.

Fig 1

Survival rate of different life stages of Grapholita molesta reared on punctured and unpunctured apples.

No significant differences were found in the preoviposition period (t = 0.38, df = 5, P = 0.721), oviposition period (t = 0.49, df = 5, P = 0.643), lifetime fecundity (t = 0.22, df = 5, P = 0.833), or adult female longevity (t = 0.45, df = 5, P = 0.671) between insects reared on punctured versus unpunctured apples (Table 3). Pupal weights (t = 1.36, df = 28, P = 0.183) were also similar between treatments (Fig 2).

Table 3

Fecundity (Mean ± SE) of female adult of Grapholita molesta reared on punctured and unpunctured apple fruits under laboratory conditions.

Treatment	Preoviposition period	Oviposition period	Lifetime fecundity	Longevity
Punctured	3.50 ± 0.29 a	17.30 ± 1.31 a	238.50 ± 8.97 a	20.80 ± 1.25 a
Unpunctured	3.33 ± 0.33 a	18.33 ± 1.86 a	234.33 ± 18.41 a	21.67 ± 1.67 a

Means within a column with different letters are significantly different (P < 0.05).

Fig 2

Pupal weights of Grapholita molesta reared on punctured and unpunctured apples.

Life history parameters of Grapholita molesta

Significant differences were observed between punctured and unpunctured apple in several life table parameters, including doubling time (DT), finite rate of increase (λ), intrinsic rate of increase (rm), and net reproductive rate (R) (Table 4). Intrinsic rate of increase (rm) was significantly higher on punctured apples, and the doubling time was significantly shorter (i.e., faster population growth) on punctured apples than in moths reared on unpunctured apples. However, mean generation time (T) was similar between punctured and unpunctured apples (Table 4).

Table 4

Life table parameters of G. molesta reared on punctured and unpunctured apples.

Treatment	Parameters (mean ± SE)
	DT	λ	r m	R o	T (Day)
Punctured	5.02 ± 0.21 b	1.15 ± 0.01 a	0.14 ± 0.01 a	87.15 ± 3.28 a	32.34 ± 1.15 a
Unpunctured	5.96 ± 0.13 a	1.12 ± 0.00 b	0.12 ± 0.00 b	47.69 ± 3.75 b	33.25 ± 0.62 a

Means followed by the same letter in a column are not significantly different from Student’s t-test for pairwise group comparison at P < 0.05.

DT: Doubling time

λ: Finite rate of increase

r: Intrinsic rate of increase

R: Net reproductive rate

T: Mean generation time.

Discussion

Grapholita molesta has been mass-reared in artificial diets for many years [5,25,30,31]. Green immature apples have also been used for rearing larvae of G. molesta and are a good food source for the development and reproduction [23,24,32-34]. First and second instars of G. molesta are highly cannibalistic when grown on an artificial diet lacking protein and vitamins, while they are not cannibalistic when grown on green apples even under crowded conditions [35]. Furthermore, Lӧfstedt et al. [36] found that the amount of ethyl trans-cinnamate is higher on adult males reared on apple fruits rather than on artificial diet. Ethyl trans-cinnamate is an important component of the sex pheromone of G. molesta [37], and G. molesta males sequester compounds from apples to produce ethyl trans-cinnamate for close-range attraction of calling females [37,38]. In addition, males reared in apple fruits can attract females from a longer distance than those reared on artificial diet [36].

Vetter et al. [25] compared an artificial diet with the use of punctured apples for mass rearing of G. molesta and found that the punctured apple diet yielded more pupae than the artificial diet. However, this study did not indicate the number of punctures, the larval developmental period, or adult fecundity or female longevity. Furthermore, Vetter et al. [25] made no comparison for G. molesta survival in apples with punctures versus unpunctured apples.

In our study, the durations of the egg, larval, prepupal, pupal, and immature stages were not statistically different between punctured and unpunctured apples. The larval developmental times on punctured and unpunctured apple were 15.5 and 15.4 days, respectively. According to Bisognin et al. [30], the larval period of G. molesta reared on apple (cv. Fuji) is 15.9 days, while it is shorter on artificial diet. Generally, the larval developmental time was longer in apple (the preferred host) than in peach or plum fruits [23,24].

