Investigating the Effect of Cold on the Possibility of Storing Pupae and Whole Insects of Parasitoid Bees

INTRODUCTION

The wasp Bracon hebetor Say is one of the most well-known and effective parasitoids that is used in most parts of the world as a biological agent to control scaly pests, especially Noctuidae Pyrallidae and Gelchiidae families [1-4]. This bee first paralyzes the last instar larvae of the host by injecting poison and then lays eggs in groups on their body surface [5, 6]. This parasitoid bee multiplies every year in a large number of insectariums, and to control various pests, especially the cotton bollworm, it is released on the surface of various fields, including cotton, tomato, and soybean [7, 8].

Low-temperature (cold) storage, the history of which dates back to more than 85 years ago [9], is an essential step in the process of mass and commercial breeding of natural enemies in insectariums [10]. In this stage, the natural enemy is stored at a low temperature (usually between zero and 15 degrees Celsius) before being released in the field and until its population reaches a sufficient number so that its metabolism and feeding are temporarily stopped [10, 11]. This technique increases the shelf life of the cultured natural enemy, and as a result, a sufficient and stable stock of it is provided for use in biological control programs [10, 12]. In addition, mass storage provides this possibility, during the outbreak of the pest in the field, the release of the natural enemy is done quickly and in sufficient numbers, and thus, the chance of success of the biological control program increases significantly [13, 14].

Although cold storage is an important part of the mass breeding process of natural enemies in insectariums and has many benefits for breeders [10], severe and long-term cold may affect various aspects of life. Natural enemies, especially parasitoids, also have negative effects [11]. The negative effect of exposure to long-term cold on the sex ratio, lifespan, fertility, length of development period, and the profile of parasitoids has been reported in different studies [10, 15]. In general, the resistance of parasitoids to cold is a complex process that is affected by a variety of external (non-living) factors such as temperature and storage duration, and internal (biological) factors such as nutritional status, sex, age, and developmental stage [11, 16]. The constant or fluctuating storage temperature has also been reported to be effective on the severity of injuries caused by cold so that if a parasitoid regularly and periodically experiences a temperature higher than the storage temperature during storage, the negative effects of cold on him will be reduced. Therefore, to maintain the quality of parasitoids stored in the cold, it is necessary to choose the right temperature for storage, the duration of cooling, and the appropriate growth stage for storage. However, the efficiency of parasitoid storage at low temperatures is often determined by the result of two factors, temperature and duration of storage [13, 17], which in some sources is called the term “cold exposure dose” to show the result of these two factors [18].

Several reports have been published on the storage of B. hebetor bees in the cold. The results of a study showed that after a month of storing the female insects of this bee in the refrigerator, their parasitism power decreased, but no change was observed in the parasitism rate of the offspring of the next generation. Also, the storage of female bees in the cold hurt the development period of the immature stages, but it did not have a significant effect on the sex ratio of the offspring of the first and second generations [17]. The results of another study showed that the females in diapause of this bee were more resistant to cold than the females without diapause, and it was possible to store them in the refrigerator for up to 8 weeks [13]. In another study, after three months of storage of whole insects, more than 50% of this parasitoid died at a temperature of 12 degrees Celsius. In contrast, the longevity of female bees, the survival of immature stages, and the sex ratio of the offspring of the next generation were not affected by cold [19]. In addition to the temperature and duration of storage, the effect of the development stage of this bee has also been studied on its resistance to cold and its eggs, larvae, and pupae have been reported to be more sensitive to cold compared to whole insects [20, 21].

Considering the necessity of storing breeding populations of parasitoid bees in insectariums and the need to investigate the response of populations to cold, this research was conducted to comprehensively investigate the lethal and sub-lethal effects of cold on whole insects and pupae of this bee. The results of this research can be used in the storage of complete insects and pupae of this bee in insectariums by supplementing the previous information.

MATERIALS AND METHODS

In this study, the larvae of Anagasta kuniella Zeller were used as an alternative host for the breeding of B. hebetor bees. Pans containing a mixture of flour and wheat bran after being infected with moth eggs of the host in room conditions (temperature 2±26 degrees, the relative humidity was 10%±60%, and the photoperiod was 16 hours of light to 8 hours of darkness). The larvae of the late stages of moths (fourth and fifth instars) were collected from the rearing pans and were given to parasitoid bees as hosts.

To breed parasitoid bees, plastic containers (height 25 cm and diameter 17 cm) were used. For this purpose, the initial population of parasitoids was confined inside these containers and their opening was blocked using white net cloth (40 mesh). Then, large host larvae (fourth and fifth instars) were provided to the bees in the container on a sheet of paper. Every 24 hours, the old host larvae were placed in closed containers with newly replaced larvae and old parasitized larvae and were kept in the rearing room until the full bees left. After five generations of rearing pupae and complete insects, the required number of bees were collected from the colony using an aspirator or manually and were gradually used in the desired experiments.

