Cold Storage Possibilities of Pupae and Adults of a Parasitoid Wasp, Bracon hebetor Say

Document Type : Research Article


1 Gorgan University of Agricultural Sciences and Natural Resources, Gorgan,

2 Gorgan University of Agricultural Sciences and Natural Resources


Introduction: Bracon hebetor Say is a well-known cosmopolitan ectoparasitoid that attacks larvae of the various lepidopteran pests, in both grain storage and field conditions. This parasitoid mass-reared on Anagasta kuehniella (Zeller) larvae in many insectaries of Iran and released annually into cotton, soybean, tomato and maize fields especially to control Helicoverpa armigera (Hübner) in augmentative biological control programs. Cold storage has been considered as an important part in augmentative biological control programs. This technique enables insectaries to store a sufficient number of biocontrol agents for a prolonged period, thus allowing the release of natural enemies concurrent with the critical stages of the pest and minimizing the cost of insect colony maintenance when they are not in demand. In an effort to improve the techniques used in the mass rearing and release of this parasitoid we assessed the storage feasibility of pupa and adult stages of this wasp at a low temperature and the effects of  cold storage on its biological and reproductive parameters.
Materials and Methods: B. hebetor adults were initially obtained from a commercial insectarium in Gorgan, northern Iran and reared on Anagasta kuehniella (Zeller) larvae as factitious host for about five generations. Cold storage experiments were carried out on pupal and adult stages of parasitoid separately. Five hundred 5-days old pupae and five hundred couples of both sexes (female and male) of one-day-old adult parasitoids were collected from the colony and divided into ten groups of 50 parasitoids. Each group (considered as a replication) was released into plastic vials separately, fed adults for 24 hours with a 30% honey solution and then stored in a refrigerator (5±1˚C, and full darkness) for 7, 14, 21, 30, 45, and 60 days. Vials of non-refrigerated control groups were kept at 26±1˚C, 60±5% RH, and a photoperiod of 16L: 8D h. After each storage period, the pupa eclosion percent and adult mortality percent were subsequently assessed. To evaluate the effect of cold storage on longevity and fecundity of parasitoid, 30 female-male pairs were chosen randomly from the emerging adults (in pupae cold storage experiment) or surviving adults (in adults cold storage experiment), and each pair was placed in a plastic vial separately. Daily, the paired wasps in each container were provided 10 last instars A.kuehniella larvae on a piece of paper, as well as several drops of honey solution. The number of eggs laid was recorded on a daily basis until the female parasitoids died. To evaluate the effect of cold storage on parasitoid performance in the next (F1) generation, 50 newly laid eggs of parasitoid were removed from parasitized host larvae, placed in 10 cm diameter petri dishes individually (one egg/dish) and allowed to develop to adult stage. Developmental time (the period from egg to adult emergence) was recorded for male and female progeny separately and sex ratio (female percentage) of emerging adults was then determined. All experiments were carried out using a completely randomized design (CRD) and data were analyzed using one-way ANOVA and LSD test.
Results and Discussion: Eclosion rate of cold-exposed pupae decreased significantly with increasing cold storage duration. After 7 days of cold storage, about 93 percent of pupae were emerged, whereas all pupae died and no adult wasp was emerged, after 30 days of cold storage. However, cold storage of pupae even for a short duration (e.g. 7 days), had a significant adverse effects on longevity, fecundity and sex ratio of emerged adult parasitoids and means of these parameters were reduced by 55.8, 53.4 and 20%, respectively, compared to unstored control pupae. In adult cold storage experiment, survival rate of both male and female parasitoids was reduced significantly, according to cold duration. Adult mortality percent increased with storage duration and reached 100 and 97.14 % in 60 days storage of male and female parasitoids, respectively. One week storage at 5˚C resulted in 4.42 percent mortality in females which was not significantly different from that of the control. Cold storage duration had no adverse effect on the longevity of both male and female parasitoids. Reproductive parameters including mean of daily oviposition (eggs/day/female) and clutch size (eggs/larva) were also not significantly affected by cold storage durations. Low temperature storage of parental parasitoids had no significant effect on developmental time (the period from egg to adult emergence) and sex ratio in the F1 generation. Developmental time of progeny had a little variation among the treatments and changed from 11.9 to12.77 days in male and 12.07 to 12.8 days in female progeny.