Also, there were no significant differences in the rate of fruit exiting for larvae, the pupation rate, or the emergence rate between punctured and unpunctured apples. However, the overall survival rate was higher in punctured apples (60%). When rearing G. molesta larvae on apple fruits at the rate of two larvae per apple, Bisognin et al. [30] found that the total survival rate of G. molesta was only 29.9%. This is much lower than the 54.8% for artificial diet. Here, we found a similarly low survival rate (36.7%) in unpunctured apples with five larvae per apple, but in punctured apples survival was much higher (60%). The lower survival rates in unpunctured apples are most likely caused by higher number of larvae being unable to enter the fruit; punctures seem necessary to avoid this mortality and maximize G. molesta production efficiency. In other cases, Finney et al. [37] found that larvae of Phthorimaea operculella (Zeller) could enter the potato more effectively if the skin was punctured, which permitted large numbers of G. operculella to be reared in individual potatoes. Unpunctured potatoes are infested only by larvae that enter through the potato eyes, reducing the number of larvae that can be reared from each tuber [39].

We found no significant differences for G. molesta between punctured and unpunctured apples in pupal weight, the preoviposition period, oviposition duration, lifetime fecundity, and longevity. This suggests that larvae that do successfully enter apple fruits obtain the necessary nutrients. Because of differences in overall survival rate, the intrinsic rate of increase was higher in G. molesta reared in punctured apples than those reared in unpunctured apples. This difference is probably due to the higher rate of the first instar larvae successfully enter the fruit (Table 1), resulting in higher overall survival rate. However, in field condition, physical damage on apple can negatively influence the population dynamics of G. molesta by plant volatiles produced to attract natural enemies [40,41].

Larvae of G. molesta prefer green, immature apples as a food source and normally enter the apple fruit through the calyx or stem end cavities [42]. Several researchers have reported a decline over time in the quality of G. molesta reared in ripe apples because ripe apples decline quickly in quality [35,43-45]. Similarly, in our previous study, ripe plum fruit was not a good food source for G. molesta larvae due to the decline in fruit firmness [24]. Therefore, G. molesta can be simply and cheaply reared using cold-stored, green, immature apples even though obtaining large quantities of green apples, and storing them without loss of quality, can be difficult. Most importantly, we show that the efficiency of rearing OFM in green apples can be greatly increased by puncturing green immature apples to allow for better ingress of neonate larvae.

Punctured apple, unpunctured apple, life table analysis, pupal weight.

(XLSX)

Click here for additional data file.

Demonstrating the puncture to the apple fruit.

(TIF)

Click here for additional data file.

28 Feb 2022

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Reviewer #1: This study showed the needle punctured green (?) apple could be a better substrate for Oriental fruit moth larval rearing medium than not-punctured ones.

The experiments were well designed and conducted.

Writing is concise and without redundancy.

Results were reasonable.

It is difficult to judge the scientific merit of this finding in science but surely important in applied aspects.

M&M Please provide when and how did the field collection of apple was done and detial the condition of apple collected.

Table 1. egg duration was presented. However in M&M section, larvae were introduced into the experimental apples and began observation. This part could be cleared.

In Table 1, provide the actual numbers of observation for each stage.

Exiting rate was measured but not described in M&M. Provide the details of the measurement. In the box, if the larvae exit the fruit then,does this mean that the larvae became dead, on what time scale?

please check the statistics; the rate larvae exited from fruits (Zc = 1.83, P = 0.067), the pupation rate (Zc = 0.99, P = 134 0.324), and the adult emergence rate (Zc = 0.00, P = 0.000) were also not different between punctured and unpunctured apples,

Authors discussed the punctured apple could provide easy approaches to the week larvae to enter into the fruits and lowering larval mortality.

In the study, separation of larval mortality and exiting rate, and pupal mortailty and pupation rate was not explicitly detailed, and which made the overall conclusion less supporting.

In the table showing adult reproduction, please provide the numbers of female observed for oviposition.

In this study, how many pairs (male and female) were studied were not presented. If If there was uneven number of male and female produced from the treatment, did you add the male from the rearing batch or ...

