RETRACTED: Cold Responses of the Mediterranean Fruit Fly Ceratitis capitata Wiedemann (Diptera: Tephritidae) in Blueberry

Abstract

In this study, the effects of cold treatment at 1.0 ± 0.2 °C were investigated on the eggs of the 1st, 2nd, and 3rd instar larvae of Ceratitis capitata in two Australian blueberry cultivars C99-42 and C00-09. Pupariation, emerged adults, and sex ratios were examined on C. capitata after the treatment. The results showed that exposure time at low temperatures was a key factor to affect pupariation and adult emergence. Eleven days of exposure to cold treatment at 1.0 ± 0.2 °C were enough to eradicate all four immature stages in both cultivars. Cold tolerance of the four C. capitata stages was not affected when reared on two different blueberry cultivars. The third instar larva is the most tolerant stage for cold treatment in two blueberry cultivars. There were no significant differences in sex ratios from surviving C. capitata among different stages after treatment. This study improves understanding of C. capitata responses to cold treatment in blueberries, which may contribute to phytosanitary required quarantine treatment of this destructive horticulture pest species.

1. Introduction

The Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann 1824), is one of the major pests of fruits and vegetables. C. capitata originated from sub-Saharan Africa and was identified in southern parts of Europe in the early 19th century. Currently, it is present in Mediterranean Europe, the Middle East, most parts of Africa, the Indian Ocean islands, South and Central America, Western Australia, and the Pacific region. The economic losses in these regions are predicted to be more than 2 billion dollars yearly [1]. Moreover, it is a major fruit fly species of quarantine importance and capable of causing extensive damage to a broad range of cultivated and wild fruit and vegetable products [2]. Medfly has the excellent dispersive ability and a tolerance of both natural and cultivated habitats over a comparatively wide temperature range, and has successfully established itself in many parts of the world [3]. Therefore, C. capitata has a significant economic impact, affecting production, control costs, and market access.

For fruits, phytosanitary treatments are used to reduce the risk in quarantined areas and to prevent an infestation in non-infested importation areas [4]. Various areas of the world have a history of repeated detections of fruit fly species, with some resulting in quarantines that prevent the export of host commodities until the pest is declared eradicated [5], which will result in huge economic losses and reputation damage.

The cold treatment has been used as a postharvest treatment method since 1916 [6], due to its advantages of being environmentally friendly (no pesticide residue) and safe for employers and consumers. It is an effective treatment method to provide phytosanitary control [7], increase fresh plant product shelf life [8], and maintain fruit quality. It is also easy to apply, compared to other methods, such as sterilizing technology and fumigation [9].

A plethora of studies have investigated the use of cold treatment against fresh fruit and vegetable pests. These studies continue to show the impact of cold treatment on insects. The species of insect pests and stages of the same species showed different responses to the cold treatment. The differences may be due to genetics, physiology, or effects of host variation [10,11,12]. C. capitata is possibly the most studied organism in phytosanitation, with numerous studies examining cold treatment [13]. However, the cold treatment on C. capitata in blueberry has not been examined previously.

Blueberries belong to the genus Vaccinium (Vander Kloet 1988) (Ericaceae), which also includes cranberries and huckleberries, which are one of the few cultivated fruits native to North America [14]. The industry has expanded to Europe, South America, and Australasia, but North America remains the major producer [15]. Blueberries have high nutritional and health value [16]. The Australian blueberry industry is one of the premium blueberry industries in the world, and cultivation increased by 70% from 2010 to 2012, with 90% production supplied to the local markets. The Australian blueberry was successfully exported to Japan until 2011; however, the export was suspended because blueberries were hosting medfly [17].

This study aimed to evaluate cold treatment against medfly in blueberry to provide an integrated knowledge for understanding the cold response of medfly in two blueberry cultivars.

2. Materials and Methods

2.1. Ceratitis Capitata Colony

The Ceratitis capitata colony used in this experiment was initially established in 2015 from a stock ancestry kept at the Department of Primary Industries and Regional Development (DPIRD) in Western Australia, which was periodically refreshed with the introduction of more wild flies. C. capitata adults were maintained in a Bugdorm-1 cage (30 × 30 × 30 cm ‘‘BioQuip products’’) with about 300 adults per cage, with access to water in a glass vial covered with a plug of cotton and also a dry 3:1 mixture of sucrose and yeast extract in a shallow glass container [18]. Mature insects laid eggs through the cloth sidewalls of the cages, which were collected and moved to the artificial breeding medium consisting of 300 g torula yeast, 1 kg ground dehydrated carrot, 4.5 L hot tap water, 36 mL HCl, 30 g nipagin, and 500 mL boiling water [19]. After 13–16 days, pupae were collected and moved into the adult breeding cages. The emerged adult insects were reared on crystalline sugar, the yeast hydrolysate, and water. Breeding conditions were 26.0 ± 1.0 °C, 60–70% relative humidity (RH), and darkness light cycle of 16:8 h [20]. Eggs and the three larval instars (1st, 2nd, and 3rd) were used in this study.