Conclusion: Cold storage of pupae even for a short period, reduced significantly their eclosion rate and longevity and fecundity of adults emerged from these cold-exposed pupae. Cold storage of adult B. hebetor, affected negatively their survival, whereas other fitness traits such as longevity, fecundity, developmental time and sex ratio were not significantly reduced after cold storage. In conclusion, B. hebetor pupae is not recommended to be stored at low temperature, even for a short period but adult parasitoids can be cold stored for up to 7 days, with negligible mortality (4.41 %). The results of this study can be used in parasitoid mass rearing and cold storing in insectaries.


- Afshari A., and Nazari Fandokht E. 2019. Effect of sugar concentration and feeding duration on the cold tolerance of Bracon hebetor Say adults. Plant Pest Research 8(4): 55-690. (In Persian with English abstract)
2- Alam M.S., Alam M.Z., Alam S.N., Miah M.R.U., and Mian M.I.H. 2016. Effect of storage duration on the stored pupae of parasitoid Bracon hebetor (Say) and its impact on parasitoid quality. Bangladesh Journal of Agricultural Research 41(2): 297-310.
3- Aliabadi A. 2015. Feeding and mating effects on cold-storage efficacy of Bracon hebetor Say (Hymenoptera: Braconidae). M.Sc. Thesis. Gorgan University of Agricultural Sciences and Natural Resources. (In Persian with English abstract)
4- Al-Tememi N.K., and Ashfaq M. 2005. Effect of low temperature storage on the fecundity and parasitizing efficacy of Bracon hebetor (Say). Journal of Agricultural Research 43(2): 155-160.
5- Amice G., Vernon P., Outreman Y., van Alphen J., van and Baaren J. 2008. Variability in responses to thermal stress in parasitoids. Ecological Entomology 33: 701-708.
6- Amir-Maafi M., and Chi H. 2006. Demography of Habrobracon hebetor (Hymenoptera: Braconidae) on two pyralid hosts (Lepidoptera: Pyralidae). Annals of the Entomological Society of America 99: 84-90.
7- Anwar M., Zain ul Abdin Abbas S.K., Tahir M., Hussain F., and Manzoor A. 2016. Effect of cold storage on the survival, sex ratio and longevity of ectoparasitoid, Bracon hebetor (Say) (Hymenoptera: Braconidae). Pakistan Journal of Zoology 48(6): 1775-1780.
8- Attaran M.R. 1996. Effect of laboratory hosts on biological attributes of parasitoid wasp Bracon hebetor Say. M.S. thesis, Tarbiat Modarres University, Iran. (In Persian with English abstract)
9- Ayvaz A., Karasu E., Karaböِrklü S., and Tunçbilek A.S. 2008. Effects of cold storage, rearing temperature, parasitoid age and irradiation on the performance of Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae). Journal of Stored Products Research 44: 232-240.
10- Bayram A., Ozcan H., and Kornosor S. 2005. Effect of cold storage on the performance of Telenomus busseolae Gahan (Hymenoptera: Scelionidae), an egg parasitoid of Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae). Biological Control 35: 68-77.
11- Carrillo M.A., Heimpel G.E., Moon R.D., Cannon C.A., and Hutchison W.D. 2005. Cold hardiness of Habrobracon hebetor (Say) (Hymenoptera: Braconidae), a parasitoid of pyralid moths. Journal of Insect Physiology 51: 759-768.
12- Chen H., Opit G.P., Sheng P., and Zhang H. 2011. Maternal and progeny quality of Habrobracon hebetor Say (Hymenoptera: Braconidae) after cold storage. Biological Control 58: 255-261.
13- Chen H., Zhang H., Zhu K.Y., and Throne J. 2013. Performance of diapausing parasitoid wasps, Habrobracon hebetor, after cold storage. Biological Control 64: 186-194.
14- Chen M., Han Z., and Wang R. 2005. A preliminary study on effects of cold storage on pupae of Aphidius gifuensus Ashmead. Plant Protection 31: 41-43.
15- Chen W.L., Leopold R.A., and Harris M.O. 2008. Cold storage effects on maternal and progeny quality of Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae). Biological Control 46: 122-132.
16- Colinet H., and Boivin G. 2011. Insect parasitoids cold storage: A comprehensive review of factors of variability and consequences. Biological Control 58: 83-95.
17- Colinet H., and Hance T. 2010. Interspecific variation in the response to low temperature storage in different aphid parasitoids. Annals of Applied Biology 156: 147-156.
