the effects of constant and fluctuating thermal regimes for reducing chill injuries during cold storage of late-instar larva of lysiphlebus fabarum (hym., braconidae)

introduction: mass production of natural enemies has been considered a necessity for biological control programs. most beneficial insects have a relatively short shelf-life, so suppliers do not have a sufficient number of biocontrol agents. the development of storage techniques for biocontrol agents provides flexibility and efficiency in mass production. cold storage by prolonging insect development times provides a steady and sufficient supply of insects and synchronizes a desired developmental stage of biocontrol agents with times of pest outbreaks; so it reduces the cost of biocontrol programs by enlarging the production period over several months. cold storage is usually associated with major fitness costs. even if the insects remain alive after cold storage, a reduction of fitness may be observed, so the production of high quality natural enemies must be ensured. developing effective methods is necessary in mass-producing. it has been reported that using fluctuating thermal regimes (ftr) (i.e. cold exposure interrupted by periodic short pulses of high temperature) versus constant thermal regimes (ctr) can progress the quality of biological control agents and significantly reduce rates of mortality. in this study, the impact of fluctuating thermal regimes versus constant low temperatures on the emergence rate, sex ratio, post-storage development time, adult size, egg load and egg size of the parasitoid wasp, lysiphlebus fabarum (marshall) was studied. the test was examined in three cold storage periods (1, 2, or 3 weeks). lysiphlebus fabarum is an aphidiine parasitoid which attacks more than 70 species of aphids. although both sexual (arrhenotokous) and asexual (thelytokous) populations of l. fabarum have been reported in iran, the former population has been studied in this research. materials and methods: a stock colony of black bean aphid, aphis fabae scopoli (hemi., aphididae) was established from material collected in bean fields in khuzestan province, iran, in spring 2012. a sexual population of the parasitoid wasp, l. fabarum was collected from black bean aphids feeding on broad bean, vicia fabae l. the synchronous cohort of l. fabarum was produced on the 2nd instar of aphid (at 21±1 ºc, 50–60% r.h., and l14:d10 photoperiod), then 30 two-day-old mated females were introduced into aphid colony consisting of 200 2nd instar individuals. the females were removed after 10 hours and parasitized aphids were reared for 54 hours (±6) to complete development to the late-instar larvae. these parasitized aphids, which were harboring late-instar larvae of wasp, were subjected to 6 ºc (as constant temperature regime) or were stored at 6 ºc with 2 hours daily exposure to 21±1 ºc (as the fluctuating temperature regime). the experiment was replicated with three cold storage periods (1, 2, or 3 weeks) with a control (direct development at 21±1 ºc). after completion of each storage temperature treatment, insects were transferred back to 21 °c until the emergence of adult wasps. accordingly, the survival rate and the sex ratio (% females) were determined. the emerged wasps were then exposed to alcohol vapor for 2 minutes. the hind tibia of both gender was measured. subsequently, the ovaries of each female were dissected to determine the egg load and egg size. results and discussion: according to results in ctr, survival of larvae was significantly reduced after one week of storage, and there was a gradual decrease in the second week, but there was no difference between survival of wasps that were stored after one and two weeks of storage. in ftr, emergence rate was decreased after one and two weeks of storage in comparison to the control, and the least survival was observed in the third week. when the survival was compared between different thermal regimes, ftr increased the survival in the first and second weeks, whereas after three weeks of storage there was no significant difference between two thermal regimes. in ctr, the least sex ratio of wasps was observed in those stored for two weeks, although there was no significant variation in sex ratios for control, one week and three week of storage. in ftr, cold storage did not affect on sex ratios of treatments. when the sex ratio was compared between different thermal regimes, for two and three weeks of storage, it was significantly higher in ftr and ctr, respectively. in females and males, larval storage increased development time relative to controls, in ctr. while in ftr, developmental time was decreased after two and three weeks of storage, in ftr. when developmental time was compared between two thermal regimes, post-storage development was slower in ctr after two and three weeks of storage in comparison to ftr, in both genders. in both thermal regimes, cold storage of wasps as larvae resulted in smaller male and female wasps relative to controls over all the storage periods. egg load and egg size at emergence were progressively diminished as a function of the storage period, regardless of the thermal regimes. egg load of wasps stored in ctr compared with ftr, was significantly higher for three weeks of storage. for all storage periods, egg size was significantly higher in ftr than ctr. conclusion: collectively, our results confirmed the beneficial effect of ftr relative to ctr, when larvae of l. fabarum were stored for two weeks at 6 °c. although ftr reduced the development time, which could be a problem during delivery for mass release of natural enemies in the field, but the emergence rate, the sex ratio, the body size and the egg size were significantly higher in ftr than those in ctr. for three weeks of storage, ftr was more stressful than the ctr, as the wasps that were stored in ctr had significantly more post-storage development, sex ratio, body size and egg load than the fluctuating ones.