2.2. Fruits and Quality Assessment

Two blueberry cultivars used for this research were pesticide-free C00-09 and C99-42 from Western Australia. Both varieties are strong sweetness and weak acidity, which make them desirable by consumers. Another reason is C00-09 is a later availability, while C99-42 is an earlier availability [21,22]. The permanent seasonal presence of those two cultivars provides a permanent host to C. capitata. The combination reasons of quality and availability for consumers and C. capitata led us to choose these two cultivars.

Whilst at 25.0 °C, thirty fruits from each cultivar were distributed in three replicates, ten fruits per replicate, for physical quality and six replicates, five fruits per replicate for biochemical quality characteristic determination. Physical quality characteristics included weight, diameter, colour (red, grey, black), hardness, and relative humidity. Biochemical qualities included the percent of acid, degrees Brix, and sugar to acid ratio. The blueberry samples were stored at 3.0 ± 0.5 °C at Murdoch University cold storage.

2.2.1. Colour Test

Colour was measured as an RGB model (Red, Grey, Black) with a colourimeter (CS-210 Portable Precision Digital Colourimeter, China). The colourimeter was calibrated with white and black colour. Three replications were performed for each blueberry fruit.

2.2.2. Size

Fruit diameters were measured using plastic Vernier calipers to the nearest millimetre (mm) (Ningbo, China) and three replications were performed to measure the diameter of each fruit.

2.2.3. Weight

Fruit weight was measured by a digital balance (PAL-BX|ACID7, Atago (Tokyo, Japan).

2.2.4. Hardness

A small part of fruit peel was removed. The hardness of blueberry fruits was measured by a fruit hardness tester (Model No GY-2, Ningbo/Shanghai, China). The capacity of the hardness tester is 0.2–4 kg/cm² (105 pa), pressure diameter is 3.50 mm, the accuracy is ±0.02 mm, pressure depth is 10 mm, and dimensions are 140 × 60 × 30 mm.

2.2.5. Relative Humidity

Relative humidity was measured via a moisture meter (model DH-100-DELMHORST, New York, NY, USA) two pins that penetrate deeply into the blueberry fruit, measuring from 0–99.9%, with an LCD screen, 80 mm width × 35 mm high × 150 mm diameter and 245 g.

2.2.6. Degrees Brix, Acid, and Their Ratio (Sugar/Acid)

Sugar and acidity level in blueberry fruit juices were measured with a Pocket Brix-acidity meter, PAL-BX|ACID7 master kit brand Atago (Tokyo, Japan). The meter was calibrated with water for sugar content and without water for acidity.

Six replicates of five blueberries, from each cultivar, were juiced individually through a piece of mesh to measure Brix. An aliquot of undiluted juice (at least 0.3 mL) was applied to the lens with a plastic pipette.

To measure acidity, 1.0 g of fruit juice was transferred into the beaker with a dilution ratio of 1:50 with distilled water to a total weight of 50 g while stirring gently. Then diluted juice was moved by plastic dropper to the lens of the PAL-BX|ACID meter in percentage (%) and measured (first pressing for acid and the second for sugar). The sugar-acid ratio was displayed and recorded. Six replicates per cultivar were done for acidity, Brix, and their ratio.