Authors had narrowed the scope of this finding only in laboratory rearing, however this findig could influence field dynamics of infestation.

By puncturing, the host apple could provide more VOCs which would influence the host location to the larvae. Please add the possibility of this in the discussion, relative to the field situation where diverse factors could impact apple fruits and give physical damages.

In this study, can we have any data of apple physical character change over the experimental periods? Since the larvae should stay ap 2 weeks inside the host food once it entered into, physical changes of the host food could influence the outcome of the results.

Reviewer #2: In this work the authors study the effect of punctured apple fruits on survivorship of G.molesta. Based on their analysis the authors conclude that punctured fruits are more suitable for OFM laboratory rearing compared to non-punctured fruits and as it is shown by the demographic parameters that the authors have estimated including the intrinsic rate of increase.

These results support previous works done in G. molesta and are generally interesting and important for OFM rearing. In this respect the authors reaffirm the importance of their work to the fact that their results will contribute to the mass production of this pest to be used for mass rearing. On the other hand, I feel that the current work is rather simplistic and based on a rather small data set.

I cannot imagine also how practical might be the mass production of OFM in a natural host compared to artificial diet despite the better demographic performances of the species. For example, injured apples will not only improve OFM growth (at least as the present work demonstrates) but also the growth of fungal infections and ultimately cannot be used for a long time being impractical. i.e. How many times have you changed the apples during larval development? How easy it is to mechanize the technique you are proposing for OFM mass rearing?

Additionally, the study does not examine whether the ''good reproductive performance'' that the authors have observed (through the two comparisons: punctured vs unpunctured hosts) holds also for other temperature regimes. Because the intrinsic rate of increase takes in to account at least both, the net maternity along with survivorship of the individuals of a cohort, it is quite possible that the differences might vanish under different temperature regimes. In table 4, for instance, the distance in rm between the two treatments is considerable shorter compared to Ro. Actually, I consider the intrinsic rate of increase as more representative for the description of the reproductive success of species (i.e. fitness) compared to the net maternity and not exclude that this difference might diminished after a slight alteration of the temperature regime.

Finally, the authors focus on the technical part of their study for OFM rearing (which is sound and not bad), but, to my view incomplete because they have not examined in deep why they have observed such significant differences. Particularly and if I am not wrong, a part of the last lines (213-214), there is actually no hypothesis of what is the profound reason-interpretation of their result and especially what is(are) the main reason(s) that the species explicit different fitness capacities when feeded with an injured host.

If it’s just the fact that it’s easy (and not energy consuming) for the larvae to crawl insight an injured apple compared to an unpunctred one, how do they explain the differences that are observed on the successive developmental stages? Are they excluding any nutritional, physiological or any other cause?

Thus, I believe that the biological-ethological and consequently the ecological dimension of the authors' findings is of greater interest but not emphasized at all.

Line 45. If this proposition were to the maximum, i.e. biological was effective, we would not need research on pesticides or even deprivation.

Line 58. It would help the reader if you included a photo in the appendix

Figure 1. To be honest I cannot follow your chart. Why do you not just plot the survivorship curves for each stage and treatment?

Reviewer #3: Puncturing Apple Fruits Increases Survival of Grapholita molesta (Lepidoptera: Tortricidae) in Laboratory Rearing By Souvic Sarker and Un Taek Lim

General comments

This is a study where unpunctured and punctured apples were compared as diet for artificially rearing the pest Oriental fruit moth, Grapholita molesta. To determine the effect of these two diets, authors measured and analysed some demographic parameters in the reared insects. The article has some interesting results. However, the results are not novel and, results are poorly presented. The manuscript has a very narrow scope, basically in the field of entomologists. My major concern is the experimental design since there is not explanation of what the experimental unit was, the apple or the insect.

Introduction

Lines 46-47. A mass-rearing system based on apples. It is not possible. Fruit phenology and fruit variation surely will introduce many difficulties in the process.

Methods

Line 67. How many insect per cylinder?

Describe with detail the rearing method. Did the authors place the larva on apple surface? How did they do it?

Line 75. Please indicate the periodicity.