18- Coudron T.A., Ellersieck M.R., and Shelby K.S. 2007. Influence of diet on long-term cold storage of the predator Podisus maculiventris (Say) (Heteroptera: Pentatomidae). Biological Control 42: 186-195.
19- Desneux N., Pham-Delegue M.H., and Kaiser L. 2004. Effects of sub-lethal and lethal doses of lambda-cyhalothrin on oviposition experience and host-searching behavior of a parasitic wasp, Aphidius ervi. Pest Management Science 60: 381-389.
20- Farghaly H.T., and Ragab Z.A. 1993. Effect of low-temperature storage on pupae of Bracon hebetor Say (Hymenoptera: Braconidae). Bulletin of Faculty of Agriculture, Cairo University, 44: 697-706.
21- Foerster L.A., Doetzer A.K., and de Castro L.C.F. 2004. Emergence, longevity and fecundity of Trissolcus basalis and Telenomus podisi after cold storage in the pupal stage. Pesquisa Agropecuaria Brasileira 39: 841-845.
22- Frere I., Balthazar C., Sabri A., and Hance T. 2011. Improvement in the cold storage of Aphidius ervi (Hymenoptera: Aphidiinae) Europen Journal of Environmental Sciences 1: 33-40.
23- Ghimire M.N., and Phillips T.W. 2010. Suitability of different lepidopteran host species for development of Bracon hebetor (Hymenoptera: Braconidae). Environmental Entomology 39(2): 449-458.
24- Ghimire M.N., and Phillips T.W. 2010. Mass rearing of Habrobracon hebetor Say (Hymenoptera: Braconidae) on larvae of the Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae): effects of host density, parasitoid density, and rearing containers. Journal of Stored Products Research 46: 214-220.
25- Hanna A.D. 1935. Fertility and tolerance of low temperature in Euchalcidia carybori Hanna (Hymenoptera: Chalcidinae). Bulletin of Entomological Research 26: 315-322.
26- Huang X.F. 1986. Use of Habrobracon hebetor Say in granary pest control. Chinese Journal of Biological Control 2: 78-80.
27- Ismail M., Vernon P., Hance T., and van Baaren J. 2010. Physiological costs of cold exposure on the parasitoid Aphidius ervi, without selection pressure and under constant or fluctuating temperatures. BioControl 55: 729-740.
28- Johnson J.A., Valero K.A., Hannel M.M., and Gill R.F. 2000. Seasonal occurrence of postharvested dried fruit insects and their parasitoids in a culled fig house. Journal of Economic Entomology 93: 1380-1390.
29- Kostal V., Vambera J., and Bastl J. 2004. On the nature of pre-freeze mortality in insects: water balance, ion homeostasis and energy charge in the adults of Pyrrhocoris apterus. Journal of Experimental Biology 207: 1509-1521.
30- Leopold R.A. 1998. Cold storage on insects for integrated pest management. In: Hallman, G.J., Denlinger, D.L. (Eds.), Temperature Sensivity in Insects and Application in Integrated Pest Management. Westview Press, Boulder, pp. 235-267.
31- Leopold R.A. 2007. Colony maintenance and mass-rearing: using cold storage technology for extending shelf-life of insects. In: Vreysen, M.J.B., Robinson, A.S., and Hendrichs, J. (Eds.). Area-Wide Control of Insect Pests. Springer, Netherlands 149-162.
32- Mahi H., Rasekh A., Michaud J.P., and Shishehbor P. 2014. The biology of Lysiphlebus fabarum (Braconidae, Aphidiinae) following cold storage of larvae and pupae. Entomologia Expermentalis et Applicata 153: 10-19.
33- Mahi H., Rasekh A., and Shishehbor P. 2016. The effects of constant and fluctuating thermal regimes for reducing chill injuries during cold storage of late-instar larva of Lysiphlebus fabarum (Hym., Braconidae). Journal of Plant Protection 30: 270-283. (In Persian with English abstract)
34- Mahi H., Rasekh A., and Shishehbor P. 2014. Cold storage of bisexual population of Lysiphlebus fabarum (Braconidae: Aphidiinae). Plant Protection (Scientific Journal of Agriculture) 37 (2): 66-78. (In Persian with English abstract)
35- Mansour A.N. 2017. Influence of cold storage on some biological aspects of the gregarious parasitoid, bracon hebetor (Say) (hymenoptera: Braconidae). Egyptian Journal of Biological Pest Control 27: 205-210.