2.3. Natural Infestation

The natural infestation method was utilised on blueberries to avoid fruit damage by artificial infestation, which may lead to microorganisms growing and affecting C. capitata development stages. Additionally, natural infestation can avoid egg damage through the collection and transferring processes to the fruits when using artificial methods. A representative random assay of blueberry fruits from each cultivar was inspected to ensure the absence of field infestation and determine cultivar health. In preparation for conducting the infestation with medfly at 26.0 ± 1.0 °C, 60–70% RH in the rearing room, approximately 300–400 random healthy blueberry fruit of each cultivar (C00-09 and C99-42) were conditioned at 25.0 ± 1.0 °C and 60–70% RH overnight. Fruits were placed in an aluminium tray of the dimensions of 29 cm width, 92 cm length, and 2.5 cm depth with metal mesh bottom (0.6 cm). The blueberries were transferred to the aluminium tray and held in the middle of the adult rearing box (200 × 150 × 40 cm) containing around 250,000–300,000 flies with the sex ratio of 50:50. This set up allowed the flies to reach fruits easily. The natural infestation was conducted repeatedly to infest a sufficient amount of fruits. Various periods of exposing the fruits to females (30, 45, 90, and 120 min) with three replicates for each exposure period were tested to identify an ideal time for females to lay an appropriate amount of eggs in each fruit. From each replicate, 16 fruits were randomly selected six times and individually dissected to count the eggs inside the fruit.

2.4. Life History Study

To study the life history of medfly in the two blueberry cultivars, two blueberry fruits from the sample of 45-min exposure time (for each cultivar which was used for life history study) after natural infestation were randomly selected and put in a glass jar (300 mm) with sand. The jar was closed by a piece of mesh and banded with a rubber band. A total of 300 jars were prepared for both cultivars and kept at 26.1 ± 1.0 °C and 60–70% RH. After 24 h incubation, 16 jars were moved to the laboratory to dissect for recording the stages of the flies under a microscope. This step was repeated every day until the first appearance of pupae in control jars.

2.5. Cold Treatment Cabinet

The temperature (1.0 ± 0.2 °C) was selected because it is a relatively safe temperature for fruits. Lower than 1.0 °C will be likely to cause serious fruit or vegetable damage. The cold cabinet is a constant temperature and humidity incubator (model HWS, LET code 0574-88000432, Tianjin, China), which was made from stainless steel with a temperature range of 0 to 65 °C, a humidity control range of 50% to 90% RH, humidity fluctuation of ±8% RH and an inner stainless-steel mirror. The door consists of toughened metal with an outside observation window. The cold cabinet is equipped with self-diagnosis tools, a sensor failure alarm, and over-temperature protection. The temperature inside the cabinet was recorded every 30 min by putting two HOBO^® data logger units at different heights (Model number H08-004-02, Onset Computer Corporation, MA, USA, www.onsetcomp.com). We calibrated the cabinet temperature sensor and HOBO^® data logger units by comparing them with four thermometers red spirit. The thermometers were checked using an ice point check as per the NATA tech note. Then the incubator and HOBO were checked with checked thermometers [23].

After 45 min of exposure to female flies, the fruits were used for cold tolerance study in laboratory conditions. Two berries from cultivar C00-09 were randomly selected and put in a glass jar (30 mm) with sand. Three hundred and twelve jars containing C00-09 were prepared with two berries in each jar, which were divided into four groups (78 jars in each group). Each group was put on a plastic plate labelled with the insect stage (eggs, 1st instar, 2nd instar, and 3rd instar), date, and cultivar. For eggs used in the experiment, the plastic plate (group 1) with 72 jars was directly transferred to cold treatment cabinet (1.0 ± 0.2 °C chamber) and six jars to rearing cabinet as control. Life-cycle results were used to determine how long each stage remained in the rearing cabinet before transferring it to the cold cabinet (Table S1). After three days and 12 h, 72 jars were moved from the rearing cabinet (1st instar, group 2) to the cold cabinet, which was set up at 1.0 ± 0.2 °C, six jars were left in the rearing cabinet as a control. After 5 days and 12 h, the second plate (2nd instar, group 3) was moved from the rearing cabinet, with 72 jars for cold cabinet and six for a control cabinet. After 8 days, the third plate (3rd instar, group 4) was moved from the rear cabinet, with 72 jars for cold treatment and 6 for a control cabinet.

After 24 h of cold treatment, six jars from each group were moved to the rearing cabinet and labelled. Every day, six jars were collected from the 1.0 ± 0.2 °C cold cabinet and transferred to the rearing cabinet. Every six jars that were moved from cold cabinet to rearing cabinet from day one to day twelve (72 jars) were left in a rearing cabinet for 28 days and checked every day. After pupae were first seen, the sand inside each jar was sieved three times weekly; and the dry blueberries were checked well for pupae. After that, the pupae were counted and placed in a sterile Petri dish, which was left in the rearing cabinet. Additional observations on the number of emerged flies were considered. This provided information on the viability of pupae and whether they were able to develop into adults, and the sex ratios observations were recorded and analysed. Firstly, all the surviving adults after treatments from eggs and the three developmental instars were collected and the percentage of females was calculated. The same protocol was applied with cultivar C99-42. Secondly, to examine if a certain period of days treatment influences the sex development, female ratios from treated flies on different days were analysed. If the numbers of adults emerged were too low (<10), results were not included in the analysis.