Lines 84-96. Did you use 12 apples as a total experimental set? Six apples per treatment? Please clarify.

Lines 100-109. Were the apples the experimental unit? Or the larvae? Five larvae per fruit gives 30 individual insects, some of them died so how many did the authors use for determining the demographic parameters?

Results

Figures can be deleted and the values presented on them can be included in the text.

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Reviewer #2: No

Reviewer #3: No

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13 Apr 2022

A. Response to reviewer #1’s comments

1. This study showed the needle punctured green (?) apple could be a better substrate for Oriental fruit moth larval rearing medium than not-punctured ones.

The experiments were well designed and conducted. Writing is concise and without redundancy. Results were reasonable. It is difficult to judge the scientific merit of this finding in science but surely important in applied aspects.

>> Thanks for the comments.

2. M&M Please provide when and how did the field collection of apple was done and detial the condition of apple collected.

>> We provided the time and collection procedure in the revised MS. Please see L80-82 in the revised MS.

3. Table 1. egg duration was presented. However in M&M section, larvae were introduced into the experimental apples and began observation. This part could be cleared.

>> We addressed this issue in our previous MS. See L71-75 in the revised MS.

4. In Table 1, provide the actual numbers of observation for each stage.

>> Yes. Please see Table 1 in revised MS.

5. Exiting rate was measured but not described in M&M. Provide the details of the measurement.

>> We corrected the sentence by adding “exiting larvae”. Please L95-96 in the revised MS.

6. In the box, if the larvae exit the fruit then, does this mean that the larvae became dead, on what time scale?

>> We addressed this issue in our previous MS in L93-94. When a larva exits from the fruit, it transferred into another dish for pupation. Please see L95-98 in the revised MS.

7. please check the statistics; the rate larvae exited from fruits (Zc = 1.83, P = 0.067), the pupation rate (Zc = 0.99, P = 134 0.324), and the adult emergence rate (Zc = 0.00, P = 0.000) were also not different between punctured and unpunctured apples, Authors discussed the punctured apple could provide easy approaches to the week larvae to enter into the fruits and lowering larval mortality.

>> As we mentioned in our previous MS in L184-185 and L202-204, the overall survival rate and entering rate of the 1st instar was higher in punctured apples despite the lack of significance in each parameter, i.e., exit rate of larvae, pupation rate, adult emergence rate. Please see L185-186 and L203-204 in the revised MS.

Nevertheless, we deleted Figure 2 as it is redundant data of Table 2.

8. In the study, separation of larval mortality and exiting rate, and pupal mortailty and pupation rate was not explicitly detailed, and which made the overall conclusion less supporting.

>> Please see our answer to above comment.

9. In the table showing adult reproduction, please provide the numbers of female observed for oviposition.

>> Yes. Please see L110-111 in the revised MS.

10. In this study, how many pairs (male and female) were studied were not presented. If If there was uneven number of male and female produced from the treatment, did you add the male from the rearing batch or ...

>> We addressed this issue in L110-113 of the revised MS.

11. Authors had narrowed the scope of this finding only in laboratory rearing, however this findig could influence field dynamics of infestation.

>> We agree with the reviewer’s comment. We added “However, in field condition, physical damage on apple can negatively influence the population dynamics of G. molesta by plant volatiles produced to attract natural enemies” in the revised MS. Please see L205-207 in the revised MS.

12. By puncturing, the host apple could provide more VOCs which would influence the host location to the larvae. Please add the possibility of this in the discussion, relative to the field situation where diverse factors could impact apple fruits and give physical damages.

>> Please see our answer to above comment

13. In this study, can we have any data of apple physical character change over the experimental periods? Since the larvae should stay ap 2 weeks inside the host food once it entered into, physical changes of the host food could influence the outcome of the results.

>> We addressed this issue in our previous MS in L206-208. Please see L209-211 in the revised MS.

B. Response to reviewer #2’s comments

1. In this work the authors study the effect of punctured apple fruits on survivorship of G.molesta. Based on their analysis the authors conclude that punctured fruits are more suitable for OFM laboratory rearing compared to non-punctured fruits and as it is shown by the demographic parameters that the authors have estimated including the intrinsic rate of increase.