Five hundred pupae of the same age (5 days old) along with cocoons were randomly collected from the bee breeding colony and after dividing them into ten populations of 50, each population was placed separately in a transparent plastic cup with the diameter of the mouth. 10 cm and 12 cm height were transferred (each population of 50 pupae was considered as a replication). The opening of the glasses is blocked with a white lace cloth and the glasses are kept for a certain period (according to the experimental treatment, 7, 14, 21, 30, 45, and 60 days) in the refrigerator (temperature 5±1 degrees Celsius and total darkness) were kept. In the control treatment, the pupae were kept in the germinator instead of the refrigerator (temperature of 26±1 degrees Celsius, relative humidity of 60±5%, and photoperiod of 16 hours of light and 8 hours of darkness). After the period considered for each treatment, the pupae of that treatment are taken out of the refrigerator, and after being transferred into the germinator, their hatching percentage (percentage of complete insects) and the sex of the bees removed (based on the presence or absence of ovulation at the end of the abdomen) was measured and recorded separately.

To investigate the effect of cold on the lifespan and reproduction of bees that came out of pupae stored in the cold (sub-lethal effects of cold), 20 pairs of male and female bees were randomly selected from among the bees that came out of the pupae stored in the cold. Daily, ten-fourth or fifth instar larvae of the Mediterranean flour moth as a host and some 30% honey juice were provided to the bees in the glasses through a piece of wet cotton. At the end of every 24 hours, the larvae were removed from the glasses and the number of eggs laid on them was counted and recorded. This process continued until the death of the bees inside the glasses, and thus, the lifespan and daily egg-laying of the bees that came out of the cold-exposed pupae were measured and recorded.

Five hundred male bees and five hundred one-day-old female bees were collected from the bee breeding colony using an aspirator. Then, each of them was divided into ten populations of 50 and each population was transferred separately into a clear plastic cup as an experimental repetition (a total of 20 cups each containing 50 bees in each treatment). The mouths of the glasses were blocked with a white net cloth and the bees inside were fed with 30% honey water for 24 hours before entering the refrigerator. After feeding with honey, the glasses containing the bees were transferred to the refrigerator and kept there for a certain period. After the desired period in each treatment had passed, the glasses related to that treatment were taken out of the refrigerator and after being placed in room conditions, the number of dead people inside them was counted and recorded separately and by gender. Dead bees were those who were not able to move and fly normally until 3 hours after leaving the refrigerator. Accordingly, a small number of bees that were only able to move their arms and legs or their antennae after this period, but were not able to move normally, were counted among the dead. The glasses related to the control treatment were stored in the germinator instead of the refrigerator.

To investigate the effect of cold on longevity and reproduction (daily average of egg laying), queen bees stored in the refrigerator from among the surviving bees at the end of each period, 30 pairs of male and female bees were randomly selected and each pair (as a replication) were placed separately in a beaker. The mouths of the glasses were blocked using a net cloth and placed upside down on the bottom of a white plastic tray. The glasses containing parasitoid bees were kept in the germinator under the conditions mentioned above. Daily and until the death of the female bee in each glass, ten larvae of Mediterranean flour moth (fourth or fifth instars) were provided to the bees in the glass. Also, the bees in the glasses were regularly fed with honey water using a dropper and the method mentioned above. Daily and regularly, paper slices containing host larvae were replaced with slices containing fresh host larvae, and the number of eggs laid on old larvae was counted and recorded. Daily and regularly, paper slices containing host larvae were replaced with slices containing fresh host larvae, and the number of eggs laid on old larvae was counted and recorded. Thus, in addition to the lifespan of male and female bees, the average daily laying of female bees in each treatment was also measured and recorded.

All experiments were conducted in a completely randomized design with seven treatments separately for pupae and whole insects. First, the normality of the data was evaluated using Minitab 13 software, and then the data were analyzed using one-way variance analysis using SAS statistical software. For statistical comparison between groups, the LSD supplemental test was used at the five percent probability level, and Excel 2007 software was used to draw graphs.

RESULTS AND DISCUSSION

Storage of pupae

The results showed that the storage of B. hebetor bee pupae in the refrigerator had a significant effect on the percentage of emergence of whole insects (P<0.0001, F_{6, 35} = 253.2). With the increase in storage time, the percentage of full insects emerging from the pupae decreased, so that in 30 days and more, all the pupae were destroyed and no complete insects came out of them. The percentage of losses (no emergence of complete insects) of pupae that were stored in the refrigerator for 7 days (7.33%) was not significantly different from the control (pupae kept at 26 degrees Celsius), but in periods of more than one During the week, the amount of pupae losses was significantly higher than the control. The results showed that storing the pupae in the cold causes a significant decrease in the lifespan of male and female bees that come out of them (P<0.0001, F_{3, 76} = 39.2; P<0.0001, F_{3, 76} = 49.8, respectively). In addition, the storage of pupae in the refrigerator significantly reduced the sex ratio (percentage of female individuals) and reproduction of released bees (P<0.001, F_{3, 20} = 12.02; P<0.0001, F_{3, 76} = 55.7, respectively). The average lifespan of male bees in the control treatment was 12.45 days, but with the increase in the storage period, their lifespan decreased and reached 2.4 days in the 21-day treatment. The average lifespan of female bees in the control treatment was 26.8 days, but with the increase in the duration of pupae storage in the refrigerator, the lifespan of the released female bees decreased and reached 4.7 days in the 21-day treatment. In the 14-day treatment, the average lifespan of female bees was 7.55 days, which was not significantly different from the 21-day treatment, but it was significantly less than the control and 7-day treatments (Table 1).

Table 1. Average fertility, sex ratio, and lifespan of full-fledged bee insects after emerging from pupae stored at five degrees Celsius.

P < 0.05, LSD-test