2.6. Statistical Analysis

The control pupariation and adult emergence rates were used to normalize treatment pupariation and adult emergence by Schneider–Orelli’s formula [24]. Firstly, the test of normal distribution was conducted via both the Kolmogorov–Smirnov test and Shapiro–Wilk test (Table S2). After the data met normal distribution, the pupariation and emerged adults’ rates of the insects under cold treatment were statistically estimated following the general linear model-univariate. The mortality rate of the insect under cold treatment was statistically estimated following the Median Lethal Time method (LT). The 90%, 95%, and 99% mortality (LT₉₀, LT_95, and LT₉₉) were estimated by using the selected models. According to the results of the normality test, the regression models (probit analysis) were selected. Pupariation and emerged adults were counted as the survival (and mortality) to calculate the lethal time (LT). If a treated fruit fly egg or larva can successfully develop to a pupa or an adult, it is a survived fly; otherwise, it is a dead fly. The LT value estimated under a generalized linear model with probit link function on cold treatment days. The model can be written as:

η = β0 + β1 x

(1)

where η is the response or proportion mortality, x is the dose, β0 is the intercept, and β1 is the coefficient of the dose. An ANOVA single factor test was used to compare the sex ratio of adult insects emerging from different stages after cold treatment in two blueberry cultivars. The fruit quality was statistically estimated following the comparing means independent samples t-test. In all the statistical methods, the probability level was ≤ 0.05 and SPSS software (SPSS, IBM version 24 Armonk, New York, NY, USA) was used.

3. Results

3.1. Fruit Quality Assessment

To investigate the effect of different blueberry variety’s impact on the fruit fly response to cold treatment, the fruit qualities of two blueberry varieties, C00-09 and C99-42, were compared. The results of biochemical tests of two blueberry cultivars (

Table 1. Fruit quality assessment includes biochemical and physical quality of blueberry cultivars C00-09 and C99-42 before cold treatment.

Fruit Quality	Test	Blueberry Cultivars	Mean ± SE	Unit	Significance
Biochemical	Sugar	C00-09	13.5 ± 0.14	%	0.3
		C99-42	11.3 ± 1.9	%
	Acid	C00-09	0.5 ± 0.03	%	0.1
		C99-42	0.4 ± 0.04	%
	Sugar to acid	C00-09	26.1 ± 1.6	%	0.05
		C99-42	34.1 ± 3.3	%
	Water content	C00-09	92.7 ± 0.7	%	0.0
		C99-42	79.4 ± 1.4	%
Physical	Weight	C00-09	3.2 ± 0.1	gm	0.0
		C99-42	2.0 ± 0.1	gm
	Diameter	C00-09	20.6 ± 0.3	mm	0.0
		C99-42	17.9 ± 0.3	mm
	Colour R	C00-09	91.1 ± 21.5	nm	0.5
		C99-42	104.5 ± 1.9	nm
	Colour G	C00-09	90.8 ± 4.5	nm	0.7
		C99-42	92.7 ± 2.3	nm
	Colour B	C00-09	108.3 ± 4.2	nm	0.0
		C99-42	92.2 ± 2.5	nm
	Hardness	C00-09	3.1 ± 0.1	Kg/cm	0.3
		C99-42	6.2 ± 3.5	Kg/cm

) indicated that there were no significant differences in percentages of acid, Brix, and the ratio of sugar to acid between the two cultivars (p > 0.05), while there was a significant difference in water content between the cultivars (F = 13.600, p < 0.0005) (Table 1). The water content in cultivar C00-09 was higher than cultivar C99-42, where the water content was 92.4 at C00-09 while 79.7 in C99-42. The physical measurements of cultivars C00-09 and C99-42 (Table 1) showed that there were significant differences in weight (F = 13.600, p < 0.0005), diameter (F = 133.000, p < 0.0005), and density blue colour (F = 9.180, p = 0.002) while there were no significant differences in density red colour, density green colour, and hardness between both cultivars.