These results support previous works done in G. molesta and are generally interesting and important for OFM rearing. In this respect the authors reaffirm the importance of their work to the fact that their results will contribute to the mass production of this pest to be used for mass rearing. On the other hand, I feel that the current work is rather simplistic and based on a rather small data set.

1. I cannot imagine also how practical might be the mass production of OFM in a natural host compared to artificial diet despite the better demographic performances of the species. For example, injured apples will not only improve OFM growth (at least as the present work demonstrates) but also the growth of fungal infections and ultimately cannot be used for a long time being impractical. i.e. How many times have you changed the apples during larval development? How easy it is to mechanize the technique you are proposing for OFM mass rearing?

>> We agree with the reviewer’s concern that this rearing method using natural host can’t be appropriate for commercial mass-production. This is why we just suggested that punctured apple is better than unpunctured apple throughout MS although Vetter et al. [25] found that the punctured-apple diet produced more pupae than the artificial diet.

Nevertheless, we added a sentence in result section regarding fungal contamination on punctured apple. See L143-144 in the revised MS.

2. Additionally, the study does not examine whether the ''good reproductive performance'' that the authors have observed (through the two comparisons: punctured vs unpunctured hosts) holds also for other temperature regimes. Because the intrinsic rate of increase takes in to account at least both, the net maternity along with survivorship of the individuals of a cohort, it is quite possible that the differences might vanish under different temperature regimes. In table 4, for instance, the distance in rm between the two treatments is considerable shorter compared to Ro. Actually, I consider the intrinsic rate of increase as more representative for the description of the reproductive success of species (i.e. fitness) compared to the net maternity and not exclude that this difference might diminished after a slight alteration of the temperature regime.

>> We agree with the comments of the reviewer. Different temperature regimes may have an impact on G. molesta's development as well as its intrinsic rate of increase. However, our goal was to see whether puncturing apple fruit might improve the intrinsic rate of increase or not. That is why we maintained the same environmental and fruit conditions.

3. Finally, the authors focus on the technical part of their study for OFM rearing (which is sound and not bad), but, to my view incomplete because they have not examined in deep why they have observed such significant differences. Particularly and if I am not wrong, a part of the last lines (213-214), there is actually no hypothesis of what is the profound reason-interpretation of their result and especially what is(are) the main reason(s) that the species explicit different fitness capacities when feeded with an injured host.

>> We changed “Therefore, in this study our goal was to compare the effects of punctured versus non-puncture apple fruit on the development, reproduction, and life table parameters of G. molesta.” to “Therefore, in this study, we hypothesized that punctured apple fruit may increase fitness of G. molesta by enhancing development and reproduction. We compared biological and life table parameters of G. molesta reared on between punctured and non-punctured apple fruits.” Please see L54-57 in the revised MS.

4. If it’s just the fact that it’s easy (and not energy consuming) for the larvae to crawl insight an injured apple compared to an unpunctred one, how do they explain the differences that are observed on the successive developmental stages? Are they excluding any nutritional, physiological or any other cause? Thus, I believe that the biological-ethological and consequently the ecological dimension of the authors' findings is of greater interest but not emphasized at all.

>> We thought punctured apple should be artefact thus it may not easily applicable to natural interpretation. Anyway, we added a discussion on impact on field population as suggested by reviewer 1. See L205-207 in the revised MS.

5. Line 45. If this proposition were to the maximum, i.e. biological was effective, we would not need research on pesticides or even deprivation.

>> We corrected the sentence by adding “potentially” in the revised MS. Please see L45 in the revised MS.

6. Line 58. It would help the reader if you included a photo in the appendix

>> We added a photo in the appendix demonstrating the puncture to the apple fruit. Please see L372-374 in the revised MS.

7. Figure 1. To be honest I cannot follow your chart. Why do you not just plot the survivorship curves for each stage and treatment?

>> Yes, we revised the figure and caption. Please see Figure 1 and L363-364 in the revised MS.