36- Mousapour Z., Askarianzadeh A., and Abbasipour H. 2014. Effect of cold storage of pupae parasitoid wasp, Habrobracon hebetor (Say) (Hymenoptera: Braconidae), on its efficiency. Archives of Phytopathology and Plant Protection 47(8): 966-972.
37- Mousapour Z., Askarianzadeh A., and Abbasipour H. 2015. Cold storage of adult parasitoid wasp, Habrobracon hebetor (Say) (Hymenoptera: Braconidae) and the flour moth larvae, Anagasta kuehniella (Zeller) at 12°C. Plant Pest Research 5(3): 17-29. (In Persian with English abstract)
38- Nadeem S., Ashfaq M., Hamed M., and Ahmed S. 2010. Optimization of short and long term storage duration for Trichogramma chilonis (Ishii) (Hymenoptera: Trichogrammatidae) at low temperatures. Pakistan Journal of Zoology 42(1): 63-67.
39- Najafi Navaei I., Taghizadeh M., Javanmoghaddam H., Oskoo T., and Attaran M.R. 2002. Efficiency of parasitoid wasps, Trichogramma pintoii and Habrobracon hebetor against Ostrinia nubilalis and Helicoverpa sp. on maize in Moghan. Proceedings of the 15th Iranian Plant Protection Congress. Razi University of Kermanshah, Iran, p.327.
40- Okine J.S., Mitchell E.R., and Hu G.Y. 1996. Low temperature effect on viability of Diadegma insulare (Hymenoptera: Ichneumonidae) pupae and effect of this parasitoid on feeding rate of diamondback moth larvae (Lepidoptera: Plutellidae). Florida Entomologist 79: 503-509.
41- Ozder N. 2004. Effect of different cold storage periods on parasitization performance of Trichogramma cacoeciae (Hymenoptera, Trichogrammatidae) on eggs of Ephestia kuehniella (Lepidoptera, Pyralidae). Biocontrol Science and Technology 14(5): 441-447.
42- Pitcher S.A., Haffmann M.P., Gardner J., Wright M.G., and Kuhar T.P. 2002. Cold storage of Trichogramma ostriniae reared on Sitotroga cerealella eggs. BioControl 47: 525-535.
43- Rundel B.J., Thomson L.J., and Hoffmann A.A. 2004. Effects of cold storage on field and laboratory performance of Trichogramma carverae (Hymenoptera: Trichogrammatidae) and the response of three Trichogramma spp. (T. carverae, T. nr. Brassicae, and T. funiculatum) to cold. Journal of Economic Entomology 97(2): 213-221.
44- Saadat D., Bandani A.R., and Dastranj M. 2014a. Comparison of the developmental time of Bracon hebetor (Hymenoptera: Braconidae) reared on five different lepidopteran host species and its relationship with digestive enzymes. European Journal of Entomology 111(4): 495-500.
45- Saadat D., Seraj A.A., Goldansaz S.H., and Karimzadeh J. 2014b. Environmental and maternal effects on host selection and parasitism success of Bracon hebetor. BioControl 59:297-306.
46- Salt R.W. 1961. Principles of insect cold-hardiness. Annual Revew of Entomology 6: 55-74.
47- SAS Institute Inc. 2002. SAS System for Windows Version 9.0, Cary, NC, USA.
48- Saxena H., Ponnusamy D., and Iquebal M.A. 2012. Seasonal parasitism and biological characteristics of Habrobracon hebetor (Hymenoptera: Braconidae): a potential larval ectoparasitoid of Helicoverpa armigera (Lepidoptera: Noctuidae) in a chickpea ecosystem. Biocontrol Science and Technology 22(3): 305-318.
49- Tezze A.A., and Botto E.N. 2004. Effect of cold storage on the quality of Trichogramma nerudai (Hymenoptera: Trichogrammatidae). Biological Control 30: 11-16.
50- Uwais A., Xu J.J., Yang X.R., He J., Tursun Guo W.C., Xu Y.Q., and Wei Y.Q. 2006. Preliminary test of controlling Helicoverpa armigera and Ostrinia furnacalis with Habrobracon hebetor in fields. Chinese Journal of Biological Control 22: 155-157.
51- Yazdanian M., Haddad Irani Nejad K., and Mashhadi Jafarloo M. 2005. Determining the number of larval instars of the Mediterranean flour moth, Anagasta kuehniella (Lepidoptera, Phycitidae) in laboratory conditions. Agricultural Science 15: 45-54. (In Persian with English abstract)