3.2. Natural Infestation

Four time-points (30, 45, 90, and 120 min) for the natural infestations were tested on two blueberry cultivars. The results of this experiment (average ± standard error) indicated that 30 min only produced 0.6 ± 0.3 and 0.8 ± 0.3 egg/fruit, in cultivars C00-09 and C99-42, respectively. At 90 and 120 min, natural infestation yielded 78.1 ± 0.7 and 76.3 ± 0.6 eggs/fruit in cultivar C00-09 while 93.3 ± 0.6 and 91.6 ± 0.4 eggs/fruit in C99-42 respectively. This high population of larvae will result in larval competition on a limited food amount (one fruit). Therefore, 45 min was selected as the average egg number per fruit is optimal, 6.8 ± 0.4 and 6.5 ± 0.4 eggs/fruit in cultivars C00-09 and C99-42, respectively. There were no significant differences in the rate of eggs between the two cultivars (p > 0.16), while there was a significant difference in the egg rates between the exposure times (F = 50866.1, p < 0.00) (Table S3)

3.3. Life History Study

To prepare for the cold treatment, the life history of C. capitata immature stages in blueberry at 26.1 ± 1.0 °C and 60–70% RH were examined (Table S1). Two days after infestation, eggs started to hatch. Four and a half days after the infestation, the maximal 1st instar was 231 and 201 for cultivar C00-09 and C99-42, respectively. Five days after the infestation, 2nd instar larvae were observed. Six days after the infestation, suitable numbers 208 and 152 for cultivar C00-09 and C99-42, respectively, of 2nd instar were obtained. Nine days later, suitable numbers 146 and 125 for cultivar C00-09 and C99-42, respectively, of 3rd instar larvae were ready to use, and pupae started to be observed on day 10.

3.4. Responses (Mortality) to Cold Treatment

The temperature and humidity inside the cabinet during cold treatment were 1.0 ± 0.2 °C and 60–70% RH. Significant differences in pupariation ratios (F = 323.567, p < 0.0005) and adult emergence (F = 387.389, p < 0.0005) were found among the four stages (eggs and three larval instars) that were treated with cold treatment (1.0 ± 0.2 °C) for 12 days (

Table 2. Pupariation rates and emerged adults of four immature stages of C. capitata after exposure to cold treatment (1.0 ± 0.2 °C) for 12 days in two Western Australian blueberry cultivars (C00-09 and C99-42). SE means standard error.