C. Response to reviewer #3’s comments

General comments

1. This is a study where unpunctured and punctured apples were compared as diet for artificially rearing the pest Oriental fruit moth, Grapholita molesta. To determine the effect of these two diets, authors measured and analysed some demographic parameters in the reared insects. The article has some interesting results. However, the results are not novel and, results are poorly presented. The manuscript has a very narrow scope, basically in the field of entomologists. My major concern is the experimental design since there is not explanation of what the experimental unit was, the apple or the insect.

>> Apple fruits were the experimental unit and we revised the sentence. See L81 in the revised MS.

Introduction

2. Lines 46-47. A mass-rearing system based on apples. It is not possible. Fruit phenology and fruit variation surely will introduce many difficulties in the process.

>> We agree with the reviewer’s concern that this rearing method using natural host can’t be appropriate for commercial mass-production. This is why we just suggested that punctured apple is better than unpunctured apple throughout MS although Vetter et al. [25] found that the punctured-apple diet produced more pupae than the artificial diet. Anyway, we deleted “mass” from the revised MS. Please see L48 in the revised MS.

Methods

3. Line 67. How many insect per cylinder?

>> We deleted “they” and added “10-15 pairs of adult moths” in the revised MS. Please see L68 in the revised MS.

4. Describe with detail the rearing method. Did the authors place the larva on apple surface? How did they do it?

>> We corrected a sentence in L92 in the revised MS.

5. Line 75. Please indicate the periodicity.

>> We changed “periodically” to “2-3 times in a year”. Please see L77 in the revised MS.

6. Lines 84-96. Did you use 12 apples as a total experimental set? Six apples per treatment? Please clarify.

>> Yes. We added “with six replications (5 insects/ apple fruit).” Please see L95 in the revised MS.

7. Lines 100-109. Were the apples the experimental unit? Or the larvae?

>> Apple fruits were the experimental unit and we revised the sentence. See L81 in the revised MS.

8. Five larvae per fruit gives 30 individual insects, some of them died so how many did the authors use for determining the demographic parameters?

>> We added the information in L110-113 in the revised MS.

Results

9. Figures can be deleted and the values presented on them can be included in the text.

>> We deleted Figure 2 as it is redundant data of Table 2.

D. Author’s corrections (Line numbers are from the first MS)

L114: We added “rate” after “survival” in L118 in the revised MS.

L115: We deleted “the percentages of larval and pupal mortality” in L119 in the revised MS.

L115: We changed “were” to “was” in L119 in the revised MS.

L134: We corrected “0.000” to “1.000” in L137 in the revised MS. It was type error.

L139-140: We changed “Age- and stage-specific survival rates of G. molesta larvae were highest on 20th day in both punctured and unpunctured apple fruits (Fig. 1)” to “The survival rates of G. molesta larvae reared on punctured and unpunctured apples were highest on 23rd and 21st day, respectively (Fig. 1)”. See L142-143 in the revised MS.

L150: We changed “Fig. 3” to “Fig. 2” in L151 in the revised MS.

L186: We deleted “because the larval and pupal mortality rates were lower in punctured fruits” in L187 in the revised MS.

L201: We changed “larval and pupal mortality” to “overall survival rate” in L202 in the revised MS.

L203: We added “(Table 1)” in L204 in the revised MS.

L204: We changed “larval” to “overall” in L205 in the revised MS.

L226: Changed “wprs” to “WPRS” in L229 in the revised MS.

L239: Changed “Sinica” to “Sin” in L242 in the revised MS.

L245 and L266: Changed “Manage” to “Manag” in L248 and L269 in the revised MS.

L312: Deleted “and”. See L315 in the revised MS.

L321: Changed “Chern” to “Chem” in L324 in the revised MS.

Submitted filename: Gm Response to reviewer 13Apr.docx

Click here for additional data file.

19 Apr 2022

Puncturing Apple Fruits Increases Survival of Grapholita molesta (Lepidoptera: Tortricidae) in Laboratory Rearing

PONE-D-22-02750R1

Dear Dr.  Un Taek Lim ,

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Reviewer #2: All comments have been addressed

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22 Apr 2022

PONE-D-22-02750R1

Puncturing Apple Fruits Increases Survival of Grapholita molesta (Lepidoptera: Tortricidae) in Laboratory Rearing

Dear Dr. Lim:

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