Blueberry	Treatment	Egg		1st Instar		2nd Instar		3rd Instar
	(Days)	Pupariation (%) Mean (SE)	Emerged Adult (%) Mean (SE)	Pupariation (%) Mean (±SE)	Emerged Adult (%) Mean (±SE)	Pupariation (%) Mean (±SE)	Emerged adult (%) Mean (±SE)	Pupariation (%) Mean (±SE)	Emerged Adult (%) Mean (±SE)
C00-09	0	100 (0)	98 (0.2)	100 (0)	99 (0.1)	100 (0)	99 (0.1)	100 (0)	100 (0)
	1	52.6 (0.4)	48.0 (0.4)	59.2 (0.3)	54.8 (0.3)	74.8 (0.3)	76.3 (0.2)	82.7 (0.4)	83.9 (0.4)
	2	37.5 (0.5)	32.8 (0.6)	37.7 (0.3)	30.8 (0.4)	56.2 (0.6)	58.0 (0.6)	79.6 (0.3)	763 (0.3)
	3	37.5 (0.5)	31.2 (0.4)	29.6 (0.6)	28.5 (0.6)	56.2 (07)	53.4 (0.4)	75.9 (0.4)	74.8 (0.3)
	4	33.8 (0.8)	26.7 (0.5)	29.6 (0.5)	24.8 (0.6)	54.0 (0.5)	48.4 (0.5)	69.9 (0.3)	68.7 (0.3)
	5	27.0 (0.5)	25.1 (0.3)	26.6 (0.8)	22.5 (0.3)	51.8 (0.7)	45.8 (0.5)	62.4 (0.9)	61.0 (0.8)
	6	19.5 (0.4)	17.5 (0.4)	207.(0.6)	19.5 (0.6)	48.1 (0.6)	42.7 (0.4)	58.6 (0.4)	50.3 (0.6)
	7	12.0 (0.2)	9.9 (0.2)	19.2 (0.6)	17.2 (0.5)	34.5 (0.4)	29.0 (0.4)	45.1 (0.2)	42.7 (0.3)
	8	4.5 (0.2)	3.8 (0.2)	17.0 (0.5)	13.5 (0.3)	19.7 (0.4)	17.5(0.5)	37.5 (0.3)	32.8 (0.4)
	9	0	0	11.8 (0.4)	4.5 (0.2)	11.1 (0.2)	6.1 (0.1)	15.7 (0.3)	15.2 (0.2)
	10	0	0	0	0	0	0	9.7 (0.4)	7.6 (0.2)
	11	0	0	0	0	0	0	0	0
	12	0	0	0	0	0	0	0	0
C99-42	0	100 (0)	99 (0.1)	100 (0)	100 (0)	100 (0)	98 (0.2)	100 (0)	99 (0.1)
	1	49.6 (0.3)	46.1 (0.3)	58.6 (0.6)	53.8 (0.7)	70.4 (0.3)	72.0 (0.2)	81.4 (0.3)	81.4 (0.3)
	2	37.5 (0.2)	33.0 (0.3)	34.5 (0.7)	30.7 (0.9)	58.4 (0.3)	56.(0.5)	78.5 (0.6)	77.7 (0.5)
	3	33.8 (0.3)	27.6 (0.2)	27.0 (0.7)	25.3 (0.7)	53.8 (0.5)	48.8 (0.6)	74.8 (0.4)	76.3 (0.3)
	4	30.0 (0.5)	30.0 (0.5)	26.3 (0.7)	23.0 (0.7)	52.3 (0.4)	48.0 (0.4)	66.6 (0.3)	66.6 (0.3)
	5	22.5 (0.3)	19.2 (0.2)	23.3 (0.6)	21.5 (0.6)	50.0 (0.3)	48.0 (0.2)	62.9 (0.2)	56.2 (0.2)
	6	17.2 (0.5)	12.3 (0.4)	19.5 (0.8)	15.3 (0.6)	46.1 (0.2)	38.4 (0.4)	52.5 (0.4)	45.1 (0.6)
	7	9.7 (0.3)	6.1 (0.1)	18.7 (0.8)	13.8 (0.6)	31.5 (0.4)	33.3 (0.2)	40.7 (0.6)	37.0 (0.5)
	8	4.5 (0.2)	2.3 (0.2)	15.0 (0.3)	12.3 (0.2)	20.0 (0.3)	14.4 (0.4)	33.3 (0.2)	26.6 (0.3)
	9	0	0	9.7 (0.3)	4.6 (0.5)	10 (0.2)	4.8 (0.2)	13.3 (0.4)	11.8 (0.4)
	10	0	0	0	0	0	0	8.1 (0.3)	4.4 (0.3)
	11	0	0	0	0	0	0	0	0
	12	0	0	0	0	0	0	0	0

).

3.4.1. Cold Treatment Bioassay

In the control group of pupariation, the natural mortality was considered zero, following common practice in calculating mortality where large populations of insects are tested inside the host fruits [25]. Based on pupariation ratios as survival indicator, the stage with the highest survival rate was 3rd instar then 2nd instar and 1st instar, while the egg was the most susceptible in both blueberry cultivars.

There was a significant difference between the pupariation rates of eggs and the three larval instars in both blueberry cultivars, which were decreasing with increased exposure time to cold (Table 2). The exposure times to the low temperature, which completely eradicated eggs and larvae, were different for different fly stages. In both cultivars, nine-day treatment was enough to reach 0.0 survival in egg samples. Ten-day treatment to 1st and 2nd larvae instars can reach 0.0 survival flies, while 11-day treatment was needed for 3rd instar larvae to achieve 100% mortality. The results of the statistical analysis showed that there were significant differences during cold treatment days in pupariation rates (F = 380.335, p < 0.0005) and adult emergence (F = 320.710, p < 0.0005). It was concluded that a longer duration of exposure to cold resulted in fewer opportunities for insect survival in both blueberry varieties.

Based on emerged adults as survival indicators, fly eggs were the most susceptible stage to the cold treatment because nine-day treatment leads to zero adults in both blueberry cultivars. The 3rd instar is the most tolerant stage in both cultivars, which required 11-day treatment to achieve zero adults. There was a significantly negative relationship between exposure time to cold and survival emerged rates in eggs and larvae instars in both cultivars. The control mortality of emerged adults was below 5%; therefore, it was ignored [26].

There was no significant effect between blueberry cultivars on pupariation means among the four stages during all exposure times (Table 2), while there were significant effects on emerged adults (F = 23.962, p < 0.0005) (survival insects) (Table 2). Blueberry cultivar C00-09 had a significant impact on an increase of emerged adults in eggs and all larvae instar compared to cultivar C99-42. However, the results showed that the survival rates in cultivar C00-09 were higher than those in the C99-42.

3.4.2. Modelling Analysis

When using the recovered pupariation as end-point for mortality modelling, the results appeared that the 3rd instar is the most tolerant stage with LT₉₀, LT_95, and LT₉₉ values being 10.3, 11.6, and 14.1 days respectively in cultivar C00-09, while 10.0, 11.3, and 13.8 days in cultivar C99-42 (

Table 3. Cold treatment duration to induce 90%, 95%, and 99% mortality of C. capitata.

Development	Blueberry	Mortality	Pupae Recovery as End Point			Adults Recovery as and Point
Stage	Cultivars	(LT)%	Treatment (Days)	95% Confidence Limits		Treatment (Days)	95% Confidence Limits
				Lower	Upper		Lower	Upper
Eggs	C00-09	90	6.7	6.0	7.7	6.3	5.8	6.9
		95	8.1	7.2	9.4	7.8	7.2	8.6
		99	10.8	9.5	12.9	10.7	9.7	11.9
	C99-42	90	6.4	5.9	7.0	5.7	5.3	6.2
		95	7.8	7.2	8.6	7.1	6.5	7.8
		99	10.5	9.6	11.7	9.7	8.8	10.9
1st	C00-09	90	8.0	6.9	9.6	7.1	6.2	8.5
		95	9.8	8.5	12.1	9.0	7.8	10.9
		99	13.3	11.3	17.0	12.4	10.6	15.6
	C99-42	90	7.6	6.6	9.2	6.9	6.0	8.8
		95	9.5	8.2	11.7	8.6	7.6	10.3
		99	13.0	11.0	16.6	12.0	10.3	14.7
2nd	C00-09	90	9.1	8.0	11.1	8.7	7.7	10.1
		95	10.7	9.2	13.2	10.1	8.9	11.9
		99	13.6	11.5	17.2	12.7	11.1	15.3
	C99-42	90	9.2	8.0	10.9	8.7	7.6	10.3
		95	10.7	9.3	12.9	10.1	8.8	12.2
		99	13.4	11.5	16.7	12.8	10.9	15.9
3rd	C00-09	90	10.3	9.3	11.8	10.0	9.1	11.2
		95	11.6	10.4	13.5	11.3	10.3	12.8
		99	14.1	12.5	16.9	13.7	12.3	15.8
	C99-42	90	10.0	9.1	11.4	9.5	8.8	10.5
		95	11.3	10.2	13.0	10.7	9.8	11.9
		99	13.8	12.3	16.1	13.1	11.9	14.7

). The 2nd instar larvae were the second most tolerant stage. Eggs were more susceptible (LT₉₉ = 10.8 and 10.5 in cultivar C00-09 and cultivar C99-42 respectively) than larvae (Table 3). Interestingly, based on the emerged adult ratios in cultivar C00-09, LT₉₉ was 10.7, 12.4, 12.7, and 13.7 days for eggs, 1st instar, 2nd instar, and 3rd instar respectively, further confirming that 3rd instar was the most cold-tolerant stage. While in cultivar C99-42 LT₉₉ values were 9.7, 12.0, 12.8, and 13.1 days for eggs, 1st instar, 2nd instar, and 3rd instar respectively. The results confirmed that the 3rd instar was the most cold-tolerant stage and the egg was the most susceptible (Table 3).

3.5. Sex Ratios

To determine, if the cold treatment affected the sex development of the emerged adults, the sex ratios of the treated eggs and larval instars were compared. There were no significant differences at each stage in both blueberry cultivars, which were all close to 50% (Figure 1). From Day 0 to Day 3, there were no significant differences between eggs, 1st, 2nd, and 3rd larval instars on each daily treatment (Figure S1).

4. Discussion

The life history study results were consistent with a previous study, in which 30 hosts (including the blueberry) were used to analyse the life history of C. capitata at 30.0 °C, RH 65% ± 10%, and 12:12 L:D (12-hour light: 12-hour dark photoperiod) [27].

The 3rd instar larvae showed a higher survival rate than the other stages. A previous study of cold susceptibility and disinfestation of Bactrocera invadens (syn. B. dorsadis) (Dew 2003) in oranges showed that the 1st instar is more susceptible than 2nd and 3rd [28]. However, this result disagreed with another study on Bactrocera tryoni (Froggatt 1897) in cold storage (1.0 °C for 12 days) in three cultivars of Australian blueberry, in which the 1st instar was the most cold-tolerant stage to cold treatment (1.0 °C for 12 days) [29]. This current study results also disagree with a study on C. capitata and B. tryoni in five Australian citruses (Citrus spp.) in which the 2nd instar was reported the most cold-tolerant stage at 2.0 and 3.0 °C [30]. Hallman et al. (2011) reported that the 3rd instar C. capitata is the most cold-tolerant stage (1.5 ± 0.5 °C) when eggs and 1st, 2nd, and 3rd instar larvae were tested in oranges [31]. In comparison with a previous study, when the C. capitata were treated by 0.0 °C with different exposure durations (0–12 days) and fed on a lab diet; the eggs were the more susceptible immature stage, while the 1st instar and 3rd instar had the highest survival rates [32]. All these studies demonstrated that different fruits fly species, fruits or temperatures may show different tolerance responses during cold treatment, which should be studied case by case.

Eleven-day cold treatment is enough to get no survival from any of the four medfly stages (eggs and larvae instars) in both blueberry cultivars at 1.0 ± 0.2 °C. Results were similar to those of Mason et al. (1934), who confirmed that 11 days were enough to kill the four stages of medfly at 1.0 ± 0.5 °C [33]. These results disagreed with those of Hill et al. (1998), who found that after 16 days of exposure to 0.0–1.5 °C there was no insects survival in disinfection of C. capitata in oranges [34]. The difference in fruit may affect the results of the same phytosanitary treatment [13].

It was shown there was no significant effect of cold treatment on sex ratios among the four stages of C. capitata throughout all exposure time. Similar results have been reported by Maurizio et al. (2019) on Queensland fruit fly B. tryoni (eggs and larvae) [35].

To understand the influences of blueberry cultivars on the survival of medfly during cold treatment, pupariation rates, emerged adults, and sex ratio were used as indicators. There were no significant effects of varieties on pupariation means (Table 2). Where in both cultivars, the survival was progressive from the lowest (eggs) to the highest (3rd instar). The time required to eradicate each stage also was the same in both cultivars, where 9 days was enough to eradicate eggs, 10 days for 1st instar and 2nd instar, and 11 days for 3rd instar, respectively. These results agreed with the results from previous studies, where the blueberry cultivars had no effect of 1st instar survival of B. tryoni at 1.0 °C. In their experiment, three cultivars, Premier blueberries, Climax blueberries, and Sharpe Blue blueberries were used. In all these cultivars, the time required to ensure no survival of the 1st instar was ten days [29]. Further, they found that the eradication time of 2nd instar and 3rd instar of B. tryoni at 1.0 °C was different in some blueberry cultivars. Jessup et al. (1998), reported that the Premier blueberries and Sharpe Blue blueberries have the same effect on eradicating time of 2nd and 3rd instars where they found that eight days of exposure to cold were enough to ensure no survival insect while Climax blueberries increased the eradication time of 2nd and 3rd instars to ten days.

Interestingly, results of this research indicated that there was a significant impact of blueberry cultivars on emerged adult rates although there were no significant differences in biochemical quality such as sugar, acid, and their ratio between the two cultivars. It is possibly attributed to the significant difference in the water content, which was higher in the cultivar C00-09 (92.7) than in C99-42 (79.4), as shown in Table 1. All living organisms require available water to grow and function [36]. For insects, the water content of the blueberry affected the consumption of diet [37].

There were significant differences in certain physical qualities including weight, diameter, and blue light density (Table 1). In cultivar C00-09, the larger weight and diameter provided a larger amount of food and area for insects comparing to cultivar C99-42. The reflection intensity of blue light in cultivar C00-09 was higher than cultivar C99-42. These results were consistent with those by Shibuya et al. (2018). They found blue light (Short-wavelength visible light 400–500 nm) affected the survival of fruit fly Drosophila melanogaster and increased mortality of eggs and larvae stage [38].

5. Conclusions

In this study, we provided the first evidence of the medfly responses to the cold treatment at 1 °C in two blueberry cultivars. Eleven days of exposing C. capitata to 1 °C in both blueberry cultivars was enough to achieve a survival rate of 0. The 3rd instar was the most cold-tolerant stage of C. capitate in blueberries. This small-scale treatment study is a preliminary investigation for medfly cold treatment in blueberries, which will help develop or refine the large-scale cold treatment of medfly for the blueberry trade industry. This study also provided information for the cold treatment of C. capitate in other fruits, especially small fruits such as strawberries, raspberries, blackberries, and grapes.